Handbook of Fermented Meat and Poultry Meat and Poultry Handbook of Fermented

An internationally respected editorial team and array of chapter contributors have developed the Handbook of Fermented Meat and Poultry, an updated and comprehensive hands-on reference book on the science and technology of processing fermented meat Handbook of and poultry products. Beginning with the principles of processing fermented meat and ending with discussions of product quality, safety, and consumer acceptance, the book takes three approaches: background and principles, product categories, and product quality and safety. Fermented The historical background on the fermentation of meat and poultry products is followed by a series of discussions on their science and technology: curing, fermentation, drying and smoking, basic ingredients (raw product, additives, spices, and casings), and starter cultures. Coverage of product categories details the science and technology of making Meat and various fermented meat and poultry products from different parts of the world, including: semidry-fermented sausages (summer sausage), dry-fermented sausages (salami), sausages from other meats, and ripened meat products (ham). Poultry Product quality and safety is probably the most important aspect of making fermented meat and poultry because it addresses the question of consumer acceptance and public health safety. While a processor may produce a wonderful sausage, the product must ultimately satisfy the consumer in terms of color, texture, taste, flavor, packaging, and so on. In the current political and social climate, food safety has a high priority. Coverage includes issues such as spoilage microorganisms, pathogens, amines, toxins, HACCP, Fidel Toldrá and disease outbreaks. Editor

About the Editor associate Editors Fidel Toldrá, Ph.D., is a Research Professor and Head of the Meat Science Laboratory, Instituto de Agroquímica y Tecnologia de Alimentos (CSIC), Spain. Dr. Toldrá has co-edited and/or authored more Y. H. Hui than 14 books in food chemistry and food biochemistry, food flavors, and meat and poultry processing. Fidel Toldrá Iciar Astiasarán Associate Editors Editor Wai-Kit Nip Joseph G. Sebranek Y. H. Hui, Ph.D., Science Technology System, West Sacramento, California Hui Iciar Astiasarán, Ph.D., Expedito-Tadeu F. Silveira University of Navarra, Pamplona, Spain Astiasarán Wai-Kit Nip, Ph.D., University of Hawaii at Manoa, Honolulu, Hawaii Nip Louise H. Stahnke Joseph G. Sebranek, Ph.D., Iowa State University, Ames, Iowa Sebranek Régine Talon Expedito-Tadeu F. Silveira, Ph.D., Instituto de Tecnología de Alimentos, Campinas – SP, Brazil Silveira Louise H. Stahnke, Ph.D., Chr. Hansen A/S, Hørsholm, Denmark Stahnke Régine Talon, Ph.D., Institut National de la Recherche Agronomique, Saint-Genès Champanelle, France Talon

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 Handbook of Fermented 15 16 17 Meat and Poultry 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 S54 N55 40454 FM 00i-xviii r3.qxd 7/21/07 2:04 PM Page iii

1 2 3 4 5 6 7 8 9 10 11 12 13 14 Handbook of Fermented 15 16 17 Meat and Poultry 18 19 20 21 22 Editor 23 Fidel Toldrá 24 25 26 Associate Editors 27 Y. H. Hui 28 29 Iciar Astiasarán 30 Wai-Kit Nip 31 32 Joseph G. Sebranek 33 Expedito-Tadeu F. Silveira 34 35 Louise H. Stahnke 36 Régine Talon 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 S54 N55 40454 FM 00i-xviii r3.qxd 7/21/07 2:04 PM Page iv

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Fidel Toldrá, Ph.D., is a Research Professor and Head of the Laboratory of Meat Science, Instituto de Agroquímica y Tecnologia de Alimentos (CSIC), Valencia, Spain. Dr. Toldrá has co-edited and/or authored more than 14 books in 16 food chemistry and food biochemistry, food technology, and meat and poultry processing. 17 18 ©2007 Blackwell Publishing 19 All rights reserved 20 21 Blackwell Publishing Professional 22 2121 State Avenue, Ames, Iowa 50014, USA 23 24 Orders: 1-800-862-6657 25 Ofﬁce: 1-515-292-0140 Fax: 1-515-292-3348 26 Web site: www.blackwellprofessional.com 27 28 Blackwell Publishing Ltd 29 9600 Garsington Road, Oxford OX4 2DQ, UK 30 Tel.: +44 (0)1865 776868 31 32 Blackwell Publishing Asia 33 550 Swanston Street, Carlton, Victoria 3053, Australia 34 Tel.: +61 (0)3 8359 1011 35 Authorization to photocopy items for internal or personal use, or the internal or personal use of speciﬁc clients, is granted 36 by Blackwell Publishing, provided that the base fee is paid directly to the Copyright Clearance Center, 222 Rosewood 37 Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license by CCC, a separate 38 system of payments has been arranged. The fee codes for users of the Transactional Reporting Service is 39 ISBN-13: 978-0-8138-1477-3/2007. 40 41 First edition, 2007 42 43 Library of Congress Cataloging-in-Publication Data 44 Handbook of fermented meat and poultry/editor, Fidel Toldrá; associate editors, Y. H. Hui ... [et al.].—1st ed. 45 p. cm. 46 Includes bibliographical references and index. 47 ISBN 13: 978-0-8138-1477-3 (alk. paper) 48 ISBN 10: 0-8138-1477-4 (alk. paper) 49 1. Fermented foods—Handbooks, manuals, etc. 2. Meat—Preservation—Handbooks, manuals, etc. 3. Fermentation—Handbooks, manuals, etc. I. Toldrá, Fidel. II. Hui, Y. H. (Yiu H.) 50 51 TP371.44.H357 2008 52 664'.9028—dc22 53S 2007013113 54N The last digit is the print number: 9 8 7 6 5 4 3 2 1 40454 FM 00i-xviii r3.qxd 7/21/07 2:04 PM Page v

1 2 3 Contents 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Contributors List ix 22 Preface xvii 23 24 Part I. Meat Fermentation Worldwide: History and Principles 25 1. A Historical Perspective of Meat Fermentation 3 26 Peter Zeuthen 27 2. Production and Consumption of Fermented Meat Products 9 28 Herbert W. Ockerman and Lopa Basu 29 3. Principles of Curing 17 30 Karl O. Honikel 31 4. Principles of Meat Fermentation 31 32 Esko Petäjä-Kanninen and Eero Puolanne 33 5. Principles of Drying and Smoking 37 34 Ana Andrés, José M. Barat, Raúl Grau, and Pedro Fito 35 36 Part II. Raw Materials 37 6. Biochemistry of Meat and Fat 51 38 Fidel Toldrá 39 7. Ingredients 59 40 Jorge Ruiz 41 8. Additives 77 42 Pedro Roncalés 43 9. Spices and Seasonings 87 44 Suey-Ping Chi and Yun-Chu Wu 45 10. Casings 101 46 Yun-Chu Wu and Suey-Ping Chi 47 48 Part III. Microbiology and Starter Cultures for Meat Fermentation 49 11. Microorganisms in Traditional Fermented Meats 113 50 Isabelle Lebert, Sabine Leroy, and Régine Talon 51 12. The Microbiology of Fermentation and Ripening 125 52 Margarita Garriga and Teresa Aymerich 53 S54 N55 v 40454 FM 00i-xviii r3.qxd 7/21/07 2:04 PM Page vi

vi Contents

1 13. Starter Cultures: Bacteria 137 2 Pier Sandro Cocconcelli 3 14. Starter Cultures: Bioprotective Cultures 147 4 Graciela Vignolo and Silvina Fadda 5 15. Starter Cultures: Yeasts 159 6 M-Dolores Selgas and M-Luisa Garcia 7 16. Starter Cultures: Molds 171 8 Elisabetta Spotti and Elettra Berni 9 17. Genetics of Microbial Starters 177 10 Marie Champomier-Vergès, Anne-Marie Crutz-Le Coq, Monique Zagorec, 11 Sabine Leroy, Emilie Dordet-Frisoni, Stella Planchon, and Régine Talon 12 18. Inﬂuence of Processing Parameters on Cultures Performance 187 13 Louise H. Stahnke and Karsten Tjener 14 15 Part IV.Sensory Attributes 16 19. General Considerations 197 17 Asgeir Nilsen and Marit Rødbotten 18 20. Color 203 19 Jens K. S. Møller and Leif H. Skibsted 20 21. Texture 217 21 Shai Barbut 22 22. Flavor 227 23 Karsten Tjener and Louise H. Stahnke 24 25 26 Part V.Product Categories: General Considerations 27 23. Composition and Nutrition 243 28 Daniel Demeyer 29 24. Functional Meat Products 257 30 Diana Ansorena and Iciar Astiasarán 31 25. International Standards: USA 267 32 Melvin C. Hunt and Elizabeth Boyle 33 26. International Standards: Europe 273 34 Reinhard Fries 35 27. Packaging and Storage 289 36 Dong U. Ahn and Byungrok Min 37 38 Part VI. Semidry-fermented Sausages 39 28. U.S. Products 303 40 Robert E. Rust 41 29. European Products 307 42 Kálmán Incze 43 44 45 Part VII. Dry-fermented Sausages 46 30. Dry-fermented Sausages: An Overview 321 47 Fidel Toldrá, Wai-Kit Nip, and Y. H. Hui 48 31. U.S. Products 327 49 Robert Maddock 50 32. Mediterranean Products 333 51 Juan A. Ordóñez and Lorenzo de la Hoz 52 33. North European Products 349 53S Jürgen Schwing and Ralf Neidhardt 54N 40454 FM 00i-xviii r3.qxd 7/21/07 2:04 PM Page vii

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Part VIII. Other Fermented Meats and Poultry 1 34. Fermented Poultry Sausages 361 2 Sunita J. Santchurn and Antoine Collignan 3 35. Fermented Sausages from Other Meats 369 4 Halil Vural and Emin Burçin Özvural 5 6 Part IX. Ripened Meat Products 7 36. U.S. Products 377 8 Kenneth J. Stalder, Nicholas L. Berry, Dana J. Hanson, and William Mikel 9 37. Central and South American Products 387 10 Silvina Fadda and Graciela Vignolo 11 38. Mediterranean Products 393 12 Mario Estévez, David Morcuende, Jesús Ventanas, and Sonia Ventanas 13 39. North European Products 407 14 Torunn T. Håseth, Gudjon Thorkelsson, and Maan S. Sidhu 15 40. Asian Products 415 16 Guang-Hong Zhou and Gai-Ming Zhao 17 18 Part X. Biological and Chemical Safety of Fermented Meat Products 19 41. Spoilage Microorganisms: Risks and Control 421 20 Jean Labadie 21 42. Pathogens: Risks and Control 427 22 Panagiotis Skandamis and George-John E. Nychas 23 43. Biogenic Amines: Risks and Control 455 24 M. Carmen Vidal-Carou, M. Teresa Veciana-Nogués, M. Luz Latorre-Moratala, 25 and Sara Bover-Cid 26 44. Chemical Origin Toxic Compounds 469 27 Fidel Toldrá and Milagro Reig 28 45. Disease Outbreaks 477 29 Colin Pierre 30 31 Part XI. Processing Sanitation and Quality Assurance 32 46. Basic Sanitation 483 33 Stefania Quintavalla and Silvana Barbuti 34 47. Processing Plant Sanitation 491 35 Jordi Rovira and Dorota Puszczewicz 36 48. Quality Control 503 37 Fidel Toldrá, M-Concepción Aristoy, Mónica Flores, and Miguel A. Sentandreu 38 49. HACCP 513 39 Maria Joao Fraqueza, Antonio S. Barreto, and Antonio M. Ribeiro 40 50. Quality Assurance Plan 535 41 Friedrich-Karl Lücke 42 43 Index 545 44 45 46 47 48 49 50 51 52 S53 N54 40454 FM 00i-xviii r3.qxd 7/21/07 2:04 PM Page ix

1 2 3 Contributors List 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 EDITOR 1955 East-West Road 22 Honolulu, HI 96822, USA 23 Fidel Toldrá, Ph.D. Phone: 1-808-955-6942 24 Instituto de Agroquímica y Tecnología de Email: [email protected] 25 Alimentos (CSIC) 26 P.O. Box 73, 46100 Burjassot 27 Joseph G. Sebranek, Ph.D. Valencia, Spain 28 Iowa State University Phone: +34 96 3900022, ext. 2112 29 Department of Food Science and Fax: +34 96 3636301 30 Human Nutrition E-mail: [email protected] 31 2312 Food Sciences Building 32 Ames, IA 50011, USA ASSOCIATE EDITORS 33 Phone: 1-515-294-1091 34 Iciar Astiasarán, Ph.D. Fax: 1-515-294-5066 35 University of Navarra E-mail: [email protected] Faculty of Pharmacy 36 Department of Food Science, Technology 37 and Toxicology Expedito-Tadeu F. Silveira, Ph. D. 38 C/Irunlarrea s/n, 31080 Pamplona, Spain Instituto de Tecnología de Alimentos 39 Phone: +34 948 425600, ext. 6405 Centro de Pesquisa e Desenvolvimento de 40 Fax: +34 948 425649 Carnes—CTC/ITAL Av. Brasil 41 E-mail: [email protected] 2880—Caixa Postal 139—CEP 42 13073-001 Campinas—SP, Brazil 43 Phone: +55 19 3743 1886 44 Y. H. Hui, Ph.D. Fax: +55 19 3743 1882 45 Science Technology System E-mail: [email protected] 46 P.O. Box 1374, West Sacramento, CA 95691, USA 47 Phone: 1-916-372-2655 48 Fax: 1-916-372-2690 Louise H. Stahnke, Ph.D. 49 E-mail: [email protected] Chr. Hansen A/S 50 Boege Allé 10-12 51 Wai-Kit Nip, Ph.D. DK-2970 Hoersholm, Denmark 52 University of Hawaii at Manoa Phone: +45 45 74 74 74 53 Department of Molecular Biosciences & Fax: +45 45 74 88 16 S54 Bioengineering E-mail: [email protected] N55 ix 40454 FM 00i-xviii r3.qxd 7/21/07 2:04 PM Page x

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1 Régine Talon, Ph. D. E-17121 Monells (Girona), Spain 2 Unité Microbiologie Phone: +34 972 630052 3 Institut National de la Recherche Fax: +34 972 630373 4 Agronomique (INRA) E-mail: [email protected] 5 Centre de Clermont-Ferrand-Theix 6 63122 Saint-Genès Champanelle, France José M. Barat 7 Phone: +33 4 73 62 41 70 Polytechnical University of Valencia 8 Fax: +33 4 73 62 42 68 Institute of Engineering for 9 E-mail: [email protected] Food Development 10 P.O. Box 22012 11 Camino de Vera s/n, 46020 Valencia, Spain 12 CONTRIBUTORS Phone: +34 96 3877365 13 Dong U.Ahn Fax: +34 96 3877956 14 Iowa State University E-mail: [email protected] 15 2276 Kildee Hall 16 Ames, IA 50011-3150, USA Shai Barbut 17 Phone: 1-515-294-6595 University of Guelph 18 Fax: 1-515-294-9143 Department of Food Science 19 E-mail: [email protected] Guelph, Ontario, CAN 1G2W1 20 Phone: +1519 824 4120, ext. 53669 21 Ana Andrés Fax: +1519 824 6631 22 Polytechnical University of Valencia E-mail: [email protected] 23 Institute of Engineering for 24 Food Development Silvana Barbuti 25 P.O. Box 22012, Camino de Vera s/n Department of Microbiology, SSICA 26 46020 Valencia, Spain Viale Tanara 31/A, 43100 Parma, Italy 27 Phone: +34 96 3877364 Phone: +39 0521 795 267 28 Fax: +34 96 3877956 Fax: +39 0521 771829 29 E-mail: [email protected] E-mail: [email protected] 30 31 Diana Ansorena Antonio S. Barreto 32 University of Navarra FMV, Av. da Universidade, Universidade Tecnica, Polo 33 Faculty of Pharmacy Universitário Alto da Ajuda 34 Department of Food Science, Technology 1300-477 Lisboa, Portugal 35 and Toxicology Phone: +35 121 365 2880 36 C/Irunlarrea s/n, 31080 Pamplona, Spain Fax: +35 121 365 2880 37 Phone: +34 948 425600, ext. 6405 E-mail: [email protected] 38 Fax: +34 948 425649 39 E-mail: [email protected] Lopa Basu 40 The Ohio State University 41 M-Concepción Aristoy 015 Animal Science Building 42 Instituto de Agroquímica y Tecnología de 2029 Fyffe Road, 43 Alimentos (CSIC) Columbus, OH 43210, USA 44 Department of Food Science Phone: 1-614-292-4317 45 P.O. Box 73 Fax: 1-614-292-2929 46 46100 Burjassot E-mail: [email protected] 47 Valencia, Spain 48 Phone: +34 96 3900022, ext. 2103 Elettra Berni 49 Fax: +34 96 3636301 Stazione Sperimentale per L’Industria delle Conserv 50 E-mail: [email protected] Viale F. Tanara 31/A 51 43100 Parma, Italy 52 Teresa Aymerich Phone: +39 0521 1795262 53S Centre de Tecnologia de la Carn, IRTA Fax: +39 0521 1771829 54N Finca Camps i Armet s/n E-mail: [email protected] 40454 FM 00i-xviii r3.qxd 7/21/07 2:04 PM Page xi

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Nicholas L. Berry 97408 Saint Denis cedex 9, France 1 Iowa State University Phone: +33 02 62 92 24 47 2 Department of Animal Science Fax: +33 02 62 92 24 31 3 109 Kildee Hall, Ames, IA 50011-3151, USA E-mail: [email protected] 4 Phone: 1-515-295-4683 5 Fax: 1-515-294-5698 Anne-Marie Crutz-Le Coq 6 INRA Jouy-en-Josas 7 Sara Bover-Cid Domaine de Vilvert Unite Flore Lactique et 8 Universitat de Barcelona Environment Carné 9 Departament de Nutrició i Bromatologia 78350 Jouy-en-Josas, France 10 Facultat de FarmaciaAvinguda E-mail: [email protected] 11 Avinguda Joan XXIII s/n 12 08028 Barcelona, Spain Lorenzo de la Hoz 13 Phone: +34 93 402 45 13 University Complutense 14 Fax: +34 93 402 18 96 Department of Nutrition, Bromatology and 15 E-mail: [email protected] Food Technology 16 Faculty of Veterinary 17 Elizabeth Boyle Avda Puerta del Hierro s/n 18 Animal Sciences & Industry 28040 Madrid, Spain 19 224 Weber Hall, Manhattan, KS 66506, USA Phone: +34 913943744 20 Phone: 1-785-532-1232 Fax: +34 913943743 21 Fax: 1-785-532-7059 E-mail: [email protected] 22 E-mail: [email protected] 23 Daniel Demeyer 24 Marie Champomier-Vergès University of Ghent 25 INRA Jouy-en-Josas Department Animal Production 26 Domaine de Vilvert Unite Flore Lactique et Proefhoevestraat 10, B-9090 Melle, Belgium 27 Environment Carné Phone: +32 9 2649000 28 78350 Jouy-en-Josas, France Fax: +32 9 2649099 29 Phone: +33 01 34 65 22 89 E-mail: [email protected] 30 E-mail: Marie-Christine.Champomier- 31 [email protected] Emilie Dordet-Frisoni 32 INRA Clermont-Ferrand-Theix Unité Microbiologie 33 Suey-Ping Chi 63122 Saint-Genès Champanelle, France 34 Chung Hwa University of Phone: +33 04 73 62 41 70 35 Medical Technology Fax: +33 004 73 62 42 68 36 College of Human Science and Technology E-mail: [email protected] 37 Department of Restaurant and 38 Hospitality Management Mario Estévez 39 89, Wunhwa 1st Street University of Extremadura 40 Jen-Te County, Tainen, Taiwan Faculty of Veterinary 41 E-mail: [email protected] Ctra. Trujillo s/n, 10071 Cáceres, Spain 42 Phone: +34 927 257122 43 Pier Sandro Cocconcelli Fax: +927 257110 44 Universita Cattolica del Sacro Cuore E-mail: [email protected] 45 Istituto di Microbiologia 46 via Emilia Parmense 84, 29100 Piacenza, Italy Silvina Fadda 47 Phone: +39 523 599251 Centro de Referencia para Lactobacillos 48 Fax: +39 523 599246 CERELA, CONICET 49 E-mail: [email protected] Chacabuco 145 50 40000 Tucuman, Argentina 51 Antoine Collignan Phone: +54 381 4311720 52 Pôle agroalimentaire, CIRAD MRST Fax: +54 381 4005600 S53 Station de la Bretagne BP 20 E-mail: [email protected] N54 40454 FM 00i-xviii r3.qxd 7/21/07 2:04 PM Page xii

xii Contributors List

1 Pedro Fito Raúl Grau 2 Polytechnical University of Valencia Polytechnical University of Valencia 3 Institute of Engineering for Food Development Institute of Engineering for Food Development 4 P.O. Box 22012 P.O. Box 22012, Camino de Vera s/n 5 Camino de Vera s/n, 46020 Valencia, Spain 46020 Valencia, Spain 6 Phone: +34 96 3877364 Phone: +34 96 3877365 7 Fax: +34 96 3877956 Fax: +34 96 3877956 8 E-mail: pﬁ[email protected] E-mail: [email protected] 9 10 Mónica Flores Dana J. Hanson 11 Instituto de Agroquímica y Tecnología de North Carolina State University 12 Alimentos (CSIC) Food Science Department 13 Department of Food Science Raleigh, NC 27695-7624, USA 14 P.O. Box 73 E-mail: [email protected] 15 46100 Burjassot, Valencia, Spain 16 Phone: +34 96 3900022, ext. 2116 Torunn T. Haseth 17 Fax: +34 96 3636301 Norwegian Meat Research Centre 18 E-mail: mﬂ[email protected] P.O. Box 396 Økern 19 N-0513 Oslo, Norway 20 Maria Joao Fraqueza Phone: +47 22092399 21 FMV, Faculdade de Medicina Fax: +47 22220016 22 Veterinária de Lisboa E-mail: [email protected] 23 CIISA, DTIA, Rua Prof 24 Cid dos Santos, Polo Universitário Karl O. Honikel 25 Alto da Ajuda Institute for Chemistry and Physics 26 1300-477 Lisboa, Portugal Bundesforschungsanstalt für Ernährung 27 Phone: +35 121 3652880 EC Baumann-Strasse 20 28 Fax: +35 121 3652880 D-95326 Kulmbach, Germany 29 E-mail: [email protected] Phone: +49 9221 803200 30 Fax: +49 9221 803303 31 Reinhard Fries E-mail: [email protected] 32 Institut fur Fleischhygiene und Technologie 33 Meat Hygiene and Technology Melvin C. Hunt 34 Free University of Berlin Animal Sciences & Industry 35 Brummerstr, 10 224 Weber Hall 36 14195 Berlin, Germany Manhattan, KS 66506, USA 37 Phone: +49 83852791 Phone: 1-785-532-1232 38 Fax: +49 83852792 Fax: 1-785-532-7059 39 E-mail: [email protected] E-mail: [email protected] 40 41 M-Luisa García Kálmán Incze 42 University Complutense Hungarian Meat Research Institute 43 Dep. Nutrición Gubacsi ut 6/b 44 Bromatología y Tecnología de Alimentos 1097 Budapest, Hungary 45 28040 Madrid, Spain Phone: +36 1 2150946 46 Phone: +34 91 3943745 Fax: +36 1 2150626 47 Fax: +34 91 3943743 E-mail: [email protected] 48 49 Margarita Garriga Jean Labadie 50 Centre de Tecnologia de la Carn, IRTA INRA Clermont-Ferrand-Theix 51 Finca Camps i Armet s/n Unité Microbiologie 52 E-17121 Monells (Girona), Spain 63122 Saint-Genès Champanelle, France 53S Phone: +34 972 630052 Phone: +33 04.73.62.41.57 54N Fax: +34 972 630373 Fax: +33 04.73.62.42.68 E-mail: [email protected] E-mail: [email protected] 40454 FM 00i-xviii r3.qxd 7/21/07 2:04 PM Page xiii

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M. Luz Latorre-Moratalla Byungrok Min 1 Universitat de Barcelona Iowa State University 2 Departament de Nutrició 2276 Kildee Hall 3 i Bromatologia Ames, IA 50011-3150, USA 4 Facultat de Farmacia Phone: 1-515-294-6595 5 Avinguda Joan XXIII s/n Fax: 1-515-294-9143 6 08028 Barcelona, Spain E-mail: [email protected] 7 Phone: +34 93 402 45 13 8 Fax: +34 93 402 18 96 Jens K.S. Møller 9 University of Copenhagen 10 Isabelle Lebert Faculty of Life Science 11 Unité Microbiologie Department of Food Science, Food Chemistry 12 INRA Centre Clermont-Ferrand-Theix Rolighedsvej 30 13 63122 Saint-Genès Champanelle, France Frederiksberg C, 1958, Denmark 14 Phone: +33 04 73 62 41 70 E-mail: [email protected] 15 Fax: +33 04 73 62 42 68 16 E-mail: [email protected] David Morcuende 17 University of Extremadura, 18 Sabine Leroy Faculty of Veterinary 19 Unité Microbiologie Ctra. Trujillo s/n, 20 INRA Centre Clermont-Ferrand-Theix 10071 Cáceres, Spain 21 63122 Saint-Genès Champanelle, France Phone: +34 927 257122 22 Phone: +33 04 73 62 41 70 Fax: +34 927 257110 23 Fax: +33 04 73 62 42 68 24 E-mail: [email protected] 25 Ralf Neidhardt 26 Chr. Hansen GmbH Friedrich-Karl Lücke 27 Giessener Strasse 94 University of Applied Sciences 28 Pohlheim, D-35415 Germany FB Oecotrophologie 29 Phone: +49 6403 95010 Department of Household Management 30 Fax: +49 6403 9501 30 Nutrition and Food Quality 31 E-mail: [email protected] Fachhochschule Fulda Marquardstr. 35 32 D-36039 Fulda, Germany 33 Phone: +49 661 9640376 Asgeir Nilsen 34 Fax: +49 661 9640399 Matforsk AS, Osloveien 1 35 E-mail: [email protected] Aas, 1430, Norway 36 Phone: +47 64970100 37 Robert Maddock Fax: +47 64970333 38 North Dakota State University E-mail: [email protected] 39 Department of Animal & Range Sciences 40 Hultz 174 George-John E. Nychas 41 P.O. Box 5727, Fargo, North Dakota 58105, USA Agricultural University of Athens 42 Phone: 1-701-231-8975 Department of Food Science and Technology 43 Fax: 1-701-231-7590 Iera Odos 75 44 E-mail: [email protected] Athens 11855, Greece 45 Phone/fax: +30 210 5294693 46 William Mikel E-mail: [email protected] 47 Mississippi State University 48 Department Food Science, Nutrition and Herbert W. Ockerman 49 Health Promotion The Ohio State University 50 P.O. Box 9805 015 Animal Science Building 51 Mississippi State, MS 37962-9805, USA 2029 Fyffe Road, Columbus, OH 43210, USA 52 Phone: 1-662-325-5508 Phone: 1-614-292-4317 S53 Fax: 1-662-325-8728 Fax: 1-614-292-2929 N54 E-mail: [email protected] E-mail: [email protected] 40454 FM 00i-xviii r3.qxd 7/21/07 2:04 PM Page xiv

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1 Juan A. Ordoñez Stefania Quintavalla 2 University Complutense Department of Microbiology, SSICA 3 Department of Nutrition, Bromatology and Viale Tanara 31/A, 43100 Parma, Italy 4 Food Technology Phone: +39 0521 795 267 5 Faculty of Veterinary Fax: +39 0521 771829 6 Avda Puerta del Hierro s/n E-mail: [email protected] 7 28040 Madrid, Spain 8 Phone: +34 913943744 Milagro Reig 9 Fax: +34 913943743 Instituto de Agroquímica y Tecnología de 10 E-mail: [email protected] Alimentos (CSIC) 11 Department of Food Science 12 Emin Burçin Özvural P.O. Box 73 13 Hacettepe University 46100 Burjassot 14 Department of Food Engineering Valencia, Spain 15 Beytepe Ankara, 06532, Turkey Phone: +34 96 3900022, ext. 2111 16 Phone: +90 312 2977116 Fax: +34 96 3636301 17 Fax: +90 312 2992123 E-mail: [email protected] 18 Esko Petäjä-Kanninen 19 Antonio M. Ribeiro Viikki Food Science 20 FMV, AV. da Universidade, Universidade Department of Food Technology, Viikki EE 21 Tecnica, Polo Universitário Alta da Ajuda P.O. Box 66, FIN-00014 Helsinki, Finland 22 1300-477 Lisboa, Portugal Phone: +358 5 046 27402 23 Phone: +35 121 365 2880 Fax: +358 9 191 58460 24 Fax: +35 121 365 2880 25 Colin Pierre E-mail: [email protected] 26 AFSSA (site de Brest) 27 Technopôle Brest-Iroise, Pedro Roncalés 28 29280 Plouzane, France Department Productión Animal y 29 Phone: +33 (0)2 98 22 45 34 Ciencia de los Alimentas 30 Fax: +33 (0)2 98 05 51 65 Facultad de Veterinaria 31 E-mail: [email protected] University of Zaragoza 32 Miguel Servet 177 33 Stella Planchon 50013 Zaragoza, Spain 34 INRA Clermont-Ferrand-Theix Unité Microbiologie Phone: +34 976 761582 35 63122 Saint-Genès Champanelle, France Fax: +34 976 761612 36 Phone: +33 04 73 62 41 70 E-mail: [email protected] 37 Fax: +33 04 73 62 42 68 38 Jordi Rovira 39 Eero Poulanne Department of Biotechnology and 40 Viikki Food Science Food Science 41 Department of Food Technology Plaza Misael Bañuelos s/n 42 Viikki EE 09001 Burgos Spain 43 P.O. Box 66 Phone: +34 947 258814 44 FIN-00014 Helsinki, Finland Fax: +34 947 258831 45 Phone: +358 9 191 58458 E-mail: [email protected] 46 Fax: +358 9 191 58460 47 E-mail: eero.puolanne@helsinki.ﬁ 48 Marit Rødbotten 49 Dorota Puszczewicz Matforsk AS 50 Department of Biotechnology and Food Science Osloveien 1 51 Plaza Misael Bañuelos s/n Aas, 1430, Norway 52 09001 Burgos, Spain Phone: +47 64970100 53S Phone: +34 947 258814 Fax: +47 64970333 54N Fax: +34 947 258831 E-mail: [email protected] 40454 FM 00i-xviii r3.qxd 7/21/07 2:04 PM Page xv

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Jorge Ruiz Fax: +34 96 3636301 1 Tecnología de los Alimentos E-mail: [email protected] 2 Facultad de Veterinaria UEx 3 University of Extramadura Maan S. Sidhu 4 Campus Universitario Norwegian Meat Research Centre 5 10071 Cáceres, Spain P.O. Box 396 Økern 6 Phone: +927 257 122 N-0513 Oslo, Norway 7 Fax: +927 257 110 Phone: +47 22092399 8 E-mail: [email protected] Fax: +47 22220016 9 E-mail: [email protected] 10 Robert E. Rust 11 118 E. 16th St. 12 Panagiotis Skandamis 13 Ames, IA 50010-5561, USA Agricultural University of Athens Phone/fax: 1-515-232-1575 (H) 14 Department of Food Science & Technology 15 1-515-233-9207 (O) Iera Odos 75 1-515-450-0533 (M) 16 Athens 11855, Greece 17 E-mail: [email protected] Phone/fax: +30 210 5294693 [email protected] 18 E-mail: [email protected] 19 20 Sunita J. Santchurn Leif H. Skibsted 21 Pôle agroalimentaire, CIRAD MRST University of Copenhagen 22 Station de la Bretagne BP 20 Faculty of Life Science 23 97408 Saint Denis cedex 9, France Department of Food Science, 24 Phone: +33 02 62 92 24 47 Food Chemistry 25 Fax: +33 02 62 92 24 31 Rolighedsvej 30 26 E-mail: [email protected] Frederiksberg C, 1958, Denmark 27 Phone: +45 35 33 3212 28 Jürgen Schwing Fax: +45 35 33 3190 29 Chr. Hansen GmbH E-mail: [email protected] 30 Giessener Strasse 94 31 Pohlheim, D-35415 Germany Elisabetta Spotti 32 Phone: +49 6403 95010 Stazione Sperimentale per L’Industria delle Conserv 33 Fax: +49 6403 9501 30 Viale F. Tanara 31/A 34 E-mail: [email protected] 43100 Parma, Italy 35 Phone: +39 0521 1795262 36 37 M-Dolores Selgas Fax: +39 0521 1771829 38 University Complutense E-mail: [email protected] 39 Facultad de Veterinaria 40 Dep. Nutrición Kenneth J. Stalder 41 Bromatología y Tecnología de Alimentos Iowa State University 42 28040 Madrid, Spain Department of Animal Science 43 Phone: +34 91 3943745 109 Kildee Hall 44 Fax: +34 91 3943743 Ames, IA 50011-3151, USA 45 E-mail: [email protected] Phone: 1-515-295-4683 46 Fax: 1-515-294-5698 47 Miguel A. Sentandreu E-mail: [email protected] 48 Instituto de Agroquímica y Tecnología de 49 Alimentos (CSIC) Gudjon Thorkelsson 50 Department of Food Science Iceland Fisheries Laboratories 51 P.O. Box 73 Skulagata 4, 101 Reykjavik, Iceland 52 46100 Burjassot, Valencia, Spain Phone: +354 414 8087 S53 Phone: +34 96 3900022, ext. 2113 E-mail: [email protected] N54 40454 FM 00i-xviii r3.qxd 7/21/07 2:04 PM Page xvi

xvi Contributors List

1 Karsten Tjener Chacabuco 145 2 Chr. Hansen A/S 40000 Tucuman, Argentina 3 Boege Alle 10-12 Phone: +54 381 4311720 4 Hoersholm, 2970, Denmark Fax: +54 381 4005600 5 Phone: +45 45747474 E-mail: [email protected] 6 Fax: +45 45748816 7 E-mail: [email protected] Halil Vural 8 Hacettepe University Department of Food Engineering 9 M. Teresa Veciana-Nogués Beytepe Ankara, 06532, Turkey 10 Universitat de Barcelona Phone: +90 312 2977116 11 Departament de Nutrició i Bromatologia Fax: +90 312 2992123 12 Facultat de Farmacia E-mail: [email protected] 13 Avinguda Joan XXIII s/n 14 08028 Barcelona, Spain Yun-Chu Wu 15 Phone: +34 93 402 45 13 16 Tunghai University Fax: +34 93 402 18 96 College of Agriculture 17 E-mail: [email protected] 18 Taichung, Taiwan, ROC 19 Phone: +886 0 3573 1866 Jesús Ventanas 20 E-mail: [email protected] University of Extremadura 21 Faculty of Veterinary Monique Zagorec 22 Ctra. Trujillo s/n, 10071 Cáceres, Spain INRA Jouy-en-Josas, Domaine de Vilvert Unité 23 Phone: +34 927 257122 Flore Lactique et Environment Carné 24 Fax: +34 927 257110 78350 Jouy-en-Josas, France 25 E-mail: [email protected] Phone: +33 01 34 65 22 89 26 Fax: +33 01 34 65 21 05 27 Sonia Ventanas E-mail: [email protected] 28 University of Extremadura 29 Faculty of Veterinary Peter Zeuthen 30 Ctra. Trujillo s/n Hersegade 7G 31 10071 Cáceres, Spain Roskilde, DK 4000, Denmark 32 Phone: +34 927 257122 Phone: +45 46355665 33 Fax: +34 927 257110 E-mail: [email protected] 34 E-mail: [email protected] 35 Gai-Ming Zhao 36 Henan Agricultural University 37 M. Carmen Vidal-Carou College of Food Science and Technology 38 Universitat de Barcelona Zhengzhou 450002, China 39 Departament de Nutrició i Bromatologia Phone: +86 25 84395376 40 Facultat de Farmacia Fax: +86 25 84395939 41 Avinguda Joan XXIII s/n E-mail: [email protected] 42 08028 Barcelona, Spain 43 Phone: +34 93 402 45 13 Gian H. Zhou 44 Fax: +34 93 402 18 96 Nanjing Agricultural University 45 E-mail: [email protected] College of Food Science and Technology 46 Nanjing 210095, PR China 47 Graciela Vignolo Phone: +86 25 84395376 48 Centro de Referencia para Lactobacillos Fax: +86 25 84395939 49 CERELA, CONICET E-mail: [email protected] 50 51 52 53S 54N 40454 FM 00i-xviii r3.qxd 7/23/07 3:35 PM Page xvii

1 2 3 Preface 4 5 6 7 8 9 10 11 12 13 14 15 16 Fermented meat products have been consumed for This handbook provides an updated and compre- 17 centuries in many different parts of the world and hensive overview of meat fermentation. It includes 18 constitute one of the most important groups of food. important developments that have occurred during the 19 Based on the natural meat flora, a wide range of last few decades including the role of microorganisms 20 products have been prepared since ancient times by naturally present or added as starter cultures; impor- 21 varying the mixture of meats and salt as well as the tant safety aspects in today’s world; a description of 22 addition of spices and seasonings. Thus, fermented the primary chemical, biochemical, physical, and 23 meat products represent a great variety of flavors and microbiological changes that occur during process- 24 textures and are receiving increased interest from ing; and a summary on how they influence the final 25 consumers all over the world who are seeking new product quality. The book also provides a detailed 26 gustatory experiences. Most of these products still description of the major typical fermented meat prod- 27 rely primarily on local, traditional manufacturing ucts around the world and the processing technologies 28 processes since little scientific information is avail- currently applied in meat processing plants. 29 able; but scientific knowledge has become an impor- This book is the result of the expertise of 93 inter- 30 tant tool for consistent production of high quality national contributors from 17 different countries. 31 and safe products. These experts from industry, government, and aca- 32 This book contains 50 chapters that are grouped demia have been led by an editorial team of 8 mem- 33 into 11 parts covering most aspects of fermented meat bers from 5 different countries. The editorial team 34 and poultry. Part I deals with the history, production, wishes to thank all the contributors for making this 35 and consumption of fermented meats as well as gen- book possible. We also thank the production team at 36 eral aspects and principles of processing (curing, fer- Wiley-Blackwell with special recognition to Susan 37 mentation, drying, and smoking). Part II describes the Engelken, senior editorial assistant and coordinator 38 main characteristics and uses of raw materials and of the project, Judi Brown, the production project 39 ingredients. Part III is focused on the microbiology manager, and Nancy Albright, the copy editor. 40 involved in meat fermentation and describes the most We sincerely hope that you will find this book 41 commonly applied starter cultures. Part IV looks into enlightening and that the information provides a 42 the sensory properties of fermented meat products. better understanding of fermented meat and poultry 43 The composition and nutritional quality, packaging, products. 44 and international standards are covered in Part V. The 45 description of manufacturing processes and charac- 46 teristics of semidry-fermented and dry-fermented Fidel Toldrá 47 sausages are covered in Parts VI and VII, respectively. Y. H. Hui 48 Fermented poultry sausages and other fermented Iciar Astiasarán 49 meats are described in Part VIII. Part IX covers the Wai-Kit Nip 50 manufacture and characteristics of ripened meat prod- Joseph G. Sebranek 51 ucts, especially dry-cured hams. Part X covers biolog- Expedito-Tadeu F. Silveira 52 ical and chemical safety aspects, and, finally, Part XI Louise H. Stahnke S53 is focused on sanitation and quality assurance. Régine Talon N54

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 Part I 15 16 17 18 19 Meat Fermentation Worldwide: 20 21 22 History and Principles 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 S54 N55 40454 01 001-008 r2.qxd 7/20/07 11:55 AM Page 3

1 2 3 1 4 5 6 A Historical Perspective of 7 8 Meat Fermentation 9 10 Peter Zeuthen 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION meat products. However, because it was poorly 22 understood, this frequently resulted in faulty produc- 23 One of the most important prerequisites for the tion. This happens today. When the author of this 24 development of civilization was to devise methods chapter visited a factory for producing Parma Hams 25 on how to preserve foods for storage and transport in about 25 years ago, the production manager 26 order to meet the need for food for increasing admitted that up to 25% of the raw material never 27 concentration in communities. Drying of foodstuffs reached a stage qualifying for the stamp of Parma 28 was probably the first development in this direction, Ham because it was putrid before it was ready 29 followed by smoking, which in many cases was a for sale. 30 natural consequence; drying often was accelerated Today, some uncertainties still exist regarding 31 by hanging up the raw material near the open fire. how to define different fermented meats. Adams 32 Nobody had the idea that other processes such as (1986) thus describes fermented food in three cate- 33 enzymatic breakdown and product changes caused gories: 1) those in which microorganisms play no or 34 by microorganisms or endogenous enzymes could little part, such as tea fermentation; 2) those in which 35 also be the reason for extension of product life. microbial growth, though an essential feature, does 36 However, the transformation of raw materials to not involve fermentative metobolism, such as the 37 more-or-less stable foods by drying and fermenta- production of tempeh; and 3) the true fermentations, 38 tion is well known in many ancient cultures and used which produce, e.g., lactic acid in products such 39 for many different foods. Actually, drying as an ini- as salami. If Adams had included raw ham, he 40 tial step of preservation, or the “wet way,” such as would probably have placed hams under category 2, 41 steeping often followed by heating, is well known in although it now is under discussion whether it 42 beer production, for example, where the breakdown rightly should be placed under category 1. Lücke 43 of raw materials takes place because the enzymes are (1986) writes that fermented sausages, and to some 44 brought into a suitable environment regarding pH extent raw ham, are produced through the participa- 45 and water activity. tion of microorganisms. Both products should there- 46 The very word fermentation, derived from the fore be considered fermented meat. The history of 47 Latin (to boil), means among other things to simmer fermented meat products is very old. In the opinion 48 or bubble, or leaven as a process, was probably not of the author, it includes both raw, dried ham and 49 well understood, except that the effect was certainly sausages, because both categories of products alter 50 used when it came to baking, wine making, beer qualities during production and storage due to 51 brewing, and production of dairy products or certain fermentation. 52 53 S54 N55 3 40454 01 001-008 r2.qxd 7/20/07 11:55 AM Page 4

4 Meat Fermentation Worldwide: History and Principles

1 EARLY RECORDS OF the blend into skins, and placed these in special 2 FERMENTED MEAT PRODUCTS rooms to dry. It was their experience that their 3 sausages would keep better if stored and dried that 4 RAW CURED HAM way. Presumably, besides the favorable extrinsic fac- 5 The Chinese (Houghton 1982) are claimed to be the tors, the sausages were inoculated by lactobacilli and 6 first ones who mentioned the production of raw micrococci (now we know that those are a mixture of 7 cured ham, but other authors of the ancients also Kocuria and staphylococci) from equipment and 8 discussed the process. Cato (234–149 B.C.) (1979) shelves, etc., and a fermentation took place. Sausage 9 explains in details in his book On Agriculture how making thus spread throughout the Roman Empire. 10 raw ham was manufactured and stored: After 5 days Leistner (1986a) describes a Chinese type of 11 of dry-cure, the hams are rotated and restacked in the sausage, Lup Cheong from the North and Southern 12 vat for 12 days. The remaining salt is brushed off. Dynasty (589–420 B.C.) made from goat and lamb 13 They are then hung for 2 days in a draught. Then, meat with salt and flavored with green onion, bean 14 after a further cleaning they are finally rubbed with sauce, ginger, and pepper. The early type of Chinese 15 oil and smoked for 2 days. They are then rubbed with sausage can hardly be regarded as a fermented 16 a mixture of oil and vinegar to avoid “flees and sausage, but the contemporary type of Lup Cheong 17 worms” and hung in a meat house. has a comparatively long keepability, mainly because 18 Similarly, Leistner (1986b) gives an account about of a high content of lactobacilli—so high that it is 19 how the Roman Emperor Diocletian in 301 A.D. in a considered sour by many (Ho and Koh, cited by 20 public notification distinguishes between highly Leistner 1986a). 21 cured and smoked hams and the lightly salted and The art of producing fermented sausages with a 22 dried (unsmoked) hams. Also, Leistner, citing Lissner long keepability spread to the rest of Europe. In 23 (1939), mentions that Varro, who lived in the 1st Germany, the manufacture of fermented sausages 24 century B.C., wrote about a substantial import to commenced only some 150 years ago. Most of the fer- 25 Rome from Gaul of hams, sausages, and other meat mented sausages are smoked; in the Mediterranean 26 products. Leistner is also of the opinion that the countries, France, Hungary, and the Balkan countries 27 technology of salted ham production was derived they are air dried. Spicy sausages predominate. Other 28 from the Romans. types of fermented sausages emerged later as a conse- 29 quence of advanced meat processing techniques and 30 the availability of refrigeration (Lücke 1985). DRY SAUSAGE 31 One of the most famous types of fermented 32 From ancient times sausages were invented as a sausages was developed in Hungary from 1830 to 33 means of making the most of leftovers of meat and 1840 by Italians (Incze 1986). Although the climate in 34 entrails. One of the most common names today for Hungary is less ideal for fermenting and drying, the 35 fermented sausages is salami. The name appears to Italians succeeded in producing their type of dry 36 have originated in the ancient Greek town of Salamis, sausage during the winter months. Characteristic for 37 on the Cyprian east coast (Pederson 1979). Although the original Hungarian sausage is that it is matured for 38 Salamis was destroyed about 450 B.C., the salami a long time at a relatively low temperature—around 39 sausage style was apparantly widely known and 10°C. It is heavily smoked and is covered by a layer of 40 appreciated by then, and clearly seems to have been mold. The latter has caused some problems because of 41 the forerunner of the many popular European vari- the danger of toxin-producing molds, a problem which 42 eties (Smith 1987). today by and large has been overcome. 43 Homer (about 800–700 B.C.) talks about 44 “sausages” in the Odyssey. Sausages were also well 45 known in the Roman Empire (Leistner 1986a). TECHNICAL AND 46 Those sausages were mainly made of blood, fat, and TECHNOLOGICAL 47 meat scrap and were cooked, so they had nothing to DEVELOPMENTS 48 do with dry, fermented sausages. Pederson (1979) RAW CURED HAM 49 citing Breasted (1938) stated that the success of 50 Caesar’s legions in the conquest of Gaul can be As earlier mentioned, drying, as a means of extend- 51 attributed to their use of dry sausage for their meat ing shelf life of food, was probably the earliest tech- 52 supply and that this aided the retention of their vigor nique employed by man. This method, however, 53S and health. The Roman butchers cut their beef and could be used only if the food was very easy to dry 54N pork into small pieces, added salt and spices, packed or semidry to begin with, e.g. seeds, or if the climatic 40454 01 001-008 r2.qxd 7/20/07 11:55 AM Page 5

A Historical Perspective of Meat Fermentation 5

conditions were suitable, such as charque and carne dry sausage, which had at least some keepability, the 1 seca, which are South American air-dried meat summer sausage was developed (Leistner 1986b). 2 specialities. In most other parts of the world, some The name summer sausage is still used in some 3 kind of salting had to be used in addition to prevent countries; in others, it is not. Thus, Kinsman (1980) 4 rot of the raw material. The combination was well writes that in the U.S., dry and/or semidry sausages 5 known in several ancient civilizations throughout the are often called summer sausages, but the term cer- 6 world—Rome and even Gaul and China (Leistner tainly covers a lot of names, which in other parts 7 1986a, 1986b). It is a method used today for the of the world would be called typical fermented 8 production of traditional dried hams. sausages, meant to have shelf lives far beyond a few 9 Several attemps have been made to speed up the weeks. Rather, the classical dry sausage will have a 10 process. Thus, Puolanne (1982) reports on the pickle keepability of a year or more. 11 injection of raw ham. The method is also used in sev- The modern way of production of dry sausage 12 eral other countries today. It reduces the production with the use of starter cultures and full control of 13 time considerately, from up to a year to a couple of temperatures and humidity has completely changed 14 weeks. Puolanne further reports that these kinds of the manufacture of today’s fermented sausage. 15 ham have much less aroma than hams manufactured 16 Classification Systems for Keepable Sausages the traditional way. This observation is reported 17 from several countries. The attempts to hasten the Several authors have attempted to classify the abun- 18 production and improve the safety precautions in dance of sausage varieties. However, when trying to 19 ham production have led to the development of inoc- do so, the confusion is increased because sausages of 20 ulation of microorganisms in dry ham. same or similar names are very different according 21 To store dry ham at a higher temperature or to to the geographical area in which they are produced. 22 expose it at a higher temperature for a short time dur- According to Kinsman (1980), a cervelat sausage in 23 ing the later part of manufacture is also used in some the U.S. is listed as five different types, with some of 24 cases. Puolanne (1982) thus reports on a Finnish- them fermented, some uncooked, others cooked. In 25 made dry-cured ham, called a sauna ham, which was Ireland, a cervelat sausage is always uncooked and 26 manufactured earlier. These hams were made as smoked. In Germany, a mettwurst from Westfalia 27 follows: After slaughter, which took place in the is fermented and dry, whereas a mettwurst from 28 autumn, the hams were dry-salted and placed in Braunschwchweig is unfermented and classified 29 wooden barrels for 1 to 4 months. Then they were hot- moist. Kinsman presents several other examples, 30 smoked at approximately 40–70°C or applied a cold showing that the name of a certain sausage in differ- 31 smoke at approximately 15–20°C for several days. ent countries is not unambiguous. 32 Smoking, a cold smoke at 20–25°C, has tradition- The tradition for consuming fermented sausages 33 ally been used in many countries. Thus, Cato (1979) in Europe varies, and it relates traditionally to the 34 mentions smoking. Various types of German dry area. Thus, probably because of the climate, fer- 35 hams are smoked, whereas the Parma type ham is mented sausages were practically not produced or 36 never smoked; neither is the Iberian ham (Garcia consumed on the British Isles, and in Ireland in 37 et al. 1991). earlier times, only whole, dried meats such as ham 38 were known. On the European continent, fermented 39 sausages were produced abundantly, a tradition start- 40 DRY SAUSAGE ing in Southern Europe. 41 Cutting up meat in smaller pieces to enable a more 42 uniform distribution of the salt and other ingredients 43 has been a well-known technique since the Greek STARTER CULTURES 44 and Roman days. However, to comminute meat 45 also opened it up for a thorough contamination, not The use of starter cultures in various types of foods 46 unly of a desired flora, but also of bacteria, which has been used for a long time, probably for as long as 47 sometimes could be pathogens. It was especially fermentation has been used by man; but it was used 48 important to exclude air. This often caused faulty simply as a kind of backslopping of remains of ear- 49 productions, especially blowing and discolorations. lier production charges. No doubt the dairy industry 50 Before artificial refrigeration and control of humid- has been using backslopping purposely, e.g., for 51 ity was available, it was almost impossible to pro- making junket and other kinds of fermented milk 52 duce dry sausages of satisfactory quality. Because of long before anybody knew anything about bacteria. S53 these difficulties, and because of the demand for a The process was derived from simple experience in N54 40454 01 001-008 r2.qxd 7/20/07 11:55 AM Page 6

6 Meat Fermentation Worldwide: History and Principles

1 the home. As Pederson (1979) describes it, “the PEDIOCOCCI 2 mothers observed that when milk soured with a Bacus and Brown (1985) report that before the intro- 3 smooth curd and pleasing acid odor, the milk could duction and subsequent use of the strains of pedio- 4 be consumed without causing distress or illness. cocci as the predominant meat starter cultures, most 5 When instead, the milk had an unpleasing odour and natural isolates from fermented meats consisted of 6 was spoiled or gassy or oherwise exhibited unsatis- various species and strains of lactobacilli. They add 7 factory character, the infant sometimes became ill that lactobacilli are still the predominant microflora 8 after consuming such milk.” Pasteurization made in products that are made from chance inoculation 9 even backslopping much more safe, but the use of (or backslopping) and that chance inoculation was 10 defined starter cultures made the production a much depended on for nitrate reduction. However, the 11 more foolproof processes. Americans (and other researchers) working with 12 With meat, it was different. Microorganisms were pure cultures failed to achieve successful lyophilized 13 added through natural contamination, and to this preparations of lactobacillus isolates with the tech- 14 very day backslopping is still commonly used in nology used at that time, so the lactobacilli inocula- 15 many places and regions of the world (Bacus and tion was replaced with Pediococcus strains that were 16 Brown 1981). resistant to lyophilization. When frozen culture con- 17 Lücke (1985) states that many productions are centrates for sausage were introduced, the interest in 18 still made successfully without addition of starter Lactobacillus strains was renewed. Lücke (1985) 19 cultures or reinoculation—backslopping of finished noted that in Europe, where low fermentation tem- 20 sausages. This is due to sausage makers being able to peratures are common and nitrate is used much more 21 design formulations and ripening conditions that frequently than nitrite, sausage makers are faced 22 favor the desired microorganisms so strongly that with a problem: A fast lactic acid fermentation sup- 23 the products are safe and palatable, even if only few presses the organisms containing catalase and nitrate 24 lactobacilli and micrococci (i.e., Kocuria and reductase to such an extent that defects in color 25 staphyloccoci) are present in the fresh mixture. No and flavor are common. For a long time, European 26 doubt the interest in the use of starter cultures arose researchers and sausage manufacturers therefore 27 parallel with the trend toward industrial production, tried to avoid lactobacilli, and lactobacilli were con- 28 short ripening times, and standardization of the sidered detrimental in spite of their ability to form 29 mode in which the sausages were made. It was also lactic acid. Not until later did they begin experiment- 30 very helpful that contemporary refrigeration and air- ing with suitable micrococci (i.e., Kocuria and 31 conditioned facilities became available. However, staphylococci). A Finnish researcher, Nurmi (1966), 32 due to the increasing use of starter cultures, which isolated a strain of Lactobacillus plantarum from 33 had taken place with such great effect in the dairy fermented sausage that turned out to be superior to 34 industries, attempts were made to develop starter Pediococcus cerevisiae at 20–22°C, a temperature 35 cultures for other foods, such as meats. normal for fermentation of dry sausage in Northern 36 Europe. 37 Pediococci became very popular in the U.S. 38 LACTOBACILLI because of the difficulties there with the shelf life of 39 Jensen and Paddock (1940) were the first to have freeze-dried lactobacilli some years ago. Another 40 published and patented the idea. The inventors used reason was that the employed fermentation tempera- 41 lactobacilli in their patent, which was a mixture of ture was usually higher than temperatures used in 42 Leuconostoc mesenterioides, Leuconostoc dextran- Europe, which meant that it was imperative that the 43 icum, and Leuconostoc pleofructi, but they also set onset of the pH decrease took place very fast during 44 forth a whole range of organisms, all lactobacilli, fermentation in order to avoid growth and toxin for- 45 which would be satisfactory for the purposes of the mation from Staphylococcus aureus. In fact, the 46 invention. They included Lactobacillus casei, Lacto- problem of staphylococcal food poisoning caused by 47 bacillus plantarum, Lactobacillus pentosus, Lacto- defective, fermented dry or semidry sausage was 48 bacillus arabinosus, Lactobacillus leichmannii, and very well known in the U.S. (USDA 1977, cited by 49 several other species. In their claims, they directly Bacus and Brown 1985). According to these authors, 50 warned against gas-forming lactobacilli. The use of a pediococcus starter cultures have proven very effec- 51 defined microbial culture thus originated in the U.S. tive in inhibiting not only S. aureus, but also other 52 with a series of patents issued during the period undesirable microorganisms, such as Salmonellae, 53S 1920–1940. Clostridium botulinum, and other species, including 54N 40454 01 001-008 r2.qxd 7/20/07 11:55 AM Page 7

A Historical Perspective of Meat Fermentation 7

bacilli, gram-negative enterics, and yeasts. Control STARTER CULTURES FOR 1 of the natural fermentation in sausages also appears CURED HAM 2 to prevent histamine accumulation. Higher hista- 3 mine levels have been found in dry sausage where a Regarding dry-cured hams, the use of bacterial 4 natural fermentation process is employed for an starter cultures is very different. This is partly 5 extended aging period (Rice et al. 1975; Rice and because the production takes place for a long time at 6 Koehler 1976). a much lower temperature than is used for fermented 7 sausages and partly because the dry-cured ham is 8 subjected to a much more salty environment on the 9 STAPHYLOCOCCI/MICROCOCCI (KOCURIA) surface. The untreated ham is not opened, so essen- 10 In his thesis, Niinivaara (1955) proposed a micro- tially the internal ham is sterile from the beginning. 11 coccus strain, which he called M53. It was selected Contamination can therefore take place only during 12 because it rapidly reduced nitrate, improved color the liquid exchange that takes place. 13 and flavor, and inhibited undesired microorgan- Giolitti et al. (1971) conducted a large investigation 14 isms. The strain was later replaced by a fermentative on dry ham. They concluded that the ripening of hams 15 micrococcus isolated by Pohja (1960). Gram- seems to follow a completely different pattern from 16 negative bacteria have also been attempted to be that of dry sausages. In their opinion, the microflora, 17 used as starter cultures in fermented meats. Keller at least in Italian hams, exerts only a minor role in 18 and Meyer (1954) reported that gram-negative bac- ripening. Souring seems to depend mainly on enzy- 19 teria have a favorable effect on dry sausage aroma. matic hydrolysis, and this does not lead to end prod- 20 Buttiaux (1957) isolated Vibrio costicolus, which ucts qualitatively different from those present in 21 was used in meat curing in France, primarily as a normal ham. True putrefaction, however, is the result 22 nitrate reducer, but also because the strain was con- of microbial growth, starting from internal and exter- 23 sidered important for aroma and flavor formation. nal surfaces. Buscailhon et al. (1993) were of the 24 These were properties that also were emphasised by opinion that the aroma of dry-cured ham muscles 25 Hawthorn and Leich (1962). could be traced back to lipid oxidation or amino acid 26 degradation. However, Hinrichsen and Pedersen 27 (1995) concluded in their study on Parma hams, rep- 28 YEASTS AND MOLDS resenting six different stages in the manufacturing 29 process, that microorganisms are important for the 30 Yeasts are also used as starter cultures. Following development of flavor in Parma ham because all of the 31 some investigations by Hammes et al. (1985), in correlating volatile compounds they found can be 32 which Debaryomyces hansenii was examined, other generated by secondary metabolism of microorgan- 33 investigators also identified D. kloeckeri as useful in isms, especially amino acid catabolism. 34 dry sausage ripening. 35 One might say that to use molds as starter cultures 36 is rather to make a virtue out of a necessity. How- CONCLUDING REMARKS 37 ever, with the frequent occurrence of molds on 38 sausages, especially at a time where climate rooms It is impossible to cover the whole history of 39 were not available during cold and humid weather, fermented meats in one short chapter, but going 40 undesirable mold growth was a frequent menace. In through some of the major epochs, the technical dis- 41 southeast Europe, it was a well known problem. coveries have resulted in far more rational proce- 42 Incze (1986) mentions that Hungarian salami was dures and thus changed many fermented meat 43 originally made without molds when it was manu- products. These items sometimes were successful, 44 factured in Italy. Originally, the so-called house flora but far from always, because many of the secrets of 45 just contaminated the product, but when the signi- fermentation were unveiled. On the other hand, 46 ficance of mycotoxins was realized, development many discoveries still wait to be made, especially as 47 proceeded to avoid the use of toxiogenic molds. far as ways in which more refined aroma and taste 48 Much development work took place, especially in compounds are formed during processing. A good 49 Germany at the Federal Centre for Meat Research. example is shown in an article by Bolzoni et al. 50 Mintzlaff and Leistner (1972) reported on a strain of (1996), which shows the development of volatiles in 51 Penicillium nalgiovense as a starter culture, later Parma ham. No doubt there is still much to discover 52 known as Edelschimmel Kulmbach, which does not in the future about the mechanisms and the control S53 form mycotoxins. of volatile development. N54 40454 01 001-008 r2.qxd 7/20/07 11:55 AM Page 8

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1 REFERENCES DM Kinsman. 1980. Principal Characteristics of 2 Sausages of the World. Connecticut: Storrs College 3 MR Adams. 1986. Fermented flesh foods. Progr Indust of Agriculture and Natural Resources, University of 4 Microbiol 23:159–198. Connecticut. 5 JN Bacus, WL Brown. 1981. Use of microbial cultures: L Leistner. 1986a. Allgemeines über Rohschinken. 6 Meat products. Food Technol 47(1):74–78. Fleischwirtsch 66:496–510. 7 ———. 1985. The pediococci: Meat products. In: SE ———. 1986b. Allgemeines über Rohwurst. Fleis- 8 Gilliland, ed. Bacterial Starter Cultures for Foods. chwirtsch 66:290–300. 9 Boca Raton, Florida: CRC Press, pp. 86–96. ———. 1995. Fermented meats. London: Chapman & 10 L Bolzoni, G Barbieri, R Virgili. 1996. Changes in Hall, pp. 160–175. 11 volatile compounds of Parma ham during matura- E Lissner. 1939. Wurstologia oder Es geht um die Wurst. 12 tion. Meat Sci 43:301–310. Frankfurt am Main: Hauserpresse Hans Schaefer. 13 JH Breasted. 1938. The Conquest of Civilization. New FK Lücke. 1985. Fermented sausages. In: BJB Wood, 14 York: Harper & Row, pp. 542–556. ed. Microbiology of Fermented Foods Vol II. 15 S Buscailhon, JL Berdagué, G Monin. 1993. Time- London: Elsevier Applied Science, pp. 41–83. 16 related changes in volatile compounds of lean tissue ———. 1986. Mikrobiologische Vorgänge bei der Her- 17 during processing of French dry-cured ham. J Sci stellung von Rohwurst und Rohschinken. Fleis- 18 Food Agric 63:69–75. chwirtsch 66:302–309. 19 R Buttiaux. 1957. Technique simple d’examens bacteri- HJ Mintzlaff, L Leistner. 1972. Untersuchungen zur 20 ologique des saumures de jambon et ses résultats. Selektion eines technologisch geeigneten und toxikol- 21 Proceedings II International Symposium on Food ogisch unbedenklichen Schimmelpilz-Stammes für 22 Microbiology, HMSO, London, 1957, pp. 137–146. die Rohwurst-Herstellung. Zeitblatt Veterinär Medi- 23 MP Cato. 1979. De agricultura (On Agriculture). Trans- cin B 19:291–300. 24 lated by WD Hooper. Harvard University Press, FP Niinivaara. 1955. über den Einfluss von Bakterien- 25 pp. 155–157. Reinkulturen auf die Reifung und Umrötung der 26 C Garcia, JJ Berdagué, T Antequera, C López-Bote, Rohwurst. Acta Agrialia Fennica 85:1–128. 27 JJ Córdoba, J Ventanas. 1991. Volatile components of E Nurmi. 1966. Effect of bacterial inoculation on char- 28 dry cured Iberian ham. Food Chem 41:23–32. acteristics and microbial flora of dry sausage. Acta 29 G Giolitti, CA Cantoni, MA Bianchi, P Renon. 1971. Agralia Fennica 108:1–77. 30 Microbiology and chemical changes in raw hams of CS Pederson. 1979. Microbiology of Food Fermenta- 31 Italian type. J Appl Bacteriol 34:51–61. tion. 2nd ed. Westport, Connecticut: The AVI Pub- 32 WP Hammes, I Röl, A Bantleon. 1985. Mikrobiologische lishing Company Inc, p. 212. 33 Untersuchungen der auf dem deutschen Markt vorhan- MS Pohja. 1960. Micrococci in fermented meat prod- 34 denen Starterkulturpräparate für die Rohwurstbere- ucts. Acta Agralia Fennica 96:1–80. 35 itung. Fleischwirtsch 65:629–636, 729–734. E Puolanne. 1982. Dry cured hams—European style. 36 J Hawthorn, JM Leitch. 1962. Recent advances in Food Proceedings of the Reciprocal Meat Conference 37 Science Vol 2. London: Butterwords, pp. 281–282. 35:49–52. 38 LL Hinrichsen, SB Pedersen. 1995. Relationship among SL Rice, RR Eitenmiller, PE Koehler. 1975. Histamine 39 flavour, volatile compounds, chemical changes, and and tyramine content of meat products. J Milk Food 40 microflora in Italian type dry cured ham during pro- Technol 38:256–258. 41 cessing. J Agric Food Chem 43:2932–2940. SL Rice, PE Koehler. 1976. Tyrosine and histidine 42 Homer. The Odyssey. XX:24–27. decarboxylase activities of Pediococcus cerevisiae 43 CG Houghton. 1982. The Encyclopedia Americana and Lactobacillus species and the production of tyra- 44 International edition Vol 24. Danbury, Connecticut: mine in fermented sausages. J Milk Food Technol 45 Grolier Inc, p. 309. 39:166–169. 46 K Incze. 1986. Technnologie und Mikrobiologie der DR Smith. 1987. Sausage—A food of myth, mystery 47 ungarischen Salami. Fleischwirtsch 66:1305–1311. and marvel. CSIRO Food Research Quarterly 48 LB Jensen, LS Paddock. 1940. Sausage treatment. US 47:1–8. 49 Patent 2,225,783. USDA. 1977. The staphylococcal enterotoxin problem 50 H Keller, E Meyer. 1954. Die bakterielle Aromati- in fermented sausage, Task Force report, Food Safety 51 seierung von Rohwurst. Fleischwirtsch 6:125–126, and Wuality Service, USDA, Washington, D.C. 52 453–454. 53S 54N 40454 02 009-016 r1.qxd 7/20/07 11:57 AM Page 9

1 2 3 2 4 5 6 Production and Consumption of 7 8 Fermented Meat Products 9 10 Herbert W. Ockerman and Lopa Basu 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION are both microbial hurdles that aid in producing a 22 safe product. Fermented sausages often have a long 23 Meat fermentation is a low-energy, biological acidu- storage life due to added salt, nitrite, and/or nitrate; 24 lation, preservation method, which results in unique low pH due to lactic acid production by LAB organ- 25 and distinctive meat properties, such as flavor and isms in the early stages of storage; and later drying, 26 palatability, color, microbiological safety, tender- which reduces the water activity. 27 ness, and a host of other desirable attributes of this Production and composition figures for fermented 28 specialized meat item. Changes from raw meat to a products are difficult to obtain, particularly because 29 fermented product are caused by “cultured” or many of these products are produced and consumed 30 “wild” microorganisms, which lower the pH. locally and quantities are not recorded. The limited 31 Because this is a biological system, it is influenced number of references available would suggest that 32 by many environmental pressures that need to be the production and consumption is sizeable. 33 controlled to produce a consistent product. Some of 34 these factors include a fresh, low-contaminated, con- 35 sistent raw material; a consistent inoculum; strict CURRENT PRODUCTS 36 sanitation; control of time, temperature, and humid- DEFINITIONS 37 ity during production; smoke; and appropriate 38 additives. The characteristics and types of fermented products 39 Lactic acid, which accounts for the antimicrobial can be found in Tables 2.1 and 2.2. Guidelines pro- 40 properties of fermented meats, originates from the posed in the U.S. (American Meat Institute 1982; 41 natural conversion of glycogen reserves in the car- Hui et al. 2004) for making fermented dry or semi- 42 cass tissues and from the added sugar during product dry sausages include a definition of dry sausage as 43 fermentation. A desirable fermentation product is chopped or ground meat products, which, due to 44 the outcome of acidulation caused by lactic acid pro- bacterial action, reach a pH of 5.3 or less. The drying 45 duction and lowering the water activity (a ) caused removes 20 to 50% of the moisture resulting in a w 46 by the addition of salt (curing) and drying. Both nat- moisture to protein ratio (MP) of no greater than 2.3 47 ural and controlled fermentations involve lactic-acid to 1.0. Dry salami (U.S.) has an MP ratio of 1.9 to 1, 48 bacteria (LAB). Their growth must be understood to pepperoni 1.6 to 1, and jerky 0.75 to 1. 49 produce a safe and marketable product. Most starter Semidry sausages are similar except that they have 50 cultures, today, consist of lactic acid bacteria and/or a 15 to 20% loss of moisture during processing. 51 micrococci, selected for their metabolic activity, Semidry sausages also have a softer texture and a dif- 52 which often improves flavor development. The ferent flavor profile than dry sausages. Because of the 53 reduction of pH and the lowering of water activity higher moisture content, semidry sausages are more S54 N55 9 40454 02 009-016 r1.qxd 7/20/07 11:57 AM Page 10

10 Meat Fermentation Worldwide: History and Principles

1 Table 2.1. Characteristics of different types of fermented sausages. 2 3 Type of Sausage Characteristics 4 Dry; long ripening, e.g., dry or hard Chopped and ground meat 5 salami, saucission, pepperoni; Commercial starter culture or back inoculum 6 shelf-stable U.S. fermentation temperature 15–35°C for 1–5 days 7 Not smoked or lightly smoked 8 U.S. bacterial action reduces pH to 4.7–5.3 (0.5–1.0% lactic 9 acid; total acidity 1.3%, which facilitates drying by denaturing 10 protein resulting in a firm texture; moisture protein ratio 11 Ͻ2.3:1, moisture loss 25–50%, moisture level ,35% 12 European bacterial action reduces pH to 5.3–5.6 for a more 13 mild taste than U.S.; processing time 12–14 weeks 14 Dried to remove 20–50% of moisture; contains 20–45% 15 moisture, fat 39%, protein 21%, salt 4.2%, aw 0.85–0.86, 16 yield 64% 17 Moisture protein ratio no greater than 2.3:1.0 18 Less tangy taste than semidry 19 20 Semidry; sliceable, e.g., summer Chopped or ground meat 21 sausage, Holsteiner, Cervelat Bacterial action reduces pH to 4.7–5.3 (lactic acid 0.5–1.3%, 22 (Zervelat), Tuhringer, Chorizos; total acidity 1%), processing time 1–4 weeks 23 refrigerate Dried to remove 8–30% of moisture by heat; contains 30–50% moisture, 24% fat, protein 21%, salt 3.5%, a 0.92–0.94, 24 w 25 yield 90% 26 Usually packaged after fermentation/heating 27 Generally smoked during fermentation 28 No mold 29 Moisture protein ratio no greater than 2.3–3.7 to 1.0 30 Moist; undried; spreadable, e.g., Contains 34–60% moisture, production time 3–5 days ϳ 31 Teewurst, Mettwurst, Frishe Weight loss 10%, aw 0.95–096 32 Braunschweiger Usually smoked 33 No mold 34 Highly perishable, refrigerate, consume in 1–2 days 35 36 Source: Modified from American Meat Institute (1982); Gilliland (1985); Campbell-Platt and Cook (1995); Doyle et al. 37 (1997); Farnworth (2003). 38 39 susceptible to spoilage and are usually fermented to a rapidly (12 hours or less) at a relatively high temper- 40 lower pH to produce a very tangy flavor. Semidry ature (32–46°C). In Europe, fermentation is slower 41 products are generally sold after fermentation (pH of (24 hours or more) at a lower temperature and results 42 5.3 or less). These are heated, and they do not go in a higher pH. These differences in speed of fer- 43 through a drying process (water activity is usually mentation and final pH, result in products having 44 0.92 or higher). They are also usually smoked during different flavors. 45 the fermentation cycle and have a maximum pH of 46 5.3 in less than 24 hours. If the semidry sausage has PRODUCT INGREDIENTS a pH of 5.0 or less and a moisture protein ratio of 47 Raw Meat 48 3.1 to 1 or less, it is considered to be shelf-stable, but 49 most semidry products require refrigeration (2°C). In Beef, mechanically separated beef (up to ϳ5%), pork, 50 Europe, fermented meat with a pH of 5.2 and a water lamb, chicken, mechanically separated chicken (up to 51 activity of 0.95 or less is considered shelf-stable ϳ10%), duck, water buffalo, horse, donkey, reindeer, 52 (USDA FSIS Food Labeling Policy Manual). gazelle, porcupine, whale, fish, rabbit, by-products, 53S To decrease the pH (below 5.0) with limited dry- and other tissue from a variety of species are used to 54N ing, the U.S semidry products are often fermented make fermented meat products. Fermented meat is 40454 02 009-016 r1.qxd 7/20/07 11:57 AM Page 11

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Table 2.2. Types of fermented sausages and areas of production. 1 2 Type Area Production Sausages Sensory 3 Dry fermented, Southern and Eastern 40–60 mm, pork, 2–6 mm Italian Salami Fruity, sweet 4 mixed Europe, particle, nitrate, starters, Spanish odor, 5 cultures psychotrophic fungi; ferment 10–24°C, Salchichon, medium 6 lactic acid, 3–7 days; ripen Chorizo buttery, 7 optimum 10–18°C, 3–6 weeks; (usually no sour and 8 growth 10–15ºC weight loss higher than starter) pungent, 9 30%, low water activity, French more 10 higher pH, 5.2 to 5.8, Saucisson, mature 11 lower lactate, 17 mmol/ 9 mg/g of 12 100 g of dry matter lactate/g 13 14 Northern Europe, 90 mm pork/beef, 1–2 mm German Salami, Buttery, sour 15 LAB (Lactobacillus particle, nitrite, starters; 15 mg/g of odor, low 16 plantarum or smoked, ferment lactate/g levels of 17 Pediococcus) and 20–32°C, 2–5 days; Hungarian Salami spice and 18 Micrococcaceae rapid acidiﬁcation to Nordic Salami fruity 19 (Staphylococcus below 5, smoking ripen notes, 20 carnosus or 2–3 weeks; weight loss more acid 21 Micrococcus) higher than 20%, lower More acid 22 pH, 4.8 to 4.9, higher More acid 23 lactate 20–21 mmol/ 24 100 g of dry matter 25 Mold ripened Europe, U.S. Usually with starters French Salami 26 Usually with starters, bowl Germany Salami 27 chopper Hungarian Salami 28 Without starters, bowl Italian Salami 29 chopper California Salami 30 Usually with starters, Yugoslavian 31 ground Salami 32 Usually with starters 33 Usually with starters 34 Semidry U.S. Usually with starters Summer sausage, 35 M/P ratio 36 2.0–3.7:1.0 37 Thuringer 38 Beef sticks 39 Dry Usually with Pediococcus Beef sticks 40 acidilactici Pepperoni, M/P 41 Usually with Pediococcus ratio 1.6:1.0 42 acidilactici 43 44 Source: Hui et al. (2004); Schmidt and Berger (1998); Demeyer et al. (2000); Stahnke et al. (1999). 45 46 47 often divided into two groups: products made from aerobic rods of psychrotrophic pseudomonads along 48 whole pieces of meat, such as hams; and products with psychrotrophic Enterobacteriaceae, small num- 49 made from meat chopped into small pieces, such as bers of lactic acid bacteria, and other gram-positive 50 various sausage types. Details on producing these bacteria. The lactic and other gram-positive bacteria 51 products will be treated in succeeding chapters. become the dominant ﬂora if oxygen is excluded 52 The predominant bacteria that appear in fresh and is encouraged during the fermentation stages. S53 meat are typically gram-negative, oxidase-positive, Because the production of fermented meats depends N54 40454 02 009-016 r1.qxd 7/20/07 11:57 AM Page 12

12 Meat Fermentation Worldwide: History and Principles

1 on microorganism growth, it is essential that these Nitrite is used at 80 to 240 mg/kg for antibacterial, 2 products are hygienically processed and chilled prior color, and antioxidant purposes. Nitrate and nitrite 3 to use and maintained under refrigeration prior and are often used in combination, but nitrate is usually 4 during the curing operation. not necessary except as a reservoir for nitrite, which 5 could be useful in long-term processing. Nitrite is 6 Starter Cultures also a hurdle, which inhibits bacterial growth and 7 retards Salmonellae multination. Fermentation can 8 Traditionally, fermented products depend on wild be produced with only salt, but there is a greater 9 inoculum, which usually do not conform to any spe- microbial risk if no nitrite is used. 10 cific species but are usually related to Lactobacilli Simple sugars such as glucose (dextrose, 0.5 11 plantarum. However, other species such as Lb. casei total %, a minimum of 0.75% is often recommended), 12 and Lb. leichmanii, as well as many others, have is the fermentation substrate that can be readily uti- 13 been isolated from traditionally fermented meat lized by all lactic acid bacteria. The quantity of sugar 14 products (Anon 1978). In the U.S., Lb. plantarum, influences the rate and extent of acidulation, and also 15 Pediococcus pentosaceus, or P. acidilactici are the contributes favorably to flavor, texture, and product 16 most commonly used starter cultures. In Europe, the yield properties. The amount of dextrose added will 17 starter cultures used most include Lb. sakei, Lb. directly influence the final product pH, and addi- 18 plantarum, Pediococcus pentosaceus, Staphylococ- tional sugar will not decrease pH further since bacte- 19 cus xylosus, S. carnosus, and to a lesser extent rial cultures cannot grow in excess acid. 20 Micrococcus spp. Reliance on natural flora results in Spices (e.g., black, red, and white pepper; car- 21 products with inconsistent quality. The advantage of damom; mustard; allspice; paprika; nutmeg; ginger; 22 a starter culture is that the same microorganisms can mace; cinnamon; garlic; and various combinations) 23 be used repeatedly, which cuts down on variation of are often included in the fermented meat formula. 24 the finished product, and a larger number of organ- Spices are used for flavor, antioxidant properties, 25 isms can be added. Now, combined starter cultures and to stimulate growth of lactic bacteria. 26 are available in which one organism produces lactic Sodium ascorbate (also in the U.S., sodium ery- 27 acid (e.g., Lactobacilli) and another improves thorbate) or ascorbic acid (also in the U.S. erythorbic 28 desirable flavors (Micrococcaceae, Lb. brevis, Lb. acid) is used for improvement and stability of color 29 buchneri). This translates into a lot of very good and and retardation of oxidation. 30 acceptable product and almost no undesirable fermented product. However, very little extremely 31 PROCESSING 32 excellent product is produced, because most starter Formulations are numerous, even for products with 33 cultures are a combination of just a few species the same name, and some are held in strict security. 34 of microorganisms and they cannot produce as Examples of formulations and processing proce- 35 balanced a flavor as sometimes can be obtained dures can be found in Komarik et al. (1974), Rust 36 when many species are included. (1976), Ockerman (1989), Campbell-Platt and Cook 37 Particularly in the south European countries, dry (1995), and Klettner and Baumgartner (1980). Time, 38 sausages are applied with atoxinogenic yeast and temperature, humidity, and smoke are also variables 39 fungi to produce products with specific flavor notes. that control the quality of the final product. 40 This is done by dipping or spraying. Mold cultures Types of casings available include natural casings, 41 tend to suppress natural molds and, consequently, artificial casings (made from cotton linters), colla- 42 reduce the risk of mycotoxins. Due to the extended gen casings, fibrous casings (synthetic, inedible) for 43 ripening and drying for these products, the final pH use in the smokehouse or cooker and which are 44 is usually higher (pH >5.5), even if the pH was lower sometimes netted or prestuck (pin-pricked) to allow 45 after fermentation, because molds can utilize lactic for better smoke penetration and elimination of air 46 acid and produce ammonia. This requires the final pockets, and cloth bags. 47 water activity to be low enough for preservation. Temperature, time, and relative humidity combi- 48 nations are quite variable in industrial productions. 49 Other Ingredients In general, the higher the fermentation temperature 50 Salt is the major additive in fermented meat prod- and water activity, the faster the lactic acid produc- 51 ucts. It is added in levels of 2–4% (2% minimum for tion. In Europe, the fermentation temperatures range 52 desired bind, up to 3% will not retard fermentation), from 5 to 26°C, with lower temperatures used in the 53S which will allow lactic acid bacteria to grow and will Mediterranean area and higher temperatures in 54N inhibit several unwanted microorganisms. northern Europe. In the U.S., semidried products are 40454 02 009-016 r1.qxd 7/20/07 11:57 AM Page 13

Table 2.3. Nutritive composition of examples of fermented meat products. 1 2 Salami, Pork, Pepperoni, 3 Cervelat Beef Pork, Beef 4 Soft Dry Dry Dry 5 6 Moisture 48.4% 29.4% 30.5% 7 Calorie (kcal) 307 451 424 466 8 9 Protein 18.6% 24.6% 23.1% 20.3% 10 Fat 25.5% 37.6% 34.8% 40.3% 11 —Monounsaturated 13.0 g/100 g – 17 g/100 g 19 g/kg 12 —Polyunsaturated 1.2 g/100 g – 3.2 g/100 g 2.6 g/100 g 13 —Saturated 12.0 g/100 g 12.4 g/100 g 16.1 g/100 g 14 Ash 6.8% 6.7% 5.5% 4.8% 15 16 Fiber 0 0 – 1.5% 17 Carbohydrate 1.6% 1.7% – 5 g/100 g 18 Sugar 0.85% – 0.7% 19 Calcium 11 mg/100 g 14 mg/100 g – 21 mg/110 g 20 21 Iron 2.8 mg/100 g 2.7 mg/100 g 1, 5 mg/100 g 1.4 mg/100 g 22 Magnesium 14.0 mg/100 g 17.8 mg/100 g 18 mg/100 g 23 Phosphorus 214 mg/100 g 294 mg/100 g 143 mg/100 g 176 mg/100 g 24 Potassium 260 mg/100 g – 379 mg/100 g 315 mg/100 g 25 26 Sodium 1242 mg/100 g – 1881 mg/100 g 1788 mg/100 g 27 Zinc 2.6 mg/100 g – 3.3 mg/100 g 2.7 mg/100 g 28 Copper 0.15 mg/100 g – 0.07 mg/100 g 0.07 mg/100 g 29 30 Manganese – – 0.4 mg/100 g 0.3 mg/100 g 31 Selenium 20.3 mg/100 g – – 21.8 ␮/100 g 32 Vitamin C (ascorbic acid) 16.6 mg/100 g – – 0.7 mg/100 g 33 Thiamine (B ) 0.3 mg/100 g – 0.6 mg/100 g 0.5 mg/100 g 34 1 35 Riboﬂavin (B ) 0.2 mg/100 g – 0.3 mg/100 g 0.2 mg/100 g 2 36 Niacin (B3) 5.5 mg/100 g – 4.9 mg/100 g 5.4 mg/100 g 37 38 Pantothenic Acid (B5) – – 1.1 mg/100 g 0.6 mg/100 g Vitamin B 0.26 mg/100 g – 0.5 mg/100 g 0.4 mg/100 g 39 6 40 Folate 2 DFE/100 g – – 6 DFE/100 g 41

Vitamin B12 – – 1.9 mg/100 g 1.6 mg/100 g 42 Vitamin E (alpha–tocopherol) 5.5 mcg/100 g – 0.3 ␮/100g 43 44 Cholesterol 75 g/100 g – 78 mg/100 g 118 mg/100 g 45 – ϭ data not reported. 46 Source: Calorie-Counter.net (2003); Nutrition Info (2005); United States Department of Agriculture (1963); National 47 Livestock and Meat Board (1984). 48 49 50 51 52 S53 N54

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14 Meat Fermentation Worldwide: History and Principles

1 usually fermented with slowly rising temperatures to quality, but they must be evaluated on a case-by-case 2 over 35°C to shorten the fermentation time, which is basis. Genetic manipulation to “tailor-make” cultures 3 frequently 12 hours or less. In the U.S., half-dried to be used as starters and fungal starter cultures are 4 summer sausage is fermented 3 days at 7°C, 3 days being evaluated for elimination of toxin production, 5 at 27 to 41°C, and 2 days at 10°C, and then heated to regulation of fermentation including proteolytic and 6 58°C for 4 to 8 hours. Smoking depends on tradition lipolytic activity, and biopreservative properties 7 and product type in the area the product is produced, such as production of natural antimicrobial com- 8 and it can vary to from no smoke to heavy smoke. If pounds for elimination of pathogens. 9 smoked, it is used to contribute flavor and to retard Muscle enzyme activity of a carcass and its influ- 10 surface bacteria, molds, and yeast. Many semidry ence on meat quality has been demonstrated (Toldrá 11 sausages are heated after fermentation and/or smok- and Flores 2000; Russo et al. 2002). Therefore, 12 ing and this often increases the pH. Often, an inter- enzymes and genes can be used to select breeding 13 nal temperature of 58.3°C is used. European drying animals and raw material for production of fermented 14 temperatures of 14°C, and 78 to 88% relative sausages. For more value-added fermented products, 15 humidities are often used. Air velocity of 1 m/s is muscle protease, muscle and fat lipase (activities), 16 also used. This results in a final water activity of and spice influence on fermented sausage flavor 17 <0.90 in the finished fermented product (Klettner (Fournaud 1978; Claeys et al. 2000) will be evalu- 18 and Baumgartner 1980). ated. If these can be altered, a flavor modification is 19 probably possible. 20 New product developments with new ingredients 21 COMPOSITION will evolve. For example, making fermented product 22 Composition varies widely due to the number of fer- from ostrich (Bohme et al. 1996), carp (Arslan et al. 23 mented product types. Products with the same name 2001), and utilization of olive oil (Muguerza et al. 24 vary considerably when made in different countries 2001) have been suggested. Acceptance of fermented 25 or even in the same country or made by the same meat products in additional international food items 26 company in different locations. The major factors seems to be gaining in popularity, such as increased 27 influencing composition of the finished product are consumption of pizza containing fermented meat in 28 the composition and ratio of raw materials used, Asia. With the past progress and the possible future 29 processing procedures utilized, and the quantity of developments, the future looks bright for fermented 30 drying. Because most meat composition data are meat products. 31 usually expressed on a percentage basis, the dryer 32 (less moisture) the sausage, the higher will be the REFERENCES 33 other individual ingredients in the finished product. American Meat Institute. 1982. Good Manufacturing 34 A few selected examples of average composition can Practices, Fermented Dry and Semi-dry Sausages. 35 be found in Table 2.3. 36 Washington D.C.: American Meat Institute AMI. 37 Anon. 1978. Some aspects of dry sausage manufacture. 38 FUTURE Fleischwirtsch 58:748–749. 39 A Arslan, AH Dincoglu, Z Gonulalan. 2001. Fermented 40 Health and spoilage will be the center focus of Cyrinus carpio L. sausage. Turkish J Vet Anim Sci 41 research in the future and a better understanding of 25:667–673. 42 the geological mechanisms will help tremendously. HM Bohme, FD Mellett, LMT Dicks, DS Basson. 43 Hazard Analysis Critical Control Point (HACCP) and 1996. Production of salami from ostrich meat with 44 hurdle technology control are becoming common- strains of Lactobacillus sakei, Lactobacillus curva- 45 place in the food industry, and both are particularly tus and Micrococcus. Meat Sci 44:173–180. 46 important in fermented sausages (see succeeding Calorie-Counter.net. 2003. http://calorie-net/meat- 47 chapters of this volume). Easily controlled fermenta- calories/pepperoni.htrm. 48 tion chambers are helpful and gaining in popularity G Campbell-Platt, PE Cook. 1995. Fermented Meats. 49 to accurately control the environmental conditions London, UK: Blackie Academic and Professional. 50 during processing. E Claeys, S De Smet, D Demeyer, R Geers, N Buys. 51 Genetic determinants and transfer mechanisms 2000. Effect of rate of pH decline on muscle enzyme 52 will be utilized to develop superior lactic acid bacte- activities in two pig lines. Meat Sci 57:257–263. 53S ria strains. These genetically modified microorgan- PE Cook. 1995. Fungal ripened meats and meat prod- 54N isms have the potential to improve food safety and ucts. In: G Campbell-Platt, PE Cook, eds. Fermented 40454 02 009-016 r1.qxd 7/20/07 11:57 AM Page 15

Production and Consumption of Fermented Meat Products 15

Meats. Glasgow: Blackie Academic and Profes- with pre-emulsified olive oil on lipid fraction and 1 sional, pp. 110–129. sensory quality of Chorizo de Pamplona. Meat Sci 2 D Demeyer, M Raemaekers, A Rizzo, A Holck, 59:251–258. 3 Ad Smedt, Bt Brink, B Hagen, C Montel, E Zanardi, National Livestock & Meat Board. 1984. Nutritive 4 E Murbrekk, F Leroy, F Vandendriessche, K Value of Muscle Foods. Chicago. 5 Lorentsen, K Venema, L Sunesen, L Stahnke, L Nutrition Info. 2005. http://www.nutri.info/nutrition_ 6 Vuyst, R Talon, R Chizzolini, S Eerola. 2000. Con- facts/Pepperoni,%20pork,%20beef.htm. 7 trol of bioflavour and safety in fermented sausages: HW Ockerman. 1989. Sausage and Processed Meat 8 First results of a European project. Food Res Int formulations. New York: Van Nostrand Reinhold, 9 33:171–180. pp. 1–603. 10 MP Doyle, LR Deuchat, TJ Montville. 1997. Food V Russo, L Fontanesi, R Davoli, LN Costa, 11 Microbiology. Washington D.C.: American Society M Cagnazzo, l Buttazoni, R Virgili, M Yerle. 2002. 12 for Microbiology, pp. 610–629. Investigation of genes for meat quality in dry-cured 13 ER Farnworth. 2003. Handbook of Fermented Func- ham production: The porcine cathepsin B (CTSB) and 14 tional Foods. Boca Raton, Florida: CRC Press, cystatin B (CSTB) genes. Anim Genet 33:123–131. 15 pp. 251–275. RE Rust. 1976. Sausage and Processed Meat Manufac- 16 J Fournaud. 1978. La microbiologie du saucisson sec. turing. Washington, D.C.: American Meat Institute, 17 L’Alimentation et la Vie 64:382–392. pp. 97–101. 18 SE Gilliland. 1985. Bacterial Starter Cultures for S Schmidt, RG Berger. 1998. Microbial formed aroma 19 Foods. Boca Raton, Florida: CRC Press, pp. 57–183. compounds during the maturation of dry fermented 20 YH Hui, LM Goddik, AS Hansen, WK Nip, PS sausages. Adv Food Sci 20:144–152. 21 Stanfield, F Toldrá. 2004. Handbook of Food and LH Stahnke, LO Sunesen, A de Smedt. 1999. Sensory 22 Beverage Fermentation Technology. New York: characteristics of European dried fermented sausages 23 Marcel Dekker, Inc., pp. 353–368, 385–458. and the correlation to volatile profile. Thirteenth 24 PG Klettner, PA Baumgartner. 1980. The technology of Forum for Applied Biotechnology. Med Fac Land- 25 raw dry sausage manufacture. Food Technol Austral bouw Univ 64/5b, pp. 559–566. 26 32:380–384. F Toldrá, M Flores. 2000. The use of muscle enzymes 27 SL Komarik, DK Tressler, L Long. 1974. Food Prod- as predictors of pork meat quality. Food Chem 28 ucts Formulary. Westport, Connecticut: The AVI 69:387–395. 29 Publishing Company Inc, pp. 33–51. United States Department of Agriculture. 1963. Com- 30 E Muguerza, O Gomeno, D Ansorena, JG Bloukas, I position of Foods. Agricultural Handbook 8. 31 Astiasarán. 2001. Effect of replacing pork back fat Washington D.C. 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 S53 N54 40454 03 017-030 r2.qxd 7/20/07 12:01 PM Page 17

1 2 3 3 4 5 6 Principles of Curing 7 8 Karl O. Honikel 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION agents penetrate slowly into the muscular tissue. 22 This dry-curing process lasts up to several weeks. 23 Curing meat means the addition of nitrite and/or The process can be accelerated by wet curing, in 24 nitrate together with salt to meat. The effective sub- which the meat cuts are either inserted into a brine of 25 stance is nitrite or its derivatives. Nitrate becomes an salt and curing agents and/or brine is injected into 26 active curing agent only after reduction to nitrite. the meat by needles or via blood vessels. 27 Nitrite at the usual pH value range of meat and dur- In small pieces and in minced or comminuted 28 ing meat processing at about pH 5–6.5 forms NO meat like sausages, salt and the curing agent are 29 which binds to myoglobin. The NO-myoglobin or mixed in or comminuted with the meat in a bowl 30 NO-hem is heat-stable and gives the meat the red chopper. This way of curing is fast, and within a 31 curing color. Nitrite oxidizes to nitrate in meat prod- short time (1–2 days) depending on the processing 32 ucts and acts as an oxygen scavenger or as an antiox- technique, leads to the characteristic appearance of a 33 idative substance. cured product like red color and a “cured” flavor. 34 By this and other reactions, the nitrite concen- 35 tration is reduced in a ready-to-eat meat product HISTORY OF CURING 36 to less than half of the ingoing amount. During 37 storage the concentration is further reduced. Due to In ancient times, salt was used as one way of preser- 38 the lack of the reaction partner of secondary amines vation of many foods, meat cuts, and fish. This 39 in fresh meat, the low nitrite concentration and the process was usually combined with drying and/or 40 high pH-value, cooked meat products do not contain smoking. Salt addition and drying result in a reduc- 41 nitrosamines. Raw, long-aged meat products may tion of the free available water for microorganisms, 42 contain traces of various nitrosamines. lowering the water activity (aw). Smoking preserves 43 by covering the surface of meat with bacteriostatic 44 DEFINITION OF CURING and mycostatic compounds. Thus, smoking also 45 adds to the preservation process. 46 Curing meat means the addition of nitrite and/or Salt in its pure state is sodium chloride (NaCl). 47 nitrate together with salt (NaCl) to meat in various Many sources of salt, such as rock salt or marine salt, 48 degrees of comminution and at different processing contain small amounts of other salts. In the 19th cen- 49 steps. tury, it was discovered that saltpeter (potassium and 50 The most basic method of curing is the covering sodium nitrate) was a constituent of many preserving 51 of meat cuts (e.g., part of hind legs of pigs) in a mix- salts. The formation of the red curing color was iden- 52 ture of salt and curing agent. The salt and curing tified as depending on the presence of nitrate. 53 S54 N55 17 40454 03 017-030 r2.qxd 7/20/07 12:01 PM Page 18

18 Meat Fermentation Worldwide: History and Principles

1 Polenske, from the German Imperial Health Office, NO formed the bright red cured meat color. The pH of 2 published a paper in 1891 where he tried to explain meat is usually around 5.5; the sequence of events 3 the red compound in meat products. He had observed known in the second decade of the 20th century is 4 that cured meat and pickling solutions contained described in Figure 3.1. 5 nitrite even though only saltpeter (potassium nitrate) So the principle of the mechansim for the red 6 had been used in the fresh solution of the salt. color of cured meat was understood. The antimicro- 7 A heated sterilized solution did not produce nitrite, bial action was still thought to be due to the salt. 8 but an unheated solution did so after a few days of Nitrite/nitrate were not considered to be responsible 9 storage. The solution was simple but nevertheless for the preservation itself. − 10 revolutionary: Nitrate (NO3 ) was reduced to nitrite 11 (NO −) by microorganisms. 2 LEGISLATION 12 This observation was followed in 1899 by 13 Lehmann. He observed that an acidified nitrite solu- After it was discovered that nitrite was the genuine 14 tion turned meat to a bright red color. Nitrate and curing agent, it took only a few years until nitrite was 15 nitric acid did not produce the bright red color known introduced into meat products manufacturing. But 16 from cured meat. Kisskalt (1899) observed in the nitrite itself is rather toxic in comparison to nitrate. 17 same year that cooking meat in nitrite solution turned As a rule of thumb, nitrite is 10 times more toxic 18 it red, but not if nitrate was applied. Nitrate turned than nitrate. The lethal oral doses for human beings 19 meat bright red only after the meat was left in nitrate are established in 80–800 mg nitrate/kg body 20 solution at ambient temperatures for a few days. weight and 33–250 mg nitrite/kg body weight 21 So three scientists confirmed within a decade that (Schuddeboom 1993). 22 nitrite was responsible for the curing color and not Nitrite was added to meat products sometimes in too 23 nitrate. high amounts and—e.g., in Germany—some people 24 Haldane (1901) brought light into the chemistry died in the 3rd decade of the 20th century due to intox- 25 of the process. He found out that the change in color ication by nitrite in meat products. Germany solved the 26 by cutting the surfaces of fresh meat with its forma- problem in 1934 with the Nitrit-Pökelsalz-Gesetz 27 tion of oximyoglobin turned back to the less-red (nitrite-curing salt law). It says that nitrite is allowed 28 state on covering it afterward with a glass plate. This only in premixes with table salt; its content should be 29 behavior was different when meat was treated with 0.5% and must not exceed 0.6%. Only nitrate could be 30 nitrite. So, salt (nitrate/nitrite containing)-treated added directly to meat batters. 0.5% means that with 31 meat in the raw state was bright red after a fresh cut. 20 g nitrite-curing salt/kg of batter (2%) 100 mg 32 It turned brown upon exposure to air. Covering this nitrite/kg batter (100 ppm) would be added. 33 meat surface with a glass plate turned it bright red In the 1950s, the Fleisch-Verordnung (meat regu- 34 again. Extracting the colored substances, he found lation) limited the residual amount to 100 mg sodium 35 that the bright red salted/cured color was nitrogen nitrite/kg in ready-to-eat meat products. In raw hams,

36 oxide–myoglobin (NO-myoglobin). NO-myoglobin 150 mg NaNO2/kg were permitted. Nitrate restric- 37 was also existing in cured and cooked meat. tions also were applied. In the Fleisch-Verordnung of 38 Hoagland (1910, 1914) found out that only an acid- 1982, nitrate was limited to some nonheated products 39 iﬁed solution led to the red cured color. He concluded with ingoing amounts of 300–600 mg/kg and residual

40 that the nitrous acid (HNO2) or a metabolite of it like amounts of 100–600 mg/kg product. 41 42 43 44 45 (KNO3) nitrate reduction by micro organisms nitrite (KNO2) 46 + + 47 KNO2 + H HNO2 + K 48 2HNO N O + H O 49 2 2 3 2 50 N2O3 NO + NO2 51 52 NO + myoglobin NO-myoglobin 53S 54N Figure 3.1. Scheme of the proposal of Hoagland (1910, 1914) for the action of nitrate in cured meat products. 40454 03 017-030 r2.qxd 7/20/07 12:01 PM Page 19

Principles of Curing 19

This regulation was followed by the European Par- The food additive sodium nitrite may be safely 1 liament and Council Directive 95/2/EC on food addi- used in or on specified foods in accordance with 2 tives other than colors and sweeteners (Official Journal the following prescribed conditions: 3 EU 1995) where indicative ingoing amount 150 mg 3) As a preservative and color fixative, with 4 nitrite/kg and 300 mg nitrate/kg were permitted in sodium nitrate, in meat curing preparations for 5 more-or-less all meat products (Table 3.1). The resid- the home curing of meat and meat products 6 ual amounts could go up to 50 mg nitrite/kg in non- (including poultry and wild game), with direc- 7 heat–treated meat products and 100 mg nitrite/kg in all tions for use which limit the amount of sodium 8 other meat products, except Wiltshire bacon and some nitrite to not more than 200 parts per million in 9 others, to 175 mg nitrite/kg. With nitrate the residual the finished meat product, and the amount of 10 amounts were 250 mg nitrate/kg in all meat products. sodium nitrate to not more than 500 parts per 11 Denmark opposed this and excluded the use of million in the finished meat product. 12 nitrate from all meat products except Wiltshire bacon 13 All regulations, directives, and laws take into and some other raw hams with ingoing 300 mg 14 account that nitrite is a toxic substance and that, nitrate/kg. Nitrite up to 150 mg ingoing amounts of 15 contrary to other additives, nitrite does not remain nitrite/kg were written down in the Danish regulation 16 unchanged in the product during processing. Some 1055/95 of December 18, 1995 (DRL 1995A). 17 of the nitrite is converted to nitrate. The European Court in Luxembourg decided on 18 Also the discoveries of the early 20th century are March 20, 2003, that the European union must 19 taken into consideration. Nitrate is effective only reconsider their regulation 95/2/EC. 20 after being reduced to nitrite. This happens only in A new directive 2006/52/EC (official J. of the EU 21 products that are not heat treated early after manu- 2006) amending directive 95/2/EC (official J. of the 22 facturing; i.e., in raw hams and raw sausages. The EU 1995) on food additives other than colors and 23 intake of nitrite plus nitrate is thus limited to the nec- sweeteners restricts the use of nitrates to unheated 24 essary minimal requirements. products with 150 mg/kg. Nitrate may be added with 25 100 or 150 mg/kg to all meat products. For both 26 compounds a number of exceptions for named prod- CHEMISTRY OF NITRITE AND 27 ucts with residual amounts are permitted. NITRATE 28 In summary: In the European Union the use of 29 nitrite and nitrate will be limited to about 150 mg/kg CHEMICAL AND PHYSICAL CHARACTERISTICS OF 30 for each salt. Nitrate is permitted in nonheated NITRITES AND NITRATES AND RELATED COMPOUNDS 31 products only. 32 Nitrite and nitrate are used in cured meat products in Many countries have similar regulations. As an 33 the form of their sodium and potassium salts: example, the regulations of the U.S. should be 34

reported here. In their Code of Federal Regulations Sodium nitrite, NaNO2, sodium salt of nitrous acid, 35 (2005) they state: CAS No. 7632-00-0 36 37 Table 3.1. Nitrate and nitrite in meat products; extract from EU Directive 95/2/EC (1995). 38 39 Indicative Amounts that May Be 40 E No Name Foodstuff Added During Manufacturing 41 42 E 249 Potassium nitrite Meat products 150 mg/kg 43 E 250 Sodium nitrite Sterilized meat products (e >3.00) 100 mg/kg 44 Wiltshire cured bacon and ham 175 mg/kg as a residue 45 Dry cured bacon and ham 175 mg/kg as a residue 46 Cured tongue, jellied veal, brisket 10 mg/kg as a residue expressed 47 as NaNO2 48 E 251 Sodium nitrate Nonheat–treated meat products 150 mg/kg 49 E 252 Potassium nitrate Wiltshire cured bacon and ham 250 mg/kg as a residue 50 Dry cured bacon and ham 250 mg/kg as a residue 51 Cured tongue, jellied veal, brisket 10 mg/kg as a residue expressed 52 as NaNO S53 2 N54 40454 03 017-030 r2.qxd 7/20/07 12:01 PM Page 20

20 Meat Fermentation Worldwide: History and Principles

1 molecular weight 69 Dalton melting point 334°C 2 white or yellowish white crystals stable under normal conditions 3 odorless may oxidize organic matter and react 4 density 2.17 g/cm3 vigorously with ammonia salts 5 solubility 85.2 g/100 g water at 20°C, 6 163 g/100 g water at 100°C Nitrous acid, HNO2, known only in solution 7 pH in aqueous solution around 9 molecular weight 47.02 Dalton 8 melting point 271°C anhydride is N2O3, which exists only in the 9 hygroscopic on air solid state at temperatures below −102°C 10 oxidizes in air slowly to nitrate above the liquid is a mixture of → ϩ 11 strong oxidizing agent with organic matter and N2O3 NO NO2 12 inorganic material, especially ammonia middle strong acid, pK 3.37 13 compounds 14 toxicity oral rat LD50: 85 mg/kg; for humans Nitric acid, HNO3, CAS No. 7697-37-2 15 50 mg/kg body weight; irritating to skin and molecular weight 63.01 Dalton 16 other body surfaces colorless liquid in closed containers − 17 melting point 42°C 18 Potassium nitrite, KNO2, potassium salt of nitrous boiling point 86°C 19 acid, CAS No. 7758-09-0 density: 1.52 g/ ml at 20°C 20 molecular weight 85.1 Dalton mixable in any quantity with water 21 white or yellowish white crystals strong acid; totally dissociated in water 22 odorless 3 23 density 1.92 g/cm Nitric oxide, Nitrogenoxid NO, CAS No. 10102-43-9 24 solubility ca. 300 g/100 g water at 20°C, molecular weight 30 Dalton 25 413 g/100 g water at 100°C colorless gas pH in aqueous solution around 9 melting point −163.6°C 26 − 27 melting point 387°C; unstable above 350°C boiling point 151.8°C oxidizes in air 2NO ϩ O → N O → 2NO 28 strong oxidizing agent with organic matter 2 2 4 2 toxicity as NaNO 29 2 NO can bind to myoglobin forming NO-myoglobin. 30 Also this compound (NO) can easily loose an Sodium nitrate (saltpeter), NaNO3, the sodium salt 31 of Nitric acid, CAS No. 7631-99-4, used as gun electron and form a NOϩ cation which may react 32 powder with amines to nitrosamines. NO is formed in our 33 molecular weight 84.99 Dalton body to function as a physiological messenger play- 34 white crystals ing a role in cardiovascular, neurologic, and immune 35 odorless systems by suppressing pathogens, vasodilation, and 36 hygroscopic on moist air neurotransmission (Lowenstein and Snyder 1992; 37 density 2.16 g/cm3 Moncada et al. 1991; Nathan 1992; Luscher 1990, 38 solubility 73 g/100 ml water at 0°C, 180 g/ 1992; Marletta 1989; Ignarro 1990; Stamler et al. 39 100 ml water at 100°C 1992). 40 pH in aqueous solution around 7 41 melting point 307°C Nitrogendioxide NO2 (N2O4) 42 stable under normal conditions molecular weight NO2 46.01 Dalton 43 may oxidize reduced organic matter N2O4 is a colorless solid, turns brown by 44 (gun powder) dissociation into NO2 on warming up, liquid 45 at ambient temperature − 46 Potassium nitrate (saltpeter), KNO3, the potassium melting point 11.2°C 47 salt of nitric acid, CAS No. 7757-79-1 boiling point 21.15°C

48 molecular weight 101.1 Dalton N2O4 is formed in the solid state only, at 49 white crystals melting point and higher, more and more

50 odorless, salty taste NO2 is formed 3 51 density 2.11 g/cm N2O4 reacts with KI: 52 solubility 31.6 g/100 ml water at 20°C, ϩ − ϩ ϩ 53S 247 g/100 ml water at 100°C 4 1 5 2 O± ϩ → ϩ ϩ 54N pH in aqueous solution around 7 2N2O4 2KI 2KNO3 2NO I2 40454 03 017-030 r2.qxd 7/20/07 12:01 PM Page 21

Principles of Curing 21

Iodide is oxidized to iodine, NO is oxidized to 1 2 2HNO2 N2O3 + H2O nitrate and reduced to NO. For this reason the 2 addition of iodide as a iodine fortifying substance to N2O3 NO + NO2 3 − NO + ½ O NO salt is not possible. Iodate (IO3 ) must be used. 2 2 4 5 2NO2 + H2O HNO2 + HNO3 6 NITRITE AND NITRATE IN MEAT 7 If nitrites are added to meat, the salt is dissolved due overall: 2HNO2 + ½ O2 HNO2 + HNO3 8 to its good solubility in the aqueous solution of pH 9 around 5.5 of the meat. Due to the pK of nitrous acid 10 of 3.37, which means that a pH 3.37 around 50% of Figure 3.3. Reaction of nitrous acid and its 11 the acid is dissociated, it can be expected that about derivatives. 12 − 13 99% of the nitrite exists of pH 5.5 as an anion, NO2 (Figure 3.2). The small amount of undissociated 14 nitrous acid is in equilibrium with its anhydride cooked hams are manufactured with nitrite only, but 15 16 N2O3, which again is in equilibrium with the 2 oxides they contain a mean of 20–30 mg nitrate/kg, as also nitric oxide and nitric dioxide (Figure 3.3). shown in a very recent survey (Table 3.2). Nitrite is 17 The small amount of NO can react with oxygen in most cases lower than nitrate in the ﬁnished 18 forming NO . In the sum it means that from two product, with concentration below 20 mg nitrite/kg 19 2 20 HNO2 molecules one HNO2 and one HNO3 mole- in the mean value. Only a few samples of cooked and cule are formed (Figure 3.3). This reaction is rather raw sausages and raw ham contain above 60 mg 21 important in meat batter because nitrite acts in this nitrite/kg (Figure 3.4), which also have higher nitrate 22 way as an antioxidant. Metal ions seem to accelerate concentrations. In the raw products nitrate may have 23 the oxidation process. Nitrate added to meat will been added, in cooked (liver and blood sausages) the 24 fully dissociate into Naϩ(Kϩ) ϩ NO −. No detectable ingoing nitrite is apparently strongly oxidized to 25 3 26 amount of undissociated HNO3 will be found. nitrate by meat enzyme systems or metal ions due to their high pH values between 6.0 to 6.8. 27 In the last three decades the use of ascorbic acid 28 NITRITE AND NITRATE IN MEAT or ascorbate or isoascorbate (erythrothorbate) has 29 PRODUCTS become common practice. There are added to meat 30 batters with 500 mg/kg. Ascorbates and isoascorbates 31 CONCENTRATION IN MEAT PRODUCTS are antioxidative substances, which may also sequester 32 The oxidation of nitrite to nitrate in meat also oxygen (Figure 3.6). In this way, they retard the oxida- 33 34 explains why in meat products to which only nitrite tion of NO to NO2 and the formation of nitrate. has been added nitrate will be found in considerable But ascorbate seems also to react with nitrite or 35 concentrations. In Figures 3.4 and 3.5, the nitrite and one of its metabolites. Dahn et al. (1960), Fox et al. 36 nitrate concentrations of German meat products are (1968), and Izumi et al. (1989) show that ascorbate 37 shown. The emulsion-type and cooked sausages and reacts with nitrite and binds the resulting NO. The 38 39 40 41 42

–3 43 Meat with 100 ppm (mg/kg) of nitrite added = 1.45 x 10 M 44 45 + + + Na + NO2– + H HNO2 + Na (a) 46 47 pH of meat 5.5 = 3 x 10–6 M H+ 48 49 pK of nitrous acid = 3.37; K = 4.27 x 10–4 50 51 reaction (a) is with more than 99 % at the left side at pH 5.5 52 S53 Figure 3.2. State of nitrite in meat homogenates. N54 40454 03 017-030 r2.qxd 7/20/07 12:01 PM Page 22

22 Meat Fermentation Worldwide: History and Principles

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Figure 3.4. Nitrite in German meat products (1996Ð2001) (Dederer 2006). 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Figure 3.5. Nitrate in German meat products (1996Ð2001) (Dederer 2006). 41 42 43 44 nitrite is reduced to NO, the ascorbate oxidized to low residual amounts of nitrite in ready-to-eat meat 45 dehydroascorbate. The bound NO seems to be products. 46 able to react as NO with other meat ingredients. After the reported reactions it could be assumed 47 Ascorbate is also added to reduce the formation of that the concentration of nitrate in a sausage 48 nitrosamines, which is discussed later. where only nitrite is added is related to the nitrite 49 But not only ascorbate and myoglobin react with content. 50 nitrite derivatives; amino acids (Figure 3.7) and Figure 3.8 shows that with emulsion-type 51 unsaturated fatty acids also react. These products are sausages (only nitrite curing salt is used) the 52 rather unstable and either release NO or are oxidized residual amounts of nitrite and nitrate exhibit no 53S to NO2 and/or nitrocompounds. These reactions of relationship above 20 mg residual nitrite/kg. There 54N NO with other substances are another reason for the is no generally recognizable increase of nitrate with 40454 03 017-030 r2.qxd 7/20/07 12:01 PM Page 23

Principles of Curing 23

Table 3.2. Nitrite and nitrate concentrations in time of storage. The reduction during storage is 1 sausages of Germany (2003Ð2005) (Dederer 2006). slower with increasing pH. 2 Table 3.5 shows the influence of different heat 3 Median mg/kg treatment. The higher the heating, the greater the 4 Year n Nitrite Nitrate loss of nitrite. The formation of nitrate is also 5 reduced. A higher pH value and/or a different 6 Emulsion- 2003 30 13.2 23.4 muscle show less nitrite loss and a higher nitrate 7 type sausage 2004 32 12.65 20.5 concentration. The addition of ascorbate and 8 2005 29 19.9 30.0 polyphosphate show that the disappearance of nitrite 9 Raw sausages 2003–2005 15 17.9 59.2 is accelerated by ascorbate in the raw batter 10 Raw ham 2003–2005 14 19.2 16.9 (Table 3.6). Heating for 7 minutes to 80°C leads to a 11 slower loss of nitrite. Heating for additionally 1 hour 12 Liver/blood 2003–2005 16 12.1 43.3 at 70°C retards the loss even longer. This is probably 13 sausages due to the inactivation of microorganisms and inacti- 14 (cooked vation of enzymes by heating. With ascorbate and 15 sausages) even more with polyphosphates, the retarding by 16 heating is also observed. 17 18 19 INFLUENCE OF HIGH PRESSURE ON NITRITE 20 AND NITRATE CONCENTRATION increasing residual amounts of nitrite. Without 21 nitrite addition, a residual amount of nitrate is The main loss of nitrite occurs in the heating 22 probably due to the added drinking water and spices. process and without heating by microorganisms. The 23 application of high pressure up to 800 MPa, which 24 inactivates microorganisms but does not exceed 25 CHANGES WITH TIME OF STORAGE 35°C shows that nitrite is oxidized to about 20% to 26 When does the nitrite disappear in the product? nitrate, but nitrite ϩ nitrate add up nearly to the 27 Table 3.3 shows results from Russian colleagues added amount of nitrite (Table 3.7). During storage, 28 (Kudryashow 2003). The largest decrease is observed the nitrite decreases to about half of the ingoing 29 during the manufacturing up to the end of the heating amount, and the nitrate increases to about 40%. The 30 process. This early loss exceeds usually 65% inde- sum of nitrite plus nitrate is even at 21 days of stor- 31 pendent of the ingoing concentrations. Within 20 age at 90% of the ingoing amount of nitrite. These 32 days of cold storage the concentrations drop further results show, in comparison to the results of Tables 33 to a third of the concentration after heating.` 3.3 through 3.5, that only small amounts of nitrite 34 The disappearance continues until 60 days of react with other substances than oxygen to nitrate, 35 cold storage. Table 3.4 confirms the results. It on applying high pressure. The reason for this obser- 36 furthermore shows that a higher pH value retards the vation is unknown. 37 disappearance of nitrite. It also confirms the results Nitrite is in all cases described here partially oxi- 38 of Table 3.3 that nitrate is already high at day 0 after dized to nitrate. In many experiments (see Tables 3.4, 39 heating. Nitrate also falls in concentrations with 3.5 and 3.7), about 10–40% of the nitrite is oxidized 40 41 42 43 44 45 46 47 48 49 50 51 52 S53 Figure 3.6. Oxygen sequestering by ascorbate (ascorbic acid). N54 40454 03 017-030 r2.qxd 7/20/07 12:01 PM Page 24

24 Meat Fermentation Worldwide: History and Principles

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 Figure 3.7. Reactions of NO with amino acid. 40 41 42 to nitrate. But only in the results of Table 3.7 nitrite Myoglobin exists in a muscle in three states, in 43 plus nitrate add up to about 90% of more of the which the cofactor hem, a porphyrin ring with an 44 nitrite addition. This has been known for decades; in iron ion in its center binds different ligands or in 45 1978, Cassens et al. postulated that nitrite is bound which the iron exists in the Fe2ϩ or Fe3ϩ state. In the 46 to various meat constituents (Table 3.8). native myoglobin, the porphyrin moiety (Figure 3.9) 47 is supported in the ligand binding by amino acids of 48 the protein in the neighborhood. NITROSOMYOGLOBIN ϩ 49 In the “original” state, myoglobin with Fe2 in the 50 The red color of cured meat products is one of porphyrin cofactor does not bind any ligand except 51 the important effects of nitrite in meat products. maybe a water molecule. In the presence of oxygen 52 The red color is developing in a number of compli- the porphyrin can bind an O2 molecule and it 53S cated reaction steps until NO-myoglobin (Fe2ϩ) is becomes bright red. The iron ion is in the Fe2ϩ state. 54N formed. But oxygen and other oxidizing agents like nitrite 40454 03 017-030 r2.qxd 7/20/07 12:01 PM Page 25

Principles of Curing 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Figure 3.8. Concentration of nitrite and nitrate in emulsion-type sausages (N = 48) (Dederer 2006). 16 17 ϩ ϩ Table 3.3. Remaining nitrite (mg/kg) during storage can oxidize the Fe2 to Fe3 (Figure 3.10). This met- 18 at 2¡C of an emulsion-type sausage (Kudryashow myoglobin is brown. 19 2003). The “original” myoglobin (Mb), the oximyoglobin 20 (MbO2) and the metmyoglobin are occurring Concentration of Nitrite 21 together in meat. In a muscle in a live animal, there Added (mg/kg) 22 is very little metmyoglobin, which increases post- 23 Storage 75 100 150 200 mortem with the disappearance of oxygen except 24 when meat is MAP-packed with high oxygen. 25 After heating 21.9 30.5 59.5 53.7 The three states of myoglobin have three charac- 26 20 days 7.5 9.3 10.2 15.4 teristic absorbance spectra between 400 and 700 nm. 27 40 days 3.6 6.4 7.6 7.7 As the three are in a kind of equilibrium to each 28 other, the spectra have an isospestic point at 525 nm 29 60 days 0.5 0.9 4.0 5.8 where all three absorption curves cross each other. 30 31 Table 3.4. Nitrite breakdown and nitrate appearance after nitrite addition to meat of various pH values after 32 heating and storage (Dordevic et al. 1980). 33 Nitrite Nitrate 34 35 Days of 100 mg/kg 200 mg/kg 100 mg/kg 200 mg/kg 36 pH Storage Added Added Nitrite Added Nitrite Added 37 38 5.3 After 39 heating 40 028702050 41 620411627 42 12 5 18 9 20 43 5.8 After 44 heating 45 0 45 120 30 64 46 6 24 110 22 40 47 12 13 21 8 17 48 6.3 After 49 heating 50 0 58 135 18 40 51 6 41 112 17 30 52 12 31 90 10 22 S53 N54 40454 03 017-030 r2.qxd 7/20/07 12:01 PM Page 26

26 Meat Fermentation Worldwide: History and Principles

1 Table 3.5. Mean values of nitrite and nitrate content in pasteurized and sterilized groups of homogenates 2 (Dordevic et al. 1980). 3 After Heat Treatment After 12 Days at 2–4°C 4 Homogenate 5 100 mg/kg of Way of NaNO2 NaNO3 NaNO2 NaNO3 6 * NaNO2 Added Heat Treatment mg/kg mg/kg mg/kg mg/kg 7 8 M. longissimus dorsi P 38.6 27.4 10.9 7.9 9 pH 5,8 S 12.9 13.1 3.5 6.0 10 M. quadriceps femoris P 49.2 35.8 26.7 12.8 11 12 pH 6,15 S 15.6 19.4 7.4 9.8 13 *P = pasteurization (75°C). S = sterilization (>110°C). 14 15 16 17 Table 3.6. Approximate number of days for residual nitrite to fall below 10 mg/kg in a pork slurry of pH 5.5Ð6.2 18 at 2.5Ð4.5% NaCl at a storage temperature of 15¡C; (N = 5) (Gibson et al. 1984). 19 Heat Treatment 20 21 80°C/7 min ϩ 22 Nitrite Added (mg/kg) Unheated 80°C/7 min 70°C/1 hour 23 24 Days Days Days 25 A) No further addition 26 100 5 12 63 27 200 10 12 68 28 300 21 21 >168 29 B) As in A ϩ ascorbate 30 (1000 mg/kg) 31 100 5 9 10 32 200 5 9 9 33 300 5 21 48 34 ϩ 35 C) As in A ascorbate ϩ 36 (1000 mg/kg) 37 polyphosphate 0.3% (w/v) 38 100 5 10 21 39 200 10 21 21 40 300 5 5 12 41 42 43 The absorbance of this wavelength can be used for ring is exchanged with Zn2ϩ, which gives a pleasant 44 detecting the percentage of each form in meat. red color. Nitrite addition prevents the exchange 45 Nitrosomyoglobin has a spectrum that has similar (Morita et al., 1996; Møller et al., 2003; Parolari et al., 46 maxima as oximyoglobin (Figure 3.11). 2003; Wakamatsu et al., 2004; Christina et al., 2006). 47 Oxygen and NO are biatomic molecules. A simi- By reducing enzymes or chemical reactions with a 48 lar biatomic molecule CO also binds to myoglobin, reducing agent like ascorbate, the Fe3ϩ is reduced to and is also very tight. In some countries, MAP pack- 2ϩ 49 Fe (Figure 3.12). The NO formed from N2O3 can 50 aging of meat with 1–2% CO is permitted. bind to the myoglobin (Fe2ϩ) and forms a heat-stable 51 In the last 2 years the riddle about the red color of NO-myoglobin. Oximyoglobin is not heat-stable and 52 non-nitrite/nitrate–cured raw hams like Parma ham dissociates. The meat turns grey or brown. 53S or Jamon Iberico has been solved. Various authors The NO-myoglobin is heat-stable. On heating the 54N could show and prove that the Fe2ϩ in the porphyrin protein moiety is denatured, but the red NO-porphyring 40454 03 017-030 r2.qxd 7/20/07 12:01 PM Page 27

Table 3.7. Nitrite and nitrate concentration in emulsion-type sausage (Lyoner) (72 ppm nitrite added) after 1 ultra-high-pressure application and storage of the unheated batter. 2 3 Days 0 7 14 21 0 7 14 21 0 7 14 21 4 Nitrite ϩ 5 Treatment Nitrite (mg/kg) Nitrate (mg/kg) Nitrate (mg/kg) 6 7 Control 54,3 47,1 42,8 39,3 15,15 19,5 23,8 26,2 69,45 66,55 66,6 65,5 8 400 MPa 53,3 46,4 41,5 37,7 15,8 22 25,9 26,95 69,05 68,4 67,4 64,6 9 10 600 MPa 53,0 44,8 40,7 37,2 16,15 23,85 27,35 29,1 69,1 68,65 68 66,25 11 800 MPa 52,3 44,7 41,6 37,7 17,5 23,95 26,55 26,6 69,75 68,65 68,15 64,25 12 13 Source: Ziegenhals (2006). 14 15 16 Table 3.8. Nitrite and metabolites in meat products. + 3 17 + 2+ Bound to or From % of Totala % of Totalb 2H + NO2¯ + Fe - myoglobin 18 19 2+ Nitrite 5–20 3+ 20 H2O + NO + Fe - myoglobin(metmyoglobin) Nitrate 1–10 10–40a 21 22 Myoglobin 5–15 Figure 3.10. Nitrite reaction with myoglobin in meat 23 Bound to ϪSH 1–15 at pH 5.5. 24 Bound to lipids 1–15 25 Bound to proteins 20–30 26 27 Gas 1–5 28 Sum ϳ70 90 MbO2 Mb 29 30 a Cassens et al. (1978). 31 b Assumption realized by the author (K. O. Honikel) MetMb 32 according to results presented in Figures 3.4, 3.5, and 3.6. MbNO 33 34 35 36 Ð Ð COO COO 37

CH2 CH2 38 39 CH2 H CH2 40 C C C 41 H C C C CH3 3 C C 42 450 470 490 510 530 550 570 590 610 630 43 C NNC nm 44 HC Fe CH 45 Figure 3.11. Spectra of myoglobin. C N N C 46 H C 47 2 C C CH C C C 3 48 H C C C 49 H 3+ 50 CH3 C metmyoglobin (Fe ) + NO + ascorbate H CH2 51 Haem 2+ NO-myoglobin (Fe ) + dehydroascorbate 52 (Fe-protoporphyrin IX) S53 Figure 3.9. Hem in the myoglobin molecule. Figure 3.12. Formation of nitrosomyoglobin. N54

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28 Meat Fermentation Worldwide: History and Principles

1 system (often called nitroso-haemochromogen) still Table 3.9. NO-dimethylamine in foods (µg/kg) 2 exists and is found in meat products heated to (Deierling et al. 1997). 3 120°C. Content 4 This heat-stable red color will change on bacterial 5 spoilage, and it fades on UV light. The ﬁrst one is Food N >0.5 Min Max 6 advantageous because the consumer recognizes 7 spoilage, such as in fresh meat, which also changes Beer 195 3 0.5 1.2 8 color on spoilage. Pizza 57 6 0.5 8.7 9 Meat products 17 0 0 0 10 NITROSAMINE FORMATION 11 Milk products 6 0 0 0 12 In the 1970s in the U.S., a discussion came up about 13 the formation of nitrosamines in cured meat products, 14 especially fried bacon. Fiddler et al. (1978) showed nitrosamines can be formed. Bacon frying, cured 15 that bacon and its cookout on frying contained con- sausage, grilling, or frying cured meat products, such 16 siderable amounts of nitrosopyrrolidine. as pizza toppings, may result in such conditions that 17 Nitrosamines are formed by amines with nitrite at nitrosamines are formed. Table 3.9 shows the results 18 higher temperatures according to the reactions in of an investigation by Deierling et al. (1997). In Ger- 19 Figure 3.13. But there are some prerequisites: man foods, only beer and pizza exhibited dimethyl- 20 nitrosamine in detectable amounts. Thus nitrosamines 21 • Amines must be present. In fresh meat, there are occur only in small amounts and are easily avoidable 22 very minute amounts of amines, which are the by proper frying, grilling, and pizza baking. 23 decarboxylation products of amino acids. During 24 aging and fermentation amines are formed. 25 • Only secondary amines form stable nitrosamines CONCLUSIONS 26 (Figure 3.13). Primary amines are immediately Curing meat is a process known since ancient times. 27 degraded to alcohol and nitrogen. Tertiary amines The curing agents nitrite and nitrate react due to 28 cannot react. Most amines in meat are primary easily varying oxidation status of nitrogen into many 29 amines derived from α-amino acids. derivatives with meat ingredients. 30 • The pH must be low enough to produce NOϩ or Both are those additives in foods that change to a 31 metal ions must be engaged to form NO considerable extent. The curing agents give the 32 (Figure 3.13). products an esteemed and stable red color; they act 33 as antioxidants, nitrite prevents or retards microbial 34 As heated meat products are produced from fresh growth, and the curing agents give a pleasant ﬂavor. 35 meat (chilled or frozen) no amines are available. In The positive effects are overwhelming against the 36 raw meat products, the nitrite concentration is rather small possibility of the formation of nitrosamines. 37 low (see Figure 3.4) and thus the formation of NOϩ is The intake of curing agents by meat products is 38 rather unlikely. In products heated above 130°C, small (few percent) in comparison with other foods. 39 40 41 + + 42 NaNO2 + H HNO2 + Na 43 + + HNO2 + H NO + H2O 44 45 2HNO2 N2O3 + H2O 46 N2O3 NO + NO2 47 NO + M+ NO+ + M 48 + + primary amine RNH2 + NO RNH-N = O + H ROH + N2 49 + + 50 secondary amine R2NH + NO R2N-N = O + H 51 tertiary amine R3N + NO+ no nitrosamine formation 52 53S Figure 3.13. Formation of nitrosamines, chemical reactions (M/Mϩ are transition metal ions such as 54N Fe2ϩ/Fe3ϩ). 40454 03 017-030 r2.qxd 7/20/07 12:01 PM Page 29

Principles of Curing 29

REFERENCES K Izumi, RG Cassens, ML Greaser. 1989. Reaction of 1 nitrite with ascorbic acid and its significant role in 2 RG Cassens, I Ito, M Lee, D Buege. 1978. The Use of nitrite-cured food. Meat Sci 26:141–153. 3 Nitrite in Meat. Biosci 28, 10:633–637. K Kisskalt. 1899. Beiträge zur Kenntnis der 4 E Christina, JKS Adamsen, K Møller, K Laursen, LH Ursachen des Rotwerdens des Fleisches beim 5 Olsen, Skibsted. 2006. Zn-porphyrin formation in Kochen nebst einigen Versuchen über die Wirkung 6 cured meat products: Effect of added salt and nitrite. der schwefeligen Säure auf der Fleischfarbe. Arch 7 Meat Sci 72:672–679. Hyg Bakt 35:11–18. Kudryasho. 2003. personal 8 Code of Federal Regulations (USA). 2005. Title 21, communication. 9 Volume 3, Food and Drugs. Food Additives, KB Lehmann. 1899. Über das Haemorrhodin. Ein 10 21CFR170.60, 172.175, 172.170. neues weit verbreitetes Blutfarbstoffderivat. Über 11 H Dahn, Lotte Loewe, CA Bunton. 1960. 42. Über die Phys Med Gesellschaft Würzburg 4:57–61. 12 Oxydation von Ascorbinsäure durch salpetrige CJ Lowenstein, SH Snyder. 1992. Nitric oxide, a novel 13 Säure. Teil VI: Übersicht und Diskussion der biologic messenger. Cell 70:705–707. 14 Ergebnisse. 18. Mitteilung über Reduktone und TF Luscher. 1990. Endothelial control of vascular tone 15 1,2,3-Tricarbonylverbindungen. Helvetica Chimica and growth. Clin Exp Hypertens (A) 12:897–902. 16 Acta 53:320–333. ———. 1992. Endogenous and exogenous nitrates and 17 Danish Regulation Lovtidende. 1995A. p. 5571 of their role in myocardial ischaemia. Br J Clin Pharma- 18 18.12.1995. col 34(Supp. 1):29–35. 19 I Dederer. 2006. Personal communication. MA Marletta. 1989. Nitric oxide: Biosynthesis and bio- 20 H Deierling, U Hemmrich, N Groth, H Taschan. 1997. logical signifcance. Trends Biochem Sci 21 Nitrosamine in Lebensmitteln. Lebensmittelchemie 14:488–492. 22 51:53–61. JKS Møller, CE Adamsen, LH Skibsted. 2003. Spectral 23 V Dordević, B Vukksan, P Radetić, H Durdica, M characterisation of red pigment in Italian-type dry- 24 Mitković. 1980. Prilog ispitivanju uticaja pojedinih cured ham. Increasing lipophilicity during process- 25 faktora na promene sadrzaja nitrita u mesu. ing an maturation. European Food Res Technol 26 Tehnologija mesa 21, 10:287–290 216:290–296. 27 W Fiddler, JW Pensabene, EG Piotrowski, JG Philips, S Moncada, RM Palmer, EA Higgs. 1991. Nitric oxide: 28 J Keating, WJ Mergens, HL Newmark. 1978. Inhibi- Physiology, pathophysiology and pharmacology. 29 tion of formation of volatile nitrosamines in fried Pharmacol Rev 43:109–142. 30 bacon by the use of cure-solubilized α-tocopherol. H Morita, J Niu, R Sakata, Y Nagata. 1996. Red pig- 31 J Agric Food Chem 26, 3:653–656. ment of Parma ham and bacterial influence on its for- 32 JB Fox, JR, SA Ackerman. 1968. Formation of nitric mation. J Food Sci 61(5):1021–1023. 33 oxide myoglobin: Mechanisms of the reaction with C Nathan. 1992. Nitric oxide as a secretory product of 34 various reductants. J Food Sci 33:364–370. mammalian cells. FASEB J 6:3051–3064. 35 AM Gibson, TA Roberts, A Robinson. 1984. Factors Official Journal of the EU. 1995. Directive 95/2/EC. 36 controlling the growth of Clostridium botulinum L61, 18.3. Food Additives Other Than Colours and 37 types A and B in pasteurized cured meats. VI. Nitrite Sweeteners, pp. 1–40. 38 monitoring during storage of pasteurized pork slur- ———. 2006. Directive 2006/52/EC. L204, 26.7. Food 39 ries. J Food Technol 19:29–44. Additives Other than Colours and Sweeteners, pp. 40 J Haldane. 1901. The red color of salted meat. 10–22. 41 J Hygiene I:115–122. G Parolari, L Gabba, G Saccani. 2003. Extraction prop- 42 R Hoagland. 1910. The action of saltpeter upon the erties and absorption spectra of dry cured hams made 43 color of meat. In: 25th Annual Report of the Bureau with and without nitrite. Meat Sci 64:483–490. 44 of Animal Industry. U.S. Department of Agriculture. E Polenske. 1891. Über den Verlust, welchen Rind- 45 Washington, D.C., Government Printing Office, fleisch und Nährwert durch das Pökeln erleidet sowie 46 1910, pp 301–316. über die Veränderungen salpeterhaltiger Pökellaken. 47 ———. 1914. Coloring matter of raw and cooked Arbeiten aus dem kaiserlichen Gesundheitsamt 48 salted meats. J Agric Res III, Secretary of Agricul- 7:471–474. 49 ture. Washington, D.C., 3 (1914/15) 3. 211–225, LJ Schuddeboom. 1993. Nitrates and nitrites in food- 50 2 Taf., Heft 3, 15.12.1914. stuffs. Council of Europe Press, Publishing and 51 LJ Ignarro. 1990. Biosynthesis and metabolism of Documentation Service. ISBN 92-871-2424-6. 52 endothelium-derived nitric oxide. Ann Rev Pharma- JS Stamler, DJ Singel, J Loscalzo. 1992. Biochemistry S53 col Roxicol 30:535–560. of nitric oxide and its redox-activated forms. Sci N54 40454 03 017-030 r2.qxd 7/20/07 12:01 PM Page 30

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1 258:1898–1902.Verordnung über Fleisch und Fleis- J Wakamatsu, J Okui, Y Ikeda, T Nishimura, A Hattori. 2 cherzeugnis (Fleisch-Verordnung) in der Fassung der 2004b. Establishment of a model experiment 3 Bekanntmachung vom 21. Januar 1982. Bundesge- system to elucidate the mechanism by which Zn- 4 setzblatt I. 1982. Nr. 3, 89–101. protoporphyrin IX is formed in nitrite-free dry-cured 5 J Wakamatsu, T Nishimura, A Hattori. 2004a. A ham. Meat Sci 68 (2):313–317. 6 Zn-porphyrin complex contributes to bright red K Ziegenhals. 2006. Personal communication. 7 colour in Parma ham. Meat Sci 67:95–100. 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53S 54N 40454 04 031-036 r2.qxd 7/20/07 12:03 PM Page 31

1 2 3 4 4 5 6 Principles of Meat Fermentation 7 8 Esko Petäjä-Kanninen and Eero Puolanne 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION muscular tissue partly melts and at the end of chop- 22 ping, a homogeneous, slightly sticky mince is 23 The process of fermenting and drying is believed to formed which is then stuffed into casings. 24 be one of the oldest techniques for preserving meat. During the first hours of processing, the tem- 25 First notions about fermented sausages are from perature of the sausage mince will increase from 26 3000 B.C., and more information is from China and approximately Ϫ2°C to the level of 20°C or more 27 the Mediterranean area from 2000 years ago depending on the sausage type. In the initial fermen- 28 (Varnam and Sutherland 1995). Although the inner 29 parts of animal tissues are sterile, in practical tation step, sugars in the mince are converted into lactic acid by lactic acid bacteria, and water starts 30 terms it is impossible to keep meat sterile during 31 slaughtering and cutting. Once contaminated, the migrating to the surface where it evaporates. Later on during drying and ripening, staphylococci, yeasts, 32 deterioration of quality and health hazards advance 33 quickly unless the meat is consumed or preserved. and molds form compounds from other sources than sugars that influence taste and aroma. These 34 Consequently, meat has been preserved since ancient 35 times by cooking, salting, drying, and—mostly in processes result in a characteristic flavored sausage with a lowered pH and a reduced water activity that 36 colder areas—by smoking, alone or in combination. 37 Early, it was also discovered that preservation makes the final sausage stable with long shelf life, even if not being subjected to heat treatment. 38 resulted in meat products with sensory properties 39 that are well liked. Fermentation of meat means, in the context of this chapter, the action of microorganisms and meat 40 A fermented dried sausage consists of muscular 41 enzymes resulting in the desired quality and keepa- tissue, from which fat and thick connective tissue 42 bility. Along with fermentation of dry-fermented membranes have been trimmed off, and fatty tissue, 43 products (fermentation at ambient temperatures) dry- which is most often pork back fat. The other ingredi- 44 ing and/or smoking are usually applied. In semidry ents are salt (NaCl) 2.2% or more, sugars (glucose, 45 products (quick fermentation at high temperatures), sucrose, oligosaccharides derived from starch, 46 cooking is used to stop the fermentation, especially sometimes lactose), and occasionally foodstuffs of 47 in the U.S. (Varnam and Sutherland 1995). vegetable origin (like tomato or onion) or cheese. 48 Spices, nitrite, and/or nitrate and ascorbate are used The utilization of fermentation, drying, and smok- 49 as well. Traditionally, the fermentation relied on the ing may have developed originally by accident. 50 natural microbial flora, but in modern production Spontaneous fermentation made the meat stable at 51 pure microbial cultures are added. The ingredients ambient temperatures and improved the sensory 52 are chopped, meat and fat in frozen stage, in a quality of the product. At first, salt, which included 53 chopper more-or-less coarsely. As a result of salt, natural nitrate, was added to meat; later, more S54 N55 31 40454 04 031-036 r2.qxd 7/20/07 12:03 PM Page 32

32 Meat Fermentation Worldwide: History and Principles

1 sophisticated comminuting-smoking-drying proce- BIOCHEMISTRY OF FERMENTATION 2 dures were introduced. Adenosine triphosphate (ATP) is a form of chemical 3 A well-known practice has been to add a piece of energy that is necessary for life. The primary reason 4 good sausage from a previous batch to the ingredi- for fermentation is to produce energy anaerobically, 5 ents of a new batch (backslopping). In the early e.g., to produce ATP using the substrate level phos- 6 1900s it was discovered that it was beneficial bacte- phorylation. In the reaction sequence, there is for 7 ria that caused the positive reactions in the sausage each reduction a corresponding oxidation, and no 8 batter, and the utilization of inoculated bacteria, oxygen or any other molecule is needed for terminal 9 starter cultures, was introduced in the 1930s and electron recipient. Practically any organic substance 10 1940s; lactobacilli (Jensen and Paddock 1940; that contains oxygen and hydrogen can be used as 11 Nurmi 1966), and later in the 1950s pediococci starting material, but usually carbohydrates or amino 12 (Niven et al. 1955) and staphylococci (Niinivaara acids are fermented. Substances such as lactate, 13 1955) opened the way for industrial applications. acetate, ethanol, acetoin, ammonia, etc., will be pro- 14 The fundamental role of microorganisms was then duced as end products, but they are not used any fur- 15 clearly shown. ther inside the cells and will be secreted to the 16 The general principles of the fermentation of dry outside (Tortora et al. 2001). 17 sausages have remained the same over the millennia, Biochemically, fermentation is defined as a 18 and the aspects of preparation been reviewed many reaction where ATP is produced in substrate level 19 times (e.g., recently by Toldrá [2004] and Demeyer phosphorylation, i.e., oxygen is not needed, and con- 20 and Toldrá [2004]). The purpose of this chapter is sequently no carbon is lost as CO . Microorganisms 21 only to present the basic principles of meat fermenta- 2 utilize ATP for maintaining cell functions and for 22 tion. Fermentation results in acid production influ- growth (Tortora et al. 2001). The main part of the use 23 encing, e.g., firmness, keepability/safety, color, is related to the pH gradient (proton motive force, 24 lipolysis, proteolysis, and formation of aroma and which is “a biological accumulator”) across the cell 25 taste compounds. These aspects will be reviewed membrane, i.e., the proton flow into the cell fuels 26 also in other chapters of the book. the ATP synthesis. Protons are also excreted from 27 the cells to establish the pH difference between the 28 FERMENTATION external and internal surface. The energy required to 29 maintain this increases, when pH is below 5.0, 30 SAUSAGE FERMENTATION because it becomes difficult to transport protons out 31 The fermentation step in the sausage production of the cell against a proton concentration gradient of 32 includes the period in the sausage process where pH higher than 100 (Konings 2002). In meat systems, it is 33 decreases from approximately 5.7 to its lowest value, especially difficult to obtain much lower pHs, because 34 which could vary from 5.5 (hard salamis) to 4.6 (or the buffering capacity of meat increases quickly at 35 even 4.2 in high-temperature fermented sausages) low pHs (buffering capacity is about 30 mmmol 36 depending on the sausage type. The fermentation Hϩ/(pH*kg) at pH 5.5 and about 70 mmmol 37 lasts from less than 12 hours to several days depend- Hϩ/(pH*kg) at pH 4.8 (Puolanne and Kivikari 2000). 38 ing on the sausage style. Sausages fermented at high The amount of ATP [expressed as moles ATP/(g*h)] 39 temperatures (37°C or higher) reach quickly the low- needed for growth is, however, much higher than the 40 est pH value (Varnam and Sutherland 1995), but consumption required for maintenance, the relation- 41 temperatures of around 24°C result in a pH of 4.6 to ship depending on growth rate. 42 5.0, and lower temperatures usually in a higher pH Each microbial strain has its own way of utilizing 43 at a slower speed. The amount of added sugar is the glucose and other substrates for energy production. 44 parameter of major influence on the lowest attain- The most frequent is glycolysis, i.e., the Embden- 45 able pH but not on the speed (Puolanne 1977). Meyerhof mechanism (see textbooks of bio- 46 Generally, pH increases during the subsequent matu- chemistry or microbiology for the detailed reaction 47 ration period, which lasts from a couple of weeks to cascades). Glucose is converted to pyruvate (2 CH 48 3 several months. Fermentation is achieved by natural COCOOϪ), which is in turn converted into 49 contaminating bacteria or by addition of lactic acid lactate (2 CH CHOHCOOϪ), and simultaneously 2 50 3 bacteria. In former times, backslopping was used to (NADHϩHϩ) are regenerated to 2 NADϩ. It should 51 start the fermentation, but now well-defined starter be mentioned here that pyruvate and lactate remain 52 cultures of lactic acid bacteria in combination with dissociated (not in the form of an undissociated 53S other microbial pure cultures are applied. acid) during these conversions and that the above 54N 40454 04 031-036 r2.qxd 7/20/07 12:03 PM Page 33

Principles of Meat Fermentation 33

reactions bind two protons. The protons that reduce tation is much more influential than simply acid 1 pH during fermentation are derived from the reac- production. 2 tion ATP ➝ ADP ϩ Pi ϩ Hϩ (Robergs et al. 2004). The LAB most commonly used as starter cultures 3 Other carbohydrates can also be utilized, but the rel- in sausage fermentation belong to the genera Lacto- 4 ative availability of carbohydrates other than added bacillus and Pediococcus (Demeyer and Toldrá 5 glucose is low. Sometimes other sugars, such as lac- 2004). The main product of glucose fermentation is 6 tose, are added. Meat itself contains glycogen and its lactate, and to a lesser extent acetic acid, propanoic 7 derivatives, mainly glucose phosphates. Glycogen is acid, or ethanol. The genera are facultative or 8 hardly decomposed by meat bacteria, and very little microaerophilic. Lactobacillus and Pediococcus are 9 is known about the degradation of glycogen by meat catalase-negative and do not reduce nitrate, and they 10 enzymes in salted meat. may thus cause discoloration during fermentation if 11 they produce hydrogen peroxide (Nurmi 1966), which 12 is not always the case. In recent years, there has been 13 MICROBIAL FERMENTATION interest also on the probiotic properties of LAB starter 14 cultures for meat products (Erkkilä and Petäjä 2000). 15 The starter cultures used in meat products are Staphylococci used as starter cultures are weak 16 homofermentative lactic acid bacteria (LAB), i.e., acidulants, produce catalase, and are able to reduce 17 the main end product of carbohydrate fermentation is nitrate, which makes them important for color for- 18 lactate. The amounts of other end products produced mation and gives them some degree of antioxidative 19 by LAB are usually very small, but on the other hand, capacity (see Chapter 20). In addition, Staphylococci 20 the flora may provide a wide diversity of substances, is deeply involved in the flavor-forming reactions of 21 thus influencing the flavor and aroma of the product fermented meat products (see Chapter 22). 22 (Tortora et al. 2001; Sunesen et al. 2001). Even 23 greater diversity will result from the fermentation of 24 other substrates (amino acid, fatty acid), as well as FACTORS INFLUENCING 25 the secondary reactions of these metabolic products FERMENTATION 26 in the food matrix outside the microbial cells. In 27 sausage, the fermentation usually stops after lactic The fermentation process is more complex than 28 acid fermentation, and subsequent fermentations most other processes in meat technology. The fer- 29 such as propanoic (propionic) acid production in mentation, the energy-producing metabolism in 30 cheese are not that relevant in sausages. In sausages microorganisms, although taking place inside the 31 prepared with molds or yeasts, however, these organ- microbial cell, is not an isolated procedure, but is 32 isms further metabolize lactate. One factor that stops closely related to intrinsic and extrinsic factors and 33 fermentation is low pH, but the decrease of a will their interactions in the ripening sausage. There is no w 34 also inhibit further fermentation. decisive factor ruling the fermentation/ripening 35 Microbial and meat enzymes also affect other (unlike in cooked sausage where cooking stabilizes 36 constituents of meat than carbohydrates. The meta- the gel and destroys most microorganisms in a very 37 bolic activity of microorganisms aims at fulfilling consistent way). The natural laws provide the limits 38 the energy and nutrient requirements of the microor- of the activities, and each individual aspect will fol- 39 ganisms, as well as creating a favorable environment low these. The complexity is, however, a result of so 40 for further growth, such as production of biogenic many independent and dependent factors function- 41 amines (may be health hazards) that increase pH ing at the same time. The main influencing factors 42 thus resulting in a more favorable pH, or the produc- are intrinsic, and only little can be done after the 43 tion of bacteriocins that inhibit growth of microor- preparation of the sausages, except extrinsically vary- 44 ganisms of similar nutritional requirements, or lactic ing the temperature-humidity-air velocity program- 45 acid that prevents the growth of acid-sensitive ming and smoking. Individual, and more or less 46 strains (see the section “Antagonistic Effects”). interrelated, factors such as meat enzymes, the 47 Microbial exo-enzymes, aimed at providing small dynamic microbial flora, salt, nitrate/nitrite, temper- 48 molecular weight substances to be absorbed by the ature, drying, carbohydrates, smoke, etc., have an 49 microorganisms, cause proteolytic and lipolytic influence on the fermentation/ripening. The process 50 changes during fermentation, and other enzymatic is characterized by lactate production resulting in a 51 and nonenzymatic reactions increase the complexity decrease in pH, an increase in salt content, 52 of the ripening process (see sections “Proteolysis” nitrate/nitrite reduction, and changes in color, firm- S53 and “Lipolysis”). As a consequence, the fermen- ness, taste, aroma, weight loss, etc. N54 40454 04 031-036 r2.qxd 7/20/07 12:03 PM Page 34

34 Meat Fermentation Worldwide: History and Principles

1 The process can be controlled by careful selection catepsin D) are the main agents of proteolysis early 2 of various ingredients, including meat raw materials, in fermentation. The pH 4.8–5.2 of fermented 3 with special attention paid to their natural microbial sausage and the ripening temperature of 15–20°C 4 flora, sugar, food additives, and spices. Reliable were optimal for the activity of such enzymes; this 5 control of the atmosphere and air circulation, as well fact confirms the role of tissue proteases in meat 6 as temperature in the ripening chamber, suitable fermentation. 7 casings, and smoking conditions are necessary for 8 successful and safe production. These aspects are LIPOLYSIS 9 exceptionally difficult to control theoretically, but 10 long experience makes possible the fine-tuning of Lipolysis is also taking place during fermentation 11 the process for good results. Finally, the utilization and ripening of dry sausages. Lipolysis means the 12 of bacterial pure cultures and surface yeasts hydrolyzation of fat, the hydrolysis products being 13 and molds provides the final technological security free fatty acids, glycerol, monoglycerides, and diclyc- 14 and also safety concerning health hazards. There will, erides. Release of long-chain fatty acids from neu- 15 always remain large uncontrolled areas. A large part tral lipids and phospholipids and from cholesterol 16 of the process remains more or less uncontrolled, but has long been a central theme in investigations on 17 the processors try to keep the uncontrolled part as chemical changes during sausage fermentation 18 small as possible. (Nurmi and Niinivaara 1964; Cantoni et al. 1966; 19 Starter cultures are not used only for safety rea- Demeyer et al. 1974; Roncales et al. 1989). Levels of 20 sons, but they also provide significantly added value. up to about 5% of total fatty acid content have typi- 21 The process is accelerated compared to the case cally been encountered in fermented meat products. 22 where only natural (accidental) flora is responsible 23 for fermentation. Starter cultures create faster fer- ANTAGONISTIC EFFECTS 24 mentation and nitrate/nitrite reduction, and usually 25 better aroma and color will be the result. The microorganisms influence the growth of other 26 For successful fermentation, suitable raw materi- species, and there is a hard competition in the micro- 27 als should be selected, especially microbial flora and bial ecological environment during fermentation. 28 pH (no meat with high pH). Sugar content (natural The antagonism is caused by the competition for 29 carbohydrates and added glucose with derivates) and nutrients as well as the effects of antimicrobial 30 other nutrients, like amino acids, fatty acids, miner- substances produced by the microorganisms. The 31 als, vitamins, etc., create the driving forces that starter cultures or natural dominating flora need 32 cause the fermentation. Starter cultures (and natural antagonism to maintain their dominance. 33 flora) and meat enzymes start the fermentation, the The microbiological flora of meat raw material 34 rate of which is controlled by external factors such primarily consists of gram-negative bacteria, most 35 as temperature and, to a lesser extent, by humidity, air being psychrotrophs. The predominating genera 36 circulation, and smoking. The above-mentioned are Pseudomonas, Acinetobacter, and Moraxella. 37 external factors and intrinsic factors, such as salt Brochothrix thermosphacta, enterobacteria, staphy- 38 content and nitrate/nitrite contents (Puolanne 1977), lococci, micrococci, spore-forming bacteria, lactic 39 can be used as regulators of the process. Temperature acid bacteria (LAB), and yeasts are also encoun- 40 is a decisive factor, because it has a strong and also tered, but the counts are several orders of magnitude 41 selective effect on the microorganisms present lower than the counts of the predominating genera 42 (Puolanne 1977). As a consequence, the relative (Lawrie 1998). Because the added salt contents may 43 numbers of different strains in the flora may vary, as vary from 2.2% to over 3% in fermented sausages 44 well as the end products of the meat and microbial (in fermented whole meat products, from 1.7% to 45 metabolism. very high concentrations), this is enough to suppress 46 growth of the most bacteria, especially gram- 47 PROTEOLYSIS negative ones (Petäjä 1977). On the other hand, 48 many LAB and staphylococci and micrococci are 49 During ripening of fermented sausage a small por- not sensitive to the salt concentrations prevailing 50 tion of the proteins is hydrolyzed (Niinivaara et al. during the fermentations in meat products. These 51 1964). According to Diedrick et al. (1974), nonpro- bacteria also can grow at fermenting temperatures of 52 tein nitrogen increases 20% in smoked sausages. The 20–25°C. The counts of these bacteria increase to 53S results of Verplaetse et al. (1989) and Demeyer the level of over 8 log CFU/g of LAB and to over 54N (1992) indicate that tissue proteases (especially 5 log CFU/g of staphylococci and micrococci. Often, 40454 04 031-036 r2.qxd 7/20/07 12:03 PM Page 35

Principles of Meat Fermentation 35

they hardly grow unless the production is very tradi- N Diedrick, P Vandekerchove, D Demeyer. 1974. 1 tional, but they are added in levels of 6–7 logs. The Changes of non-protein nitrogen-compounds during 2 pH decreases to 5.0 or below, which ﬁnally sup- dry sausage ripening. J Food Sci 39:301–304. 3 presses the growth of most other bacteria, but not S Erkkilä, E Petäjä. 2000. Screening of commercial 4 staphylococci and micrococci, meaning that bacte- starter cultures at low pH and in the presence of bile 5 rial ﬂora is formed of LAB and staphylococci and salts for potential probiotic use. Meat Sci 6 micrococci after the fermentation of about 1 week. 55:297–300. 7 Hydrogen peroxide production of LAB may also S Erkkilä, M Venäläinen, S Hielm, E Petäjä, E Puolanne, 8 suppress the growth of other bacteria (Lücke 1986). T Mattila-Sandholm. 2000. Survival of Escherichia 9 In conclusion, LAB and staphylococci/micrococci coli O157:H7 in dry sausage fermented by probiotic 10 compete well with and thus suppress the growth of lactic acid bacteria. J Sci Food Agric 80:2001–2004. 11 other bacteria, but also thrive better in the circum- L Jensen, L Paddock. 1940. US Patent 2,225,783. (Ref. 12 stances of meat fermentation than the other bacteria. Niinivaara 1955.) 13 The growth or survival of pathogens, such as N Konings. 2002. The cell membrane and struggle for 14 Salmonella, Yersinia enterocolitica, and Campy- life of lactic acid bacteria. Antonie van Leeuwenhoek 15 lobacter, are mainly suppressed in the fermented 82:3–27. 16 meat products because of low pH value in combina- RA Lawrie. 1998. Meat Science. Cambridge, UK: 17

tion with the produced lactate and the low aw value. Woodhead Publ. Ltd. Sixth ed., pp. 119–142. 18 Listeria survives better, but in most cases they also FC Lücke. 1986. Microbiological processes in the man- 19 disappear during drying. In a dry sausage, listeria ufacture of dry sausage and raw ham. Fleishwirtsch 20 disappeared at latest during the third week of ripen- 66:1505–1509. 21 ing (Työppönen et al. 2003). Neurotoxin production FP Niinivaara. 1955. Über den Einﬂuss von Bakterien- 22 and growth of Clostridium botulinum are controlled reinkulturen auf die Reifung and Umrötung der 23

by nitrite, low pH, and low aw value in these products Rohwurst. Acta Agralia Fennica 84:1–126. 24 (Peck and Stringer 2005). The only pathogens, FP Niinivaara, MS Pohja, SE Komulainen. 1964. Some 25 which could be harmful, are Staphylococcus aureus aspects about using bacterial pure cultures in the 26 and Escherichia coli, such as O157:H7. The entero- manufacture of fermented sausages. Food Technol 27 toxin production and growth of S. aureus are sup- 18:25–31. 28 pressed by using starter cultures (LAB) (Niskanen A Niskanen, E Nurmi. 1976. Effects of starter culture 29 and Nurmi 1976). If inoculated into dry sausage, the on staphylococcal enterotoxin and thermonuclease 30 counts of E. coli O157:H7 (EHEC) decrease 3 log production in dry sausage. Appl Environ Microbiol 31 CFU/g to the level of 2 log CFU/g, but because of 31:10–12. 32 low level of contamination, it can be regarded that C Niven, R Deibel, Wilson. 1955. The use of pure bac- 33 risk in fermented meat products is not particularly terial cultures in the manufacture of summer sausage. 34 large (Erkkilä et al. 2000). Annual Meeting of American Meat Institute. 5p. 35 (Ref. Nurmi 1966.) 36 E Nurmi. 1966. Effect of bacterial inoculation on charac- REFERENCES 37 teristics and microbial flora of dry sausage. Thesis, 38 C Cantoni, MR Molnar, P Renon, G Giolitti. 1966. University of Helsinki. Acta Agralia Fennica 108:1–77. 39 Investigations on the Lipids of Dry Sausages. Pro- E Nurmi, FP Niinivaara. 1964. Lipid changes of fats in 40 ceedings of the 12th European Meeting of Meat dry sausages. Proceedings of the 10th European 41 Research Workers, Sandefjord, E-4. Meeting Meat Research Workers, Roskilde, G-8. 42 DI Demeyer. 1992. Meat fermentation as an inte- MW Peck, SC Stringer. 2005. The safety of pasteurised 43 grated process. In: FJM Smulders, F Toldrá, J Flores, in-pack chilled meat products with respect to the 44 M Prieto, eds. New Technologies for Meat and Meat foodborne botulism hazard. Meat Sci 70: 461–475. 45 Products. Nijmegen, NL: Audet Tijdschriften, E Petäjä. 1977. The effect of some gram-negative bacte- 46 pp. 21–36. ria on the ripening and quality of dry sausage. J Sci 47 D Demeyer, J Hoozee, H Mesdom. 1974. Specificity of Agric Soc of Finland 49:107–166. 48 lipolysis during dry sausage ripening. J Food Sci E Puolanne. 1977. Der Einfluss von verringerten 49 39:293–296. Nitrit und Nitratzusätzen auf den Eigenschaften der 50 D Demeyer, F Toldrá. 2004. Fermentation. In: Rohwurst. J Sci Agric Soc of Finland 49:1–106. 51 WK Jensen, C Devine, M Dikeman, eds. Encyclope- E Puolanne, R Kivikari. 2000. Determination of the 52 dia of Meat Sciences. Amsterdam: Elsevier Academic buffering capacity of postrigor meat. Meat Sci S53 Press, pp. 467–474. 56:7–13. N54 40454 04 031-036 r2.qxd 7/20/07 12:03 PM Page 36

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1 RA Robergs, F Ghiasvand, D Parker. 2004. Biochem- G Tortora, R Funke, C Case. 2001. Microbiology. An 2 istry of exercise-induced metabolic acidosis. Am J of Introduction. Chapter 5. Microbial metabolism. 3 Physiology. Regulatory, Integrative and Comparative San Francisco: Addison Wesley Longman Inc., 4 Physiology 287:R502–516. pp. 113–150. 5 P Roncales, M Aguilera, JA Beltran, I Jaime, JM Piero. S Työppönen, A Markkula, E Petäjä, M-L Suihko, 6 1989. Effect of the use of natural or artiﬁcial casings on T Mattila-Sandholm. 2003. Survival of Listeria 7 the ripening and sensory quality of dry sausage. Pro- monocytogenes in North European type dry sausage 8 ceedings 35th International Congress Meat Science fermented by bioprotective meat starter cultures. 9 and Technology, Copenhagen, p. 825–832. Food Contr 14:181–185. 10 LO Sunesen, V Dorigoni, E Zanardi, L Stahnke. 2001. AH Varnam, JP Sutherland. 1995. Meat and Meat Prod- 11 Volatile compounds released during ripening in ucts: Technology, Chemistry and Microbiology. 12 Italian dried sausage. Meat Sci 58:93–97. London, UK: Chapman & Hall, pp. 314–354. 13 F Toldrá. 2004. Meat: Fermented meats. In: JS Smith, A Verplaetse, M De Bosschere, D Demeyer. 1989. Pro- 14 YH Hui, eds. Food Processing. Principles and teolysis during dry sausage ripening. Proceedings of 15 Applications. Ames, Iowa: Blackwell Publishing, the 35th International Congress of Meat Science and 16 pp. 399–415. Technology, Copenhagen, pp. 815–818. 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53S 54N 40454 05 037-048 r2.qxd 7/20/07 12:04 PM Page 37

1 2 3 5 4 5 6 Principles of Drying and Smoking 7 8 Ana Andrés, José M. Barat, Raúl Grau, and Pedro Fito 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION BASIC PRINCIPLES OF DRYING 22 AND SMOKING 23 Food drying is one of the oldest methods of preserv- 24 ing food for later use that is simple, safe and easy to Drying and/or dehydration is a method of food 25 learn. It removes enough moisture from the food so preservation wherein the moisture is evaporated by 26 bacteria, yeasts and molds can’t grow. Drying also exposure of the product to heat (solar, microwave, 27 slows down the action of enzymes because it etc.) and air. It is a process governed by the funda- 28 removes moisture, the food shrinks, and it becomes mental principles of physics and physical chemistry. 29 lighter in weight (Gould 1920). Drying is an energy-intense process using heat 30 Sun drying is the most ancient method for food applied externally or generated internally by micro- 31 preservation and even now is still used for fruit dry- wave or radio frequency energy to evaporate water 32 ing. In the 18th century, drying included the use of on the surface and drive trapped moisture to the 33 smoke to accelerate the drying processes, but this surface. Efficient drying is maintained only by heat 34 system implies some important flavor changes that penetrates to the center of the food, forcing 35 (Gould 1920). moisture to the surface for evaporation or the point 36 Drying is used in the food industry as a method to of lower vapor pressure area of the food particle 37 preserve a large amount of products. The great (Mujumdar 1995). 38 weight and volume reduction of the dried products There are different types of driers depending on 39 imply an important reduction of storage and ship- the way of heating (convection, radiation, dielectric, 40 ping costs. However, drying is a process that largely or conduction). The most common driers used in the 41 affects the sensorial properties of the food products food industry are convective or air driers, working 42 (Baker 1997; Heldman and Hartel 1997). continuously or by batches depending on the type of 43 Because the presence of water in foods directly product. Designing a drier requires understanding 44 affects its spoilage, removing water from a product the mechanism and the kinetics of the food product 45 will reduce the possibility of its biological alteration, to be dried—that is, it is necessary to establish the 46 as well as the kinetics of other spoilage mechanisms. quantitative relationships between the time of the 47 Removing water from a food implies two impor- process and the drying conditions (Bimbenet and 48 tant problems: it carries the risk of altering the nutri- Lebert 1990). The variables that determine the time 49 tional and sensorial quality, and it is sometimes a of drying of a material are variables related to air- 50 high energy-consuming process. The lack of selec- drying (temperature, velocity, humidity, flow charac- 51 tivity in water-removing processes can produce an teristics, etc.) and variables related to the product 52 important loss of aromas, even more when working (moisture, size, shape, structure, etc.). For this rea- 53 at vacuum pressures (Bimbenet and Lebert 1990). son, the experimental determination of the drying S54 N55 37 40454 05 037-048 r2.qxd 7/20/07 12:04 PM Page 38

38 Meat Fermentation Worldwide: History and Principles

1 curves (moisture versus time) for ﬁxed experimental remains wet and behaves like a liquid mass. As the 2 conditions is necessary. Those experimental condi- drying process proceeds, the product dries and the 3 tions must be as close as possible to the industrial water transport rate toward the surface decreases. 4 device in terms of heat transfer mechanism, airﬂow, This is the end of the constant rate period, and the 5 product density, etc. In other words, the industrial product moisture at this point is known as critical

6 process must be imitated to make the scaling-up eas- moisture content (Xc) and the time needed to reach 7 ier (Baker 1997). this point is called critical time (tc). The constant rate 8 Different types of experimental data are necessary period is the drying step where the most important 9 to design a drying process; some of them are related changes in terms of volume reduction take place. 10 to quality aspects (evolution of color, shape and size, Internal moisture is more difficult to remove because 11 texture, flavor, etc.) and others are related to the it is more tightly bound and is protected by the insu- 12 kinetics of the process (evolution of the mass and lating effect of the already dried material close to the 13 moisture throughout the drying process). The deter- surface. The water in the center is very difficult to 14 mination of the evolution of moisture in a food prod- remove and the removal of this water is done by dif- 15 uct under a drying process gives the well-known fusion. Thus, it is called the falling rate period or dif- 16 drying curve. Figure 5.1 shows a typical drying fusion period of drying—that is, this water must be 17 curve for wet products, where three zones can be dis- diffused from the center outward for evaporation to 18 tinguished corresponding to three typical steps accomplish a dried product. The drying rate in this 19 occurring in a drying process: induction period, con- period is very low as a typical diffusion process, and 20 stant rate period, and falling rate period of drying. product moisture decreases to reach equilibrium

21 The induction period corresponds to the begin- moisture (Xe) or in other words, the equilibrium of 22 ning of the drying process during which a heat trans- water activity with the surrounding airﬂow. 23 fer occurs from the air to the product, increasing the These typical drying periods can also be 24 surface temperature until the wet-bulb temperature. explained taking into account the shape of the sorp- 25 The duration of this period depends on many factors, tion isotherm of a high-moisture food product 26 but it is negligible as compared to the following (Figure 5.2). It is well known that the drying rate 27 periods, and for this reason it is not taken into depends on the driving force acting during the 28 account for design purposes. process, that is, the difference between the water

29 When the surface reaches the wet-bulb tempera- activity (aw) of the product and the water activity of 30 ture the total amount of heat from the air is used to the drying air. Another consideration to take into 31 evaporate water, and because the rate of evaporation account is that the equilibrium condition is accom- 32 is lower than the rate of water transport to the sur- plished at the interface; the water activity at the 33 face, the drying rate is constant (constant rate product surface equals the water activity of the dry- 34 period). In this period, it is assumed that the surface ing air. During the constant rate period, the water 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53S 54N Figure 5.1. Drying periods in a typical drying curve. 40454 05 037-048 r2.qxd 7/20/07 12:04 PM Page 39

Principles of Drying and Smoking 39

activity of the product is near 1, and as the product center outward is hindered. Some of the food compo- 1 dries, big changes on the moisture product lead to nents like pigments, vitamins, etc., are temperature- 2 small changes on the water activity, as can be appre- sensitive and these quality aspects will limit the 3

ciated in the isotherm (Figure 5.2). Because aw maximum air temperature usable in the drying 4 remains nearly constant, the driving force can be process. In some cases, the drying process has other 5 considered constant as well as the drying rate. purposes than removing water, allowing some bio- 6 However, during the falling rate period, small chemical reactions to develop taste and ﬂavor, and it 7 changes in moisture content lead to big changes in is known as a curing process. Because these reac- 8 water activity. tions are time-dependent, low air temperatures are 9 Food properties (composition, structure, etc.) used. This is the case of ﬁsh and meat products, and 10

influence the aw parameter, which is much related to sometimes a mixture of air and smoke is used to get 11 water distribution in the different food phases, and a characteristic smoked flavor. 12 will influence on the drying kinetics. For this reason, Smoke curing is a typical combined treatment, 13 it is interesting to know the relationship between based on the concerted action of enzymes and heat, 14

aw and equilibrium moisture content (sorption which promote protein and lipid changes in the 15 isotherm). previously salted raw material. The treatment has 16 The driving force for water evaporation is the nutritional implications and affects also the sensory 17 water activity gradient between the food and the quality, safety, and shelf life of the product, but the 18 forced hot air, but other properties of drying air will extension of these changes will depend on many 19 also affect the drying kinetics, as was mentioned factors, such as the type of smoking; the relative 20 before. One of the variables affecting the drying rate humidity, velocity, temperature, density, and compo- 21 is the air velocity; the higher the air velocity, the sition of the smoke; and the time of smoking (Yean 22 higher the drying rate. However, above a certain et al. 1998). 23 value of air velocity, the drying rate will not depend The smoking technology has experienced an 24 on the air velocity because the factor controlling the important evolution in the last years. In the past, 25 drying kinetic is the water diffusion rate from the products to be smoked were directly exposed to the 26 center to the surface. smoke in the same chamber where it was produced. 27 On the other hand, it can be said that the higher the In the modern systems, the chamber for smoke pro- 28 air temperature, the higher the drying rate, although duction and the chamber for food smoking are dif- 29 sometimes, if water is removed too fast, the food ferent devices. The main advantage of separating 30 material may case harden, that is, seal up the outer these two steps is based on the smoke cooling during 31 surface area so that the water diffusion from the the pathway from the smoke production chamber to 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 S53 Figure 5.2. Relationship between the drying periods and the sorption isotherm. N54 40454 05 037-048 r2.qxd 7/20/07 12:04 PM Page 40

40 Meat Fermentation Worldwide: History and Principles

1 the smoking chamber; thus, the aromatic polycyclic product, reducing the smoking time, although the 2 hydrocarbons (nondesirable substances from the quality of the products obtained with this method 3 point of view of consumers’ health, with a demon- differs from the traditional products. 4 strated carcinogen action), together with the tars, 5 settle in the conduction walls, thereby avoiding in TREATMENT WITH SMOKE AROMAS OR 6 some extent the meat products. CONDENSATES 7 Today, the smoking chambers are air-conditioned 8 rooms useful for meat processes other than smoking, This process is considered a revolutionary smoking 9 like curing, drying, cooking, etc. There are various procedure because it allows giving the typical prop- 10 smoking procedures that are classified in the follow- erties of a smoked product to new meat products or 11 ing sections (Cava-López and Andrés-Nieto 2001). some meat products to which the traditional method 12 is difficult to apply, without existing contact between 13 the smoke and the meat product. TRADITIONAL SMOKING 14 15 Traditional smoking is divided into two categories: 16 HURDLE TECHNOLOGY APPLIED • Cold smoking, with smoke temperatures between 17 TO DRIED AND SMOKED MEAT 20–25°C and relative humidity of 70–80%. The 18 AND POULTRY PRODUCTS duration of this process can be days or weeks 19 because the smoke used is poor in aromatic and As stated in the previous section, meat drying and 20 preserving components. smoking are techniques used originally for meat 21 • Hot smoking, usually applied to meat products preservation due to the water activity reduction 22 previously cooked or blanched. Temperatures can and additionally the bacteriostatic effect of sub- 23 be about 75–80°C, and the relative humidity must stances present in smoke, in the case of smoking 24 be high to avoid dehydration. With this procedure, (Doe et al. 1998). 25 the smoking time is reduced to hours. If drying and smoking should have to be used as 26 unique meat preservation techniques, the final mois- 27 ture of the dried products should have to be very low. 28 ELECTROSTATIC SMOKING That moisture would be below a “comfortable” value 29 from a sensory point of view. Figure 5.3 shows the 30 In this procedure, an electric field is applied between water activity value limiting the microbial growth 31 the smoke particles and the meat product. Electri- and the typical water activity of the most common 32 cally charged particles quickly precipitate over the 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53S Figure 5.3. Water activity values limiting the growth of the most common spoilage microorganisms and of 54N some meat products. 40454 05 037-048 r2.qxd 7/20/07 12:04 PM Page 41

Principles of Drying and Smoking 41

meat products (Smith et al. 1989; IFT/FDA 2003; is within a narrow range from 3–20°C. When using 1 Hoz et al. 2004; Garriga et al. 2004; Serra et al. cold, smoking the maximum temperature increases 2 2005; Soriano et al. 2006; Muthukumarasamy and to 30°C. 3 Holley 2006; Rubio et al. 2006a). Figure 5.3 shows In the case of Spanish cured ham, the piece begins 4

that a water activity (aw) value of 0.8 seems to be a to dry during the postsalting stage and the process 5 reasonable value to get a shelf-stable product. temperature is kept close to 3°C at the beginning of 6 For instance, to get that value at room temper- the postsalting. Further increases of temperature 7 ature (25°C) (Figure 5.4) for smoked chicken throughout the processing occur associated with the 8 sausages, the moisture value should have to be water activity decrease, until a shelf-stable product 9 around 24%, which is obviously too low for direct at room temperatures is achieved at the end of the 10 consumption. process (Barat et al. 2005; Toldrá 2002). 11 That’s the reason why drying and smoking are 12 usually employed in combination with other pre- 13 CURING serving techniques, and thus the hurdle technology 14 principles (Leistner 1995) are present in most of the Curing is basic in order to obtain an adequate shelf 15 meat products developed by those techniques. life. The curing process is well known and explained 16 The main preserving techniques employed in in Chapter 3. The most active components of the 17 combination with drying and/or smoking are curing salts are the nitrites, which prevent the micro- 18 described in the following sections. bial growth, mainly the Chlostridium botulinum and 19 Listeria monocytogenes. 20 21 REFRIGERATION 22 SALTING All the raw materials are perishable at room temper- 23 atures in their original form, because of the use of Salting is always present in case of meat products. 24 low temperatures during storage, transportation, and NaCl plays many technologic roles: salty taste and 25 processing. In some cases, the use of low tempera- ﬂavor perception, control of microbial growth and 26 tures at the beginning of the drying process is neces- enzymatic activity, and modiﬁcation of the adher- 27 sary, and is more important the higher the meat ence and the water-holding capacity of the protein 28 sample size. The temperature range used for meat matrix. In the case of sausages, NaCl is added during 29 products drying varies depending on the product, but the mixing phase; in the case of whole meat pieces 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 S53 Figure 5.4. Moisture desorption isotherms of smoked chicken sausages (adapted from Singh et al. 2001). N54 40454 05 037-048 r2.qxd 7/20/07 12:04 PM Page 42

42 Meat Fermentation Worldwide: History and Principles

1 processing, salt can be added by means of dry or wet FUNDAMENTALS OF DRYING AND 2 salting. The use of dry salting without drainage with SMOKING MEAT AND POULTRY 3 a small food/salt ratio could imply that at the end of PRODUCTS 4 the salting period the product could be surrounded 5 by a brine. In this former case it would be considered The general principles related with food drying and 6 as pickling (Barat et al. 2006). smoking were introduced in the Introduction of the 7 chapter. Nevertheless, some particular aspects must 8 be taken into account when working with meat and SPICING 9 poultry products. 10 The use of spices is very frequent in the case of The protein matrix suffers dramatic changes at a 11 sausages. The addition of spices not only contribute certain temperature, which implies the protein 12 to color and flavor development, but also become a denaturation. These changes are usually undesirable 13 source of external contamination and antioxidant except in certain smoked products where proteins 14 effect, and in some cases they have an inhibitory coagulate as a consequence of the high tempera- 15 activity on microbial growth (Shelef 1983; Zaika tures. The before mentioned temperatures are in the 16 1988; Martínez et al. 2006; Careaga et al. 2003; range of 40–80°C (Bertram et al. 2006; Stabursvik 17 García et al. 2001). and Martens 1980). 18 Drying and/or smoking not only contributes to 19 decrease the a of the product but also affects the FERMENTATION AND BIOPRESERVATION w 20 hardness and stability of the protein matrix as a con- 21 Fermentation is one of the operations involved in sequence of the moisture reduction. 22 many manufactured food products (Adams and Another of the phenomenon associated to the 23 Robert-Nout 2001; Lücke 1999). The key point of drying process is the case hardening (Gou et al. 24 fermentation consists in the decrease of the pH of the 2005), which not only implies an increase in the 25 sausage as a consequence of the conversion of sugar hardness of the surface, but also a dramatic decrease 26 into lactic acid due to the action of bacteria. The in the drying rate and thus leads to a heterogeneous 27 decrease of pH in fermented meat products not only dried product with a highly dried surface and a 28 improves its preservation but also the drying process poorly dried core. Recent studies have been done to 29 due to the decrease in the water-holding capacity of correlate the moisture content and texture to charac- 30 the meat proteins when pH approaches the isoelec- terize the crust formation and prevent case harden- 31 tric value. ing by controlling the drying conditions (Beserra 32 Specific bacteriostatic activity of some of the et al. 1998b; Gou et al. 2005; Ruiz-Ramírez et al. 33 microorganisms added through the starter culture 2005; Serra et al. 2005). 34 has been described (e.g., lactic acid bacteria [LAB]), The presence of fat in meat products contributes to 35 contributing to the microbial stabilization of the a decrease in the drying rate because of its barrier 36 final product (Hammes and Hertel 1998; Aymerich effect, but on the other hand prevents the food surface 37 et al. 2000; Coffey et al. 1998). from an excessive drying. In fact, in the case of ham 38 drying, a layer of fat is usually applied on its surface 39 in the final processing period—among other reasons, PACKAGING AND STORAGE 40 for preventing drying. The fat content of the sausage 41 Vacuum and modified atmosphere packaging (MAP) products range is very wide, ranging from 5–70%. 42 are the most frequent ways for packaging. These The higher the fat content, the lower the drying 43 techniques employ gas mixtures and packaging rate. Another thing that must be taken into account is 44 materials technology to extend the shelf life of food. that the excessive mincing of fat and lean gives place 45 For meat products, the atmosphere in the package is to a phenomenon called smearing, which leads to 46 modified by pulling a vacuum and then replacing the case hardening—a product with a highly moist core 47 package atmosphere with a gas mixture of oxygen region.

48 (O2) and carbon dioxide (CO2) or nitrogen (N2) and One of the strategies followed by the industry to 49 carbon dioxide mixtures. In dry-cured meat prod- prevent case hardening is to ﬂuctuate from lower to 50 ucts, the gas mixture more utilized is formed by 0% higher water activity of the air and vice versa. When

51 O2, 20–35% CO2, and 65–80% N2 (García-Esteban the water activity of the surrounding air decreases 52 et al. 2004; Rubio et al. 2006b; Valencia et al. 2006). (aw = RH/100) the drying process accelerates, while 53S In these conditions, the time of storage at 10–15°C is the subsequent aw increase enables the water to mig- 54N 3 months. rate from the interior to the surface fast enough to 40454 05 037-048 r2.qxd 7/20/07 12:04 PM Page 43

Principles of Drying and Smoking 43

1 2 3 4 5 6 7 8 9 10 11 12 13 Figure 5.5. Relative humidity changes throughout a typical sausage drying process (adapted from 14 Comaposada et al. 2004). 15 16 17 increase again the moisture of the surface. Figure 5.5 DRYING KINETICS MODELING 18 shows the relative humidity changes throughout a 19 typical sausage-drying process. Modeling drying behavior of meat and poultry prod- 20 In general, the risk of case hardening increases ucts is a very complex task (Kottke et al. 1996). 21 when the demand of water increases from the prod- First, the phase controlling the drying process has 22 uct by air. This demand mainly depends on the air to be defined (surrounding air, meat product, or both 23 relative humidity and the air speed inside the cabi- of them). Under real processing conditions, both 24 net. The lower the relative humidity of the air, the the internal and external phase control the drying 25 higher the water activity gradient between the meat process (Simal et al. 2003). The Biot number for 26 product surface and the bulk air, which implies an mass transfer should have to be used to define under 27 increase in the drying rate. The other parameter which conditions the process is accomplished. Nev- 28 affecting the drying rate is the air velocity; when the ertheless, it is usually assumed that the internal 29 air rate increases, a higher turbulent flow is created phase is controlling the drying process, and the dif- 30 in the neighborhood of the product, which implies a fusion equations obtained by integrating the Fick’s 31 decrease of the boundary layer thickness, which also law for Diffusion (Crank 1975) are used to model the 32 experimental results. implies an increase in the effective aw gradient in the 33 surroundings of the product. Water diffusion inside the food depends on many 34 On the other hand, the temperature increase can factors: temperature (Gou et al. 2004), structure 35 contribute to a decrease in the tendency for case (Gou et al. 2002), fat and moisture content (Kottke 36 hardening. The effective diffusion of water inside the et al. 1996), pH (Gou et al. 2002), and shrinkage 37 meat product usually increases exponentially with (Trujillo et al. 2007). It even depends on the equa- 38 temperature, as predicted by Arrhenius law (Palmia tion used to obtain such value (Merts et al. 1998; 39 et al. 1993; Mittal 1999). The higher the transport Trujillo et al. 2007) and the size and shape of the 40 rate of water inside the product, the later the case sample. That implies that a wide range of effective 41 diffusion (D ) values can be found in literature, rang- hardening will occur or the less intensity it will have, e 42 ϫ −11 2 ϫ −10 2 because surface water is constantly replaced by ing from 0.9 10 m /s to 5.3 10 m /s. (Motar- 43 water migration from inside the product, keeping the jemi 1988; Mittal 1999), even for the same product 44 product surface moist enough to avoid that problem working under the same drying conditions (Trujillo 45 (Beserra et al. 1998a). Nevertheless, the higher the et al. 2007). 46 temperature, the higher the microbial risks and the 47 possibility of protein denaturing due to temperature. AIR-CONDITIONING AND 48 Another possible effect of the increase in tempera- CIRCULATING IN MEAT DRYING 49 tures is the fat melting, which can contribute to AND SMOKING 50 creating a hydrophobic barrier in the product, favor- 51 ing the smearing effect. This is the reason why low As stated in the previous sections, in addition to the 52 melting point temperature fats are avoided in the temperature and the speed of the air, the air relative S53 manufacturing of this kind of product. humidity control is fundamental to accomplish an N54 40454 05 037-048 r2.qxd 7/21/07 8:13 AM Page 44

44 Meat Fermentation Worldwide: History and Principles

Figure 5.6. Scheme of the Mollier diagram.

Heating (HE1) Humidifying (HU) Cooling (CO) Heating (HE2)

Figure 5.7. Basic scheme for air-conditioning.

adequate drying process—one that is fast, safe, and heating. By means of the combination of all or any minimizes the case hardening. of them, any combination of relative humidity and The aim of this section is to give the fundamentals temperature in the regular range of work can be in air-conditioning for a better understanding of the achieved. Figure 5.7 shows a basic scheme of the drying chambers used in the processes. combination of those operations that enables the All the given explanations are supported on a desired T and RH combination inside the drying psychometric chart, in this case the Mollier diagram chamber. shown schematically in Figure 5.6. The x axis indi- When working the ﬁrst heater (HE1), temperature cates the absolute humidity of the air (kg water/kg dry of the air increases, through a vertical line in the

air), the curves in the diagram indicate the relative Mollier diagram (X constant, iG increasing, and RH humidity of the air (RH), being the highest possible decreasing) (Figure 5.8). This operation is usually value corresponding to saturation (100%). The lines accomplished to increase the water retaining capac- crossing the diagram diagonally from above the left to ity of air. below the right correspond to isoenthalpic values The following step consists of increasing air mois- (kcal/kg dry air), and the lines close to the horizontal ture (HU). The usual way of introducing water vapor ones correspond to isotherms (°C or °K). in the air consists of spraying water (mechanically or In air-conditioning, there are four main opera- using ultrasound humidiﬁers). This way of humidiﬁ- N tions: humidifying, dehumidifying, cooling, and cation is isoenthalpic, which means that no net energy 40454 05 037-048 r2.qxd 7/20/07 12:04 PM Page 45

Principles of Drying and Smoking 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Figure 5.8. Scheme of the changes in the air conditions throughout a conditioning process. 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 Figure 5.9. Normal way of air circulation inside a drying chamber. 38 39 40 changes in the air exist, and thus the changes in the air after the humidifying is not possible; the tempera- 41 properties occur following an isoenthalpic line. ture control of the air doesn’t imply any problem at 42 The following operation is air cooling (CO). This industry. 43 is the way to reach a relative humidity equal to It must be kept in mind that these are the basic 44 100%, reaching the X value of the final air condi- principles of air-conditioning, and at an industrial 45 tions by controlling the final air temperature (dehu- level more complex situations exist because the air 46 midifying process). recycling and the air-conditioning devices vary 47 Finally, the air is heated (HE2) to reach the desired depending on the equipment supplier. 48 relative humidity. The homogeneity of air conditions inside the 49 The combination of these operations (HE1+HU+ drying chambers is very important. The regular way 50 CO++HE2) is needed when the initial air conditions of circulation is to introduce the air downstream 51 are placed on the left of the final air conditions in the through orifices joined to the walls and to suction the 52 Mollier diagram (Figure 5.8). The overhumidifying air through holes placed at the center of the ceiling S53 is needed because the accurate control of the X value (Figure 5.9). N54 40454 05 037-048 r2.qxd 7/20/07 12:04 PM Page 46

46 Meat Fermentation Worldwide: History and Principles

1 qualities. A combined FT-IR spectroscopy and low- 2 field NMR relaxometry study. J Agric Food Chem 3 54(5):1740–1746, 4 FJ Beserra, P Fito, JM Barat, A Chiralt, J Martínez- 5 Monzó. 1998a. Drying kinetics of Spanish 6 Salchichón. In: Proceedings of the 44th International 7 Congress of Meat Science and Technology, Vol. II, 8 872–873, Barcelona, Spain. 9 FJ Beserra, P Fito, A Chiralt, JM Barat, J Martínez- 10 Monzó. 1998b. Influence of ripening conditions on 11 the pH, colour and texture development of Spanish 12 Salchichón. In: Proceedings of the 44th International 13 Congress of Meat Science and Technology, Vol. II, 14 870–871, Barcelona, Spain. 15 JJ Bimbenet, A Lebert. 1990. Some practical questions 16 and remarks about drying. In: Engineering and Food 17 Preservation Processes and Related Techniques. 18 Figure 5.10. Air circulation arrangement in very Vol.2. WEL Spiess, H Schubert, eds. London: Else- 19 high drying chambers. vier Applied Science. 20 MO Careaga, E Fernández, L Dorantes, L Mota, ME 21 Jaramillo, H Hernandez-Sanchez. 2003. Antibacter- 22 ial activity of Capsicum extract against Salmonella 23 Higher homogeneous drying conditions can be typhimurium and Pseudomonas aeruginosa inocu- 24 achieved by changing the air direction in the cham- lated in raw beef meat. Intl J Food Microbiol 83(3): 25 ber, by introducing the air in the chamber at two 331–335. 26 different points (in case of very high chambers) R Cava-López, A Andrés-Nieto. 2001. EL ahumado de 27 (Figure 5.10), or by alternating the holes used to suc- los productos cárnicos. In: Enciclopedia de la carne y 28 tion the air through the ceiling. de los productos cárnicos, Vol. II. M Bejarano, Plasen- 29 Finally, most of the air in the chamber is reused, cia, eds. Cáceres: Martín & Macías, pp. 983–993. 30 and some is permanently renewed. A Coffey, M Ryan, RP Ross, C Hill, E Arendt, 31 G Schwarz. 1998. Use of a broad-host-range 32 REFERENCES bacteriocin-producing Lactococcus lactis transconju- 33 gant as an alternative starter for salami manufacture. 34 MR Adams, MJ Robert-Nout. 2001. Fermentation and Intl J Food Microbiol 43(3):231–235. 35 food safety. Dordretch: Kluwer Academic Publish- J Comaposada, P Gou, I Muñoz, J Arnau. 2004. Carac- 36 ers, pp. 300. terización y análisis de distribución de temperaturas, 37 MT Aymerich, M Garriga, JM Monfort, I Nes, M humedades relativas y velocidades de aire en un 38 Hugas. 2000. Bacteriocin-producing lactobacilli in secadero industrial de embutidos. Eurocarne 1–13. 39 Spanish-style fermented sausages: Characterization J Crank. 1975. The Mathematics of Diffusion. London: 40 of bacteriocins. Food Microbiol 17(1):33–45. Oxford University Press, Ely House. 41 CGJ Baker. 1997. Industrial drying of foods. London: P Doe, Z Sikorski, N Haard, J Olley, BS Pan. 1998. 42 Blackie Academic & Professional. Basic principles. In: Fish Drying and Smoking. Pro- 43 JM Barat, R Grau, P Fito, A Chiralt. 2006. Vacuum salt- duction and Quality. PE Doe, ed. Lancaster, Pennsyl- 44 ing treatment for accelerating processing of dry- vania: TECHNOMIC, Pub. Co., Inc., pp. 13–45. 45 cured ham. In: Advanced Technologies for Meat SJ Eilert. 2005. New packaging technologies for the 46 Processing. L Nollet, F Toldrá, eds. Marcel Dekker, 21st century. Meat Sci 71:122–127. 47 Inc., pp. 353–369. S García, F Iracheta, F Galván, N Heredia. 2001. 48 JM Barat, R Grau, JB Ibáñez, P Fito. 2005. Post-salting Microbiological survey of retail herbs and spices 49 studies in Spanish cured ham manufacturing. Time from Mexican markets. J Food Protect 64(1):99–103. 50 reduction by using brine thawing-salting. Meat Sci M García-Esteban, D Ansorena, I Astiasarán. 2004. 51 69:201–208. Comparison of modified atmosphere packaging and 52 HC Bertram, A Kohler, U Böcker, R Ofstad, HJ vacuum packaging for long period storage of dry- 53S Andersen. 2006. Heat-induced changes in myo- cured ham: effects on colour, texture and microbio- 54N fibrillar protein structures and myowater of two pork logical quality. Meat Sci 67:57–63. 40454 05 037-048 r2.qxd 7/20/07 12:04 PM Page 47

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M Garriga, N Grèbol, MT Aymerich, JM Monfort, M binding and water diffusivity in minced meat prod- 1 Hugas. 2004. Microbial inactivation after high- ucts. PhD Thesis. Lund University, Lund, Sweden. 2 pressure processing at 600 MPa in commercial meat AS Mujumdar. 1995. Handbook of industrial Drying. 3 products over its shelf life. Innov Food Sci Emerg Vol.1. New York: Marcel Dekker Inc. 4 Technol 5:451–457. P Muthukumarasamy, RA Holley. 2006. Microbiologi- 5 PJ Gou, J Comaposada, J Arnau. 2002. Meat pH and cal and sensory quality of dry fermented sausages 6 meat ﬁbre direction effects on moisture diffusivity in containing alginate-microencapsulated Lactobacillus 7 salted ham muscles dried at 5°C. Meat Sci reuterim. Intl J Food Microbiol 111:164–169 8 61(1):25–31. F Palmia, M Pecoraro, S Ferri. 1993. Essiccazione di 9 ———. 2004. Moisture diffusivity in the lean tissue of prodotti carnei: calcolo del coefﬁciente di diffusione 10

dry-cured ham at different process times. Meat Sci effettivo (De) dell’acqua in fette di lombo suino. 11 67(2):203–209. Industria Conserve 68:238–242. 12 P Gou, J Comaposada, J Arnau, Z Pakowski. 2005. B Rubio, B Martínez, C González-Fernández, MD 13 On-line determination of water activity at the lean García-Cachán, J Rovira, I Jaime. 2006a. Influence 14 surface of meat products during drying and its rela- of storage period and packaging method on sliced 15 tionship with the crusting development. Drying dry-cured beef “Cecina de Leon”: Effects on micro- 16 Technol 23(8):1641–1652. biological, physicochemical and sensory quality. 17 WA Gould. 1920. Fundamentals of Food Processing Meat Sci 74:710–717 18 and Technology. Maryland: CTI Publications, Inc. ———. 2006b. Development of an n-3 fatty acid and 19 WP Hammes, C Hertel. 1998. New developments in ␣-tocopherol enriched dry fermented sausage. Meat 20 meat starter cultures. Meat Sci 49(SUPPL. 1): Sci 67:485–495. 21 S125–S138. J Ruiz-Ramírez, X Serra, J Arnau, P Gou. 2005. Pro- 22 DR Heldman, RW Hartel. 1997. Principles of Food Pro- files of water content, water activity and texture in 23 cessing. New York: Chapman and Hall. crusted dry-cured loin and in non-crusted dry-cured 24 L Hoz, MD Arrigo, I Cambero, JA Ordóñez. 2004. loin. Meat Sci 69(3):519–525. 25 Development of an n-3 fatty acid and ␣-tocopherol X Serra, J Ruiz-Ramírez, J Arnau, P Gou. 2005. Texture 26 enriched dry fermented sausage. Meat Sci parameters of dry-cured ham m. biceps femoris 27 67:485–495. samples dried at different levels as a function of 28 IFT/FDA. 2003. Factors that Influence Microbial water activity and water content. Meat Sci 69: 29 Growth. Comp Rev Food Sci Food Saf (2):21–32. 249–254. 30 V Kottke, H Damm, A Fischer, U Leutz. 1996. Engi- LA Shelef. 1983. Antimicrobial effects of spices. 31 neering aspects in fermentation of meat products. J Food Saf 6:29–44. 32 Meat Sci 43(1):243–255. S Simal, A Femenia, P Garcia-Pascual, C Rosselló. 33 L Leistner. 1995. Principles and Applications of Hurdle 2003. Simulation of the drying curves of a meat- 34 Technology. In: GW Gould , ed. New methods of based product: Effect of the external resistance to 35 food preservation. London: Blackie Academic & mass transfer. J Food Eng 58(2):193–199. 36 Professional, pp. 1–21. RRB Singh, KH Rao, AR Anjaneyulu, GR Patil. 2001. 37 FK Lücke. 1999. Utilization of microbes to process Moisture sorption properties of smoked chicken 38 and to preserve meat. Proceedings 45th ICoMST, sausages from spent hen meat. Food Res Inter 39 pp. 538–547. 34:143–148. 40 L Martinez, I Cilla, JA Beltrán, P Roncalés. 2006. HJ Smith, S Messier, F Tittiger. 1989. Destruction of 41 Effect of Capsicum annuum (red sweet and cayenne) Trichinella spiralis spiralis during the preparation of 42 and piper nigrum (black and white) pepper powders the “dry cured” pork products proscuitto, proscuittini 43 on the shelf life of fresh pork sausages packaged in and Genoa salami. Can J Vet Res 53:80–83. 44 modified atmosphere. J Food Sci 71(1):S48–S53. A Soriano, B Cruz, L Gómez, C Mariscal, A García- 45 GS Mittal. 1999. Mass diffusivity of meat food prod- Ruiz. 2006. Proteolysis, physicochemical character- 46 ucts. Food Rev Intl 15(1):19–66. istics and free fatty acid composition of dry sausages 47 I Merts, SJ Lovatt, CR Lawson. 1998. Diffusivity of made with deer (Cervus elaphus) or wild boar (Sus 48 moisture in whole muscle meat measured by a drying scrofa) meat: A preliminary study. Food Chem 49 curve method. In IIR Proceedings Series, Refrigera- 96:173–184. 50 tion Science and Technology, Sofia, Bulgaria, E Stabursvik, H Martens. 1980. Thermal denaturation 51 pp. 473–479. of proteins in post rigor muscle tissue as studied by 52 Y Motarjemi. 1988. A study of some physical proper- differential scanning calorimetry. J Sci Food Agric S53 ties of water in foodstuffs. Water activity, water 31:1034–1042. N54 40454 05 037-048 r2.qxd 7/20/07 12:04 PM Page 48

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1 F Toldrá. 2002. Dry-Cured Meat Products. Trumbull, atmosphere packaging with carbon monoxide on the 2 Connecticut: Food & Nutrition Press, pp. 27–62. storage quality of master-packaged fresh pork. Meat 3 FJ Trujillo, C Wiangkaew, QT Pham. 2007. Drying Sci 73:605–610. 4 modeling and water diffusivity in beef meat. J Food YS Yean. R Pruthiarenun, P Doe, T Motohiro, 5 Eng 78(1):74–85. K Gopakumar. 1998. Dried and smoked ﬁsh prod- 6 I Valencia, D Ansorena, I Astiasarán. 2006. Stability of ucts. In: Fish Drying and Smoking. Production and 7 linseed oil and antioxidants containing dry fermented Quality. PE Doe, et al., eds. Lancaster, Pennsylvania: 8 sausages: A study of the lipid fraction during differ- TECHNOMIC, Pub. Co., Inc., pp. 47–87. 9 ent storage conditions. Meat Sci 73:269–277. LL Zaika. 1988. Spices and herbs: Their antimicro- 10 BHP Wilkinson, JAM Janz, PCH Morel, RW bial activity and its determination. J Food Saf 9: 11 Purchas, WH Hendriks. 2006. The effect of modiﬁed 97–118. 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53S 54N 40454 06 049-058 r2.qxd 7/20/07 12:07 PM Page 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 Part II 15 16 17 18 19 Raw Materials 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 S54 N55 40454 06 049-058 r2.qxd 7/20/07 12:07 PM Page 51

1 2 3 6 4 5 6 Biochemistry of Meat and Fat 7 8 Fidel Toldrá 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION: MUSCLE MEAT COMPOSITION 22 STRUCTURE 23 Meat is basically composed of water, protein, lipid, 24 Skeletal muscle constitutes the most important part minerals, and trace amounts of carbohydrate. An 25 of meat. This is a voluntary and striated muscle with example of typical composition of pork muscle is 26 several nuclei located peripherally in the cell. The given in Table 6.1. Water is the major constituent, 27 muscle is divided into sections by thin connective followed by proteins, but proportions can vary 28 tissue layers, named perimysium, and each section is depending on the amount of fattening. So, the con- 29 divided into fibers by thin collagen fibers, named tent of protein and water decrease when the amount 30 endomysium. Each fiber contains about 1,000 of fat increases. Proteins, lipids, and enzymes as 31 myofibrils, arranged in a parallel way, which are major components of meat and fat are briefly 32 responsible for the contraction of the muscle. The described in the next sections. 33 length of a muscle fiber may reach several centime- 34 μ ters and its diameter is about 50 m. Myofibrils are MUSCLE PROTEINS 35 composed of thick and thin filaments, which overlap 36 in different alternative regions known as dark (A) Proteins constitute the major fraction of meat, 37 and light (I) bands. These filaments are composed of approximately from 15–22%. There are three main 38 proteins that are responsible for muscle contraction groups of proteins in the muscle: myofibrillar, sar- 39 and relaxation. The Z and M lines bisect each I and coplasmic, and stromal, which are briefly described 40 A band, respectively. The sarcomere is the distance in the next sections. Main proteins for each group 41 between two consecutive Z lines; its length is usually and its localization are summarized in Table 6.2. 42 around 2–3 μm. Muscle fibers can be red and white, Myofibrillar Proteins 43 and each type of fiber has different biochemical 44 properties. Depending on the proportion of both These proteins are the main constituents of the struc- 45 types of fibers, the appearance and properties of the ture of the myofibrils. Major proteins are myosin and 46 muscle will vary significantly. Red muscle, which is actin, which provide the structural backbone of the 47 rich in red fibers, has a larger content of myoglobin myofibril. Regulatory proteins like tropomyosin and 48 and lipid and higher oxidative enzymatic activity; troponins are involved in muscle contraction and 49 white muscle, which is rich in white fibers, has relaxation. The Z line proteins serve as bridges 50 larger glycogen content and glycolytic enzyme between the thin filaments of adjacent sarcomeres. 51 activity (Toldrá 2006a). Red muscle is more aerobic Desmin connects adjacent myofibrils at the level of 52 in metabolism but slower in contraction than white the Z line. Longitudinal continuity and integrity of 53 muscle. muscle cells are achieved with two very large S54 N55 51 40454 06 049-058 r2.qxd 7/20/07 12:07 PM Page 52

52 Raw Materials

1 proteins, titin and nebulin, that run in parallel to the proteins are quite diverse, including myoglobin (the 2 long axis of the myofibril (Bandman 1987). natural pigment of meat) and many metabolic 3 enzymes present in mithocondrias, lysosomes, micro- Sarcoplasmic Proteins 4 somes, and nucleus and free in the cytosol. The 5 These proteins are water-soluble and comprise about amount of myoglobin depends on the fiber type, the 6 30–35% of the total protein in muscle. Sarcoplasmic age of the animal, and the animal species (Kauffmann 7 2001). 8 Table 6.1. Example of the approximate composi- 9 tion of pork lean muscle (expressed as g/100 g). Stromal Proteins 10 Fibers and muscles are surrounded by connective tis- Mean Range 11 sue (epimysium, perimysium, and endomysium). of Variation 12 Collagen, which is the basic protein of this tissue, 13 Moisture 72–76 provides strength and support to the muscle struc- 14 ture. Elastin is found in lower amounts, usually in Proteins 15–22 Myofibrillar 9–10 15 arterial walls. 16 Sarcoplasmic 9–10 17 Stromal 2–3 18 Lipids 1.5–4.0 Triacylglycerols PEPTIDES AND FREE AMINO ACIDS 19 1.5–3.5 Three natural dipeptides, carnosine (β-alanyl-L- 20 Phospholipids histidine), anserine (β-alanyl-L-1-methylhistidine), 21 0.5–0.6 and balenine (β-alanyl-L-3-methylhistidine) are 22 Free fatty acids present in the muscle. Some physiological properties 23 <0.05 of these dipeptides in the muscle are buffering, 24 Cholesterol antioxidant, and activity as neurotransmitter and as 25 <0.05 modulators of enzyme action (Chan and Decker 26 Carbohydrates <1 1994; Gianelli et al. 2000). Its content is specially 27 Ash <1 higher in muscles with glycolytic metabolism but it 28 varies with the animal species, age, and diet (Aristoy 29 30 Table 6.2. Description and localization of major muscle proteins (from Bandman 1987; Pearson and Young 31 1989; Toldrá 2002). 32 33 Proteins Group Main Proteins Main Localization in Muscle 34 35 Myofibrillar Myosin Thick filaments 36 Actin Thin filaments 37 Tropomyosin Between thick and thin filaments 38 Troponins T, C, I Between thick and thin filaments 39 Titin and nebulin Between Z-line and thin filaments ␣ ␤ ␥ 40 , , , and eu-actinin Z-line 41 Filamin; synemin; vinculin; zeugmatin; Z-line 42 Z nin; C, H, X, F, and I proteins 43 Desmin Between adjacent myofibrils at Z-lines 44 Myomesin, creatin kinase, and M protein M-line 45 Sarcoplasmic Mitochondrial enzymes Mitochondria 46 Lysosomal enzymes Lysosomes 47 Calpains Z-line 48 Lipases, glucohidrolases Sarcoplasm 49 ATP-ase Thick filament 50 Myoglobin Sarcoplasm 51 Hemoglobin Remaining blood in sarcoplasm 52 Stromal Collagen Connective tissue 53S Elastin Capillaries, nerves, tendons, etc. 54N 40454 06 049-058 r2.qxd 7/20/07 12:07 PM Page 53

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and Toldrá 1998). Beef and pork contain higher its location. So, intramuscular lipids are mainly 1 amounts of carnosine and lower amounts of anser- composed of triacylglycerols, which are stored in fat 2 ine, lamb contains similar amounts of carnosine and cells, and phospholipids, which form part of cell 3 anserine, and poultry is very rich in anserine. Bale- membranes. Intermuscular and adipose tissue lipids 4 nine is present in minor amounts in pork muscle and are mainly composed of triacylglycerols, with small 5 at very low concentration in the rest of animals amounts of cholesterol (40–50 mg/100 g). 6 (Aristoy and Toldrá 2004). 7 Triacylglycerols Free amino acids are continuously generated in 8 living muscle. The content of amino acids is signifi- Triacylglycerols are the major constituents of fat. 9 cantly higher in oxidative muscles than in glycolytic The fatty acid content mainly depends on the age 10 ones (Aristoy and Toldrá 1998). The content of free of the animal, type of feed, and environment. In fact, 11 amino acids is rather poor just after death but is sub- the fatty acids composition of triacylglycerols is 12 stantially increased during postmortem storage as a very important, because it defines its properties for 13 consequence of the proteolytical chain during meat processing. So, when the fat is rich in polyunsatu- 14 aging. rated fatty acids, such as linoleic acid (typical of 15 feeds rich in corn or barley) or eicosapentanoic and 16 docosahexanoic acids (feeds rich in fish oil), the fat 17 MUSCLE AND ADIPOSE TISSUE LIPIDS appears to be soft (oily appearance) and prone to 18 The amount of lipids in skeletal muscle is highly oxidation (rancid aromas and yellowish colors) (Irie 19 dependent on the degree of fattening and amount of and Sakimoto 1992; Morgan et al. 1992). The com- 20 adipose tissue and may range from 1–13% of total position in fatty acids somehow reflects the diet of 21 muscle. Lipids are mainly found within a muscle the animal, especially in pork and poultry (see some 22 (intramuscular), between adjacent muscles (inter- examples in Table 6.3). In the case of ruminants, the 23 muscular), or around the external muscle (adipose nutrients are somehow standardized due to the 24 tissue). The composition of lipids, mainly triacyl- action of the microbial population of the rumen 25 glycerols and phospholipids, changes depending on (Jiménez-Colmenero et al. 2006). 26 27 28 29 Table 6.3. Example of total fatty acids composition of pork muscle lipids expressed as % of total fatty acids as 30 affected by type of feed (from Irie and Sakimoto 1992; Morgan et al. 1992; Miller et al. 1990). 31 32 Fatty Acids 6% Fish Barley ϩ Soya Bean Meal Canola Oil 33 Myristic acid (C 14:0) 1.93 – 1.6 34 35 Palmitic acid (C 16:0) 26.89 23.86 20.6 36 Estearic acid (18:0) 16.30 10.16 9.8 37 Palmitoleic acid (C 16:1) 2.56 3 3.6 38 Oleic acid (C 18:1) 37.31 39.06 45.9 39 40 C 20:1 – – 41 Linoleic acid (C 18:2) 7.78 17.15 12.3 42 C 20:2 0.59 – 0.4 43 Linolenic acid (C 18:3) 2.08 0.91 3.0 44 45 C 20:3 0.08 0.21 0.1 46 Arachidonic acid (C 20:4) 0.46 4.26 0.74 47 Eicosapentanoic acid (C 22:5) 0.91 0.64 – 48 49 Hexadecanoic acid (C 22:6) 1.13 0.75 – 50 Total SFA 45.12 34.02 33.6 51 Total MUFA 39.87 42.06 49.5 52 Total PUFA 13.03 23.92 16.6 S53 N54 40454 06 049-058 r2.qxd 7/20/07 12:07 PM Page 54

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1 Phospholipids of the main substrates and products for these 2 enzymes is summarized in Table 6.4. Even though phospholipids are present in minor 3 amounts, they have a strong influence on flavor devel- 4 opment and oxidation. The reason is that phospho- ENDOPEPTIDASES 5 lipids are relatively rich in polyunsaturated fatty 6 Lysosomal Proteinases acids in comparison to triacylglycerols. Phosphatidyl- 7 choline (lecithine) and phosphatidylethanolamine are The main lysosomal proteinases are cathepsins B and 8 the major constituents. The amount of phospholipids L (cysteine proteinases that require a reducing envi- 9 depends on the genetic type of the animal and ronment for optimal activity and are very active at 10 anatomical location of the muscle and is higher in red pH 6.0) and cathepsin D (aspartate proteinase with 11 oxidative muscles than in white glycolytic muscles optimal acid pH in the range 3.0–5.0). These 12 (Hernández et al. 1998). enzymes are small (20–40 KDa) and can penetrate 13 into the myofibrillar structure (Etherington 1987). 14 Cathepsin H, which is also a cystein proteinase with 15 optimal pH at 6.8, may degrade myosin, although its 16 MUSCLE PROTEASES AND main function seems to be as an aminopeptidase 17 LIPASES (Okitani et al. 1981). Cathepsins B, D, and L have 18 Muscle contains a large and varied number of shown good ability to degrade different myofibrillar 19 enzymes, responsible for most of the biochemical proteins (Matsakura et al. 1981; Zeece and Katoh 20 changes observed during the processing of meat and 1989) and are partially inhibited by sodium chloride 21 meat products (Toldrá 2006b). Some of the most (Toldrá et al. 1992). Cathepsin D was found to 22 important muscle enzymes are related with protein be major contributor to proteolysis in fermented 23 breakdown (endopeptidases, also known as pro- sausages due to its stability and optimal activity at 24 teinases), generation of small peptides (peptidases), acid pH (Molly et al. 1997). 25 or free amino acids (aminopeptidases and carbxypep- 26 Neutral Proteinases: Calpains tidases) and lipids breakdown (lipases) (Toldrá 27 1992). The location of the enzymes may differ; some Calpains I and II are cystein endopeptidases located in 28 are located in lysosomes and others remain free in the the cytosol but, very especially, in the Z-disc region. 29 cytosol or attached to membranes. A brief description Calpains I requires 50–70 μM of Ca2ϩ for activity; 30 31 32 33 Table 6.4. Brief description of main substrates and products for most important muscle proteolytic and lipolytic 34 enzymes. 35 Enzymes Main Substrate Main Product 36 37 Cathepsins Myofibrillar proteins Protein fragments 38 Calpains Myofibrillar proteins Protein fragments 39 40 20S proteasome Myofibrillar proteins Protein fragments 41 Tripeptidylpeptidases Polypeptides Tripeptides 42 Dipeptidylpeptidases Polypeptides Dipeptides 43 Dipeptidases Dipeptides Free amino acids 44 45 Aminopeptidases Peptides (amino termini) Free amino acids 46 Carboxypeptidases Peptides (carboxy termini) Free amino acids 47 Lysosomal acid lipase Triacylglycerols Free fatty acids 48 49 Acid phospholipase Phospholipids Free fatty acids 50 Esterases Triacylglycerols Short chain free fatty acids 51 Hormone sensitive lipase Triacylglycerols Free fatty acids 52 Monoacylglycerol lipase Monoacylglycerols Free fatty acids 53S 54N 40454 06 049-058 r2.qxd 7/20/07 12:07 PM Page 55

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calpain II requires 1–5 mM of Ca2ϩ. Both enzymes Gly-Arg, These enzymes are able to generate a wide 1 are active at neutral pH, around 7.5, but with poor range of dipeptides during the processing of meat 2 activity below pH 6.0 (Etherington 1985). Calpains products (Sentandreu and Toldrá 2002). 3 are heterodimers of 110 KDa composed of an 80 KDa 4 catalytic subunit and a 30 KDa subunit of unknown Dipeptidases 5 function (Bond and Butler 1987). Calpains are able to 6 degrade a wide variety of myofibrillar proteins except Dipeptidases catalyze the hydrolysis of dipeptides 7 myosin and actin (Goll et al. 1983; Koohmaraie into two single free amino acids. They receive differ- 8 1994). Calpain I has poor stability in postmortem ent names depending on the preference for certain 9 muscle; calpain II seems to be stable for a few weeks amino acids. This is the case of glycylglycine dipep- 10 (Koohmaraie et al. 1987). In addition, the acid pH val- tidase, which is very specific for dipeptides contain- 11 ues of fermented sausages makes rather unlikely any ing glycine, and cysteinylglycine dipeptidase, which 12 calpain activity after a few days of processing. The is specific for the dipeptide Cys-Gly (McDonald and 13 activity of calpains is regulated in postmortem muscle Barrett 1986). 14 by the endogenous inhibitor calpastatin (Ouali 1991), 15 which is destroyed by autolysis after a few days post- Aminopeptidases 16 slaughter (Koohmaraie et al. 1987). 17 There are five different aminopeptidases reported in 18 muscle: Leucyl, arginyl, alanyl, pyroglutamyl, and 19 Proteasome methionyl aminopeptidases. They are active at neu- 20 tral or basic pH and receive different names depend- The proteasome complex constitutes a large prote- 21 ing on the preference or requirement for a specific olytic enzyme exerting multiple activities such as a 22 N-terminal amino acid. However, these enzymes chymotrypsin-like activity, a trypsin-like activity 23 are able to hydrolyze other amino acids, although and a peptidyl-glutamyl hydrolyzing activity (Coux 24 at a lower rate (Toldrá and Flores 1998). Alanyl et al. 1995). The 20S proteasome has shown ability 25 aminopeptidase is the aminopeptidase with higher to degrade myofibrils and affect M and Z lines, espe- 26 activity in postmortem muscle and accounts for most cially in high pH and slow-twitch oxidative muscles. 27 of the release of free amino acids (Toldrá 2002). The So, this enzyme could have a role on tenderness in 28 role of aminopeptidases in protein and peptide those specific muscles (Dutaud et al. 1996; Ouali 29 degradation during the processing of dry meat prod- and Sentandreu 2002). 30 ucts has received attention in recent years due to the 31 accumulation of free amino acids in the final prod- 32 EXOPEPTIDASES ucts and its influence on flavor (Toldrá 2006b,c). 33 Tripeptidylpeptidases 34 Carboxypeptidases Tripeptidylpeptidases (TPP) are enzymes that 35 hydrolyze the amino termini of polypeptides and Carboxypeptidases are located in lysosomes, having 36 release different tripeptides. TPP I is located in the optimal activity at acid pH. These enzymes generate 37 lysosomes and has an optimal acid pH (4.0); TPP II, free amino acids from the carboxy termini of peptides 38 which is located in the cytosol, has an optimal neu- and proteins. Carboxypeptidase A has preference 39 tral pH (7.0) and a wide substrate specificity (Toldrá for hydrophobic amino acids, and carboxypeptidase 40 2006c). B has a wide spectrum of activity (McDonald and 41 Barrett 1986). 42 43 Dipeptidylpeptidases 44 MUSCLE LIPASES Dipeptidylpeptidases (DPP) are enzymes that 45 hydrolyze the amino termini of polypeptides and There are several lipases in muscle, especially 46 release different dipeptides. DPP I and II are located located in the lysosomes where lysosomal acid 47 in the lysosomes and have optimal acid pH (5.5). lipase and phospholipase A are located. Both 48 DPP III is located in the cytosol and DPP IV is enzymes are active at acid pH (4.5–5.5) and are able 49 linked to membranes. Both have basic optimal pH to release long chain free fatty acids from triacyl- 50 (7.5–8.0). DPP II and DPP IV have special prefer- glycerols and phospholipids, respectively. Lysoso- 51 ence for Gly-Pro. DPP III has preference for Arg- mal acid lipase hydrolyzes preferentially primary 52 Arg and Ala-Arg and DPP I for Ala-Arg and ester bonds of try-acylglycerols (Fowler and Brown S53 N54 40454 06 049-058 r2.qxd 7/20/07 12:07 PM Page 56

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1 1984; Imanaka et al. 1985). Phospholipase A is enzyme has preference for the hydrolysis of fatty 2 responsible for the hydrolysis of phospholipids, at acids at position 1 (Fielding and Fielding 1980). 3 positions 1 or 2, at the water-lipid interface (Alasnier Acid and neutral esterases are also present in adi- 4 and Gandemer 2000). Acid and neutral esterases are pose tissue (Motilva et al. 1992) and can participate 5 located in the lysosomes and cytosol, respectively in the degradation of lipoprotein cholesteryl esters 6 (Motilva et al. 1992). These enzymes hydrolyze (Belfrage et al. 1984). 7 short chain fatty acids from tri-, di- and mono- 8 acylglycerols, but their activity is restricted due to 9 scarce available substrates. POSTMORTEM MUSCLE 10 METABOLISM AND QUALITY 11 MUSCLE OXIDATIVE AND ANTIOXIDATIVE ENZYMES 12 Several enzymatic reactions that can affect the meat 13 Oxidative Enzymes quality take place during early postmortem. Just 14 after death, glucose is hydrolyzed to lactic acid by Lipoxygenase catalyzes the incorporation of molec- 15 glycolytic enzymes. Due to the absence of blood cir- ular oxygen in polyunsaturated fatty acids giving a 16 culation, lactic acid is accumulated into the muscle conjugated hydroperoxide as final product (Marczy 17 and produces a pH drop to values about 5.6–5.8 in a et al. 1995). The different names of lipoxigenases 18 few hours. The pH drop rate depends on the glucose depend on the position where oxygen is introduced. 19 concentration, temperature of the muscle, and meta- These enzymes are activated by mM concentrations 20 bolic status of the animal previously to slaughter of Caϩ2 and by ATP (Yamamoto 1992). Lipoxyge- 21 (Toldrá 2006a). nase is stable during frozen storage and can acceler- 22 The rate and extent of pH drop has a great inci- ate rancidity development in meats like poultry, rich 23 dence on the quality of pork and poultry meats. So, in unsaturated fats (Grossman et al. 1988). 24 pigs with accelerated metabolism as a consequence 25 Antioxidative Enzymes of some type of premortem stress result in rapid 26 pH drops, below 5.8 after 2 hours postmortem, and 27 Antioxidative enzymes constitute a defense system almost full disappearance of ATP (Batlle et al. 28 against oxidative susceptibility and physical stress 2001), resulting in exudative meats. This is the case 29 (Young et al. 2003). Superoxide dismutase and cata- of the pale, soft, exudative pork meats (PSE) and red, 30 lase protect against the prooxidative effects by cat- soft, exudative pork meats (RSE) (Warner et al. 31 alyzing the dismutation of hydrogen peroxide to less 1997). Normal meat, known as red, firm, and normal 32 harmful hydroxides. Glutathione peroxidase, which meat (RFN), experiences a progressive pH drop 33 has higher activity in glycolytic muscles than in down to values around 5.8–6.0 at 2 hours post- 34 oxidative muscles, catalyzes the dismutation of alkyl mortem. Finally, the dark, firm, dry pork meat 35 hydroperoxides by reducing agents like phenols (DFD) and dark cutting beef meat are produced from 36 (Daun et al. 2001). exhausted animals (poor content of carbohydrates 37 before slaughter), where no lactic acid can be gener- 38 ADIPOSE TISSUE LIPASES ated during early postmortem. Glycolysis is almost 39 negligible in this type of meats and the pH remains 40 Adipose tissue contains three important lipolytic high—higher than 6.0, constituting a risk from the 41 enzymes, hormone-sensitive lipase, monoacylglyc- microbiological point of view. 42 erol lipase, and lipoprotein lipase, which are active Pork meat classification is usually based on pH 43 in the neutral/basic pH range (Toldrá 1992). The measurement at early postmortem, color, and drip 44 hormone-sensitive lipase hydrolyzes stored adipocyte loss. Exudative meats, specifically PSE, which have 45 lipids, with special preference for primary ester L values higher than 50 and drip losses higher than 46 bonds of tryacylglycerols, and releases free fatty 6%, must be controlled and detected when received 47 acids (Belfrage et al. 1984). The monoacylglycerol at the factory because they can affect the production 48 lipase, mainly located in the adipocytes, is able of fermented and ripened meat products. Some of 49 to hydrolyze medium and long chain monoacyl- the most important defects consist of excessive salti- 50 glycerols with no positional specificity (Tornquist ness (in the case of ripened pieces where salt is 51 et al. 1978). Lipoprotein lipase, which is located added on the surface), poor red color (paleness), pro- 52 in the capillary endothelium, hydrolyzes the acyl- tein denaturation, and inadequate drying (Toldrá 53S glycerol components at the luminal surface of the 2002). On the other hand, DFD meats that have a 54N endothelium (Smith and Pownall 1984). This drip loss lower than 2%, may cause microbiological 40454 06 049-058 r2.qxd 7/20/07 12:07 PM Page 57

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1 MF Miller, SD Shackelford, KD Hayden, JO Reagan. M Prieto. Nijmegen, The Netherlands: Audet, 2 1990. Determination of the alteration in fatty acid pp. 209–231. 3 profiles, sensory characteristics and carcass traits of ———. 1998. Proteolysis and lipolysis in flavour 4 swine fed elevated levels of monounsaturated fats in development of dry-cured meat products. Meat Sci 5 the diet. J An Sci 68:1624–1631. 49:s101–s110. 6 K Molly, DI Demeyer, G Johansson, M Raemaekers, ———. 2002. Dry-cured Meat Products. Trumbull, 7 M Ghistelinck, I Geenen. 1997. The importance of Connecticut: Food & Nutrition Press, pp. 1–238. 8 meat enzymes in ripening and flavor generation in ———. 2006a. Meat: Chemistry and biochemistry. 9 dry fermented sausages. First results of a European In: Handbook of Food Science, Technology and 10 project. Food Chem 54:539–545. Engineering. YH Hui, JD Culbertson, S Duncan, I 11 CA Morgan, RC Noble, M Cocchi, R McCartney. 1992. Guerrero-Legarreta, ECY Li-Chan, CY Ma, CH 12 Manipulation of the fatty acid composition of pig Manley, TA McMeekin, WK Nip, LML Nollet, MS 13 meat lipids by dietary means. J Sci Food and Agric Rahman, F Toldrá, YL Xiong, eds. Volume 1. Boca 14 58:357–368. Raton, Florida: CRC Press, pp. 28-1–28-18. 15 MJ Motilva, F Toldrá, J Flores. 1992. Assay of lipase and ———. 2006b. Biochemistry of processing meat and 16 esterase activities in fresh pork meat and dry-cured poultry. In: Food Biochemistry and Food Processing. 17 ham. Z Lebensmittel Untersuchung und Forschung YH Hui, WK Nip, ML Nollet, G Paliyath & BK 18 195:446–450. Simpson, eds. Ames, Iowa: Blackwell Publishing, 19 A Okitani, T Nishimura, H Kato. 1981. Characteriza- pp. 315–335. 20 tion of hydrolase H, a new muscle protease possess- ———. 2006c. Biochemical proteolysis basis for 21 ing aminoendopeptidase activity. Eur J Biochem improved processing of dry-cured meats. In: 22 115:269–274. Advanced Technologies for Meat Processing. LML 23 A Ouali. 1991. Sensory quality of meat as affected by Nollet, F Toldrá, eds. Boca Raton, Florida: CRC 24 muscle biochemistry and modern technologies. In: Press, pp. 329–351. 25 Animal Biotechnology and the Quality of Meat Pro- F Toldrá, M Flores. 1998. The role of muscle proteases 26 duction. LO Fiems, BG Cottyn, DI Demeyer, eds. and lipases in flavor development during the process- 27 Amsterdam, The Netherlands: Elsevier Science Pub, ing of dry-cured ham. Crit Rev Food Sci Nutrit 38: 28 BV, pp. 85–105. 331–352. 29 A Ouali, MA Sentandreu. 2002. Overview of muscle F Toldrá, E Rico, J Flores. 1992. Activities of pork muscle 30 peptidases and their potential role in meat texture proteases in cured meats. Biochimie 74:291–296. 31 development. In: Research Advances in the Quality H Tornquist, P Nilsson-Ehle, P Belfrage. 1978. 32 of Meat and Meat Products. F Toldrá, ed. Trivan- Enzymes catalyzing the hydrolysis of long-chain 33 drum, India: Research Signpost, pp. 33–63. monoacylglycerols in rat adipose tissue. Biochimica 34 AM Pearson, RB Young. 1989. Muscle and Meat Bio- Biophysica Acta 530:474–486. 35 chemistry. San Diego, California: Academic Press, RD Warner, RG Kauffman, RL Russell. 1997. Muscle 36 pp. 1–261. protein changes post mortem in relation to pork qual- 37 MA Sentandreu, G Coulis, A Ouali. 2002. Role of ity traits. Meat Sci 33:359–372. 38 muscle endopeptidases and their inhibitors in meat S Yamamoto. 1992. Mammalian lipoxygenases: molec- 39 tenderness. Trends Food Sci Technol 13:398–419. ular structures and functions. Biochimica Biophysica 40 MA Sentandreu, F Toldrá. 2002. Dipeptidylpeptidase Acta 1128:117–131. 41 activities along the processing of Serrano dry-cured JF Young, K Rosenvold, J Stagsted, CL Steffensen, 42 ham. European Food Res Technol 213:83–87. JH Nielsen, HJ Andersen. 2003. Significance of pres- 43 LC Smith, HJ Pownall. 1984. Lipoprotein lipase. In: laughter stress and different tissue PUFA levels on 44 Lipases. B. Borgström, HL Brockman, eds. Amster- the oxidative status and stability of porcine muscle 45 dam, The Netherlands: Elsevier, pp. 263–305. and meat. J Agric Food Chem 51:6877–6881. 46 F Toldrá. 1992. The enzymology of dry-curing of MG Zeece, K Katoh. 1989. Cathepsin D and its effects 47 meat products. In: New Technologies for Meat and on myofibrillar proteins: a review. J Food Biochem 48 Meat Products. FJM Smulders, F Toldrá J Flores, 13:157–161. 49 50 51 52 53S 54N 40454 07 059-076 r2.qxd 7/21/07 9:55 AM Page 59

1 2 3 7 4 5 6 Ingredients 7 8 Jorge Ruiz 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION high as 150–160 kg are also used for fermented meat 22 production (Mayoral et al. 1999). This leads to a 23 Meat and lard are the two major ingredients in most much higher fat content both in the carcass and in 24 fermented meat products and, consequently, their the lean (Figure 7.1). 25 characteristics strongly influence the features of the Bovine meat was frequently used as a supplement 26 final product, including sensory, nutritional, safety, for pig meat in many fermented meat products. 27 and health aspects. For the purpose of this text, meat However, due to the high prices of this type of meat 28 can be defined as the skeletal muscle of livestock, and to the better technological and sensory proper- 29 poultry, and game, mainly including muscle, fat, and ties of pork for dry-fermented processing, the use of 30 connective tissues, but also others such as vascular, bovine meat for elaboration of fermented sausages 31 lymphatic, or nervous tissues. Nevertheless, most fer- has decreased. Ovine and goat meat are used to a 32 mented meat products are produced using pork. As far lesser extent for the production of fermented prod- 33 as lard is concerned, it is constituted by the subcuta- ucts, although there is still a significant production 34 neous fat from swine. Only in a few regional types of of sausages from old ovine livestock whose meat 35 fermented sausages are ovine and bovine subcuta- cannot be aimed toward fresh meat consumption. 36 neous fat added. Besides these two major compo- The problem with these two types of meats (espe- 37 nents, salt is a main constituent in almost all types of cially when animals are not young) is their strong 38 fermented sausages, and only in recent years a few and identifiable flavor, which is usually rejected by 39 salt-free (or salt-reduced) products have been devel- consumers. Nevertheless, in Muslim countries, fer- 40 oped directed to potentially high blood pressure con- mented meat products are mainly based on bovine 41 sumers. Other minor ingredients include offal, plant and ovine meats mixed with ovine fat, because pork 42 materials or water. is not allowed for religious reasons (Leistner 1992). 43 The production of sausages with horse, mule, and 44 LEAN donkey meats are strange and limited to some 45 regional specialities. Finally, there is a growing pro- 46 Lean refers to commercial pieces of meat, including duction of fermented sausages made from game 47 muscle, lard, and other tissues. Most fermented meat, especially those from wild boar and deer. 48 products are produced using pork obtained from bar- After decades in which frozen-thawed meat was 49 rows and gilts. Animals are usually slaughtered at the major form in which lean was used for fermented 50 90–100 kg live weight when around 6 months old. sausage production, there is currently a growing ten- 51 Nevertheless, for some kind of products, heavier dency to use fresh raw material (Solignat 1999). An 52 pigs are preferred and, thus, pigs with live weights as inappropriate freezing process, frozen storage, or 53 S54 N55 59 40454 07 059-076 r2.qxd 7/21/07 9:55 AM Page 60

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1 12 Intramuscular Fat Content 2

3 9 LD BF 4 Muscle 60.00 5 6 Fat 6 3 7 Bone 8 g fresh muscle g fat/100 0 100 200 300 400 500 9 Age (days) 10 11 40.00 12 13 14

15 % of weight 16 17 20.00 18 19 20 21 22 23 0.00 24 0 100 200 300 400 500 25 Age (days) 26 Figure 7.1. Mean (ϮSD) of the proportion of weight of muscle, dissectible fat, and bone in left hemicarcass of 27 heavy pigs at slaughter throughout lifetime.The inset shows intramuscular fat content (g fat/100 g fresh muscle). 28 LD: Longissimus dors; BF: Biceps femoris (taken from Mayoral et al. 1999). 29 30 31 thawing, might lead to protein damages which, in Fat is an essential component for fermented 32 turn, produce drip and defects in meat texture sausages, providing several features positively 33 (Miller et al. 1980; Lawrie 1995), affecting the final related to their sensory and technological quality, 34 quality of fermented products. Moreover, frozen namely easing chewing, enhancing juiciness, con- 35 storage slows down oxidative processes, but does tributing to aroma development and release, and 36 not totally avoid them (Novelli et al. 1998: Hansen slowing down the dehydration process (Wirth 1988). 37 et al. 2004). Thus, frozen meats stored for a long The fat used for production of fermented sausages 38 time might speed up lipid oxidative processes during is mainly that from pig, especially lard, which is the 39 the processing of fermented products. For these rea- subcutaneous adipose tissue. Other anatomical loca- 40 sons, when using frozen meats for fermented meat tions of fat, such as the fat associated with the kidney 41 products, it is generally recommended that storage at (perinephric) or the omental fat, are rarely used for 42 Ϫ18°C is not longer than 6 months for beef and elaboration of fermented meat products. It is currently 43 3 months for pork (Prändl et al. 1988). getting more common to use fresh lard for fermented 44 sausage production, although it is usually ground at Ϫ 45 FAT 1.0°C to avoid melting and to achieve a well- 46 defined cutting surface. When using frozen lard, stor- 47 Adipose tissue is a specialized type of connective tis- age should not be longer than 3 months, for the same 48 sue, constituted by fat cells called adipocytes, which reasons explained for frozen lean (Prändl et al. 1988). 49 show a drop of triglycerides comprising almost all In the last decades, there has been a growing inter- 50 the volume of the cell. As any connective tissue, adi- est in producing healthier meat products. Some of 51 pose fat also shows connective fibers of collagen, the most common strategies have been directed to a 52 although the number and strength of these fibers is reduction of fat content and a decrease of saturated 53S much lower than in other types of connective tissues fatty acids with a concomitant increase in polyunsat- 54N (such as those in tendons, fasciae, or muscle). urated fatty acids or monounsaturated fatty acids. 40454 07 059-076 r2.qxd 7/21/07 9:55 AM Page 61

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60 1 2 (a) 50 3 4 40 5 6 30 7 8 20 9

Weight loss (%) Weight 10 10 LSD = 10.04 11 12 0 13 14 15 15 14 (b) 16 13 17 12 18 11 19 20 10 21 Brine (%) 9 22 8 23 LSD = 2.06 7 24 25 6 26 1.00 27 (c) 28 29 0.95 30 31 0.90 32 33 LSD = 0.02 34 Water activity Water 0.85 35 36 37 0.80 38 0714 21 39 40 Storage Time (Days) 41 10% fat, in CS 20% fat, in CS 30% fat, in CS 42 43 10% fat, in RR 20% fat, in RR 30% fat, in RR 44 Figure 7.2. Effect of fat level and storage conditions on (a) weight loss, (b) brine content, and (c) water activity 45 during storage of traditional sausages. CS ϭ cold storage, RR ϭ ripening room (taken from Papadima and 46 Bloukas 1999). 47 48 49 The latter has been achieved through pig dietary tion and poor sensory features of the ﬁnal product. 50 means and by replacing lard with other oils during Thus, Papadimas and Bloukas (1999) have shown 51 sausage production (discussed later in this chapter). much faster dehydration in 10% added fat sausages 52 As far as fat reduction is concerned, excessive reduc- than in 30% ones, leading to huge differences in the S53 tion of fat content may lead to a very rapid dehydra- water activity of the ﬁnal product (Figure 7.2). Fat N54 40454 07 059-076 r2.qxd 7/21/07 9:55 AM Page 62

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1 8 Low-fat Medium-fat High-fat 2 3 4 5 7 6 7 8 9 6 10 11 12 5 13 14 15 16 4 17 Juiciness Tenderness Spiciness Saltiness Taste Overall acceptancy (consumers) 18 sensory traits 19 20 Figure 7.3. Sensory characteristics and overall acceptability of dry-cured fermented sausages with low 21 (6.5%), medium (12.5%), and high (25%) added fat (elaborated with data from Mendoza et al. 2001). 22 23 24 1.4 25 26 1.2 27 28 1.0 29 30 0.8 31 32 0.6 33 0.4 34 (mg MA/kg) TBA value 35 36 0.2 37 38 0.0 02 46810 39 Time (days) 40 41 Figure 7.4. Effect of fat level and temperature on the TBA value during the ripening period of fermented 42 sausages. Solid lines, 20–22°C, dashed lines, 24–26°C; • 10% fat added, ▲ 20% fat added, ■ 30% fat added 43 (taken from Soyer et al. 2005). 44 45 46 content is usually positively related to sensory fea- FACTORS AFFECTING THE 47 tures of the ﬁnal product, as evidenced by Mendoza FEASIBILITY OF LEAN AND FAT 48 et al. (2001) comparing low, medium, and high fat FOR PROCESSING 49 content sausages (Figure 7.3) However, Soyer (2005) 50 observed higher levels of free fatty acids and oxida- The technological quality of the meat includes those 51 tion in fermented sausages elaborated with greater features that make the meat optimum for production 52 amounts of added fat (Figure 7.4). This author found of good-quality fermented sausages, with as little 53S the highest overall acceptability for sausages with spoilage as possible, as high yield as possible, and 54N intermediate (20%) levels of added fat. leading to a ﬁnal product pleasant for consumers. 40454 07 059-076 r2.qxd 7/21/07 9:55 AM Page 63

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The economical profitability of the whole produc- The use of exudative meats for fermented meat 1 tion depends on all these factors: directly on the products processing may lead to several problems. 2 yield and the proportion of spoiled product, and First, as a consequence of the low water-holding 3 indirectly on the consumer acceptability, which in its capacity of these meats, dehydration occurs very fast 4 turn determines how often the consumer buys the (Prändl et al. 1988). It could be thought that this is 5 product and, thus, the total sales (Aaslying 2002). not a problem for dry and semidry-cured fermented 6 sausages, because dehydration is one of the main 7 physical processes involved in the stabilization of the 8 PH AND WATER-HOLDING CAPACITY products. In fact, some authors have proposed the use 9 Muscle contains approximately 75% water. The of PSE meat for accelerating the drying during pro- 10 majority of water in muscle is held within the struc- duction of fermented sausages (Townsend et al. 11 ture of the muscle itself, either within the myofibrils, 1980), and Honkavaara (1988) has found slightly bet- 12 between the myofibrils themselves, and between the ter sensory properties in dry-fermented sausages pro- 13 myofibrils and the cell membrane. Most of this water duced with PSE meat. However, if dehydration in the 14 is physically retained by capillarity within myofibri- surface is too fast, water migration from the depth is 15 lar proteins; only a small amount is bound by the not quick enough to equalize the moisture content 16 sarcoplasmic proteins and the connective tissue between the surface and the depth of the product. If 17 (Wismer-Pedersen 1987). such a situation occurs, proteins in the surface may 18 Water-holding capacity of meat is defined as the get too desiccated, forming a crust (personal observa- 19 ability of the postmortem muscle (meat) to retain tion). As a compromise for using PSE meat for the 20 water even though external pressures (e.g., gravity, production of dry-fermented sausages, Wirth (1985) 21 heating) are applied to it. Once the animal is slaugh- recommended a maximum of 30% PSE meat in the 22 tered, the amount of retained water in muscle can meat batter. 23 change, depending on numerous factors related to Another potential problem when using exudative 24 the tissue itself and how the meat is handled. Some meats is the formation of weak meat gels, due to the 25 of these factors are the net charge of myofibrillar damage suffered by myofibrilar proteins. This leads to 26 proteins and the structure of the muscle cell and its low consistency products with poor consumer accept- 27 components (myofibrils, cytoskeletal linkages, and ance (Kuo and Chu 2003) (Figure 7.5). Moreover, due 28 membrane permeability), as well as the amount of to the low consistency of exudative meats, the surface 29 extracellular space within the muscle itself (Huff- of the ground meat obtained during the grinding step 30 Lonergan 2002). Changes undergone during rigor is not neat, giving rise to a fuzzy surface. As far as 31 mortis (pH decline and contraction) lead to a whole muscle products are concerned, the use of PSE 32 decrease in the amount of water that can be retained shows many problems, including an excessive salting 33 by the myofibrilar structure, due to a reduction of the of hams, defective texture, and two-toning appearance 34 space within such structure, which in turn is due to (Guerrero et al. 1996; Garcia-Rey et al. 2004). 35 both a pH close to the isoelectrical point of proteins There is little scientific information about the 36 (reducing the net charge of the proteins) and to a potential problem of using DFD meats in fermented 37 smaller size of the sarcomere as a consequence of sausage production. Although water retention is 38 contraction. quite high in DFD meats, and protein functionality is 39 Due to different rates of pH decline during rigor very good for gel formation and emulsification, their 40 mortis, and of the final pH reached during this pH value above 6.2 makes them more prone to 41 process, marked differences in the ability for retain- microbial spoilage. This might not be a problem in 42 ing water in muscle are observed (Huff-Lonergan fermented sausage production, because microbial 43 2002). Briefly, fast rates of pH decline lead to very growth in the first steps of processing will lead to a 44 exudative meats called PSE (pale, soft, and exuda- pH decline that will probably compensate the intrin- 45 tive, which summarize their features). Likewise, sic high pH of DFD meat. However, when starter 46 those meats with a low final pH (5.2–5.4) also show cultures are not used for the production of fermented 47 similar characteristics and exude a great amount of sausages, such as in small plants for traditional 48 water. On the other hand, if the final pH value is sausage production, the use of DFD meat could lead 49 higher than 6.2 (usually due to a low initial glycogen to microbial spoilage in the first steps of sausage 50 concentration when rigor mortis initiates), the production. With respect to whole meat fermented 51 amount of retained water is higher than in normal and dry-cured products, the use of high ultimate pH 52 meats, leading to dark cuts with scarce exudates meats should be avoided because it leads to either S53 (DFD meats: dark, firm, and dry). texture problems or spoilage (Guerrero et al. 1999). N54 40454 07 059-076 r2.qxd 7/21/07 9:55 AM Page 64

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1 8 100% normal meat 50% normal meat+50% PSE 100% PSE 2 3 4 7 5 6 7 8 6 9 10 core (sensory)

11 S 5 12 13 14 15 4 16 Texture Flavor Overall acceptablity 17 Figure 7.5. Sensory features of Chinese fermented sausages made with either normal or PSE pork. Hedonic 18 scale: 1 ϭ extremely dislike in texture, flavor, or overall acceptability; 5 ϭ neither like nor dislike; 9 ϭ extremely 19 like in texture, flavor, or overall acceptability (elaborated with data from Kuo & Chu 2003). 20 21 22 LEAN COLOR remaining pigment, which has not reacted with 23 nitrite, leading to dark greyish colors in the surface. The intensity of meat color is dependent upon the 24 This process is very common during slicing and concentration of the sarcoplasmic protein myoglobin 25 packaging, because the surface is exposed to the air (Giddings 1977). The chemical state of iron in myo- 26 and light, promoting such oxidation processes. In globin determines the color of the pigment, and, 27 this sense, the use of meats with high levels of thus, the color of the meat (Govindarajan 1973). 28 vitamin E through supplementation of pig diets with The bright red fresh color of meat is due to the pres- 29 α-tocopherol is an effective strategy to reduce dis- ence of the reduced form of myoglobin binding an 30 coloration during the slicing of dry-fermented prod- oxygen molecule in the heme group. This form of 31 ucts (Ruiz and López-Bote 2002). 32 the pigment is called oxymyoglobin. In the absence 33 of oxygen, the pigment can also be in a reduced 34 state, giving rise to the so-called deoxymyoglobin INTRAMUSCULAR FAT CONTENT 35 form, which is purple-red. Oxidation of heme iron to 36 the ferric (Fe3+) form results in the formation of met- Intramuscular fat refers to the adipose tissue between 37 myoglobin, which has a brownish color negatively muscle fiber bundles that constitutes marbling. The 38 valued by consumers. The oxidation of the reduced positive effect of marbling on the sensory quality of 39 forms of myoglobin to metamyoglobin occurs as a the meat is well known (Bejerholm and Barton-Gade 40 consequence of several prooxidant factors, such as 1986). Moreover, in dry-cured ham intramuscular fat 41 compounds from lipid oxidation, light, temperature, has been pointed out as the main factor influencing 42 or metals. juiciness (Ruiz et al. 2000), which in turn was high- 43 During the curing process myoglobin is converted lighted as the main sensory trait related to acceptabil- 44 into nitrosomyoglobin in the presence of nitrite, ity (Ruiz et al. 2002). However, given that in most 45 forming a red stable color (Skibstead 1992). For dry-cured fermented sausages supplementary fat is 46 most processed fermented sausages, intense colors added, intramuscular fat is not so important as in 47 are preferred to pale ones. This leads to the utiliza- fresh meat or dry-cured hams. Nevertheless, in those 48 tion of heavier animals in some cases, because ani- dry-cured fermented meat products in which the 49 mal age is directly related to muscle myoglobin muscle is not ground, or in those in which the pieces 50 content (Mayoral et al. 1999). of meat are of an appreciable size, intramuscular fat 51 During storage the color might further change plays an important role in controlling the rate of des- 52 depending on both the package material and on the iccation, in the development of the flavor during the 53S storage conditions (Andersen et al. 1988). This can processing and in the sensory characteristics of the 54N cause discoloration due to the oxidation of the elaborated product (Muriel et al. 2004). 40454 07 059-076 r2.qxd 7/21/07 9:55 AM Page 65

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A decrease in water activity in fermented meat migration in dry-cured meat products (Girard et al. 1 products is one of the main factors contributing to 1989; Ruiz and López-Bote 2002). In this sense, the 2 their microbial stability. Such a decrease is achieved levels of polyunsaturated fatty acids (PUFA) in fatty 3 essentially by adding sodium chloride to the meat tissues of pigs used for production of fermented 4 batter and by dehydration during the processing. sausages are critical, because an excessive propor- 5 Water losses imply two different processes: water tion will lead to impaired dehydration and poorer 6 migration from the depth to the surface of the product texture and microbial stability. Accordingly, Girard 7 and water evaporation in the surface. Diffusion coef- et al. (1989) fed diets to pigs in which the main 8 ﬁcients of salt and water in meat products are energy source was provided by barley and copra oil 9 inversely correlated to the fat content (Palumbo et al. (saturated fat), or maize and maize oil (unsaturated 10 1977), due to lower diffusion in the fat than in the fat). These diets produced a range of linoleic acid 11 lean tissue (Wood 1966). Therefore, the higher the (C18:2 n-6) concentration in pig adipose tissue from 12 intramuscular fat content in those products in which 7.58% to 30.95% (expressed as the percentage of 13 muscle is intact, the longer the time required for salt total fatty acids). Subsequent processing of dry- 14 diffusion and for the appropriate moisture loss. fermented sausages from these pigs revealed a major 15 We have recently found a close relationship effect of fat unsaturation on the drying process. Dry 16 between intramuscular fat content and most appear- pork sausages manufactured from pigs fed highly 17 ance and texture sensory traits of dry-cured loin, a unsaturated fatty acids did not permit adequate dry- 18 dry-fermented meat product typical from Spain, ing. According to these authors, impaired moisture 19 pointing to a direct inﬂuence of this parameter on the loss became evident from the 10th day of manufac- 20 eating quality of the product, and probably to others ture and was most pronounced during the later 21 in which the whole muscle is processed (Ventanas stages of drying (Figure 7.6). Low weight losses dur- 22 et al. 2007). ing processing, higher water activity values (0.88 23 versus 0.75), and higher moisture content in the fat- 24 free product (55% versus 45%) revealed impaired 25 FATTY ACID COMPOSITION drying in the group of pigs fed highly unsaturated 26 Not only the amount of fat, but its consistency is fats (maize and maize oil). 27 essential for an appropriate rate of moisture loss dur- This effect is most likely due to the physical state 28 ing the processing of fermented sausages (Ruiz and of the fat during the processing. The physical state of 29 López-Bote 2002). In fact, there are some indica- the fat mostly depends on the fatty acid melting 30 tions that dietary fat unsaturation affects water point, which is dependent on its turn on the number 31 32 33 75 1.00 34 CONTROL (DM) 35 70 36 0.95 37 65 PUFA (DM) 38 39 0.90 60 40 PUFA (aw) 41 55 42 0.85

Water activity Water 43

Dry matter (%) 50 44 45 0.80 CONTROL (a ) 45 w 46 47 40 0.75 48 0102030 40 49 Time (Days) Girard et al. (1989) 50 51 Figure 7.6. Variation in dry matter and water activity throughout the processing of dry-fermented sausages 52 produced with meat from pigs fed a diet either high in SFA (CONTROL) or high in PUFA (PUFA) (elaborated with S53 data from Girard et al. 1989). N54 40454 07 059-076 r2.qxd 7/21/07 9:55 AM Page 66

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1 of double bonds per fatty acid residue, the energy when the cellular mechanisms for controlling lipid 2 needed to melt the fat being lower as the number of oxidation no longer work. The rate and extent of lipid 3 unsaturations rises. Among fatty acids present in pig oxidation depends on the presence of PUFA and the 4 tissues, stearic acid (C18:0) and linoleic acid (C18:2 ratio between prooxidant and antioxidant factors. 5 n-6) show the highest correlations (positive and Among the formers, the main one in muscle is the 6 negative, respectively) with fat consistency (Wood presence of heme proteins (myoglobin). Major antiox- 7 1984; Davenel et al. 1999). However, from a practi- idants in muscle include several enzymes, some pro- 8 cal point of view, linoleic acid is the main fatty acid teins, carnosine, tocopherols, ascorbic acid, and 9 determining fat consistency in pig fat. polyphenols. 10 Fat consistency may also affect firmness of dry- Phospholipids are the primary substrates of lipid 11 cured ham. It has been shown that an increased oxidation in muscle foods, and triglycerydes seem to 12 polyenoic fatty acid content in pig diets leads to sig- play a minor role (Igene and Pearson 1979). The 13 nificantly reduced product firmness in dry-cured fer- high sensitivity of phospholipids to oxidation has 14 mented sausages (Stiebing et al. 1993; Warnants primarily two causes: the high proportion of long 15 et al. 1998; Houben and Krol 1980). Due to this chain PUFA, which are very susceptible to oxida- 16 effect, some researchers (Stiebing et al. 1993; tion, and the close contact of phospholipids with cat- 17 Boulard et al. 1995) have recommended a maximum alysts of lipid oxidation located in the aqueous phase 18 proportion of linoleic acid of 12% when meat is used of the muscle cell, because they are the major cell 19 for sausage processing. membrane components. 20 Fatty acid melting point also affects lard color: The overall mechanism of fatty acid oxidation 21 solid fat shows a white color, and melted fat shows a includes three steps: initiation, propagation, and ter- 22 grey-dark appearance, probably because it becomes mination (Frankel 1984). Primary products of lipid 23 transparent and therefore allows the observation of autoxidation are hydroperoxides, which can further 24 capillary tubes and connective tissue (Zhou et al. decompose to form a large variety of volatiles includ- 25 1993). This is generally considered a drawback ing alkanes, aldehydes, ketones, alcohols, esters, car- 26 because consumers relate darker colors in the pieces boxylic acids, and so on. Both the nature and the 27 of fat to rancidity (Ruiz et al. 2002). relative proportion of the volatile compounds in the 28 Finally, fat consistency shows important conse- muscle food depend on several factors, among which 29 quences on the sensory features of the final product. the fatty acid profile of the raw material is the main one 30 Products with fats rich in PUFA and MUFA show a (Grosch 1987). Among the numerous volatiles formed 31 bright and oily surface when they are sliced and may from unsaturated fatty acids, the most important 32 have an excessively soft texture (Townsend et al. aroma compounds are aldehydes and several unsatu- 33 1980). This can be either a drawback or a quality rated ketones and furan derivatives (Grosch 1987). 34 characteristic depending on the type of product and the They include C3–C10 aldehydes, C5–C8 unsaturated 35 country: for most consumers worldwide, oiliness is not ketones, and pentyl or pentenyl furans. In fact, satu- 36 a desirable characteristic, but it is in dry-fermented rated aldehydes have been extensively used as markers 37 sausages from Iberian pigs in Spain (Ruiz et al. 1998). for lipid oxidation in food (Shahidi et al. 1987). 38 Among PUFA, the n-3 fatty acids have been 39 reported to more severely affect the susceptibility of OXIDATIVE STATUS 40 fresh meat to lipid oxidation than n-6 fatty acids, 41 Lipid oxidation is one of the main causes of deteriora- probably because some n-3 fatty acids are highly 42 tion in the quality of meat products during storage and unsaturated (e.g., eicosapentaenoic acid or docosa- 43 processing, due to generation of compounds showing hexaenoic acid). In fact, the susceptibility of fatty 44 rancid flavor (Grosch 1987). On the other hand, some acids to oxidation increases with the number of 45 of the volatile compounds with higher impact on the double bonds: the relative oxidation rates of fatty 46 aroma found in meat and meat products arise from acids containing 1, 2, 3, 4, 5, or 6 double bonds are 47 oxidation of unsaturated fatty acids (Gasser and 0.025, 1, 2, 4, 6, and 8, respectively (Horwitt 1986). 48 Grosch 1988). Furthermore, compounds from autoxi- This explains why even small differences in the con- 49 dation of unsaturated fatty acids may further react with centration of these long chain PUFA (particularly in 50 amino compounds, leading to formation of very low the polar fraction of the fat) may be critically impor- 51 threshold volatiles, which have been highlighted tant in the development of oxidation. Accordingly, 52 as being responsible for pleasant flavor notes in Eichenberger et al. (1982) revealed that lipid oxida- 53S cooked meat and meat products (Mottram 1998). tion in isolated membranes occurred mainly in n-3 54N Lipid oxidative phenomena in muscle-based foods fatty acids containing 5 or 6 double bonds. More- occur immediately after slaughter (even preslaughter), over, using a triangle test, de la Hoz et al. (2004) 40454 07 059-076 r2.qxd 7/21/07 9:55 AM Page 67

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evidenced noticeable sensory differences between great attention in the last decades (Ruiz and López- 1 dry-fermented sausages made with meat from pigs Bote 2002). The main advantage of this practice is 2 fed either on a diet rich in linolenic acid (C18:3 n-3) that the effect is much higher than the direct use of 3 or on a control diet. the antioxidant in the meat product, since the antiox- 4 Warnants et al. (1998), studying the effect of feed- idant is distributed to every tissue and subcellular 5 ing diets rich in PUFA on the sensory characteristics structures. For example, de la Hoz et al. (2004) 6 of salami, found that enriched products were easily found that dry-fermented sausages produced using 7 detectable due to higher oxidative rancidity. These meat enriched in n-3 fatty acids were easily detected 8 authors suggested a limit of 21% PUFA for fat aimed by panelists. However, when animal feeding was 9 toward production of fermented sausages (equiva- supplemented with 250 mg/Kg of α-tocopherol, 10 lent to the inclusion of 26 g PUFA/kg concentrate panelists were unable to detect such differences, and 11 used for pig feeding). Nevertheless, other authors lipid oxidation was not signiﬁcantly different to the 12 have suggested considerably lower levels (14% control sausages. Such a procedure is especially 13 PUFA) for fat dedicated to production of these kinds interesting when producing whole dry-cured fer- 14 of products (Stiebing et al. 1993). A similar behavior mented products, like hams or loins, in which the 15 has been evidenced in other dry products, and, thus, direct addition of antioxidants is not possible or it is 16 linoleic acid content in fresh meat is limited to 15% scarcely effective (Ruiz and López-Bote 2002). 17 in Parma ham and to 9% in Iberian ham. Direct addition of antioxidants to the meat batter 18 The use of diets rich in n-3 fatty acids shows simi- also contributes to a better control of oxidative 19 lar or even more accentuated consequences. The deterioration of fermented meat products. In fact, 20 main problem in this case is that products from oxi- Valencia et al. (2006) achieved similar levels of lipid 21 dation of these fatty acids show low olfaction thresh- oxidation in sausages enriched with n-3 PUFA 22 old and particular objectionable ﬂavor notes. Thus, through addition of a mixture of BHT and BHA. 23 Pastorelli et al. (2003) found easily detectable those Nevertheless, the presence of such additives is usu- 24 samples from Parma hams from pigs fed linseed ally not pleasing for some consumers and, thus, the 25 enriched diets. use of other type of plant extracts with antioxidative 26 Some other compounds in the muscle may experi- properties is getting more common for elaboration 27 ence oxidative reactions, such as proteins or heme of fermented meat products. The reduction in the 28 pigments. Very little is known about protein oxida- number of “E” additives for elaborating meat prod- 29 tion in fermented meat products. In a recent experi- ucts is currently a goal for many producers; this 30 ment (Ventanas et al. 2006), evidence of protein practice is known as “cleaning the label.” Thus, in a 31 oxidation throughout processing of dry-cured loins recent paper, Sebranek et al. (2005) showed that the 32 was detected. This process has been linked to rosemary extract at 2500 ppm was equally effective 33 impaired water retention and decreased texture in as BHA/BHT for controlling lipid oxidation in 34 other meat products (Xiong 2000), and so its impor- sausages during refrigerated storage (Figure 7.7). 35 tance in the quality of dry-fermented products 36 should not be dismissed. 37 Compounds with antioxidative properties may 38 reduce overall lipid oxidative phenomena, avoiding 39 or decreasing the presence of rancid notes in fer- Control BHA/BHT Rosemary 40 mented products. There are two possible strategies: 3 41 feeding pigs with mixed diets rich in antioxidants or 2.5 42 directly adding antioxidants to the meat batter. As far 43 as pig dietary strategies are concerned, phenolic 2 44 antioxidants (BHT, BHA, ethoxyquine) are exten- 1.5 45 sively used in animal feeding due to their reducing 46 lipid oxidation properties. However, only a small 1 47 amount of these antioxidants are absorbed by ani- TBARs (mg/kg) 0.5 48 mals and, therefore, they only show the effect of sta- 49 0 bilizing fat in the feeding but not in animal tissues 01428425670 84 98 112 50 lipids. Some other antioxidants added to the feeding 51 Time (days) may be absorbed and accumulated in animal tissues, 52 showing their effects in vivo and after slaughter. In Figure 7.7. TBARS values during frozen storage of S53 fact, the use of α-tocopherol for controlling oxida- raw pork sausage with different antioxidants (taken N54 tive reactions in meat and meat products has gained from Sebranek et al. 2005). 40454 07 059-076 r2.qxd 7/21/07 9:55 AM Page 68

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1 CONNECTIVE TISSUE product. Thus, those sausages produced using the 2 meat from the foreleg are considered of higher qual- Connective tissue in muscle is organized at different 3 ity, and cuts including more fibrous muscles are levels: epimysium surrounding the whole muscle, 4 more frequently used for lower-quality products. At perymisium enclosing muscle fiber bundles, and 5 any rate, legislation of some countries clearly estab- endomysium encircling individual muscle fibers 6 lishes a limit for the hidroxyproline content of some (Lawrie 1995). This tissue is essentially formed by 7 type of sausages to control the addition of ten- collagen and elastin. It seems that from a meat qual- 8 dons, fasciae, or highly fibrous meat to high-quality ity point of view the former is more directly related 9 sausages. In this way, consumers are protected with texture. Collagen is formed by tropocollagen 10 against those who could potentially use low-quality subunits crosslinked by stable bonds forming highly 11 cuts to produce expensive sausages through carrying resistant fibers. As a consequence of a higher num- 12 out a very intense grinding of fresh lean. ber of stable bonds, collagen becomes more ther- 13 The processing of fermented products does not mally stable and less soluble (Sims and Bailey 14 produce significant changes in the structure of colla- 1981). These features have been linked to meat 15 gen, and, thus, does not reduce the fibrousness and toughness. The number and stability of collagen 16 toughness occasioned by this structural component crosslinking bridges is dependent upon a number of 17 (Cordoba 1990). factors, such as animal age, sex, type of muscle, or 18 exercise (Sims and Bailey 1981; Mayoral et al. 1999) 19 (Figure 7.8). 20 OTHER INGREDIENTS From a practical point of view, real variations in 21 the collagen stability between meats from commer- Although meat and fat constitute most of the total 22 cial pigs of different ages are not relevant, nor that volume of the fermented products, other ingredients 23 occasioned by sex, because pigs are slaughtered are also crucial for their elaboration. In fact, from 24 when they are not old enough for these factors to ancient times these products were produced through 25 show any influence. However, there are large varia- adding sodium chloride to lean meat, and, thus, salt 26 tions in collagen content between muscles, leading is a key component for achieving the conservation 27 to great differences in texture. This in turn influences and sensory characteristics in fermented meat prod- 28 the texture and the quality of the final dry-fermented ucts. Only in the last years have some attempts been 29 30 31 32 33 Total collagen Insoluble collagen 34 35 20 36 37 38 15 39 a 40 41 10 42 b 43 b 44 45 5 46 47 c 48 moisture-free tissue) (fat-free, mg collagen/g muscle 0 49 0 100 200 300 400 500 50 Time (days) 51 52 Figure 7.8. Means (ϮSD) of total and insoluble collagen content in longissimus dorsi throughout lifetime of pigs 53S slaughtered at heavy weights.Means of the insoluble collagen with different letter differ significantly (P < 0.05) 54N (taken from Mayoral et al. 1999). 40454 07 059-076 r2.qxd 7/21/07 9:55 AM Page 69

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made to produce sausages with reduced salt content Salty taste is an essential taste note in all fer- 1 or even the total absence of this ingredient, but their mented meat products. Saltiness is sensory deﬁned 2 presence in the market is still scarce. Other common as the taste occasioned by sodium chloride. In fact, 3 ingredients are discussed latter in this book. Finally, the saltiness of other salts is not as pure as that of 4 some other ingredients, such as offal, blood, veg- sodium chloride, and this is a major drawback when 5 etable starch, or exogenous proteins are less usual in trying to reduce salt content in fermented meat prod- 6 common commercial sausages, but there are some ucts by using substitutes. For salt to produce a salty 7 regional varieties that may include them. taste in the tongue it should be dissolved in the 8 mouth. In dry-fermented products, chloride and 9 sodium ions are tightly bound to proteins, and prod- 10 WATER ucts with 5–6% salt do not show a deep salty taste; 11 Water is a common ingredient for most cooked meat with lower salt concentration, other fresh products 12 products because it allows obtaining an adequate show a stronger saltiness. The high concentration of 13 meat gel and increases yield. However, given that in a compound necessary for partial replacement of 14 fermented meat products there is a process of desic- NaCl gives rise in general to sensory problems. 15 cation, water addition has none of these goals, but A saturated sodium chloride solution in water 16 enables or makes possible a correct mixing of ingre- (more than 26.5 g/100 g solution) produces a water 17

dients or dissolving of some of them, such as exoge- activity (aw) of 0.753. Therefore, the addition of salt 18 nous proteins or starches. Thus, in some whole to fermented meat products decreases aw in the ﬁrst 19 fermented meat products, salting, seasoning, and steps of processing. Variation in the quantity of 20 additive addition is achieved through dissolving all water and in the salt/water ratio during fermentation 21 these ingredients in water forming a brine and and storage of fermented sausages has an important 22

immersing the product in it for several days. At any role on microbial multiplication. Decreased aw due 23 rate, water does not exert any other technological to salt addition controls microbial spoilage of fer- 24 function in fermented products, and, thus, any edible mented products in the ﬁrst steps of processing, 25 water can be used for the cited purposes. when other hurdles to microbial growth still are not 26 enough for limiting it. However, salt added to raw 27 fermented meat products is usually below 4% (w/v), 28 SALT so it just slightly contributes to the global decrease in 29

For ages, meat and salt have been the only two aw. Nevertheless, such a slight decrease is crucial in 30 strictly necessary ingredients for production of fer- fermented sausages stabilization because it takes 31 mented meat products. Sodium chloride shows a place in the beginning of the process, contributing 32 number of effects on meat products, most of them to the selection of a microbial population which will 33 desirable, which include imparting salty taste, the thereafter help stabilization through decreasing the 34 reduction of water activity and hence the control of pH. A derived effect of different microbial growth 35 microbial spoilage, the increase of water retention due to the different salt content is that concerning 36 ability, the facilitation of proteins to get dissolved, ﬂavor formation by microorganisms, especially 37 the reduction of the activity of some enzymes, and Staphylococcus (Tjener et al. 2004; Olesen et al. 38 the enhancement in the occurrence of oxidative 2004). Similarly, Gardini et al. (2001) observed 39 processes. lower biogenic amine production due to lower 40 The increasing occurrence of high blood pressure microbial counts in sausages with higher salt con- 41 and cardiovascular diseases in developed countries tent. In fact, with levels of 5% salt, presence of bio- 42 together with a trend to foods with a milder taste genic amines in the products was almost avoided. 43 during the last 50 years, have led to a considerable One of the problems when using a salt substi- 44 reduction in salt content in fermented meat products, tute in fermented meat products is that the initial 45

and even to the production of salt-free products in decrease in aw is not as intense: the aw of a saturated 46 the last decade, because sodium intake has been KCl solution is 0.843 (Greenspan 1977), and that of 47 related to high blood pressure (Frost et al. 1991). a saturated glycine solution is 0.921 (Na et al. 1994). 48 Such a reduction might produce technological Thus, selective growth of halotolerant microorgan- 49 and sensory problems because the relevance of NaCl isms, such as Lactobacillaceae or Microccocaceae 50 in solubilization and binding capacity of proteins, bacteria, could be impaired, and other bacteria can 51 preservation, and taste makes it especially difﬁ- reach higher counts (Gelabert et al. 2003). However, 52 cult to reduce its concentration in dry-fermented several authors have shown that with an appropriate S53 sausages. mixture of salts such a problem can be solved N54 40454 07 059-076 r2.qxd 7/21/07 9:55 AM Page 70

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1 (Gimeno et al. 2001a). Vanburik and Dekoos (1990) authors have reported that a high concentration of 2 noted the preservative qualities of the lactate ion, NaCl could inhibit lipolytic activity, showing that 3 and suggested that it may be possible to use potas- when the NaCl level decreases, the release of 4 sium lactate (K-lactate) as a substitute for salt, fatty acids increases (Stahnke 1995). On the other 5 although the abnormal taste encountered could limit hand, proteolytic activity seems to be inhibited 6 its use. Another approach has been the use of cal- by high levels of salt. Accordingly, Toldrá et al. 7 cium ascorbate for substituting sodium chloride, (1992) pointed out that the activity of cathepsins 8 with no detrimental effect on microbial counts and could be affected by different salt levels, cathep- 9 limited variations in instrumental measurements sin H and D activities being substantially inhib- 10 (Gimeno et al. 2001b), but these authors did not ited by salt. Thus, Ibañez et al. (1997) showed a 11 measure sensory traits. higher degradation of sarcoplasmic and myofibrilar 12 Muscle water retention ability is directly related to protein and higher nonprotein nitrogen content in 13 pH and the extent to which myofibrilar proteins are sausages with partial replacement of sodium chlo- 14 denatured. As commented before, at the isoelectrical ride with potassium chloride, most likely due to a 15 point of muscle proteins (around 5.0–5.2), water lower inhibition of the proteolytic activity (Figure 16 retention ability reaches the minimum. For a pH 7.9). Accordingly, Waade and Stahnke (1997) 17 value that can be naturally found in meat (above observed a negative influence of salt content on the 18 5.4), WHC is increased by the presence of sodium amount of several free amino acids in fermented 19 chloride, probably due to strong binding between the sausages. 20 chloride ion and positive charged groups of meat Several authors consider that sodium chloride acts 21 proteins, leading to electrostatic repulsion of pro- as a prooxidant in meat and meat products (Coutron- 22 teins, which in turn leads to a higher swelling of the Gambotti et al. 1999; Kanner et al. 1991), although 23 muscle structure, conducting to a higher water reten- other authors have not evidenced such an effect in a 24 tion (Durand 1999). long-processing dry-cured product (Andrés et al. 25 Myofibrilar protein solubility is directly related to 2004). Salt accelerates lipid oxidation, although the 26 the ionic strength of the solution. The presence of mechanism is not fully understood. Sodium chloride 27 sodium chloride increases ionic strength and hence may enhance the activity of oxidases (Lea 1937). 28 increases protein solubilization, leading to better Salt could modify the heme proteins catalysing lipid 29 gelling and emulsifying properties of meat proteins. oxidation (Love and Pearson 1974), or this lipid oxi- 30 This is especially important for the formation of a dation may be due to the action of the reactive chlo- 31 firm protein gel during subsequent acidification and ride ion on lipids. Osinchak et al. (1992) reported 32 dehydration processes undergone by meat fermented that a chloride ion may solubilize iron ions and stim- 33 products, leading to their final desirable texture. ulate the lipid peroxidation in liposomes. By con- 34 Myosin solubilization was good enough for meat trast, sodium chloride has been also considered as an 35 products processed at levels above 2% of NaCl and antioxidant (Chang and Watts 1950; Mabrouk and 36 extremely good when initial salt content was around Dugan 1960). 37 4%, although this would lead to very salty products 38 (Durand 1999). MEAT BY-PRODUCTS 39 In order to substitute salt in meat products without 40 affecting protein functionality, some authors have The use of animal by-products for production of fer- 41 proposed modifications in the processing, such as mented meat products is very limited. Nevertheless, 42 tumbling (Frye et al. 1986), emulsion coating (Thiel there are a number of regional varieties in which dif- 43 et al. 1986), or adding potassium sorbate (Sofos ferent offal, blood, or porcine skin are used as ingredi- 44 1986) or certain protein hydrolysates on a collagen ents. For example, in pepperoni, finely ground cooked 45 basis (Hofmann and Maggrander 1989), which com- porcine skin is added to the mixture to provide desir- 46 pensate for the loss of binding produced by a reduc- able textural characteristics. The procedure usually 47 tion of NaCl. used for this inclusion is to cook the pork skins to a 48 The activity of several muscle and microbial point in which they are somewhat translucent; subse- 49 enzymes involved in the development of the flavor in quently skins are drained and chilled, and then ground 50 fermented meat products can be affected by the con- to the desirable texture to be included in the finished 51 centration of sodium chloride. Thus, as far as lipases product and mixed in a proportion usually lower than 52 are concerned, Toldrá et al. (1997) showed that NaCl 10% (Rust 1988). Osburn et al. (1997) have studied 53S concentrations from 25 to 60 g/l activate the acid the use of connective tissue from the rind of pigs for 54N esterase but inhibit lysosomal acid lipase. Other reducing costs in bologna sausages. Visessanguan 40454 07 059-076 r2.qxd 7/21/07 9:55 AM Page 71

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40 1 Sarcoplasmic-control Sarcoplasmic-modified 2 Myofibrillar-control Myofibrillar-modified 3 NPN-control NPN-modified 4 30 5 6

–1 7 8 20 9 10

mg N NT 11 12 10 13 14 15 16 0 17 0 51015 20 25 Time (Days) 18 19 Figure 7.9. Evolution of sarcoplasmic, myofibrillar, and nonprotein nitrogen (NPN) throughout the ripening of 20 fermented sausages made with 3% NaCl (control) and with 1.5% NaCl ϩ 1.5% KCl (modified) (elaborated with 21 data from Ibáñez et al. 1997). 22 23 24 25 et al. (2005) found that a minced pork to cooked rind formation of a polysaccharide gel through heating in 26 ratio of 5:5 was the best for production of Nham, a the presence of water. However, the scientific infor- 27 Thai fermented pork sausage. mation regarding the effects of these ingredients on 28 Blood is used in some Spanish fermented sausages, the characteristics of the final product is scarce. 29 providing binding properties, due to its high protein Magra et al. (2006) claimed leek (Allium porrum) in 30 content, and some color, due to hemoglobin. These Greek fermented sausages is a source of nitrates as 31 types of sausages usually include some vegetables, well as a rich source of sulphur volatiles such as 32 such as cooked potatoes or pumpkin. Much less com- thiopropanal S-oxide, thiosulphinates, and related 33 mon is the use of offal, such as liver, heart, or lungs, compounds (zwiebelanes, capaenes) in minor quan- 34 for production of fermented sausages, although some tities, which participate in the flavor (Ferary and 35 minor varieties include them in their formula in dif- Auger 1996; Mondy et al. 2002). Moreover, flavonol 36 ferent proportions. As meat, these organs usually glycosides present in leek show antioxidant activity 37 show a high protein content. However, their protein (Fattorusso et al. 2001). In addition, allium plants 38 structure, protein functionality, final pH, or fat distri- also possess antifungal activity due to the chitinaces 39 bution, are considerably different from those of meat (Vergawen et al. 1998; Yin and Tsao 1999) and show 40 (except, perhaps, in the heart). high contents of inulin-type fructans with potential 41 effects on texture (Mendoza et al. 2001). 42 In the last decade there has been an increasing 43 VEGETABLES,FRUITS,BEANS, AND OTHER interest in plant ingredients—or at least part of their 44 PLANT INGREDIENTS components, such as protein, starch, or fiber—as fat 45 Only some fermented meat product specialities replacers and functional ingredients for meat prod- 46 include vegetables, fruits, or beans. In most cases, ucts. Thus, Garcia et al. (2002) reported dry-cured 47 these plant materials are boiled before added to the fermented sausages with similar physical and sen- 48 mixture of ingredients. These plant ingredients sory properties to commercial ones with reduced 49 include onion, garlic (which could be also consid- added fat (only 10%) and added fruit fiber (1.5%). 50 ered spices or condiments), potato, pumpkin, leeks, These authors claimed that using this procedure is 51 different types of nuts, and mushrooms. Their roles possible to obtain healthier fermented sausages. 52 in meat products are to impart a characteristic flavor Papavergou et al. (1999) used lupin flour and lupin S53 and achieve a distinguishing texture, attained by the protein isolate as ingredients in fermented sausages. N54 40454 07 059-076 r2.qxd 7/21/07 9:55 AM Page 72

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1 Although flour-added sausages showed very different to contribute to the overall taste of the product, coun- 2 properties to those of control sausages, the use of teracting salty taste (Prändl et al. 1988). 3 lupin protein isolate allowed the production of 4 sausages with similar properties as those of control 5 ones, and even lower lipid oxidation development. REFERENCES 6 Other plant (and animal) proteins used for fermented 7 meat products will be discussed later in this book. MD Aaslying. 2002. Quality indicators for raw meat. 8 The addition of different types of vegetable oils to In: J Kerry, J Kerry, D Ledward, eds. Meat Pro- 9 the mixture of ingredients in fermented sausages cessing: Improving Quality. Boca Raton, Florida: 10 production constitutes another approach to achieve a CRC Press LLC and Woodhead Publishing Ltd. 11 healthier fatty acid profile in this type of product Chapter 8. 12 (Muguerza et al. 2004). This procedure has been H Andersen, G Bertelsen, L Boegh-Soerensen, CK 13 used for ages in the production of some dry-cured Shek, L Skibsted. 1988. Effect of light and package 14 sausage varieties, but the amount of oil was rather conditions on the colour stability of sliced ham. Meat 15 low and the objective was not to modify the fatty Sci 22:283–292. 16 acid profile but to facilitate mixing and slow down AI Andrés, R Cava, J Ventanas, E Muriel, J Ruiz. 17 the subsequent dehydration process. Compared to 2004. Lipid oxidative changes throughout the ripen- 18 changing the fatty acid profile of pig lard by dietary ing of dry-cured Iberian hams with different salt 19 means, this procedure is more effective, is cheaper, contents and processing conditions. Food Chem 20 and does not imply the coordination between farm- 84:375–381. 21 ers and meat sausages producers. However, the phys- C Bejerholm, P Barton-Gade. 1986. 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partial replacement of NaCl with KCl and CaCl2 on teins. Meat Sci 46:277–284. 50 microbiological evolution of dry fermented sausages. JO Igene, AM Pearson. 1979. Role of phospho- 51 Food Microbiol 18:329–334. lipids and triglycerides in warmed-over ﬂavor devel- 52 ———. 2001b. Calcium ascorbate as a potential partial opment in meat model systems. J Food Sci S53 substitute for NaCl in dry fermented sausages: Effect 44:1285–1290. N54 40454 07 059-076 r2.qxd 7/21/07 9:55 AM Page 74

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1 J Kanner, S Harel, R Joffe. 1991. Lipid peroxidation of HS Na, A Arnold, AS Myerson. 1994. Cluster forma- 2 muscle food as affected by NaCl. J Agric Food Chem tion in highly supersaturated solution droplets. J 3 39:1017–1024. Cryst Growth 139:104–112. 4 CC Kuo, CY Chu. 2003. Quality characteristics of E Novelli, E Zanardi, GP Ghiretti, G Campanini, G 5 Chinese sausages made from PSE pork. Meat Sci Dazzi, G Madarena, R Chizzolini. 1998. Lipid and 6 64:441–449. Cholesterol Oxidation in Frozen Stored Pork, Salame 7 R Lawrie. 1995. The structure, composition and preser- Milano and Mortadella. Meat Sci 48:29–40. 8 vation of meat. In: G Campbell-Platt, PE Cook, eds. PT Olesen, AS Meyer, LH Stahnke. 2004. Generation of 9 Fermented Meats, London: Blackie Academic & flavour compounds in fermented sausages—The 10 Professional, pp. 39–52. influence of curing ingredients, Staphylococcus starter 11 CH Lea. 1937. TITULO. J Soc Chem Ind 56:376. culture and ripening time. Meat Sci 66:675–687. 12 F Leistner. 1992. The essential of production stable and WN Osburn, RW Mandigo, KM Eskridge. 1997. Pork 13 safe raw fermented sausages. In: FJM Smulders, skin connective tissue gel utilization in reduced-fat 14 F Toldrá, J Flores, M Prieto, eds. New Technologies bologna. J Food Sci 62:1176–1182. 15 for meat and meat products. Utrecht: ECCEAMST, JE Osinchak, HO Hultin, TZ Olver, S Kelleher, CH 16 pp. 1–18. Huang. 1992. Effect of NaCl on catalysis of lipid oxi- 17 JD Love, AM Pearson. 1974. Metmyoglobin and non- dation by the soluble fraction of fish muscle. Free 18 heme iron as pro-oxidants in cooked meat. J Agric Rad Biol Med 12:35–41. 19 Food Chem 22:1032–1034. SA Palumbo, M Komanowsky, V Metzger, JL Smith. 20 AF Mabrouk, LR Dugan. 1960. A kinetic study of the 1977. Kinetics of pepperoni drying. J Food Sci 21 autoxidation of methyl linoleate and linoleic acid 42:1029–1033. 22 emulsions in the presence of sodium chloride. J Am SN Papadimas, JG Bloukas. 1999. Effect of fat level 23 Oil Chem Soc 37:486–490. and storage conditions on quality characteristics of 24 TI Magra, G Bloukas, GA Fista. 2006. Effect of frozen traditional Greek sausages. Meat Sci 51: 103–113. 25 and dried leek on processing and quality characteris- EJ Papavergou, JG Bloukas, G Doxastakis. 1999. Effect 26 tics of Greek traditional sausages. Meat Sci of lupin seed proteins on quality characteristics of 27 72:280–287. fermented sausages. Meat Sci 52:421–427. 28 AI Mayoral, M Dorado, MT Guillén, A Robina, JM G Pastorelli, S Magni, R Rossi, E Pagliarini, P Baldini, 29 Vivo, C Vázquez, J Ruiz. 1999. Development of meat P Dirinck, F Van Opstaele, C Corino. 2003. Influence 30 and carcass quality characteristics in Iberian pigs of dietary fat on fatty acid composition and sensory 31 reared outdoors. Meat Sci 52:315–324. properties of dry-cured Parma ham. Meat Sci 32 E Mendoza, ML García, C Casas, MD Selgas. 2001. 65:571–580. 33 Inulin as fat substitute in low fat, dry fermented O Prändl, A Fischer, T Schmidhofer, HJ Sinell. 1988. 34 sausages. Meat Sci 57:387–393. Fleisch. Technologie und Hygiene der Gewinnung 35 AJ Miller, SA Ackerman, SA Palumbo. 1980. Effect of und Verarbeitung. Stuttgart: Ulmer. 36 frozen storage on functionality of meat for process- J Ruiz, C García, E Muriel, AI Andres, J Ventanas. 37 ing. J Food Sci 45:1466–1471. 2002. Influence on sensory characteristics on the 38 N Mondy, D Duplat, JP Christides, I Arnault, J Auger. acceptability of dry-cured ham. Meat Sci 61: 39 2002. Aroma analysis of fresh and preserved onions 247–354. 40 and leek by dual solid phase microextraction-liquid J Ruiz, CJ López-Bote. 2002. Improvement of dry- 41 extraction and gas chromatography-mass spectrome- cured ham quality by lipid modification through 42 try. J Chromatogr A 963:89–93. dietary means. In: F Toldrá, ed. Research Advances 43 DS Mottram. 1998. The chemistry of meat flavour. In: in the Quality of Meat and Meat Products. Kerala, 44 F Shahidi, ed. Flavor of Meat, Meat Products and India: Research Signpost, pp. 255–271. 45 Seafoods, London: Blackie Academic & Professional, J Ruiz, J Ventanas, R Cava, AI Andrés, C García. 2000. 46 pp. 5–26. Texture and appearance of dry-cured ham as affected 47 E Muguerza, O Gimeno, D Ansorena, I Astiasarán. by fat content and fatty acid composition. Food Res 48 2004. New formulations for healthier dry fermented Int 33:91–95. 49 sausages: A review. Trends Food Sci Technol J Ruiz, J Ventanas, R Cava, ML Timon, C García. 1998. 50 15:452–457. Sensory characteristics of Iberian ham: Influence of 51 E Muriel, J Ruiz, L Martin, MJ Petron, T Antequera. processing time and slice location. Food Res Int 52 2004. Physico-chemical and sensory characteristics 31:53–58. 53S of dry-cured loin from different Iberian pig lines. RE Rust. 1987. Sausage products. In: JF Price, BS 54N Food Sci Technol Int 10:117–123. Schweigert, eds. The Science of Meat and Meat 40454 07 059-076 r2.qxd 7/21/07 9:55 AM Page 75

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Products (3rd ed.) Westport, Connecticut: Food and fermented meat model system. Int J Food Microbiol 1 Nutrition Press, Inc., pp. 457–485. 97:31–42. 2 RE Rust. 1988. Formulated meat products using edible F Toldrá, M Flores, Y Sanz. 1997. Dry-cured ham 3 meat by-products. In: AM Pearson, TR Dutson, eds. flavour: Enzymatic generation and process influence. 4 Edible Meat By-products. Advances in Meat Food Chem 59:523–530. 5 Research 5. London: Elsevier Applied Science, F Toldrá, E Rico, J Flores. 1992. Activities of pork 6 pp. 341–357. muscle proteases in model cured meat systems. 7 JG Sebranek, VJH Sewalt, KL Robbins, TA Houser. Biochimie 74:291–296. 8 2005. Comparison of a natural rosemary extract and WE Townsend, CE Davis, CE Lyon, SE Mescher. 1980. 9 BHA/BHT for relative antioxidant effectiveness in Effect of pork quality on some chemical, physical, 10 pork sausage. Meat Sci 69:289–296. and processing properties of fermented dry sausage. 11 F Shahidi, J Yun, LJ Rubin, DF Wood. 1987. The hexa- J Food Sci 45:622–626. 12 nal content as an indicator of oxidative stability and I Valencia, D Ansorena, I Astiasarán. 2006. Nutritional 13 flavor acceptability in cooked ground pork. Can Inst and sensory properties of dry fermented sausages 14 Food Sci Technol 20:104–106. enriched with n-3 PUFAs. Meat Sci 72:727–733. 15 TJ Sims, AJ Bailey. 1981. Connective Tissue. In: AMC Vanburik, JT Dekoos. 1990. Sodium lactate in 16 R Lawrie, ed. Developments in Meat Science 2. meat products. Fleischwirtsch 70:1266–1268. 17 London: Applied Science Publishers, pp. 29–60. S Ventanas, M Estevez, JF Tejeda, J Ruiz. 2006. Protein 18 LH Skibstead. 1992. Cured meat products and their and lipid oxidation in Longissimus dorsi and dry 19 oxidative stability. In: DA Ledward, DE Johnston, cured loin from Iberian pigs as affected by cross- 20 MK Knight, eds. The Chemistry of Muscle-Based breeding and diet. Meat Sci 72:647–655. 21 Foods. 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Influence of minced pork and 31 ture on biochemical and sensory characteristics of rind ratios on physico-chemical and sensory quality 32 naturally fermented Turkish sausages (sucuk). Eur of Nham—A Thai fermented pork sausage. Meat Sci 33 Food Res Technol 221:412–415. 69:355–362. 34 A Soyer, AH Ertas, U Uzumcuoglu. 2005. Effect of pro- C Waade, LH Stahnke. 1997. Dried sausages fermented 35 cessing conditions on the quality of naturally fer- with Staphylococcus xylosus at different tempera- 36 mented Turkish sausages (sucuks). Meat Sci tures and with different ingredient levels. Part IV. 37 69(1):135–141. Amino acid profile. Meat Sci 46:101–114. 38 LH Stahnke. 1995. Dried sausages fermented with N Warnants, MJ Van Oeckel, CC Boucque. 1998. Effect 39 Staphylococcus-xylosus at different temperatures and of incorporation of dietary polyunsaturated fatty 40 with different ingredient levels 2. Volatile compo- acids in pork backfat on the quality of salami. Meat 41 nents. Meat Sci 41:193–209. Sci 49:435–445. 42 A Stiebing, D Kuhne, W Rodel. 1993. Fat qualityI—Its F Wirth. 1985. The technology of processing meat not 43 influence on the storage stability of firm dry sausage. of standard quality. Fleischwirtsch 66:998. 44 Fleischwirtsch 73:1169–1172. ———. 1988. Technologies for making fat-reduced 45 LF Thiel, PJ Bechtel, FK McKeith, J Novakofski, TR meat products. Fleischwirtsch 68:1153–1156. 46 Carr. 1986. Effect of salt reduction on the yield, J Wismer-Pedersen. 1987. Water. In: JF Price, BS 47 breaking force, and sensory characteristics of Schweigert, eds. The Science of Meat and Meat 48 emulsion-coated chunked and formed ham. J Food Products. Westport, Connecticut: Food & Nutrition 49 Sci 51:1439–1443. Press Inc., pp. 141–154. 50 K Tjener, LH Stahnke, L Andersen, J Martinussen. FW Wood. 1966. Diffusion of salt in pork muscle and 51 2004. The pH-unrelated influence of salt, tempera- fat tissue. J Sci Food Agric 17:138–140. 52 ture and manganese on aroma formation by Staphy- JD Wood. 1984. Fat deposition and the quality of S53 lococcus xylosus and Staphylococcus carnosus in a fat tissue in meat animals. In: J Wiseman, ed. Fats N54 40454 07 059-076 r2.qxd 7/21/07 9:55 AM Page 76

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1 in Animal Nutrition. London: Butterworths, pp. MC Yin, SM Tsao. 1999. Inhibitory effect of seven 2 407–435. Allium plants upon three Aspergillus species. Int J 3 YL Xiong. 2000. Protein oxidation and implications for Microbiol 49:49–56. 4 muscle foods quality. In: EA Decker, C Faustman, CJ GH Zhou, A Yang, RK Tume. 1993. A relationship 5 López-Bote, eds. Antioxidants in Muscle Foods. between bovine fat color and fatty-acid composition. 6 New York: Wiley, pp. 85–111. Meat Sci 35:205–212. 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53S 54N 40454 08 077-086 r1.qxd 7/20/07 12:37 PM Page 77

1 2 3 8 4 5 6 Additives 7 8 Pedro Roncalés 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION properly as additives, such as nonmeat proteins, sug- 22 ars, hydrolysates, and smoke extracts, are also used 23 Dry-cured and/or fermented sausages are traditional depending upon particular regulations. This chapter 24 meat products that may be stored even at ambient deals with the brief description of usage, advantages, 25 temperatures without risk of spoilage. This means and shortcomings of the most commonly used addi- 26 that the use of additives either for manufacturing pur- tives and supplementary ingredients in the manu- 27 poses or for preservation is not necessary. In fact, a facturing of dry-cured and/or fermented sausages. 28 high percentage of sausages made following tradi- Sugars are addressed in Chapter 7, which deals with 29 tional procedures do not contain any additives. How- ingredients. For further information on the use and 30 ever, a larger part of commercial dry-cured or properties of additives, see Ash and Ash (2002), 31 fermented sausages do include a variable number of Martín-Bejarano (2001), Ranken (2000), Saltmarsh 32 additives in their formulation. Why is this? Manufac- (2000), and Toldrá (2002). 33 turers pursue one or more of the following advan- 34 tages with the use of additives: (1) to facilitate, 35 enhance, accelerate or standardize the manufacturing ACIDS AND RELATED ADDITIVES 36 processes; (2) to ensure product safety regarding 37 microbial toxins; (3) to avoid the presence of undesir- Acidification is common in most dry/fermented 38 able surface molds; (4) to meet consumer require- sausages; indeed, they usually have pH values within 39 ments regarding sensory attributes; and (5) to increase the range 4.5–5.5. Reasons underlying its conve- 40 the product yield. Only the latter purpose may be nience are many and varied: It selects the microbiota, 41 associated with low-quality products, but this is not inhibiting pathogens and undesirable nonpatho- 42 usually a preferential aim in this type of sausages. genic microorganisms; it favors the emulsifying and 43 In accordance with the statements of the previous gelling ability of meat proteins, enhancing particle 44 paragraph, most legal regulations concerning the use binding and soft texture perception; it facilitates the 45 of additives in dry-cured and fermented sausages are formation of curing pigments; it enhances proteolysis 46 rather restrictive; the number of permitted items is and lipolysis involved in maturation; and it provides a 47 normally low. An example of this is provided by light acid taste. In summary, acidification greatly 48 European Union regulations (EPC 1995), which helps to conduct and accelerate the whole process, as 49 allow the use of a few acids, antioxidants, colorants, well as to achieve product quality and safety. A con- 50 flavor enhancers, and preservatives, as well as the venient acidification may be obtained following 51 multipurpose phosphates. It appears that such a sugar fermentation by lactic acid bacteria (LAB), 52 short additive list fulfills the requirements of both either naturally present in the meat or added as starter 53 industrials and consumers. In addition, a few nontra- cultures to the ingredients mixture (see Part III). This S54 ditional supplementary ingredients not considered is the way chiefly used for acidifying sausages by N55 77 40454 08 077-086 r1.qxd 7/20/07 12:37 PM Page 78

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1 Table 8.1. Acids and related additives: CAS (Chemical Abstract Service) and EU numbers, and common 2 concentration range added to sausage formulations. 3 Common 4 Additive Molecule/Form CAS Number EU Number Concentration Range 5 6 Lactic acid 50-21-5 E270 0.5–3 g/100 g 7 L- 79-33-4 8 D- 10326-41-7 9 DL- 598-82-3 10 sodium salt 72-17-3 E325 11 potassium salt 996-31-6 E326 12 Citric acid Anhydrous 77-92-9 E330 0.5–3 g/100 g 13 monohydrate 5949-29-1 14 15 Glucono-delta-lactone 90-80-2 E575 0.5–2 g/100 g 16 17 18 19 20 21 both traditional and large industries. Nevertheless, in no other limitation to the concentration added than 22 many cases, particularly when no starter cultures are good manufacturing practices. 23 used, direct addition of acids is preferred in order to Problems regarding the addition of acids, or 24 assure pH lowering within a very short time, i.e., as even GDL, have been related to the high speed of 25 initial value. acidification, which may result in early dehydration, 26 Common organic acids are used for this purpose, particularly within the outer part of the sausage mass. 27 mainly lactic and citric acids, as well as their sodium This early drying will cause a rapid mass shrinkage 28 and potassium salts, which show much less ability and, as a consequence, separation and wrinkling of 29 to lower pH values. Besides these, an acid-related the casing, together with undesirable crust forma- 30 molecule may also be used: glucono-delta-lactone tion. Therefore, strict control of temperature and rel- 31 (GDL). GDL is the cyclic 1,5-intramolecular ester of ative humidity is needed in order to avoid these 32 D-gluconic acid. In aqueous media, it is hydrolyzed shortcomings. 33 to an equilibrium mixture of D-gluconic acid 34 (55–66%) and the delta- and gamma-lactones. ANTIOXIDANTS 35 Therefore, GDL was envisaged as a slower acid- 36 releasing agent, with a view to mimicking gradual Oxidative reactions may be deleterious for dry/ 37 acidification by LAB fermentation. fermented sausages; in fact, intense oxidation leads 38 Table 8.1 gives the Chemical Abstract Service to rancidity development. However, most synthetic 39 (CAS) (JECFA 2006) and EU (EPC 1995) numbers antioxidant additives are not permitted by legal reg- 40 of molecules most commonly used as acidification ulations, so rancidity becomes usually the shelf-life 41 additives, as well as the common concentration range limiter of many meat products. Nevertheless, a lim- 42 added to sausage formulations. Lowering of pH by ited number of “soft” antioxidants are allowed by 43 added acids depends chiefly on the concentration most regulations. The reason for this is that sausage 44 used, but the presence of other molecules that may curing, particularly concerning nitrate/nitrite action, 45 act as buffering agents, such as phosphates, must cannot be achieved under prevalent oxidant condi- 46 also be taken into account because they decrease tions; well on the contrary, reducing conditions are 47 their acidification ability. Lactic acid concentrations needed for color formation and antimicrobial activ- 48 of 1–3% are used for a pH lowering of 0.5–1.5 units. ity. Accordingly, the permitted “soft” antioxidants find 49 Sodium and potassium salts of lactate cause only a their use rather as reductant agents than as proper 50 minimum pH decrease; nevertheless, addition of antioxidants. 51 those salts has been described to be very effective in In agreement with this view, the antioxidants most 52 microbial inhibition (Shelef 1994). Most legal regu- commonly used include only ascorbic acid (vitamin C) 53S lations allow the addition of all those additives on and tocopherols (vitamin E) and some related mole- 54N the basis of the quantum satis principle, i.e., there is cules, such as erythorbic acid, as well as their salts. 40454 08 077-086 r1.qxd 7/20/07 12:37 PM Page 79

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Table 8.2. Antioxidant additives: CAS (Chemical Abstract Service) and EU numbers, and common concentra- 1 tion range added to sausage formulations. 2 3 Common 4 Additive Molecule/Form CAS Number EU Number Concentration Range 5 Ascorbic acid 50-81-7 E300 Quantum satis 6 Sodium salt 134-03-2 E301 ϳ500 mg/kg 7 Calcium salt 5743-27-1 E302 8 Fatty acid esters 25395-66-8 E304 9 10 Ͻ Erythorbic acid 89-65-9 E315 500 mg/kg 11 Sodium salt 6381-77-7 E316 12 Tocopherols Extract mix 59-02-9 E306 Quantum satis 13 Alpha- 1406-18-4 E307 ϳ500 mg/kg 14 Gamma- E308 15 Delta- E309 16 Extracts of a variety Phenols Quantum satis 17 of herbs and spices Highly variable 18 19 20 21 22 All these molecules have been proven to exert an both types of reductants in order to assure both ini- 23 effective steady reducing effect while they suffer tial and midterm antioxidant effects. Besides this, 24 oxidation themselves. Some fatty acid esters of seasoning with herbs and spices, either raw or in the 25 ascorbic acid (palmitate and stearate) may also be form of extracts, also contributes to a midterm 26 considered as reductant additives, particularly for antioxidant effect. 27 midterm reducing ability. Furthermore, the fact that No deleterious or negative effects have been 28 many spices and condiments possess antioxidant described following the addition of those reductants 29 properties must not be overlooked, because they within the common concentration range. Conversely, 30 contribute to the oxidative stability of meat products, almost all formulations for industrial manufacturing 31 too (Sánchez-Escalante et al. 2003; Martínez et al. of dry/fermented sausages include one or more of 32 2006a). Phenolic constituents present in those sea- those additives (Balev et al. 2005). 33 sonings protect them with varying intensity against 34 oxidative deterioration. There is the opportunity, too, COLORANTS 35 of using a variety of industrial extracts of those herbs 36 and spices (rosemary, oregano, tea, citrus fruits, etc.) Color is by far the most important sensory attribute in 37 as antioxidant additives; furthermore, they are not relation to the visual appearance and acceptability of 38 considered as proper additives by legal regulations. most food items. Dry/fermented sausages are not an 39 Table 8.2 gives the CAS (JECFA 2006) and EU exception to this; appealing red colors of cured meat 40 (EPC 1995) numbers of molecules most commonly strongly determine product acceptance and purchase 41 used as antioxidant additives, as well as the common decision. Appropriate nitrate and/or nitrite curing 42 concentration range added to sausage formulations. provides a rather stable nitroso-compound of myo- 43 As in the case of acids, those antioxidants are allowed globin, but the stability of this pigment is not guaran- 44 on the basis of “the quantum satis” principle, with teed for long periods of time if prooxidant conditions 45 the exceptions of erythorbic acid and sodium ery- are prevailing. Addition of colorants may afford a 46 thorbate, which are limited to 500 mg/kg. In any convenient solution to this problem. Seasoning with 47 case, this amount appears to be the average of those red pepper powder has been traditional in many 48 used ordinarily. Some of the antioxidants are more countries (Spain, Hungary, Mexico, etc.), not only 49 indicated for steady midterm reducing ability, partic- because of its desired ﬂavor, but also because it 50 ularly the fatty acid esters of ascorbic acid and the strongly helps to avoid sausage discoloration. A huge 51 tocopherols; formulations with ascorbic acid and its number of natural and artiﬁcial colorants have been 52 salts are designed to provide mainly a reliable and proposed for use in food coloring, although only a S53 rapid reducing capacity. Many formulations include few of them were approved in legal regulations. N54 40454 08 077-086 r1.qxd 7/20/07 12:37 PM Page 80

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1 Table 8.3. Colorant additives: CAS (Chemical Abstract Service) and EU numbers, and common concentration 2 range added to sausage formulations. 3 Common 4 Additive Molecule/Form CAS Number EU Number Concentration Range 5 6 Cochineal extract Extract powder 1343-78-8 E120 Ͻ200 mg/kg 7 Carminic acid 1260-17-9 8 Paprika extract Oleoresin 68917-78-2 E160c Ͻ10 mg/kg 9 10 Ponceau 4R Trisodium-2-hydroxy- 2611-82-7 E124 Ͻ250 mg/kg 11 (cochineal red) 1-(4-sulfonato-1-naphthylazo)- 12 6,8-naphthalenedisulfonate 13 Beet red Betanine 7659-95-2 E162 Quantum satis 14 Ͻ200mg/kg 15 16 17 18 In the case of dry/fermented sausages, the variety of establish, with a few exceptions, maximum concen- 19 useful colorants is rather short; in fact, only those trations (see Table 8.3). Among the colorants consid- 20 mimicking the red color of cured-meat pigment ered for dry/fermented sausages, only beet red is 21 should be considered, despite the fact that some yel- allowed without any limit other than good practices. 22 low hues or caramels are also useful, but only in spe- The use of natural colorants is usually preferred for 23 cial products. traditional sausage manufacture, although there 24 Only a few red colorants are commonly used in appear to be no perceptible differences in the final 25 dry/fermented sausage manufacture. These include products; all of them provide a stable red color of 26 both natural and artificial. Cochineal and paprika convenient appeal for consumers quite similar to that 27 extracts are from natural origin. Cochineal extract is of cured meat. Taking into account that the price of 28 the powder of dried cochineals (Dactylopius coc- artificial colorants is much lower, manufacturers pre- 29 cus), an insect growing on nopal cactuses (Opuntia fer its use in formulations designed for mid-price 30 ficus indica). Mexico and Spain are large producers sausages; natural colorants are used mainly in tradi- 31 of this dye, well known and appreciated for its tional and high-price sausages. Paprika extract seems 32 extremely high content of intense-red carmine (or to be more appropriate for use only in paprika- 33 carminic acid) in America since the pre-Hispanic sausages because its addition results in an orange- 34 times. Paprika extract is the oleoresin of any of the red color typical of this meat products. 35 varieties of red pepper (Capsicum annuum), also of No shortcomings derived from the use of colorants 36 American origin, which are currently grown in large have been described. Nevertheless, it is recommended 37 amounts all over the world. This extract is a rich that they be used at rather low concentrations in order 38 source of red colorants capsorubin and capsanthin, to provide a natural appearance, as similar as possible 39 together with capsaicin, and some varieties may also to colorant-free, nitrite-cured sausages. 40 contain hot-pungent capsaicinoids. Cochineal red 41 (Ponceau 4R) and beet red (betanine) are from artifi- EMULSIFIERS 42 cial origin. Ponceau 4R is an industry-synthesized 43 carmine, a closely related molecule to the red col- Dry/fermented sausages, unlike most cooked 44 orant of cochineal. Its color is quite similar to that of sausages, are not essentially made of an emulsion 45 cochineal extract, but its price is significantly lower. but of meat and fat fragments of varying sizes within 46 Betanine is also an industry-synthesized colorant, the range of, approximately, 2 to 20 mm. However, 47 which is known as a major constituent of red beet a certain amount of emulsion is needed for binding 48 root (Beta vulgaris) (Martínez et al. 2006b). and tightening those pieces into forming a proper 49 Table 8.3 gives the CAS (JECFA 2006) and EU mass. The emulsifying ability is provided by meat 50 (EPC 1994) numbers of molecules or extracts most proteins, especially myosin, which are first extracted 51 commonly used as colorant additives, as well as the from the tissue and solubilized by added salt; follow- 52 common concentration range added to sausage for- ing mechanical work, they form thereafter an aque- 53S mulations. All the colorants normally have limita- ous phase able to emulsify droplets of solid fat. The 54N tions for their use; as an example, EU regulations emulsion formed is stabilized by slow gellation of 40454 08 077-086 r1.qxd 7/20/07 12:37 PM Page 81

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Table 8.4. Emulsifier nonmeat proteins: CAS (Chemical Abstract Service) and EU numbers, and common 1 concentration range added to sausage formulations. 2 3 Common 4 Additive Molecule/Form CAS Number EU Number Concentration range 5 Milk proteins Defatted milk powder 5–30 g/kg 6 Whey proteins 9005-46-3 7 Caseinates (sodium) 8 9 Blood proteins Red blood cells powder 5–30 g/kg 10 Serum powder 11 Soja proteins Soja meal 5–30 g/kg 12 Soja proteins 13 Cereal proteins Wheat proteins 5–30 g/kg 14 Corn proteins 15 16 17 proteins throughout the sausage-drying process. they are also added frequently with the aim of 18 This imperfect gel fills all the spaces among meat increasing the product yield by embedding meat and 19 and fat chunks, giving rise to a sliceable mechani- fat chunks in a large amount of a protein-water 20 cally homogeneous mass. Naturally, the higher the matrix. In any case, addition of nonmeat proteins is 21 amount of soluble protein, the larger the volume of be related to the manufacturing of medium- to low- 22 interchunk matrix. quality products. 23 There exists a variety of emulsifiers of very differ- The preferred nonmeat proteins are those having a 24 ent origins available for their use in foods and particu- higher emulsifying ability, which are mainly of ani- 25 larly in meat products. Among them, only proteins are mal origin, particularly caseinates. Nevertheless, the 26 of interest for manufacturing dry/fermented sausages; price of those proteins is rather high, and the higher 27 the rest find their use chiefly in cooked meat emul- the degree of protein isolation, the higher the price. 28 sions. Emulsifying nonmeat proteins may proceed This is why many industrials are using defatted milk 29 from a large number of animal and vegetal sources. powder; actually, it is used not only because of its 30 Major animal sources are milk and blood, either raw low price but also because milk powder contains lac- 31 preparations or isolated proteins or protein groups. tose, a sugar that may serve both as a selective sub- 32 They possess an emulsifying ability close to that of strate for lactic acid bacteria and as a fairly good 33 meat proteins, so they are well liked by industrials. humectant. All proteins from vegetal sources have a 34 Major vegetal sources are soja and cereals, either raw medium emulsifying ability, but their low prices are 35 preparations or isolated protein groups. They also highly convenient for industrials. Protein concen- 36 possess a good emulsifying ability, although it is trates are a better choice than raw meals because the 37 lower than that of animal proteins (Stiebing 1998). latter may confer extraneous flavors to sausages. 38 Table 8.4 points out that most emulsifier nonmeat Furthermore, new protein preparations are regularly 39 proteins are natural products containing a variable appearing in the additive market. Addition of non- 40 number of proteins and even other nonrelated mole- meat proteins must be based on sausage quality cri- 41 cules, as well as that they are not considered as addi- teria; therefore, the concentrations used must remain 42 tives by EU and many other regulations. Their use is low in any case, ranging between 0.5% and 3%. No 43 normally regulated in local dispositions for the shortcomings other than quality lowering have been 44 diverse meat products, often varying according to described. 45 product quality categories. In a strict sense, added 46 proteins are not at all necessary for dry/fermented FLAVOR ENHANCERS 47 sausage manufacture; meat proteins should be enough 48 for forming the small amount of emulsion required Flavor is the most appreciated sensory attribute of 49 for sausage sliceability. However, small amounts of foods, and this is particularly true regarding meat 50 exogenous proteins may be sometimes convenient products. Consumers expect dry/fermented sausages 51 for assuring rapid emulsion formation and early sta- to be very savory. Normally, this is achieved by 52 bilization, as well as for achieving a homogeneous traditional seasonings and appropriate processing; S53 and soft texture of the sausage even after drying. But flavor of dry/fermented sausages is the result of the N54 40454 08 077-086 r1.qxd 7/20/07 12:37 PM Page 82

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1 interaction of spice and condiment flavors and those many derivatives of guanylic acid, inosinic acid, and a 2 generated by the fermentation-curing process of variety of ribonucleotides. So, guanylates, inosinates, 3 meat and fat ingredients. On many occasions, how- and ribonucleotides greatly broadened the spectrum 4 ever, one or more of the following problems occur: of available flavor enhancers. 5 seasoning is poor, ingredients are not of the best qual- Table 8.5 gives the CAS (JECFA 2006) and EU 6 ity, or the fermenting-curing process is not sufficiently (EPC 1995) numbers of molecules most commonly 7 intense. Consumers also may demand extremely used as flavor enhancers, as well as the common con- 8 savory products, hardly achieved by traditional manu- centration range added to sausage formulations. 9 facturing. In all those cases, industrials may solve the Glutamic acid and its salts, particularly monosodium 10 problem, at least in part, by using flavor enhancers. glutamate (MSG), have been used for many years as 11 These additives, very well known in oriental cuisines, exclusive flavor enhancers in meat products. Results 12 have the ability of making more intense basic flavors have been good, but criticism arose regarding the fla- 13 of meat, meat products, and other foods of animal vor homogeneity brought about in quite different 14 origin. Most researchers acknowledge those mole- meat products. Currently, the large number of avail- 15 cules as actors of the perception of a particular taste able flavor enhancers helps overcome this problem; 16 known by its Japanese name umami. the diverse molecules contribute with fairly different 17 Glutamic acid is the best known flavor enhancer; flavors to overall sausage flavor. So, flavor spectra 18 this common amino acid, as well as some of its salts, have increased, especially with the use of mixtures 19 has been used for this purpose for many years and it of variable but small amounts of some of the avail- 20 is a principal ingredient in oriental cooking practices able flavor enhancers. The added concentrations of 21 and the oriental food industry. Now it is a highly used all the additives included in this group are normally 22 food additive all over the world. More recent devel- limited by most legal regulations, but they are high 23 opments in food additive research demonstrated that enough to achieve intense flavors. In fact, they are 24 a number of compounds very active in biological very commonly used at low concentrations in a 25 systems, such as nucleosides and nucleotides, also variety of medium-quality dry/fermented sausages. 26 had the ability to be perceived as umami by taste No shortcomings other than flavor homogeneity 27 receptors, i.e., to be flavor enhancers. These include have been so far described. 28 29 30 Table 8.5. Flavor enhancers: CAS (Chemical Abstract Service) and EU numbers, and common concentration 31 range added to sausage formulations. 32 33 Common 34 Additive Molecule/Form CAS Number EU Number Concentration Range 35 Glutamic acid 56-86-0 E620 36 Monosodium salt 142-47-2 E621 Ͻ10 g/kg 37 Monopotassium salt 19473-49-5 E622 38 Calcium diglutamate 19238-49-4 E623 39 Magnesium diglutamate 18543-68-5 E625 40 Monoammonium glutamate 7558-63-6 E624 41 42 Guanylic acid 85-32-5 E626 Ͻ500 mg/kg 43 Calcium salt 38966-30-2 E629 44 Dipotassium salt 3254-39-5 E628 45 Disodium salt 5550-12-9 E627 46 Inosinic acid 131-99-7 E630 Ͻ500 mg/kg 47 Calcium salt 38966-29-9 E633 48 Dipotassium salt 20262-26-4 E632 49 Disodium salt 4691-65-0 E631 50 Ribonucleotides Mixtures of calcium E634 Ͻ500 mg/kg 51 or disodium salts E635 52 of guanylate and 53S inosinate 54N 40454 08 077-086 r1.qxd 7/20/07 12:37 PM Page 83

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FLAVORING AGENTS exception of liquid smoke. This extract contains a 1 large number of molecules formed through wood 2 The convenience of using flavor enhancers has been combustion. Because uncontrolled combustion may 3 discussed in the previous section. The use of flavor- lead to formation of carcinogenic benzopyrenes, 4 ing agents is partly related to the same arguments, the smoke extracts must be prepared under conditions 5 need of fulfilling consumer demands on intense fla- avoiding the presence of those molecules, especially 6 vor. This is the case for protein or yeast hydrolysates, by using low-temperature combustion. 7 which might be considered as either flavor enhancers 8 or flavoring agents. These hydrolysates are very com- 9 plex mixtures of amino acids and different-sized pep- PRESERVATIVES 10 tides in the first case, and of amino acids, peptides, 11 Dry/fermented sausages are the result of processing nucleotides, nucleosides and many other biologically 12 meat by using technologies that ensure a convenient active compounds in the second one. But, besides 13 preservation and safety from a microbial perspective. this, there are specific flavor deficiencies that can be 14 In agreement with this view, legal regulations are very overcome by using particular flavoring agents; those 15 restrictive in preservative additives allowance. Only agents are mainly the following: replacing raw prepa- 16 two groups of preservatives are usually considered for rations of spices and condiments with their extracts 17 use: 1) those inhibiting specifically toxin-producing, or replacing natural smoking with smoke extracts. 18 spore-former microbes; and 2) those inhibiting sur- So, both herb and spice extracts, chiefly oleoresins, 19 face molds and yeasts. The former have been used and liquid smoke preparations are to be included in 20 traditionally for meat curing; they are highly recog- this additive group. 21 nized for their ability to effectively avoid risks associ- Table 8.6 points out that most flavoring agents are 22 ated to microbial toxins produced by Clostridium industrial preparations of natural products contain- 23 and Bacillus. In addition, they are essential substrates ing a highly variable number of many different mol- 24 for the development of cured-meat color and fla- ecules, as well as that they are not considered as 25 vors (see Chapter 3). Regarding mold and yeast additives by EU and many other regulations, with 26 inhibitors, their use must be considered if growth of the exception of paprika oleoresin. Their use is 27 those organisms on sausage surface is not desired, sometimes regulated in local dispositions for the 28 particularly when sausages are not subject to smoking. diverse meat products, often varying according to 29 It is well known that smoke components have an product quality categories, but they are more fre- 30 intense fungistatic effect, but many dry/fermented quently considered as mere ingredients within the 31 sausages, especially in Southern Europe, are not group of seasonings. In summary, regulations on fla- 32 smoked. vorings are rather confusing. In any case, all of them 33 Nitrates and nitrites are the only integrants of the confer high-intensity flavors, together with color in 34 first group of antimicrobials. These inorganic salts, the case of paprika, smoke, and other extracts, so 35 especially nitrates, are present as contaminants in that the concentrations added are subjected to 36 nonpurified crystals of common salt, sodium chlo- achieving the desired level of flavor and, therefore, 37 ride. They have been present in sausage formulations to good industrial practices. No shortcomings fol- 38 in this form since ancient times, giving rise to the lowing their use have been described, with the 39 40 41 42 43 Table 8.6. Flavoring agents: CAS (Chemical Abstract Service) and EU numbers, and common concentration range added to sausage formulations. 44 45 Common 46 Additive Molecule/Form CAS Number EU Number Concentration Range 47 48 Ͻ Herbs and spices Paprika oleoresin 68917-78-2 E160c 10 mg/kg 49 extracts Many other oleoresins 50 Many other extracts 51 Smoke extracts Liquid smoke Highly variable 52 Hydrolysates Protein Yeast Ͻ1000 mg/kg S53 N54 40454 08 077-086 r1.qxd 7/20/07 12:37 PM Page 84

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1 desired, though involuntary, curing. Modern scien- beneficial effects; but maximum concentrations are 2 tific evidence led to current knowledge on the continuously subject to revision, with a steady trend 3 involvement of nitrates/nitrites in curing chemistry to being diminished. Usually, unlike most other addi- 4 and resulted in the use of those salts as technological tives, both an indicative added concentration and a 5 additives (see Chapter 3). residual maximum concentration are established. 6 Fungistatic additives for surface treatment of This is due to the well-known fact that both nitrates 7 sausages include a variety of molecules. Most of them and nitrites are very reactive. In fact, nitrate is trans- 8 are organic acids such as sorbic, benzoic, and para- formed into nitrite under reducing conditions and 9 hydroxybenzoic acids, as well as their salts. These had nitrite, in turn, may form a variety of molecules. 10 been used in meat products as broad-spectrum antimi- Some of them will react to produce nitroso-myoglobin 11 crobials, and they still have this use in many other if reducing conditions prevail, together with many 12 foods. But they have been demonstrated to be highly other nitroso-compounds, including those exerting 13 effective fungistatic agents, too; so this is their only the antimicrobial effect (Cassens 1990). Therefore, 14 use in dry/fermented sausages according to many legal both nitrates and nitrites may be used as curing/pre- 15 regulations, in which they are allowed to be used only servative agents. Nitrates must be added at higher 16 on the sausage surface. Two other fungistatic agents concentrations and their action is delayed throughout 17 come usually into consideration: nisin and natamicin. curing; nitrites must be added at lower concentrations 18 They are both antibiotics of microbial origin and they and their effect is achieved earlier. So, industrials 19 are very active against molds and yeasts. prefer nitrates for long-curing sausages and nitrites 20 Table 8.7 gives the CAS (JECFA 2006) and EU for short-curing ones, but the use of mixtures of both 21 (EPC 1995) numbers of molecules most commonly salts is common, too. 22 used as preservatives, as well as the common concen- Regarding fungistatic preservatives, there is a large 23 tration range added to sausage formulations. The use number of available molecules, but not all of them 24 of nitrates and nitrites is highly controversial. They have the same interest in dry/fermented sausages. 25 have been demonstrated to be related to some forms They are all highly effective against mold and yeast 26 of cancer, particularly to stomach tumors, due to the growth, but they do not have the same pH optimum 27 formation of nitrosamines (see Chapter 43). How- for activity. P-hydroxybenzoates (parabens) are more 28 ever, all legal regulations allow use of nitrates and efficient at higher pH values, i.e., at 6.0 or above; sor- 29 nitrites in meat products on the basis of their highly bates are more active at relatively acidic pH values, 30 31 32 Table 8.7. Preservatives: CAS (Chemical Abstract Service) and EU numbers, and common concentration 33 range added to sausage formulations. 34 Common 35 Additive Molecule/Form CAS Number EU Number Concentration Range 36 37 Nitrates/nitrites Potassium nitrate 7757-79-1 E252 250 (300) mg/kg* 38 Sodium nitrate 7631-99-4 E251 250 (300) mg/kg* 39 Potassium nitrite 7758-09-0 E249 50 (150) mg/kg* 40 Sodium nitrite 7632-00-0 E250 50 (150) mg/kg* 41 Sorbic acid 110-44-1 E200 Quantum 42 Potassium salt 24634-61-5 E202 satis 43 Calcium salt 7492-55-9 E203 (surface) 44 45 Benzoic acid 65-85-10 E210 Quantum 46 Potassium salt 582-25-2 E212 satis 47 Sodium salt 532-32-1 E211 (surface) 48 Calcium salt E213 49 P-hydroxybenzoates Ethyl-PHB 120-47-8 E214 Quantum 50 Methyl-PHB 99-76-3 E218 satis 51 Propyl-PHB 94-13-3 E216 (surface) 52 Natamicin Pimaricin 7681-93-8 E235 Ͻ1 mg/dm2 53S 54N *Figures in parentheses are recommended maximum added concentrations. 40454 08 077-086 r1.qxd 7/20/07 12:37 PM Page 85

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i.e., at about 5.0, and benzoates are more active at meat products, in which they perform a valuable 1 lower pH values (Calvo 1991). Therefore, sorbates enhancing effect on water-holding capacity, but also 2 are more appropriate for their use in most dry/ in dry/fermented sausage manufacturing. Why is 3 fermented sausages. The antibiotic natamicin is also this? The answer is easily understandable: Addition 4 active within the usual pH range of these sausages. of phosphates results in a better binding of meat and 5 Because all these preservatives are mainly used on fat particles, a more equilibrated drying, a longer 6 the sausage surface (this is compulsory in EU regula- shelf life, a smoother texture, and a higher juiciness. 7 tion), they are not added to the mass mix; they are So, processing and product quality benefits appear to 8 applied by immersion in—or nebulization with— be doubtless. Then, this is the question: Are they nec- 9 a concentrated preservative solution. essary? The answer is not so easily understandable, 10 but it is simple: No, they are not necessary. All those 11 apparent benefits may be achieved without adding 12 MULTIPURPOSE ADDITIVES: phosphates by carefully optimizing every phase of 13 PHOSPHATES the manufacturing process. In conclusion, working 14 without phosphates is hard work; but the ingredients 15 Some additives are not easy to classify according to label will be free of an extra additive. 16 their possible uses and benefits as additives in meat The number of available phosphate molecules is 17 products; this is the case with phosphates. They may high. All are inorganic salts of single, double, triple, 18 have activity as thickeners, stabilizers, humectants, or multiple phosphate anion groups. They can be 19 gelling agents, or even bulking agents; in fact, their classified in mono (orto-), di- (pyro-), tri- and poly- 20 function includes all of these features. Accordingly, phosphates. All of them have the ability to bind 21 they may be qualified as multipurpose additives. Phos- strongly to the outer parts of proteins, causing a sort 22 phates are considered convenient additives by many of “swelling” of the protein matrix, which results 23 industrials, not only for the manufacturing of cooked in an enhancement of the beneficial actions of meat 24 25 26 Table 8.8. Multipurpose phosphates: CAS (Chemical Abstract Service) and EU numbers, and common 27 concentration range added to sausage formulations. 28 29 Common 30 Additive Molecule/Form CAS Number EU Number Concentration Range 31 32 Monophosphates Sodium salts 7558-80-7 E339 (i, ii, iii) Ͻ5 g/kg 33 (mono, di and tri) 7601-54-9 usually ca.1 g/kg 34 Potassium salts 7778-77-0 E340 (i, ii, iii) (total phosphate) 35 (mono, di and tri) 7778-53-2 36 Calcium salts 7757-93-9 E341 (i, ii, iii) 37 (mono, di and tri) E343 (i, ii) 38 Magnesium salts 7757-86-0 39 (mono and di) 40 Diphosphates Sodium salts 7758-16-9 E450 (i-vii) Ͻ5 g/kg 41 (di, tri and tetra) 7722-88-5 usually ca.1 g/kg 42 Potassium salt 7320-34-5 (total phosphate) 43 (tetra) 44 Calcium salts 14866-19-4 45 (mono and di) 35405-51-7 46 Triphosphates Sodium salt (penta) 7758-29-4 E451 (i, ii) Ͻ5 g/kg 47 Potassium salt (penta) 13845-36-8 usually ca.1 g/kg 48 (total phosphate) 49 Polyphosphates Sodium salt 68915-31-1 E452 (i-iv) Ͻ5 g/kg 50 Potassium salt 7790-53-6 usually ca.1 g/kg 51 Calcium salt (total phosphate) 52 Sodium and calcium salt S53 N54 40454 08 077-086 r1.qxd 7/20/07 12:37 PM Page 86

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1 proteins on sausage evolution and properties. Natu- D Balev, T Vulkova, S Dragoev, M Zlatanov, S 2 rally, the different phosphate types confer different Bahtchevanska. 2005. A comparative study on the 3 properties to the protein matrix, depending upon the effect of some antioxidants on the lipid and pigment 4 degree of polymerization and the type of salt. Besides oxidation in dry-fermented sausages. Intern J Food 5 this, all of them act as buffering agents, showing a Sci Technol 40:977–983. 6 trend to increasing pH values to different degrees. M Calvo. 1991. Aditivos Alimentarios; Propiedades, 7 Table 8.8 gives the CAS (JECFA 2006) and EU Aplicaciones y Efectos Sobre la Salud. Zaragoza: 8 (EPC 1995) numbers of phosphates most commonly Mira Editores. 9 used, as well as the common concentration range R Cassens. 1990. Nitrite-cured Meat; A Food Safety 10 added to sausage formulations. It has been said previ- Issue in Perspective. Trumbull, Connecticut: Food 11 ously that the diverse phosphates interact differently and Nutrition Press. 12 with meat proteins; this means that final effects on EPC (European Parliament and Council). 1994. Direc- 13 sausage properties also will be different. In summary, tive No 94/36/EC, of 30/06/94, on colours for use in 14 though highly simplifying, the higher the degree of foodstuffs. Official Journal L237, of 10/09/94, 15 polymerization, the higher the additive “multipur- pp. 13–29. 16 pose” effect and the better the results. Of course, the ———. 1995. Directive No 95/2/EC, of 20/02/95, on 17 effect of phosphates is concentration-dependent. As a food additives other than colours and sweeteners. 18 consequence, polyphosphates are much more valued Official Journal L061, of 18/03/95, pp. 1–40. 19 than monophosphates, with tri- and diphosphates in JECFA (Joint FAO/WHO Expert Committee on Food 20 intermediate ranking. Nevertheless, it has been Additives). 2006. Compendium of Food Additives 21 demonstrated that the mixture of two or more kind of Specifications. FAO. http://www.fao.org/ag/agn/jecfa/. 22 phosphates has synergistic effects (Klettner 2001). PG Klettner. 2001. Effect of phosphates on technologi- 23 Therefore, industrials tend to use mixtures contain- cal parameters by dry fermented sausage. Fleis- 24 ing poly- and any of tri-, di- or monophosphates. chwirtshaft 81 (10):95–98. 25 The concentration of added phosphates is normally S Martín-Bejarano. 2001. Enciclopedia de la Carne y de 26 limited by most regulations. As an example, the EU los Productos Cárnicos. Plasencia: Martín & Macías. 27 regulations establish a maximum residual concentra- L Martínez, I Cilla, JA Beltrán, P Roncalés. 2006a. 28 tion of 5 mg/kg in any meat product. This seems to be Effect of Capsicum annuum (red sweet and cayenne) 29 a sufficient amount to obtain the benefits of phos- and Piper nigrum (black and white) pepper powders 30 phates addition, but it must not be overlooked that on the shelf-life of fresh pork sausages packaged in 31 meat itself contains a large amount of phosphates. In modified atmosphere. J Food Sci 71:48–53. 32 fact, the concentration of inorganic phosphate may ———. 2006b. Comparative effect of red yeast rice 33 pile up to circa 4 mg/kg, which proceeds from the (Monascus purpureus), red beet root (Beta vulgaris) 34 postmortem degradation of organic phosphates ATP, and betanin (E-162) on colour and consumer accept- 35 ADP, and IMP, among others. Consequently, industri- ability of fresh pork sausages packaged in modified 36 als have a narrow concentration range at free dis- atmosphere. J Sci Food Agric 86:500–508. 37 posal. In any case, relatively low concentrations of MD Ranken. 2000. Handbook of Meat Product Tech- 38 about 1 mg/kg, particularly in the form of mixtures of nology. Oxford: Blackwell. 39 poly- and other phosphates, are enough to bring M Saltmarsh. 2000. Essential Guide to Food Additives. 40 about the desired beneficial effects on dry/fermented Leatherhead: Leatherhead Publishing. 41 sausages. In conclusion, industrials may decide the A Sánchez-Escalante, D Djenane, G Torrescano, JA 42 use of phosphates or not as a function of their techno- Beltrán, P Roncalés. 2003. Antioxidant action of 43 logical needs and the display of its presence in the borage, rosemary, oregano and ascorbic acid in beef 44 product label. No shortcomings have been reported patties packaged in modified atmosphere. J Food 45 related to the use of phosphates as a food additive, Sci 68:339–344. 46 despite some consumer groups resistance to its use. LA Shelef. 1994. Antimicrobial effect of lactates; A 47 review. J Food Protect 57:445–450. 48 REFERENCES A Stiebing. 1998. Influence of proteins on the 49 ripening of fermented sausages. Fleischwirtshaft 78: 50 M Ash, I Ash. 2002. Handbook of Food Additives. 1140–1143. 51 Endicott, New York: Synapse Information Resources F Toldrá. 2002. Dry-cured meat products. Ames, Iowa: 52 Inc. Blackwell Publishing. 53S 54N 40454 09 087-100 r1.qxd 7/20/07 12:39 PM Page 87

1 2 3 9 4 5 6 Spices and Seasonings 7 8 Suey-Ping Chi and Yun-Chu Wu 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION dehydrated vegetables such as onions, garlic 22 powder, and celery powder from the spice list. 23 The terms spices and herbs are usually used together According to the FDA’s definition, a spice is any 24 to describe a group of aromatic plant parts, e.g., bark aromatic vegetable substance in the whole, 25 (cinnamon), buds (cloves), flowers (saffron), leaves broken, or ground form that is used to season 26 (bay leaf, sage), fruit (allspice, chilies), bulbs (gar- food rather than to contribute nutrients. This 27 lic, onion), roots (ginger), or seeds (caraway, mus- definition also requires spices to be true to the 28 tard). In general, spices are highly aromatic due to name and unmodified so that no volatile oil or 29 their high contents of essential oils, whereas herbs other flavoring principle has been removed. FDA 30 are low in essential oils and are usually used to regulations state that spices may be labeled as 31 produce delicate or subtle flavors in food preparation spices; however, color-contributing spices— 32 in contrast to the aromatic flavors imparted by paprika, turmeric and saffron—must be declared 33 spices. as spice and coloring or by their common names. 34 Spices and herbs are not clearly differentiated Essential oils, oleoresins, and their natural plant 35 because various countries have differences of opin- extractives containing flavor constituents may 36 ion on their definitions. Even within the same coun- be declared as natural flavor. Dehydrated 37 try, different agencies and trade organizations have vegetables must be declared by their usual 38 different definitions for them: or common names. 39 40 • The American Spice Trade Association define This chapter discusses (a) ethnic preferences of 41 spices in very broad terms—dried plant products spices and herbs (collectively called spices below), 42 used primarily to season food. (b) botanical properties of spices, (c) product forms 43 • The USDA defines the term spice as any and appearance of spices, (d) sensory properties 44 aromatic vegetable substance in the whole, of spices, (e) chemical properties of spices, and 45 broken, or ground forms, with the exception (f) application of spices in fermented meats and 46 of onions, garlic, and celery, whose primary poultry with emphasis on meat and poultry products. 47 function in food is seasoning rather than 48 nutritional and from which no portion of any ETHNIC PREFERENCES 49 volatile oil or other flavoring principle has been OF SPICES 50 removed. 51 • The USFDA also has a broad definition for Spices and herbs have been used by different cul- 52 spices with one important exception: it excludes tures for centuries to season their foods. Originally, 53 S54 N55 87 40454 09 087-100 r1.qxd 7/20/07 12:39 PM Page 88

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1 they used the spices and herbs available in their own COMMONLY USED SPICES IN 2 regions. Later, spices and herbs from other regions PROCESSED MEATS 3 penetrated into other cultures. Only until the last two 4 to three centuries, with the frequency of international In general, about 50 different spices are commonly 5 trade and preference, have spices and herbs available used in the food industry (Charalambous 1994; 6 in one culture become easily accessible to another Farrell 1990; Hirasa and Takemasa 1998; Lewis 7 culture. Historically, Europeans sailed out to the 1984; Tainter and Grenis 2001; Uhl 2000; Under- 8 “Indies” searching for tropical spices, with Colum- rinder and Hume 1994). About 25 to 35 of these 9 bus “discovering” America as the best known spices are used in the processing of meats and poul- 10 example. However, various cultures still maintain try. Table 9.1 lists the names of these spices and their 11 their own cultural heritage with typical preferences applications in meat processing. 12 for spices and herbs, even though some of these 13 spices are not native to their region. The following BOTANICAL PROPERTIES OF 14 are some of the spices preferred by selected cultures 15 (Giese 1994b; Lewis 1984; Moore 1979): SPICES 16 • Chinese: Anise, cinnamon, clove, garlic, onion, ORIGINS OF VARIOUS SPICES 17 red pepper, star anise 18 The spices used in processing meats originate from • East Indian: Black pepper, coriander, 19 many countries in Asia, Europe, Africa, and the • French: Black pepper, marjoram, thyme 20 Americas (Table 9.2). They have spread all over • German: Ginger, nutmeg, onion, paprika, white 21 the world and are now cultivated in their original pepper 22 countries as well as in other countries that can • Greek: Black pepper, cinnamon, garlic, onion, 23 produce them in large quantities for international thyme 24 trade. • Hungarian: Cinnamon, dill seeds, garlic, onion, 25 paprika, white pepper 26 • Indonesia: Cinnamon, clove, curry, garlic, ginger, BOTANICAL CLASSIFICATION OF SPICES 27 red pepper, cardamom, celery, cinnamon, clove, 28 Spices we use now come from various plant families nutmeg 29 with growth characteristics that include evergreens, • Italian: Anise, black pepper, garlic, marjoram, 30 annuals, biannuals, or perennials. Plant parts may be onion, oregano, red pepper, sage, thyme 31 berries, seeds, leaves, inner bark, fruits, fruit peri- • Jamaican: Allspice, garlic, red pepper, thyme, 32 carp, fruit pods, unopened flower buds, bulbs, rhi- onion 33 zomes, aril of seed, or flowing tops. Table 9.3 is a • Mexican: Allspice, anise, bay leave, chili pepper, 34 summary of the botanical characteristics of common cinnamon, cloves, coriander, garlic, onion, cumin 35 spices used in processing meat. seeds, red pepper 36 • Moroccan: Anise, cinnamon, coriander, cumin 37 seeds, red pepper PRODUCT FORMS AND 38 • Southwestern U.S.: Chili pepper, cinnamon, APPEARANCES OF SPICES 39 clove, cumin, garlic, onion 40 Spices used in processing meat and poultry may • Spanish: Cinnamon, cumin seeds, garlic, hot 41 exist in various forms depending on the product to be pepper, nutmeg, onion, paprika, sweet pepper, 42 processed. They may be whole seeds, whole ground, thyme 43 whole pods, leaf flakes, slices, chopped, granules, or • Spanish Caribbean: Black pepper, cinnamon, 44 powder. They can also be applied in the form of cloves, cumin seed, garlic, ginger, nutmeg, onion, 45 spice oleoresin, essential oil, encapsulated oil or ole- paprika, thyme 46 oresin, or essential oil plus oleoresin (Anonymous • Thai: Black, white, and red peppers; cardamom; 47 1981; Charalambous 1994; Deline 1985; Farrell cinnamon; cloves; coriander root; curry paste (red 48 1990; Giese 1994a; Hirasa and Takemasa 1998; and green); garlic; ginger 49 Lewis 1984; Pszzola 1999; Tainter and Grenis 2001; • Vietnamese: Chilies; black, white and red peppers; 50 Uhr 2000, Underrinder and Hume 1994). Table 9.4 clove; curry powder; five spice powder; star anise. 51 is a summary of product forms and appearance of 52 This cultural heritage offers typical flavors for food some common spices used in processing meats and 53S products of individual ethic backgrounds. poultry. 54N 40454 09 087-100 r1.qxd 7/20/07 12:39 PM Page 89

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Table 9.1. Spices commonly used in processed meats. 1 2 Spice Use 3 Allspice Bologna, pickled pigs feet, head cheese 4 5 Anise seed Dry sausage, mortadella, pepperoni 6 Bay (laurel) leaves Pickle for pigs feet, lamb, pork tongue 7 Cardamom Frankfurters, liver sausage, head cheese 8 9 Cassia Bologna, blood sausage 10 Celery seed Pork sausage 11 Cinnamon Bologna, head sausage 12 Clove Bologna, head cheese, liver sausage 13 14 Coriander seed Frankfurters, bologna, Polish sausage, luncheon specialties 15 Garlic Pork sausage, many types of smoked sausage 16 Ginger Pork sausage, frankfurters, corned beef 17 Mace Veal sausage, liver sausage, frankfurters, bologna 18 19 Marjoram Liver sausage, Polish sausage, head cheese 20 Mustard Good in all sausage 21 Nutmeg Veal sausage, bologna, frankfurters, liver sausage, head cheese 22 23 Onion Liver sausage, head cheese, baked loaf 24 Paprika Frankfurters, Mexican sausage, dry sausage 25 Pepper (black) Bologna, Polish sausage, head cheese 26 Pepper (Cayenne or red) Frankfurters, bologna, veal sausage, smoked sausage, smoked 27 country sausage 28 29 Pepper (white) Good in all sausage 30 Pimento (pimiento) Loaves 31 Sage Pork sausage, baked loaf 32 Savory Good in all sausages 33 34 Thyme Good in all sausages 35 Source: Coggins (2001); Forrest et al. (1975); Ockerman (1989). 36 37 38 39 40 41 42 ANTIOXIDATIVE EFFECTS CHEMICAL PROPERTIES OF 43 SPICES It is now known that some spices have antioxidative 44 effects. Some spices are more effective than the 45 MAJOR CONSTITUENTS others. The effectiveness also depends on the 46 Each spice has its own major ﬂavor constituent(s). medium in which they are tested. For example, in 47 Usually it exists in its essential oil and in some cases lard medium, sage is remarkably effective and mace, 48 also in its oleoresin. Together these constituents pro- nutmeg, and thyme are effective; in oil-in-water 49 vide the spice’s typical ﬂavor. Table 9.5 summaries medium, clove is strongest, followed by allspice, cas- 50 the principal constituents in essential oils and signif- sia, cinnamon, ginger, sage, and savory being strong, 51 icant constituents in oleoresins of various spices and anise seed, cardamom, marjoram, and pepper 52 used in processed meats and poultry. (black and white) showing some effectiveness S53 N54 40454 09 087-100 r1.qxd 7/20/07 12:39 PM Page 90

1 Table 9.2. Origin and current major sources of some common spices. 2 3 Spice Places of Origin Current Major Sources 4 Allspice Western Hemisphere Caribbean and Central America 5 6 Anise seed Middle East Turkey, Egypt, Syria, Spain, China, Mexico 7 Bay leaf Mediterranean countries Greece, Turkey 8 Caraway seed Europe and Western Asia Canada, The Netherlands, Egypt, Poland, 9 Denmark 10 Cardamom Southern India and Guatemala, India, Sri Lanka 11 Sri Lanka (Ceylon) 12 13 Guatemala 14 Cinnamon China China, Indonesia, Vietnam 15 Clove Madagascar, Zanzibar, Pemba Madagascar, Indonesia, Brazil, Zanzibar, 16 Sri Lanka 17 Coriander Southern Europe, Asia Minor, Canada, Mexico, Morocco, Rumania, Argentina 18 Southwest Russia 19 20 Cumin seed Egypt and Mediterranean region Middle Eastern countries, India, Pakistan, 21 Switzerland 22 Dill seed Mediterranean region Canada, India, Indonesia 23 Dill weed Mediterranean region Canada, India, Indonesia 24 25 Garlic Central Asia U.S., China 26 Ginger, dehydrated China India, Jamaica, Nigeria, China 27 Mace Moluccas Islands and East Indies, Indonesia, West Indies 28 East Indian Archipelago 29 Marjoram, sweet Mediterranean region and Egypt, France, 30 Western Asia 31 32 Mustard seed Middle East Canada, U.S., U.K., Denmark 33 Nutmeg Moluccas Islands and East Indies, Indonesia, West Indies 34 East Indian Archipelago 35 Onion West or Central Asia U.S. 36 Paprika Western hemisphere U.S., Spain, Hungary, Morocco 37 38 Parsley Mediterranean region U.S., Israel, Hungary 39 Pepper, black Malabar Coast of Indonesia, India, Brazil, Malaysia 40 Southern India 41 Pepper, red Western hemisphere Africa, China, India, Pakistan, Mexico 42 43 Pepper, white Malabar Coast of Indonesia, India, Brazil, Malaysia 44 Southern India 45 Sage Mediterranean region Albania, Croatia, Germany, Italy 46 Macedonia, Turkey 47 Star anise China China 48 Savory, sweet Southern Europe and Yugoslavia, France 49 summer Mediterranean region 50 51 Thyme Mediterranean region Spain, France 52 53S Source: Charalambous (1994); Curry and Nip (2005); Farrell (1990); Lewis (1984); Tainter and Grenis (2001); Uhl (2000); 54N Underrinder and Hume (1994).

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Table 9.3. Botanical characteristics of some common spices used in processed meats. 1 2 Growth 3 Spice Scientific Name Plant Family Characteristics Plant Part 4 Allspice Pimenta doica Myrtle Evergreen Berries 5 6 Anise seed Pimpineela anisum Parsley Annual Seeds 7 Basil Ochimum basilicum Mint Annual Leaves 8 Bay leaf Laurus nobilis Laurel Evergreen Leaves 9 10 Cassia Cinnamonum cassia Laurel Evergreen Inner bark 11 Cardamom Elettraia Ginger Perennial Seeds 12 cardamomum 13 Celery seed Apium graveolens Parsley Biannual or annual Fruits 14 Cinnamon Cinnamonum Laurel Evergreen Inner bark 15 zelancium 16 17 Clove Syzgium aromaticum Myrtle Evergreen Unopened 18 flower buds 19 Coriander Coriandrum sativum Parsley Annual Fruits 20 Cumin seed Cumium cyminum Parsley Annual Ripe fruits 21 Garlic, dehydrated Allium sativum Onion Perennial Bulbs 22 23 Ginger, dehydrated Zingiber officinale, Ginger Perennial Rhizomes 24 Roscoe 25 Mace Myristica fragans Nutmeg Evergreen Aril of seed 26 Marjoram, sweet Origanum majorana Mint Perennial Leaves 27 28 Mustard seed, black Brassica hira B. alba Cabbage Annual Seeds 29 Mustard seed, brown Brassica juncea Cabbage Annual Seeds 30 Mustard, white Brassica nigra Cabbage Annual Seeds 31 Nutmeg Myristica fragans Nutmeg Evergreen Seeds 32 33 Onion, dehydrated Allium cepa Onion Biannual Bulbs 34 Oregano Origanum spp. Mint Perennial Leaves 35 Paprika (pepper, red) Capsicum annum Nightshade Annual or perennial Fruit Pericarp 36 Parsley Petrosalinum crispum Parsley Biannual Leaves 37 38 Pepper, black Piper nigum Pepper Perennial Unripened berries 39 or peppercorns 40 Pepper, chili Capsicum minimum Pepper Annual or Fruit 41 Perennial 42 Pepper, red Capsicum fructescens Nightshade Perennial Fruit pods 43 (Capsicum) 44 45 Pepper, white Piper nigum Pepper Perennial Skinless, ripe 46 berries 47 Sage Salvia officinalis Mint Perennial Leaves 48 Savory, sweet Satureja indicum Mint Annual Leaves 49 summer 50 Star anise, Chinese Illicum vercum Illiciaceae Perennial Fruit 51 52 Thyme Thymus vulgaricus Mint Perennial Leaves and S53 flowering tops N54 Underrinder and Hume (1994). 91 40454 09 087-100 r1.qxd 7/20/07 12:39 PM Page 92

Table 9.4. Product forms and appearance of some common spices used in processed meats. Spice Product Forms (Dried) Product Appearance (Dried) 1 2 Allspice Whole and ground Dark reddish-brown pea-sized fruit 3 Anise seed Whole and ground Greyish brown, oval-shaped, and about 4 3/16 in long 5 6 Bay leaf Whole and ground Deep green leaves up to 3 in long 7 Caraway seed Whole only Brown, hard seeds of 3/16 in long 8 Cardamom Whole pods (bleached and Irregularly round pod with seeds about 9 green), decorticated (seed 3/32 in long 10 alone), and ground 11 Celery seed Light brown seeds up to Whole, ground, and ground mixed with 12 1/16 in long table salt (celery salt) 13 Celery flakes Flakes, granulated, powdered Light green dehydrated celery leaves 14 15 Cinnamon Whole and ground Brown bark 16 Clove Whole and ground Brown nail-shaped, 1/2 to 3/4 in long 17 Coriander Whole and ground Tan to light brown, globular with vertical 18 ridges up to 3/16 in diameter 19 Cumin seed Whole and ground Yellow-brown seeds of 1/8 to 1/4 in long 20 21 Dill, seed Whole and ground Light brown, oval-shaped, up to 1/16 in long 22 Garlic, dehydrated Large sliced, chopped, minced, Off-white 23 ground, granulated, powder; 24 also powder or granulated 25 with table salt (garlic salt) 26 Ginger, dehydrated Whole, cracked or ground Even, light buff hands or fingers 27 Mace Whole or ground Yellow-orange aril or skin 28 Marjoram, sweet Whole (bits of leaves) Grey-green leaves 29 and ground 30 31 Mustard seed, black Whole, mustard flour, and ground Black seeds 32 Mustard seed, brown Whole, mustard flour, and ground Brown seeds 33 Mustard, white Whole, mustard flour, and ground White seeds 34 Nutmeg Whole, ground Greyish-brown, oval fruit, up to 1-1/4 in long 35 36 Onion, dehydrated Sliced, chopped, diced, minced, Off-white 37 ground, granulated, powdered, 38 toasted (all sizes), powder, or 39 granulated onion with table salt 40 (onion salt) 41 Paprika Powder Bright rich red stemless pods 42 Pepper, black Whole, ground, cracked, Black seeds 43 decorticated 44 Pepper, red Whole and ground Various-sized red-colored pods 45 46 Pepper, white Whole and ground White seeds 47 Sage Whole, cut, rubbed, and ground Silver-grey leaves 48 Savory, sweet Whole (bits of leaves) Brown-green leaves less than 3/8 in long 49 summer and ground 50 Thyme Whole (bits of leaves) and Brownish-green leaves, seldom exceed 51 ground 1/4 in long 52 53S Source: Charalambous (1994); Curry and Nip (2005); Farrell (1990); Lewis (1984); Tainter and Grenis (2001); Uhl (2000); 54N Underrinder and Hume (1994).

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Table 9.5. Principal constituents in some common spices used in processed meats. % Essential Principal Constituents in Significant Constituents Spice Oil (Range) Essential Oil in Oleoresin 1 2 Allspice 3.3 to 4.5 Eugenol (60–75%), eugenol methyl None 3 ether, cineole, phellandrene, 4 caryophylene 5 Anise seed 1.5 to 3.5 Anethole (major), anisaldehyde, None 6 anisketone, methyl chavicol 7 8 Bay leaf 1.5 to 2.5 Cineole None 9 Cardamom 2 to 10 Cineole, alpha-terpinyl acetate, None 10 liminone, linalyl linalool, borneol, 11 alpha-terpineol, alpha-pinene, 12 limonene, myracene 13 Celery seed 1.5 to 3 d-Limonene, beta-selinene None 14 15 Cinnamon 1.5 to 3.0 Eugenol (clovelike flavor), cinnamic None 16 aldehyde (cinnamonlike flavor) 17 Clove Up to 20% Eugenol, eugenol acetate, None 18 beta-caryophyllene 19 Coriander 0.1 to 1.5 d-Linalool (coriandrol), Pinenes, None 20 terpinenes, geranol, vorneal, 21 decylaldehyde 22 Cumin seed 2 to 5 Cumaldehyde (cumic aldehyde), None 23 dihydrocumaldehyde, ciminyl alcohol, 24 dl-pinene, p-cumene, diterpene 25 26 Ginger ?? Gingerols (pungency) Zingerone, shogoals 27 Ginger, dehydrated 1.5 to 3.0 (−)-alpha-zingiberene, None 28 (−)-beta-bisabolene, (+)-ar-curcumene 29 (ginger flavor), farnesene, beta- 30 sequiphellandrene (ginger flavor), 31 alpha-terpineol (lemony flavor), citral 32 (lemony flavor) 33 Mace 15 to 25 Alpha-pienene, Beta-pinene, sabinene, None 34 myristicin (main flavor) 35 Marjoram, sweet 0.7 to 3 Terpen-4-ol, alpha-terpineol None 36 37 Mustard seed, black 0.6 (av.) Allyisothiocyanate None 38 Mustard seed, yellow None None Sinabin mustard oil 39 (pungent) 40 Nutmeg 6 to 15 Alpha-pienene, Beta-pinene, None 41 sabinene, myristicin (main flavor) 42 43 Onion, dehydrated Trace Ethyl and propyl disulfides, vinyl sulfide None 44 Paprika None None Capsanthin, carotene, 45 capsorubin (pigments) 46 Pepper, black 0.6 to 2.6 alpha-pinene, beta-pinene, None 47 1-alpha-pellandrene, beta- 48 caryophyllene, limonene, sabine-delta- 49 3-carene pungency:piperine, 50 piperettine, peperyline, piperolein 51 A and B, piperanine 52 S53 (Continues) N54

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94 Raw Materials

1 Table 9.5. (Continued) 2 3 % Essential Principal Constituents in Significant Constituents 4 Spice Oil (Range) Essential Oil in Oleoresin 5 Pepper, red Capsaicin, Dihydrocapsaicin None 6 7 Pepper, white 1.0 to 3.0 alpha-pinene, beta-pinene, None 8 1-alpha-pellandrene, beta- 9 caryophyllene, limonene, 10 sabine-delta-3-carene pungency: 11 piperine, piperettine, peperyline, 12 piperolein A and B, piperanine 13 Sage 1.5 to 2.5 Thujone, borneol, cineole None 14 Savory, sweet <1 Carvacrol, thymol, p-cymene None 15 summer 16 Sesame seed N/A Pyrazines (in toasted seeds) Sesamin, sesamolin 17 Star anise 5 to 8 Anethol None 18 19 Thyme 0.8 to 2.0 Thymol, p-cymene, d-linalool None 20 Source: Charalambous (1994); Deline (1985); Farrell (1990); Lewis (1984); Pszzola (1999); Tainter and Grenis (2001); Uhl 21 (2000); Underrinder and Hume (1994). 22 23 24 25 26 (Balententine et al. 1989; Charalambous 1994; Duke 2001; Uhl 2000). Consumers should not be scared 27 1994; Farrell 1990; Mandsen et al. 1997; Mansour from consuming the very minute amounts of spices 28 and Khalil 2000; Nakatani 1994; Tainter and Grenis present in processed meats and poultry, but proces- 29 2001; Uhl 2000; Underrinder and Hume 1994). sors should be careful in handling large amount of 30 These findings may explain why some of the the following spices: 31 processed meats and poultry are fairly stable during 32 storage without development of rancidity. • Anise volatile oil: Anthethole (estrogenic activity) 33 • Black pepper: Safrole (carcinogenic activity), 34 Piperine (carcinogenic activity) 35 ANTIBACTERIAL EFFECTS • Black pepper + nitrate: n-Nitropiperine 36 It is also now know that some spices provide antibac- (carcinogenic activity) 37 terial properties. Table 9.6 summarizes some • Mustard (brown): Sinalbin, then allylisocyanate 38 examples in which bacteria are being inhibited by (lacinmater, mutagenic in Ames test, carcinogenic 39 various spices. This may explain why processed in rats) 40 meats and poultry containing various spices are • Mustard (white): Glucosinolates (Goitrogenic and 41 fairly safe, provided they are properly packaged and antithroid activity), Sinalbin, then allylisocyanate 42 stored. (lacinmater, mutagenic in Ames test, carcinogenic 43 in rats) 44 • Paprika + nitrate: n-Nitrosopyrrolidine 45 NATURALLY OCCURRING TOXICANTS (carcinogenic activity) 46 • Nutmeg: Myristicin (Hallucinogen) 47 Some spices have been known for a long time to • Sage: Evidence of estrogenic activity. 48 show adverse effects if consumed in large doses or 49 when they are exposed to consumers for an extended 50 period. It is now known that some constituents in SPICE QUALITY STANDARDS 51 some spices may be estrogenic, carcinogenic, goitro- CHEMICAL TESTS USED IN QUALITY CONTROL 52 genic, mutagenic, and hallucinogenic (Ames 1983; 53S Conner 1993; Dalby 2000; Farrell 1990; Hirasa and Spices used in the food industry come from various 54N Takemasa 1998; Miloslav 1983; Tainter and Grenis parts of the world with diversified quality standards. 40454 09 087-100 r1.qxd 7/20/07 12:39 PM Page 95

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Table 9.6. Common foodborne bacterial pathogens inhibited by selected spices used in meat processing. 1 2 Staphylococcus Clostridium Clostridium Salmonella Camphobacter 3 Spice aureus botulinum perfrigens enterica E. coli jejuni 4 Allspice X X 5 6 Bay leaves X X X 7 Cinnamon X X 8 Cloves X X X X X 9 10 Cumin X X X 11 Dill X 12 Fennel X X 13 Garlic X X X X X 14 15 Lemongrass X X X 16 Mint X 17 Onion X X 18 Oregano X X X 19 20 Rosemary X X 21 Sage X X 22 Tarragon X X 23 24 Thyme X X 25 Source: Beuchat (1994); Beuchat and Golden (1989); Billing and Sherman (1998); CAST (1998); Curry and Nip (2005); 26 Davidson et al. (1983); Deans and Richie (1987); Dorman and Deans (2000); Giese (1994a); Jay and Rivers (1984); Kim 27 et al. (1995); Nakatani (1994); Shelef (1984); Smith-Palmer et al. (1998); Synder (1997); Zaika (1988). 28 29 30 31 32 33 This is expected because the spices are produced in • Pepper (Black): Piperine 34 various locations in the world. Their active con- • Residual solvent tolerance: Various solvents. 35 stituents are not the same due to different growing 36 conditions and handling practices. However, some 37 38 measures can be set up to provide some standards for SANITATION CRITERIA producers and users to follow. For example, standard 39 chemical tests can be used to determine the potency Most spices are produced in developing countries 40 of some spices. The following are some of the stan- with a poor sanitation environment. It is not uncom- 41 dard tests that can be conducted for spices common to have insect infestation and bacterial con- 42 monly used in processing meats and poultry tamination in spices arriving at the port of entry. 43 (Charalambous 1994; Farrell 1990; Hirasa and USFDA has Defective Action Levels set up for vari- 44 Takemasa 1998; Lewis 1984; Tainter and Grenis ous spices, their probable courses, and their signiﬁ- 45 2001; Uhl 2000; Underrinder and Hume 1994): cance. Readers interested in this topic should visit 46 the USFDA’s website at www.fda.gov for detailed 47 • Allspice: Total acid, acid-soluble ash, water- information. 48 soluble ash Spices are also easily contaminated with bacteria 49 • Capsicum: Scoville heat units in the production phase. Several measures are avail- 50 • Capsicum oleoresin: Capsaicin able to reduce bacterial counts in spices. Each proce- 51 • Nutmeg or mace: Phenol dure has its own advantages and disadvantages. They 52 • Paprika: Carotenoids are summarized as follows (Charalambous 1994; S53 • Paprika, defatted: Sample structure Farrell 1990; Hirasa and Takemasa 1998; Leistritz N54 40454 09 087-100 r1.qxd 7/20/07 12:39 PM Page 96

1 Table 9.7. Flavor characteristics and commercial applications of some common spices used in processed 2 meats. 3 Spice Flavor Characteristics Commercial Applications 4 5 Allspice (pimento) Warm, sweet, and slightly peppery, In almost all meats and sausages 6 reminiscent of clove 7 Clove Very aromatic, warm and astringent In hams, tongue, and pork products 8 9 Coriander Mild and pleasantly aromatic In meat stuffings 10 Cumin seed Salty sweet, similar but coarser Basic ingredient of processed meats 11 than that of caraway, bitter 12 aftertaste 13 Garlic, dehydrated Strong and characteristic With care in most types of products 14 15 Ginger, dehydrated Aromatic, hot, and biting In most meat products 16 Mace Finer than, but similar to, In processed meat loaf and sausages 17 that of nutmeg 18 Marjoram, sweet Distinctive, delicately sweet In most meat dishes, soups, and 19 and spicy with a slightly stuffings 20 bitter back note 21 Nutmeg Sweet and aromatic adding a certain In many meat products 22 richness to spice mixes 23 24 Onion, dehydrated Characteristic and penetrating In many meat dishes requiring an 25 onion flavor 26 Paprika (red pepper) Piquant and moderately pungent In all foods to give added zest 27 Paprika, rosen Characteristic and stronger than In many specialty meat products 28 normal paprika with a definite 29 pungency 30 Pepper Penetrating, strong, and characteristic In almost all dishes, a small quantity 31 of pepper improves the 32 overall flavor 33 34 Pepper, chile Intense pungent Used with discretion in any product 35 requiring heat 36 Pepper, red Hot and pungent Used with discretion in any product 37 requiring heat 38 Sage, Dalmatian Strongly aromatic, warm with With care in all pork dishes and poultry 39 slightly bitter aftertaste stuffings 40 41 Sage, English Milder and much more pleasant With care in all pork dishes and poultry 42 flavor than that of Dalmatian stuffings 43 sage, lacks the objectionable 44 “thujone” note 45 Savory, sweet summer Strongly aromatic In mixtures for sausages and patés 46 Star anise Warm, sweet, aromatic In meat dishes, also as a 47 component for Chinese 48 Five Spices 49 Thyme Penetratingly warm and pungent, In meat stuffings and many meat dishes 50 the aroma is long lasting 51 52 Source: Charalambous (1994); Curry and Nip (2005); Farrell (1990); Hirasa and Takemasa (1998); Geise (1994b); Lewis 53S (1984); Moore (1979); Tainter and Grenis (2001); Underrinder and Hume (1994); Uhl (2000). 54N

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1997; Lewis 1984; Tainter and Grenis 2001; Uhl onion, sage, and thyme can make or deodorize some 1 2000; Underrinder and Hume 1994.): undesirable flavor. Paprika is an oil-soluble colorant. 2 Table 9.7 is a summary of flavor characteristics 3 • Ethylene oxide: Proven method for whole spices and commercial applications of various spices in 4 and seeds; safety of using ethylene oxide is a processing meats and poultry. 5 concern; long treatment time; banned in some It should be noted that sensory attributes recog- 6 countries nized in one culture may be different in another cul- 7 • Irradiation: Cost effective; overall effectiveness; ture. For example, cinnamon is considered as woody 8 can process packed products; consumer concerns and sweet in the western culture, the same spice is 9 on irradiated products; not universally accepted considered bitter in Indian culture and pungent and 10 • Dry steam: Highly effective; more expensive than sweet in Chinese culture. Table 9.8 presents several 11 irradiation process, but less expensive than examples showing discrepancies in sensory attributes 12 ethylene oxide and wet steam process; absence of in various cultures. 13 residual fumigant 14 • Wet steam: Absence of residual fumigant; loss of 15 volatile oils; lost of color in some spices; APPLICATIONS OF SPICES 16 development of unaccepted flavor. IN FERMENTED MEAT 17 PROCESSING 18 SENSORY PROPERTIES OF 19 COMPONENTS IN SPICE BLENDS SPICES 20 Spice blends are available in the market for the con- 21 The major purpose of adding various spices to meats venience of consumers or processors. The composi- 22 in their preparation or processing is to provide dis- tion of each blend generally contains some 23 tinct flavor(s) to the final products. Each spice has its “standard” components that have been well recog- 24 own sensory characteristic(s), and the same flavor nized. It should be noted that producers usually have 25 characteristic may also be provided by several their proprietary formulations for their products, 26 spices, allowing choices of spices in the formulation. even though the spices may or may not be the same. 27 Adding spices to processed meats also provides Table 9.9 summarizes some published information 28 other minor purposes, such as texture and masking/ on spice blend components for groups of processed 29 deodorizing undesirable flavors. Basic flavoring meats. 30 attributes provided by various spices are summa- 31 rized as follows (Charalambous 1994; Deline 1985; 32 Farrell 1990; Giese 1994b; Hirasa and Takemasa AMOUNT OF SPICES IN VARIOUS FERMENTED 33 1998; Lewis 1984; Moore 1979; Tainter and Grenis SAUSAGES AND MEATS 34 2001; Uhl 2000; Underrinder and Hume 1994): Individual fermented sausage and meat processors 35 • Bitter: Bay leaf, celery, clove, mace, thyme usually have their own formulations to attract their 36 • Cooling: Anise customers. They either blend their own spices or 37 • Earthy: Black cumin have the spice companies provide proprietary for- 38 • Floral aromatic: Black pepper, ginger mulations for them. There are also published formu- 39 • Fruity: Coriander root, star anise lations available. Table 9.10 shows some published 40 • Herbaceous: Sage formulations for some common fermented sausages 41 • Hot: Chili pepper, mustard, black pepper, white and meats. 42 pepper, wasabi 43 • Nutty: Mustard seeds 44 • Piney: Thyme CONCLUSION 45 46 • Pungent: Garlic, ginger, onion, wasabi The blending of various spices with different sen- • Spicy: Clove, cumin, coriander, ginger 47 sory attributes provides the characteristic flavor for 48 • Sulfury: garlic, onion each product. It is this characteristic flavor that con- • Sweet: Allspice, anise, cardamom, cinnamon, 49 sumers enjoy and that provides momentum for meat 50 star anise processors to come up with new formulations to • Woody: Cassia, cardamom, clove. 51 keep their business moving. Formulation of spice 52 Garlic, mustard, and onion also provide texture to blends is still an art that challenges the processors S53 the final product. Bay leaf, clove, coriander, garlic, and spice providers. N54 40454 09 087-100 r1.qxd 7/20/07 12:39 PM Page 98

1 Table 9.8. Sensory differences of some common spices between cultures. 2 3 Basic Sensory 4 Spice Characteristics Indian Chinese 5 Cinnamon Woody, sweet Bitter Pungent, sweet 6 7 Clove Bitter Bitter Pungent 8 Coriander Spicy Sweet Pungent 9 Cumin seed Spicy Sweet Pungent 10 Garlic, dehydrated Pungent, spicy Spicy/pungent Pungent 11 12 Ginger, dehydrated Spicy Spicy/pungent Pungent 13 Pepper, black Hot Spicy/pungent Pungent 14 Pepper, chili Hot Spicy/pungent Pungent 15 16 Source: Charalambous (1994); Caurry and Nip (2005); Deline (1985); Farrell (1990); Giese (1994b); Hirasa and 17 Takemasa (1998); Lewis (1984); Lu (1986); Moore (1979); Tainter and Grenis (2001); Uhl (2000); Underrinder and 18 Hume (1994). 19 20 21 22 23 Table 9.9. Components in some common spice mixtures or blends for processed meats. 24 25 Name of Mixture (Blend) Components 26 27 Bologna or wiener seasoning Pimento, nutmeg, black pepper, clove leaf or stem, 28 capsicum, paprika, coriander, cassia, mustard, 29 onion, garlic 30 Five spices, Chinese Black pepper, star anise or anise, fennel, clove, 31 cinnamon 32 Ham seasoning Cassia, clove stem or leaf, pimento, capsicum, celery, bay 33 leaf,garlic 34 35 Italian sausage seasoning Anise, fennel, paprika, black pepper, red pepper 36 Italian seasoning Oregano, basil, red pepper, rosemary, garlic 37 (optional) 38 Japanese seven spices Perilla, basil, sesame, poppy seed, hemp, red pepper, 39 Japanese pepper 40 Mixed pickling spice Mustard seeds, bay leaves, black and white peppercorns, 41 red peppers, ginger, cinnamon, mace, allspice, coriander 42 seeds, etc. 43 44 Pork sausage seasoning Sage, black pepper, red pepper, nutmeg 45 Poultry seasoning Sage, thyme, marjoram, savory, rosemary (optional) 46 Roast beef or corn beef rub Coriander, onion, garlic, celery, oregano, basil, black 47 pepper, paprika 48 Smoked sausage seasoning Garlic powder, black pepper, coriander, pimento 49 50 Source: Charalambous (1994); Curry and Nip (2005); Farrell (1990); Giese (1994b); Hirasa and Takemasa (1998); 51 Lewis (1984); Lu (1986); Moore (1979); Pszzola (1999); Tainter and Grenis (2001); Uhl (2000); Underrinder and 52 Hume (1994). 53S 54N

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Table 9.10. Amount of spices used in selected fermented sausages and smoked meats. 1 2 Amount of Spice Used per Amount of Spice Used per 3 Sausage or Meat 100 lbs of Raw Meat Sausage or Meat 100 lbs of Raw Meat 4 Semidry or Summer Sausage Dry Sausage 5 6 Thuringer #1 Ground black pepper, Genoa Salami White ground pepper, 3 oz 7 4 oz Whole white pepper, 1 oz 8 Whole mustard seed, B. C. Salami (Hard) White pepper, 3 oz 9 2 oz Garlic pepper, 0.25 oz 10 Coriander, 1 oz Pepperoni #1 Sweet paprika, 12 oz 11 Thuringer #2 Caraway, 0.75 oz Decorticated ground 12 Celery seed, 0.75 oz pepper, 6 oz 13 Coriander, 1 oz Capsaicin, 4 oz 14 Ginger, 1oz Whole fennel seed, 4 oz 15 Mace, 1 oz Pepperoni #2 Allspice, 2 oz 16 Pepper, black, 6 oz Anise seed, 5 oz 17 Cerevelat #1 Ground black pepper, Pepper, red, 2 oz 18 4 oz 19 Ham Whole black pepper, 20 2 oz Deviled ham Mustard seed, ground, 7.5 oz 21 Paprika, 0.3 oz Cerevelat #2 Cardamom, 1 oz 22 Pepper, red, 0.4 oz Coriander, 2.8 oz 23 Pepper, white, 0.1 oz Nutmeg, 1 oz 24 Tumeric, 0.4 oz Pepper, white, 7.5 oz 25 Prosciutto All spice, 8 oz 26 Lebanon bologna #1 Mustard, 8 oz White pepper 5 oz 27 White pepper, 2 oz Black pepper, 2 oz 28 Ginger, 1 oz Nutmeg, 2 oz 29 Mace, 1 oz Mustard seed, 0.5 oz 30 Lebanon bologna #2 Allspice, 2 oz Coriander, 0.5 oz 31 Cinnamon, 1 oz Beef 32 Cloves, 1 oz 33 Nutmeg, 2 oz Corned beef Celery seed, 0.5 34 Pepper, black, 5 oz Onion powder, 2 oz 35 Pepper, white, 1 oz Pepper, black, 2 oz 36 Chorizos #1 Sweet red pepper, 6 oz Pastrami 37 38 Chili powder, 6 oz Curing solution Garlic juice, 1.5 39 Red pepper (hot), 2 oz Rub #1 Black pepper 40 Chorizos #2 Cardamom, 0.4 oz and coriander 41 Coriander, 0.4 oz Rub #2 Paprika 42 Garlic powder, 0.1 oz Rub #3 65% black pepper and 43 Ginger, 0.6 oz 35% all spice 44 Oregano, 0.1 oz Rub #4 60% coriander, 25% all 45 Paprika, 2.5 oz spice, 15% white 46 Pepper, black, 1.6 oz pepper, and garlic ﬂour 47 Source: Adapted from Kramlich et al. (1973); Ockerman (1989). 48 49 50 DA Balententine, MC Albano, MG Nair. 1999. Role of REFERENCES 51 medicinal plants, herbs, and spices in protecting 52 BN Ames. 1983. Dietary carcinogens and anticarcino- human health. Nutr Rev 57(9):S41–S45. S53 gens. Science 21:1256–1264. LR Beuchat. 1994. Antimicrobial properties in N54 Anonymous. 1981. The Oleoresin Handbook. 3rd ed. spices and their essential oils. In: VM Dillon, RG New York: Fritzsche Dodge & Olcott, Inc. Board, eds. Natural Antimicrobial Systems and Food 40454 09 087-100 r1.qxd 7/20/07 12:39 PM Page 100

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1 Preservation. Wallingford, U.K.: CAB International, J Kim, MR Marshall, C Wei. 1995. Antibacterial activities 2 pp. 167–179. of some essential oil components against five food- 3 LR Beuchat, DA Golden. 1989. Antimicrobials occur- borne pathogens. J Agric Food Chem 43: 2839–2845. 4 ring naturally in food. Food Technol 43(1):134–142. WE Kramlich, AM Pearson, FW Tauber. 1973. 5 J Billing, PW Sherman. 1998. Antimicrobial functions Processed Meats. Westport: AVI. 6 of spices: Why some like it hot. Quart Rev Biol W Leistritz. 1997. Methods of bacterial reduction in 7 73(1):3–49. spices. In: SJ Risch, and CH Ho, eds. Spices: Flavor 8 CAST. 1998. Naturally Occurring Antimicrobials in Chemistry and Antioxidant Properties. Washington, 9 Food. Ames (Iowa): Council for Agricultural Science D.C.: American Chemical Society, pp. 7–10. 10 and Technology. YS Lewis. 1984. Spices and Herbs for the Food 11 G Charalambous. 1994. Spices, Herbs, and Edible Industry. Orpington, England: Food Trade Press. 12 Fungi. New York: Elsevier. HC Lu. 1986. Chinese System of Food Cures—Preven- 13 PC Coggins. 2001. Spices and flavorings for meat tion and Remedies. New York: Sterling Publishing 14 and meat products. In: Yh Hui, WK Nip, RW Rogers, Co., Inc. 15 OA Young, eds. Meat Science and Applications. New HL Mandsen, G Beretelsen, LH Skibsted. 1997. 16 York: Marel Dekker, Inc., pp. 371–401. Antioxidative activity of spices and spice extracts. In: 17 DE Conner. 1993. Naturally occurring compounds. In: SJ Risch, and CH Ho, eds. Spices: Flavor Chemistry 18 PM Davidson, AL Bransen, eds. Antimicrobials in and Antioxidant Properties. Washington, D.C.: 19 Foods. New York: Marcel Dekker, Inc., pp. 441–468. American Chemical Society, pp. 176–187. 20 JC Curry, WK Nip. 2005. Spices and herbs. In: YH Hui, EH Mansour, AH Khalil. 2000. Evaluation of 21 JD Culbertson, S Duncan, I Guerrero-Legarreta, antioxidant activity of some plant extracts and their 22 ECY Li-Chan, CY Ma, CH Manley, TA McMeekin, application to ground beef patties. Food Chem 23 WK Nip, LML Nollar, MS Rahman, F Toldrá, YL 69:15–141. 24 Xiong, eds. Handbook of Food Science, Technology R Miloslav, Jr., ed. 1983. CRC Handbook of Naturally 25 and Engineering, Vol. 2. Boca Raton, Florida: Taylor & Occurring Food Toxicants. Boca Raton, Florida: 26 Francis Group (CRC), pp. 89–1 to 89–28. CRC Press. 27 A Dalby. 2000. Dangerous Tastes—The Story of Spices. K Moore. 1979. The correct spices—Keys to ethnic 28 Berkeley, California: University of California Press. products. Food Prod Dev May:18–23. 29 PM Davidson, LS Post, AL Branen, AR McCurd. 1983. Nat- N Nakatani. 1994. Antioxidant and antimicrobial con- 30 urally occurring and miscellaneous food antimicrobials. stituents of herbs and spices. In: Spices, Herbs, and 31 In: AL Branen, PM Davidson, eds. Antimicrobials in Edible Fungi. G. Charalombous, ed. New York: Else- 32 Food. New York: Marcel Dekker, Inc., pp. 371–419. vier, pp. 251–271. 33 SG Deans, G Richie. 1987. Antibacterial properties of HW Ockerman. 1989. Sausage and Processed Meat 34 plant essential oils. Intl J Food Microbiol. 5:165–180. Formulations. New York: Van Nostrand Reinhold. 35 GD Deline. 1985. Modern spice alternatives. Cereal D Pszzola. 1999. Engineering ingredients: Believe it or 36 Foods World 30(10):697–698, 700. not. Food Technol 53(7):98–105. 37 HJD Dorman, SG Deans. 2000. Antimicrobial agents LA Shelef. 1984. Antimicrobial effect of spices. J Food 38 from plants: Antibacterial activity of plant essential Saf 6:29–44. 39 oils. J Appl Microbiol 88:308–316. A Smith-Palmer, J Stewart, L Fyfe. 1998. Antimicro- 40 JA Duke. 1994. Biologically active compounds in bial properties of plant essential oils and essences 41 important spices. In: Spices, Herbs, and Edible Fungi. against five important foodborne pathogens. Letters 42 G Charalambous, ed. New York: Elsevier, pp. 225–250. in Appl Microbiol 26:118–122. 43 KT Farrell. 1990. Spices, Condiments, and Seasonings. OP Synder. 1997. Antimicrobial Effects of Spices and 44 New York: Van Nostrand Reinhold. Herbs. St. Paul (Minnesota): Hospitality Institute of 45 JC Forrest, ED Aberle, HB Hederick, MD Judge, RA Technology and Management. 46 Merkel. 1975. Principles of Meat Science. San Fran- DR Tainter, AT Grenis. 2001. Spices and Seasonings— 47 cisco: Freeman. A Food Technology Handbook. 2nd ed. New York: 48 J Giese. 1994a. Antimicrobials: Assuring food safety. Wiley-VCH. 49 Food Technol. 48(6):101–110, 1994. R Uhl. 2000. Handbook of Spices, Seasonings, and Fla- 50 ———. 1994b. Spices and seasoning blends: A taste for vorings. Lancaster, Pennsylvania: Technomic Pub. 51 all seasons. Food Technol 48(4):87–90, 92, 94–95, 98. EW Underrinder, IR Hume, eds. 1994. Handbook of 52 K Hirasa, KM Takemasa. 1998. Spice Science and Industrial Seasonings. London: Blackie Academic & 53S Technology. New York: Marcel Dekker, Inc. Professional. 54N JM Jay, GM Rivers. 1984. Antimicrobial activity of some LL Zaika. 1988. Spices and herbs: Their antimicrobial food flavoring compounds. J Food Saf 6:129–139. activity and its determination. J Food Saf 9:97–117. 40454 10 101-110 r1.qxd 7/20/07 12:42 PM Page 101

1 2 3 10 4 5 6 Casings 7 8 Yun-Chu Wu and Suey-Ping Chi 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION are relative newcomers to the artificial field, mainly 22 born out of market demand during the early twenti- 23 The word sausage originally came from the Latin eth century (Anonymous 2004; Forrest et al. 1975; 24 word salsus, which means salted or preserved. In INSCA 1987; Lawrie 1974; Ockerman 1996; Pearson 25 those days, people did not have ways to preserve and Tauber 1984; Pearson and Dutson 1988). 26 their meat, so making sausage was one of the ways to 27 overcome this problem. 28 Dry sausage was born as a result of the discovery TYPES OF CASINGS 29 of new spices, which helped enhance flavor and pre- NATURAL CASINGS 30 serve the meat. Different countries and different 31 cities within those countries started producing their Natural casings are made from the submucosa, 32 own distinctive types of sausage, both fresh and dry. largely a collagen layer of the intestine. The fat and 33 These different types of sausage were mostly influ- the inner mucosa lining are removed. Because small 34 enced by the availability of ingredients as well as the intestines are collagen in nature, they have many of 35 climate. the same characteristics common to all types of col- 36 Basically, people living in particular areas devel- lagen, particularly the unique characteristic of vari- 37 oped their own types of sausage and these sausages able permeability. 38 became associated with this particular area. For Natural casings are hardened and rendered less 39 example, bologna originated in the town of Bologna permeable through drying and smoking processes. 40 in Northern Italy, Lyons sausage from Lyons in Moisture and heat make casings more porous and 41 France, and Berliner sausage from Berlin in tend to soften them, which explains why smoking, 42 Germany. Casings were first used to contain and cooking, and relative humidity must be carefully 43 form chopped meat products. Ground and commin- controlled. 44 uted meat often require some type of casing to hold Animal casings have the advantage of contributing 45 the product intact while it is being processed, pre- to the old-world appearance. They often do not have 46 cooked, distributed, and finally cooked. Today, to be peeled prior to consumption, and have many of 47 casings are used not only to contain and form, but also the physical and chemical properties that make them 48 to flavor, season, and protect products. Consumers very desirable containers for sausage items; but they 49 around the world are so used to sausages being are usually the most expensive type of casings. 50 packed into some type of casings that a variety of cas- Therefore, animal casings are usually used for the 51 ings can now be found in marketed products. Today more expensive sausage items, such as fermented 52 there are numerous types of sausage casings, includ- sausages. Areas of the animal used to manufacture 53 ing natural and artificial such as collagen, cellulose, casings are the small and large intestines, weasand, S54 and plastic. Collagen, cellulose, and plastic casings urinary bladder, stomach, and rectum from most N55 101 40454 10 101-110 r1.qxd 7/20/07 12:42 PM Page 102

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1 meat-producing animals. The utilization of these • Caliper: Diameter of the casing desired is 2 products varies tremendously from country to coun- determined by the country of use and also 3 try and for different sausage types in which they are according to type of sausage ingredient that is 4 going to be utilized. This also indicates the areas that going to be placed in the casing. Modern sausage 5 are removed from the intestinal tract in order to trans- processing equipment requires, however, that the 6 fer the product into usable casings (Table 10.1). caliper of the casing be uniform in order to have 7 As received from the slaughter floor, the intestinal adequate machinability. The greatest demand for 8 tract always is highly microbiologically contami- sheep casing is in the 0.78 to 0.95 in (20 to 24 mm) 9 nated. The cleaning and removal of various internal, diameter range. In some cases, small caliber hog 10 and sometimes external layers are necessary to con- casings can be substituted for the larger sheep 11 vert this product into a useful casing. Many factors casings. The greatest demand for hog casings is in 12 influence the quality of the casing, such as health of the 1.38 in (35 mm) and over range. Beef rounds 13 the animal, species, age of the animal, breed, fodder are normally 3.09 in (33 mm) and over, and 14 consumed, conditions under which the animal was middles are 2.17 in (55 mm) and over. Oversizes 15 raised, portion of the intestinal tract utilized, and are in greatest demand. 16 how the product is handled and processed after the • Curing: Casings are normally cleaned, and then 17 animal is slaughtered. Some determinants for evalu- salted, and only in a few cases are they dried. 18 ating casings include Also, in some cases, some of them may be frozen, 19 but this primarily is utilized for surgical cat gut. • Cleanliness: Casings should be clean, free from 20 Some countries, even in this area, use salted stains, odor, fat particles, parasites, nodules, and 21 products. The curing should be accomplished with ulcers, and should be sound and free from pin 22 high-quality, fresh, and small-particle–sized salt holes. 23 grades. • Strength: Casings should be strong enough to 24 • Packaging: The type of packaging is either withstand the pressures that will be put on them 25 wooden or plastic containers or tins. Often during filling, stuffing, cooking, processing, 26 these containers are lined with plastic bags. Some- storage, and consumer cooking. Only the 27 times plastic bags are utilized and protected by submucosal part of the intestine has the required 28 sacks. However, the trend seems to be in favor of strength to make this possible. 29 plastic containers because of cost and ease of • Length: The number of pieces per hank or bundle 30 sanitation. often varies according to the country in which the 31 casings were collected. The country in which 32 Removal of the Viscera the product is going to be utilized also influences 33 the desirable length. On the average, sheep and hog The first step in casing preparation (Table 10.2) is 34 casings are 299.8 ft (91.4 m), beef rounds are pack- removing the viscera and separating them from the 35 aged in bundles of 54 ft (18 m), and tennis rackets internal organs. This is primarily a hand and/or knife 36 and surgical strings in lengths of 9.24 ft (6 m). operation. The viscera are placed on a table and 37 38 39 40 41 Table 10.1. Composition of natural casings. 42 43 Natural casings are acquired from the alimentary tract of meat animals. The construction of the intestine is 44 composed of five layers, which from inside to outside, are shown, along with the layers that are remaining 45 after it is processed into a casing. 46 47 Hog and sheep casing 1.–Mucosa glands aid in secretion, digestion, and absorption 48 Beef casing 2.–Submucosa—collagen and often fat 49 3.–Circular muscle 50 51 4.–Longitudinal muscle 52 5.–Serosa—collagen, elastin 53S 54N Source: Adapted from Ockerman (1996). 40454 10 101-110 r1.qxd 7/20/07 12:42 PM Page 103

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Table 10.2. Steps for the conversion of the digestive tract into sausage casing. 1 2 1.–Remove the intestine from the animal. 3 2.–Running—remove loose mesentery fat. 4 3.–First stripping—squeeze to force out intestinal contents. 5 6 4.–Wash and cool (50°F water). 7 5.–Mechanically strip or hand-strip. 8 6.–Use brushes to remove fat. 9 10 7.–Slimming—remove tissue layers—revolving drums and warm (115°F) water or hand-slimming. 11 8.–Use strippers or hand-remove appropriate tissue layers. 12 9.–Store overnight in ice and 15–20% saturated salt solution. 13 10.–Grade—species, size, quality. 14 15 11.–Cure—rub with salt; allow to set for 1 week. 16 12.–Remove from cure, shake free of excess salt, rub with fine salt, and pack (40% salt). 17 13.–Flush prior to use. 18 19 Source: Adapted from Ockerman (1996). 20 21 22 separated from the mesentery fat. Again, this is a bearings. Again, a great quantity of 108°F (42°C) 23 hand operation that can be facilitated by using an air- water is used in this operation. Crushing also can be 24 operated knife. The puller usually will start at the accomplished by a hand operation wherein the casing 25 stomach and pull the casing away from the ruffle fat. is scrapped with a dull-bladed knife or an oyster 26 The next step in casing manufacturing is to run the shell. The casing then goes through a mucosa strip- 27 casing through a manure stripper to squeeze out the per, which looks essentially like the manure stripper. 28 liquid and manure using large rollers similar to a Again, 108°F (42°C) water is used to keep the opera- 29 laundry ringer. These rollers are usually rubber and tion sanitary. If labor is very economical, this opera- 30 sometimes are wrapped with burlap. This operation tion also can be accomplished by hand scraping. 31 can also be done by hand and by pulling the casing Next, the casing goes through a finishing machine 32 through the fingers. In either case, a great quantity of to remove any stringlike material and remaining 33 potable water is needed to wash the casings and to mucosa. Rollers again are used in this operation. 34 keep the operation clean. The casing should be Finishing also can be accomplished by hand if a 35 soaked for approximately 30 minutes in 100–108°F great deal of attention is paid to detail. In either 36 (38–42°C) water. system, large quantities of 108°F (42°C) potable 37 In some areas of the world, casings now go water are essential to keep the operation clean. 38 through a fermentation cycle, but in many areas, After finishing, the casings are soaked again in 39 processing casings by fermentation is no longer 50–60°F (10–16°C) water and/or a salt brine tank to 40 legal (e.g., U.S.). If fermentation is to be conducted, remove excess blood. This tank normally has contin- 41 the casings usually are soaked overnight in 72°F uously flowing water to remove the stained liquid. If 42 (22°C) water or until the mucosa and muscular coat- continuously running water is used, salt normally is 43 ings are loosened. Excessive fermentation will not used at this stage. The soaking time usually 44 soften the casings. If fermentation is used, the cas- ranges from 30 minutes to overnight. 45 ings usually are stripped after fermentation, soaked, After the soaking operation, casings usually are 46 restripped, and often restripped again. Next, they are salted either by hand or again by machine. The salt- 47 run through a cleaning machine (which is often a ing and shaking of the casing usually are continued 48 drum with a revolving scraper blade). until the casings absorb 40% salt, at which point 49 If the casing is not fermented, the next step is to they are packed into a container. If the casings are 50 run it through a crushing machine and soaking tank. packed in a slush container, the container will hold 51 The purpose of this is to break the intermucosal 10–15% salt brine. The advantage of using a dry 52 membrane and separate it from the casing. This pack is that the casings become less tangled. Also, S53 machine also has two adjustable rollers with eccentric the casings will be darker in color. N54 40454 10 101-110 r1.qxd 7/20/07 12:42 PM Page 104

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1 Items such as hog bung, hog stomach, blind end or Generally speaking, the natural-casing markets 2 cecum, bladder, beef bung, and weasand usually are include hog stomachs; bungs (hog, beef, and lamb 3 handled by hand, trimmed of excess material, and cecum); beef bladders; hog, sheep, and lamb casings 4 salted again. (small intestine); beef rounds (small intestine of 5 If attention is paid to detail, a large quantity of cool cattle); beef middles (large intestine of cattle); and 6 water is used to keep the casings clean. When labor hog middles (large intestine in hogs). On the average, 7 is inexpensive and strict sanitation is maintained, the small casings used in breakfast sausages are edi- 8 suitable casings can be produced by hand operation ble, but the larger casings such as bungs, bladders, 9 and will make very desirable casings for the sausage and stomachs, are generally not considered as edible. 10 industry. However, a breakdown in any one of these The benefits of natural casings are the remarkably 11 processes will result in an unsanitary product that beautiful, smoked, and cooked appearance. They 12 has little use in modern sausage processing. take on a beautiful color when they’re smoked. 13 Table 10.2 summarizes the various steps in the Natural casings add to the nutritional value of the 14 manufacture of natural casings. product because they are a protein-based material. 15 Casing names and locations, as well as sausage They tend to be flavor-neutral, but they certainly do 16 products for which they are utilized and yield per not create off-flavors in the sausage product. 17 animal can be found in Table 10.3. Table 10.4 is a Natural casings allow products to be placed in 18 summary of how to use sausage casings in the meat upscale markets primarily due to the eye appeal of 19 packing industry. the sausage. 20 If all steps of the process are performed satisfacto- Natural casings are expensive. Unit price of 21 rily, a very gourmet sausage product can be pro- casing per tonnage output of sausage would be at a 22 duced by utilizing value-added and upgraded animal manufacturing cost disadvantage when compared to 23 casings which, in their native state, have almost no similar products manufactured in regenerated colla- 24 value but can be transformed to a very desirable con- gen and cellulose casing. Handling of natural cas- 25 tainer for high-quality sausage products. ings in preparation for sausage manufacture is more 26 27 Table 10.3. Classifications of natural casings used for meat purposes. 28 Natural Appearance 29 Casing Location and Comments Sausage Average Yield/Animal 30 31 Beef 32 Round Small intestine Ringlike, tougher, easily Ring bologna, 90 to 135 ft long 33 handled, less breakage Polish sausage 34 Bung Cecum Capocolla, salami 4 to 5 ft long 35 36 Bladder Bladder Oval or molded Minced specialty, 7 to 14 in wide 37 Mortadella 38 Middles Large intestine Sewed, most expensive, Bologna, salami 20 to 25 feet long 39 adds uniformity 40 Weasand Windpipe 18 to 26 in long 41 42 Hog 43 Round Small intestine May be eaten or peeled Large frankfurter, 42 to 52 ft long 44 hog sausage 45 Bung Cecum May be sewed Braunschweiger 30 to 72 in long 46 47 Middles Large intestine Curly Chitterlings 12 to 16 ft long 48 Stomach Stomach Head cheese, souse 49 Bladder Bladder 5 to 9 in wide 50 51 Sheep 52 Small intestine Most tender, Small frankfurter, 90 ft long 53S most breakage pork sausage 54N Source: Adapted from Ockerman (1996). 40454 10 101-110 r1.qxd 7/20/07 12:42 PM Page 105

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Table 10.4. How to use casings. 1 2 In using natural casing, there should be a drying period in the smoking cycle to obtain uniform smoke color, 3 and great care should be exercised in cooking and smoking these products. Because these products are 4 primarily collagen they behave in the following manner. 5 Dry: They become brittle and shrink; also, smoke will not penetrate. Some drying is necessary to reduce 6 smoke penetration to keep from forming a skin under the casing. 7 Wet: Smoke goes through the casing to the sausage. 8 Acid (smoke): Firm, harder, and tougher. 9 Heat in presence of moisture: They become soft and hydrolyze slowly (sheep is the worst). 10 If these casings are handled properly, the meat product can stand more abuse (less fatting out) than with 11 cellulose casings. 12 13 Source: Adapted from Ockerman (1996). 14 15 16 labor intensive and is a bit more delicate than the obtain a larger, more uniform finished product, these 17 handling of collagen or cellulose casings. Natural casings are custom-made and can be purchased in 18 casings must be rinsed because they have been almost any shape or size suitable to the needs of the 19 packed and stored with salt—the salt must be processors. Most of the products are used exclu- 20 washed out of them. It takes a little more finesse and sively for liver sausage, Genoa or Thuringer, Braun- 21 artisan skill to handle natural casings for filling schweiger, summer sausage, and Cervelats. 22 sausage product (Anonymous 2004; Dewied 2001; 23 Naghaski and Rust 1987; Naghaski 1971; Ockerman Hog Middles/Chitterlings 24 1988, 1996; Pearson and Dutson 1988). 25 Hog middles/chitterlings are put up in three calibers: 26 wide, medium, or narrow. The size is determined by HOG CASINGS 27 the location of the item within the animal. There are 28 Hog casing is the most popular casing, which can normally nine to ten 1 m pieces to a bundle. Hog 29 actually be used for almost any sausage. It is used for middles are easily recognizable by their curly appear- 30 the manufacture of cooked sausage, country-style ance. Chitterlings are also available and selected into 31 sausage, fresh pork sausage, Pepperoni, Italian 5 mm calibers. 32 sausage, large frankfurters, Kishka, Kielbasa, and 33 Bratwurst—to name just some of the best-selling BEEF AND OX CASINGS 34 items (Dewied 2001; INSCA 1987; Ockerman 1996; 35 RAMP 2004; Sausagemaking 2004). Beef casings, such as rounds, middles and bungs are 36 Hog casings are used commonly for manufactur- ideal for boiled, cooked, and cured sausages of high- 37 ing of Bratwurst, Saucisson (chitterlings), liver est quality. Beef bladders are used for specialty 38 sausage (hog bungs), pork sausage, and Polish sausages and mortadellas. Beef rounds are used for 39 sausage. Typically, hog casings are used for fresh and bolognas, liver sausage, and certain types of fer- 40 smoked sausages, as well as breakfast links. Hog mented sausages. Beef middles are used for liver 41 bungs are used for liver sausages and certain vari- sausages and dry sausages. Beef bungs are used for 42 eties of dry sausages. Hog middles are used for liver specialty pork products, bolognas, and salamis. They 43 sausage and certain types of Italian salamis. are also used for sausages that require a very thick 44 Common origins of hog casings include China, casing, such as bologna and salami. 45 Europe, and the U.S. The three most used beef casings are beef bung caps, beef rounds, and beef middles. Beef bung 46 47 Hog Bungs: Regular and Sewn, Hog Bungs, caps—are used for Capocolla, veal sausage, large 48 and Hog Bung Ends bologna, and Lebanon and cooked salami. Origins of beef bungs, rounds, and casings are 49 Regular hog bungs are sold as individual pieces South America, especially Brazil, and Europe. 50 and are used primarily for liver sausage and 51 Branschweiger. Sewn (or Sewed) hog bungs are pro- 52 Beef Rounds and Middles duced in double-walled and single-walled varieties; S53 they are made by sewing two or more pieces of These casings derive their name from their character- N54 smaller sizes of regular hog bungs together. To istic “ring” or “round” shape. Beef rounds are used 40454 10 101-110 r1.qxd 7/20/07 12:42 PM Page 106

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1 for ring bologna, ring liver sausage, Mettwurst, Pol- Ox Runners 2 ish sausage, blood sausage, Kishka, and Holsteiner. Ox runners are excellent for traditional salami. 3 Beef rounds and middles are mainly being used for 4 Servelats, round Bologna, Fleischwurst, and Hol- 5 steiner Italian salami. Beef middles can be used for LAMB AND SHEEP CASINGS 6 Leona style sausage, all other types of Bologna, dry Sheep casings are very tender, and used in the pro- 7 and semidry Cervelats, dry and cooked salami and cessing of fresh and smoked sausages. Sheep casings 8 veal sausage. are the highest-quality, small-diameter casings used 9 Beef middles are measured in sets or bundles of for the finest in sausages, such as Bockwurst, frank- 10 9 and 18 m (29–30 ft and 57–60 ft) each. Beef furters, and Port Sausage. 11 middles can be sewn so that they have a uniform Origins of lamb and sheep casings are Turkey, 12 diameter and a uniform length, with or without a Iran, China, Mongolia, Australia, New Zealand, 13 hanger (stitching loop). Afghanistan, Pakistan, Chile, Peru, Egypt, and 14 Syria. 15 Beef Bladders 16 Beef bladders, the largest diameter casings from HORSE CASINGS 17 cattle, are oval in shape, and will hold from 2.5–6.5 kg 18 (5–14 lb) of sausage. They are used chiefly for The high standards of Italian salami are best fulfilled 19 minced specialty sausage and mortadella, either in by using horse casings. 20 their natural oval form, in square molds for sand- Origins of horse casings are South America 21 wich slices, or in the flat, pear-shaped styles. (mainly Brazil and Uruguay) and Europe (mainly 22 France and Eastern Europe). 23 Natural Ox Bung 24 GLOSSARY OF TERMS FOR NATURAL CASINGS Natural ox bungs are ideal for making haggis, mor- 25 tadella, or any sausage or salami that requires a large 26 See Table 10.5 for a summary of terms for natural casing. 27 casings. 28 29 30 Table 10.5. Glossary of terms for casings. 31 32 Bundles A measured unit of casings ready for sale in salted, preflushed, or tubed form; bundles 33 will be either hog casings or sheep casings consisting of 91 m (100 yd). Bundles can 34 also refer to a customer-defined specification. 35 Green Weights Represents approximate stuffing capacity of casings before cooking or smoking, 36 per 91 m lengths. 37 Hanks Another essentially interchangeable term with the same meaning as bundles, applying 38 to hog and sheep casings. 39 40 Nodules Pimples that appear on some beef rounds or beef bung caps. 41 Sets A unit of beef casings ready for sale in salted form, consisting of 18–30 m for beef 42 rounds and 9–18 m for beef middles. 43 Shirred Refers to the result of applying a casing to a dummy transfer horn or to a flexible plastic 44 sheath to expedite the stuffing process. 45 Tierce A shipping container made of plastic with a packing volume to 208 l (approximately 46 55 gal). 47 48 Windows Damage to casings caused by overcrushing. They are approximately half the thickness 49 of the casing. 50 Whiskers The capillary that holds the intestine in the fat and provides a flow of blood to the intestine. 51 When removing the intestine with a knife, the capillary is not completely removed, 52 creating a hairlike appearance on the surface of the casing. 53S 54N Source: Adapted from INSCA (1987); Rust (1988). 40454 10 101-110 r1.qxd 7/20/07 12:42 PM Page 107

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COLLAGEN CASING CELLULOSE CASING 1 2 Collagen casings are made from the gelatinous sub- This is an artificial casing made from solubilized 3 stance (collagen) found in the connective tissue, cotton or wood pulp. It is very uniform, strong, and 4 bones, and cartilage of all mammals. The substance is not quite as susceptible to bacteria as other types of 5 harvested from the animals and reconstructed in the casings. Skinless hotdogs are made with cellulose 6 form of a casing. Most sausage in the U.S. is stuffed casings (Anonymous 2004; NOSB 2001; RAMP 7 into this kind of casing (Devro 2006; Hood 1987; 2004; Viskase 2006). 8 Mid-Western Research & Supply 2006; RAMP Small-diameter cellulose casings are used to man- 9 2004). ufacture most skinless frankfurters in the United 10 Sausages produced by this method are easier to States. They probably have captured more than 90% 11 pack than those in variable animal casings due to of the small-diameter frankfurter market in this 12 their uniform size and weight. This is advantageous country. 13 in both retail and professional markets. Large cellulose casings are larger in diameter and 14 The collagen is derived from the middle corium thicker-walled cellulose casings. They are used to 15 layer of cattle hide, obtained from hides by the contain the deli-style bolognas. These products are 16 mechanical removal of the epidermal and flesh lay- typically about 2 in diameter, shaped nicely, glossy, 17 ers. After a pretreatment, the collagen-rich corium and have an attractive appearance. 18 layer is referred to normally as the limed collagen The third type of regenerated cellulose casing is 19 split. The process involves an extensive washing, the very large casing used to shape and hold bone- 20 decalcification, and buffering process of the limed less and semiboneless hams. These very large cellu- 21 collagen split to give a purified food-grade collagen lose casings are thick-walled and will be sufficiently 22 material. This is further reduced into a fibrous slurry, robust for the ham-filling process. 23 which is blended with a food-grade acid and veg- The fourth type of regenerated cellulose casing is 24 etable cellulose fiber to produce a homogeneous col- the fibrous casing used to make dry and semidry 25 lagen protein gel. The collagen fibers are arranged to sausage. They are cellulose impregnated paper. The 26 give a crisscross structure that contributes signifi- fibrous casing is extremely strong and is used to stuff 27 cantly to the strength and caliber stability of the fin- sausage that is very tightly packed because it will not 28 ished product. break. The inside of this casing is coated with pro- 29 The following are advantages of collagen sausage tein that allows it to shrink with the meat as it dries 30 casings (Devro 2006): out. Fibrous casing is manufactured by bonding 31 regenerated soluble cellulose (viscose) to a highly • Elastic (shrinks with the product) 32 refined paper. Together they become very robust; • Digestible (edible) 33 they are resistant to stretching and breakage during • Permeable to smoke and moisture strong 34 the sausage filling process. Fibrous casings are man- • Uniform in size 35 ufactured with uniform diameters. • Nonrefrigerated storage 36 The last type of regenerated cellulose casing is • Consistent casing size equals reduced giveaway 37 called moisture-proof (MP) casing. This casing is a • Fresh casings allow the “real bloom” of product 38 fibrous casing with a barrier latex coating. It is used to show through 39 for sausage products that must be cooked or need an 40 Collagen casings can be used for any comminuted, extensive shelf life. It is an expensive but effective 41 coarse, or fine-chopped sausage products. There are product for water cooking and extending product 42 very few sectioned-and-formed meats placed in col- shelf life. MP casings, however, are expensive. 43 lagen casings. Sausage products that are manufac- Nylon casing is also a barrier and less expensive. 44 tured in regenerated collagen casings have the snap, The difference is that MP casings are coated-fibrous 45 bite, and attractiveness that natural casings bring to casings, and they offer exact-diameter control. The 46 the product. However, there is often a production nylon casing, although less expensive and a barrier, 47 cost advantage of choosing natural versus collagen doesn’t offer the same exact-diameter control. 48 casings in ease of handling and cost of product. The small-diameter cellulose casing is used to 49 There is another development in regenerated col- manufacture a variety of smoked sausages and some 50 lagen casing products brought to the market by one types of fresh sausages, such as chorizo or Italian 51 casing manufacturer. This method uses the approach sausage. The large cellulose casing would be used for 52 of a coextrusion of collagen dough with a sausage bolognas and salamis. The very large cellulose cas- S53 batter. ings are typically for boneless and semiboneless N54 40454 10 101-110 r1.qxd 7/20/07 12:42 PM Page 108

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1 hams. The fibrous casing would be used for all the with a first clip attached to begin the stuffing 2 fermented sausages, large-diameter cooked sausages, process. They are barrier casings, and they offer 3 and deli-style meats. tremendous graphics for attractiveness to the pack- 4 The small-diameter cellulose casing is manufac- age. They can be offered in various sizes. They typi- 5 tured and formed in shirred sticks, just as the small- cally range in sizes from 1–5 in diameter. The nylon 6 diameter collagen casing. The difference is that the casing’s challenge has been the inability to be 7 small-diameter cellulose is much stronger and elas- smoked because nylon/plastic casing is a barrier. 8 tic than collagen and is easier to use to manufacture Table 10.6 elucidates the advantages and disad- 9 products. vantages of different casing types. 10 Productivity is a strength for cellulose casings, 11 and the cost is lower than collagen casings. Cellulose REGULATORY COMPLIANCE 12 is a moisture-sensitive membrane, and it will allow 13 water-soluble material such as smoke to move across In the U.S., all casing suppliers are under the juris- 14 it to the surface of the sausage. Products manufac- diction of the Food and Drug Administration (FDA) 15 tured in small-diameter cellulose casings are usually because they are manufacturing a food container that 16 smoked and cooked, and the casings are removed is in direct contact with food. All components used 17 before the product goes to market. The large-cellulose in their casing materials must comply with Title 21 18 casings are used for deli bolognas, as well as bone- Code of Federal Regulation—whether it is a natural 19 less and bone-in hams. casing and the way a natural casing is manufactured; 20 Fibrous casings are strong, smoke permeable, and the collagen casings and the procedures approved for 21 can be peeled easily after products have been cooked regenerated collagen, or the regenerated cellulose 22 and cooled. All fibrous and cellulose casings must be casing and procedures approved for regenerated cel- 23 removed before product is consumed. Spent casing lulose; and all materials used to plasticize those cas- 24 is the one key downside of cellulose casings. The ings to keep them flexible, supple, and damp. 25 following are advantages of fibrous sausage casings The second step is complying with United States 26 (Viskase 2006): Department of Agriculture (USDA) regulations, 27 because casings are used in USDA establishments. • Excellent size uniformity, giving uniform product 28 The casing manufacturers must supply to the estab- and increased yields 29 lishment owner a Continuing Product Guarantee that • Consistent casing color due to a controlled dying 30 meets the requirements of Title 7. It is different from process 31 an individual guarantee in that an individual guaran- • Wide range of colors available 32 tee is for a particular item with a particular specifica- • Can be printed, clipped, string-tied, cut, and 33 tion. A Continuing Product Guarantee meets the shirred 34 requirement of a nonchanging item. • Nonrefrigerated storage 35 The USDA occasionally does a compositional • Some permeable to smoke and moisture 36 review check. USDA offices will put a call out to • Strength 37 casing suppliers for them to update their files to 38 make sure that everything is in compliance. This 39 SYNTHETIC CASING happens every so often. 40 This casing is made from alginates and requires no Once casings come into a USDA establishment, 41 refrigeration. It is used by mass producers and can the establishment must comply with USDA regula- 42 be made in different colors: red for bologna, clear for tions. Cellulose casings can not be eaten by the con- 43 some salamis, and white for liverwurst. Much like sumer and must be removed before consumption. 44 the cellulose casing, it is uniform and strong. The wording on the cellulose casing box must iden- 45 tify that the casings remaining on the product must 46 PLASTIC/NYLON CASING be removed. Any collagen casings that cannot be 47 eaten must be identified as inedible and removed. 48 Nylon casing is a relatively inexpensive and attrac- Those natural casings that should not be consumed 49 tive barrier casing. It allows a way to produce a high must also be identified To be removed. 50 volume of items that need shelf life or products that Any product that goes to the retail market cannot 51 must be water-cooked and then enter the market— mislead or cause the end-user to assume that it is 52 and the product does not have to be repackaged. something different. In other words, if a processor 53S There are both cost and high-productivity bene- chooses to send a smoked sausage to retail in a 54N fits. They are also delivered in shirred casing units fibrous casing and the color of the casing is brown, 40454 10 101-110 r1.qxd 7/20/07 12:42 PM Page 109

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Table 10.6. Advantages and disadvantages of casing types. 1 2 Natural Collagen Cellulose 3 Most expensive, cost/lb product Most expensive Less expensive Least expensive 4 5 Refrigeration storage Yes Yes No 6 Degree of tenderness Most tender Less tender Peeled 7 Break during processing Most likely Less likely Least likely 8 9 Casing preparation cost Most expensive None None 10 Soaking and flushing before use Yes No Sometimes soaking 11 Ease of smoke penetration Most penetration Less penetration Least penetration 12 Best machinability Least Less Best 13 14 Best product yield, per ft of casing Least Less Best 15 Finished product uniformity Least Less Best 16 Cost of casing removal None None Most 17 Printability None Limited Best 18 19 Old World appearance Best Less None 20 Ease of plant storage Least storage Less storage Best storage 21 22 Source: Adapted from Ockerman (1996). 23 24 25 on/eyes-on” scrutiny. The fully cleaned casings are the end-user might not necessarily associate that 26 placed in a saturated salt environment to prepare for brown color with the casing—and may think that the 27 further processing. The casings are then sorted into brown color is the color of the product. That is con- 28 various grades and diameters. The selection process sidered misleading, which the USDA does not 29 is dictated by such factors as type of animal and cri- accept (Anonymous 2004; INSCA 1987). 30 teria set by the casing processor, and ultimately the 31 sausage producers. HANDLING CASINGS 32 33 Today, casing recovery—most often done in large DETERMINING QUALITY 34 slaughtering facilities—is both a precise science and 35 an elaborate process. It requires high-level expertise, Qualities of casing are determined in several precise 36 state-of-the-art machinery, and maximum sanitation and labor-intensive ways. In sheep, for example: 37 and quality control procedures. Because the intrinsic 38 • A Quality casing is determined during selection, value of the raw material represents a large part of 39 and is defined as a casing with no holes or the finished casing product, every inch of tract needs 40 weakness. This casing can be used for the finest to be utilized. In the slaughterhouse, the viscera of 41 frankfurter emulsion. the animal are removed, and the various parts of the 42 • B Quality casings are of acceptable strength and intestinal tract are separated. This separation of parts 43 quality for coarse ground emulsions, such as is instrumental in creating a variety of products 44 those used in pork sausage. Export Quality is ranging all the way from pig chitterlings to sheep 45 sometimes used. This term describes casings as appendixes for pharmaceutical products (Anonymous 46 free of nodules (pimples) or scores (windows). 2004; INSCA 1987; Ockerman 1996; Pearson and 47 Dutson 1988). With hog casings, there is a single quality standard 48 The casings are prepared by removing the manure; with several specifications for length. Where the 49 mucosa (raw material for the anticoagulant heparin); various hog casings originate from—taking into 50 and any undesirable elements such as fat, threads, consideration factors of species, climate, and diet— 51 and animal fluids. This removal, facilitated in a will generally determine the different characteris- 52 series of both hot- and cold-water soaks, is accom- tics of the casings. Some will be white or virtually S53 plished by machine crushing under close “hands- transparent/clear; others will be darker and more N54 40454 10 101-110 r1.qxd 7/20/07 12:42 PM Page 110

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1 opaque and will have more visible veining. These and cost savings. They also offer opportunities that 2 characteristics also have an effect on the tenderness are not available to natural casings. Both types of cas- 3 or bite of the casing. ings complement each other in the manufacturing of 4 Clear hog casings are generally used for fresh prod- very acceptable products for the consumers. 5 ucts. Thicker and stronger casings such, as Chinese 6 hog casings, are generally best suited for smoked 7 products, because these casings better withstand the REFERENCES 8 smoking process and because casing appearance is Anonymous. 2004. Casings 101. The National Provi- 9 not as critical a selling feature, due to the smoking sioner. NP/2004/08, p. 26. 10 process itself (INSCA 1987). Devro. 2006. Collagen casings. http://www.devro.plc. 11 uk/products/index.htm. 12 Dewied. 2001. Casing Products. http://.www.dewied. 13 TEST PROCEDURES com/casing/htm. 14 The traditional methods for grading and testing nat- JC Forrest, ED Aberle, HB Hederick, RA Merkel. 15 ural casings are 1975. Principles of Meat Science. San Francisco: 16 Freeman. 17 • Water testing for sheep and hog casings. LL Hood. 1987. Collagen in sausage casing. In: 18 • Air testing for beef casings. Advances in Meat Research. Vol. 4. New York: 19 Elsevier. 20 The casings are appropriately filled with water or air INSCA. 1987. Facts about Natural Casings. Inter- 21 and periodically expanded under pressure to check national Natural Sausage Casings Association. 22 for size and quality. The casings are then cut to final RA Lawrie. 1974. Meat Science. New York: Pergamon. 23 sizes, and quality specifications are confirmed dur- Mid-Western Research & Supply, 2006. Collagen 24 ing quality control. sausage casings. http://www.midwesternresearch. 25 It is of utmost importance to adhere to the normal com/CSAING_COLLAGEN.htm. 26 practice of filling product, and follow proper han- J Naghaski, RE Rust. 1987. Natural casings. In: JF 27 dling procedures of Good Manufacturing Practices Price, BS Schweigert, eds. The Science of Meat and 28 (GMPs). As casings are removed from meat prod- Meat Products. Westport, Connecticut: Food and 29 ucts, there is the potential for contamination or Nutrition Press. 30 recontamination. Significant attention has been J Naghaski. 1971. By-products—Hide, skins, and natu- 31 focused on minimizing recontamination at the point ral casings. In: JF Price, BS Schweigert, eds. The 32 of casing removal of hotdogs and frankfurters in Science of Meat and Meat Products. Westport, Con- 33 meat processing plants. It has been a real issue, but necticut: Food and Nutrition Press. 34 processors have made a great effort to deal with it. NOSB. 2001. Cellulose processing. National Organic 35 There is also a risk in food preparation of Standards Board Technical Panel Review. 36 sausages. For example, almost all hotdogs are fully HW Ockerman. 1996. Meat Chemistry. Columbus, 37 cooked when they enter commercial trade. Many OH: The Ohio State University. 38 times the end-user will not reheat the hotdogs or will HW Ockerman, CL Hansen. 1988. Animal By- 39 do a quick microwave. Any low-level contamination product–processing. Ltd., Cambridge, U.K.: VCH 40 that could have occurred during packaging could Publisher. 41 create risk for the consumer eating this product AM Pearson, TR Dutson. 1988. Edible meat by- 42 (INSCA 1987; Mid-Western Research & Supply products. In: Advances in Meat Research. Vol. 5. 43 2006; Naghaski and Rust 1987). New York: Elsevier. 44 AM Pearson, FW Tauber. 1984. Processed Meats. 45 Westport: AVI. 46 CONCLUSION RAMP. 2004. Natural and artificial casings. http:// 47 Natural casings give the best flavor and appearance to ramp.ch/English/Produkte_E.htm. 48 the final product. A natural casing enhances and com- Sausagemaking. 2004. Casings. http://www.sausage- 49 plements the natural juices and quality of the meat making.org/acatalog/Casings.html. 50 and spices. Natural casings permit deep smoke pene- Viskase. 2006. Products/Applications. http://www. 51 tration. However, artificial casings offer uniformity viskase.com/products.php. 52 53S 54N 40454 11 111-124 r1.qxd 7/20/07 12:51 PM Page 111

1 2 3 4 5 6 7 8 9 10 11 12 13 14 Part III 15 16 17 18 19 Microbiology and Starter Cultures 20 21 22 for Meat Fermentation 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 S54 N55 40454 11 111-124 r1.qxd 7/20/07 12:51 PM Page 113

1 2 3 11 4 5 6 Microorganisms in Traditional 7 8 Fermented Meats 9 10 Isabelle Lebert, Sabine Leroy, and Régine Talon 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION a great diversity in the methods of production lead- 22 ing to diversity in the size of the sausages and in 23 In Europe, natural fermented sausages have a long their organoleptic characteristics. This diversity is 24 tradition originating from Mediterranean countries observed between the countries but also between dif- 25 during Roman times (Comi et al. 2005). Then the ferent regions in a country. 26 production spread to Germany, Hungary, and other As a consequence, the type of microflora that 27 countries including the United States, Argentina, and develops in traditional sausages is closely related to 28 Australia (Demeyer 2004). Today’s Europe is still the diversity in formulation, and to the fermentation 29 the major producer and consumer of dry-fermented and ripening practices. The knowledge and the con- 30 sausages (Talon et al. 2004). trol of this typical microflora during the processing 31 There are a wide variety of dry-fermented prod- are essential in terms of the microbiological quality, 32 ucts on the European market as a consequence of organoleptic characteristics, and food safety. 33 variations in the raw materials, formulations, and The objective of this paper is to review the diver- 34 manufacturing processes, which come from the sity of manufacturing practices and microflora in 35 habits and customs of the different countries and traditional fermented sausages. The variety of lactic 36 regions. But from a global viewpoint, two categories acid bacteria (LAB) and gram-positive catalase- 37 can be distinguished: Northern and Mediterranean positive cocci (GCCϩ), which are the two categories 38 products with some specific characteristics. In of bacteria that play a significant role in the final 39 Northern products, rapid acidification by lactic acid organoleptic and hygienic properties of the fer- 40 bacteria to a final pH below 5 and smoking ensure mented sausages is also reported. 41 safety, improve shelf life, and contribute largely to 42 the sensory quality. In Mediterranean products, acid- 43 ification reaches a final pH above 5. Safety and shelf TRADITIONAL SAUSAGE 44 life are mainly ensured by drying and lowered water MANUFACTURE 45 activity (Demeyer 2004). In both categories, the 46 industrial development in the second half of the Traditional fermented sausage can be defined as a 47 nineteenth century has led to using starter cultures to meat product made of a mixture of meat (often 48 control sausage manufacturing. However, consider- pork); pork fat and salt, including eventually sugar; 49 ing the recent abundant literature, it appears that in nitrate; and/or nitrite. The meat to fat ratio is 2 to 1 in 50 Mediterranean countries there are still a lot of tradi- most industrial sausages mixes, whereas in tradi- 51 tional naturally fermented meats as outlined in this tional ones, the ratio is variable. The fat percentage 52 chapter. These traditional products are often known could be very low (about 10%), or high (40%), or 53 at local or regional levels. They are characterized by difficult to evaluate in mixes prepared with raw S54 N55 113 40454 11 111-124 r1.qxd 7/20/07 12:51 PM Page 114

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1 materials in which lean and fat are not separated The relative humidity during the fermentation 2 (Fontana et al. 2005a; Lebert et al. 2007). The mix- varies from a minimum of 63–75% to maximum val- 3 ture is seasoned with various spices depending on ues of 86–95% and during drying from 64–69% 4 the recipe. Contrary to industrial products inocu- to 70–85% (from references in Tables 11.1a and 5 lated by starter culture, traditional ones are fre- 11.1b). This diversity leads to variable water content 6 quently not inoculated, and fermentation relies of traditional sausages at the end of drying. Thus, 7 on the indigenous microbiota. Backslopping, men- water activity varies from 0.83 to 0.93 in French, 8 tioned by only few authors (Leroy et al. 2006), seems Spanish, Portuguese, and Italian sausages, and it was 9 to be a marginal practice for traditional producers. higher (approximately 0.95) for most of the Greek 10 The mixture is stuffed usually into natural casings. traditional sausages (Talon 2006). 11 The resulting sausages are often processed in two 12 consecutive and separate stages, referred to as 13 fermentation and ripening/drying, respectively DESCRIPTION OF ECOSYSTEMS 14 (Demeyer 2004). Greek sausages are often smoked TECHNOLOGICAL MICROFLORA 15 (Drosinos et al. 2005; Papamanoli et al. 2003; 16 Samelis et al. 1998). In industrial production, lactic acid bacteria (LAB) 17 Industrial manufacturers, to standardize their pro- and Staphylococcus or Kocuria (GCCϩ) are usually 18 duction, carry out the fermentation and the drying used as starter cultures. They have been developed to 19 in rooms with well-controlled conditions of tem- improve the hygienic and sensory qualities of the 20 perature, relative humidity (RH), and air velocity. final product (Talon and Leroy-Sétrin 2006; Talon 21 Traditional producers carried out the process in less- et al. 2002). 22 controlled rooms for temperature and RH (Parente In traditional sausages, LAB constitute the major 23 et al. 2001). Some of these producers dry their microflora at the end of the ripening stage and their 24 sausage in a natural dryer depending on local cli- final levels are similar in the different sausages pre- 25 matic conditions (Corbiere Morot-Bizot et al. 2006; sented in Tables 11.1a and 11.1b. Even if their initial 26 Lebert et al. 2007). levels vary and are lower than that of industrial 27 Conditions of fermentation vary in terms of tem- sausages inoculated, their final levels are close to the 28 perature and duration. When the fermentation is car- one of industrial manufacturers. LAB usually 29 ried out at high temperature (between 18–24°C), increase the very first days of fermentation (Comi 30 usually it lasts between 1 and 2 days (Cocolin et al. et al. 2005), and they remain constant during ripen- 31 2001a; Comi et al. 2005; Mauriello et al. 2004). ing at about 7 to 9 log cfu/g (Cocolin et al. 2001a; 32 However, even at high temperature, the fermentation Comi et al. 2005). Rantsiou et al. (2005b) observed 33 can last 7 days for Greek (Drosinos et al. 2005; that in one fermentation of naturally fermented 34 Papamanoli et al. 2003; Samelis et al. 1998), Argen- northeast Italian sausages, LAB population increased 35 tinean (Fontana et al. 2005a; Fontana et al. 2005b), more slowly. The initial population can be low, as 36 and some Italian sausages (Cocolin et al. 2001b). observed in Salame Milano in Italy (Rebecchi et al. 37 Fermentation can be carried out at low tempera- 1998) and in a French sausage manufactured at low 38 ture (10–12°C), and lasts approximately 1 week temperature (Chevallier et al. 2006), but it is generally 39 (Chevallier et al. 2006; Lebert et al. 2007; Mauriello comprised of between 3.2 and 5.3 log cfu/g (Tables 40 et al. 2004). 11.1a and 11.1b). LAB growth is often correlated 41 Similarly, conditions of drying vary in terms of with the decrease in pH in the first stage of matura- 42 temperature and duration. The variation of tempera- tion (Cocolin et al. 2001a; Cocolin et al. 2001b). 43 ture is less important than for fermentation; it ranges Because of the good adaptation of the LAB to the 44 mainly between 10–14°C for French (Chevallier et al. meat environment and their fastest growth rate dur- 45 2006; Lebert et al. 2007), Italian (Cocolin et al. ing fermentation and ripening of sausages, they 46 2001a; Cocolin et al. 2001b; Comi et al. 2005; become the dominant microflora. 47 Coppola et al. 2000; Rantsiou et al. 2005b), and In traditional sausages, GCCϩ constitute the sec- 48 Greek sausages (Papamanoli et al. 2003). The dura- ond microflora at the end of ripening. In the majority 49 tion of drying for these products varies from 4 to 12 of the sausages, GCCϩ have a population of 6–8 log 50 weeks. Higher drying temperature between 15–19°C cfu/g, which is generally inferior to that of the LAB 51 can be observed for Argentinean (Fontana et al. and close to the population reached in industrial 52 2005a; Fontana et al. 2005b) or Greek sausages sausages. GCCϩ are poor competitors in the pres- 53S (Drosinos et al. 2005; Samelis et al. 1998); it is ence of active aciduric bacteria. Their initial level 54N accompanied by short ripening from 1 to 3 weeks. varies from 3.1 to 4.4 log cfu/g (Tables 11.1a and 40454 11 111-124 r1.qxd 7/20/07 12:51 PM Page 115 Data are expressed in log cfu/g in log Data are expressed Clostridia. : Sulfite reducing Clostridia : Gram-positive catalase-positive cocci; catalase-positive : Gram-positive ϩ Dry FuetProduct Chorizo Product : number of positive samples out of total samples or abs: absence in 25 g; ±: standard deviation; (1) Drosinos et al. (2005); samples out of total or abs: absence in 25 g; ±: standard deviation; : number of positive <2<1 <2 <1 1.3 ± 1.6 0.7 ± 1.3 <1 <2 <1 <2 <1 <2 0.3 ± 0.5 <2 <2 <2 0/99/9 0/9 0/9 abs 4/4 abs abs abs 1/10 abs 2/7 abs abs abs abs abs abs abs abs L. monocytogenes 4.4 ± 0.6 3.0 ± 1.45.1 ± 4.4 ± 0.8 0.5 5.2 ± 0.7 <2 7.0 ± 0.9 4.5 ± 1.0 6.3 ± 0.9 <2 3.5 6.6 4.2 6.3 4.4 4.0 5.2 4.5 and Microbial ecosystems of traditional fermented sausages. fermented Microbial ecosystems of traditional Salmonella ϩ except for except et al. (2003); (4) Lebert et al. (2007). Aymerich (2) Samelis et al. (1998); (3) Sausages of Time Salami F01 F07 Table 11.1a. Table CountryReference GreeceProcess (1)TVCLABGCC Yeasts/molds 0 Day Enterococci GreecePseudomonas 5.3 ± 0.8 28 Days (2) 4.1 ± 0.6Enterobacteria 4.5 ± 1.0 7.6 ± 1.1 0 Day S. aureus 4.1 ± 1.2 2.8 ± 1.4 7.8 ± 0.5Clostridia 6.1 ± 0.5 2.5 ± 0.3 5.1 ± 3.9 ± 0.6 28 DaysSalmonella 0.3 5.3 ± 0.7 8.1 ± 0.1 Spain 2.8 ± 4.2 ± 0.5 0.6 8.0 ± Final <1 0.2 5.0 ± 0.2 Count; LAB: Lactic acid bacteria; GCC Viable 7.9 ±Total TVC: 0.7 (3) Final 3.2 ± 1.0 2.7 ± 1.0 8.5 ± 0.7 2.6 ± 0.5 <2 0 Day France 3.5 88 Days 3.3 0.3 ± 0.6 0 Day 8.6 0.8 ± (4) 1.0 3.3 55 Days 4.0 4.2 3.1 2.6 5.8 7.9 1.0 5.7 4.5 4.4 5.9 2.6 L. monocytogenes L. monocytogenes pH 6.3 ± 0.1 4.9 ± 0.2 6.2 ± 0.1 5.1 ± 0.1 6.0 ± 0.2 5.5 ± 0.1 5.6 5.4 5.7 5.8

115 40454 11 111-124 r1.qxd 7/20/07 12:51 PM Page 116 Data are expressed in log cfu/g in log Data are expressed Clostridia. : Sulﬁte reducing Clostridia : Gram-positive catalase-positive cocci; catalase-positive : Gram-positive ϩ absabs abs abs abs abs NP NP abs abs NP NP abs abs NP NP 1.5 ± 2.7 NP : number of positive samples out of total or abs: absence NP: not performed; ±: number of positive d.: days : standard deviation; 4.0 5.5 4.0 ± 0.7 5.2 ± 0.8 3.2 ± 0.1 5.2 ± 0.2 3.1 ± 0.2 4.6 ± 0.2 3.7 ± 0.1 5.0 ± 0.1 L. monocytogenes abs abs <2 <2 <2 <2 <2 <2 <2 <2 and Microbial ecosystems of traditional fermented sausages. fermented Microbial ecosystems of traditional Salmonella ϩ except for except (5) Fontana et al. (2005a); (6) Comi (2005); (7) Rantsiou (2005b). (5) Fontana Table 11.1b. Table CountryReferenceSausagesDay ArgentinaTVC (5)LABGCC 0 Day Italy 5.3 14 Days 4.1 (6) 0 Day 7.2 28 Days 7.1L. monocytogenes 5.4 ± 0.7 0 Day 4.6 ± 0.8 7.8 ± 1.2pH 120 Days 8.4 ± 0.0 (7) 4.5 ± 0.3 Count; LAB: Lactic acid bacteria; GCC Viable Total TVC: 0 Day 3.2 ± 0.9 7.0 ± 0.0 8.8 ± Product C 0.2 4.7 ± 45 Days 0.4 5.3 ± 0 Day 0.2 4.2 ± 5.9 0.2 8.3 ± 0.1 5.1 ± 0.2 28 Days 4.2 ± 0.2 Product L 9.1 ± 0.5 5.2 8.5 ± 0.1 Product U 5.7 ± 0.1 5.5 5.6 5.6 5.7 5.8 5.6 Yeasts/moldsEnterococciPseudomonas 3.9 2.4 4.7 5.5 2.7 ± 1.6 3.9 ± 1.6 ± 0.2 1.4 6.1 ± 2.0 ± 0.2 0.0 2.1 ± 0.3 6.7 ± 0.1 4.1 ± 3.6 ± 0.2 0.8 3.2 ± 0.1 <2 5.5 ± 0.5 3.7 ± 3.0 ± 0.4 0.4 6.3 ± 2.3 ± 0.3 0.3 EnterobacteriaS. aureus Clostridia 2.5Salmonella 2.7 3.2 ± 0.9 3.8 ± 1.5 1.7 ± 0.2 <1 1.9 ± 0.2 2.6 ± 0.1 2.4 ± 0.1 4.7 ± 0.5

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11.1b). In a French sausage, their level was lower in the type of sausages (Tables 11.1a and 11.1b). They 1 winter (low temperature) than in spring (higher tem- ranged from 1.7 to 4.4 log cfu/g for enterobacteria 2 perature) (Corbiere Morot-Bizot et al. 2006). GCCϩ and from 1.5 to 5.2 log cfu/g for Pseudomonas. In 3 sometimes grew during the fermentation period to Greek sausages, they were progressively eliminated 4 about 106–108 cfu/g (Mauriello et al. 2004) but they regardless of their initial population (Drosinos et al. 5 usually grew during ripening (Comi et al. 2005). An 2005; Samelis et al. 1998). Other authors found that 6 increase in the pH to 5.5–5.7 and sometimes 6.2 at enterobacteria and Pseudomonas increased during 7 the end of the ripening period (Chevallier et al. the fermentation (Comi et al. 2005). Then enter- 8 2006) could be explained by the proteolytic activity obacteria remained constant until the end while 9 of various microorganisms including the GCCϩ. Pseudomonas remained constant or disappeared 10 (Chevallier et al. 2006; Comi et al. 2005; Lebert 11 et al. 2007). 12 OTHER MICROFLORA 13 Yeasts and Molds 14 PATHOGENIC MICROFLORA 15 Yeast and molds are usually detected in all sausages 16 in the batter at levels varying from 2.0 to 4.5 log Salmonella and sulphite-reducing clostridia were 17 cfu/g (Tables 11.1a and 11.1b). Some authors not detected in any traditional sausages. 18 observed slow growth during the ripening period, Pathogenic staphylococci were detected only in 19 with levels not increasing above 5 log cfu/g (Drosinos some Italian and French sausages. In Salame Milano, 20 et al. 2005), and then a decrease in the population Staphylococcus aureus was observed at the begin- 21 (Comi et al. 2005). Yeasts and molds were not ning of the production process and decreased during 22 detected in some products at the end of ripening ripening until it was undetectable at the end of the 23 (Rebecchi et al. 1998). process (Rebecchi et al. 1998). It was still enumerated in sausages produced in Italian artisanal plants 24 Enterococci in levels ranging from 2 to 4 log cfu/g (Blaiotta et al. 25 26 Enteroccocci have an initial level between 2 and 4 2004). In French sausages, S. aureus was present in 27 log cfu/g (Tables 11.1a and 11.1b). Enterococci usu- sporadic cases, as, for example, presence in winter 28 ally grow during early fermentation and remain con- samples and not detected in spring (Chevallier et al. 29 stant at a level of 4–6 log until the end of the whole 2006). 30 process (Comi et al. 2005; Drosinos et al. 2005; Listeria monocytogenes was sometimes present in 31 Rebecchi et al. 1998). In few cases their counts initial samples, but it was diminished or was not 32 declined. Chevallier et al. (2006) observed that the detected by the end of fermentation in Greek 33 counts were dependent of the season. In some sausages (Drosinos et al. 2005; Samelis et al. 1998). 34 French sausage, their levels reached 6 to 7.5 log In the European project Tradisausage (Talon 2006), 35 cfu/g (Lebert et al. 2007). The persistence of entero- L.monocytogenes was enumerated in 3 French tradi- 36 cocci during ripening can be attributed to their wide tional producers out of the 10 studied. In the first 37 range of growth temperature and to their high toler- one, the level of L. monocytogenes decreased from 38 ance to salt. Moreover, enterococci are poor acidi- the batter until the end of the ripening. In the second 39 fiers, and in traditional sausages of high pH they find producer, L. monocytogenes was detected in the final 40 good conditions for survival and growth (Hugas product at a level under the hygienic tolerable level 41 et al. 2003). There is still controversy over consider- (2.0 log CFU/g), and in the third producer L. mono- 42 ing them as GRAS (Generally Recognized as Safe) cytogenes level was above this limit (Lebert et al. 43 microorganisms (Giraffa 2002). However, studies 2007). In Spanish sausages, sporadic cases of con- 44 point out that meat enterococci, especially Entero- tamination by L. monocytogenes were mentioned 45 coccus faecium, have a much lower pathogenicity (Tables 11.1a and 11.1b [Aymerich et al. 2003]). 46 potential than clinical strains, and some strains of 47 E. faecium are already used as starter culture or pro- 48 biotic (Hugas et al. 2003; Martin et al. 2005). IDENTIFICATION OF THE FLORA 49 In the last decade, it was shown that classical micro- 50 biological techniques did not give a real view of SPOILAGE MICROFLORA 51 microbial diversity. To improve identification of 52 Spoilage bacteria such as Pseudomonas and enter- LAB and GCCϩ, molecular methods have been S53 obacteria have far different initial levels according to developed (Aymerich et al. 2003; Comi et al. 2005; N54 40454 11 111-124 r1.qxd 7/20/07 12:51 PM Page 118

1 Table 11.2. LAB species identiﬁed in traditional fermented dry sausages at the end of ripening. 2 3 Country (Reference)/Product Species Percentage 4 Spain (1) L. sakei 74.0 5 L. curvatus 21.2 6 Leuc. mesenteroïdes 4.8 7 8 Greece (2) L. plantarum 37.2 9 L. plantarum/pentosus 25.0 10 LAB n.i. 9.0 11 L. curvatus 7.3 12 L. pentosus 5.9 13 Other species <5.0 14 L. rhamnosus, L. sakei, L. paracasei, 15 L. salivarius, L. brevis 16 Greece (3)/Product I L. sakei 42.8 17 L. curvatus 18.7 18 L. buchneri 13.3 19 L. plantarum 8.0 20 Other species <5.0 21 L. paracasei, L. casei, L. coryniformis 22 L. rhamnosus 23 Greece (3)/Product II L. paracasei 29.2 24 L. sakei 23.6 25 L. buchneri 16.7 26 L. curvatus 13.8 27 Other species <5.0 28 L. plantarum, L. casei, L. paraplantarum 29 Greece (4) L. curvatus 48.2 30 L. sakei 19.2 31 L. plantarum 14.9 32 L. paraplantarum 5.3 33 L. casei/paracasei 5.3 34 L. alimentarius <5.0 35 36 Hungary (4) L. sakei 70.8 37 W. viridescens 7.3 38 L. curvatus 7.3 39 W. paramesenteroides/hellenica 5.8 40 Other species <5.0 41 Leuc. mesenteroïdes, L. paraplantarum/ 42 plantarum, L. plantarum 43 Italy (4) L. sakei 48.8 44 L. curvatus 29.8 45 L. plantarum 6.6 46 L. paraplantarum 5.8 47 Other species <5.0 48 L. paraplantarum/pentosus, 49 W. paramesenteroides/hellenica 50 51 52 53S 54N

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Table 11.2. (Continued) 1 2 Country (Reference)/Product Species Percentage 3 Italy (5)/Product 1 L. sakei 42.0 4 L. curvatus 22.0 5 L. plantarum 18.0 6 L. paraplantarum/pentosus 8.0 7 W. paramesenteroides/hellenica 6.0 8 Other species <5.0 9 L. paraplantarum, Leuc. citreum 10 11 Italy (5)/Product 2 L. curvatus 52.0 12 L. sakei 34.0 13 L. paraplantarum 12.0 14 W. paramesenteroides/hellenica <5.0 15 Italy (5)/Product 3 L. sakei 52.0 16 L. curvatus 34.0 17 Leuc. mesenteroïdes 8.0 18 L. brevis <5.0 19 Italy (6) L. sake* 34.0 20 L. sakei 28.8 21 Leuc. mesenteroïdes 9.4 22 L. curvatus 8.4 23 L. plantarum 5.2 24 Other species <5.0 25 L. casei, LAB spp., L. alimentarius, 26 L. sanfranciscensis, L. maltaromicus 27 28 Italy (7) L. sakei 43.3 29 L. plantarum 16.6 30 L. carnis 15.0 31 L. curvatus 13.3 32 Other species <5.0 33 L. casei, L. farciminis, L. sharpeae, 34 L. delbrueckii, L. amilophylus 35 Italy (8)/Product C L. sakei 88.8 36 L. casei 5.9 37 L. curvatus <5.0 38 Italy (8)/Product L L. sakei 85.5 39 L. curvatus 8.3 40 L. plantarum <5.0 41 Italy (8)/Product U L. sakei 52.0 42 L. curvatus 31.0 43 L. plantarum 6.7 44 L. brevis <5.0 45 L. paraplantarum <5.0 46 47 L. Lactobacillus; Leuc. Leuconostoc; LAB: Lactic Acid bacteria;* L. sake former L. bavaricus; W. Weissella. n.i. not identiﬁed. 48 (1) Aymerich et al. (2006); (2) Drosinos et al. (2005); (3) Papamanoli et al. (2003); (4) Rantsiou et al. (2005a); (5) Comi 49 et al. (2005); (6) Coppola et al. (2000); (7) Greco et al. (2005); (8) Urso et al. (2006). 50 51 52 S53 N54

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1 Table 11.3. GCCϩ species identiﬁed in traditional fermented dry sausages at the end of ripening. 2 3 Country (Reference)/Product Species Percentage 4 5 Greece (1) S. saprophyticus 31.1 6 S. xylosus 19.2 7 S. simulans 11.4 8 S. capitis 8.2 9 S. haemolyticus 7.8 10 S. sciuri 7.8 11 S. caprae 5.9 12 Other species <5.0 13 S. aureus/intermedius, S. hominis, 14 S. auricularis, S. warneri, S. cohnii, 15 S. epidermidis, S. carnosus 16 Greece (2)/Product I S. saprophyticus 27.6 17 S. carnosus 25.9 18 S. cohnii 13.8 19 S. xylosus 5.2 20 S. epidermidis 5.2 21 S. hominis 5.2 22 K. varians 5.2 23 Other species <5.0 24 S. capitis, S. warneri, S. auricularis, 25 S. hyicus, S. sciuri 26 Greece (2)/Product II S. xylosus 16.7 27 S. saprophyticus 14.3 28 S. carnosus 11.9 29 S. haemolyticus 11.9 30 S. simulans 9.5 31 S. epidermidis 7.1 32 Other species <5.0 33 S. cohnii, S. auricularis, S. hyicus, 34 K. varians, S. lentus 35 Spain (3)/Fuet S. xylosus 83.3 36 S. warneri 10.8 37 S. epidermidis 5.0 38 S. carnosus <5.0 39 40 Spain (3)/Salchichon S. xylosus 72.9 41 S. warneri 12.5 42 S. epidermidis 12.5 43 K. varians <5.0 44 Spain (3)/Chorizo S. xylosus 80.8 45 S. warneri 8.3 46 S. epidermidis 5.8 47 S. carnosus <5.0 48 Spain (4)/Chorizo S. xylosus 94.6 49 Other species <5.0 50 S. intermedius, S. saprophyticus, S. hominis 51 S. epidermidis, S. aureus 52 53S 54N

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Table 11.3. (Continued) 1 2 Country (Reference)/Product Species Percentage 3 Italy (5)/Salami Naples S. xylosus 44.8 4 S. saprophyticus 17.2 5 S. lentus 17.2 6 S. warneri 13.8 7 S. succinus <5.0 8 9 Italy (5)/Soppressata Ricigliano S. saprophyticus 32.0 10 S. xylosus 29.5 11 S. succinus 14.1 12 S. equorum 11.5 13 S. vitulus 5.1 14 Other species <5.0 15 S. pasteuri, S. warneri, S. haemolyticus 16 Italy (5)/Sopressata Gioi S. xylosus 25.4 17 S. saprophyticus 24.8 18 S. equorum 20.9 19 S. succinus 14.7 20 Other species <5.0 21 S. warneri, S. lentus, S. vitulus, S. pasteuri, 22 S. epidermidis, S. haemolyticus 23 Italy (6)/Salame Milano S. xylosus 65.0 24 S. sciuri 35.0 25 26 Italy (7) S. xylosus 100.0 27 Italy (8)/Plant C S. warneri 27.0 28 S. xylosus 25.0 29 S. pasteuri 25.0 30 M. caseolyticus 12.0 31 S. epidermidis 5.0 32 S. saprophyticus, S. equorum <5.0 33 Italy (8)/Plant L S. xylosus 62.0 34 S. equorum 12.0 35 S. warneri 8.0 36 M. caseolyticus 8.0 37 S. pasteuri 7.0 38 S. epidermidis, S. saprophyticus <5.0 39 Italy (8)/Plant U S. xylosus 40.0 40 S. equorum 18.0 41 S. carnosus 11.0 42 S. warneri 8.0 43 S. epidermidis 8.0 44 M. caseolyticus 8.0 45 S. pasteuri 5.0 46 S. saprophyticus, S. cohnii <5.0 47 48 Italy (9)/sausages from Basilica S. xylosus 51.2 49 S. pulvereri/vitulus 13.4 50 S. equorum 10.2 51 S. saprophyticus 10.0 52 Other species <5.0 S53 S. pasteuri, S. succinus, S. epidermidis N54 (Continues) 121 40454 11 111-124 r1.qxd 7/20/07 12:52 PM Page 122

122 Microbiology and Starter Cultures for Meat Fermentation

1 Table 11.3. (Continued) 2 3 Country (Reference)/Product Species Percentage 4 France (10) S. equorum 82.0 5 S. succinus 12.0 6 Other species <5.0 7 S. warneri, S. saprophyticus 8 9 (1) Drosinos et al. (2005); (2) Papamanoli et al. (2002); (3) Martin et al. (2006); (4) Garcia-Varona et al. (2000); (5) Mau- 10 riello et al. (2004); (6) Rebecchi et al. (1998); (7) Cocolin et al. (2001b); (8) Iacumin et al. (2006); (9) Blaiotta et al. (2004); 11 (10) Corbière Morot-Bizot et al. (2006). 12 13 14 Corbiere Morot-Bizot et al. 2004; Giammarinaro et al. products, S. equorum and S. succinus were also 15 2005; Rantsiou and Cocolin 2006; Urso et al. 2006). isolated (Table 11.3). In a French small producer, 16 Corbiere Morot-Bizot et al. (2006) showed that the 17 staphylococal microflora of the products and LACTIC ACID BACTERIA 18 the environment was dominated by S. equorum 19 Table 11.2 shows the diversity of LAB identified in (49% of the isolates) and S. succinus (33%) of 20 naturally fermented sausages at the end of ripening the processing unit. Many other minor species 21 originated from different countries, regardless of the were identified in the different traditional sausages 22 identification method. The LAB species most com- (Table 11.3). 23 monly identified in traditional fermented sausages 24 are Lactobacillus sakei, Lactobacillus curvatus, and CONCLUSION 25 Lactobacillus plantarum, with L. sakei as the domi- 26 nant one. When L. sakei is dominant, it represents This review outlines the diversity in the manufac- 27 42–88.8% of the isolates (Table 11.2). Aymerich turing and the microflora of naturally fermented 28 et al. (2006) showed that L. sakei was identified in all sausages, but also the similarity in the microorgan- 29 of the Spanish sausages and represented 89.3% in ism development. Indeed, LAB and GCCϩ always 30 chorizo and 76.3% in fuet. In a French sausage, became the dominant flora in the final products. 31 L. sakei represented 100% of the isolates on the final Traditional fermented sausages generally did not 32 products, although it was minor in the raw materials present sanitary risk. This study also outlined the 33 (Ammor et al. 2005). L. curvatus is the second diversity in the LAB and GCCϩ species, which was 34 species identified; it is dominant in some Greek or certainly due to the formulation, fermentation, and 35 Italian sausages. L. plantarum is the third one; it ripening practices. 36 dominates the LAB flora in a Greek sausage. Many 37 other species are identified, as listed in Table 11.2, REFERENCES 38 but represent a minor population. 39 S Ammor, C Rachman, S Chaillou, H Prévost, X Dousset, 40 M Zagorec, E Dufour, I Chevallier. 2005. Phenotypic GRAM-POSITIVE CATALASE-POSITIVE COCCI 41 and genotypic identification of lactic acid bacte- 42 Staphylococcus carnosus and Staphylococcus xylo- ria isolated from a small-scale facility producing 43 sus are used as starter cultures for manufacturing traditional dry sausages. Food Microbiol 22: 44 industrial dry sausages in Europe. In traditional 373–382. 45 products at the end of ripening, S. xylosus is the most T Aymerich, B Martín, M Garriga, M Hugas. 2003. 46 common species and is identified in all the sausages Microbial quality and direct PCR identification of 47 (Table 11.3). It represents from 17–100% of the lactic acid bacteria and nonpathogenic Staphylococci 48 isolates according to the type of sausages. Even if it from artisanal low-acid sausages. Appl Environ 49 was not the dominant species in the batter, S. xylosus Microbiol 69:4583–4594. 50 became rapidly dominant in Salame Milano (Rebecchi T Aymerich, B Martín, M Garriga, MC Vidal-Carou, 51 et al. 1998). S. saprophyticus is the second dominant S Bover-Cid, M Hugas. 2006. Safety properties and 52 species identified. It is dominant in some Greek and molecular strain typing of lactic acid bacteria from 53S Italian sausages (Drosinos et al. 2005; Mauriello slightly fermented sausages. J Appl Microbiol 54N et al. 2004; Papamanoli et al. 2002). In the Italian 100:40–49. 40454 11 111-124 r1.qxd 7/20/07 12:52 PM Page 123

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Int J Food Microbiol 106:270–285. 31 turing traditional dry fermented sausages. Int J Food B Martin, M Garriga, M Hugas, T Aymerich. 2005. 32 Microbiol 108:210–217. Genetic diversity and safety aspects of enterococci 33 S Corbiere Morot-Bizot, R Talon, S Leroy. 2004. from slightly fermented sausages. J Appl Microbiol 34 Development of a multiplex PCR for the identifica- 98:1177–1190. 35 tion of Staphylococcus genus and four staphylococ- B Martín, M Garriga, M Hugas, S Bover-Cid, MT 36 cal species isolated from food. J Appl Microbiol Veciana-Nogues, T Aymerich. 2006. Molecular, 37 97:1087–1094. technological and safety characterization of gram- 38 D Demeyer. 2004. Chapter 20: Meat fermentation: positive catalase-positive cocci from slightly 39 Principles and applications. In: YH Hui, L Meunier- fermented sausages. Int J Food Microbiol 40 Goddik, AS Hansen, J Josephsen, W-K Nip, PS Stan- 107:148–158. 41 field, F Toldrá, eds. 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1 E Parente, M Martuscelli, F Gardini, S Grieco, MA microbiological ecology study. Int J Food Microbiol 2 Crudele, G Suzzi. 2001. Evolution of microbial pop- 44:69–82. 3 ulations and biogenic amine production in dry R Talon. 2006. Assessment and improvement of safety 4 sausages produced in Southern Italy. J Appl Micro- of traditional dry sausages from producers to 5 biol 90:882–891. consumers “Tradisausage” European project, QLK1 6 K Rantsiou, L Cocolin. 2006. New developments in the CT-2002-02240. Summary available at: http://www. 7 study of the microbiota of naturally fermented clermont.inra.fr/tradisausage/index.htm. 8 sausages as determined by molecular methods: A R Talon, S Leroy-Sétrin. 2006. Chapter 16: Latest 9 review. Int J Food Microbiol 108:255–267. developments in meat bacterial starters. In: LML 10 K Rantsiou, EH Drosinos, M Gialitaki, R Urso, J Nollet, F Toldrá, eds. Advanced Technologies for 11 Krommer, J Gasparik-Reichardt, S Toth, I Metax- Meat Processing. New York: CRC Press, Taylor and 12 opoulos, G Comi, L Cocolin. 2005a. Molecular char- Francis group, pp. 401–418. 13 acterization of Lactobacillus species isolated from R Talon, S Leroy-Sétrin, S Fadda. 2002. Chapter 10: 14 naturally fermented sausages produced in Greece, Bacterial starters involved in the quality of fermented 15 Hungary and Italy. Food Microbiol 22:19–28. meat products. In: F Toldrá, ed. Research Advances 16 K Rantsiou, R Urso, L Iacumin, C Cantoni, P Cattaneo, in the Quality of Meat and Meat Products. Research 17 G Comi, L Cocolin. 2005b. Culture-dependent Signpost, pp. 175–191. 18 and -independent methods to investigate the micro- ———. 2004. Chapter 23: Dry Fermented sausages. In: 19 bial ecology of Italian fermented sausages. Appl YH Hui, L Meunier-Goddik, AS Hansen, J Josephsen, 20 Environ Microbiol 71:1977–1986. W-K Nip, PS Stanﬁeld, F Toldrá, eds. Handbook of 21 A Rebecchi, S Crivori, PG Sarra, PS Cocconcelli. 1998. Food and Beverage Fermentation Technology, 1st Ed. 22 Physiological and molecular techniques for study of New York: Marcel Dekker Inc., pp. 397–416. 23 bacterial community development in sausage fer- R Urso, G Comi, L Cocolin. 2006. Ecology of lactic 24 mentation. J Appl Microbiol 84:1043–1049. acid bacteria in Italian fermented sausages: isolation, 25 J Samelis, J Metaxopoulos, M Vlassi, A Pappa. 1998. identiﬁcation and molecular characterization. Syst 26 Stability and safety of traditional Greek salami—A Appl Microbiol 29(8):671–690. 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53S 54N 40454 12 125-136 r1.qxd 7/20/07 12:53 PM Page 125

1 2 3 12 4 5 6 The Microbiology of Fermentation 7 8 and Ripening 9 10 Margarita Garriga and Teresa Aymerich 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION earthquake in 450 B.C. Sausage, saucisse, and 22 salchicha (English, French, and Spanish, respec- 23 Raw meat is a nutrient matrix rich in protein with a tively) derive from the Italian salsiccia. 24 higher water activity (a ϭ 0.96–0.97) and a favor- w Although typical preparations based on indige- 25 able pH (5.6–6.0) for optimal microbial growth. It nous microflora still exist, fermentations should now 26 allows a rapid spoilage process when microorganism be considered more than just a preservation method. 27 contamination occurs and preservation measures are Across the centuries, people learned by empirical 28 not correctly applied. Gastrointestinal tract, feet, practice and experimentation how to control these 29 hides, or skins of slaughtered animals are primary processes, and fermented sausages became an inde- 30 sources of contamination. Instruments and surfaces pendent class of foodstuff with different regional 31 in the slaughterhouse, infected personnel, or healthy specialities, appreciated for their rich diversity in 32 carriers may contaminate the meat at all stages of sensorial attributes. They comprise a great variety of 33 meat processing. The contaminating microflora products that vary between countries and different 34 includes technologically important microorganisms regions of the same country in the batter composi- 35 but also spoilage and pathogenic bacteria, resulting tion, fermentation, and ripening processes. Europe is 36 in a varied and complex microflora. the major producer of fermented sausages (Campbell- 37 Fermentation and drying are one of the oldest Platt 1995). Germany, which is known to have been 38 methods used to preserve food for long periods of manufacturing fermented sausages since the 1850s, 39 time. The origin of fermented sausages seems to have is probably the country producing the greatest vari- 40 come from Mediterranean countries in Roman times ety, with differences in ripening times, flavor, firm- 41 (Lücke 1974), although a fermented pork sausage ness, acidification, meat/fat grinding, and diameter 42 from Thailand called Nham has also been reported as of products. In general, Mediterranean countries 43 an example of other areas of local production (Adams produce naturally dried, nonsmoked, spiced sausages 44 1986). Production was established in America, South with a limited sour taste. 45 Africa, and Australia by immigrants from European 46 countries. In France in the fifth century, the char- 47 cutiers prepared different pork specialities; in fact the THE MANUFACTURE OF 48 chair cuite, which originated from the French word FERMENTED SAUSAGES 49 charcuterie, is closely related to the Italian word 50 salsamenteria/salumeria. The word salami comes Pork and pork/beef, and rarely mutton or chicken 51 from salumen, which in old Latin means a conjuga- meat, are used in the manufacture of fermented 52 tion of salted things (Cantoni et al. 1987). Pederson sausages. Fat comes from pork, backfat being the 53 (1971) suggested the name could come from choice for producing high-quality products. The pro- S54 Salamis, a city on the cost of Cyprus destroyed by an portion of meat and fat varies depending on the N55 125 40454 12 125-136 r1.qxd 7/20/07 12:53 PM Page 126

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1 product as well as the method and degree of minc- shelf life. The intrinsic factors of the sausage mix,

2 ing. Fermented sausage consists of comminuted together with the final aw (generally under 0.9) and 3 meat and fat, mixed with salt, sugar, curing agents, pH (between 6.2 and 4.5) exert a natural selectivity 4 and spices, and then stuffed into casings and sub- for promoting the development of the desired salt- 5 jected to fermentation by indigenous microflora or tolerant microflora, which contributes to the com- 6 by selected starter cultures. After the fermentation plexity of the reactions that take place in order to 7 period, which lasts between 1–3 days depending on fulfil these objectives. 8 the temperature and relative humidity applied, a 9 ripening-drying process transforms a highly perish- TECHNOLOGICAL MICROFLORA 10 able product into a stable cured product that does not 11 need refrigeration. The first studies on the ecology of fermented sausages 12 The main additives added to the meat batter are back in the 1970s (Lücke 1974) established two 13 carbohydrates, curing salts, and spices: groups of microorganisms as being mainly res- 14 ponsible for the transformation involved during fer- • Carbohydrates: Glucose is usually added to the 15 mentation and ripening: lactic acid bacteria (LAB), mixture because it is the primary growth substrate 16 in particular Lactobacillus, and the gram-positive for all bacteria. Generally it is dosed at 1–8 g/kg 17 coagulase-negative cocci (GCC+), especially Staphy- to reach a pH value under 5.2 after the 18 lococcus. Yeasts and molds were also often isolated fermentation period, which ensures firmness and 19 from fermented meats. LAB are responsible for lactic microbiological stability. Excess amounts of 20 acid production and the subsequent decrease of pH, easily fermentable sugars may result in a product 21 which causes the coagulation of meat proteins and the that is too sour and the development of undesired 22 tangy flavor of sausages for the necessary reactions Lactic Acid Bacteria (Kröckel 1995). 23 for color formation and for microbiological stability. • Curing salts: Sodium chloride is added to the 24 They also produce small amounts of acetic, ethanol, mixture at 20–30 g/kg. Together with the 25 acetoin, carbon dioxide, and pyruvic acid that are pro- antimicrobial effect against spoilage bacteria by 26 duced during fermentation, depending on the endo- the decrease of the a , NaCl participates in the 27 w genous microflora, the carbohydrate used, and the solubilization of meat proteins, which determine 28 sources of meat proteins and additives used (Demeyer the cohesiveness of the product. Current meat- 29 et al. 2000; Thornill and Cogan 1984; Bacus 1986). curing practice involves the addition of nitrate 30 Some LAB strains may also act as bioprotective cul- and/or nitrite salts, which contribute to the 31 tures by the production of antimicrobial compounds formation of the characteristic cured-meat color 32 (bacteriocins), thus enhancing the safety of fermented and flavor. Nitrite participates in the inhibition of 33 sausages (Hugas 1998). Some authors have also oxidative processes, thus preventing rancidity as 34 pointed out their ability to hydrolyze the pork muscle well as the inhibition of pathogens like 35 sarcoplasmic proteins, releasing peptides that could Clostridium botulinum. The added salt and sugars 36 play a role in the taste (Fadda et al. 1999). Staphylo- in the comminuted meat favors the growth of 37 coccus and Kocuria are important for nitrite and indigenous fermentative microorganisms, which 38 nitrate reductase activity, promoting the desired red will grow at relatively high osmotic pressure. 39 color development and its stabilization (Liepe 1983), Sodium ascorbate is usually added at 0.5 g/kg. 40 decomposition of peroxides (Schleifer 1986; Montel It is an antioxidant substance that favors the 41 et al. 1998), limiting lipid oxidation and preventing transformation of nitrite into nitric oxide, 42 rancidity (Barrière et al. 1998; Talon et al. 1999), and improving the stability of the cured-meat color. 43 contributing to the flavor by formation of esters and • Spices: Black and white pepper are usually added 44 other aromatic compounds from amino acids through to practically all types of fermented sausages. 45 their proteolytic and lipolytic activities (Cantoni et al. Other spices such as paprika and garlic are used 46 1967; Debevere et al. 1976; Berdagué et al. 1993; in several specialities, such as the Spanish 47 Johansson et al. 1994; Miralles et al. 1996; Montel chorizo. Wine is used in some specialities. 48 et al. 1998; Cai et al. 1999). Yeast and molds may also 49 According to Buckenhüskes (1993), the main objec- contribute to the product quality by the development 50 tives of the fermentation and ripening process are to of typical flavors and tastes through the lactate 51 achieve the formation of the desired and typical oxidation, proteolysis, degradation of amino acids, 52 cured color and fermentation flavor, the develop- and lipolysis (Sorensen and Samuelsen 1996; 53S ment of firmness to permit sliceability, the inhibition Sunesen and Stahnke 2003). The interconnection of 54N of pathogenic and spoilage bacteria, and an extended their metabolic activities is considered essential to 40454 12 125-136 r1.qxd 7/20/07 12:53 PM Page 127

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healthiness, texture, desired flavor, and color of fer- during the first half of the ripening and then 1 mented sausages. decreased at different rates (Mauriello et al. 2004). 2 3 Lactic Acid Bacteria 4 BIODIVERSITY AND BEHAVIOR OF THE Although meat batter composition, together with final 5 TECHNOLOGICAL MICROFLORA pH and process technology, may influence the 6 Sausage fermentation is characterized by an increase microflora, the species of LAB most commonly found 7 in the number of LAB from 103–105 CFU/g to in different kinds of fermented sausages processed 8 106–109 CFU/g within the first days of fermentation with different technologies in Germany, Hungary, 9 (1–3 days) or maturation/ripening (up to 14 days), Greece, Spain, Italy, and Argentina were L. sakei and 10 which generally remains stable for the rest of the L. curvatus. In the majority of the studies, L. sakei 11 process and becomes the dominant microflora. The was the dominating species in the final products, 12 evolution of the LAB population on traditional exceeding 55% of the total LAB population. Some 13 Spanish fermented sausage is reported in Figure other species, such as L. plantarum, L. alimentarius, 14 12.1. The initial number of GCCϩ, between L. casei, L. paraplantarum, L. pentosus, L. paracasei, 15 103–105 CFU/g, increased to 105–108 CFU/g during Pediococcus spp., Lactococcus lactis, Lactococcus 16 the first 14–20 days of fermentation and ripening garviae, Leuconostoc citreum, Leuconostoc mesen- 17 (Schillinger and Lücke 1987; Torriani et al. 1990; teroides, Weisella paramesenteroides/hellenica, 18 Hugas et al. 1993; Samelis et al. 1994; Cocolin et al. Weissella viridescens, Enterococcus faecium, Entero- 19 2001; Metaxopoulos et al. 2001; Aymerich et al. coccus faecalis, Enterococcus durans, and Entero- 20 2003; Fontana et al. 2005). Evolution of the GCCϩ coccus pseudoavium have also been described 21 population in traditional Spanish fermented sausages (Schillinger and Lücke 1987; Torriani et al. 1990; 22 is reported in Figure 12.1. Short ripening time and Montel et al. 1991; Hugas et al. 1993; Samelis et al. 23 higher fermentation temperature lead to higher num- 1994; Santos et al. 1998; Cocolin et al. 2000; Parente 24 bers of lactobacilli from the early stages of fermenta- et al. 2001; Aymerich et al. 2003; Rantsiou et al. 25 tion and produce a final product with acid flavor and 2005a, Rantsiou et al. 2005b, Aymerich et al. 2006). 26 little aroma. In contrast, sausages with longer matu- In some slightly fermented Italian sausages, Entero- 27 ration times and low fermentation temperatures con- coccus and the lactobacilli population have been 28 tain higher numbers of GCC+ in the early stages and reported to be highly balanced (Dellapina et al. 1994). 29 produce a less tangy product with more flavor Although only a few species seem to be able to 30 (Demeyer et al. 2000). Yeasts showed slow growth implant and dominate at the end of the ripening 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 Figure 12.1. Microflora of traditional Spanish fermented sausages (pH 5.79) during the process. Values are S53 the median of 10 different products from different producers (TRADISAUSAGE QLK1-CT-2002-02240). N54 40454 12 125-136 r1.qxd 7/20/07 12:54 PM Page 128

128 Microbiology and Starter Cultures for Meat Fermentation

1 process, biodiversity at strain level inside the intraspecificic variability, 9 profiles out of 12 iso- 2 predominant species has been reported. In Figure lates, and L. sakei showed 112 different strains out of 3 12.2, biodiversity of 12 different strains isolated 185 isolates. L. curvatus presented the lower intra- 4 from a slightly fermented Spanish sausage is specific variability. 5 reported, and 6 different clusters at 70% homology Enterococci are ubiquitous microorganisms that 6 could be differentiated through a composite RAPD- inhabit the gastrointestinal tract of humans and ani- 7 profile. Santos et al. (1998) detected 4 different bio- mals. They are frequently isolated from fermented 8 chemical groups of L. sakei and 4 different groups of meat products with counts of up to 106 CFU/g (Papa 9 L. curvatus among the isolates from different kinds et al. 1995; Samelis et al. 1998; Metaxopoulos et al. 10 of chorizo, the group S1 of L. sakei being the most 2001; Aymerich et al. 2003; Fontana et al. 2005) due 11 competitive during the whole fermentation/ripening to their tolerance to sodium chloride and nitrite, 12 process. Rantsiou et al. (2005a) reported a high bio- allowing them to survive and even to multiply during 13 diversity inside the 295 isolates analyzed from the the first period of fermentation and ripening (Cocolin 14 predominant LAB species found in different kinds et al. 2001; Giraffa 2002). The enterococci evolution 15 of sausages produced in Hungary, Italy, and Greece. through the fermentation/ripening process of the tra- 16 The clustering analysis at 70% of homology gave 5 ditional Spanish fermented sausages is reported in 17 different clusters for the 27 isolates of L. plantarum, Figure 12.1. The species most frequently isolated are 18 9 clusters for the 100 isolates of L. curvatus and 19 Enterococcus faecium and Enterococcus faecalis, 19 clusters for the 168 isolates of L. sakei. The majority followed by Enterococcus hirae and Enterococcus 20 of the clusters grouped country-specific. Aymerich durans. (Devriese et al. 1995; Peters et al. 2003; 21 et al. (2006) were able to distinguish 144 different Martín et al. 2005). A high intraspecific biodiversity 22 RAPD/plasmid profiles among the 250 isolates from has been reported in the enterococci population of 23 21 different Spanish slightly fermented sausages. fermented sausages by RAPD-PCR. Sixty strains 24 Leuconostoc mesenteroides displayed the higher out of 106 isolates were identified from 21 different 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53S 54N Figure 12.2. Strain biodiversity of LAB and GCCϩ population of a traditional Spanish fermented sausage. 40454 12 125-136 r1.qxd 7/20/07 12:54 PM Page 129

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fermented Spanish sausages by RAPD profiles, population is reported in Figure 12.2, where 12 dif- 1 thus indicating a high genetic variability (Martín ferent clusters could be differentiated at 70% of 2 et al. 2006). homology when a composite RAPD profile was con- 3 The presence of enterococci in foods is highly considered. By genotypic clustering based on compari- 4 troversial. Although some authors consider them son of RAPD-PCR profiles, Rossi et al. (2001) were 5 undesirable, indicators of fecal contamination, and able to distinguish 22 different clusters at 70% of 6 responsible for the spoilage of meat products (Franz homology. The combination of RAPD-PCR and 7 et al. 1999), they are also producers of toxic sub- plasmid profiling allowed the discrimination of 208 8 stances such as biogenic amines (Tham 1988), and different profiles among the 240 GCC+ character- 9 sausage alterations (Knudtson and Hartman 1992); ized from the slightly fermented sausages (Martín 10 others report their important role in flavor develop- et al. 2005). Fontana et al. (2005) reported a high 11 ment and bioprotection (Coppola et al. 1988; Centeno variability at the first stage of Argentinean sausage 12 et al. 1996; Aymerich et al. 2000; Callewaert et al. processing, and a strong selection of the microflora 13 2000). They also produce L(+) lactate, which con- occurred after day 5 with only two strains of 14 tributes to pH drop. In recent decades, enterococci S. saprophyticus that became dominant. 15 have unfortunately acquired clinical relevance, 16 although foodborne enterococci had never been Yeasts and Molds 17 proved to be the direct cause of clinical infections 18 (Adams 1999). The resistance of enterococci to a Yeast microbiota in sausages depends on the nature 19 wide variety of antimicrobials (Murray 1990; Mundy and the processing conditions of the product. The 20 et al. 2000) together with their ability to exchange main role of yeasts in fermented sausages seems to 21 genes by conjugation (Clewell 1990; Franz et al. be related to the increase of ammonia and reduction 22 1999) are a cause for concern to the health authorities. of lactic acid contents (Gehlen et al. 1991) together 23 with proteolytic activity (Durá et al. 2004), thus con- 24 Gram-positive Coagulase-negative Cocci tributing to the final sensory properties of the fer- 25 Regarding GCC+, Staphylococci, mainly Staphylo- mented sausages. 26 coccus xylosus, have been described as the predom- In traditional Spanish fermented sausages, an 27 inant species in Spanish, German, and Italian increase in the population from 4 log CFU/g to nearly 28 fermented sausages (Fischer and Schleifer 1980; 6 log CFU/g at the end of ripening has been observed 29 Simonetti and Cantoni 1983; Seager et al. 1986; (Figure 12.1). Rantsiou et al. (2005b) observed three 30 Coppola et al. 1996; García-Varona et al. 2000; different behaviors of the yeast population in three 31 Aymerich et al. 2003; Martín et al. 2006). In some fer- different local products. In the first product, the yeast 32 mented Greek and Argentinean sausages, a dominance population decreased during the central period of fer- 33 of S. saprophyticus has been reported (Samelis et al. mentation with an increase in the counts at the last 34 1998; Papamanoli et al. 2002; Fontana et al. 2005). sampling point, reaching 106 to 107 CFU/g. In the 35 A codominance of S. saprophyticus, S. xylosus, and second product yeasts were detected only on the first 36 S. equorum has been reported in three different types 3 days. An undulating pattern was observed in the 37 of Italian fermented sausages by Mauriello et al. third, with initial counts of 103–104 CFU/g turning 38 (2004). Other species such as S. carnosus, S. succinus, out to be undetectable for a 2-week period; they 39 S. warneri, S. lentus, S. vitulus, S. pasteuri, S. epider- increased to 103–104 CFU/g at day 14 and decreased 40 midis and S. haemolyticus, S. intermedius, S. pulverei, to 102–103 CFU/g at the end of ripening. 41 S. sciuri, S. cohnii, S. hyicus, Kocuria varians, Kocuria Debaryomyces, Rhodotorula, Hansenula, and 42 kristiniae, Micrococcus luteus, and Macrococcus Torulopsis have been described as being the most 43 caseolyticus have been described (Fischer and representative genera (Comi and Cantoni 1980). 44 Schleifer 1980; Seager et al. 1986; Cocolin et al. D. hansenii has been reported as the dominant species 45 2001; Aymerich et al. 2003; Rantsiou et al. 2005b, in Spanish products and has been isolated from 46 Martín et al. 2006). Underestimation of some staphy- all the stages of processing (Encinas et al. 2000). In 47 lococci species such as S. equorum has been sug- other products, the D. hansenii population has been 48 gested by misidentification when only biochemical reported to decrease during processing (Gehlen et al. 49 methods were used (Blaiotta et al. 2003). 1991; Olesen and Stahnke 2000; Rantsiou et al. 50 A high biodiversity inside staphylococci from 2005b). Other species such as Candida tropicalis, 51 fermented sausages has been described by genotyp- Candida krisii, Candida sake, Candida fermentati, 52 ing and/or biochemical methods. The strain biodi- Willopsis saturnus, and Entyloma dahliae have also S53 versity of the 12 different isolates from the GCCϩ been described (Rantsiou et al. 2005b). N54 40454 12 125-136 r1.qxd 7/20/07 12:54 PM Page 130

130 Microbiology and Starter Cultures for Meat Fermentation

1 The development of fungal mycelia is commonly food handling is essential to avoid hazards such as 2 observed on the casings of sausages, mainly those of Salmonella, Shigella, E. coli, Staphylococcus aureus, 3 Southern Europe, which have a long ripening period. and Clostridium perfringens. Nevertheless, some 4 Fungi have been described as protecting against foodborne pathogens such as Pseudomonas, Clostrid- 5 lipid oxidation and extending the volatile profile ium botulinum type E, Listeria monocytogenes can 6 of the final product (Hierro et al. 2005). The species even grow. 7 of molds most commonly found are Penicillium, Salmonella spp. and L. monocytogenes have been 8 Aspergillus, Mucor, and Cladosporium. reported as the major causes of deaths by foodborne 9 pathogens, followed by Campylobacter and entero- 10 SPOILAGE MICROFLORA hemorragic E.coli (Mead et al. 1999; Anonymous 11 2001; Le Loir et al. 2003). Salmonella spp. has fre- 12 Gram-negative aerobic bacteria (e.g., Pseudomonas, quently been isolated on pork carcasses and pork cuts 13 Acinetobacter) may be involved in the spoilage of (Giovannacci et al. 2001; Pala and Sevilla 2004). Sal- 14 the products. Proteolytic activity and/or catabolism monella Arizonae have also been isolated from black 15 of sulphur-containing amino acids can cause defects pepper (Kneifel and Berger 1994). Absence of 16 in texture and flavor. High counts of Pseudomonas Salmonella in final products has been observed in the 17 up to 104 CFU/g have been reported in freshly study of 10 traditional Spanish workshops, as has 18 prepared sausages (Metaxopoulos et al. 2001) also been reported in other products from Greece, 19 (Figure 12.1), but they are reported to disappear Spain, and Italy (Metaxopoulos et al. 2001; Aymerich 20 (Metaxopoulos et al. 2001) or decrease (Figure 12.1) et al. 2003). S. aureus and L. monocytogenes are 21 during fermentation and ripening. Brochothrix ther- ubiquitous in processing environments (Salvat et al. 22 mosphacta, a gram-positive bacterium that causes 1995; Borch et al. 1996) and their presence in raw pork 23 cheesy odors, can grow to 104 CFU/g during the fer- and meat products has been reported, (Anonymous 24 mentation period, but it decreases during ripening. 1999; Atanassova et al. 2001; Pala and Sevilla 2004) 25 The gram-negative facultative anaerobic Enterobac- although the reported levels in the final products are 26 teriaceae may grow to values of up to 103–106 CFU/g generally low (Aymerich et al. 2003; Chevallier et al. 27 during the first days of fermentation/ripening when 2006). In a survey of 10 traditional Spanish work-

28 pH, aw, and salt/moisture coefficient are compatible shops, L. monocytogenes was detected in some of the 29 with these microorganims. From days 5–7, they pro- workshop equipment and products, but generally 30 gressively drop down to 102 CFU/g (Figure 12.1) with counts of less than 1.0 log CFU/100 cm2 and 0.7 31 (Lücke 1986; Domínguez et al. 1989; Lizaso et al. log CFU/g, respectively. Counts of S. aureus through 32 1999; Castaño et al. 2002). Moreover, Enterobacteri- the ripening process of traditional Spanish fermented 33 aceae are aminogenic bacteria that may produce sausages are reported in Figure 12.1. Levels under 34 biogenic amines as cadaverin, putrescine, and hista- 100 CFU/g are usually reported in final products 35 mine, thus being a concern in relation to food quality (Metaxopoulos et al. 2001; Aymerich et al. 2003; 36 (Vidal-Carou et al. 1990; Mariné-Font et al. 1995). Rantsiou et al. 2005b). 37 Escherichia coli, Hafnia alvei, Serratia liquefaciens, Few outbreaks associated with Salmonella and 38 Serratia odorifera and Enterobacter amnigenus, S. aureus have been reported in fermented sausages 39 Enterobacter intermedius, Providencia alcalifaciens, from Europe (Smith and Palumbo 1980; van Netten 40 Proteus vulgaris, Providencia penneri, and Mor- et al. 1986; Hartog et al. 1987). In 1994, an outbreak 41 ganella morganii (Silla-Santos 1998; Castaño et al. of E.coli O157:H7 related to sliced salami was 42 2002; Mavromatis and Quantick 2002; Chevallier et al. reported in the U.S. (Anonymous 1995). 43 2006) have been isolated from fermented sausages. 44 STARTER CULTURES 45 FOODBORNE PATHOGENS 46 Although fermented sausages can be produced with- 47 Veterinary inspection can detect and remove raw out the use of inoculated strains, the use of starter 48 meat affected by Bacillus anthracis, Mycobacterium cultures for sausage production is increasing so as to 49 tuberculosis, or Brucella abortis, but infections pro- guarantee safety and standardize product properties, 50 duced by Salmonella spp., Listeria monocytogenes, including consistent flavor and color, and to shorten 51 Campylobacter jejuni, Escherichia coli O157:H7, the ripening period. Nevertheless, proper hygienic 52 and Yersinia enterocolitica are not readily detected in and safety strains, properly identified at species and 53S vivo and have been involved in several outbreaks. strain level, which preserve typical characteristics, 54N Proper cooling of carcasses at chill temperatures and are essential in order to assure the hygienic and 40454 12 125-136 r1.qxd 7/20/07 12:54 PM Page 131

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safety quality together with the regional tradition for Atanassova V, Meindl A, Ring C. 2001. Prevalence of 1 fermented sausages. A positive implantation of the Staphylococcus aureus and staphylococcal entero- 2 starter culture used has to be controlled and guaran- toxins in raw pork and uncooked smoked ham—a 3 teed. In recent years, molecular methods such as comparison of classical culturing detection and 4 Pulse Field Gel Electrophoresis (PFGE), plasmid RFLP-PCR. Int J Food Microbiol 68:105–113. 5 profiling, amplified fragment length polymorphisms Aymerich MT, Garriga M, Jofré A, Martín B, Monfort 6 (AFLP), and randomly amplified polymorphic DNA JM. 2006. The use of bacteriocins against Meat-borne 7 (RAPD)-PCR have been used as efficient molecular pathogens. In:Advanced technologies for meat pro- 8 techniques that enable strain typing in a rapid and cessing. LML Nollet, F Toldrá. Marcel Dekker, Inc. 9 sensitive way (Cocconcelli et al. 1995; Farber et al. 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1 Miralles MC, Flores J, Perez-Martinez G. 1996. Bio- Rebecchi A, Crivori S, Sarra PG, Cocconcelli PS. 1998. 2 chemical tests for the selection of Staphylococcus Physiological and molecular techniques for the 3 strains as potential meat starter cultures. Food Micro- study of bacterial community development in 4 biol. 13:227–236. sausage fermentation. J Appl Microbiol 84: 5 Montel MC, Masson F, Talon R. 1998. Bacterial role in 1043–1049. 6 flavour development. Meat Sci 49:S111–S123. Rossi F, Tofalo R, Torriani S, Suzzi G. 2001. Identifica- 7 Montel MC, Talon R, Fournaud J, Champomier MC. tion by 16S–23S rDNA intergenic region amplifica- 8 1991. A simplified key for identifying homofermen- tion, genotypic and phenotypic clustering of 9 tative Lactobacillus and Carnobacterium spp. from Staphylococcus xylosus strains from dry sausages. J 10 meat. J. Appl. Bacteriol. 70:469–472. Appl Microbiol 90:365–371. 11 Mundy LM, Sahm DF, Gilmore MS. 2000. 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1 2 3 13 4 5 6 Starter Cultures: Bacteria 7 8 Pier Sandro Cocconcelli 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION The first generation of starter bacteria, generally 22 derived from cultures for vegetable fermentation, 23 Bacteria have long been an unrecognized agent such as L. plantarum and pediococci, was selected 24 responsible for fermentation and aroma formation of mainly for their acidification properties. The second 25 meat products. Traditional meat fermentation occurs generation has been developed isolating strains of 26 without the addition of selected microbial starter cul- meat origin, such as L. sakei and coagulase-negative 27 tures, and fermentation was driven by strains from the staphylococci, harboring phenotypic traits of techno- 28 indigenous microbiota deriving from raw materials, logical relevance. Increased knowledge on the phys- 29 mainly meat, and from the environment. Still today iological properties of meat-fermenting bacteria, 30 several fermented meat products are produced with- comprising information derived from genomic and 31 out starter cultures, and indigenous homofermentative proteomic analysis, and the use of molecular tools for 32 lactobacilli and coagulase-negative staphylococci strain characterization, now allows the development 33 (CNS), selected by the physicochemical conditions of a third generation of functional starter cultures, 34 imposed by production technology, dominate the fer- selected for the presence of technologically relevant 35 mentation process. The identification and the selec- traits. The aim of this chapter is to provide the most 36 tion of bacteria and their use as starter cultures began recent information on bacterial starters, and to delin- 37 in the second half of the twentieth century. In 1940, eate their functional properties to improve the quality 38 Jensen and Paddock (1940, US Patent 2,225,783) of fermented meat products. 39 developed the idea of inoculating the raw material for 40 fermented sausages using lactobacilli, with the aim to 41 govern and accelerate meat fermentation. BACTERIAL STARTER CULTURES 42 In the fifties, cultures of Micrococcus sp. and FOR FERMENTED MEAT: 43 Pediococcus cerevisiae were developed in Europe by CLASSIFICATION AND 44 Niinivaara (1955) and in the U.S. by Deibel and Niven PHYSIOLOGY 45 (1957). The combination of these first experiences on 46 meat starter cultures led Nurmi (1966) to develop the The bacterial starter cultures used for meat fermen- 47 first mixed culture composed of lactobacilli and tations are preparations of viable bacteria that 48 micrococci. Now, in the industrial process for fer- develop desired metabolic activities, primarily acidi- 49 mented sausage production, bacterial starter cultures fication and the development of aroma, during meat 50 are widely used to drive the fermentation process in fermentation. They are composed of lactic acid bac- 51 the desired direction, reducing the variability in the teria strains, primarily belonging to Lactobacillus or 52 quality of the product, limiting the growth of spoilage Pediococcus genus, of coagulase-negative staphylo- 53 bacteria by accelerating fermentation, and improving cocci (CNS) and members of Micrococcaceae. The S54 the sensorial properties of fermented meat products. most frequently used bacterial species and their N55 137 40454 13 137-146 r1.qxd 7/20/07 12:56 PM Page 138

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1 Table 13.1. Bacteria species most frequently used as meat starter culture. 2 3 Functional Properties 4 Species for Meat Fermentation Safety Qualification—Lack of 5 Lactobacillus sakei Acidification Acquired antimicrobial resistance** 6 Catalase activity Biogenic amine production 7 Flavor development 8 Amino acid metabolism 9 Antioxidant properties: 10 –catalase and SOD 11 Bacteriocin production* 12 ** 13 Lactobacillus curvatus Acidification Acquired antimicrobial resistance 14 Proteolytic activity Biogenic amine production 15 Antioxidant properties: catalase * 16 Bacteriocin production 17 Lactobacillus plantarum Acidification Acquired antimicrobial resistance** 18 Antioxidant properties: catalase Biogenic amine production 19 Bacteriocin production* 20 Lactobacillus rhamnosus Probiotic Acquired antimicrobial resistance** 21 Biogenic amine production 22 Pediococcus acidilactici Acidification Acquired antimicrobial resistance** 23 — Bacteriocin production* Biogenic amine production 24 ** 25 Pediococcus pentosaceus Acidification Acquired antimicrobial resistance * 26 Bacteriocin production Biogenic amine production 27 Staphylococcus xylosus Flavor development Acquired antimicrobial resistance 28 Antioxidant properties: Enterotoxin determinants 29 catalase and SOD 30 Amino acid catabolism 31 Fatty acid metabolism 32 Nitrate reduction 33 Staphylococcus carnosus Flavor development Acquired antimicrobial resistance 34 Antioxidant properties: Enterotoxin determinants 35 catalase and SOD 36 Amino acid catabolism 37 Fatty acid metabolism 38 Nitrate reduction 39 Staphylococcus equorum Flavor development Acquired antimicrobial resistance 40 Nitrate reduction Enterotoxin determinants 41 42 Kocuria varians Nitrate reduction 43 *Bacteriocin production is discussed in Chapter 14. 44 ** 45 Heterofermentative lactobacilli and pediococci are intrinsically resistant to vancomycin. 46 47 48 functions during sausage fermentation are reported lactic acid bacteria. This genus encompasses more 49 in Table 13.1. than 100 different species with a large variety of 50 phenotypic, biochemical, and physiological proper- 51 ties (Axelsson 2004), but only few of them have LACTOBACILLUS 52 been isolated from meat fermentations and are used 53S The genus Lactobacillus is a wide and heteroge- as starter cultures. L. sakei, L. curvatus and L. plan- 54N neous taxonomic unit, composed of rod-shaped tarum, belonging to the subgroup of facultative 40454 13 137-146 r1.qxd 7/20/07 12:56 PM Page 139

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heterofermentative lactobacilli, are generally used to the L. casei –Pediococcus subcluster of the Lacto- 1 for this purpose. The main energetic metabolism of bacillus cluster. The genus consists currently of nine 2 these bacteria is the dissimilation of sugar to organic species, but only P. pentosaceus is generally used as 3 acid, by means of glycolysis and phosphoketolase a starter culture for meat fermentation. The species 4 pathways. When hexoses are the energy source, lac- P. cerevisiae,frequently mentioned as a starter cul- 5 tic acid is the major fermentation end product. ture, has been now reclassified as P. pentosaceus. 6 L. sakei is the predominant species in fermented The genome sequencing project of this species is 7 meat products and its use as a starter culture for ongoing (http://genome.jgipsf.org/draft_microbes/ 8 sausage production is widespread. The recent analy- pedpe/pedpe.info.html). 9 sis of the 1.8 Mb genome sequence (Chaillou et al. 10 2005) has revealed that this organism has evolved to 11 STAPHYLOCOCCUS AND KOCURIA GENERA adapt itself to the meat environment, harboring the 12 genetic function that confers the ability to grow and Although Micrococcaceae are frequently mentioned 13 survive in the meat environment. The adaptation to as components of meat starter cultures, this term gen- 14 the meat, an environment rich in amino acids due to erally refers to members of Staphylococcus genus, 15 the activity of endogenous proteases, has led to a which belong to the family Staphylococcaceae. 16 lack of biosynthetic pathways for amino acid synthe- Staphylococci were originally grouped with other 17 sis. Thus, L. sakei is auxotrophic for all amino acids gram-positive cocci, such as Micrococcus genus, 18 but not aspartic and glutamic acid (Champomier- because these two genera often cohabit the same 19 Vergès et al. 2002). Amino acid metabolism can pro- habitats. However, molecular taxonomy has revealed 20 vide an alternative energy source for L. sakei when that these genera are phylogenetically separate and 21 glucose is exhausted, and this affects the sensorial distant: Staphylococcus belongs to the cluster of low 22 properties of the sausage, as discussed later. More- GC gram-positive bacteria, and Micrococcus is part 23 over, this species has psychrotrophic and osmotoler- of the Actinomycetales. 24 ant properties, being able to grow at low temperature The staphylococci present a spherical shape and 25 and in the presence of up to 10% of sodium chloride cell often grouped to form clusters. Staphylococcus 26 (NaCl). The physiological features are associated to genus can be separated into 38 species and 21 sub- 27 the presence in the genome of a higher number of species, consisting of 6 coagulase-positive or vari- 28 genes coding for stress response proteins, such as ability coagulase and 32 coagulase-negative species 29 cold shock and osmotolerance proteins, when com- (CNS). 30 pared to other Lactobacilli. Staphylococci are widespread in nature; their 31 Although L. plantarum has been identified as part major habitats are skin, skin glands, and mucous 32 of the meat microbiota and it is used as starter cul- membranes of mammals and birds. Some species, 33 tures for meat fermentation, this species lacks the mainly coagulase-positive such as S. aureus, are 34 meat specialization found in L. sakei. L. plantarum pathogens responsible for infections and foodborne 35 is a versatile bacterium, encountered in a variety of intoxications. Staphylococci are facultative anaer- 36 different environment and a genome size of 3.3 Mb obes capable of metabolizing a number of different 37 (Kleerebezem et al. 2003), the wider genome of Lac- sugars. Under anaerobic conditions, the major end 38 tobacillus genus, reflects its metabolic and environ- product is lactic acid, but acetate and pyruvate and 39 mental flexibility. acetoin are also formed. These organisms show the 40 Much less information is available on the physiol- ability to survive environmental stress, such as 41 ogy and genetics of L. curvatus, other than the pro- high salt and low temperatures, encountered during 42 duction of antimicrobial peptides, discussed in meat fermentation. Moreover, they reduce nitrate 43 Chapter 14. to nitrite, a technologically relevant physiological 44 feature. 45 Many CNS, such as Staphylococcus xylosus, 46 PEDIOCOCUS S. carnosus, S. equorum and S. saprophyticus, have 47 Pediococci are gram-positive, coccus-shaped, lactic been isolated from dry-fermented sausages, but 48 acid bacteria, showing the distinctive characteristic other species occur too. The genome sequencing 49 of tetrads formation, via cell division in two perpen- of S. xylosus is ongoing and information can 50 dicular directions in a single plane. Pediococci pres- be found at www.cns.fr/externe/English/Projets/ 51 ent a homofermentative metabolism where lactic Projet_NN/NN.html. 52 acid is the major metabolic end product (Axelsson Kocuria varians, formerly classified as Micrococ- S53 2004). Phylogenetically Pediococcus species belong cus varians, is a member of Micrococcaceae and is N54 40454 13 137-146 r1.qxd 7/20/07 12:56 PM Page 140

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1 used as meat starter cultures for its nitrate reductase adaptation of this organism to the meat environment. 2 ability. Glucose, the favourite carbon source of L. sakei, is 3 rapidly consumed in meat; adenosine and inosine 4 are abundant, reaching twice the concentration of 5 FUNCTIONAL PROPERTIES OF glucose. L. sakei harbors genes coding for adenosine 6 BACTERIAL STARTER CULTURES deaminase, for inosine hydrolase, and for nucleoside 7 FOR FERMENTED MEAT phosphorilase, which allow the release of ribose 8 moiety from nucleoside and its subsequent metabo- COLONIZATION AND ESTABLISHMENT IN THE 9 lism (Chaillou et al. 2005). Moreover, the presence BACTERIAL COMMUNITY OF MEAT FERMENTATION 10 of methylglyoxal synthase, a novel genetic trait 11 One of the fundamental properties of bacterial in lactic acid bacteria, has been proposed as a path- 12 starter cultures is the ability to compete with the way to counteract frequent glucose starvation and 13 indigenous microbiota of meat, to colonize this envi- modulate metabolism of alternative carbon sources 14 ronment, and to dominate the microbial community (Chaillou et al. 2005). 15 of the fermented products. Thus successful coloniza- 16 tion of meat products by starter cultures is a prereq- 17 uisite for their contribution to sensorial qualities of NITRATE REDUCTION 18 the final product. The nitrate is added to the fermented sausages by its 19 In recent years, the development of a variety of capacity to fix and to obtain the typical color of cured 20 molecular methods has allowed the study of the products other than by its antimicrobial properties. 21 growth dynamics of bacterial strains during meat To be effective, the added nitrate must be reduced to 22 fermentation. Staphylococcal community develop- nitrite. Besides contributing to flavor, staphylococci 23 ment in traditional dry-fermented sausage has been and Kocuria have a role for their nitrate reductase 24 studied by means of Multiplex PCR, Pulse Field Gel and antioxidant activities (Talón et al. 1999). These 25 Electrophoresis (PFGE) and sequencing analysis of microorganisms reduce nitrate to nitrite, which is 26 the sodAgene (Corbière Morot-Bizot et al. 2006). important for the formation of nitrosylmyoglobin, the 27 Fontana et al. (2005) showed that RAPD is a valid compound responsible for the characteristic red color 28 method to monitor the population dynamics in of fermented meats. The nitrate reductase activity is 29 sausage fermentation, and this technique has more widespread in CNS: It has been detected in S. xylo- 30 recently been applied to assess the performance of sus, S. carnosus, S. epidermidis S. equorum, S. lentus, 31 bacterial strains from commercial starter cultures and S. simulans (Talón et al. 1999; Mauriello et al. 32 (Cocolin et al. 2006). 2004). In S. carnosus, the molecular genetic determi- 33 nants for nitrogen regulation, the nreABC genes, 34 ACIDIFICATION were identified and shown to link the nitrate reduc- 35 tase operon (narGHJI) and the putative nitrate trans- 36 Acidification in sausage manufacture is mainly the porter gene narT. The data provide evidence for a 37 result of carbohydrate fermentation by lactic acid global regulatory system with oxygen as the effectors 38 bacteria. This process has a central role for flavor, molecule (Fedtke et al. 2002). 39 color, and texture development and for control of 40 undesired microbiota during fermentation. The pro- 41 duction of lactic acid is primarily derived from the FLAVOR FORMATION 42 dissimilation of endogenous glucose or other sugars 43 added to the batter. The addition of sugar to the batter The flavor and the aroma of fermented meats is 44 and the manufacture technology (temperature, salt- formed in a combination of several elements, such 45 ing and ripening time) influence the acidification as batter ingredients, manufacture technology, activ- 46 rate, the final pH, and the sensorial properties of fer- ity of tissular enzymes, proteases and lipases, and 47 mented sausage. The level of acidification required microbial metabolism. Bacterial community con- 48 depends on the sensorial properties of the product: tributes to flavor development through carbohydrate 49 lactic acid taste is desired in north European sausages dissimilation, peptide metabolism, and lipolysis. 50 but not in typical south European fermented meats. Lactic acid bacteria produce lactic acid and small 51 Acidification could also be the result of alterna- amounts of acetic acid, ethanol, and acetoin, and 52 tive pathways. In L. sakei, the presence of genes together with staphylococci, are involved in peptide 53S involved in the energetic catabolism of nucleoside, and amino acid metabolism; only CNS show an 54N such as adenosine and inosine, is an example of the effective metabolism of lipids. 40454 13 137-146 r1.qxd 7/20/07 12:56 PM Page 141

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Metabolism of meat protein is of major impor- increase the concentration of methylketones, ethyl 1 tance for flavor characteristics of fermented meats. esters (Larrouture et al. 2000; Beck et al. 2002). 2 In these products, tissular proteases as well as micro- 3 bial enzymes are responsible for the proteolytic 4 ANTIOXIDANT PROPERTIES changes that occur during meat processing, leading 5 to the generation of small peptides and free amino The metabolism of most lactic acid bacteria, such as 6 acids, which are considered to be flavor compound the indigenous lactobacilli which contaminate raw 7 precursors (Aristoy and Toldrá 1995; Sanz and meat, could lead to the formation of hydrogen perox- 8 Toldrá 1997; Sanz et al. 1998). It has been shown ide, a compound that interferes with the sensorial 9 that several Lactobacillus spp. exhibit proteolytic properties of meat products, being involved in discol- 10 activity on porcine muscle myofibrillar and sar- oration of nitroso-heme-pigment and lipid oxidation. 11 coplasmic proteins; even the processing conditions Bacterial strains used in meat cultures can produce 12 during meat fermentation did not favor meat protein catalase, antioxidant enzymes that disproportionate 13 breakdown by bacterial proteases. Fadda et al. (2001) hydrogen peroxide to oxygen and water, preventing 14 reported the contribution of curing conditions, in the the risk of reduced quality of fermented meat. Thus, 15 generation of hydrophilic peptides and free amino catalase production is considered a relevant techno- 16 acids by the proteolytic activity of L. curvatus CRL logical property of starter cultures for meat fermented 17 705. Moreover, it has been demonstrated that L. sakei products (Leroy 2006). Production of this antioxidant 18 play an important role in amino acid generation enzyme is a common trait in aerobic bacteria, such as 19 (Fadda et al. 1999a,b; Sanz et al. 1999; Champomier- CNS. The characterization of catalase and superoxide 20 Verges et al. 2002). Sanz and Toldrá (2002) reported dismutases in S. carnosus and S. xylosus have been 21 an arginine-specific aminopeptidase activity in reported. The catalase gene katA of S. xylosus has 22 L. sakei important for the release of the free amino been studied in detail (Barrière 2001a,b; 2002). 23 acid, because it could be further channeled into the Transcriptional activity of this gene is induced upon 24 arginine deiminase pathway. The genes encoding the entry into stationary phase by oxygen and hydrogen 25 proteins required for arginine catabolism in L. sakei peroxide. Moreover, a second gene coding for heme- 26 are organized in a cluster (Züriga et al. 2002) and dependent catalase has been detected in S. xylosus. 27 their transcription is repressed by glucose and Although lactic acid bacteria have long been con- 28 induced by arginine. Arginine in particular is an sidered as a catalase-negative microorganism, in the 29 essential amino acid for L. sakei and specifically last decade two groups of catalase activity have been 30 promotes its growth in meat, being used as an energy reported in genera Lactobacillus, Pediococcus, and 31 source in absence of glucose (Champomier-Verges Leuconostoc. The first group is defined as heme cata- 32 et al. 1999). The concentration of free arginine in lase and the second group nonheme Mn-containing 33 raw meat is low, although it is relatively abundant in catalase. The presence of a heme-dependent catalase 34 muscle myofibrillar proteins. Moreover, the genome has been demonstrated in L. plantarum (Igarashi 35 analysis have shown that L. sakei harbors a second et al. 1996) and L. sakei (Noonpakdeea et al. 1996). 36 putative arginine deiminase pathway, containing two Moreover, the genome analysis of L. sakei, revealed 37 peptydil-arginine deaminases, enzymes that can that this meat organism harbors several system for 38 contribute to the metabolism of arginine residues not the protection against reactive oxygen species, such 39 released from peptides (Chaillou et al. 2005), as Mn-dependent SOD, heme-dependent catalase 40 increasing the competitiveness of L. sakei in meat (Chaillou et al. 2005). 41 environment. 42 To ensure sensory quality of fermented sausages, 43 the contribution of staphylococci with their prote- SAFETY OF BACTERIAL 44 olytic and lipolytic activities is fundamental. The STARTER CULTURES FOR 45 volatiles so far recognized as being produced by FERMENTED MEAT 46 staphylococci are primarily amino acid catabolites, 47 pyruvate metabolites, and methylketones from The safety of bacterial strains intentionally added to 48 β-oxidation of fatty acids (Stahnke et al. 2002). In food, such as starter cultures used for meat prod- 49 particular, S. xylosus and S. carnosus modulate the ucts, is becoming an issue for their application in 50 aroma through the conversion of amino acids (parti- food. Although meat starter cultures have a long 51 cularly the branched-chain amino acids leucine, history of apparent safe use, safety concerns can be 52 isoleucine, and valine) and free fatty acids. They also associated with lactic acid bacteria and, more fre- S53 were able to enhance the dry-salami odor and to quently, with CNS. N54 40454 13 137-146 r1.qxd 7/20/07 12:56 PM Page 142

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1 ANTIMICROBIAL RESISTANCE staphylococcal enterotoxins genes in CNS from 2 slightly fermented sausages was rare, detecting only A risk factor potentially associated with all bacterial 3 entC in S. epidermidis. It is essential that the CNS groups used as starter cultures for sausage is the pres- 4 strains selected to be used as starter culture do not ence of acquired genes for antimicrobial resistance. 5 produce enterotoxins and do not harbor gene coding The emergence and spread of resistance to antimi- 6 for enterotoxin or enterotoxin-like superantigens. crobials in bacteria poses a threat to human and 7 animal health, and commensal bacteria has been 8 proposed to act as reservoirs of resistance genes. BIOGENIC AMINES PRODUCTION 9 Antibiotic resistance bacteria has been isolated from 10 Biogenic amines (BA) such as cadaverine, putrescine, raw meat and meat products and genetic determi- 11 spermidine, histamine, phenethylamine, agmatine, nants for antimicrobial resistance, such as tet(M) 12 and tyramine, are basic compounds present in living conferring resistance to tetracycline, has been identi- 13 organisms at low levels where they are responsible fied in L. plantarum and L. sakei (Gevers et al. 2003). 14 for many essential functions. The presence in food of Antimicrobial resistance in CNS has been studied in 15 these bioactive compounds is concern for health, for detail, due to its clinical relevance. These bacteria 16 their biological effect, which can lead to toxicologi- display a high prevalence of antibiotic resistance 17 cal symptoms, such as pseudoallergenic reactions, (Archer et al. 1994; Agvald-Ohman et al. 2004) and 18 histaminic intoxication, and interaction with drugs. can constitute a reservoir of antibiotics resistance 19 (Mariné-Font et al. 1995). The formation of BA in genes that can be transferred to other staphylococci 20 fermented foods requires the presence of decarboxy- (Wieders et al. 2001). Moreover, antibiotic resis- 21 lase-producing microorganisms, which may be part tance strains were found in fermented meat products 22 of the associated microbiota of a particular food (Gardini et al. 2003; Martin et al. 2006) and genes 23 product or can be introduced by contamination for antimicrobial resistance to tetracycline, tet(M), 24 before, during, or after food processing. The final BA tet(K), to erythromycin, ermB and ermC, and to β- 25 contents in fermented sausages depend on the lactams (blaZ and mecA), has been detected in CNS 26 microbial composition of meat used as raw material, isolated from fermented meat (S. Gazzola, personal 27 but also on the type and activity of the starter culture communication 2006). 28 inoculated (Bover-Cid et al. 2001). High levels of An additional concern is that, even in the absence 29 BA, especially tyramine, but also histamine and of selective pressure, mobile genetic elements carry- 30 the diamines putrescine and cadaverine, have been ing antibiotic resistance can be transferred at high 31 described in fermented sausages (Hernández-Jover frequency among the microbial community during 32 et al. 1997a,b; Bover-Cid et al. 2000a,b). sausage fermentation (Cocconcelli et al. 2003). 33 The use of highly competitive decarboxylase- 34 negative starter cultures was shown to prevent the 35 growth of biogenic amine producers and leads to end ENTEROTOXINS PRODUCTION 36 products nearly free of BA, as long as the raw mate- 37 Risk concerns are related to CNS, because members rial is of sufficient quality (Bover-Cid et al. 2001). 38 of this group, primarily S. epidermidis, have been González-Fernández et al. (2003), reported that to 39 identified as the third most common cause of intra- avoid the presence of high concentrations of BA in 40 hospital infections and most frequent cause of chorizo, it is advisable to use a competitive starter 41 bacteremia (CDC NNIS System 1998). Moreover, culture of L. sakei, a negative-decarboxylate strain, 42 staphylococci could be the cause of foodborne in- which may decrease the pH quickly during the fer- 43 toxication by contaminated food consumption for mentation step and be predominant throughout the 44 the production of termostable enterotoxins (Balaban process; thus it would prevent the growth of bacteria 45 and Rasooly 2000). Although CNS of food origin that can produce BA. Similarly, the presence of a 46 have not been found to produce nosocomial infec- selected starter culture L. sakei CTC494 reduced BA 47 tions, some strains have been described that produced accumulation during ripening, but only if raw meat 48 enterotoxins, but to date, the coagulase-positive S. was characterized by good quality. Addition of the 49 intermedius is the only species in addition to S. same strain of L. sakei, along with proteolytic 50 aureus that clearly has been related to staphylo- S. carnosus and S. xylosus, decreased BA accumula- 51 coccal intoxication. Vernozy-Rozand et al. (1996) tion in the production of fuet (Bover-Cid et al. 52 reported enterotoxin E as the most frequent entero- 2000a) and reduced the total BA content 80–90% 53S toxin found in S. equorum and S. xylosus,however, with respect to the sausages without starter cultures 54N Martin et al. (2006), reported that the occurrence of added. 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possess amine oxidase activity might be a way of analysis of gene expression at genome level), and 1 further decreasing the amount of BA produced high throughput screening, combined with the 2 during meat fermentation (Martuscelli et al. 2000; increased knowledge of the interaction between 3 Fadda et al. 2001; Gardini et al. 2002; Suzzi and technology and the bacterial community, may allow 4 Gardini 2003). the identification of new strains from fermented 5 meat with improved functional properties and lack- 6 ing undesirable traits, such as virulence and antibi- 7 MEAT STARTER CULTURES AND otic resistance determinants. Moreover, the microbial 8 PROBIOTICS biodiversity of traditional high-quality fermented 9 Probiotics, live microorganisms, which when admin- meat products, characterized by a complex micro- 10 istered in adequate amounts confer a health benefit bial community highly adapted to meat environ- 11 on the host (FAO 2006), are added to a variety of ment, could be exploited to provide the new strains 12 food. Recently, attention has been directed to the use of lactic acid bacteria and CNS for development of 13 of fermented sausages as a food carrier because new functional starter cultures. 14 these products could contain high numbers of viable 15 lactic acid bacteria. To use probiotics as starter cul- REFERENCES 16 tures for fermented sausages, in addition to the 17 demonstrated probiotic features (FAO 2006), other C Agvald-Ohman, B Lund, C Edlun. 2004. Multiresis- 18 properties are demanded. The probiotic culture tant coagulase-negative staphylococci disseminati 19 should be well adapted to the conditions of fer- frequently between intubated patient into a multidis- 20 mented sausage to become dominant in the final ciplinary intensive care unit. Crit6 Care 8:42–47. 21 product, competing with other bacterial populations G Archer, D Niemeyer, J Thanassi, J Pucci. 1994. Dis- 22 from meat and from starter culture. In addition, the semination among staphylococci of DNA sequences 23 culture should not develop off-flavors in the product. associated with methicillin resistance. Antmicrob 24 Commercial probiotic cultures, such as strains Agents Chemother 38:447–454. 25 L. rhamnosus GG, L. rhamnosus LC-705, L. rham- MC Aristoy, F Toldrá. 1995. Isolation of flavour pep- 26 nosus E-97800, and L. plantarum E-98098 have tides from raw pork meat and dry-cured ham. In: 27 been tested as functional starter culture strains in G Charalambous, ed. Food Flavours: Generation, 28 Northern European sausage fermentation without Analysis and Process Influence. Amsterdam, The 29 negatively affecting the technological or sensory Netherlands: Elsevier Science B.V., pp. 1323–1344. 30 properties, with an exception for L. rhamnosus LC- L Axelsson. 2004. Lactic acid bacteria: Classification 31 705 (Erkkila et al. 2001a, b). Klingberg et al. (2005), and physiology. In: S Salminen, A Ouwehand, A Von 32 identified L. plantarum and L. pentosus strains, orig- Wright, eds. Lactic Acid Bacteria: Microbiology and 33 inated from the dominant NSLAB of fermented Functional Aspects, 3rd ed. New York: Marcel 34 meat products, as promising candidates of probiotic Dekker, Inc. 35 meat starter cultures suitable for the manufacture of N Balaban, A Rasooly. 2000. Staphylococcal enterotox- 36 the Scandinavian-type fermented sausage. ins. Int J Food Microbiol 61:1–10. 37 C Barriere, R Bruckner, D Centeno, R Talon. 2002. 38 Characterisation of the katA gene encoding a cata- 39 PROTECTIVE MEAT STARTER lase and evidence for at least a second catalase 40 CULTURES activity in Staphylococcus xylosus, bacteria used in 41 food fermentation. FEMS Microbiol Lett 216(2): 42 The production of bacteriocins and other antimicro- 277–283. 43 bial compounds, one of the most promising techno- C Barriére, D Centeno, A Lebert, S Leroy-Setrin, 44 logical features of starter cultures, is discussed in J Berdagué, R Talon. 2001a. Roles of superoxide dis- 45 Chapter 14. mutase and catalase of Staphylococcus xylosus in the 46 inhibition of linoleic acid oxidation. FEMS Micro- 47 CONCLUSION biol Lett 201:181–185. 48 C Barriére, S Leroy-Sétrin, R Talon. 2001b. Charac- 49 A new generation of starter cultures, with distinctive terization of catalase and superoxide dismutase in 50 properties for quality and safety of fermented meat Staphylococcus carnosus 833 strain. J Appl Micro- 51 products, could be expected from advancement in biol 91:514–519. 52 the study of lactic acid bacteria and CNS. New tools, H Beck, A Hansen, F Lauritsen. 2002. Metabolite S53 such as genomic, proteomic, transcriptomic (the production and kinetics of branched-chain aldehyde N54 40454 13 137-146 r1.qxd 7/20/07 12:56 PM Page 144

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1 2 3 14 4 5 6 Starter Cultures: Bioprotective Cultures 7 8 Graciela Vignolo and Silvina Fadda 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION risk of bacterial resistance, which constitutes a 22 microbiological hazard. On the basis of these data, 23 In the last few years, concerns over food safety have the need emerges for solutions to the problem of 24 increased their importance due to its dramatic food hygienic quality. Consumers are increasingly 25 impact on public health. Over the past decade, a demanding pathogen-free foods with minimal pro- 26 series of food scandals have erupted involving meat cessing, fewer preservatives and additives, high nutri- 27 and meat products, which feature prominently in the tional value, and intact sensory quality. In response to 28 food safety crisis. At present, bovine spongiform these conflicting demands, current trends in the meat 29 encephalitis and the rapid spread of avian influenza industry include the investigation of alternatives for 30 from eastern to western countries have triggered a safer and healthier products. Biopreservation has 31 sudden lack of consumer confidence in meat prod- gained increasing attention as a means of naturally 32 ucts and led to a dramatic fall in demand. The glob- controlling the shelf life and safety of meat products. 33 alization of commerce, the gradual increase in world The application of bioprotective cultures to ensure 34 population and the change in lifestyles have resulted hygienic quality is a promising tool. However, it 35 in consumer claims for safety oriented to foods of should be considered only as an additional hurdle to 36 animal origin. Undoubtedly, the major threat to food good manufacturing, processing, storage, and distri- 37 safety is the emergence of “new” pathogens. The bution practices. In the present contribution, the use 38 recent role of Listeria monocytogenes, Escherichia of antagonist microorganisms to inhibit and/or inacti- 39 coli O157:H7, Campylobacter jejeuni, Yersinia ente- vate pathogens and spoilage flora in meat fermented 40 rocolitica and Vibrio parahemolyticus as food-borne products is discussed, with particular reference to 41 microorganisms has been related to the increase in bacteriocin-forming bacterial strains. A new concept 42 food poisoning outbreaks, compared to traditional of starter cultures for fermented sausages is ana- 43 food pathogens. Changes in the food chain will con- lyzed where biopreservative and probiotic features 44 tinue to create opportunities for the emergence of complete the recently established essential criteria 45 new diseases and the reemergence of old ones (Elmi for a meat starter culture. 46 2004; Church 2004). In addition, the presence in 47 meat products of chemical additives and residues of 48 agrochemical and veterinary drugs are also perceived DRY-CURED SAUSAGES 49 by consumers as a health risk. Even when the level of MICROORGANISMS INVOLVED 50 these residues seldom exceeds regulatory limits in 51 meat products (Tarrant 1998), the use of antibiotics The fermentation of foodstuffs can be traced 52 in intensive animal production poses the additional back thousands of years. It comes forth as a food 53 S54 N55 147 40454 14 147-158 r1.qxd 7/21/07 9:56 AM Page 148

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1 preservation procedure although it was only in the TARGET PATHOGENS AND SPOILAGE MICROBIOTA IN 2 more recent past that microorganisms were recog- DRY-FERMENTED SAUSAGES:SHELF STABILITY AND 3 nized as being responsible for the fermentation THE HURDLE EFFECT 4 process. Traditional dry sausages rely on natural Fermented dry sausages are known as shelf-stable 5 contamination by environmental flora, which occurs products, this term referring to those products that do 6 during slaughtering and increases during manufac- not require refrigeration or freezing for safety and 7 turing. Each workshop has a specific “house flora” acceptable organoleptic characteristics because most 8 composed of useful microorganisms for fermenta- often they are stored at room temperature. Shelf sta- 9 tion and flavor development of sausages, but also of 10 spoilage and pathogenic flora. Different species of bility is due to a combination of factors known as the 11 lactic acid bacteria (LAB) and gram-positive, coagu- hurdle effect (Leistner 1996, 2000). Dry-fermented 12 lase-negative cocci (GCC) are the microorganisms sausages (salami and salami-type sausage) become 13 primarily responsible for sausage fermentation. stable and safe through a sequence of hurdles, some of which are specifically included (NaCl, NaNO / 14 LAB, in particular lactobacilli, contribute to the 2 NaNO , ascorbate) but others are indirectly created in 15 hygienic and sensory quality of meat products 3 16 mainly through their carbohydrate and protein catab- the stuffed mix (low Eh, antagonistic substances, low 17 olism resulting in sugar depletion, pH reduction, water activity). By means of these hurdles, spoilage 18 production of antimicrobial agents, and generation and food-poisoning bacteria are inhibited, whereas the 19 of flavor compounds (Lücke 2000; Talon et al. desirable organisms, especially LAB, are hardly 20 2002). GCC participate in color development and affected. Apart from LAB, GCC, and molds and 21 stabilization through a nitrate reductase activity that yeasts involved in sausage fermentation, beef and 22 leads to the formation of nitrosomyoglobin. The pork meat as the major components of dry-cured 23 antioxidant potential due to catalase and superoxide sausages regularly contain pathogenic bacteria and 24 dismutase activity, the role in aroma formation via are often implicated in the spread of foodborne 25 GCC proteolytic and/or lipolytic properties, and the diseases. Raw dry sausage materials (meat and cas- 26 removal of excess nitrate in the meat batter con- ings) are the principal vehicles for pathogens and 27 tribute to a stable and safe fermented product contaminating microorganisms. After dressing of 28 (Stahnke 2002). Molds and yeast traditionally play carcasses, the microbiota comprises a mixture of 29 an important role by bringing about a characteristic mesophiles and psychotrophs. During meat chilling a 30 surface appearance and flavors due to their well- selection is produced and mesophiles growth will 31 known proteolytic and lipolytic activity. Inoculation no longer occur. Because most pathogens are 32 of sausage batter with a starter culture composed of mesophiles, meat obtained in good hygienic condi- 33 selected microorganisms improves the quality and tions would presumably not be implicated in sanitary 34 safety of the final product and standardizes the pro- risk. Still, the growth of pathogenic bacterial species 35 duction process (Lücke 2000; Demeyer 2003). The overcoming the existent natural hurdles can occur. 36 use of starter cultures in meat products has Food-poisoning Staphylococcus aureus, Salmonellae, 37 increased over the past 25 years, mainly in large- and Clostridium perfringes have been traditionally 38 scale industrial processes. Commercial starter cul- implicated in fermented dry sausage contamination, 39 tures in Europe are generally made up of a balanced and species within the genera Campylobacter and 40 mixture of LAB (Lactobacillus, Pediococcus) and Yersinia as well as L. monocytogenes and shiga toxin- 41 GCC (Staphylococcus, Kocuria). In Mediterranean producing E. coli have recently emerged as pathogens 42 countries, yeasts and molds are also inoculated onto of major public health concern. Dry-fermented 43 the surface, in contrast to northern technology in sausage conditions, curing additives, and the presence 44 which smoke is applied. In spontaneously fermented of LAB starter cultures may act as significant hurdles 45 European sausages, homofermentative lactobacilli for the control of these pathogens (Table 14.1). How- 46 constitute the predominant flora throughout ripen- ever, these hurdles are not sufficient to prevent the 47 ing, Lactobacillus sakei and Lactobacillus curvatus survival of L. monocytogenes or E. coli O157:H7 dur- 48 dominating the fermentation process. Recently, ing the manufacturing process. An additional hurdle 49 studies carried out on small-scale processing plants to reduce the risk of L. monocytogenes would be the 50 confirmed on the bases of their genetic fingerprints use of competitive bacteriocin-producing starter cul- 51 that Lb. sakei was the predominant species isolated tures referred to as bioprotective cultures. Besides, 52 from artisanal fermented sausages (Fontana et al. strategies to overcome the barrier presented by the 53S 2005). outer membrane in gram-negative bacteria, such as 54N 40454 14 147-158 r1.qxd 7/21/07 9:56 AM Page 149

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Table 14.1. Effective hurdles inhibitory to pathogens objectives, respectively. Food processors face a 1 present in dry fermented sausages. major challenge, with consumers demanding safe 2 foods with a long shelf-life, but also expressing a 3 Pathogen Hurdles preference for minimally processed products, less 4 Ͻ Ͻ severely damaged by heat and freezing and not con- 5 Staphylococcus aureus pH 5.1; aw 0.86; bacteriocins taining chemical preservatives. Bacteriocins consti- 6 tute an attractive option that could provide at least 7 Salmonella pH Ͻ 5.0; a Ͻ 0.95; w part of the solution. 8 NaCl/NaNO2 9 Clostridium perfringes LAB (acid and 10 ANTIMICROBIAL PEPTIDES PRODUCED BY LAB bacteriocins) 11 ASSOCIATED WITH MEAT PRODUCTS Yersinia enterocolítica LAB (acid) 12 Campylobacter jejuni LAB (acid) Acid production as a result of carbohydrate catabo- 13 lism is a common feature among LAB, although not 14 Listeria monocytogenes a Ͻ 0.90; bacteriocins w all LAB can produce antimicrobial peptides during 15 Escherichia coli O157:H7 LAB (acid) growth. The production of these antagonistic sub- 16 stances seems to be a common phenotype among 17 LAB because numerous bacteriocins have been iso- 18 lated over the last three decades, varying in size from 19 the use of chelating agents, will improve bacteriocin small (<3 kDa), heavily post-translationally modi- 20 efficiency. fied peptides, to large heat-labile proteins. Bacteri- 21 ocins produced by LAB are a heterogeneous group 22 of peptides and proteins. The latest revised classifi- 23 BIOPROTECTIVE CULTURES cation scheme divides them into two main cate- 24 gories: the lanthionine-containing lantibiotics (class I) 25 BIOPROTECTION:DEFINITION and the non-lanthionine–containing bacteriocins 26 Preservation of foods using antagonistic microor- (class II); the large, heat-labile murein hydrolases 27 ganisms or their antimicrobial metabolites has been (formerly class III bacteriocins) constitute a separate 28 termed bioprotection or biopreservation. Antagonis- group called bacteriolysins (Cotter et al. 2005). 29 tic cultures added to foods to inhibit pathogens Even though there has been a dramatic increase in 30 and/or extend the shelf life, while changing the sen- the number of novel bacteriocins discovered in the 31 sory properties as little as possible, are referred to as past two decades, biochemical and genetic charac- 32 protective cultures (Lücke 2000). The main objec- terization has been carried out only in some of them. 33 tives of bioprotection are to extend storage life, to The majority of bacteriocins identified to date and 34 enhance food safety, and to improve sensory quali- produced by LAB belong to class I and II and the 35 ties. LAB have a major potential for use in biop- present description will principally focus on the one 36 reservation because they are safe for consumption, produced by LAB associated with meat and meat 37 and during storage they naturally dominate the products (Table 14.2). Lactococcal bacteriocins are 38 microbiota of many foods. Due to their typical asso- produced by several species of Lactococcus lactis 39 ciation with food fermentations and their long tradi- isolated from dairy, vegetable, and meat products 40 tion as food-grade bacteria, LAB are Generally (Guinane et al. 2005). In favorable conditions, nisin 41 Recognized as Safe (GRAS). In addition, antimicro- has a wide spectrum of inhibition against gram- 42 bial peptides produced by LAB can be easily broken positive microorganisms. It has been extensively 43 down by digestive proteases so as not to disturb gut characterized and the precise structure of its mole- 44 microbiota. LAB can exert a bioprotective or cule and the mechanism of action have been deter- 45 inhibitory effect against other microorganisms as a mined. Although nisin is the only commercially 46 result of competition for nutrients and/or of the pro- exploited lantibiotic to date, efforts have been made 47 duction of bacteriocins or other antagonistic com- to develop applications for other lantibiotics. Among 48 pounds such as organic acids, hydrogen peroxide, them, lacticin 3147, a two-peptide lantibiotic pro- 49 and enzymes. A distinction can be made between duced by L. lactis subsp. lactis DPC3147 isolated 50 starter cultures and protective cultures in which from Irish kefir grains, exhibits a bactericidal mode 51 metabolic activity (acid production, protein hydroly- of action against food spoilage and pathogenic bac- 52 sis) and antimicrobial action constitute the major teria. The high heat stability and broad pH range of S53 N54 40454 14 147-158 r1.qxd 7/21/07 9:56 AM Page 150

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1 activity of lacticin 3147 make it attractive for use in isolated from Greek traditional fermented sausages 2 the food industry. Even though most lactococcal bac- producing curvaticin L442, was also demonstrated 3 teriocins were isolated from dairy and vegetable to inhibit L. monocytogenes. Due to their high 4 products, several nisin-producing L. lactis strains antilisterial potential, these bacteriocin producer 5 were isolated from fermented sausages, indicating LAB are of considerable interest as biopreservative 6 the potential use of lactococci in meat fermentation. cultures (Table 14.2). The two-peptide bacteriocins 7 Nisin-producing L. lactis strains from Spanish fer- (class IIb) require the combined activity of both pep- 8 mented sausages and from traditional Thai fermented tides to exert their antimicrobial activity. Lactococ- 9 sausage were effective in inhibiting closely related cin G and lactacin F produced by L. lactis and 10 LAB, L. monocytogenes, Cl. perfringes, B. cereus, Lactobacillus johnsonii of dairy origin, respectively, 11 and St. aureus (Leroy at al. 2006). Moreover, Lb. sakei isolated and characterized in the early 1990s, are the 12 L45 isolated from Norwegian dry sausages and Lb. first reported two-peptide bacteriocins (Garneau 13 sakei 148 from Spanish fermented sausages secrete et al. 2002). These bacteriocins have a narrow 14 lactocin S, a lantibiotic whose moderate spectrum of spectrum of inhibition against other lactobacilli, 15 activity comprises LAB and Clostridium (Aymerich Clostridium and E. faecalis. Plantaricins EF and JK 16 et al. 1998). The abundance of LAB strains produc- produced by Lb. plantarum C11 from vegetable ori- 17 ing lantibiotic bacteriocins shows the special rele- gin and plantaricin S produced by Lb. plantarum 18 vance of these substances in fermented products; LPCO10 from fermented green olives are among the 19 they are present in very different products and cli- best characterized two-peptide systems. On the other 20 matic environments. Class II bacteriocins include a hand, lactocin 705, a two-peptide bacteriocin produced 21 very large group of small (<10 kDa) heat-stable pep- by Lb. curvatus CRL705 (formerly Lb. casei) iso- 22 tides but, unlike lantibiotics, they are unmodified bac- lated from Argentine fermented sausages, showed 23 teriocins. Four different groups have been recently to be antagonistic toward other LAB and Bro- 24 suggested (Cotter et al. 2005): class IIa include chothrix thermosphacta when tested in meat systems 25 pediocinlike bacteriocins, class IIb two-peptide bacte- (Castellano et al. 2004). Class IIc bacteriocins com- 26 riocins, class IIc cyclic bacteriocins, and class IId prise a few cycle peptides and class IId, also called 27 non-pediocinlike single linear peptides. Class IIa non-pediocin single linear peptides, include the 28 bacteriocins have a narrow spectrum of action remaining isolated antimicrobial substances. Bacte- 29 but display a high specific activity against L. mono- riocins from class I and II are among the best charac- 30 cytogenes. Pediocinlike bacteriocins can be consid- terized biochemically and genetically and the most 31 ered as the major subgroup among non-lantibiotic likely to be used in food applications due to their 32 peptides, not only because of their large number, high target specificity. 33 but also because of their significant biological activi- 34 ties and potential applications (Fimland et al. 2005). USE OF BACTERIOCINS AS BIOPRESERVATIVES IN 35 Pediocin PA-1 is produced by P. acidilactici isolated FERMENTED SAUSAGES 36 from America-style sausages, P. pentosaceous 37 Z102 from Spanish-style sausages, P.parvulus from As previously described, fresh meat and fermented 38 vegetable products, and Lb. plantarum WHE92 iso- meat products provide an excellent environment for 39 lated from cheese. Sakacin A and K produced the growth of pathogenic and spoilage organisms. 40 by Lb. sakei strains (Lb706 and CTC494), curvacin Properly processed meat should have a low pH and 41 A produced by Lb. curvatus LTH1174, and ente- minimum numbers of contaminating bacteria, even 42 rocin A produced by Enterococcus faecium CTC492, though spoilage will occur if methods to restrict 43 all of them strains isolated from meat and fermented microbial growth are not applied. Microbial antago- 44 sausages, demonstrated to be active against other nism is used empirically in sausage fermentations 45 LAB, L. monocytogenes and Clostridium. In addi- where LAB accumulate lactic acid that inhibits meat 46 tion, Leuconostoc gelidum and Leuconostoc foodborne pathogenic bacteria while coagulating 47 mesenteroides isolated from chill-stored vacuum- soluble meat proteins, thereby reducing water-binding 48 packaged meat secrete leucocin A, inhibitory against capacity and facilitating the drying of the product. 49 LAB, L. monocytogenes, and Enterococcus faecalis. However, the use of bacteriocinogenic strains as 50 Carnobacterium piscicola LV17B and V1 iso- starter cultures in fermented sausages has been sug- 51 lated from vacuum-packaged meat also produce gested as an extra hurdle to ensure product safety 52 bacteriocins such as carnobacteriocin B1 and and quality. Bacteriocins can be introduced into food 53S B2, which are highly effective against L. mono- in at least three different ways: (1) as purified or 54N cytogenes and other LAB. 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Table 14.2. Examples of bacteriocins produced by LAB isolated from meat and fermented sausages. 1 2 a Producer strain Bacteriocin Class Source Active Against 3 L. lactis BB24 Nisin Ia Spanish fermented sausages 4,5,7,9 4 5 L. lactis WNC20 Nisin Z Ia Thai fermented sausages 1,2,4,6,9 6 Lb. sakei 148, V18 Lactocin S Ia Spanish fermented sausages 1,4,5,6 7 Lb. sakei L45 Lactocin S Norwegian fermented sausages 1,4,5,6 8 9 Lb. sakei LTH673, 674 Sakacin K, P IIa Beef 1,6,7 10 Lb. sakei I151 Sakacin P IIa Italian fermented sausages 7 11 Lb. sakei Lb706 Sakacin A IIa Beef, meat products 1,6,7 12 Lb. sakei CTC494 Sakacin K IIa Spanish fermented sausages 1,6,7 13 14 Lb. sakei MN Bavaricin MN IIa Beef 7 15 Lb. brevis SB27 Brevicin 27 IId Fermented sausages 1,2 16 Lb. curvatus LTH1174 Curvacin A IIa German meat products 1,2,7 17 Lb. curvatus CRL705 Lactocin 705 IIb Argentine fermented sausages 1,2,3,8 18 19 Lb. curvatus FS47 Curvaticin FS47 IId Minced beef products 1,2,6 20 Lb curvatus L442 Curvaticin L442 IIa Greek fermented sausages 1,7 21 Lb. plantarum CTC305 Plantaricin A IId Spanish fermented sausages 1,7 22 23 Lc. gelidum UAL187 Leucocin A IIa Vacuum-packed meat 1,6,7 24 Lc. mesenteroides TA33a Leucocin A IIa Vacuum-packed meat 1,6,7 25 Lc. mesenteroides L124 IIa Greek fermented sausages 1,7 26 Lc. mesenteroides E131 Greek fermented sausages 7 27 28 Lc. carnosum TA11a Leucocin A IIa Vacuum-packed meat 1,6,7 29 P. acidilactici PAC1.0 Pediocin PA-1/AcH IIa American-style sausages 1,4,5,7,8 30 P. acidilactici L50 Pediocin L50 IId Spanish fermented sausages 1,4,5,6,7,8,9 31 P. pentosaceous Z102 Pediocin PA-1 IIa Spanish fermented sausages 1,4,5,7,9 32 33 C. piscicola LV17B Carnobacteriocin B2 IIa Vacuum-packed meat 7 34 C. piscicola V1 Piscicocin V1a IIa Fish 1,6,7 35 C. piscicola LV17A Carnobacteriocin A IId Processed meat 1,7 36 37 C. piscicola JG126 Ciscicolin 126 IIa Spoiled ham 7 38 C. piscicola KLV17B Carnobacteriocin IIa Vacuum-packed meat 1,6,7 39 —B1/B2 40 C. divergens 750 Divergicin 750 IId Vacuum-packed meat 1,5,6,7 41 C. divergens LV13 Divergicin A IId Vacuum-packed meat 1 42 43 E. faecium CTC492 Enterocin B IId Spanish fermented sausages 1,5,7 44 E. faecium CTC492 Enterocin A IIa Spanish fermented sausages 7 45 E. casseliﬂavus IM416K1 Enterocin 416K1 IIa Italian fermented sausages 7 46 47 a1: other LAB; 2: B. cereus; 3: B. thermosphacta; 4: Cl. botulinum; 5: Cl. perfringes; 6: E. faecalis/faecium; 48 7: L. monocytogenes; 8: Propionibacteria; 9: Staph. aureus. 49 50 51 52 S53 N54

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1 (2) as bacteriocins produced in situ by bacterial fermented dry sausages in particular, Lb. sakei 2 cultures that substitute for all or part of the starter CTC494 sakacin K producer, was able to suppress 3 culture, and (3) as an ingredient based on a fermen- the growth of Listeria, whose numbers significantly 4 tate of a bacteriocin-producing strain. At present decreased compared to the initial values and to the 5 only nisin and pediocin PA-1/AcH are widely used standard starter non-bacteriocin producer (Hugas 6 in foods. Nisin is used mostly as Nisaplin (Danisco), et al. 2002). Lb. sakei Lb706, which secretes sakacin 7 a preparation containing 2.5% nisin. Pediocin PA1 A, was also able to reduce the number of Listeria in 8 for food biopreservation has also been commercially fresh Mettwurst. On the other hand, the inoculation 9 exploited as ALTA 2431 (Quest), based on a fermen- in sausages as a starter culture of Lb. sakei LTH673, a 10 tate from a pediocin PA-1-producing strain of sakacin K producer isolated from meat, failed to lead 11 P. acidilactici, its use being covered by several U.S. the fermentation, although it reduced the number of 12 and European patents. The major criteria to be taken Listeria when compared to the non-bacteriocinogenic 13 into account for food application of bacteriocins are starter (Hugas et al. 1996). The meat isolate Lb. curva- 14 summarized in Table 14.3. Among class Ia bacteri- tus LTH1174, which secretes curvacin A, also proved 15 ocins, nisin has not been quite successful in meat to be highly competitive in sausage fermentations, 16 products because of its low solubility, uneven distri- dominating the fortuitous microbiota by reducing 17 bution, and lack of stability. Lactococcal bacteriocins counts of artificially added Listeria compared to a 18 are not particularly adapted to sausage technology nonbacteriocinogenic control (Hugas et al. 2002). 19 because they display sensitivity to some ingredients On the basis of these results, Lb. curvatus LTH1174 20 and manufacture conditions. However, the use of lac- shows a promising potential for use in European 21 ticin 3147-producing strains as starter cultures in the sausage fermentation. Pediocin-producing strains of 22 production of salami showed that the bacteriocin was P. acidilactici tested for their ability to reduce the 23 produced throughout manufacture and that final counts of L. monocytogenes during chicken summer 24 overall characteristics were similar to the control. Its sausages fermentation showed significant reductions 25 protective ability was also demonstrated in spiked compared to non-pediocin–producing strains. More- 26 sausages because there was a significant reduction of over, when the survival of L. monocytogenes was 27 L. innocua and Staph. aureus (Scannell et al. 2001). compared in German, American, and Italian-style 28 Nevertheless, nisin producers isolated from fer- fermented sausages using pediococci as a starter cul- 29 mented meat products seem to have some ecological ture, higher reductions in Listeria numbers were 30 advantages that may improve their effectiveness in observed in American-style sausages after fermenta- 31 these products. The narrow-spectrum Listeria-active tion and drying, with respect to German-style 32 bacteriocins seem to have a niche that provides a sausages prepared with a non-bacteriocinogenic cul- 33 solution for foods in which the presence of L. mono- ture. The higher fermentation temperature used in 34 cytogenes, mainly as a postcontamination event, American-style sausages (25–35°C) may have a pos- 35 can be a problem. Lb. sakei sakacin producers, have itive influence on the production of bacteriocins 36 been assayed in in situ experiments, in which they by P. acidilactici. Although the use of bacteriocin- 37 demonstrated a strong activity against Listeria. In producing enterococci in cheese manufacture is well 38 39 40 Table 14.3. Major criteria for the selection of bacteriocins and/or biopreservative cultures for dry sausage application. 41 42 GRAS status of producer strain 43 Adaptation to meat environment (preferably sausage isolates) 44 45 No gas or polysaccharide production 46 Noninhibitory to GCC involved in the mixed culture 47 Selection according to sausage formulation and technology. Recipe (NaCl, NaNO2/NaNO3, spices) 48 and temperature used 49 Broad spectrum of inhibition or high specific activity against a specific pathogen (L. monocytogenes) 50 51 Heat stability of the bacteriocin produced 52 No associated health risks 53S Activity in food must lead only beneficial effects (improved safety, quality, flavor, improved health). 54N 40454 14 147-158 r1.qxd 7/21/07 9:56 AM Page 153

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documented in the literature, information on their membrane and peptidoglycan layer of the cells. This 1 potential as biopreservatives in the meat industry is asymmetrical membrane contains glycerophospho- 2 scarce. Bacteriocin-producing E. faecium strains lipids and lipopolysaccharide (LPS) molecules in 3 have been included in the starter culture in Spanish- which anchoring divalent cations are involved. 4 style dry-fermented sausage to effectively inhibit Treatment with metal-chelating agents as EDTA 5 Listeria. The bacteriocin-producing Enterococcus generally results in removal by chelation of divalent 6 casseliflavus IM416K1 isolated from Italian caccia- cations with a consequent disruption of the outer 7 tore sausage showed a high effectiveness in L. mono- membrane (Alakomi et al. 2003). Lactic acid and its 8 cytogenes elimination; a listeriostatic effect was salts are also potent outer membrane disintegrating 9 obtained when enterocin CCM4231 produced by a agents as evidenced by their ability to cause LPS 10 strain of E. faecium was added to the sausage batter release. These permeabilizers increase the suscepti- 11 during the manufacture of Hornád salami, a dry- bility to hydrophobic substances such as bacteri- 12 fermented traditional Slovakian product (Lauková ocins by enabling them to penetrate the cell wall. 13 et al. 1999). In contrast, E. faecium CTC492, an Accordingly, food-grade permeabilizers in combina- 14 enterocin A and B producer, was unable to exert any tion with bacteriocins would be effective as part of 15 positive antilisterial effect on different meat prod- the hurdle concept in inhibiting gram-negative bac- 16 ucts compared to the batches treated with enterocins teria. Various food-grade chelators (citrate, phos- 17 A and B, this being attributed to an insufficient bac- phate, EDTA, hexametaphosphate) have been 18 teriocin production (Aymerich et al. 2000). Because examined for their ability to overcome the penetra- 19 enterococci are hardly competitive in the early tion barrier in Salmonella enterica and E. coli by 20 stages of the fermentation reducing listerial counts, rendering these species sensitive to hydrophobic 21 bacteriocin-producing enterococcal strains may be antibiotics. When the deferred addition of bacteri- 22 used as adjunct cultures during sausage manufacture. ocins produced by Lb. curvatus CRL705 (lactocin 23 Even when many bacteriocinogenic strains inhib- 705) and L. lactis CRL1109 (nisin) in combination 24 ited L. monocytogenes in vitro and in meat systems, with chelators was assayed, differences in E. coli 25 most studies on the in situ effect were carried out on strains sensitivity to bacteriocin/chelator combina- 26 single strains of the pathogen or have used high tions were reported, the sensitization by sodium lac- 27 levels of contamination. Significant variations in the tate and EDTA combined with lactocin 705 being 28 sensitivity of Listeria strains to the same bacteriocin the most effective strategy (Belfiore et al. 2006). 29 as well as the emergence of spontaneous resistant When the ability of chelators and nisin generated in 30 mutants to individual bacteriocins were reported situ to inhibit and inactivate Gram-negative bacteria 31 (Cotter et al. 2005). For improved control of target was studied, E. coli was found to be more sensitive 32 microorganisms and bacteriocin-resistant strains and than salmonellas, but Pseudomonas showed the great- 33 species inhibition, the combined use of bacteriocins est susceptibility. The combination of sodium lactate 34 was proposed. The combined effect of nisin (class and nisin also afforded increased protection against 35 Ia), lactocin 705 (class IIb), and enterocin CRL35 Staph. aureus and Salmonella species in fresh pork 36 (class IIa) against different L. monocytogenes strains sausage, providing an alternative to sulphite addition. 37 in meat slurry showed no viable counts after incuba- Moreover, the spectrum of nisin activity can be 38 tion (Vignolo et al. 2000). The simultaneous addi- extended to a variety of pathogens including gram- 39 tion of a nisin-curvaticin 13 combination also led to negative bacteria when used together with food-grade 40 the absence of viable Listeria cells in broth, prevent- enzymes such as lysozyme or combined with physical 41 ing the regrowth of bacteriocin-resistant mutants treatments. The effectiveness of high hydrostatic pres- 42 (Bouttefroy and Milliere 2000). It is generally sure to induce sublethal injuries to living foodborne 43 assumed that a mixture containing more than one bacteria cells was also assayed in combination with 44 bacteriocin will be bactericidal to more cells in a various bacteriocins (Hugas et al. 2002). 45 sensitive population, because cells resistant to one 46 bacteriocin would be killed by another. 47 INFLUENCE OF CURING ADDITIVES AND 48 Gram-negative Bacteria Inhibition FERMENTATION CONDITIONS ON BACTERIOCIN 49 EFFECTIVENESS Although lantibiotics have wider spectra of inhibi- 50 tion than nonlantibiotics, none of the bacteriocins The effect of bacteriocins as well as the bacteriocino- 51 produced by LAB were effective in killing gram- genic LAB in meat fermented products should 52 negative bacteria. This inability is due to the protec- consider the adequacy of the environment for bacteri- S53 tive outer membrane, which covers the cytoplasmic ocin stability and/or production. 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1 semipuriﬁed bacteriocins are added to fermented CTC492 was used as a bioprotective culture in fer- 2 meat, a loss of activity is often detected as a direct mented sausages and hamburgers, it did not exert any 3 consequence of meat components such as proteolytic positive antilisterial effects compared to enterocins A 4 enzymes, bacteriocin-binding protein, or fat particles. and B, this fact being attributed to a higher inhibition 5 The endogenous enzymes present in raw meat would of the producer strain by refrigeration temperatures 6 produce a degradation of the bacteriocin added or and sausage ingredients (Aymerich et al. 2000).

7 produced during ripening, thus impairing bacteriocin Although NaNO2 did not affect sakacin K produced 8 effectiveness. Inactivation by pasteurization of a by Lb. sakei CTC494, its presence decreased bacteri- 9 meat slurry used to inoculate L. monocytogenes ocin production because of its toxic effect on cell 10 showed a cell count decrease by 2 logs in the pres- growth, this being enhanced by the lactic acid pro- 11 ence of lactocin 705 compared to a nonpasteurized duced. Moreover, a protective effect of L. monocyto- 12 meat system, thus corroborating the meat protease genes against the action of lactocin 705 by NaCl μ 13 effect (Vignolo et al. 1996). Lipids and phospho- (3–5%) and NaNO2 (200 g/g) was demonstrated in 14 lipids may also interfere with bacteriocin activity, as a meat slurry at 20°C (Vignolo et al. 1998). Addition 15 observed for nisin against L. monocytogenes, the of spices to the sausage mixture also influences the 16 activity of which decreases with increasing fat con- effectiveness of bioprotective cultures. Pepper, nut- 17 tent, probably due to binding to fat globules. Fat was meg, rosemary, mace, and garlic decreased curvacin 18 also shown to be responsible for a large loss of bacte- A production by Lb. curvatus LTH1174 in vitro; 19 riocin activity in a Lb. sakei CTC494 culture super- paprika was the only spice that increased it 20 natant (Leroy and De Vuyst 2005). Even when nisin (Verluyten et al. 2004). Black pepper was shown to 21 is apparently not the bacteriocin of choice in meat enhance the inhibitory activity of sakacin K against 22 products, promising results were obtained when it L. monocytogenes in vitro and in model sausages, a 23 was used as an alternative to reduce nitrite levels in synergistic effect with nitrite, NaCl and Bac+ starter 24 cured sausages. Nisin application in meat products is cultures being observed (Hugas et al. 2002). How- 25 affected by inadequate pH as well as low tempera- ever, the addition of some spices may have a positive 26 tures, both leading to poor solubility in the food influence on the metabolism of starter cultures due to 27 matrix and less efficiency against pathogens, proba- the presence of micronutrients, especially manganese 28 bly due to a decreased cell membrane fluidity. More- (Leroy and De Vuyst 2005). 29 over, because nisin is the only bacteriocin approved 30 as a food ingredient by the Food and Drug Adminis- 31 tration, the use of bacteriocin-producing LAB may NEW TRENDS FOR STARTER 32 be a better choice for introducing bacteriocins into CULTURE UTILIZATION 33 meat products because they can provide a bacteriocin FUNCTIONAL MEAT PRODUCTS:FUNCTIONAL 34 source over a longer period of time. In this regard, it STARTER CULTURES 35 seems important to apply strains from their own envi- 36 ronment, naturally better adapted, more competitive, As discussed above, a spectacular growth in the 37 and more in agreement with the characteristics of the development of health products has occurred in 38 product and the technology used. Bacteriocin- the last decades as a result of consumer demands. 39 producing LAB associated with meat and meat fer- The concept of health products includes what is 40 mentations such as Pediococcus, Leuconostoc, known as functional foods. These are defined as 41 Carnobacterium, and Lactobacillus spp. are likely to foods that are used to prevent and treat certain disor- 42 have greater potential as meat preservatives (Table ders and diseases, in addition to their nutritional 43 14.2). Bacteriocin-producing LAB have been used value per se (Jiménez-Colmenero et al. 2001). Meat 44 successfully in a number of cases to control adventi- and meat products are important sources of proteins, 45 tious populations in meat products of consistent qual- vitamins, and minerals, which could be considered 46 ity and to delay spoilage. It has been shown that the functional compounds. However, they also contain 47 previously described sausage isolates Lb. curvatus fat, saturated fatty acids, cholesterol, and salt, which 48 LTH1174, Lb. curvatus L442 and Lb. sakei CTC494 are responsible for the unfortunate image of meat 49 optimally produce bacteriocins under conditions of and meat products because of their association with 50 pH and temperature that prevail during European cardiovascular diseases, some types of cancer, and 51 sausage fermentation. However, the performance of obesity. The use of functional ingredients to obtain 52 bacteriocinogenic LAB starter or adjuncts will be healthy meat products constitutes a suitable strategy 53S influenced by curing ingredients. When E. faecium for meat processors by eliminating or reducing 54N 40454 14 147-158 r1.qxd 7/21/07 9:56 AM Page 155

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components considered harmful or by adding ingre- feeding lactating ewes with selected strains of Lacto- 1 dients considered beneficial for health, such as mod- bacillus acidophilus caused an important drop in the 2 ified fatty acid profiles, antioxidants, dietary fiber, mean cholesterol level of meat cuts (Lubbadeh et al. 3 and probiotics (Fernández-Ginés et al. 2005). 1999). The proteolytic system of LAB can contribute 4 The performance of commercial starter cultures to the release of health-enhancing bioactive peptides 5 has been questioned because their behavior is not from food proteins, thus improving absorption in the 6 always the same when applied to other types of fer- intestinal tract. They can also stimulate the immune 7 mented meat products. It is crucial, therefore, to give system, exert antihypertensive or antithrombotic 8 traditional producers the means to produce safe and effects, display antimicrobial activity, or function as 9 standardized products while preserving their typical carriers for minerals, especially calcium. Bioactive 10 sensory quality. As a response to these needs and to peptide may be generated during meat aging as well 11 the demands for health products, the use of new gen- as during meat fermentation by LAB (Korhonen and 12 eration starter cultures has already been suggested Pihlanto 2003). The ability to produce CLA, another 13 (Hansen 2002; Leroy et al. 2006). The so-called health promoter, was described in vitro by propioni- 14 functional starter cultures contribute to food safety bacteria, bifidobacteria, and lactobacilli. However, 15 by producing antimicrobial compounds such as bac- human clinical studies are needed to confirm the 16 teriocins and also provide sensorial, technological, health-promoting effects of probiotics in sausages. 17 nutritional, and/or health advantages. Traditional fermented sausages constitute a 18 highly appreciated specialty with gastronomic value 19 and are a rich source of biodiversity, the deliberate 20 STARTER CULTURES WITH PROBIOTIC use of which in industrial processes could help to 21 POTENTIALITIES introduce quality and safety advantages. The use of 22 Probiotics represent an expanding research area and bacteriocin-producing strains that are well adapted 23 the possibilities of the use of probiotics in dry sausage to the sausage environment will provide an extra 24 manufacturing processes have been addressed hurdle in a multihurdle integrated system. Despite 25 recently (Työppönen et al. 2003). Probiotics have the above-mentioned advantages of biopreserva- 26 been used mainly in dairy products such as yogurt and tion, it should be borne in mind that bacteriocins are 27 other fermented milks. The use of fermented sausages not meant to be used as the sole means of food 28 as probiotic food carriers has been postulated because preservation. 29 these products are not heated and harbor high num- 30 bers of LAB. However, in order to use probiotics in REFERENCES 31 fermented sausages, several characteristics of the cul- 32 ture, in addition to technological, sensory, and safety H Alakomi, M Saarela, J Helander. 2003. Effect of 33 properties, should be taken into account. The evalua- EDTA on Salmonella enterica serovar Typhimurium 34 tion of probiotic cultures suitable for use as starter involves a component not assignable to lipopolysac- 35 cultures for dry-fermented sausages should involve charide release. Microbiol 149:2015–2021. 36 the ability to become dominant in the final product T Aymerich, M Artigas, M Garriga, J Monfort, M 37 because fermented meat products contain a natural Hugas.2000. Effect of sausage ingredients and addi- 38 background microbiota, this ability being mainly tives on the production of enterocins A and B by 39 achieved by a fast acid and bacteriocin production. Enterococcus faecium CTC492. Optimization of in 40 Specific probiotic properties such as bile tolerance, vitro production and anti-listerial effect in dry fer- 41 resistance to stomach acidity, and adhesion to the mented sausages. J Appl Microbiol 88:686–694. 42 intestinal epithelium in addition to antimicrobial T Aymerich, M Hugas, J Monfort. 1998. Review: Bac- 43 activity will ensure strain survival in the human gas- teriocinogenic lactic acid bacteria associated with 44 trointestinal tract. Even though LAB are considered meat products. Food Sci Technol Int 4:141–158. 45 GRAS organisms, specification of origin, nonpatho- C Belfiore, P Castellano, G Vignolo. 2007. Reduction 46 genicity, and antibiotic resistance characteristics of of Escherichia coli population following treatment 47 the strains should also be assessed. Moreover, other with bacteriocins from lactic acid bacteria and chela- 48 LAB abilities that could improve safety and impart a tors. Food Microbiol 24:223–229. 49 healthy note to meat fermented products include A Bouttefroy, J Milliere. 2000. Nisin-curvaticin 13 50 moderate cholesterol-lowering action, bioactive pep- combinations for avoiding the regrowth of bac- 51 tide generation from food proteins and conjugated teriocin resistant cells of Listeria monocytogenes 52 linoleic acid (CLA) production. In fact, studies ATCC15313. Int J Food Microbiol 62:65–75. S53 N54 40454 14 147-158 r1.qxd 7/21/07 9:56 AM Page 156

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1 P Castellano, W Holzapfel, G Vignolo. 2004. The con- F Jiménez-Colmenero, J Carballo, S Cofrades. 2001. 2 trol of Listeria innocua and Lactobacillus sakei in Healthier meat and meat products: Their role as func- 3 broth and meat slurry with the bacteriocinogenic tional foods. Meat Sci 59:5–13. 4 strain Lactobacillus casei CRL705. Food Microbiol H Korhonen, A Pihlanto. 2003. Food-derived bioactive 5 21:291–298. peptides, opportunities for designing future foods. 6 D Church. 2004. Major factors affecting the emergence Curr Pharm Des 9:1297–1308. 7 and re-emergence of infectious diseases. Clin Lab A Lauková, S Czikková, S Laczková, P Turek. 1999. 8 Med 24:559–586. Use of enterocin CCM 4231 to control Listeria 9 P Cotter, C Hill, P Ross. 2005. Bacteriocins: Develop- monocytogenes in experimentally contaminated dry 10 ing innate immunity for food. Nature Rev Microbiol fermented Hornád salami. Int J Food Microbiol 11 3:777–798. 52:115–119. 12 D Demeyer. 2003. Meat fermentation: Principles and L Leistner. 1996. Food protection by hurdle technology. 13 applications. In: Y Hui, L Goddik, A Hansen, J Bull Jpn Soc Res Food Prot 2:2–26. 14 Josephsen, W Nip, P Stanﬁeld, F Toldrá, eds. Hand- ———. 2000. Basic aspects of food preservation by 15 book of Food and Beverage Fermentation Technol- hurdle technology. Int J Food Microbiol 55:181–186. 16 ogy. New York: Marcel Dekker, pp. 353–367. F Leroy, L, De Vuyst. 2005. Simulation of the effect of 17 M Elmi. 2004. Food safety: Current situation, unad- sausage ingredients and technology on the function- 18 dressed issues and the emerging priorities. East ality of the bacteriocin-producing Lactobacillus 19 Mediterr Health J 10:794–800. sakei CTC494 strain. Int J Food Microbiol 20 J Fernández-Ginés, J Fernández-López, E Sayas- 100:141–152. 21 Barberá, J Pérez-Alvarez. 2005. Meat products F Leroy, J Verluyten, L De Vuyst. 2006. Functional 22 as functional foods: A review. J Food Sci 70: meat starter cultures for improved sausage fermenta- 23 37–43. tion. Int J Food Microbiol 106:270–285. 24 G Fimland, L Johnsen, B Dalhaus, J Nissin-Meyer. W Lubbadeh, M Haddadin, M Al-Tamimi, R Robinson. 25 2005. Pediocin-like antimicrobial peptides (class IIa 1999. Effect on the cholesterol content of fresh lamb 26 bacteriocins) and their immunity proteins: Biosyn- of supplementing the feed of Awassi ewes and lambs 27 thesis, structure, and mode of action. J Peptide Sci with Lactobacillus acidophilus. Meat Sci 52: 28 11:688–696. 381–385. 29 C Fontana, P Cocconcelli, G Vignolo. 2005. Monitor- F-K Lücke. 2000. Utilization of microbes to process 30 ing the bacterial population dynamics during fermen- and preserve meat. Meat Sci 56:105–115. 31 tation of artisanal Argentine sausages. Int J Food A Scannell, G Schwarz, C Hill, R Ross, E Arendt. 2001. 32 Microbiol 103:131–142. Pre-inoculation enrichment procedure enhances 33 S Garneau, N Martin, J Vederas. 2002. Two-peptide the performance of bacteriocinogenic Lactococcus 34 bacteriocins produced by lactic acid bacteria. lactis meat starter culture. Int J Food Microbiol 35 Biochimie 84:577–592. 64:151–159. 36 C Guinane, P Cotter, C Hill, R Ross. 2005. Microbial L Stahnke. 2002. Flavour formation in fermented 37 solutions to microbial problems; lactococcal bacteri- sausage. In: F Toldrá, ed. Research Advances in the 38 ocins for the control of undesirable biota in food. Quality of Meat and Meat Products. Kerala, India: 39 J Appl Microbiol 98:1316–1325. Research Signpost, pp. 193–223. 40 E Hansen. 2002. Commercial bacterial starter cultures R Talon, S Leroy-Sétrin, S Fadda. 2002. Bacterial 41 for fermented foods of the future. Int J Food Micro- starters involved in the quality of fermented meat 42 biol 78:119–131. products. In: F Toldrá, ed. Research Advances in the 43 M Hugas, M Garriga, M Aymerich, J Monfort. 2002. Quality of Meat and Meat Products. Kerala, India: 44 Bacterial cultures and metabolites for the enhance- Research Signpost, pp. 175–191. 45 ment of safety and quality in meat products. In: F P Tarrant. 1998. Some recent advances and future prior- 46 Toldrá, ed. Research Advances in the Quality of Meat ities in research for the meat industry. Meat Sci 47 and Meat Products. Kerala, India: Research Sign- 49(Suppl 1):S1–S16. 48 post, pp. 225–247. S Työppönen, E Petäjä, T Mattila-Sandholm. 2003. Bio- 49 M Hugas, B Neumeyer, F Pagés, M Garriga, W protectives and probiotics for dry sausages. Int J 50 Hammes. 1996. Comparison of bacteriocin-producing Food Microbiol 83:233–244. 51 lactobacilli on Listeria growth in fermented J Verluyten, F Leroy, L De Vuyst. 2004. Effects of dif- 52 sausages. Fleischwirtschaft 76:649–652. ferent spices used in production of fermented 53S 54N 40454 14 147-158 r1.qxd 7/21/07 9:56 AM Page 157

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sausages on growth of and curvacin A production by ———. 1998. Effects of curing additives on the control 1 Lactobacillus curvatus LTH1174. Appl Environ of Listeria monocytogenes by lactocin 705 in meat 2 Microbiol 70:4807–4813. slurry. Food Microbiol 15:259–264. 3 G Vignolo, S Fadda, M Kairuz, A R Holgado, G Oliver. G Vignolo, J Palacios, M Farías, F Sesma, U 4 1996. Control of Listeria monocytogenes in ground Schillinger, W Holzapfel, G Oliver. 2000. Combined 5 beef by lactocin 705, a bacteriocin produced by effect of bacteriocins on the survival of various Liste- 6 Lactobacuillus casei CRL705. Int J Food Microbiol ria species in broth and meat system. Curr Microbiol 7 29:397–402. 41:410–416. 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 S53 N54 40454 15 159-170 r1.qxd 7/20/07 12:58 PM Page 159

1 2 3 15 4 5 6 Starter Cultures:Yeasts 7 8 M-Dolores Selgas and M-Luisa Garcia 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION the yeast level is close to 102–104 cfu/g and coexists 22 with gram-negative bacteria, which range between 23 Fermentation is one of the oldest and most important 103–107 cfu/g. During low-temperature storage of 24 methods of preserving meat. The process involves the fresh meat, yeast counts may increase and eventually 25 anaerobic breakdown of carbohydrates yielding sev- dominate the microbiota, reaching values close to 26 eral compounds (alcohols, organic acids, mainly lac- 106 cfu/g. Competition between both groups of 27 tic acid) whose nature depends on the type of food and microorganisms favors the increase and predomi- 28 the correct growth of responsible microorganisms. In nance of yeasts. Although their counts are generally 29 meat, fermentation has been used especially in com- lower than those of spoilage bacteria, they can pro- 30 minuted products such as sausages, which differ from liferate and reach amounts enough to form a visible 31 fresh meat by the addition of salts or sugars. These surface slime (Cook 1995). 32 compounds select the fermentative microbiota, which Yeasts can also be considered habitual compo- 33 will grow at relatively high osmotic pressure. nents of microbiota growing on fermented sausages, 34 Dry-fermented sausages are one of the most and their origin is mainly related to the environment 35 important meat products. They have a long storage and the meat used as raw material. In fermented 36 life due to many factors, such as the presence of meats, the lactic acid produced by bacteria and the 37 salts, the lactic acid produced by lactic microbiota, low a resulting from the presence of salts or a dehy- 38 the nitrite produced from nitrate thanks to members w dration process constitute modiﬁed environmental 39 of the genus Kocuria, and the drying that occurs dur- factors that hinder the bacterial growth and favor the 40 ing the ripening process. All these factors modify the development of natural competitors. Thus, yeasts 41 original meat and contribute to the development of use all the nutrients and energy and grow quickly 42 the texture and ﬂavor and to the selection of the and easily (Dillon and Board 1991). 43 desired microbiota (Ordóñez et al. 1999). Yeasts can grow at pH, water activity, and temper- 44 However, although bacteria are the predominant ature values habitual in fermented sausages (Monte 45 factors in fermented sausages, molds and yeasts have et al. 1986; Hammes and Knauf 1994). Many species 46 also been tried for curing and fermenting sausages. can even grow as easily at pH 4 as at pH 6 and are 47 able to maintain a neutral intracellular pH when the 48 PRESENCE OF YEASTS ON MEAT external environmental is very acid. This ability has 49 SAUSAGES been attributed to a relative impermeability of the 50 plasmatic membrane or the existence of a mecha- 51 Yeasts are found naturally on fresh meat, an excel- nism that drives out the protons that enter by passive 52 lent growth media, from where they are easily spread diffusion. As a consequence, pH is not an active con- 53 during slaughtering (Dillon and Board 1991). There, troller to yeast growth in these foods. S54 N55 159 40454 15 159-170 r1.qxd 7/20/07 12:58 PM Page 160

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1 The decrease of aw in meat products, as a conse- the difficulties associated with isolation and identifi- 2 quence of either the increase in salts or the dehydra- cation and the continuous modifications in the tax- 3 tion process, scarcely influences yeasts. In this onomy. Traditionally, the identification of yeast 4 sense, it has been described as D. hansenii and has involves the use of numerous morphological and

5 been isolated from low aw foods like sugar syrup physiological tests (Encinas et al. 2000). 6 (Marquina et al. 2001). However, growth slows when Samelis et al. (1994) founded a clear predomi- 7 the external metabolite concentration is close to nance of Debaryomyces in salami; lower levels of 8 50–60% and even when the solute concentration is Candida and Pichia; and very low levels of Crypto- 9 higher than 70%. Tolerance at a high level of coccus, Torulopsis, and Trichosporon. Metaxopoulos 10 metabolites is due to the liberation in the cytoplasm et al. (1996) isolated 100 yeast strains during the fer- 11 of compatible metabolites, mainly polyalcohols mentation and ripening stages of Greek dry salami.

12 (glycerol) that balance intra/extracellular aw. The most common were ascomycetous yeasts, in 13 Although yeasts are able to grow in anaerobic both perfect and imperfect states. Debaryomyces 14 media, the majority of species have a strictly aerobic was again the most abundant and represented 66% of 15 metabolism. This favors yeast growth on the surface the total. Candida famata, C. zeylanoides, C. guil- 16 of meat products. When yeasts arrive to processed liermondii, C. parapsilosis, and C. kruisii were the 17 meats as sausages, they can grow on the outer sur- ascomycetous species identified. Basidiomicetous 18 face, where they form a firm and white external coat. yeasts were represented by Candida humicola, Cryp- 19 This coat controls water loss and permits uniform tococcus albidus, Cr. skinneri, and Trichosporon pul- 20 dehydratation. Moreover, this coat gives to the prod- lulans. Due to the prevalence of D. hansenii in all 21 ucts a peculiar and characteristic appearance that is batches, these authors suggested its potential use as 22 considered a criterion of quality due to the favorable starter culture. Wolter et al. (2000) identified the 23 whiteness aspect that it gives to sausages (Leistner same genera in salami and other dry sausages of 24 1995; Coppola et al. 2000; Selgas et al. 2003). The South Africa; they also identified Sporobolomyces, 25 oxygen requirement restricts their development to Torulaspora, and Saccharomyces. Encinas et al. 26 near the surface. However, in several cases these (2000) identified D. hansenii as the dominant 27 microorganisms have been isolated in the innermost species in Spanish fermented sausages; other species 28 parts of the products (Grazia et al. 1986; Selgas et al. like Trichosporon, Citeromyces, and Candida were 29 2003). This is mainly related to the growth of facul- also present. Coppola et al. (2000) isolated 79 yeast 30 tative anaerobic metabolism of several yeast species, strains from fermented sausages, which were 31 which require only oxygen for the production of cell grouped in four genera: Debaryomyces, Candida, 32 wall constituents such as fatty acids (Deak and Trichosporon, and Cryptococcus. Most of them 33 Beuchat 1996). It is possible to isolate them even (40%) were classified as D. hansenii, by far the most 34 into the original mince of ripened sausages. These predominant yeast, especially during the first few 35 yeasts are the most important because they can grow days of ripening. Similar data were reported by 36 at low oxygen levels, and the final metabolite of the Selgas et al. (2003), who isolated yeast strains from 37 fermentation process (gas and volatiles products) Spanish dry-fermented sausages manufactured 38 can modify the food properties (Leyva et al. 1999). without starter culture, with C. famata, T. mucoides, 39 Regarding temperature, it has been described that Y. lipolytica, and D. hansenii being the most abun- 40 yeasts are sensitive to heat from 38–40°C; at dant species. 41 50–60°C, cellular death occurs in the majority of In any case, the composition of the mycobiota 42 species. Thus, all are able to grow at the tempera- changes during ripening of fermented sausages. 43 tures used in the fermentation of meat. A change from a yeast-dominated to a mold-domi- 44 Hence, yeasts can grow on sausages. The most nated pattern has been observed. During ripening, 45 frequently described on meat and meat products are yeasts undergo a selection process mainly related to

46 the following: Candida, Cryptococcus, Debary- the decrease of the aw, but in general terms, yeast 47 omyces, Hansenula, Hypopichia, Kluyveromyces, Leu- counts are lower than those of bacteria. During early 48 cosporidium, Pichia, Rhodosporidium, Rhodotorula, ripening, yeasts are the most important component 49 Trichosporon, Torulopsis, andYarrowia, with Debary- of mycobiota, but after 2 weeks the numbers of mold 50 omyces being the most commonly isolated (Dillon and yeast are in balance (Andersen 1995). Roncalés 51 and Board 1991; Cook 1995; Coppola et al. 2000; et al. (1991) studied the evolution of yeasts and 52 Osei Abunyewa et al. 2000). molds for a period of 28 days. They observed initial 53S The presence of yeasts in fermented raw sausages values of 102–103 cfu/cm2 and an increase during 54N has been studied less than bacteria and molds due to the 10 days of ripening that reached levels of 40454 15 159-170 r1.qxd 7/20/07 12:58 PM Page 161

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105–106 cfu/cm2 after 25 days. Boissonnet et al. et al. (1996) detected the lowest yeasts counts before 1 (1994) reported levels of 103–104 yeast/g after the salting period, probably due to the predominance 2 3 weeks of ripening. Osei Abunyewa et al. (2000) of bacteria, mainly micrococci. The highest counts 3 described initial values of 103 cfu/g in commercial were founded at the end of the postsalting period and 4 salami, but this number increased after 12 days of during the drying stage, due to increase of tempera- 5 maturation, reaching a maximum of 105 cfu/g at ture. Initial counts close to 104 cfu/g increased to 6 day 20. Encinas et al. (2000) observed an initial 106–107 cfu/g during these periods. However, the 7 4 5 count close to 10 cfu/g and a final count of 10 cfu/g subsequent decrease of aw, and probably that of 8 in dry-fermented sausages. Coppola et al. (2000) some essential nutrients, led to a decrease until 9 obtained yeast from the core of the product that counts close to 104–106 cfu/g (Rodriguez et al. 10 peaked at 104 cfu/g and remained steady until the 2001). Similar results were obtained by Huerta et al. 11 end of ripening. By contrast, counts from external (1988) in other dry-cured Spanish hams. 12 parts reached a maximum of 106 cfu/g after 3 weeks. The dominant species described belong to the gen- 13 It is also important that the most common genera era Debaryomyces, Pichia, Rhodotorula, and Cryp- 14 are present in low concentrations compared with the tococcus; Candida and Saccharomycopsis occurred 15 levels of general microbiota. For that, several in low numbers (Cook 1995). Molina et al. (1990) 16 authors considered that their participation on the isolated species of Hansenula (67%), Rhodotorula 17 ripening of sausages is very low (Cook 1995). (19%), Cryptococcus (9%), and Debaryomyces (5%). 18 Although these yeast levels are relatively low, However, there were some differences with respect to 19 106 cfu/cm2 is considered equivalent to a biomass of manipulation methods, geographic area of manufac- 20 108 bacterias/cm2, a number considered sufficient to ture, and contamination of the raw meat. In any case, 21 have a considerable effect in meat products. In this due to the characteristics of this type of meat product, 22 context, it should be remembered that the biomass of all yeast growing on ham must be able to grow at 23 a yeast cell is approximately 100 times greater than high NaCl levels (8%) (Cook 1995). 24 the biomass of a bacterial cell (Gill and Newton D. hansenii and C. zeylanoides are the predomi- 25 1978; Dillon and Board 1991). nant species in the Iberian ham, although C. blankii, 26 The evolution of yeast population during the fer- C. intermedia, Pichia corsonii, and Rhodotorula 27 mented sausages manufacture process is influenced rubra have also been detected (Núñez et al. 1996). 28 by different variables in which it is possible to con- Yeast levels generally decrease during ripening, but 29 sider the type of products, the influence of the envi- it is possible to find them at the end of long ripening 30

ronment of the factory and other factors, like the time if aw is higher than 0.85. During salting and 31 degree of smoking. So, Encinas et al. (2000) postsalting, new yeasts appear on the ham surface. 32 observed that the type of manufacture (industrial Most of them disappear during ripening, except 33 and artisanal) and sausage diameter were the most Debaryomyces spp., which are the only yeasts able 34

influential variables, with differences in yeast counts to grow at aw < 0.85 (Rodriguez et al. 2001). 35 statistically significant from the second stage of Saldanha-da-Gama et al. (1997) isolated several 36 manufacture onward; they observed how the use of a species from ham, with D. hansenii being the 37 lactic acid starter culture and sorbate control the predominant strain and D. polymorphus, Cryptococ- 38 yeast growth and contribute to the decrease of their cus laurentii, and C. humicolus presented in a low 39 counts in relation to those of artisanal manufacture. number. 40 Sausage diameter is also an important variable; the Table 15.1 lists a selection of yeasts species that 41 availability of oxygen is higher in smaller sausage have frequently been isolated from meat products. 42 and explains the higher counts in these products. 43 Yeasts are sensitive to the smoking process 44 (Leistner 1995), and hence lower counts have been ROLE OF YEASTS ON MEAT 45 observed in smoked sausages than in the homolo- PRODUCTS 46 gous nonsmoked sausages (Encinas et al. 2000). 47 Several studies have been carried out in order to Fermented sausages are a very complex system. 48 know the effect of spices on yeast growth. These During fermentation and ripening, many reactions 49 studies have found an inhibitory effect of garlic occur in which the final products of some constitute 50 (Asehraou et al. 1997; Olesen and Stahnke 2000). the substrates of others. Most of the different com- 51 The presence of yeasts is also detected in dry- pounds contribute to the flavor and give to final 52 cured hams. Their evolution during the ripening has products sensory properties different from those of S53 been extensively described. In Iberian ham, Núñez the meat used as raw material. N54 40454 15 159-170 r1.qxd 7/20/07 12:58 PM Page 162

1 Table 15.1. Yeasts species frequently isolated from meat products. 2 3 Yeasts Synonym Cured Meats Fermented Sausages Ham 4 Candida spp. 1,14 16 5 6 C. blanckii 11,13 7 C. brumptii 14 8 C. catenulata 14 15 9 C. curvata 14 14 14 10 11 C. famata 1,2,3 12 C. gropengiesseri 9 13 C. guilliermondii 3 14 C. haemulonii 9 15 16 C. humicola Cryptococcus 1,3 17 humicola 18 C. iberica 14 19 C. incommunis 7 20 21 C. intermedia 5,8 11,13 22 C. kruisii 3 23 C. lipolytica 14 1,14 14 24 C. parapsilosis 14 1,3,5,14 25 26 C. rugosa 14 14 15 27 C. torulopsis 1 28 C. tropicalis 14 14 29 C. zeylanoides 1,3,5,6,9,14 11,13 30 31 Citeromyces matritensis 5 32 Cryptococcus spp. 4 16 33 C. skinneri 3 34 35 C. laurentii 6,14 10 36 C. hungaricus 6 37 C. albidus 3,7,9,14 38 C. humicola 10 10 39 40 Debaryomyces 10,14 2,3,4,5,6,7,8,9,13 10,11,13,14,15,16 41 –hansenii 42 D. kloeckeri D. hansenii 14 8,14 43 D. marama 312 44 D. matruchoti D. hansenii 8 45 46 D. occidentalis 9 47 D. nicotianae 14 14 48 D. polymorphus 3,9 10 49 50 D. subglobosus 14 14 51 D. vanriji 6,9 52 53S 54N

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Table 15.1. (Continued) 1 2 Yeasts Synonym Cured Meats Fermented Sausages Ham 3 Galactomyces geotrichum 9 4 5 Geotrichum candidum 14 6 Hansenula anomala var. Pichia anomala 14 8 7 –anomala 8 Hansenula citerii 16 9 10 H. holstii 16 11 Kluyveromyces spp. 1 12 Pichia spp. 1,4 13 P. carsonii Debaryomyces 1 11,13 14 carsonii 15 16 P. etchellsii Debaryomyces 1 17 etchellsii 18 P. farinosa 9 19 P. haplophila 14 20 P. philogaea 9 21 22 Rhodotorula spp. 1,14 16 23 R. glutinis 1,8,14 24 R. rubra R. mucilaginosa 6 11,13 25 26 R. minuta 14 9,14 27 R. mucilaginosa R. rubra 14 6,9 14 28 Saccharomyces spp. 1 29 S. rosei Torulaspore 8 30 delbrueckii 31 32 S. cerevisiae 6 33 Saccharomycopsis spp. 16 34 Sporobolomyces roseus 14 6,9,14 14 35 Sterigmatomyces halophilus 9 36 37 Torulaspora delbrueckii 6,9 38 Torulopsis spp. 4,14 39 T. apicola Candida apicola 8 40 41 T. candida 14 8,14 42 T. etchellsii 14 14 43 T. famata 14 14 14 44 T. pulcherrima Metschnikowia 8 45 pulcherrima 46 47 T. sphaerica Kluyveromyces lactis 8 48 Trichosporon spp. 4,14 49 T. mucoides 2 50 T. pullulans 14 3,7,14 51 52 S53 (Continues) N54

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1 Table 15.1. (Continued) 2 3 Yeasts Synonym Cured Meats Fermented Sausages Ham 4 T. beigelii T. cutaneum 6,9 5 6 T. terrestre Arxula terrestris 7 7 T. ovoides 5 8 T. cutaneum 8 9 Yarrowia lipolytica 2,5,6,9 15 10 11 Sources: 1: Boissonet et al. (1994); 2: Selgas et al. (2003); 3: Metaxopoulos et al. (1996); 4: Samelis et al. (1994); 12 5: Encinas et al. (2000); 6: Wolter et al. (2000); 7: Coppola et al. (2000); 8: Comi and Cantoni (1983); 9: Osei Abunyewa 13 et al. (2000); 10: Saldanha-da-Gama et al. (1997); 11: Núñez et al. (1996); 12: Monte et al. (1986); 13: Rodríguez et al. 14 (2001); 14: Dillon and Board (1991); 15: Huerta et al. (1988); 16: Molina et al. (1990). 15 16 17 18 The role played by bacteria is reasonably well Changes in flavor are assumed to be a conse- 19 known and their contribution to the flavor of fer- quence of the formation of volatiles (Ordóñez et al. 20 mented sausages has been established. In general 1999). Many species of yeasts are able to produce 21 terms, their implication in the biochemical changes volatiles esters (Stam et al. 1998). Peppard and 22 depends on their enzymatic capacity against proteins Halsey (1981) associated yeasts with the reduction of 23 and fat, the main substrate of these meat products carbonyls to alcohols, with 2-methylpropanol, 2- and 24 (Hierro et al. 1997; Ordóñez et al. 1999; Tjener et al. 3-methylbutanol, and 2-phenylethanol being the 25 2003). Similar considerations can be made in main ones produced. It is also believed that yeasts 26 relation to the role played by molds. A direct rela- delay the autoxidative reactions and the onset of ran- 27 tionship between molds and flavor has been demon- cidity that take place in the fat. Yeasts’ contribution 28 strated in both sausages and hams (Toledo et al. seems to be related to the consumption of oxygen and 29 1997; Selgas et al. 1999; Bruna et al. 2001; García the catalase production with the consequent perox- 30 et al. 2001; Rodriguez et al. 2001). ides degradation (Lücke and Hechelmann 1987). 31 Traditionally, it has been believed that the pres- There is great interest in the metabolism of 32 ence of yeasts enhances the appearance and the fla- branched-chain amino acids by yeasts, due to the 33 vor of the meat products (Boissonnet et al. 1994; formation of branched-chain alcohols and their con- 34 Cook 1995; Metaxopoulos et al. 1996), and a contri- tribution to the flavor (Flores et al. 2004). Olesen 35 bution to the color of fermented sausages has also and Stahnke (2000) studied the influence of C. utilis 36 been described, related to the consumption of oxy- on the aroma formation in fermented sausages and 37 gen that favors the curing reactions and the estab- model minces and observed high metabolic activity 38 lishment of the appealing red pigment (Lücke and producing several volatile compounds, particularly 39 Hechelmann 1987). esters. They also observed an ability to ferment 40 Lipolytic and proteolytic activities have been valine, yielding important compounds for the aroma 41 described from several yeasts isolated from fermented of sausages such as 2-methylpropanoic acid and the 42 sausages: Debaryomyces, Pichia, Trichosporon, and respective acetate and ethyl esters in addition to 43 Cryptococcus (Comi and Cantoni 1983; Huerta et al. 2-methylpropanol. This metabolic fate also seems to 44 1988; Cook 1995; Selgas et al. 2003). Miteva et al. be shared by the two other methyl branched amino 45 (1986) described how the presence of Candida utilis acids, isoleucine and leucine. These authors indi- 46 enhances the flavor of both smoked and unsmoked cated that these complicated results could be related 47 fermented sausages, due probably to its lipolytic to the microbial interaction between C. utilis and 48 activity. Molina et al. (1991) studied the lipolytic other bacteria, mainly staphylococci. 49 activity of Cryptococcus albidus in model meat D. hansenii is the most commonly isolated yeast 50 systems inoculated with several microorganisms. from sausages. This suggests that it could have an 51 The volatile and nonvolatile acidity generation important effect on flavor (Gehlen et al. 1991), but 52 observed was much lower than L. curvatus, P. pen- contradictory data have been published in this 53S tosaceous, or S. xylosus, which are commonly used regard. Jessen (1995) indicated that the presence of 54N in meat fermentation. D. hansenii influences the final flavor because of the 40454 15 159-170 r1.qxd 7/20/07 12:58 PM Page 165

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enzymatic activity against proteins and fat. This 2000). Moreover, their deaminase activity produces 1 author also reported that it leads to higher oxygen a slight increase in pH that could have influence on 2 consumption, which favors the color formation. the flavor (Gehlen et al. 1991; Larpent-Gourgaut 3 Lücke and Hechelmann (1987) related the presence et al. 1993; Durá et al. 2002). 4 of D. hansenii with a protective effect against the Other authors disagree with the results above 5 influence of light and oxygen through the formation described. Lizaso et al. (1999) reported that the 6 of a dense white superficial coat (Leistner 1995) that enzymatic action of yeasts can be considered of sec- 7 delays fat oxidation and enhances the flavor. ondary importance in the manufacture of Spanish 8 Encinas et al. (2000) found a relationship between fermented sausages because yeast counts were lower 9 the presence of this yeast and the development of the than those of other microbial groups. Selgas et al. 10 most desirable organoleptic characteristics of fer- (2003) studied the technological ability of 172 yeast 11 mented sausages. Santos et al. (2001) studied the strains isolated from the surface of artisanal 12 activity of D. hansenii on sarcoplasmic proteins iso- sausages. Four strains, belonging to Debaryomyces, 13 lated from pork muscle and observed that this yeast Trichosporon, Candida, and Yarrowia, were selected 14 showed good potential for the hydrolysis of these because of their high hydrolytic activity against fat 15 proteins by generating several polar and nonpolar and proteins isolated from pork meat. These active 16 peptides and free amino acids. Increases in Tyr, Val, strains were inoculated onto sausages or into the 17 Met, Leu, Ile, Phe, and Lys in excess of 50% were mince, and sensory properties were evaluated after 18 observed. ripening. The organoleptic characteristics, irrespec- 19 Flores et al. (2004) manufactured fermented tive of inoculation site, did not differ significantly 20 sausages inoculating Debaryomyces hansenii and a from those of the control noninoculated batches. 21 mixture of bacterial starter cultures. They observe These authors concluded that the influence of yeasts 22 significant increases in compounds like ethyl on the sensory characteristics of the products is, at 23 2-methyl-propanoate and ethyl 2- and 3-methyl least, doubtful. Hadorn et al. (2005) inoculated into 24 butanoate, related to the sausage aroma (Stahnke salami a yeast starter culture originally used for 25 1994), when sausages were inoculated with cheese. In comparison with the uninoculated con- 26 D. hansenii at the level of 106 cfu/g. They also trol, similar values were obtained in physicochemi- 27 observed that these sausages showed a marked cal parameters and microbial counts. Remarkable 28 decrease in volatile compounds that arise from lipid differences were determined in 2-heptanone and 29 oxidation, such as aldehydes and hydrocarbons. This 2-nonanone, but no differences were observed in 30 suggests that the yeast has an impact on aroma by sensory profiles. 31 inhibiting the development of rancidity and generat- In the case of dry-cured hams, numerous studies 32 ing esters that contribute to the flavor. Durá et al. have also been performed in order to find out the 33 (2004) described how the generation of volatile influence of yeasts on sensory characteristics. Comi 34 compounds from branched-chain amino acids by and Cantoni (1983) found that the lipolytic activity 35 D. hansenii was negatively affected by the presence of the yeasts isolated from Parma hams was low. 36 of salt, and the decrease of pH raised the yield of Huerta et al. (1988) observed that most of the yeasts 37 alcohols and aldehydes. These studies seem to indi- isolated from dry-cured Spanish hams were lipolytic 38 cate that the incorporation of this yeast as starter cul- but not proteolytic. Rodríguez et al. (1998) studied 39 ture could favorably modify the flavor pattern of the proteolytic activity of Debaryomyces strains iso- 40 dry-cured sausages. However, Olesen and Stahnke lated from Spanish dry-cured ham using myosin and 41 (2000) observed a little impact on the production of slices aseptically obtained from pork loins as sub- 42 volatile compounds in model minces; these authors strate. After incubation at 25°C during 30 days, free 43 pointed out the possibility that the fungistatic effect amino acid fractions were analyzed and results were 44 of garlic present on the experimental sausages was similar to those of a sterile control. Only small 45 responsible for an inhibition of growth with the cor- changes without statistical differences were 46 responding decrease of volatile compounds. observed. The authors considered that the contribu- 47 Yeasts’ influence on aroma has also been attrib- tion of this yeast to the proteolytic process occurring 48 uted to their ability to metabolize organic acids, in dry-cured hams is very poor. Martín et al. (2002) 49 mainly lactic and acetic acid. It has been observed studied a D. hansenii strain, isolated from dry-cured 50 that lactic acid levels were higher in sausages with ham, which showed proteolytic activity against 51 the lowest yeast counts; in this sense, a definite syn- myosin in culture broth. They observed that the 52 ergistic relationship between LAB and yeast has changes in the free amino acids detected on pork S53 been described (Campbell-Plat 1995; Wolter et al. loins inoculated with this yeast seem to be negligible, N54 40454 15 159-170 r1.qxd 7/20/07 12:58 PM Page 166

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1 and no statistical differences were obtained in culture (Dowell and Board 1968; Rossmanith et al. 2 relation to the uninoculated control. However, 1972). They reported that curing, color, and flavor of 3 D. hansenii seems to improve the sensory properties sausages could be improved by adding selected 4 of ham, contributing to the development of less yel- Debaryomyces strains. Coretti (1977) observed that 5 low superficial fat as an index of a low rancidity the use of a combination of D. hansenii, lactobacilli, 6 (Rodriguez et al. 2001). and micrococci resulted in better flavor and taste of 7 On the other hand, there are several studies that sausages. 8 demonstrate the importance of the muscle pro- The predominant strains used in commercial 9 teinases (cathepsins) that remain active after almost starter culture, alone or in combination with bacteria 10 9 months of curing, favoring the breakdown of pro- or molds, are classified as D. hansenii and its imper- 11 teins. This important role minimizes the contribution fect form Candida famata (Hammes and Hertel 12 of yeasts and even molds to the flavor of cured hams 1998; Toldrá 2002; Pozzi 2003). The species is char-

13 because they grow only on the surface (Toldrá and acterized by a high salt tolerance, low pH and aw 14 Etherington 1988; Molina and Toldrá 1992). resistance, and aerobic or weak fermentative metab- 15 Finally, it is important to bear in mind that yeasts olism (Wyder and Puhan 1999; Kurita and Yamazaki 16 have an antagonistic effect on different spoilage bac- 2002). Also, it can grow at temperatures close to 17 teria. This has been observed in cheese with respect those used during the ripening of fermented 18 to Clostridium tyrobutiricum and Cl. butyricum sausages (Fleet 1990). Nitrate reduction ability is 19 (Fatichenti et al. 1983). It has also been demonstrated not an important characteristic of yeast starters; 20 that D. hansenii is capable of inhibiting the growth of however, a slight inhibition of nitrate reduction in 21 Staphylococcus aureus in fermented sausages fermented sausages has been described (Meisel et al. 22 through oxygen consumption (Meisel et al. 1989). 1989). 23 Similar results have been described by Olesen D. hansenii has been used habitually as a compo- 24 and Stahnke (2000) in meat mince models. Neverthe- nent of cultures offered for exterior and interior use 25 less, this inhibitory action can also occur with other (Jessen 1995). For surface treatment, yeasts can be 26 species of the genus Staphylococcus and could be used alone or in combination with mold strains to 27 detrimental to their role in the ripening process. give an external appearance that the consumers 28 A protective effect against the growth of unfavorable judge favorably. The inoculation of the surface can 29 molds has also been described (Hadorn et al. 2005). be performed by immersion or spraying with a sus- 30 pension containing the culture. Their antioxidant 31 YEAST STARTER CULTURES effect and the possibility of contributing the final fla- 32 vor by enzymatic activity, and the degradation of 33 Starter cultures are considered one of the scientific lactic acid also justifies its use in starter cultures 34 and industry responses to the need for increase meat (Pozzi 2003). However, the amount of Debary- 35 product quality and safety. They improve sensory omyces spp. should be optimized in order to avoid a 36 quality, shelf life, and production safety, minimizing high generation of acids associated with off-flavors 37 the risk of pathogenic microorganisms. They also that can mask the positive effect. Flores et al. (2004) 38 help overcome problems with ripening, achieving observed that a concentration of 106 cfu/g increases 39 the standardization of the production pattern. acid content (acetic, propanoic, and butanoic acids) 40 Current starter culture contains mainly lactic acid to levels associated with off-flavors. However, no 41 bacteria and strains of Kocuria and Staphylococcus. off-flavors have been detected by Selgas et al. (2003) 42 The most commonly used bacteria are L. plantarum, using as inoculum a yeast suspension containing 43 L. sake, L. curvatus, Pediococcus pentosaceus, 107 cfu/g. 44 P. acidilactici, K. varians, Staph. carnosus, and Independently of the considerations made above, 45 Staph. xylosus (Toldrá 2002). the future of yeast starter cultures will depend on the 46 Pioneering works on the yeasts present in fer- knowledge of the metabolism of the yeast used as a 47 mented sausages were conducted in the first decades means of enhancing and improving their technologi- 48 of the twentieth century (Cesari 1919; Cesari and cal capability. Exploratory studies on the natural 49 Guilliermond 1920) when the importance of the diversity of wild yeast strains occurring in artisanal 50 “fleur du saucisson” was recognized and the use of products will also be very important, especially 51 pure yeast cultures for flavoring in fermented studies on genetic aspects that could build a frame- 52 sausages began to be recommended. Later works work that will lead to the generation of new and 53S established that yeasts are commonly present on enhanced starters with increased diversity, stability, 54N sausages and suggested their utilization as starter and industrial performance. This work could lead to 40454 15 159-170 r1.qxd 7/20/07 12:58 PM Page 167

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new genetically modified yeast strains with a tai- ———. 2004. Effect of growth phase and dry-cured 1 lored functionality. sausage processing conditions on Debaryomyces spp. 2 Generation of volatile compounds from branched- 3 chain amino acids. Food Chem 86:391–399. 4 REFERENCES JP Encinas, TM López-Diaz, ML García-López, 5 SJ Andersen. 1995. Compositional changes in surface A Otero, B Moreno. 2000. Yeast populations 6 mycoflora during ripening of naturally fermented on Spanish fermented sausages.Meat Sci 7 sausages. J Food Prot 58:426–429. 54(3):203–208. 8 A Asehraou, S Mobieddine, M Faid, M Serhrouchni. E Fatichenti, JL Gergere, D Pietrino, AF Giovaunnii. 9 1997. Use of antifungal principles from garlic for the 1983. Antagonistic activity of Debaryomices 10 inhibition of yeasts and moulds in fermenting green hansenii towards Clostridium tyrobutyricum and 11 olives. Grasas y aceites 48:68–73. Cl. butyricum. J Dairy Res 80:449–457. 12 JA Barnett, RW Payne, D Yarrow. 2000. Yeasts: Charac- GH Fleet. 1990. Yeast in dairy products. J Appl Bacte- 13 teristics and Identification. 3rd ed. Cambridge: riol 68:199–221. 14 Cambridge University Press. M Flores, MA Durá, A Marco, F Toldrá. 2004. Effect of 15 B Boissonnet, C Callon, M Larpent-Gourgaut, Debaryomyces spp. On aroma formation and sensory 16 O Michaux, J Sirami, P Bonin. 1994. Isolement et quality of dry-fermented sausages. Meat Sci 17 sélection des levures lipolytiques a partir de saucis- 68:439–446. 18 sons secs fermentés. Viand Prod Carnés 15:64–67. ML García, C Casas, E Mendoza, VM Toledo, MD 19 JM Bruna, EM Hierro, L de la Hoz, DS Mottram, Selgas. 2001. Effect of selected moulds on the sen- 20 M Fernández, JA Ordóñez. 2001. The contribution of sory properties of dry fermented sausages. Europ 21 Penicillium aurantiogriseum to the volatile composi- Food Res Technol 212(3):287–291. 22 tion and sensory quality of dry fermented sausages. KH Gehlen, C Meisel, A Fischer, WP Hammes. 1991. 23 Meat Sci 59:97–107. Influence of the yeast Debaromyces hansenii on dry 24 G Campbell-Platt. 1995. Fermented meats: A world sausage fermentation. Proceedings of 35th Inter- 25 perspective. In: G Campbell-Platt, PE Cook, eds. national Congress Meat Science and Technology, 26 Fermented Meats. London: Blackie Academic & Kulmbach, 1991, pp. 871–876. 27 Professional, pp. 39–52. CO Gill, KG Newton. 1978. The ecology of bacterial 28 EP Cesari. 1919. La maturation du saucisson. Acad Sci spoilage of fresh meat at chill temperatures. Meat Sci 29 Paris 168:802–803. 2:207–217. 30 EP Cesari, A Guilliermond. 1920. Les levures du L Grazia, P Romano, A Bagni, D Roggiani, G Guglielmi. 31 saucisson. Ann Inst Pasteur 34:229–248. 1986. The role of moulds in the ripening process of 32 G Comi, C Cantoni. 1983. Presenza di lieviti nei pro- salami. Food Microbiol 3:19–25. 33 sciutti crudi stagionati. Ind Alim February:102–104. R Hadorn, P Eberhard, D Guggisberg, D Isolini, 34 PE Cook. 1995. Fungal ripened meats and meat prod- P Piccinali, H Schlichtherle-Cerny. 2005. An alterna- 35 ucts. In: G Campbell-Platt, PE Cook, eds. Fermented tive surface culture for salami. Proceedings of 51st 36 Meats. London: Blackie Academic & Professional, International Congress of Meat Science and Technol- 37 pp. 39–52. ogy, Baltimore, 2005. 38 S Coppola, G Mauriello, M Aponte, G Moschetti, WP Hammes, C Hertel. 1998. New developments in 39 F Villani. 2000. Microbial succession during ripen- meat starter cultures. Meat Sci 49(suppl 1): 40 ing of Naples-type salami, a southern Italian fer- S125–S138. 41 mented sausage. Meat Sci 56:321–329. WP Hammes, HJ Knauf. 1994. Starters in the process- 42 K Coretti. 1977. Starterculturen in der fleis- ing of meat products. Meat Sci 36:155–168. 43 chwirtschaft. Fleischwirtschaft 3:545–549. E Hierro, L de la Hoz, JA Ordóñez. 1997. Contribution 44 T Deak, LR Beuchat. 1996. Handbook of Food of microbial and meat endogenous enzymes to the 45 Spoilage Yeasts. Boca Raton: CRC Press, pp. 25–26. lipolysis of dry fermented sausages. J Agric Food 46 VM Dillon, RG Board. 1991. Yeast associated with red Chem 45:2989–2995. 47 meats. J Appl Microbiol 71:93–108. T Huerta, A Querol, J Hernández Haba. 1988. Yeasts of 48 MJ Dowell, RG Board. 1968. A microbiological survey dry cured hams, qualitative and quantitative aspects. 49 of British fresh sausages. J Appl Bacteriol Microbiol Alim Nutr 6:289–294. 50 31:378–396. B Jessen. 1995. Starter culture for meat fermentations. 51 MA Durá, M Flores, F Toldrá. 2002. Purification and In: G Campbell-Platt, PE Cook, eds. Fermented 52 characterization of a glutaminase from Debary- Meats. London, Blackie Academic & Professional, S53 omyces spp. Int J Food Microbiol 76:117–126. pp. 130–159. N54 40454 15 159-170 r1.qxd 7/20/07 12:58 PM Page 168

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1 O Kurita, E Yamazaki. 2002. Growth under alkaline E Monte, JR Villanueva, A Domínguez. 1986. Fungal 2 conditions of the salt tolerant yeast Debaryomyces profiles of Spanish country-cured hams. Int J Food 3 hansenii IFO10939. Current Microbiol 45:277–280. Microbiol 3:355–359. 4 M Larpent-Gourgaut, O Michaux, C Callon, O Brenot, F Núñez, MM Rodríguez, JJ Córdoba, ME Bermúdez, 5 J Sirami, C Bonnin, B Boissonnet. 1993. Etude com- MA Asensio. 1996. Yeast population during ripening 6 pareé des flores d’aromatisation du saucisson de fab- of dry-cured Iberian ham. Int J Food Microbiol 7 rication industrielle ou artisanale. Viand Prod Carnés 29:271–280. 8 14:23–27. PT Olesen, LH Stahnke. 2000. The influence of 9 I Leistner. 1995. Stable and safer fermented sausages Debaryomyces hansenii and Candida utilis on the 10 world-wide. In: G Campbell-Platt, PE Cook, eds. aroma formation in garlic spiced fermented sausages 11 Fermented meats. London, Blackie Academic & Pro- and model minces. Meat Sci 56:357–368. 12 fessional, pp. 160–175. JA Ordóñez, EM Hierro, JM Bruna, L de la Hoz. 1999. 13 JS Leyva, M Manrique, I Prats, MC Loureiro-Dias, Changes in the components of dry-fermented 14 J Peinado. 1999. Regulation of fermentative sausages during ripening. Crit Rev Food Sci Nutr

15 CO2 production by the food spoiling yeast 39(4):329–367. 16 Zygosaccharomyces bailii. Enz Microb Technol AA Osei Abunyewa, E Laing, A Hugo, BC Viljoen. 17 24:270– 275. 2000. The population change of yeasts in commercial 18 G Lizaso, J Chasco, MJ Beriain. 1999. Microbiological salami. Food Microbiol 17:429–438. 19 and biochemical changes during ripening of TL Peppard, SA Halsey. 1981. Malt-flavour- 20 salchichón, a Spanish dry cured sausage. Food Transformation of carbonyls compounds by yeast 21 Microbiol 16(3):219–228. during fermentation. J Inst Brew 87:3860–3890. 22 FK Lücke, H Hechelman. 1987. Cultivos starter para W Pozzi. 2003. Advantages in dry sausages production. 23 embutidos secos y jamón crudo. Composición y Fleischwirtschaft Int 2:14–16. 24 efecto. Fleischwirtschaft español 1:38–48. M Rodriguez, A Martín, F Núñez. 2001. Población 25 D Marquina, P Llorente, A Santos, JM Peinado. 2001. microbiana del jamón y su contribución en la madu- 26 Characterization of the yeast population in low water ración. Cultivos iniciadores. In: J. Ventanas, ed. Tec- 27 activity foods. Adv Food Sci 23:249–253. nología del jamón ibérico. Madrid: Mundi Prensa, 28 A Martín, MA Asensio, ME Bermúdez, MG Córdoba, pp. 347–366. 29 E Aranda, JJ Córdoba. 2002. Proteolytic activity of M Rodríguez, F Núñez, JJ Córdoba, ME Bermúdez, 30 Penicillium chrysogenum and Debaryomyces MA Asensio. 1998. Evaluation of proteolytic activity 31 hansenii during controlled ripening of pork loins. of micro-organisms isolated from dry cured hams. 32 Meat Sci 62:129–137. J Appl Microbiol 85:905–912. 33 C Meisel, KH Gehlen, A Fisher, WP Hammes. 1989. P Roncalés, M Aguilera, JA Beltrán, I Jaime, JM Peiro. 34 Inhibition of the growth of Staphylococcus aureus in 1991. The effect of natural or artificial casing on the 35 dry sausages by Lactobacillus curvatus, Micrococ- ripening and sensory quality of a mould-covered dry 36 cus varians and Debaryomyces hansenii. Food sausage. Int J Food Sci Technol 26:83–89. 37 Biotechnol 3:145–168. E Rossmanith, HJ Mintzlaff, B Streng, W Christ, L 38 J Metaxopoulos, S Stravopoulos, A Kakouri, J Samelis. Leistner. 1972. Hefen als starterkulturen für 39 1996. Yeast isolated from traditional Greek dry rohwürste. Jahr der BAFF 147–148. 40 salami. Ital J Food Sci 1:25–32. A Saldanha-da-Gama, M Malfeito-Ferreira, V Loureiro. 41 E Miteva, E Kirova, D Gadjeva, M Radeva. 1986. Sen- 1997. Characterization of yeasts associated with 42 sory aroma and taste profiles of raw-dried sausages Portuguese pork-based products. Int J Food Micro- 43 manufactures with lipolitically active yeasts culture. biol 37:201–207. 44 Nahrung 30(8):829–832. J Samelis, S Stavropoulos, A Kakouti, J Metaxopoulos. 45 I Molina, P Nieto, J Flores, H Silla, H. S Bermell. 1991. 1994. Quantification and characterization of micro- 46 Study of the microbial flora in dry-cured hams. 5. bial populations associated with naturally fermented 47 Lipolytic activity. Fleischwirtschaft 71:906–908. Greek dry salami. Food Microbiol 11:447–460. 48 I Molina, H Silla, J Flores. 1990. Study of microbial NN Santos, RC Santos-Mendoça, Y Sanz, T Bolumar, 49 flora in dry cured hams. 4: Yeasts. Fleischwirtschaft MC Aristoy, F Toldrá. 2001. Hydrolysis of pork 50 70:74–76. muscle sarcoplasmic proteins by Debaryomyces 51 I Molina, F Toldrá. 1992. Detection of proteolytic activ- hansenii. Int J Food Microbiol 68:199–206. 52 ity in microorganisms isolated from dry cured ham. MD Selgas, C Casas, VM Toledo, ML García. 1999. 53S J Food Sci 57:1308–1310. Effect of selected mould strains on lipolysis in dry 54N 40454 15 159-170 r1.qxd 7/20/07 12:58 PM Page 169

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fermented sausages. Europ Food Res Technol F Toldrá. 2002. Fermentation and starter cultures. In: 1 209(5):360–365. F Toldrá, ed. Dry-cured meat products. Trumbull: 2 MD Selgas, J Ros, ML García. 2003. Effect of selected Food & Nutrition Press Inc., pp. 89–112. 3 yeast strains on the sensory properties of dry F Toldrá, DJ Etherington. 1988. Examination of cathep- 4 fermented sausages. Eur Food Res Technol sins B, D, H and L activities in dry-cured hams. Meat 5 217:475–480. Sci 23:1–7. 6 LH Stahnke. 1994. Aroma components from dried VM Toledo, MD Selgas, C Casas, JA Ordóñez, ML 7 sausages fermented with Staphylococcus xylosus. García. 1997. Effect of selected mould strains on 8 Meat Sci 38:39–53. proteolysis in dry fermented sausages. Z 9 H Stam, M Hoogland, C Laane. 1998. Food ﬂavours Lebensm.Unters. Forsch204:385–390. 10 from yeast. In: BJ Wood, ed. Microbiology of Fer- H Wolter, E Laing, BC Viljoen. 2000. Isolation and iden- 11 mented Foods (2nd ed.). London: Blackie Academic & tiﬁcation of yeasts associated with intermediate mois- 12 Professional, pp. 505–542. ture meats. Food Technol Biotechnol 38(1):69–75. 13 K Tjener, LH Stahnke, L Anmdersden, J Martinussen. MY Wyder, Z Puhan. 1999. Role of selected yeast in 14 2003. A fermented meat model system for studies of cheese ripening: An evaluation in aseptic cheese curd 15 microbial aroma formation. Meat Sci 66:211–218. slurries. Int Dairy J 9:117–124. 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 S53 N54 40454 16 171-176 r2.qxd 7/20/07 1:00 PM Page 171

1 2 3 16 4 5 6 Starter Cultures: Molds 7 8 Elisabetta Spotti and Elettra Berni 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION • Protects fat from oxidation because it metabolizes 22 and consumes peroxides, thus preventing 23 MOLDS ON NATURALLY RIPENED DRY SAUSAGES rancidity. 24

The ripening techniques used to process meat prod- • Reduces the O2 levels on the surface of the 25 ucts quickly lead to a distinctive surface colonization product, thus avoiding oxidative processes and 26 by a great number of filamentous fungi. Apart from improving meat color. 27 molds, surface microflora can also include yeasts, • Contributes to enhancing the flavor of the final 28 which play a role similar to that of molds in sausage product (especially when natural casing is used), 29 production (Samelis and Sofos 2003). because it breaks up fats, proteins, and lactic acid, 30 In general, at the beginning of the ripening thus favoring pH increase. 31 process yeasts are the predominant microorganisms • Makes sausage peeling easier thanks to the 32 on the surface, totaling about 95% of the mycoflora. differentiation of the fungal basal hyphae into a 33 After the first 2 weeks, molds and yeasts are present sort of root called rhizoid, which can penetrate 34 in equal amounts. At the end of the sausage ripening the inner part of the mixture (Grazia et al. 1986). 35 process (from the 4th to 8th week) mycoflora is 36 mainly represented by molds; yeasts may undergo 37 one logarithmic decrement. PARAMETERS AFFECTING MOLD GROWTH 38 Molds usually prevail on yeasts; this is due to the 39 The physicochemical parameters recorded in ripening progressive reduction of surface water activity in 40 environments are directly related to the microbial sausages, to the invasive way molds grow on the 41 growth. In particular, surface molding of fermented surfaces (by apical increase of cells called hyphae) 42 meat during ripening is influenced by: composition of and by substantial production of conidia, the molds 43 the sausage mince, especially the fat particle size; the reproductive units, and to the production of second- 44 relative humidity (RH) of the air and the temperature ary toxic metabolites. 45 conditions applied in industrial plants; drying profile On the surface of many fermented products from 46 (how surface water activity, a , decreases during the several European countries (Italy, Rumania, Bul- w 47 product ripening) and ventilation methods (alternat- garia, France, Hungary, Switzerland, Germany, 48 ing ventilation cycles at high air velocities with cycles Spain, Austria, and Belgium) mold growth is desir- 49 in which the product is allowed to rest favors fungal able. This is due to the fact that the mycelium: 50 growth). According to this, it has been demonstrated 51 • Prevents excessive drying, allowing water loss that matured sausages of a greater size and with 52 and therefore homogeneous dehydration of longer ripening times usually show initial growth 53 the product. mainly characterized by hydrophilic molds and S54 171 N55 40454 16 171-176 r2.qxd 7/20/07 1:00 PM Page 172

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1 toward the end of the ripening period there is a preva- ings showing that many of the Penicillium and 2 lence of xerotolerant strains (Baldini et al. 2000). Aspergillus species frequently found on the surface 3 of dry sausages have the ability to produce mycotox- 4 ins in the actual product, have stressed the impor- 5 ENVIRONMENT- tance of possibly controlling contaminating molds 6 CONTAMINATING MOLD on raw sausages during ripening by using mold 7 SPECIES starters. Starters studied in the past were selected 8 and are now available on the market (Leistner et al. For a long time, molding of sausages was left to 9 1989). They have been shown to impart a desirable contamination by environment-contaminating mold 10 appearance and good technological and sensory species, mainly belonging to the genera Penicillium 11 characteristics to the product, to be unable to synthe- and Aspergillus, which better adapt to the techno- 12 size antibiotics and/or toxic metabolites such as logical conditions to which these products are 13 mycotoxins and to not cause well-known allergies or subjected. 14 mycoses. For the reasons above, they can be used to Natural contamination results in a prevalence of 15 inoculate fermented sausages by spraying or by xerotolerant strains belonging to the genus Penicil- 16 immersion in a spore suspension. lium subgenus Penicillium over those belonging to 17 The selection of fungal starter cultures among the more xerophilic genera such as Aspergillus or 18 molds that naturally occur on the surface of fer- Eurotium. This is due to the easier growth of most 19 mented and ripened sausages allows control of Penicillium species in environments with RH ranging 20 molding in the first steps of the process and makes from 85 to 92% and with temperatures from 10–20°C 21 the starter presence predominant. (see Table 16.1). A minor percentage of growing 22 Among the starters that can be considered both molds consists of hydrophilic fungi producing darkish 23 safe and effective, P. nalgiovense has been fre- mycelium and conidia, such as Mucor spp. and some 24 quently isolated on the surface of ripened meat Scopulariopsis spp. These species must be removed 25 products as a “domesticated species” from P. chryso- because the former do not allow homogeneous drying 26 genum (wild type) (Pitt and Hocking 1997). At pres- of the product in the first steps of the process and the 27 ent it is routinely used as starter culture in many latter can produce an unpleasant ammonia odor 28 traditional productions, and so it is often present in caused by their strong proteolytic activity in the final 29 the air of environments. Apart from P. nalgiovense, step of the ripening (Dragoni et al. 1997; Lücke 2000; 30 various strains of P. gladioli are usually considered Spotti and Baldini, personal communication 2005). 31 acceptable because they are nontoxinogenic and 32 result in desirable flavor and appearance due to the 33 FUNGAL STARTER CULTURES grey color of mycelium (Grazia et al. 1986). Even 34 P. camemberti should be considered a starter 35 The ripening techniques considered positive and culture for the meat industry because of its ability 36 normally applied in industrial practice usually allow to improve the sensory characteristics of dry- 37 for the growth of characteristic whitish molds, which fermented sausages (Bruna et al. 2003). 38 can also inhibit the multiplication of other molds, P. chrysogenum has been mentioned too as a suit- 39 especially those that prove to be potentially toxino- able starter for mold-fermented products (Hammes 40 genic and/or with a mycelium having undesirable and Knauf 1994; Ministero della Sanità 1995), 41 color (Baldini et al. 2000). In particular, recent find- because some nontoxic strains have been shown to 42 43 44 Table 16.1. Fungal species isolated from typical salami from Northern Italy. 45 46 Most Significant Species Isolated Uncommon Species 47 A. candidus (22) P. chrysogenum (7) P. nalgiovense (15) A. ochraceus (3) 48 E. rubrum (11) P. commune (3) P. olsonii (3) Mucor spp. (4) 49 P. aurantiogriseum (18) P. gladioli (7) P. solitum (7) Eupenicillium spp. (10) 50 P. brevicompactum (24) P. griseofulvum (15) P. verrucosum (30) S. brevicaulis (2) 51 P. camemberti (5) P. implicatum (5) P. waksmanii (9) S. candida (5) 52 53S Source: E Spotti, Department of Microbiology, SSICA, Parma, Italy. Unpublished data. 54N Note: The number in parentheses indicates the occurrence in the past years. 40454 16 171-176 r2.qxd 7/20/07 1:00 PM Page 173

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produce proteases that contribute to the generation the two temperatures tested. Despite their different 1 of characteristic ﬂavor compounds in dry-fermented metabolisms, P. gladioli used as starter culture in the 2 sausages (Rodríguez et al. 1998; Martín et al. 2002). SSICA pilot plants and in industrial practice was 3 However, P. chrysogenum is not always considered shown to supply ripened products with good techno- 4 acceptable in industrial practice because its albino logical and sensory features, as P. nalgiovense does. 5 mutant may start producing green conidia, and most This fact highlights that lipolytic activity affects the 6 of the strains tested may produce antibiotics and molding process more than the proteolytic one, which 7 toxic substances such as roquefortine C (Pitt and seems to be exerted also by bacteria and proteases 8 Hocking 1997). Other strains belonging to Penicil- native to the meat. 9 lium species have also been excluded because they 10 produce dark-green conidia that may impair sausage 11 outward appearance. GROWTH AND COMPETITION 12 TESTS 13 Recently studies were carried out in the SSICA pilot 14 LIPOLYTIC AND PROTEOLYTIC 15 ACTIVITIES OF STARTERS plant in Parma, Italy, in order to evaluate a range of commercial and noncommercial fungal starter cul- 16 In the last few years a lot of studies have been carried tures for growth and competitiveness towards vari- 17 out throughout the world concerning the lipolytic and ous undesirable species. 18 proteolytic activity of molds involved in the ripening 19 process, including P. nalgiovense, P. chrysogenum and 20 GROWTH P. camemberti (Geisen et al. 1992; Rodríguez et al. 21 1998; Ockerman et al. 2001; Bruna et al. 2003). A A research work was carried out in model systems 22 recent in vitro experiment focused on the biochemical reproducing a traditional Italian ripening process, 23 activity of two starters, P. nalgiovense and P. gla- and the studies were focused on the growth of ﬁve 24 dioli, in comparison with that of the environment- starter cultures (P. camemberti M and P, P. gladioli 25 contaminating molds grown on ripened products P and B, and P. nalgiovense) and four environment- 26 (P.griseofulvum, P. brevicompactum, P. olsonii, P.impli- contaminating strains (P. solitum, P. verrucosum, 27 catum, P.verrucosum, Talaromyces wortmannii, Mucor P. chrysogenum, and A. ochraceus) as a function of 28 spp.) (Spotti and Berni 2004). With regard to lipid temperature (from 8–22°C) and water activity (from 29 breakdown, both P. nalgiovense and P. gladioli proved 0.78 to 0.92) (Spotti et al. 1999). For each strain, the 30 to have good lipolytic activity: The lipase production of average of growth radial rate (in mm/day, obtained 31 the former was increased by lower temperature (more from the linear correlation between colony diame- 32 marked at 14°C than at 20°C), whereas that of the latter ters and growth time during linear phase) and the 33 was greater at the higher temperatures. P. nalgiovense average duration of lag time (time during which no 34 showed a strong proteolytic activity both at 14°C and at growth occurs) were determined. The most impor- 35 20°C, whereas P. gladioli did not show this activity at tant results are reported in Tables 16.2 and 16.3. 36 37 38 Table 16.2. Optimal growth conditions with corresponding values of minimum lag time and growth rate for 39 different fungal strains. 40 Optimum Minimum Lag Growth Rate 41 Strain Temperature (°C) Optimum RH (%) Time (days) (mm/day) 42 43 P. camemberti M 17 91 2 1.7 44 P. camemberti P 12 91 5 1.2 45 P. gladioli P 19 88 4 1.0 46 P. gladioli B 17 90 4 1.2 47 P. nalgiovense 14 90 4 2.0 48 P. solitum 16 89 5 1.4 49 P. verrucosum 20 88 4 1.3 50 P. chrysogenum 16 89 3 1.1 51 A. ochraceus 19 86 1 2.2 52 S53 Source: Spotti et al. (1999). N54 40454 16 171-176 r2.qxd 7/20/07 1:00 PM Page 174

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1 Table 16.3. Lag time and growth rate at 15¡C for 3rd Stage—Ripening 2 different fungal strains. This last phase is carried out at temperatures ranging 3 Lag Time Growth Rate from 10–15°C, whereas product surface a remains 4 w Strain (Days) (mm/day) within the same range as in the drying phase. 5 Temperature must therefore be regarded as the 6 P.camemberti M 4 1.6 parameter discriminating between the two process- 7 P.camemberti P 5 1.2 ing stages. 8 P. gladioli P 5 0.7 On the basis of the temperature for optimum 9 P. gladioli B 5 1.0 growth, as reported in Table 16.2, in particular 10 P. nalgiovense 4 2.0 P.nalgiovense and P. camemberti are able to prolifer- 11 P. solitum 5 1.3 ate during ripening. However, for a better compari- 12 P. verrucosum 6 1.0 son of the various strains, lag time and growth rate 13 P. chrysogenum 3 1.0 values were determined at 15°C, keeping the relative 14 A. ochraceus 14 1.1 humidity at the optimal value for each individual 15 strain (see Table 16.3). 16 Source: Spotti et al. (1999). Table 16.3 shows that the strain for which domi- 17 nance over the “undesired species” is predictable 18 is P. nalgiovense, because it has the highest growth 19 In order to make the results transferable to the rate under the temperature conditions indicated. 20 industry, the results of the trial were described for Even though P. chrysogenum has shorter lag time 21 the three primary stages of the salami production, (3 days) as compared to P. nalgiovense (4 days), 22 taking into account the wide variability of tempera- its growth rate is half that of P. nalgiovense, and its 23 ture and RH applied in the industrial practice. mycelium is therefore less invasive. As to the 24 other strains, evaluated by the same criterion, the 25 1st Stage—Heating following rating scale is obtained: P. nalgiovense > 26 This phase is carried out at 20–24°C, at environmen- P. camemberti M > P. chrysogenum (wild type) > 27 tal RH values for a few hours (depending on salami P.solitum > P. camemberti P > P. gladioli B > P.ver- 28 size) until the temperature of the inner product rucosum > P. gladioli P > A. ochraceus. 29 reaches 18–20°C. This phase does not seem to have The starters therefore show different dominance 30 a significant effect on subsequent surface mold over “undesired molds.” In particular, together with 31 growth; instead, it is very important for the onset of P.camemberti and the two P. gladioli strains, assum- 32 correct product fermentation by lactic acid bacteria, ing equal contamination levels, also P. chrysogenum, 33 staphylococci, and micrococci. P. solitum, and P. verrucosum may be present on the 34 same product. Instead, P. nalgiovense and P.camem- 35 2nd Stage—Drying berti M prove to dominate during ripening. 36 This phase allows the fermentation to go on because Relative humidity proves to have greater influence 37 the RH is maintained at high values. Processing tem- than temperature for almost all strains except 38 peratures usually range from 15–20°C, whereas sur- A. ochraceus; this accounts for the lag time at opti- 39 face a values are much more variable, ranging from mum temperature in Table 16.3 not being too dif- 40 w 0.80 to 0.95, and most frequently between 0.88 and ferent from those in Table 16.2. The only exception 41 0.91. Surface molding begins to appear in this phase, is A. ochraceus, which is much more sensitive 42 which lasts for a few days. The molds most likely to to temperature changes than to humidity changes, 43 grow during this phase, based on temperature and as is shown by its long lag time at 15°C (14 44 relative humidity for optimum growth, are P. gladi- days), whereas under optimum conditions it is only 45 oli, P. solitum, P. verrucosum, and P. chrysogenum 1day. 46 (see Table 16.2). However, it is also possible to find Recently, the course of growth rate of the fungal 47 P. camemberti and A. ochraceus (Mutti et al. 1988). species that more frequently occur on the surface of 48 Under these conditions, the strains have a lag time of molded meat products during ripening has also been 49 1–5 days and a growth rate of 1–2.2 mm/day (Table investigated (Table 16.4) (Spotti and Berni 2005). 50 16.2); product surface can thus be indifferently Table 16.4 clearly shows how the growth of starter 51 invaded by P. camemberti and A. ochraceus, and the cultures such as P. nalgiovense and P. gladioli are 52 suitability of initial treatment of the products with favored by higher relative humidity in the first days 53S starters cultures proves recommendable. of ripening. 54N 40454 16 171-176 r2.qxd 7/20/07 1:00 PM Page 175

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Table 16.4. Trend in growth rate of most frequent fungal species during ripening at 14Ð15¡C as a function of 1

the surface water activity (aw). 2 3 Fungal Species a = 0.85 a = 0.86 a = 0.88 a = 0.90 a = 0.92 w w w w w 4 P. gladioli →→→→MG 5 P. nalgiovense →→→MG ← 6 P.chrysogenum MG ←←←← 7 P. griseofulvum MG ←←←← 8 P. implicatum MG ←←←← 9 P. verrucosum MG ←←←← 10 P. solitum MG ←←←11 12 Source: E Spotti. Presentation at 2nd Annual Conference on the Research Project “Salumi piacentini,” 25 October 2005, 13 SSICA, Parma, Italy. 14 Note: MG = Maximum growth rate. → = Growth rate tends to increase if surface water activity increases. ← = growth rate 15 tends to decrease if surface water activity increases. 16 17 18 COMPETITION species. The above-mentioned rating scale, relating 19 to the individual starters reported, appears to be valid According to techniques tested by Wheeler and 20 and useful in the industrial practice only on the basis Hocking (1993), several competition trials were car- 21 of these remarks. ried out, where each starter was inoculated in combi- 22 Nevertheless, the use of selected cultures is not a nation with the undesired strain, each at a time 23 widespread practice. In fact, it is not applied when (Spotti et al. 1994, 1999; Spotti and Berni 2004). 24 the producers expect that the final product will The results of these experiments showed that P. nal- 25 obtain satisfactory technological and sensory results. giovense may be overgrown by A. ochraceus during 26 In that case, the presence of occasional indigenous drying (T Ն 15°C), whereas P. nalgiovense will dom- 27 mycoflora on the surface of ripened products is inate this undesired mold during ripening (T Յ 15°C) 28 favorably considered. Regarding this, Williams as previously referred. On the contrary, P. nal- 29 et al. (1985) observed that most spontaneous molds giovense is unable to dominate over P. chrysogenum 30 species subjected to consecutive subcultures, as it (wild type), P. solitum, and P. verrucosum in the case 31 frequently occurs in the ripening rooms within sub- of similar initial contamination levels. During the 32 sequent productive cycles, proved to rapidly degen- drying phase, P. gladioli is inhibited by A. ochraceus 33 erate or to change their morphological appearance and P. solitum, whereas during ripening it prevails 34 and adapt to environmental conditions. This is due to over A. ochraceus, but not over P. chrysogenum (wild 35 the fact that the genome of the species belonging type), P. solitum, and P. verrucosum. A. ochraceus 36 to the genus Penicillium subgenus Penicillium iso- may therefore dominate over the starter strains during 37 lated from sausages appears to be unstable and tends drying and may grow invasively throughout the prod- 38 to cause a rapid adaptation of the above species to uct surface even during ripening, if present in equal 39 the nutritional niches available in order to take amounts. The results of the trials also suggested that 40 advantage of them. the starter cultures could coexist with P. chrysogenum 41 To conclude, when the producers choose not to (wild type), P. solitum, and P. verrucosum on the 42 use starters, it is necessary to identify the fungal product, if their initial levels are the same. 43 species occurring on the surface of the ripened prod- 44 uct, regardless of the good appearance and the high 45 sensory quality of the final product. Besides, the CONCLUSIONS 46 control on the growth of the selected species and 47 The poor competitiveness of surface starter cultures their actual predominance over undesirable molds 48 such as P. nalgiovense and P. gladioli compared with should be periodically planned by routine laboratory 49 other contaminants requires a low rate of environ- tests (i.e., isolation and identification of the species 50 mental contamination and higher average relative applied in the industrial process) to avoid the unex- 51 humidity; therefore, starter molds should be used at pected setting up of environment-contaminating 52 high inoculation levels on sausages to be ripened in mold species, which are morphologically similar to S53 order to avoid possible development of undesirable the selected ones. N54 40454 16 171-176 r2.qxd 7/20/07 1:00 PM Page 176

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1 REFERENCES P Mutti, MP Previdi, S Quintavalla, E Spotti. 1988. 2 Toxigenity of mold strains isolated from salami as a 3 P Baldini, E Berni, C Diaferia, A Follini, S Palmisano, function of culture medium. Industria Conserve 4 A Rossi, PG Sarra, GL Scolari, E Spotti, M Vescovo, 63:142–145. 5 C Zacconi. 2005. Materia prima flora microbica nella HW Ockerman, FJ Céspedes Sánchez, MA Ortega 6 produzione dei salumi piacentini. Rivista di Suini- Mariscal, F León-Crespo. 2001. The lipolytic activity 7 coltura 4:183–187. of some indigenous Spanish molds isolated from 8 P Baldini, E Cantoni, F Colla, C Diaferia, L Gabba, meat products. J Muscle Foods 12:275–284. 9 E Spotti, R Marchelli, A Dossena, E Virgili, S Sforza, JI Pitt, AD Hocking. 1997. Fungi and Food Spoilage. 10 P Tenca, A Mangia, R Jordano, MC Lopez, L Med- 2nd ed. Cambridge: University Press. Blackie Aca- 11 ina, S Coudurier, S Oddou, G Solignat. 2000. Dry demic & Professional. 12 sausages ripening: Influence of thermohygrometric M Rodríguez, F Núñez, JJ Cordoba, ME Bermúdez, 13 conditions on microbiological, chemical and MA Asensio. 1998. Evaluation of proteolytic activity 14 physico-chemical characteristics. Food Res Int of micro-organisms isolated from dry cured ham. 15 33:161–170. J Appl Microbiol 85:905–912. 16 JM Bruna, EM Hierro, L de la Hoz, DS Mottram, J Samelis, JN Sofos. 2003. Yeasts in meat and meat 17 M Fernández, JA Ordoñez. 2003. Changes in products. In: Yeasts in Food. Cambridge: Woodhead 18 selected biochemical and sensory parameters as Publishing Limited, pp. 243–247. 19 affected by the superficial inoculation of Penicillium RA Samson, JC Frisvad. 2004. Polyphasic taxonomy of 20 camemberti on dry fermented sausages. Int J Food the Penicillium. A guide to identification of food and 21 Microbiol 85:111–125. air-borne terverticillate Penicillia and their mycotox- 22 I Dragoni, C Cantoni, A Papa, L Vallone. 1997. Muffe ins. In: Penicillium subgenus Penicillium: New taxo- 23 alimenti e micotossicosi. 2nd ed. Milano: CittàStud- nomic Schemes and Mycotoxins and Other 24 iEdizioni di UTET, p. 109. Extrolites. Stud Mycol 49:1–173. 25 R Geisen, FK Lücke, L Kröckel. 1992. Starter and pro- E Spotti, E Berni. 2004. Il controllo della flora fungina 26 tective cultures for meat and meat products. Fleis- superficiale. Proceedings of 1st Annual Conference 27 chwirtsch 72:894–898. on the Research Project “Salumi piacentini,” Univer- 28 L Grazia, P Romano, A Bagni, D Roggiani, D sità Cattolica del Sacro Cuore, Piacenza, Italy, 27 29 Guglielmi. 1986. The role of molds in the ripening October 2004. 30 process of salami. Food Microbiol 3:19–25. E Spotti, E Berni. 2005. Variazione della flora micro- 31 WP Hammes, HJ Knauf. 1994. Starter in the processing bica superficiale nei salumi oggetto della ricerca. 32 of meat products. Meat Sci 36:155–168. Proceedings of 2nd Annual Conference on the 33 L Leistner, R Geisen, J Fink-Gremmels. 1989. Mold- Research Project “Salumi piacentini”, SSICA, 34 fermented foods of Europe: Hazards and Develop- Parma, Italy, 25 October 2005. 35 ments. In: S Natori, K Hashimoto, Y Ueno, eds. E Spotti, C Busolli, F Palmia. 1999. Sviluppo di colture 36 Mycotoxins and Phycotoxins 1988. Amsterdam: fungine di importanza rilevante nell’industria dei 37 Elsevier Science Publishers B.V., pp. 145–160. prodotti carnei. Industria Conserve 74:23–33. 38 F-K Lücke. 2000. Fermented meats. In: BM Lund, TC E Spotti, P Mutti, F Scalari. 1994. P. nalgiovense, 39 Baird-Parker, GW Gould, eds. The Microbiological P. gladioli, P. candidum e A. candidus: possibilità 40 Safety and Quality of Food. Vol 1. Maryland: Aspen d’impiego quali colture starter. Industria Conserve 41 Publishers Inc, pp. 420–444. 69:237–241. 42 A Martín, MA Asensio, ME Bermúdez, MG Cordoba, KA Wheeler, AD Hocking. 1993. Interactions among 43 E Aranda, JJ Cordoba. 2002. Proteolytic activity of xerophilic fungi associated with dried salted fish. 44 Penicillium chrysogenum and Debaryomyces J Appl Bacteriol 74:164–169. 45 hansenii during controlled ripening of pork loins. AP Williams, JI Pitt, AD Hocking. 1985. The closely 46 Meat Sci 62:129–137. related species of subgenus Penicillium—A phylo- 47 Ministero della Sanità. 1995. Decreto 28 Dicembre genic exploration. In: RA Samson, JI Pitt, eds. 48 1994. Gazzetta Ufficiale Repubblica Italiana. Serie Advances in Aspergillus and Penicillium Systemat- 49 generale n. 89, pp. 4–5, 15.04.1995. ics. New York: Plenum Press, pp. 121–128. 50 51 52 53S 54N 40454 17 177-186 r2.qxd 7/21/07 9:57 AM Page 177

1 2 3 17 4 5 6 Genetics of Microbial Starters 7 8 Marie Champomier-Vergès, Anne-Marie Crutz-Le Coq, Monique Zagorec, 9 Sabine Leroy, Emilie Dordet-Frisoni, Stella Planchon, and Régine Talon 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION Pulse Field Gel Electrophoresis (PFGE), was esti- 22 mated to be 1,845 kbp (Dudez et al. 2002), and a first 23 The main bacterial species used in meat fermentation chromosome map was obtained by the use of 47 24 are Lactobacillus sakei, Staphylococcus xylosus, and genetic loci available in 2002. In 2005, Chaillou 25 Staphylococcus carnosus. These species have been et al. published the complete genome sequence of 26 applied for more than 50 years alone or in combina- L. sakei 23K. The exact size of this circular chromo- 27 tion with each other or other microorganisms as some is 1,884,661 bp and the knowledge of the 28 starter cultures for the manufacturing of fermented 1,883 putative genes highlighted the main functions 29 sausages. Studies of these starter cultures were of the species, especially those linked to the adapta- 30 mainly focused on their role in the sensory quality tion to technological environments encountered dur- 31 or in the microbial safety of fermented meat prod- ing sausage production (see also Eijsink and 32 ucts. Genetic studies have provided basic knowledge Axelsson 2005). The main features of this genome, 33 on main metabolic activities and stress-resistance when compared to three other lactobacilli genomes, 34 functions. Appropriate genetic tools for molecular are summarized in Table 17.1. From the known 35 genetic analyses were developed, allowing the involvement of L. sakei as sausage starter, the 36 exchange of chromosomal genes or plasmids in these genome analysis confirmed that the main role of 37 naturally nontransformable bacteria. Global appro- L. sakei is to ferment sugars into lactic acid, and that 38 aches based on proteomics and transcriptomics were it lacks main aroma production pathways. L. sakei 39 developed or are in progress in order to better under- 23K also lacks genes responsible for biogenic amine 40 stand their adaptation to the meat environment and production. A battery of functions were identified, 41 their interactions with the ecosystem and the meat which might explain adaptation or resistance of this 42 substrate. species to stressing environmental conditions used 43 during sausage manufacturing (presence of curing 44 CHROMOSOMAL ELEMENTS agents, spices, smoke, low temperature). 45 46 LACTOBACILLUS SAKEI 47 STAPHYLOCOCCUS CARNOSUS AND STAPHYLOCOCCUS Although numerous L. sakei strains have been stud- 48 XYLOSUS ied in different laboratories, most of the genetic 49 information has been obtained from one strain, The size of the circular chromosome of the 50 L. sakei 23K, initially isolated from a fermented dry S. carnosus TM300 strain was estimated by PFGE to 51 sausage (Berthier et al. 1996; Champomier-Vergès be 2,590 kb (Wagner et al. 1998). The complete 52 et al. 2002). The size of its genome, determined by genome sequence of this strain allowed establishing 53 S54 177 N55 40454 17 177-186 r2.qxd 7/21/07 9:57 AM Page 178

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1 Table 17.1. Summary of the main characteristics of L. sakei genome with comparison to three other 2 lactobacilli genomes. 3 L. sakei L. plantarum L. johnsonii L. acidophilus 4 23K WCFS1 NCC533 NCFM 5 6 Chromosome size 1,884,661 3,308,274 1,992,676 1,993,564 7 GC (%) 41.25 45.60 34.60 34.71 8 9 Coding sequences 1,883 3,009 1,821 1,864 10 Unique genes 254 293 150 186 11 rRNAs 7 5 4 4 12 13 tRNAs 63 62 79 61 14 IS elements 12 15 14 17 15 PTS1 transporters 6 25 16 20 16 Proteinase/peptidases 0/22 0/19 PrtP/25 PrtP/20 17 18 Amino-acid transport/biosynthesis 24/2 22/17 25/3 24/10 19 Redox2 functions 55 60 20 29 20 Compatible solute Transport3 43 1 1 21 CspA (cold shock Proteins A) 4 3 1 1 22 23 Source: from Chaillou et al. (2005). 24 1 = Phosphoenol pyruvate : carbohydrate phosphotransferase transport systems. 25 2 = Proteins involved in redox balance adaptation. 26 3 = Transporters of osmolytes compatible solutes are involved in osmolarity and cold temperature resistance. 27 28 the exact size of the chromosome to 2.56 Mb comparative studies showed a high conservation 29 (Rosenstein et al. 2005). The size of the chromo- in gene order between S. carnosus and other 30 some of S. xylosus strain C2a was estimated to be sequenced staphylococci. However, comparison of the 31 2,868 kb (Dordet-Frisoni et al. 2007). So the size of gene products revealed that about 20% of them have 32 the genome of S. carnosus and S. xylosus is compa- no staphylococcal homolog (Rosenstein et al. 2005). 33 rable to that of other staphylococci. The size vari- A physical and genetic map of S. xylosus C2a was 34 ability within the staphylococcal genomes is about established by locating 47 restriction fragments and 35 14%, ranging from 2,499 kb (Staphylococcus epi- 33 genetic markers. Twenty-three previously identi- 36 dermidis ATCC 12228, Zhang et al. 2003) to 2,903 fied loci mainly concerned carbohydrate utilization 37 kb (Staphylococcus aureus MRSA252, Holden and carbon catabolite repression (Brückner et al. 38 et al. 2004). Inter- and intraspecies variations of 1993; Egeter and Brückner 1995; Bassias and 39 genome size were observed as summarized Table Brückner 1998; Fiegler et al. 1999) and antioxidant 40 17.2. S. xylosus exhibited also a relatively large capacities (Barrière et al. 2001, 2002), and 10 were 41 intraspecies variation (11%) (Dordet-Frisoni et al. newly identified (Dordet-Frisoni et al. 2007). The 42 2007). In contrast, S. carnosus species constituted a S. xylosus C2a chromosome contains six rrn operons. 43 relatively homogeneous genetic group (Martin et al. As also observed in other staphylococcal species, 44 2006; Planchon et al. 2007). this number varies among strains from five to six 45 A physical and genetic map of S. carnosus TM300 (Dordet-Frisoni et al. 2007; Kuroda et al. 2001, 2005; 46 was constructed (Wagner et al. 1998). Thirty-one Baba et al. 2002; Holden et al. 2004; Gill et al. 2005; 47 restriction fragments obtained from rare cutting Zhang et al. 2003; Takeuchi et al. 2005). A first 48 restriction enzymes were positioned and 18 genetic comparison of S. xylosus C2a map with S. aureus, 49 markers were located, including four ribosomal S. epidermidis, S. haemolyticus, and S. saprophyticus 50 operons (rrn) and genes related to sugar metabolism sequenced genome showed that the relative position 51 and to nitrate and nitrite reduction important of rrn operons and several genetic markers are well 52 for meat fermentation (Wagner et al. 1998). The conserved between S. xylosus and the other stap- 53S chromosome of S. carnosus TM300 comprises 2,454 hylococci (Figure 17.1). The sequencing of the 54N ORFs and the annotation of the completed sequence complete genome of S. xylosus is in progress (http:// is under way (Rosenstein et al. 2005). The first www.genoscope.cns.fr). 40454 17 177-186 r2.qxd 7/21/07 9:57 AM Page 179

Table 17.2. Intra- and interspecies variation of chromosome size among staphylococcal 1 sequenced genomes. 2 3 Chromosome 4 Species Strains Size (kb) References 5 S. aureus Mu50 2,879 Kuroda et al. 2001 6 N315 2,815 Kuroda et al. 2001 7 MW2 2,820 Baba et al. 2002 8 MRSA252 2,903 Holden et al. 2004 9 MSSA476 2,800 Holden et al. 2004 10 COL 2,809 Gill et al. 2005 11 USA300 2,873 Diep et al. 2006 12 13 S. epidermidis ATCC 12228 2,499 Zhang et al. 2003 14 RP62A 2,617 Gill et al. 2005 15 S. saprophyticus ATCC 15305 2,517 Kuroda et al. 2005 16 S. haemolyticus JCSC1435 2,685 Takeuchi et al. 2005 17 S. carnosus Tm300 2,560 Rosenstein et al. 2005 18 19 20 21 22 23 katA 24 ilvE ilvE 25 cysE 26 cysE cysE cysE ilvE 27 gap ilvE ilvE cysE hprK hprK 28 dltA hprK hprK dltA hprK 29 gap gap gap gap dltA 30 dltA dltA katA 31 femA katA 32 33 katA sodA femA katA 34 femA glkA sodA femA femA glkA sodA 35 ccpA sodA glkA 36 sodA2 ccpA glkA ccpA ccpA 37 ccpA 38 39 40 ureG scrA scrA scrA2 41 fbp scrA1 scrA 42 ureG fbp fbp scrA cudB cudB 43 ureG ureG 44 fbp cudB cudB 45 cudB 46 gyrA gyrA gyrA gyrA gyrA dnaA dnaA dnaA dnaA dnaA 47 48 S. aureus S. epidermidis S. haemolyticus S. saprophyticus S. xylosus 49 MW2 RP62A JCSC1435 ATCC 15305 C2a 2.82 Mb 2.64 Mb 2.69 Mb 2.52 Mb 2.87 Mb 50 51 Figure 17.1. Comparison of the genetic map of S. xylosus C2a with the chromosomes of S. aureus MW2, 52 S. epidermidis RP62a, S. haemolyticus JCSC1435, and S. saprophyticus ATCC 15305.The alignment was done S53 from the putative origin of replication labeled by dnaA and gyrA genes. The arrows correspond to ribosomal N54 operons.

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1 CRYPTIC PLASMIDS DNA TRANSFER,VECTORS,AND 2 GENETIC TOOLS 3 LACTOBACILLUS SAKEI 4 L. sakei strains usually contain 1–5 plasmids, the size LACTOBACILLUS SAKEI 5 of which can range from about 1.5 to more than 50 Plasmid or gene transfer exists naturally among 6 kbp. The two main types of replication mode, rolling- bacteria and is also of great importance for genetic 7 circle (RC) and theta replication, exist. Plasmids studies. Natural transformation essentially relies on 8 homologous to the 2.1 kbp pLP1 from Lactobacillus competence and conjugation. Natural competence 9 plantarum or to the 2.6 kbp pLC2 from Lactobacillus was never reported in L. sakei, although the com- 10 curvatus (RC type) are known to be present in L. sakei plete genome sequence of L. sakei 23K revealed 11 (Bringel et al. 1989, Vogel et al. 1992). The replication genes homologous to the well-known competence 12 region of different groups of plasmids has been genes of Bacillus subtilis, but their exact function is 13 sequenced: pSAK1 (theta-type, Genbank: Z50862), unknown. Conjugative plasmids required for conju- 14 pLS141-1 (RC type, Genbank: AB109041), and gation were not yet isolated from L. sakei. Neverthe- 15 pRV500, a 13 kbp theta-replicating plasmid (Alpert less, conjugative transfers were observed under 16 et al. 2003). The latter is the only plasmid isolated laboratory conditions with the broad host-range con- 17 from L. sakei that has been totally sequenced so far. jugative plasmid pAMβ1 (Langella et al. 1996), and 18 Gene products other than those involved in replica- during sausage fermentation in the closely related 19 tion have been identified by sequence homology, species Lactobacillus curvatus (Vogel et al. 1992). 20 such as a restriction/modification system on pRV500 For laboratory research, in the objective of under- 21 (Alpert et al. 2003) and a general stress protein on standing the molecular basis of L. sakei physiology, 22 pLS141-1. The first correlation between the presence genetic tools and transformation protocols have been 23 of plasmids and a phenotype was evidenced in 1988 by developed. Electroporation protocols give relative 24 Shay et al. for cystein transport. Tetracycline resistance good efficiency, depending of the strains (Aukrust 25 genes, detected in L. sakei isolated from fermented and Blom 1992, Berthier et al. 1996). A list of vectors 26 sausages, were mainly plasmid-borne (Gevers et al. developed for L. sakei’s molecular biology is given in 27 2003). Bacteriocin production, an important techno- Table 17.3. Construction of chromosomal mutants 28 logical trait, has been given high attention. Although relies on homologous recombination and utilization 29 many gene clusters characterized so far are chromoso- of integrative or delivery vectors. This allowed the 30 mal, plasmid-encoded genes were reported for sakacin construction of point mutations for which no exoge- 31 P (Vaughan et al. 2003), sakacin A (Axelsson and nous DNA persists (Stentz and Zagorec 1999), or 32 Holck 1995), sakacin G (Simon et al. 2002), and lac- deletion mutants (Malleret et al. 1998, Stentz et al. 33 tocin S (Skaugen et al. 1997). To our knowledge, no 2000). No genetic tools based on phage or transposi- 34 other potentially noteworthy technological traits have tion are yet available. This is the consequence of the 35 been linked to plasmids in L. sakei so far. lack of knowledge of these elements in L. sakei. 36 Indeed, only one phage has been described, but its 37 sequence was not determined (Leuschner et al. 1993) STAPHYLOCOCCUS CARNOSUS AND STAPHYLOCOCCUS 38 and the presence of IS elements is strain-dependent 39 XYLOSUS (Skaugen et al. 1997; Skaugen and Nes 2000; Chail- 40 One cryptic plasmid, pSX267, issued from S. xylosus lou et al. 2005). Inducible promoters to study gene 41 DSM20267, was described. It is a 29.5 kbp multi- functions (Stentz et al. 2000) or to overexpress pro- 42 resistance plasmid, which contains an antimonite, teins (Sorvig et al. 2005) have been described and 43 arsenite, and arsenate operon (Rosenstein et al. 1992). reporter genes can also be used in L. sakei such as 44 The pSX267 replication region (1.8 kb) was sequenced lacZ (Stentz et al. 2000), gusA (Hertel et al. 1998), 45 and defined a new family of theta-replicating plasmid and GFP (Gory et al. 2001), the activity of the latter 46 in staphylococci (Gering et al. 1996). Utilization of the being detected by fluorescence and allowing to fol- 47 pSX267 replicon was considered to develop a novel low strains in complex environments. 48 narrow host range vector for gram-positive bacteria 49 (Gering et al. 1996). A large diversity of plasmid pro- STAPHYLOCOCCUS CARNOSUS AND STAPHYLOCOCCUS 50 files was recently reported for S. xylosus strains isolated 51 from slightly fermented sausages (Martin et al. 2006). XYLOSUS 52 Although the presence of plasmids was recently Natural competence has never been reported in 53S reported for this species (Martin et al. 2006), none S. xylosus and S. carnosus. An efficient transfor- 54N was well described for S. carnosus. mation system was first reported for the S. carnosus 40454 17 177-186 r2.qxd 7/21/07 4:57 PM Page 181

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Table 17.3. Some of the plasmids developed for molecular genetics of L. sakei. 1 2 Plasmid Name Replicon Selection Host-range Function/Use 3 pRV3001 colE14 erm NA Integrative vector 4 Insertional mutagenesis 5 6 2 pRV566 colE1, pRV500 erm E. coli lactobacilli Shuttle vector 7 Replicative 8 pSIP vector3 series p256 including erm L. sakei, Expression vectors 9 or not colE1 L. plantarum with sakacin inducible 10 promoters 11 12 1 Leloup et al. (1997). 13 2 Alpert et al. (2003). 14 3 Sorvig et al. (2005). 4 15 colE1 is a replicon of Escherichia coli that does not sustain replication in gram-positive bacteria. 16 17 18 TM300 strain using competent protoplasts (Götz tool to analyze protein expression under various 19 et al. 1983; Götz and Schumacher 1987). S. carnosus environmental conditions (Figure 17.2). 20 TM300 can be considered as a wild-type plasmid As an example of analysis of process adaptation, 21 and cloning host strain (Götz, 1990) and has been proteins differentially expressed at cold temperature 22 used in many studies. The transformation procedure or in the presence of NaCl were identified. By the 23 of this strain was simplified by the development of a use of mutants constructed with the genetic tools 24 procedure for electroporation (Augustin and Götz described above, the involvement of these proteins in 25 1990). This method could be applied to other staphy- this adaptation was demonstrated. Two proteins 26 lococcal species but not to S. xylosus. A specific pro- belong to carbon metabolism and four could be clus- 27 cedure for electroporation of S. xylosus C2a strain tered as stress proteins. Five of them appeared to be 28 was developed (Brückner 1997). In the same time, a necessary for a correct survival during stationary 29 system for gene replacement in S. carnosus and phase (Marceau et al. 2004). Proteomics also 30 S. xylosus was described using the temperature- revealed that high-pressure stress survival mecha- 31 sensitive E. coli—Staphylococcus shuttle plasmids, nisms of Listeria monocytogenes and L. sakei 32 pBT1 and pBT2 (Brückner 1997). Transposon muta- elicited different responses through the involvement 33 genesis can be used to obtain S. carnosus and of different set of proteins. Stress proteins were 34 S. xylosus mutants (Wagner et al. 1995; Neubauer induced in L. sakei, whereas L. monocytogenes 35 et al. 1999). Several expression vectors have been induced proteases to overcome high-pressure stress 36 created for the display of heterologous proteins to (Jofré et al. 2007). 37 the surface of S. carnosus (Samuelson et al. 1995; 38 Wernérus and Ståhl 2002), and S. xylosus (Hansson STAPHYLOCOCCUS XYLOSUS 39 et al. 1992). A reporter gene system based on the β- Similarly, the knowledge of the whole chromosome 40 galactosidase gene of S. xylosus has been described 41 (Jankovic et al. 2001). So, many genetic tools can be sequence of S. xylosus C2a will allow the development of proteomic and transcriptomic studies for a 42 used to study the molecular basis of S. carnosus and 43 S. xylosus physiology. better understanding of the physiology of S. xylosus. A proteomic approach to study cell-envelope pro- 44 teins of S. xylosus has been developed (Planchon 45 TOWARD POSTGENOMICS 2006; Planchon et al. 2007). This method was based 46 on lysostaphin treatment giving a first fraction of 47 LACTOBACILLUS SAKEI protein, and a second membrane protein fraction was 48 The determination of the whole chromosome efficiently recovered in a procedure involving a 49 sequence of L. sakei 23K allows the development of delipidation and solubilization steps. Proteins were 50 proteomics and transcriptomics, through large scale separated by 2-DE and identified by mass spectrom- 51 and global analyses of the response of this bacterium etry. A total of 90 distinct proteins were identified in 52 to its environment. The use of two-dimensional gel the two fractions. Proteins with transmembrane S53 electrophoresis (2-DE) revealed to be a powerful domains, predicted as lipoproteins or homologous to N54 40454 17 177-186 r2.qxd 7/21/07 9:57 AM Page 182

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1 MM 2 90 3 4 70 5 6 7 8 35 9 10 11 12 20 13 14 15 16 17 18 10 19 47 20 Figure 17.2. Two-dimensional gel electrophoresis of proteins of the cytoplasmic fraction after Commassie blue 21 staining. Proteins expressed by L. sakei 23K during growth in MCD (Chemically Defined Medium, Lauret et al. 22 1996) at 30¡C are shown. About 420 spots could be detected in the pI range 4Ð7 on the 1116 predicted proteins 23 encoded by the genome in this range. Peptide Mass Finger Print analysis by MALDI Tof combined with L. sakei 24 23K genome database searching allowed protein identification. 25 26 27 28 29 components of membrane protein complex were 30 identified in the membrane fraction (Figure 17.3). 31 Proteins potentially secreted or primarily predicted 32 as cytoplasmic, but that could interact temporarily 33 with membrane components, were also identified. 34 The method, allowing to recover a significant set of 35 cell envelope proteins from S. xylosus, could be 36 applied to study the physiology of this bacterium in 37 different environmental meat conditions. 38 These proteomic approaches combined with the 39 future analysis of the transcripts will allow having a 40 more integrated and comprehensive knowledge of 41 the mechanisms by which L. sakei and S. xylosus 42 contribute to the fermentation of meat and how these 43 bacteria interact with each other. 44 45 46 REFERENCES 47 Figure 17.3. Two-dimensional gel electrophoresis 48 CA Alpert, A-M Crutz-Le Coq, C Malleret, of proteins of the membrane fraction from S. xylosus M Zagorec. 2003. Characterization of a theta-type 49 C2a grown in tryptic soya broth medium supple- 50 mented with 0.6 g/L yeast extract and 20 g/L sodium plasmid from Lactobacillus sakei: A potential basis 51 chloride under shaking (150 rpm) at 30¡C during for low-copy vectors in lactobacilli. Appl Environ 52 48 h. Protein extracts were separated by IEF over a Microbiol 69:5574–5584. 53S nonlinear pH gradient 3 to 10 followed by a 10% J Augustin, F Götz. 1990. Transformation of Staphylo- 54N SDS-PAGE and revealed with silver staining. coccus epidermidis and other staphylococcal species 40454 17 177-186 r2.qxd 7/21/07 9:57 AM Page 183

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with plasmid DNA by electroporation. FEMS Micro- E Dordet-Frisoni, R Talon, S Leroy. 2007. Physical and 1 biol Lett 54:203–207. genetic map of the Staphylococcus xylosus C2a chro- 2 T Aukrust, H Blom. 1992. Transformation of Lacto- mosome. FEMS Microbiol Lett, 266:184–195. 3 bacillus strains used in meat and vegetable fermenta- AM Dudez, S Chaillou, L Hissler, R Stentz, 4 tion. Food Res Int 25:253–261. M Champomier-Vergès, CA Alpert, M Zagorec. 5 L Axelsson, A Holck. 1995. The genes involved in pro- 2002. Physical and genetic map of the Lactobacillus 6 duction of and immunity to sakacin A, a bacteriocin sakei 23K chromosome. Microbiol 148:421–431. 7 from Lactobacillus sake Lb706. J Bacteriol O Egeter, R Brückner. 1995. Characterization of a 8 177:2125–2137. genetic locus essential for maltose-maltotriose 9 T Baba, F Takeuchi, M Kuroda et al. 2002. Genome and utilization in Staphylococcus xylosus. J Bacteriol 10 virulence determinants of high virulence commu- 177:2408–2415. 11 nity-acquired MRSA. Lancet 359:1819–1827. VGH Eijsink, L Axelsson. 2005. Bacterial lessons in 12 C Barrière, R Brückner, D Centeno, R Talon. 2002. sausage making. Nature Biotechnol 23:1494–1495. 13 Characterization of the katA gene encoding a catalase H Fiegler, J Bassias, I Jankovic, R Brückner. 1999. 14 and evidence for at least a second catalase activity in Identification of a gene in Staphylococcus xylosus 15 Staphylococcus xylosus, bacteria used in food fer- encoding a novel glucose uptake protein. J Bacteriol 16 mentation. FEMS Microbiol Lett 216:277–283. 181:4929–4936. 17 C Barrière, R Brückner, R Talon. 2001. Characteriza- M Gering, F Götz, R Brückner. 1996. Sequence and 18 tion of the single superoxide dismutase of Staphylo- analysis of the replication region of the Staphylococ- 19 coccus xylosus. Appl Environ Microbiol 67: cus xylosus plasmid pSX267. Gene 182:117–122. 20 4096–4104. D Gevers, M Danielsen, G Huys, J Swings. 2003. 21 J Bassias, R Brückner. 1998. Regulation of lactose uti- Molecular characterization of tet(M) genes in 22 Lactobacillus isolates from different types of fer- lization genes in Staphylococcus xylosus. J Bacteriol 23 mented dry sausage. Appl Environ Microbiol 69: 180:2273–2279. 24 1270–1275. F Berthier, M Zagorec, M Champomier-Vergès, 25 SR Gill, DE Fouts, GL Archer et al. 2005. Insights on SD Ehrlich, F Morel-Deville. 1996. High-frequency 26 evolution of virulence and resistance from the transformation of Lactobacillus sake by electropora- 27 complete genome analysis of an early methicillin- 28 tion. Microbiol 142:1273–1279. resistant Staphylococcus aureus strain and a biofilm- 29 F Bringel, L Frey, JC Hubert. 1989. Characterization, producing methicillin-resistant Staphylococcus 30 cloning, curing, and distribution in lactic acid bacte- epidermidis strain. J Bacteriol 187:2426–2438. 31 ria of pLP1, a plasmid from Lactobacillus plantarum L Gory, MC Montel, M Zagorec. 2001. Use of green 32 CCM 1904 and its use in shuttle vector construction. fluorescent protein to monitor Lactobacillus sakei in 33 Plasmid 22:193–202. meat fermented products. FEMS Microbiol Lett 34 R Brückner. 1997. Gene replacement in Staphylococ- 194:127–133. 35 cus carnosus and Staphylococcus xylosus. FEMS F Götz. 1990. Staphylococcus carnosus. A new host for 36 Microbiol Lett 151:1–8. gene cloning and protein production. Soc Appl Bac- 37 R Brückner, E Wagner, F Götz. 1993. Characterization teriol Symp Ser 19:49S–53S. 38 of a sucrase gene from Staphylococcus xylosus. F Götz, B Kreutz, KH Schleifer 1983. Protoplast trans- 39 J Bacteriol 175:851–857. formation of Staphylococcus carnosus by plasmid 40 S Chaillou, M Champomier-Vergès, M Cornet, A-M DNA. Mol Gen Genet 189:340–342. 41 Crutz Le Coq, A-M Dudez, V Martin, S Beaufils, R F Götz, B Schumacher. 1987. Improvements of proto- 42 Bossy, E Darbon-Rongère, V Loux, M Zagorec. plast transformation in Staphylococcus carnosus. 43 2005. Complete genome sequence of the meat-born FEMS Microbiol Lett 40:285–288. 44 lactic acid bacterium Lactobacillus sakei 23K. M Hansson, S Stahl, TN Nguyen, T Bachi, A Robert, 45 Nature Biotechnol 23:1527–1533. H Binz, A Sjolander, M Uhlen. 1992. Expression 46 M Champomier-Vergès, S Chaillou, M Cornet, of recombinant proteins on the surface of the 47 M Zagorec. 2002. Lactobacillus sakei: Recent devel- coagulase-negative bacterium Staphylococcus xylo- 48 opment and future prospects. Res Microbiol 153: sus. J Bacteriol 174:4239–4245. 49 115–123. C Hertel, G Schmidt, M Fischer, K Oellers, 50 BA Diep, SR Gill, RF Chang et al. 2006. Complete WP Hammes. 1998. Oxygen-Dependent Regulation 51 genome sequence of USA300, an epidemic clone of of the Expression of the catalase gene katA of Lacto- 52 community-acquired meticillin-resistant Staphylo- bacillus sakei LTH677 Appl Environ Microbiol S53 coccus aureus. Lancet 367:731–739. 64:1359–1365. N54 40454 17 177-186 r2.qxd 7/21/07 9:57 AM Page 184

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1 MT Holden, EJ Feil, JA Lindsay et al. 2004. Complete S Morot-Bizot, R Talon, S Leroy-Sétrin. 2003. Devel- 2 genomes of two clinical Staphylococcus aureus opment of specific PCR primers for a rapid and accu- 3 strains: evidence for the rapid evolution of virulence rate identification of Staphylococcus xylosus, a 4 and drug resistance. Proc Natl Acad Sci USA species used in food fermentation. J Microbiol 5 101:9786–9791. Methods 55:279–286. 6 I Jankovic , O Egeter, R Brückner. 2001. Analysis of H Neubauer, I Pantel, F Götz. 1999. Molecular charac- 7 catabolite control protein A-dependent repression in terization of the nitrite-reducing system of Staphylo- 8 Staphylococcus xylosus by a genomic reporter gene coccus carnosus. J Bacteriol 181:1481–1488. 9 system. J Bacteriol 183:580–586. S Planchon 2006. Aptitude de Staphylococcus carnosus 10 A Jofré, MC Champomier-Vergès, P Anglade, et Staphylococcus xylosus à former des biofilms— 11 F Baraige, B Martin, M Garriga, M Zagorec, Étude d’une souche biofilm positif par une approche 12 T Aymerich. 2007. Proteomic analysis of the protéomique. Thèse de Docteur de l’Université 13 response of lactic acid and pathogenic bacteria to Blaise Pascal, Clermont-Ferrand II. 14 high hydrostatic pressure treatment, submitted. S Planchon, C Chambon, M Desvaux, I Chafsey, 15 M Kuroda, T Ohta, I Uchiyama et al. 2001. Whole S Leroy, R Talon, M Hébraud. 2007. Proteomic 16 genome sequencing of meticillin-resistant Staphylo- analysis of cell envelope proteins from Staphylococ- 17 coccus aureus. Lancet 357:1225–1240. cus xylosus C2a. J Proteomic Res, submitted. 18 M Kuroda, A Yamashita, H Hirakawa et al. 2005. Whole R Rosenstein, C Nerz, A Resch, F Götz. 2005. Compar- 19 genome sequence of Staphylococcus saprophyticus ative Genome Analyses of Staphylococcal Species. 20 reveals the pathogenesis of uncomplicated urinary 2nd European Conference on prokaryotic genomes. 21 tract infection. Proc Natl Acad Sci USA Prokagen. 22 102:13272–13277. R Rosenstein, A Peschel, B Wieland, F Götz. 1992. 23 P Langella, M Zagorec, SD Ehrlich, F Morel-Deville. Expression and regulation of the antimonite, arsenite, 24 1996. Intergeneric and intrageneric conjugal transfer and arsenate resistance operon of Staphylococcus 25 of plasmids pAMβ1, pIL205 and pIP501 in Lacto- xylosus plasmid pSX267. J Bacteriol 174: 3676–3683. 26 bacillus sake. FEMS Microbiol Lett 139:51–56. P Samuelson, M Hansson, N Ahlborg, C Andreoni, 27 R Lauret, F Morel-Deville, F Berthier, M Champomier- F Götz, T Bachi, TN Nguyen, H Binz, M Uhlen, 28 Vergès, P Postma, SD Ehrlich, M Zagorec. 1996. S Stahl. 1995. Cell surface display of recombinant 29 Carbohydrate utilization in Lactobacillus sake. Appl proteins on Staphylococcus carnosus. J Bacteriol 30 Environ Microbiol 62:1922–1927. 177:1470–1476. 31 L Leloup, SD Ehrlich, M Zagorec, F Morel-Deville. BJ Shay, AF Egan, M Wright, PJ Rogers. 1988. Cystein 32 1997. Single cross-over integration in the Lactobacil- metabolism in an isolate of Lactobacillus sake: plas- 33 lus sake chromosome and insertional inactivation of mid composition and cystein transport. FEMS 34 the ptsI and lacL genes. Appl Environ Microbiol Microbiol Lett 56:183–188. 35 63:2127–2133. L Simon, C Frémaux, Y Cenatiempo, J-M Berjeaud. 36 RGK Leuschner, EK Arendt, WP Hammes. 1993. 2002. Sakacin G, a new type of antilisterial bacteri- 37 Characterization of a virulent Lactobacillus sake ocin. Appl Environ Microbiol 68:6416–6420. 38 phage PWH2, Appl Microbiol Biotechnol 39: M Skaugen, CI Abildgaard, IF Nes. 1997. Organization 39 617–621. and expression of a gene cluster involved in the 40 C Malleret, R Lauret, SD Ehrlich, F Morel-Deville, biosynthesis of the lantibiotic lactocin S Mol Gen 41 M Zagorec. 1998. Disruption of the sole ldhL gene in Genet 253:674–686. 42 Lactobacillus sakei prevents the production of both M Skaugen, IF Nes, 2000. Transposition in Lactobacil- 43 L- and D-lactate. Microbiol 144:3327–3333. lus sakei: inactivation of a second lactocin S operon 44 A Marceau, M Zagorec, S Chaillou, T Méra, by the insertion of IS1520, a new member of the IS3 45 M Champomier-Vergès. 2004. Evidence for the family of insertion sequences. Microbiol 146: 46 involvement of at least six proteins in Lactobacillus 1163–1169. 47 sakei adaptation to cold temperature and addition of E Sorvig, G Mathiesen, K Naterstad, VG Eijsink, 48 NaCl. Appl Environ Microbiol 70:7260–7268. L Axelsson. 2005. High-level, inducible gene expres- 49 B Martin, M Garriga, M Hugas, S Bover-Cid, sion in Lactobacillus sakei and Lactobacillus plan- 50 MT Veciana-Nogues, T Aymerich. 2006. Molecular, tarum using versatile expression vectors. Microbiol 51 technological and safety characterization of gram- 151:2439–2449. 52 positive catalase-positive cocci from slightly R Stentz, C Loizel, C Malleret, M Zagorec. 2000. 53S fermented sausages. Int J Food Microbiol 107: Development of genetic tools for Lactobacillus 54N 148–158. sakei: disruption of the β-galactosidase gene and use 40454 17 177-186 r2.qxd 7/21/07 9:57 AM Page 185

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of lacZ as a reporter gene to study regulation of the RF Vogel, M Lohmann, AN Weller, M Hugas, 1 putative copper ATPase, AtkB. Appl Environ Micro- WP Hammes. 1991. Structural similarity and distri- 2 biol 66:4272–4278. bution of small cryptic plasmids of Lactobacillus 3 R Stentz, M Zagorec. 1999. Ribose utilization in Lacto- curvatus and Lactobacillus sake. FEMS Microbiol 4 bacillus sakei: analysis of the regulation of the rbs Lett 84:183–190. 5 operon and putative involvement of a new trans- E Wagner, J Doskar, F Götz. 1998. Physical and genetic 6 porter. J Mol Microbiol Biotechnol 1:165–173. map of the genome of Staphylococcus carnosus 7 F Takeuchi, S Watanabe, T Baba et al. 2005. Whole- TM300. Microbiol 144:509–517. 8 genome sequencing of Staphylococcus haemolyticus E Wagner, S Marcandier, O Egeter, J Deutscher, F Götz, 9 uncovers the extreme plasticity of its genome and the R Brückner. 1995. Glucose kinase-dependent catabo- 10 evolution of human-colonizing staphylococcal lite repression in Staphylococcus xylosus. J Bacteriol 11 species. J Bacteriol 187:7292–7308. 177:6144–6152. 12 A Vaughan, VG Eijsink, D van Sinderen. 2003. Func- H Wernérus, S Ståhl. 2002. Vector engineering to 13 tional characterization of a composite bacteriocin improve a staphylococcal surface display system. 14 locus from malt isolate Lactobacillus sakei 5. Appl FEMS Microbiol Lett 212:47–54. 15 Environ Microbiol 69:7194–7203. YQ Zhang, SX Ren, HL Li et al. 2003. Genome-based 16 RF Vogel, M Becke-Schmid, P Entgens, W Gaier, WP analysis of virulence genes in a non-bioﬁlm–forming 17 Hammes. 1992. Plasmid transfer and segregation in Staphylococcus epidermidis strain (ATCC 12228). 18 Lactobacillus curvatus LTH1432 in vitro and during Mol Microbiol 49:1577–1593. 19 sausage fermentations. Syst Appl Microbiol 15: 20 129–136. 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 S53 N54 40454 18 187-194 r2.qxd 7/21/07 1:19 PM Page 187

1 2 3 18 4 5 6 Influence of Processing Parameters on 7 8 Cultures Performance 9 10 Louise H. Stahnke and Karsten Tjener 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION concentration are still of utmost importance to keep 22 in control—although for controlling the spoilage 23 During the processing of cured meat products the flora rather than for enhancing the growth of a starter 24 applied starter cultures—and the natural contami- culture. 25 nating flora, not to forget—are affected by an Parameters that affect the activity of starter cul- 26 immense number of changing parameters that tures in the production of cured meat products are 27 invariably will influence their biochemical activity temperature; curing salts (NaCl, nitrate, nitrite, 28 and subsequently be of importance for the quality of ascorbate); carbohydrates; and, to a lesser extent, 29 the final product. Thus, in order to produce cured microbial contamination and initial pH of the raw 30 meat products of high reproducible quality it is materials; spices; and other additives such as phos- 31 essential that intrinsic and extrinsic parameters that phate or binders. For dry sausages, the diameter is 32 may influence the progress of the applied starter cul- also of some importance, and for mold-inoculated 33 tures during the different processing steps be well products, the casing type, smoke, air speed, and air 34 controlled. humidity (Roncales et al. 1991; Incze 1992; 35 As touched upon in previous chapters, the primary Dossmann et al. 1996). 36 objective of using starter cultures is to ensure repro- 37 ducible and correct microbial growth to avoid faulty 38 productions caused by contaminating spoilage bac- TEMPERATURE 39 teria, pathogens, or other undesired microorganisms. 40 In particular, it is essential to ensure that the fermen- Fermentation and ripening temperature is a factor of 41 tation step, and thereby the growth of the added most importance for all genera of starter cultures 42 lactic acid bacteria, progresses correctly, because the applied to cured meat products, because the meta- 43 acidification process directly and indirectly influences bolic pathways and regulation systems within the 44 the texture, color, and flavor formation as well as the microbial cells are highly temperature-dependent 45 drying-out (Bacus 1984; Lücke and Hechelmann due to temperature’s strong influence on the involved 46 1987). enzymes. Bacteria grow within a temperature range 47 In the production of fermented sausages, particu- whose average is approximately 30°C, with each 48 lar mince ingredients and temperature could vary species having a well-defined upper and lower limit 49 quite a lot depending on the actual recipe and pro- (Nester et al. 1983). However, since the optimum 50 cessing technology, and this will influence the cul- temperature for growth of a species is also close to 51 ture growth. Processing parameters do not vary quite the upper limit of its growth, meat fermentation 52 as much within different recipes for cured whole always takes place far below or just below the opti- 53 muscles, but factors such as temperature and salt mum temperature for the applied lactic acid bacteria. S54 N55 187 40454 18 187-194 r2.qxd 7/21/07 1:19 PM Page 188

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1 LACTIC ACID BACTERIA STAPHYLOCOCCUS AND KOCURIA 2 Table 18.1 shows the recommended fermentation The optimum growth temperature for Staphylococ- 3 temperatures for different species of lactic acid cus and Kocuria strains for meat fermentation is 4 bacteria from various culture manufacturers. Apart around 21–32°C (Texel 1998; Chr. Hansen 2003), 5 from Pediococcus acidilactici being best suited for which is well suited for most fermentation steps dur- 6 very high fermentation temperature, such as those ing which the nitrate reductase activity must be 7 applied in the U.S., there is no general trend showing optimal for color formation. However, in sausages 8 that specific species are more suited than others at applied with an acidifying culture, such high tem- 9 European fermentation temperatures. It is strain- peratures could lower pH too fast, depending on the 10 dependent rather than species-dependent and relies acidifying strain and the sugar content, having a 11 on the actual technology applied. However, it has detrimental effect on the nitrate reductase activity 12 been shown that sausages made with Lactobacillus (color formation) and the flavor-forming activity of 13 are able to reach a lower pH than sausages fermented the Staphylococcus/Kocuria. Both Staphylococcus 14 with Pediococcus (Landvogt and Fischer 1991b; and Kocuria are very sensitive to pH lowering 15 Chr. Hansen 2006). (Sørensen and Jakobsen 1996; Guo et al. 2000; 16 For lactic acid bacteria, an increase in temperature Søndergaard and Stahnke 2002), and even in 17 below the optimum temperature may increase the sausages fermented at a low speed Staphylococcus 18 fermentation rate greatly. It has been found that a often begin to die after a few days in the process 19 5°C increase in temperature approximately doubles (Johansson et al. 1994; Tjener et al. 2003, 2004a). 20 the rate of acid formation (Baumgartner et al. 1980). Undoubtedly also the enzymatic activities of the 21 Temperature affects the lag phase as well as the cells are influenced negatively because the optimum 22 actual speed of acidification during the exponential pH of a cell represents the best average for the func- 23 growth phase (Landvogt and Fischer 1991 a,b; Chr. tion of all its cellular enzymes (Nester et al. 1983). 24 Hansen 2006). With respect to flavor formation by Staphylococcus 25 Figure 18.1 shows typical curves of the it has been shown that temperature and, thus, pH- 26 temperature-related pH reduction caused by a com- lowering both affect the level of important flavor 27 mercial strain of Lb. sakei. Similar temperature compounds (Tjener et al. 2004b). 28 dependencies have been shown as actual growth 29 curves, acidification curves, or fitted mathematical 30 YEAST AND MOLD models for other starter cultures in broth or in sausage 31 fermentations (Baumgartner et al. 1980; Blickstad The optimum temperature of Debaryomyces and 32 and Molin 1981; Landvogt and Fischer 1991a,b; Penicillium strains used in meat processing is around 33 Raccach 1992; Huang and Lin 1993; Wijtzes et al. 18–25°C in a wide pH range (Texel 1998; Chr. 34 1995; Balduino et al. 1999; Leroy and De Vuyst Hansen 2003), but the species will grow well down 35 1999; Messens et al. 2003; Leroy et al. 2005). to 8–15°C, which is most often applied during the 36 ripening process for yeast- and mold-inoculated 37 sausages (Texel 1998; Masoud and Jakobsen 2005). 38 However, to ensure a fast onset of Penicillium 39 growth on the sausage surface, both temperature and 40 Table 18.1. Recommended fermentation temperatures of typical commercial lactic acid bacteria for humidity must be high at the very beginning: above 41 meat fermentation. 20°C and around 90% relative humidity, respec- 42 tively. If water activity in sausage surface drops 43 Recommended below 0.80, neither germination nor growth will take 44 Fermentation place (Sunesen and Stahnke 2003). 45 Genera Species Temperature (°C) 46 47 Lactobacillus sakei 21–32 CURING SALTS 48 plantarum 25–35 NACL 49 farciminis 22–32 50 curvatus 22–37 The primary curing agents include common salt, 51 Pediococcus pentosaceus 20–37 sodium nitrite, and potassium nitrate, as well as 52 acidilactici 25–45 sodium ascorbate or sodium erythrobate (Lücke 53S 1998; Adams 1986). Usually 2.0–3.5% common salt 54N Source: Texel (1998); Quest (1999); Chr. Hansen (2003). (NaCl) is added to the sausage mince, giving an 40454 18 187-194 r2.qxd 7/21/07 1:19 PM Page 189

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initial water activity around 0.97–0.96 (salt-in-water bacteriocin-positive lactic acid bacteria is lowered 1 of 5–7%) depending on the amount of fat or added by increased salt concentration (Leroy and De Vuyst 2 binders. The lowered water activity inhibits growth 1999). Among homofermentative lactobacilli, 3 of the spoilage flora normally associated with chilled strains of Lb. curvatus seem more sensitive to NaCl 4 meats, though undesired microorganisms such as than Lb. sakei (Korkeala et al. 1992). 5 Staphylococcus aureus, Listeria monocytogenes, The most commonly applied yeast in the meat 6 Salmonella, and E. coli O157:H7 are still able to sur- industry, Debaryomyces hansenii, is quite salt- 7 vive unless other hurdles are applied (Lücke 1998; tolerant but species differences do exist. Optimum is 8 Adams 1986; Roca and Incze 1990; Leistner 1991). normally around 0–4% NaCl, with a decline at 9 Just as for the contaminating flora, the activity of higher salt levels (Prista et al. 1997; Sørensen and 10 the applied starter cultures is affected by the concen- Jakobsen 1997; Texel 1998; Olesen and Stahnke 11 tration of NaCl. The inhibition brought about 2000; Chr. Hansen 2003). Molds are very salt- 12 depends on the involved species and their sensitivity tolerant and the most typical used strains of Penicil- 13 to the water activity (Landvogt and Fischer 1991a). lium nalgiovense will grow at water activities as low 14 In general, decreasing the water activity of a sausage as 0.85, though optimum growth occur at approxi- 15 mince, either by adding salt or decreasing the water mately 0.90 or above (Lopez Diaz et al. 2002; see 16 content by drying, results in a continues decline in also Chapter 16, “Starter Cultures: Molds,” in this 17 growth for most bacterial meat cultures (Landvogt volume). Table 18.2 shows the maximum recom- 18 and Fischer 1991a; Søndergaard and Stahnke 2002; mended salt concentration for typical starter cul- 19 Leroy and De Vuyst 2005), though some staphylo- tures; species differences are not outlined. Most 20 cocci seem to have maximum growth within cultures do have some activity at higher salt levels, 21 3.5–10% salt-in-water depending on strain but their function is seriously hampered. 22 (McMeekin et al. 1987; Hammes et al. 1995). In par- 23 ticular, the lag phase is prolonged by increased salt 24 NITRITE AND NITRATE concentration, whereas the growth rate in the expo- 25 nential phase is much less affected (Leroy et al. Nitrite alone or together with nitrate is added to the 26 2005). Also, the specific bacteriocin production of sausage mince in order to produce the characteristic 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 Figure 18.1. Influence of fermentation temperature on pH lowering by a commercial Lb. sakei strain growing in S53 sausage mince, added 0.5% glucose (from Chr. Hansen 2003). N54 40454 18 187-194 r2.qxd 7/21/07 1:19 PM Page 190

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1 Table 18.2. Maximum recommended salt concen- Table 18.3. Lactic acid production by the meat 2 trations for typical meat starter cultures. starter culture Lb. pentosus during growth in MRS- 3 broth at 30¡C for 12 hours. Maximum Recommended 4 Genera NaCl-concentration (%) Carbohydrate (1%) Lactic Acid (%) 5 6 Lactobacillus 9–13 Glucose 0.98 7 Pediococcus 7–10 Saccharose 0.86 8 Staphylococcus 15–16 Maltose 0.72 9 Kocuria No data available Maltodextrin 0.54 10 Debaryomyces 16 Galactose 0.31 11 Penicillium 18–20 12 Source: Chr. Hansen (2003). 13 Source: Texel (1998); Chr. Hansen (2003). 14 15 cured color and to inhibit the growth of undesired mince as fermentation substrates for the lactic acid 16 bacteria. Nitrite together with salt promotes the bacteria because the natural content of glucose is too 17 establishment of Lactobacillus and Micrococcaceae/ low and variable to be reliable in modern sausage 18 Staphylococcaceae by inhibiting species of Enter- production (Lücke 1998). The sugar concentration 19 obacteriaceae and other bacteria (Lücke 1998; and the type of sugar are directly related to the 20 Vösgen 1992). If present in large amounts, nitrite lowest pH achieved and the rate of the pH drop, 21 could inhibit growth of lactic acid bacteria, in partic- respectively. 22 ular at low pH, but this is not the case with the Glucose is fast utilized by all lactic acid bacteria, 23 amounts regularly used (Blickstad and Molin 1981; but other sugars, such as saccharose, lactose or malt- 24 Korkeala et al. 1992; Dossmann et al. 1996; Leroy ose, are less easily fermented (Klettner and List 25 and De Vuyst 1999; Leroy et al. 2005). If inhibition 1980; Bacus 1984; Liepe et al. 1990; Huang and Lin 26 takes place, nitrite seems to affect the lag phase rather 1994) or not fermentable at all by some strains (Chr. 27 than the exponential growth rate (Blickstad and Hansen 2003). The enzymes involved in the gly- 28 Molin 1981). Staphylococcus and Kocuria are not colytic pathway that degrades glucose into lactic 29 affected by the nitrite levels used in meat fermenta- acid are constitutive, whereas proteins for trans- 30 tion (Guo et al. 2000; González and Díez 2002). portation and degradation of sugars such as lactose 31 In the levels used for meat fermentation, nitrate or maltose are inducible, i.e., they are synthesized 32 does not influence the growth of lactic acid bacteria; only when those sugars are present (Nester et al. 33 however, Staphylococcus and Kocuria may exploit 1983). However, these differences make it possible 34 nitrate as an electron acceptor for respiratory growth to control the profile of the pH drop. A mixture of 35 at low redox potential, in this way retaining their fast and slowly fermentable carbohydrates can 36 metabolic activity during sausage processing (Metz ensure a rapid, but relatively small, pH decrease at 37 and Hammes 1987; Neubauer and Götz 1996). the beginning of the fermentation cycle—in order to 38 Nitrate has a significant effect on the free amino acid inhibit unwanted bacteria without suppressing the 39 profile and the aroma compound development of dry staphylococci—followed by a slower fermentation 40 sausages through its influence on staphylococcal step to achieve the final pH. Table 18.3 shows the 41 metabolism (Waade and Stahnke 1997; Olesen et al. amount of lactic acid produced from different sugars 42 2004). within a certain time span. 43 Neither nitrite nor nitrate seems to have a signifi- 44 cant effect on growth of yeast and Penicillium SUGAR CONTENT 45 species used as starter culture, but literature on the 46 subject is very scarce (Lopez Diaz et al. 2002; Dura Depending on the type, up to 2% carbohydrate is 47 et al. 2004). added to the sausage mince (Lücke 1998), but usually 48 0.3–0.8% glucose or saccharose proves sufficient 49 (Pyrcz and Pezacki 1981; Leistner 1991). Some tra- 50 CARBOHYDRATES ditional sausages ripened for a very long time with 51 very low water activities have no added sugar at all SUGAR TYPE 52 (Lois et al. 1987; Incze 1992). However, these days, 53S Various sugars, such as glucose, sucrose, lactose, industrial-produced sausages typically demand pH 54N corn syrups, etc., are commonly added to sausage drops that make it essential to optimize the extent of 40454 18 187-194 r2.qxd 7/21/07 1:19 PM Page 191

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Figure 18.2. Influence of glucose concentration on pH lowering by a commercial P.pentosaceus strain growing 26 in sausage mince at 24¡C (from Chr. Hansen 2003). 27 28 29 acidification with the exact right amount of sugar. Staphylococci are also able to degrade fer- 30 For Mediterranean sausages, pH is typically lowered mentable sugars into lactic acid, though part of the 31 to 5.0–5.1, whereas for North European- and U.S.- lactic acid is converted to acetic acid at high redox 32 style sausages, pH is commonly reduced to around potential (Arkoudelos and Nychas 1995). However, 33 4.8, though specific sausage types reach even lower the amount of acid produced by staphylococci is 34 values. Examples are some Swedish and Danish fer- probably insignificant because meat fermentation 35 mented sausages with final pH of 3.9–4.2. conditions in general are less favorable for staphylo- 36 When glucose concentration is above a certain coccal growth. No studies seem to have looked into 37 level, increased amounts of fermentable sugar this. It is possible, though, that sugar fermentation 38 primarily affect the final pH but neither the lag phase could matter for staphylococcal metabolism in tradi- 39 nor the rate of lactic acid formation (see Figure 18.2). tionally fermented sausages with high pH and 40 Landvogt and Fischer (1991b) found the critical high amounts of residual sugars, such as certain 41 level to be 0.15% glucose in their study. However, Mediterranean-style sausages to which are added 42 the extent of the pH drop also depends on the relatively high concentrations of lactose or skim 43 specific lactic acid bacteria and the water activity milk powder. In basic investigations using broth and 44 of the drying sausage. In general, for the same meat models, glucose seemed to have no, or even a 45 amount of fermentable sugar, pH will drop to a lower slight negative effect on staphylococcal growth, but 46 value in a fast fermentation than in a slow fermen- probably this was due to the lack of buffering capacity 47 tation using the same strain (Chr. Hansen 2003). enabling the staphylococci to produce acid and reduce 48 The less-pronounced pH drop in the slower fermen- their own growth (Sørensen and Jakobsen 1996; 49 tation is due to sugar being consumed by competing Stahnke 1999; Søndergaard and Stahnke 2002). 50 background flora producing other metabolic prod- To the authors’ knowledge, the direct effect of 51 ucts than lactic acid or due to inhibition of the cul- sugars on the growth of yeasts and molds in sausages 52 ture growth by the lowering in water activity over has not been studied. Yeasts and molds grow slowly S53 time. as compared to bacteria in fermented meat products, N54 40454 18 187-194 r2.qxd 7/21/07 1:19 PM Page 192

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1 and typically they develop late in the process where be limiting if higher amounts of acid must be 2 most sugar has been converted to acids unless very produced to reach the wanted pH. Meat pH is influ- 3 large amounts were added. However, yeasts and enced by factors such as animal type and breed, 4 molds assimilate the produced lactic acid, in this way slaughtering technology, and degree of spoilage 5 maintaining their metabolism and increasing the pH (Lawrie 1998). 6 of the product. The lowering in pH that takes place Sausage casing diameter has an effect on the 7 during sausage fermentation is insignificant for their growth of lactic acid bacteria because increasing 8 growth (Sørensen and Jakobsen 1997; Lopez Diaz diameter decreases the availability of oxygen for the 9 et al. 2002; Masoud and Jakobsen 2005). microorganisms inside the sausage. With increasing 10 diameter, lactic acid production increases due to less 11 SPICES oxidative metabolism by the microorganisms 12 involved (Incze 1992). Casing type is important for 13 A number of different spices are used for fermented molded sausages because mycelium growth is sig- 14 meat products, the most typical being black and nificantly better on natural casings as compared to 15 white pepper. Apart from the obvious function as fla- cellulose fiber and collagen casings (Roncales et al. 16 vorings, some spices have been found to stimulate 1991; Andersen 1994; Toledo et al. 1997). 17 the growth of lactic acid bacteria and thereby the rate 18 of lactic acid formation (Nes and Skjelkvåle 1982; REFERENCES 19 Zaika and Kissinger 1984). Black pepper, white pep- 20 per, mustard, garlic, allspice, nutmeg, ginger, corian- MR Adams. 1986. Fermented flesh foods. In: MR 21 der, clove, mace, cinnamon, cardamom, and red Adams, ed. Progress in Industrial Microbiology, 22 pepper all stimulate acid production to varying vol 23. Amsterdam: Elsevier, pp. 159–198. 23 degrees depending on the concentration and the bac- SJ Andersen. 1994. Anti-microbial characteristics of 24 terial strain (Nes and Skjelkvåle 1982; Zaika and white mold cultures for fermentation of sausages. 25 Kissinger 1984; Adams 1986). The content of man- PhD thesis. Lyngby: Technical University of 26 ganese ions in the spices is the primary factor (Zaika Denmark, pp. 1–165. 27 and Kissinger 1984; Hagen et al. 2000). In addition, JS Arkoudelos, GJE Nychas. 1995. Comparative 28 spices such as paprika contain amounts of fer- studies of the growth of Staphylococcus carnosus 29 mentable sugars so high that the final pH drop may with or without glucose. Letters Appl Microbiol 30 be significantly altered in sausages like the Spanish 20:19–24. 31 Chorizo (Lois et al. 1987; Incze 1992). J Bacus. 1984. Utilization of Microorganisms in Meat 32 Processing. A Handbook for Meat Plant Operators. 33 OTHER PARAMETERS Letchworth, England: Research Studies Press Ltd, 34 pp. 1–170. 35 In order to obtain a safe dominance of the starter cul- R Balduino, AS de Oliveira, MC de O Hauly. 1999. 36 ture the size of inoculum must be 106–107 CFU/g so Influence of carbon source and temperature on the 37 that the level of background flora is at least 2 log lactic acid fermentation developed by starter cul- 38 units lower than the added culture (Bacus 1984; tures. English Summary. Ciencia Tecnol Alim 39 Lücke 1998; Hammes et al. 1990). The performance 19:363–366. 40 of the starter lactic acid bacteria may be seriously PA Baumgartner, PG Klettner, W Rödel. 1980. The 41 affected if a strongly competitive population of influence of temperature on some parameters for dry 42 another lactic acid bacteria is present in the raw sausage during ripening. Meat Sci 4:191–201. 43 materials. This is, for example, the case if vacuum- E Blickstad, G Molin. 1981. Growth and lactic acid pro- 44 packed, cold-stored, nonfrozen meat is used. An duction of Pediococcus pentosaceus at different gas 45 increased level of inoculum brings about a shorter environments, temperatures, pH values and nitrite 46 lag phase and thereby a shorter acidification time. concentrations. Europ J Appl Microbiol Biotechnol 47 However, size of inoculum does not affect the final 13:170–174. 48 pH unless water activity decreases very fast and Chr. Hansen. 2003. Bactoferm™ Meat Manual, vol. 1. 49 inhibits the starter growth too soon (Landvogt and Production of Fermented Sausages with Chr. Hansen 50 Fischer 1991b). Starter Cultures. 1st ed. Hørsholm, Denmark: Chr. 51 Initial pH of the meat could influence the fermen- Hansen A/S, pp. 1–31. 52 tation time because high pH means higher buffering ———. 2006. Brief Characterization of Bactoferm® 53S capacity and an extension of time to reach final pH Starter Cultures from Chr. Hansen. Bulletin no. 3. 54N (Incze 1992). In addition, the amount of sugar could vs1. Hørsholm, Denmark: Chr. Hansen A/S, pp. 1–20. 40454 18 187-194 r2.qxd 7/21/07 1:19 PM Page 193

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MU Dossmann, RF Vogel, WP Hammes. 1996. Mathe- RA Lawrie. 1998. Lawrie’s Meat Science, 6th ed. 1 matical description of the growth of Lactobacillus Cambridge, UK: Woodhead Publishing, pp. 1–352. 2 sake and Lactobacillus pentosus under conditions L Leistner. 1991. Stability and safety of raw sausage, 3 prevailing in fermented sausages. Appl Microbiol part I. Fleischerei 42:III. 4 Biotechnol 46:334–339. F Leroy, L De Vuyst. 1999. The presence of salt and 5 M-A Dura, M Flores, F Toldrá. 2004. Effects of curing curing agent reduces bacteriocin production by Lac- 6 agents and the stability of a glutaminase from tobacillus sakei CTC 494, a potential starter culture 7 Debaryomyces spp. Food Chem 86:385–389. for sausage fermentation. Appl Environ Microbiol 8 B González, V Díez. 2002. The effect of nitrite 65:5350–5356. 9 and starter culture on micrological quality of ———. 2005. Simulation of the effect of sausage 10 “chorizo”—A Spanish dry cured sausage. Meat Sci ingredients and technology on the functionality of 11 60:295–298. the bacteriocin-producing Lactobacillus sakei CTC 12 HL Guo, MT Chen, DC Liu. 2000. Biochemical char- 494 strain. Int J Food Microbiol 100:141–152. 13 acteristics of Micrococcus varians and Staphylococ- F Leroy, K Lievens, L De Vuyst. 2005. Modeling bacte- 14 cus xylosus and their growth on Chinese-style beaker riocin resistance and inactivation of Listeria innocua 15 sausages. Asian-Australasian J Anim Sci 13: LMG 13568 by Lactobacilllus sakei CTC 494 under 16 376–380. sausage fermentation conditions. Appl Environ 17 BF Hagen, H Næs, AL Holck. 2000. Meat starters have Microbiol 71:7567–7570. 18 individual requirements for Mn2+. Meat Sci H-U Liepe, E Pfeil, R Porobic. 1990. Influence of sug- 19 55:161–168. ars and bacteria on dry sausage souring. Fleis- 20 WP Hammes, A Bantleon, S Min. 1990. Lactic acid chwirtsch 70:189–192. 21 bacteria in meat fermentation. FEMS Microbiol Rev AL Lois, LM Gutierrez, JM Zumalacarregui, A Lopez. 22 87:165–174. 1987. Changes in several constituents during the 23 WP Hammes, I Bosch, G Wolf. 1995. Contribution of ripening of “Chorizo”—A Spanish dry sausage. 24 Staphylococcus carnosus and Staphylococcus pis- Meat Sci 19:169–177. 25 cifermentans to the fermentation of protein foods. TM Lopez Diaz, CJ Gonzalez, B Moreno, A Otero. 26 J Appl Bacteriol Symp Suppl 79:76S–83S. 2002. Effect of temperature, water activity, pH and 27 C-C Huang, C-W Lin. 1993. Drying temperature and some antimicrobials on the growth of Penicillium 28 time affect quality of Chinese-style sausage inocu- olsonii isolated from the surface of Spanish fer- 29 lated with lactic acid bacteria. J Food Sci mented meat sausages. Food Microbiol 19:1–7. 30 58:249–253. F-K Lücke. 1998. Fermented sausages. In: BJB Wood, 31 ———. 1994. Utilization of sugars in sausage meat by ed. Microbiology of Fermented Foods, vol 2, 2nd ed. 32 lactic acid bacteria. English Summary. J Chinese Soc London: Blackie Academic & Professional, 33 Anim Sci 23:209–220. pp. 441–483. 34 K Incze. 1992. Raw fermented and dried meat products. F-K Lücke, H Hechelmann. 1987. Starter cultures for 35 Fleischwirtsch 72:58–62. dry sausages and raw ham, composition and effect. 36 G Johansson, J-L Berdagué, M Larsson, N Tran, Fleischwirtsch 67:307–314. 37 E Borch. 1994. Lipolysis, proteolysis and formation W Masoud, M Jakobsen. 2005. The combined effects of 38 of volatile components during ripening of a fer- pH, NaCl and temperature on growth of cheese 39 mented sausage with Pediococcus pentosaceus and ripening cultures of Debaryomyces hansenii and 40 Staphylococcus xylosus as starter cultures. Meat Sci coryneform bacteria. Int Dairy J 15:69–77. 41 38:203–218. TA McMeekin, RE Chandler, PE Doe, CD Garland, 42 P-G Klettner, D List. 1980. Beitrag zum einfluss der J Olley, S Putros, DA Ratkowsky. 1987. Model for 43 kohlenhydratart auf den verlauf der rohwurstreifung. combined effect of temperature and salt concentra- 44 Fleischwirtsch 60:1589–1593. tion/water activity on the growth rate of Staphylococ- 45 H Korkeala, T Alanko, T Tiusanen. 1992. Effect of cus xylosus. J Appl Bacteriol 62:543–550. 46 sodium nitrite and sodium chloride on growth of lac- W Messens, J Verluyten, F Leroy, L De Vuyst. 2003. 47 tic acid bacteria. Acta Vet Scand 33:27–32. Modelling growth and bacteriocin production by 48 A Landvogt, A Fischer. 1991a. Dry sausage ripening, Lactobacillus curvatus LTH 1174 in response to tem- 49 targeted control of the acidification achieved by perature and pH values used for European sausage 50 starter cultures. Fleischwirtsch 71:902–905. fermentation processes. Int J Food Microbiol 51 ———. 1991b. Dry sausage ripening, targeted control 81:41–52. 52 of the acidification achieved by starter cultures. Part 2. M Metz, WP Hammes. 1987. Optimum conditions for S53 Fleischwirtsch 71:1055–1056. growth and nitrate reductase activity of Micrococcus N54 40454 18 187-194 r2.qxd 7/21/07 1:19 PM Page 194

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1 varians. In: H Chmiel, WP Hammes, JE Bailey, eds. culture Staphylococcus xylosus. Food Microbiol 2 Biochemical Engineering: A Challenge for Interdis- 13:265–274. 3 ciplinary Cooperation. New York: VCH Publishers ———. 1997. The combined effects of temperature, 4 Inc, pp. 355–360. pH and NaCl on growth of Debaryomyces hansenii 5 IF Nes, R Skjelkvåle. 1982. Effect of natural spices and analyzed by flow cytometry and predictive microbi- 6 oleoresins on Lactobacillus plantarum in the fermen- ology. Int J Food Microbiol 34:209–220. 7 tation of dry sausage. J Food Sci 47:1618–1625. LH Stahnke. 1999. Volatiles produced by Staphylococ- 8 EW Nester, CE Roberts, ME Lidstrom, NN Pearsall, cus xylosus and Staphylococcus carnosus during 9 MT Nester. 1983. Microbiology, 3rd ed. Philadelphia: growth in sausage minces. Part II. The influence of 10 Saunders College Publishing, pp. 1–875. growth parameters. Lebensm-Wiss u-Technol 11 H Neubauer, F Götz. 1996. Physiology and interaction 32:365–371. 12 of nitrate and nitrite reduction in Staphylococcus LO Sunesen, LH Stahnke. 2003. Mould starter cultures 13 carnosus. J Bacteriol 178:2005–2009. for dry sausages—Selection, application and effects. 14 PT Olesen, AS Meyer, LH Stahnke. 2004. Generation Meat Sci 65:935–948. 15 of flavour compounds in fermented sausages—The Texel. 1998. Product information material. Groupe 16 influence of curing ingredients, Staphylococcus Rhône-Poulenc Denmark, February 1998. 17 starter culture and ripening time. Meat Sci K Tjener, LH Stahnke, L Andersen, J Martinussen. 18 66:675–687. 2003. A fermented meat model system for studies of 19 PT Olesen, LH Stahnke. 2000. The influence of microbial aroma formation. Meat Sci 66:211–218. 20 Debaryomyces hansenii and Candida utilis on the ———. 2004a. Growth and production of volatiles by 21 aroma formation in garlic spiced fermented sausages Staphylococcus carnosus in dry sausages: Influence 22 and model minces. Meat Sci 56:357–368. of inoculation level and ripening time. Meat Sci 23 C Prista, A Almagro, MC Loureiro-Dias, J Ramos. 67:447–452. 24 1997. Physiological basis for the high salt tolerance ———. 2004b. The pH-unrelated influence of salt, 25 of Debaryomyces hansenii. Appl Environ Microbiol temperature and manganese on aroma formation by 26 63:4005–4009. Staphylococcus xylosus and Staphylococcus carnosus 27 J Pyrcz, W Pezacki. 1981. Einfluss sauerer gärungspro- in a fermented meat model system. Int J Food Micro- 28 dukte der kohlenhydrate auf die sensorische qualität biol 97:31–42. 29 von rohwurst. Fleischwirtsch 61:446–454. VM Toledo, MD Selgas, MC Casa, JA Ordonez, ML 30 Quest. 1999. Product Information. Quest International, Garcia. 1997. Effect of selected mould strains on 31 The Netherlands, July 1999. proteolysis in dry fermented sausages. Zeitschr 32 M Raccach. 1992. Some aspects of meat fermentation. Lebensm-Untersuch-Forsh A 204:385–390. 33 Food Microbiol 9:55–65. W Vösgen. 1992. Curing. Are nitrite and nitrate 34 M Roca, K Incze. 1990. Fermented sausages. Food Rev superfluous as curing substances? Fleischwirtsch 72: 35 Int 6:91–118. 1675–1678. 36 P Roncales, M Aguilera, JA Beltran, I Jaime, JM Peiro. C Waade, LH Stahnke. 1997. Dried sausages fermented 37 1991. The effect of natural or artificial casing on the with Staphylococcus xylosus at different tempera- 38 ripening and sensory quality of mould-covered dry tures and with different ingredient levels. Part IV. 39 sausage. Int J Food Sci Technol 26:83–89. Amino acid profile. Meat Sci 46:101–114. 40 AK Søndergaard, LH Stahnke. 2002. Growth and aroma T Wijtzes, JC de Wit, JHJ Huis in ’t Veld, K van’t Riet, 41 production by Staphylococcus xylosus, S. carnosus, MH Zwietering. 1995. Modelling bacterial growth of 42 S. equorum—A comparative study in model systems. Lactobacillus curvatus as a function of acidity and 43 Int J Food Microbiol 75:99–109. temperature. Appl Environ Microbiol 61:2533–2539. 44 BB Sørensen, M Jakobsen. 1996. The combined effects LL Zaika, JC Kissinger. 1984. Fermentation enhance- 45 of environmental conditions related to meat fermen- ment by spices: Identification of active component. 46 tation on growth and lipase production by the starter J Food Sci 49:5–9. 47 48 49 50 51 52 53S 54N 40454 19 195-202 r2.qxd 7/20/07 1:40 PM Page 195

1 2 3 4 5 6 7 8 9 10 11 12 13 14 Part IV 15 16 17 18 19 Sensory Attributes 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 S54 N55 40454 19 195-202 r2.qxd 7/20/07 1:40 PM Page 197

1 2 3 19 4 5 6 General Considerations 7 8 Asgeir Nilsen and Marit Rødbotten 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION answer the question being asked about the product. 22 This is also true when sensory parameters are to be 23 Microbiological and nutritious quality are essential measured. Thus, the sensory tests are often classified 24 parameters when the value of food is considered, and according to their primary purpose and most valid 25 so is the sensory quality. Several instrumental tech- use. First, the sensory evaluation specialist has to 26 niques are able to measure important quality decide upon the choice between analytical or affec- 27 attributes in meat products with impressive accu- tive analysis. Because the evaluation is conducted 28 racy, and some of the measurements have a high with human subjects as assessors or instrument, the 29 correlation to sensory measurements. To this day, panel (i.e., the sensory instrument) can be used to 30 however, no instrument can fully replace the human give analytical, objective information about the 31 subject when the purpose is to achieve informa- product, i.e., which sausage is the juiciest, or how 32 tion that is relevant to understand the consumers’ juicy one sausage is compared to other sausages. 33 preferences. This task will demand specific sensory methods and 34 Sensory evaluation has been defined as a scientific also special training of the panelists. A sensory panel 35 method used to evoke, measure, analyze, and inter- may also give affective, subjective information about 36 pret those responses to products as perceived through the meat product, i.e., which meat sample is pre- 37 the senses of sight, smell, touch, taste, and hearing ferred and whether the sample is acceptable. This 38 (Stone and Sidel 1993). Amerine et al. (1965) have task will also demand specific sensory methods, but 39 given an overview of the history of sensory science in this situation the panel should be consumers 40 up to 1965, and Meilgaard et al. (1991), Lawless and untrained in sensory analysis. 41 Klein (1991), Stone and Sidel (1993), and Lawless In product development, the use of sensory analy- 42 and Heymann (1998) have published other recog- sis today is recognized worldwide, both the objective 43 nized sensory textbooks more recently. and the subjective methods. The quality of a product 44 Sensory evaluation is a quantitative science in is often measured by the consistency of the per- 45 which numerical data are collected to establish spe- ceived attributes of the product. The consumer 46 cific relationships between product characteristics expects the product he/she buys to be of the same (or 47 and human perception. Sensory practitioners are close to) sensory quality tomorrow as it was when 48 concerned both with analytical specification of per- bought yesterday. Quality control by means of sen- 49 ceived product differences as well as predicting con- sory analysis conducted by a well-trained panel that 50 sumer acceptance of the product. One major concern is supervised by a well-trained panel leader will 51 of anyone who is responsible for a scientific analysis inform the producer about the degree of perceived 52 is to ensure that the test method is appropriate to variation of the product. The quality factor is of the 53 S54 N55 197 40454 19 195-202 r2.qxd 7/20/07 1:40 PM Page 198

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1 greatest importance to the consumer, and conse- be presented in a paired comparison test (ISO 2 quently of economic value for the producer. 5495:2005), triangular test (ISO 4120:2004), or duo- 3 trio test (ISO 10399:2004). These tests are used for 4 revealing slight differences between samples and 5 SENSORY METHODS can also be used in selecting and training assessors 6 It is important to discriminate between sensory eval- for sensory panels. Difference tests can be inter- 7 uations made by trained assessors in laboratories or preted by using statistical tables where significant 8 production facilities and the preference evaluation levels are easily found. Other methods for difference 9 made by consumers. The Standards from the Inter- testing, such as “2-out-of-five” and “A—not A” 10 national Organization for Standards (ISO) describe tests, exist, but they are not commonly used in the 11 all the difference test and descriptive tests com- industry. 12 monly used. The brief overview of sensory and con- 13 sumer testing in Figure 19.1 shows which sensory Ranking Tests 14 methods are proper for different tasks. Exact infor- Ranking tests (ISO 8587:1988) are used to place 15 mation on how they are carried out is among other samples in order of a defined sensory stimulus, e.g., 16 aspects described in Lawless and Heymann (1998). increasing color intensity or increasingly ripened fla- 17 vor in a fermented meat product. An advantage of 18 this test is that more than two samples can be com- ANALYTICAL METHODS 19 pared to each other in the same test. Friedman’s sta- 20 Analytical sensory evaluations are performed with tistical analysis determines the smallest difference 21 trained assessors that give analytical, objective infor- between samples that are necessary to claim signifi- 22 mation about the product. Regular training regarding cant differences (Friedman 1937). 23 relevant products and maintenance of the assessors 24 are necessary to trust the results from a sensory panel Profile Tests (Descriptive Tests) 25 performing analytical methods. In the following sec- Profile tests are powerful sensory tools used in prod- 26 tions, the major analyses are described further. 27 uct control and development to describe the sensory profile of products. Descriptive analysis is widely 28 Difference Tests 29 used to specify quantitative differences in appear- 30 Difference tests indicate whether a difference can ance, odor, flavor, and texture characteristics of 31 be found between two samples. The samples can products. The “Sensory Spectrum” method (Lawless 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53S Figure 19.1. Overview of methods used in sensory analyses and consumer tests (from A. B. Tecator, Box 70, 54N S-263 01, Höganäs, Sweden, 1985). 40454 19 195-202 r2.qxd 7/20/07 1:40 PM Page 199

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and Heymann 1998), “Quantitative Descriptive however, describe the products in more detail—for 1 Analysis” (Lawless and Heymann 1998), and “Fla- example, with the following objective attributes: 2 vor Profile” method (ISO 6564:1985) are examples 3 • Appearance: whiteness, color hue, color intensity, of other methods used for descriptive testing. 4 shiny, uneven, greasy (Berdagué et al. 1993; Common for all descriptive methods is the use of 5 Helgesen and Næs 1995; Andrés et al. 2004) statistical methods such as analysis of variance 6 • Odor: meat (i.e., pork, goat, poultry), acidic, sour, (ANOVA) (Lea et al. 1997), Principal Component 7 gamy, off-odor, metallic, rancid, moldy, cheesy Analysis (Martens and Martens 2001), and others to 8 (Mielnik et al. 2002; Cosnenza et al. 2003a,b) describe significant differences between different 9 • Taste: salty, sweet, sour, bitter, umami (Rødbotten product profiles. 10 et al. 2004) 11 • Flavor: meat (i.e., pork, goat, poultry), spicy, 12 AFFECTIVE METHODS (PREFERENCE TESTS) gamy, metallic, acidic, moldy, rancid, off-flavor, 13 matured, nutty, cheesy, smoky (Mielnik et al. Affective tests are performed with untrained con- 14 2002, Cosnenza et al. 2003a,b) sumers who give information about preference, 15 • Texture: juicy, dry, hard, fatty, sticky, grainy, acceptance, liking, or appropriateness of products. 16 mellow, firm, fibrous (Stahnke et al. 2002). ISO 6658:2005 gives general guidance on the use of 17 sensory analysis and indicates which tests can be 18 APPEARANCE AND COLOR used for determining preferences. 19 The first impression of a fermented meat product is 20 usually perceived by sight. Depending on the actual 21 TEST ENVIRONMENT product, the size, shape, color, and degree of gloss 22 It is suggested that evaluation of food products give the visual impression of the quality. When, for 23 should be performed in specially designed lab instance, slices of fermented, dry sausages are evalu- 24 booths. Assessors in a sensory panel are then free ated, it is important to consider the particle size, 25 from outside distractions and in a room with con- uniformity of particles, glistening of fat, color, stick- 26 trolled lighting, heating, and ventilation. Detailed iness, and more (Meilgaard et al. 1991). Sensory dif- 27 guidelines for the layout of general sensory laborato- ference tests and descriptive tests have been used in 28 ries have been published in ISO 8589:1988. This both hedonic and analytical evaluation of appear- 29 standard includes suggested design for the testing ance (Berdagué et al. 1993; Helgesen and Næs 1995; 30 room and lighting requirements. General guidance Andrés et al. 2004). 31 for the selection, training, and monitoring of asses- Color can be evaluated by describing degree of 32 sors can be found in ISO 8586-1:1993. whiteness and blackness, hue and color intensity, 33 and it is recommended, whenever possible, to use 34 references to both attributes and samples (Mielnik et 35 SENSORY ANALYSIS OF al. 2002). An example of an attribute reference is the 36 FERMENTED MEAT PRODUCTS Natural Color System (SS 19102:2004), a system 37 based on resemblance of sample color to the six ele- 38 During development of a fermented meat product or mentary colors: white, black, yellow, red, blue, and 39 during research investigating the influence of differ- green. The intensity of the color may vary depending 40 ent parameters on sensory quality of meat products, on the gloss of the product surface. For reliable 41 various sensory analytical methods come in handy. results, the assessors should be instructed to evaluate 42 A preference test, preferably in addition to a sensory every sample placed at the same distance and angle 43 profile test, gives essential information about the from the light source and the same distance from the 44 product attributes of importance to the consumers, eyes of the assessor. 45 whereas a profile test—or in simpler cases, difference One of the challenges when evaluating appear- 46 tests—are valuable to show objective differences dur- ance of fermented meat products is the lack of uni- 47 ing research and development. Thus, a number of formity in the product. A fermented sausage may 48 publications show the use of sensory analysis on fer- vary, in all sensory aspects, from the center to the 49 mented meat products where appearance, flavor, outside part, and a smoked, cured ham is naturally 50 and/or texture are described in various degrees. The very uneven from the inside to the outside and from 51 attributes most often used scientifically are color, the thin to the thick part. When samples are uneven 52 flavor, rancid flavor, salty taste, and texture, in addi- like the described examples, a decision has to be S53 tion to overall acceptance. Some research work does, taken on whether the entire slice of the sausage or N54 40454 19 195-202 r2.qxd 7/20/07 1:40 PM Page 200

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1 ham is going to be evaluated or whether the inner Mielnik et al. 2002). In most situations, a rancid fla- 2 part and the outer part should be evaluated sepa- vor is an undesirable flavor in the product, but in 3 rately. Usually the whole sample is of interest, but many of the smoked and cured ham products, a ran- 4 the producer or the scientist may want to investigate cid flavor is a desired and an important attribute of 5 effects of special processes or treatments on parts of the product. Most consumers would probably not 6 the samples, and thus detailed evaluations have to be describe a matured ham to be rancid even if we know 7 undertaken. that the fat in the product is partly oxidized. A cer- 8 tain degree of rancidity is a part of the flavor profile 9 of cured ham and is consequently a positive attribute ODOR AND FLAVOR 10 when present in the “correct” intensity. 11 Odor and flavor are often highly correlated, but in Meat from some species is more naturally acidic 12 some situations it is observed that a specific flavor is in flavor than other meats (Rødbotten et al. 2004). In 13 present where no corresponding odor can be the fermentation and ripening process of meat prod- 14 detected. The volatile compounds are dependent on ucts, additional flavors add to the sensory profile and 15 moisture to be perceived as odor by the human nose one is a sour flavor. It is important to discriminate 16 (Engen 1982). A newly cut slice of ham or sausage between sour and acidic. The intensity of the two 17 will most presumably be more intense in odor than a attributes can give valuable information about the 18 slice being cut some time ago. It is therefore impor- fermentation process, if it is successful or not. 19 tant when comparing various samples to ensure Boar-taint is a sensory phenomenon well known 20 equal preparation time for all samples before serving in the pig industry. The flavor is very unpleasant and 21 to the panel. During the eating process, flavor is much stronger in intensity in warm products than 22 released from the product and the flavor is often per- cold products. One should be aware of the fact that 23 ceived differently from the beginning to the end of the boar-taint is not recognizable to everyone 24 the masticating process. This is partly explained by (Amoore et al. 1977), so it is important to check 25 the fact that the various flavor compounds in the whether the participants in the sensory panel con- 26 product are released over time due to the mechanical ducting sensory quality control are able to detect this 27 and chemical process in the mouth while chewing, in unpleasant flavor (Lundström and Bonneau 1996). 28 addition to the increased activation of the taste buds 29 in the mouth. Thus it is often difficult to find a high TEXTURE 30 correlation between sensory and instrumental meas- 31 urements of the “same flavor” attribute. No instru- The mouth is a very special instrument not only for 32 ment can fully simulate the process in the mouth. measuring flavor, but also for measuring texture. The 33 Flavor wheels are developed for groups of foods jaw muscles, the molars, and the saliva give a unique 34 and beverages, for example, for wine, cheese, and setting for measuring texture. The jaw muscles are 35 beer. The starting point (the center of the wheel) uses necessary to measure the force required to penetrate 36 few descriptors, i.e., the following for meat descrip- the sample with the molars. The geometric surface of 37 tion: meat flavor, acidic flavor, and rancid flavor. the molars is important for the typical cutting and 38 These words or attributes are not very detailed; each crushing of the product. The saliva adds to the product 39 of them can be described in more detailed words. in the mouth and may change the sensory perception. 40 “Meat flavor” is a description to be used in a general Saliva together with melting fat in the mouth can 41 meaning, and it gives no information about the change the textural sensation of a product from the 42 special species being described. Assessors may be start of eating a product to when it is ready for 43 trained in recognizing the specific flavor of the swallowing. 44 species, i.e., pork, goat, or poultry (Mielnik et al. When evaluating texture properties of fermented 45 2002; Cosnenza et al. 2003a,b), as well as discrimi- meats it is important that each participant of the sen- 46 nating between various species without using the sory panel has the same understanding of the various 47 species specific attribute, i.e., not using the attribute’s texture attributes. Definition of texture attributes are 48 pork flavor or goat flavor, but rather acidic, metal, bit- given in ISO 5492:1992. Several mechanical devices 49 ter, gamy flavor, and more (Rødbotten et al. 2004). exist that can measure texture properties in fer- 50 A sensory panel may also be trained in discrimi- mented meats, based on principles such as shearing, 51 nating between various origins of fat and the sensory biting, compression, and structural measures. The 52 variation between, for example, fresh contra-stored instrumental measurements are often necessary in 53S pork fat, lamb fat, and so on (Buscailhon et al. 1994; practical evaluation of a product and will often show 54N Coutron-Gambatti et al. 1999; Muguerza et al. 2001; a high correlation to the sensory measurements. 40454 19 195-202 r2.qxd 7/20/07 1:40 PM Page 201

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However, a high correlation is not always the case ISO 5495:2005. Sensory analysis—Methodology— 1 and sensory evaluations must be used to develop Paired comparison test. International Organization 2 a product that can obtain customer acceptance. for Standardization, Geneve. 3 Meat consumers rate tenderness as the most ISO 6658:2005. Sensory analysis—Methodology— 4 important factor determining the quality of meat. General guidance. International Organization for 5 For fermented meat products the tenderness is Standardization, Geneve. 6 important but not the most important issue because ISO 6564:1985. Sensory analysis—Methodology— 7 fermented products are known to be tender products Flavour profile methods. International Organization 8 in general. for Standardization, Geneve. 9 ISO 8586-1:1993. Sensory analysis—General guidance 10 for the selection, training and monitoring of 11 REFERENCES assessors—Part 1: Selected assessors. International 12 Organization for Standardization, Geneve. 13 MA Amerine, RM Pangborn, EB Roessler. 1965. Prin- ISO 8587:1988. Sensory analysis—Methodology— 14 ciples of Sensory Evaluation of Food. New York: Ranking. International Organization for Standardiza- 15 Academic Press, pp. 1–2. tion, Geneve. 16 JE Amoore, P Pelosi, LJ Forrester. 1977. Specific anos- ISO 8589:1988. Sensory analysis—General guidance 17 mia to 5-androst-16-ene-3one and pentadecalactone: for the design of test rooms. International Organiza- 18 The urinous and musky primary odors. Chem Senses tion for Standardization, Geneve. 19 Flav 3:401–425. ISO 10399:1991. Sensory analysis—Methodology— 20 AI Andrés, R Cava, J Ventanas, V Thovar, J Ruiz. 2004. Duo—Trio test. International Organization for Stan- 21 Sensory characteristics of Iberian ham: Influence of dardization, Geneve. 22 salt content and processing conditions. Meat Sci HT Lawless, H Heymann. 1998. Sensory Evaluation of 23 68:45–51. Food: Principles and Practices. New York: Chapman 24 JL Berdagué, P Monteil, MC Montel, R Talon. 1993. & Hall, pp. 1–26, pp. 358–362, pp. 351–356. 25 Effect of starter cultures on the formation of flavor HT Lawless, PB Klein. 1991. Sensory Science Theory 26 compounds in dry sausages. Meat Sci 35:275–287. and Applications in Foods. Chicago: Marcel Dekker, 27 S Buscailhon, JL Berdagué, J Bousset, M Cornet, G pp. 1–31. 28 Gandemer, C Touraille, G Monin. 1994. Relations P Lea, T Næs, M Rødbotten. 1997. Analysis of Variance 29 between compositional traits and sensory quality of for Sensory Data. Aas, Norway: John Wiley & Sons 30 French dry-cured ham. Meat Sci 37:229–243. Ltd., pp. 23–27. 31 GH Cosnenza, SK Williams, DD Johnson, C Sims, CH K Lundström, M Bonneau. 1996. Off-flavour in meat 32 McGowan. 2003a. Development and evaluation of a with particular emphasis on boar taint. In: S Taylor, 33 fermented cabrito snack stick product. Meat Sci A Raimundo, M Severini and FJM Smulders, eds. 34 64:51–57. Meat Quality and Meat Packaging. Utrecht, The 35 ———. 2003b. Development and evaluation of a cabrito Netherlands: ECCEAMST, pp. 137–143. 36 smoked sausage product. Meat Sci 64:110–124. H Martens, M Martens. 2001. Multivariate Analysis of 37 C Coutron-Gambatti, G Gandemer, S Rousset, O Quality. An introduction. Chichester, UK: J Wiley & 38 Maestrini, F Casabianca. 1999. Reducing salt content Sons Ltd., pp. 93–110. 39 of dry-cured ham: effect on lipid composition and M Meilgaard, GV Civille, BT Carr. 1991. Sensory 40 sensory attributes. Food Chem 64:13–19. Evaluation Techniques, 2nd ed. Boca Raton, Florida: 41 T Engen. 1982. The Perception of Odors. New York: CRC Press, pp. 1–5. 42 Academic Press Inc, pp. 17–34. MB Mielnik, K Aaby, K Rolfsen, MR Ellekjær, A Nils- 43 M Friedman. 1937. The use of ranks to avoid the son. 2002. Quality of comminuted sausages formu- 44 assumption of normality implicit in the analysis of lated from mechanically deboned poultry meat. Meat 45 variance. J Am Statist Assoc 32:675–701. Sci 61:73–84. 46 H Helgesen, T Næs. 1995. Selection of dry fermented E Muguerza, O Gimeno, D Ansorena, JG Bloukas, I 47 lamb sausages for consumer testing. Food Qual Pref Astiasarán. 2001. Effect of replacing pork back fat 48 6:109–120. with pre-emulsified olive oil on lipid fraction and 49 ISO 4120:2004 Sensory analysis—Methodology— sensory quality of Chorizo de Pamplona—A tradi- 50 Triangle Test. International Organization for Stan- tional Spanish fermented sausage. Meat Sci 59: 51 dardization, Geneve. 251–258. 52 ISO 5492:1992. Sensory analysis—Vocabulary. Inter- Norsk Institutt for Næringsmiddelforskning (NINF) S53 national Organization for Standardization, Geneve. 1985. Norge, Tecator AB, Sverige. N54 40454 19 195-202 r2.qxd 7/20/07 1:40 PM Page 202

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1 M Rødbotten, E Kubberød, P Lea, Ø Ueland. 2004. maturity and ﬂavor compounds. J Food Sci 67: 2 A sensory map of the meat universe. Sensory proﬁle 1914–1921. 3 of meat from 15 species. Meat Sci 68:137–144. H Stone, JL Sidel. 1993. Sensory Evaluation Practices, 4 LH Stahnke, A Holck, A Jensen, A Nilsen, E Zanardi. 2nd ed. San Diego: Academic Press, pp. 1–17. 5 2002. Maturity acceltration of Italian dried sausage SS 19102:2004. NCS Color Atlas. SIS Swedish Stan- 6 by Staphylococcus carnosus—Relationship between dards Institute, Stockholm, Sweden. 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53S 54N 40454 20 203-216 r2.qxd 7/20/07 1:42 PM Page 203

1 2 3 20 4 5 6 Color 7 8 Jens K. S. Møller and Leif H. Skibsted 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION of parameters that have been found to have an effect 22 on color formation and stability in fermented sausage 23 The manufacturing of fermented meat products is shown in Figure 20.1. Here, additional postpro- 24 relies on centuries of tradition and transference of cessing aspects are also considered and will be fur- 25 knowledge over generations. However, these types ther dealt with in later sections. 26 of meat products also represent a serendipitous uti- 27 lization of biotechnological principles and specific 28 processing conditions in order to obtain unique COLOR-FORMING COMPOUNDS 29 attributes of the final product in relation to texture, 30 flavor, and color. During the fermentation or drying The amount of nitrate/nitrite added in the curing 31 process numerous chemical reactions take place process will primarily affect the initial color inten- 32 including enzymatic and bacterial processes, and sity, as an increasing amount of NO sources will 33 II this section is dedicated to the pathways involved in increase the yield of nitrosylmyoglobin, MbFe NO, 34 color formation in fermented meat products. The eventually resulting in higher intensities of reddish 35 color formation and color stability of sausages, or pink color (Froehlich et al. 1983). In relation to 36 hams, and related meat products are, however, color development of fermented meat products, 37 closely related to hydrolytic and specifically oxida- additional aspects have to be considered in relation 38 tive processes in the meat matrix. to activities of specific bacterial cultures each pres- 39 In raw, cured meat products to which are added ent in very high numbers. 40 either nitrite or nitrate, as is common practice for For the meat processing industry, alternative 41 many bulk products, the nitrosylated form of myo- methods to obtain stable color of meat products with- 42 globin (Mb) primarily determines the color of the out the use of nitrite are of continuing interest, also in 43 final product; in heat-treated products it is a dena- relation to the growing markets for organically pro- 44 tured derivative (Killday et al. 1988). The stability of duced foods. Non-nitrite curing can be achieved by 45 the NO complex of Mb is quite poor, especially when various strategies (Shahidi and Pegg 1995), including 46 exposed to light in the presence of air. Therefore, a color formation by adding specific microorganisms 47 number of systematic investigations have been per- or by formation of alternative chromophores in the 48 formed on several interacting factors during produc- meat matrix. The latter can be achieved either by 49 tion, packaging, storage, and display of nitrite-cured addition of specific compounds to the product or 50 meats in order to identify factors important for qual- even more interestingly through induction of natural 51 ity deterioration. These studies have resulted in for- endogenous chemical transformations. The chemical 52 mulation of guidelines for meat producers, packers, identification of Mb derivatives in certain dry-cured 53 and retailers in order to ensure optimal production products have presented a challenge for the food S54 quality and increased shelf life. A schematic outline chemist and is also of specific interest to the industry, N55 203 40454 20 203-216 r2.qxd 7/20/07 1:43 PM Page 204

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Figure 20.1. Schematic outline of product/ingredient flow and critical process parameters in different steps of 27 fermented sausage manufacturing. OTR is the abbreviation for the oxygen transmission rate of the given pack- 28 aging material. 29 30 31 because a deep red color is formed in the meat during presence of reductants in the meat, where substances 32 the maturation process without adding nitrate or such as thiol-groups in peptides and enzymatic cofac- 33 nitrite (Parolari 1996). tors may reduce added nitrite. In fermented products, 34 the initial reduction of added nitrate is influenced by the choice of starter culture (Krotje 1992), as will be 35 CHEMISTRY OF MEAT COLOR 36 discussed in more detail later. Addition of reductants, 37 Nitrate and nitrite have been used for centuries for cur- especially ascorbate or erythorbate, in excess during 38 ing and preserving meat and fish, and in some types of the curing process facilitate the color formation. The 39 cheeses (Cammack et al. 1999). For cured, fermented reaction steps involving myoglobins and the curing 40 sausages, in which nitrate and/or nitrite are added dur- agents nitrite and nitrate, which is often referred to 41 ing the curing process, flavor, appearance, and oxida- as the NO generating pool, are summarized in 42 tive stability are directly related to the conversion of Figure 20.2. 43 the curing agent nitrite to NO and the following reac- The final products often contain salt in the con- 44 tion with meat pigments, forming MbFeIINO. Nitrate centration range of 2–5%. Of the initial nitrite addi- 45 is converted to nitrite by endogenous or added reduction, typically ranging between 60–150 ppm, only a 46 tants prior to formation of the nitrosation agents, small fraction of the nitrite can be detected even 47 which transfer the NO moiety to Mb or other proteins. shortly after production due to the numerous reac- 48 MbFeIINO has a bright red color in its native form tions in which nitrite participate, as also illustrated 49 and is pinker when the protein moiety is denatured by in Figure 20.2 (Cassens et al. 1979). 50 mild heat treatment yielding nitrosylmyochrome, 51 dMbFeIINO. However, both forms are very labile FORMATION AND REACTIVITY OF NO 52 toward degradation, which causes formation of met- 53S myoglobin, MbFeIII, giving the meat surface a dull The high reactivity of added nitrite in the meat batter 54N grayish color. The color formation is affected by the will result in an initial discoloration due to oxidation 40454 20 203-216 r2.qxd 7/20/07 1:43 PM Page 205

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Figure 20.2. The NO generating pool in nitrite-cured meat can participate in numerous reactions with various 25 meat constituents yielding modified proteins, altered pigments, and other nitrosyl products. 26 27 28 II II of oxymyoglobin (MbFe O2) and deoxymyoglobin mechanism behind MbFe NO formation from nitrite 29 (MbFeII), present in the raw meat, to the brown met- and MbFeIII has been suggested, as shown in Figure 30 myoglobin, MbFeIII as indicated in Equations 20.1 20.3 (Bonnett et al. 1980). The coordination of NO 31 and 20.2 (Möhler 1974; Killday et al. 1988). These to Fe(II) in Mb involves a change from high spin 32 reactions occur rapidly after meat batter mixing and (h.s.) to low spin (l.s.) and an increase in coordina- 33 addition of the additives, and the discoloration per- tion number of Fe(II), which may be monitored by 34 sists for hours. Subsequently, and under the influ- ESR spectroscopy: 35 ence of added reducing agents, both MbFeIII and the Heme-FeII(h.s.) ϩ NO(g)(Sϭ1/ ) → 36 formed nitrate will be reduced into species that are 2 —Heme-FeII(i.s.)ϪNO (Sϭ1/ ) (20.3) 37 active in the color formation. 2 38 A number of comprehensive reviews based on MbFeII ϩ 2Hϩ ϩ NOϪ → 39 2 intensive research during several decades can be con- —MbFeIII ϩ NO(g) ϩ H O (20.1) 40 2 sulted for further details concerning various practical 41 and theoretical aspects of meat curing, including the 4MbFeIIO ϩ 4NOϪϩ 4Hϩ → 42 2 2 structure of pigments in cured meat (Tarladgis 1962; —4MbFeIII ϩ 4NOϪϩ O ϩ 2H O (20.2) 43 3 2 2 Bonnett et al. 1980; Pegg and Shahidi 1997), chemi- 44 The complex chemistry occurring during nitrite cal reactions of nitrite and chloride under various 45 curing of meat has been studied extensively over the pH-conditions (Sebranek and Fox 1985), interaction 46 years, and recently reviewed (Møller and Skibsted of nitrite and ascorbic acid leading to nitrosylating 47 2002), and will not be discussed in further detail. agents, and oxidative processes in cured meats affect- 48 Nitrosylated heme proteins were early identified as ing pigments and lipids (Cassens et al. 1979; Skibsted 49 the primary pigment in nitrite-cured meats, and the 1992; Cornforth 1996). The physicochemical features 50 exact chemical structure as a mononitrosyl complex of both nitrite-cured meat pigment and pigment in 51 was fully elucidated by the use of Electron Spin Res- dry-cured meat products (Sakata 2000) have also 52 onance (ESR) spectroscopy (Bonnett et al. 1980; been discussed in detail, together with alternatives to S53 Pegg et al. 1996). From kinetic studies, a reaction nitrite in meat (Shahidi and Pegg 1995). N54 40454 20 203-216 r2.qxd 7/20/07 1:43 PM Page 206

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1 2 3 4 5 6 7 8 9 10 11 Figure 20.3. Reaction steps involved in the formation of nitrosylmyoglobin in nitrite-cured meat, including a 12 cationic protein radical intermediate (modified from Bonnett et al. 1980). 13 14 15 FORMATION OF COLOR WITHOUT NO light absorption of the isolated pigment in com- 16 parison to the absorption spectra of more well- Dry-cured ham is a traditional and important meat 17 characterized Mb derivatives. However, recently a product in many countries, but it is crucial to distin- 18 Japanese group reported that the main chromophore guish between dried ham variants to which only salt 19 in Parma ham is a complex of Zn and protoporphyrin is added, such as Parma ham and most variants of 20 IX (Wakamatsu et al. 2004). Subsequently, this Zn Iberian ham, and other dry-cured hams, such as the 21 complex has been detected in other meat products, Spanish Serrano ham to which are added either 22 but notably addition of nitrite apparently seems to nitrate or nitrite together with salt. In dry-cured hams 23 completely inhibit the formation of zinc proto- with added nitrate or nitrite, the complex MbFeIINO 24 porhyrin IX (Adamsen et al. 2005), possibly through will be formed (Perez-Alvarez et al. 1999), which, 25 an increased stabilization of the iron binding to por- however, seems to have a slightly different structure 26 phyrin in heme. Nitrite has previously been sug- than the native complex found in brine-cured bacon 27 gested to bind at the entrance to the heme cavity or the denatured form dMbFeIINO present in cooked 28 protecting Mb against degradation by heat and oxida- ham. Such subtle structural differences are indicated 29 tive processes (Arendt et al. 1997). 30 in the ESR spectra (Figure 20.4), which have the 31 same hyperfine splitting pattern due to the paramag- 32 netic ligand NO (Møller et al. 2003a), but which also INFLUENCE OF FERMENTATION PARAMETERS ON 33 show varying contribution from the protein moiety COLOR 34 as observed from other spectral features, e.g., the 35 anisotropic characteristics. Notably by comparing Optimal color formation in fermented sausages 36 ESR spectra of raw and heat-treated bacon, similar depends strongly on the actual fermentation and 37 spectral observations indicate the same type of differ- resulting pH decrease and oxygen depletion. For one 38 ences in protein association to the chromophore product an initial conversion of 70% of the total pig- 39 (Bonnett et al. 1980). ment into NO derivatives in the meat mince was 40 Nitrate and nitrite have been banned from the man- shown to increase to a 90% conversion in the final 41 ufacturing of Parma ham since 1993 (Parolari 1996), product following fermentation (Chasco et al. 1996). 42 and the unique color formation and stability observed Fermented sausages added both nitrate and nitrite 43 for Parma ham is intriguing because the stable color together with erythorbate were found to have 44 seems to be formed without the action of nitrate/ approx. 80% pigment conversion into MbFeIINO 45 nitrite. Analysis for nitrate and nitrite content in the after 8 days of dehydration when the initial fermen- 46 sodium chloride of marine origin used for process- tation period applied was between 12–21 hours and 47 ing of Parma ham showed a negligible and insuffi- with final pH around 5.2. A shorter fermentation and 48 cient amount to account for the uniform color a higher final pH provided a lower degree of pigment 49 observed in the final product. Only a few studies are conversion (Acton and Dick 1977). The nitrite and 50 available regarding the nature of the Mb derivatives nitrate have been shown to convert to each other 51 formed in Parma ham, and spectral changes have dynamically during dry-fermented sausage produc- 52 been reported to occur gradually during processing tion (Stahnke 1995), and nitrate concentration has a 53S (Parolari et al. 2003; Møller et al. 2003a), and some tendency to decrease while nitrite concentration 54N unique features were observed regarding a red-shifted increases as higher fermentation temperatures and 40454 20 203-216 r2.qxd 7/20/07 1:43 PM Page 207

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 Figure 20.4. Electron spin resonance (ESR) spectra of hypervalente perferrylmyoglobin, two forms of nitrosy- 40 lated myoglobin and the metal center in the major Parma ham chromophore, Zn porphyrin complex, which is 41 ESR-silent. The detailed chemical structure of various types of metal coordination spheres responsible for the observed ESR spectra are shown in conjunction with the actual spectrum. 42 43 44 45 higher sugar levels are applied in combination with whereas nitrate did not react in this type of product, 46 lower salt concentration. Bacterial activities clearly possibly due to lack of active micrococci as seen as a 47 affect the distribution between nitrate and nitrite and poor color formation (Alley et al. 1992). Another 48 also the dynamics of interconversion and in residual study in which color formation in fermented and 49 nitrite. Application of backslopping and varying nonfermented sausages was compared showed a 50 levels of nitrite and nitrate with or without ascorbate higher degree of pigment conversion into MbFeIINO 51 addition for fermented sausage production led to for the fermented product (Demasi et al. 1989). For 52 the conclusion that optimal color formation was fermented sausages, the conversion further depends S53 obtained when nitrite was combined with ascorbate, on pH because a more acidic product was found to N54 40454 20 203-216 r2.qxd 7/20/07 1:43 PM Page 208

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1 result in more efficient nitrite utilization. In the from Micrococcus halodenitrificans is an iron protein 2 processing of Turkish-type fermented sausages that containing molybdenum (Equation 20.5); the 3 included two fermentations and two ripening stages, Pseudomonas enzyme is a cytochrome enzyme 4 the optimal color was present already after the first (Equation 20.6). The NADPH dependent variant of 5 ripening; no color changes occurred during the sec- nitrate reductase (EC 1.7.1.2) further has FAD or 6 ond ripening and final production stage (Uren and FMN, heme, and molybdenum as cofactors; another 7 Babayigit 1997). variant of the enzyme (EC 1.7.1.3) requires FAD, 8 heme, molybdenum, and an iron-sulfur cluster. 9 NOϪ ϩ NADPH ϩ Hϩ → 10 3 BACTERIAL ROLE IN MEAT —NOϪϩ NADPϩ ϩ H O (20.5) 11 COLOR 2 2 12 As mentioned, a cytochrome nitrate reductase 13 In dry-fermented sausages, flavor and oxidative sta- (EC 1.9.6.1) also exists for which the reaction is the 14 tus depend both on endogenous chemical changes in following: the meat batter, mainly due to enzymes, and on the 15 2[cyt]FeII ϩ 2Hϩ ϩ NOϪ → nature of the starter cultures added according to the 3 16 —2[cyt]FeIII ϩ NOϪ ϩ H O (20.6) 17 actual recipe. Starter cultures are added in order to 2 2 18 ensure a decrease in pH, reduction of nitrate, and It is generally recognized that most bacterial 19 flavor formation during product maturation. Some strains of the Micrococcaceae and Staphylococ- 20 bacterial cultures, however, also seem to affect other caceae families are positive for nitrate reductase 21 chemical processes in the final meat product, activity in standard biochemical tests, and a study of 22 because it was found that microbial metabolism led more than 400 isolates, predominantly Staphylococ- 23 to color changes or prevented oxidative processes cus xylosus, from chorizo also found 97% to show 24 (Montel et al. 1998). nitrate reduction (Garcia-Varona et al. 2000). The 25 number of staphylococci has been found to increase 26 only slightly during the fermentation period of NITRATE REDUCTASE 27 sausages from approximately 103 up to 106 CFU/g, 28 The nitrogen cycle found in many biological sys- while, for instance, lactic acid bacteria reach num- 29 tems, including some bacteria, results in numerous bers as high as 108–109 CFU/g during processing for 30 N-containing compounds as intermediates in the more than 35 days (Mauriello et al. 2004). Another 31 complex enzymatic metabolism related to nitrifica- study performed to follow ripening of Spanish-type 32 tion and denitrification (Richardson 2001). Some of chorizo shows no effect of sodium nitrite level 33 these transient nitrogen species can react with meat (50 and 150 ppm) and of an added lactobacilli starter 34 Mb and thereby affect color. Prokaryotic nitrate culture on the survival of Micrococcaceae, although 35 reduction serves a number of metabolic functions these factors significantly inhibited Enterobacteri- 36 and depends on a variety of nitrate reductases, as aceae (Gonzalez and Diez 2002). It is a prerequisite 37 found in nitrogen assimilation and anaerobic respira- for ripening that these bacteria strains exhibit 38 tion (Richardson et al. 2001). The role of bacterial metabolic activity during sausage fermentation; 39 nitrate reductases has been acknowledged and uti- however, the information about this class of micro- 40 lized for many decades; however, very little is still bial enzymes is quite limited with respect to impor- 41 known regarding expression or variations in this tance for color formation in fermented meats. 42 enzymatic activity for either bacteria present in A study including seven staphylococci strains iso- 43 the environment or commercial starter cultures. The lated from either sausage or cheese discovered that 44 enzymes belong to the class of oxidoreductases, and isolates of S. xylosus and S. carnosus were able to 45 it is the general reaction of Equation 20.4 that is synthesize nitrate reductase, and a S. warneri had no 46 being catalyzed during the microbial anaerobic res- activity of this enzyme under static conditions (Talon 47 piratory metabolism. et al. 1999). However, during the exponential growth phase, the synthesis was maximal, whereas under 48 Nitrate ϩ reduced acceptor → shaking condition, the synthesis was maximal at the 49 —nitrite ϩ acceptor (20.4) 50 beginning of the stationary phase of growth. In all 51 The assimilatory nitrate reductases can be divided cases, nitrate reductase activity was increased in pres- 52 into at least three subgroups depending on the nature ence of nitrate, which was particularly pronounced for 53S and number of cofactors for electron transfer present the two S. carnosus strains exhibiting the highest 54N in the enzymes (Richardson et al. 2001). The enzyme activity. 40454 20 203-216 r2.qxd 7/20/07 1:43 PM Page 209

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Götz and co-workers have performed a number NITRITE REDUCTASE 1 of extensive investigations with respect to the pres- 2 The nitrite reductases (NIR) and nitrate reductases ence and activity of nitrate/nitrite reductases in a 3 are active at low oxygen tension using nitrogen S. carnosus strain (Fast et al. 1996; Neubauer and 4 oxides as electron acceptors in the metabolism of the Götz 1996; Pantel et al. 1998; Neubauer et al. 1999; 5 bacteria, and the products originating from such Fedtke et al. 2002). Nitrate reductase is of interest 6 metabolism are ammonia, nitrogen oxides, and dini- also in other scientific areas such as soil processes, 7 trogen (NH , NO, N O, N ). It has recently been and this enzyme class has been studied in many 3 2 2 8 shown that NO exists as a free intermediate in deni- details relating to anaerobic respiration of several 9 trifying bacteria with steady state concentrations in bacterial species. The enzyme contains a molybde- 10 the range 10−7–10−9 M (Watmough et al. 1999). In num atom in the active site and several species pos- 11 the enzymatic reaction behind NO formation from sess a membrane-bound type of the enzyme, which 12 nitrite in Equation 20.7, two His residues each for this specific isoform has been found to consist of 13 donate a H+ to the ligand followed by dehydration three subunits (Pinho et al. 2005). The Pseudomonas 14 and reduction of the nitrosyl complex to yield a fer- nitrate reductase is a cytochrome enzyme, and the 15 ric NO complex from which NO can escape. enzyme from Micrococcus halodenitrificans is an 16 iron protein-containing molybdenum. A critical II + 17 [NIR]Fe NO2 + 2H → parameter regarding this type of enzyme and its III 18 —[NIR]Fe + NO(g) + H2O (20.7) activity is induction and expression of the enzyme, 19 which is achieved by the presence of nitrate in the NIR activity has been found in certain Lactobacil- 20 growth environment of the bacteria. Figure 20.5 pro- lus species normally employed as starter cultures for 21 vides a simplified reaction cycle taking into account raw fermented sausages (Wolf and Hammes 1988), 22 steps that are either catalyzed by bacterial enzymes and is found (as well as catalase activity) to depend 23 or occurring spontaneously. on the presence of heme-containing compound 24 However, it may be concluded that despite the (hematin, Mb and Hb) (Wolf et al. 1991). Another 25 many studies devoted to bacterial nitrate reductase, study showed a significant activity of two types of 26 our knowledge regarding molecular biological NIR in various lactic acid bacteria: Type I is heme- 27 aspects of the nitrate reductase and the activity in dependent and produces ammonia, whereas type II 28 bacterial species commonly applied as starter cul- is heme-independent and produces NO and N2O 29 ture for fermented meat products is very limited. (Wolf et al. 1990). A periplasmatic cytochrome 30 More research regarding conditions affecting c nitrite reductase from E. coli has been studied in 31 enzyme activity in starter culture should accordingly detail in relation to the respiratory reduction of 32 be undertaken, because more detailed knowledge nitrite. This enzyme was also found capable of 33 would help optimize color formation and the use of reducing nitric oxide (NO) in vitro, and in vivo 34 additives in meat products such as fermented assessments also demonstrate that this particular 35 sausages. enzyme confers NO detoxification or control in 36 oxygen-limited environments (Poock et al. 2002). 37 A heme protein in Clostridium botulinum, found to 38 display femto-molar sensitivity toward NO, has been 39 suggested to be important for a mechanism in which 40 this bacterium recognizes and avoids NO in the 41 growth environment (Nioche et al. 2004). This heme 42 protein may very well be the ultimate target for 43 sodium nitrite inhibition of growth of this heat- 44 resistant and extremely toxic bacteria species 45 responsible for botulism. 46 47 48 NO SYNTHASE (NOS) Figure 20.5. Cycle of nitrogen oxides occurring in 49 the meat matrix depending on bacterial and/or chem- Efforts have been made to identify and apply bacte- 50 ical conversions resulting in the bright red pigment, rial strains capable of actively changing the color 51 nitrosylmyoglobin, MbFeIINO, that subsequently can of meat products during fermentation. A number of 52 undergo autooxidation into metmyoglobin, MbFeIII, bacteria are found capable of reducing MbFeIII to S53 and nitrate, which may reenter the cycle. MbFeII, which subsequently binds atmospheric N54 40454 20 203-216 r2.qxd 7/20/07 1:43 PM Page 210

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1 oxygen giving a bright red complex identified as COLOR STABILITY OF CURED 2 MbFeIIO (Arihara et al. 1993, 1994), although only 2 MEAT PRODUCTS 3 a few strains have been identified, which without the 4 presence of nitrite/nitrate can form MbFeIINO Conditions during storage, transportation, and retail 5 (Arihara et al. 1993; Morita et al. 1994, 1998). The display can jeopardize sound precautions taken ear- 6 identification of bacterial strains forming MbFeIINO lier in the manufacturing of cured meat products to 7 has even led to the suggestion that the pigment maintain color stability. Brine-cured products have 8 formed in Parma ham during the lengthy process of been studied in most details and only a limited num- 9 ripening was MbFeIINO resulting from microbial ber of studies of packaging conditions for fermented 10 activity (Morita et al. 1996), although the involve- sausages or dry-cured meat products in relation to 11 ment of bacteria in the color formation of Parma their chemical stability are available. 12 ham subsequently was questioned (Sakata 2000). 13 The bacterial generation of MbFeIINO depends on INFLUENCE OF OXYGEN 14 more than a single pathway with respect to the For nitrite-cured meat products, packaging should 15 microbial metabolism leading to the nitrosyl deriva- aim at excluding oxygen completely from the prod- 16 tive of Mb. The interaction between bacteria and NO uct, and this is under practical conditions obtained 17 has mostly been described in relation to enzymatic either by vacuum or modified atmosphere packaging 18 reductase activity occurring as part of denitrification (MAP) (Eidt 1991). However, the problem of color 19 processes in bacteria (Cutruzzola 1999; Watmough fading in nitrite-cured meat products has not fully 20 et al. 1999). As already mentioned, recent studies been solved by the industry, which in part is due to 21 have found NOS (nitric oxide synthetase) activity in the implementation of new and, in other respects, 22 bacteria such as Nocardia (Chen and Rosazza 1994), improved packaging technologies that usually cause 23 Staphylococcus aureus (Choi et al. 1997), Bacillus new problems to emerge. For fermented sausages, a 24 subtilis (Pant et al. 2002), and possibly even in flow-packaging machine is often applied and this 25 L. fermentum (IFO3956) (Morita et al. 1997). A results in higher levels of residual oxygen compared 26 recent review dealing with lactic acid bacteria and to other methods. The use of MAP in relation to fer- 27 NO also emphasizes that these bacteria on several mented sausages has mainly been for presliced 28 occasions have been found to produce NO, a bacte- sausages in convenient dome packages, which has 29 rial activity that could be exploited for color devel- largely been driven by retailers trying to meet con- 30 opment in meat products (Karahan et al. 2005). sumer demands for chilled products with high safety 31 However, other studies of L. fermentum (IFO 3956) and quality. This packaging method may impose 32 species showed less promising results with respect to problems because the residual oxygen content is 33 non-nitrite color formation in salami sausages difficult to maintain at a sufficient low level, and 34 (Møller et al. 2003). The NOS enzyme found in S. because the residual oxygen level in the package 35 aureus has been described in more detail, and it was immediately after closure is clearly a vital parameter 36 concluded that it behaved similarly to mammalian in preventing discoloration of MA-packaged cured 37 NOS with respect to L-arginine binding and depend- meat (Muller 1990); even low concentrations of 38 ence on cofactors (Chartier and Couture 2004). The gaseous oxygen present in the package combined 39 discovery of this particular bacterial NOS, although with light exposure rapidly cause substantial discol- 40 in a highly pathogenic species, could imply the oration of the product (Møller et al. 2003b). Resid- 41 presence of similar types of enzymes in other non- ual oxygen will also be affected by the oxygen 42 pathogenic staphylococci species, which could be transmission rate (OTR) of the packaging material, 43 useful for meat fermentation. and a low OTR, which notably is dependent on tem- 44 In contrast to the activity of NIR, the enzymatic perature and relative humidity, is crucial for the sta- 45 formation of NO by NOS uses two equivalents of bility of the color during storage. A study including 46 oxygen for every molecule of NO formed from 5 packaging materials with OTR ranging from 1–90 47 L-arginine, and this reaction has been found to occur cm2 O /m2/atm/24h established a similar relation- 48 in Nocardia sp. (Chen and Rosazza 1995) and in 2 ship between color stability and OTR for vacuum- 49 S. aureus (Choi et al. 1997). Another study has found packaged salami (Yen et al. 1988). The critical value 50 L. fermentum (IFO3956) to incorporate 15N into for oxygen concentration is mainly affected by 51 nitrite/nitrate (the oxidation product of NO) from 52 guanidino-15N labeled L-arginine to increase when • Headspace to product volume ratio

53S various cofactors of the enzyme (NADPH, H4B, FAD • Storage period in the dark prior to light exposure 54N and FMN) are present (Morita et al. 1997). • The amount of cured pigment in the meat product 40454 20 203-216 r2.qxd 7/20/07 1:43 PM Page 211

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Increasing the amount of nitrite in the meat prod- the animal had been fed a vitamin E supplemented 1 uct will stabilize the product color, allowing a higher diet (Houben and Van Dijk 2001). A concentration- 2 level of residual oxygen in the package headspace dependent effect of nitrite to inhibit development of 3 without having detrimental effects on color stability, rancidity (TBARS or other secondary lipid oxidation 4 because residual nitrite may regenerate MbFeIINO products) has been confirmed in meat originating from 5 from MbFeIII as long as the meat still has reducing various species, e.g., fish, chicken, pork, and beef 6 capacity. (Morrissey and Tichivangana 1985). Several mecha- 7 nisms for this well-described antioxidative effect of 8 nitrite have been presented, which are often related to 9 INFLUENCE OF LIPID OXIDATION the interaction of nitrite or compounds derived from 10 The flavor of meat products with added nitrite has nitrite with metal ions or heme compounds otherwise 11 been described as a pure meat flavor, because no con- acting as prooxidants in meat (Carlsen et al. 2005). 12 tribution from lipid oxidation during processing, Møller et al. (2002) have suggested a possible mecha- 13 cooking, or storage adds off-flavors, and nitrite-cured nism for the role of MbFeIINO as a chain-breaking 14 meats do not develop Warmed-Over-Flavor (WOF) antioxidant where MbFeIINO inactivate peroxyl radi- 15 (Cross and Ziegler 1965). In meat model systems, cals (Skibsted 1992). 16 nitrite alone has been found to act prooxidative at The role of NO in lipid peroxidation is more com- 17 higher levels (Ͼ25 ppm), but did inhibit oxidation plex, as seen in Figure 20.6, in which the small radi- 18 initiated by Fe ions (Macdonald et al. 1980). Notably, cal is described both as a prooxidants and an 19 in brine-cured meat products a reducing agent is also antioxidant depending on the concentration and 20 added, and this ensures optimal conditions for color other available reagents. 21 formation yielding MbFeIINO including regeneration 22 of MbFeII from MbFeIII prior to binding of NO. Like- 23 Microbial Effect on Lipid Oxidation wise, nitrite itself has been shown to act as an antiox- 24 idant in model systems with MbFeIII as a prooxidant In general, both lipolysis and lipid oxidation con- 25 (Arendt et al. 1997). The well-documented antioxi- tribute to development of the characteristic flavor of 26 dant effect of nitrite in cooked meat may be related to dry-cured, fermented sausages (Chizzolini et al. 27 a blocking of release of iron ions from heme pig- 1998). It is, however, intriguing how certain types of 28 ments (Adamsen et al. 2005). traditional meat products originating from Southern 29 No significant oxidation, as measured by TBARS, Europe maintain their wholesomeness during lengthy 30 was observed in nitrite (60 ppm) cured beef during 28 maturation periods and afterward during nonrefriger- 31 days illuminated storage at 8°C, regardless of whether ated storage. Conditions such as high concentration 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 Figure 20.6. Reaction pathways of the small diatomic, radical nitric oxide, NO, and various non-heme biomole- S53 cules resulting in nitrosative/oxidative stress, radical scavenging, or stabilization of the antioxidative NO. N54 40454 20 203-216 r2.qxd 7/20/07 1:43 PM Page 212

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1 of salt and low moisture content help select a benefi- products from Southern Europe in which extensive 2 cial microflora and at the same time retard microbial formation of free fatty acids takes place during mat- 3 spoilage in dry-cured meat products. Lipid oxidation uration, in part due to activities of microbial derived 4 in sausages with added starter cultures was found to enzymes. However, it is still largely unknown 5 be lower in comparison to control sausages produced whether these factors also help protect the lipids 6 without starter culture. Several of the bacterial strains and preserve the color of dry-cured ham and dry- 7 commonly applied as starter cultures in fermented fermented sausage. 8 meat products seem—in addition to acidification, 9 color, and flavor formation—also to be involved in INFLUENCE OF OTHER PARAMETERS 10 retarding lipid oxidation and development of rancid- 11 ity. A screening of 19 strains of lactic acid bacteria At retail display, several conditions such as storage 12 (species represented: Lactobacillus acidophilus; temperature and illumination of the surface of the 13 L. bulgaricus; Streptococcus thermophilus and Bifi- product will affect the chemical stability during the 14 dobacterium longum) for antioxidative activity storage period. Both the spectral characteristics of 15 included effect on rate of ascorbate autoxidation, the illumination and the light intensity are important 16 metal ion chelation, scavenging of reactive oxygen for the detrimental effect of light on meat quality. 17 species (ROS), enzyme inhibition, and reducing Klettner & Terstiege (1999) have examined various 18 activity of intracellular cell-free extract (Lin and Yen types of illumination during display, and they found 19 1999). All strains investigated chelated metals, scav- raw sausage to discolor within 1–3 hours when 20 enged free radicals, and showed general reducing stored at 5°C and exposed to 800 lux. One thousand 21 capacities; no activity of superoxide dismutase lux seems a typical exposure at the package surface 22 (SOD) could be detected and was not induced by in the retail trade in many countries. The wavelength 23 availability of Mn2+, Fe2+, Cu2+, or Zn2+. Catalase and dependence for degradation of the pigment of cured II 24 SOD has been characterized in S. carnosus using meat is less significant than for MbFe O2 present in 25 model systems (Barriere et al. 2001), and the SOD in fresh meat. The use of packaging material with 26 a S. xylosus also has been investigated (Barriere et al. incorporated UV filter has accordingly little or no 27 2001). In a system with linoleic acid as oxidation beneficial effect on the color stability of nitrite-cured 28 substrate, more details have been obtained with meat products (Andersen et al. 1988). A level for

29 respect to individual roles of catalase and SOD in residual O2 between 0.2–0.5% (depending on other 30 S. xylosus. It was concluded that the wild-type strain packaging conditions) at the time of packaging has 31 with both enzymes intact had the highest antioxida- been identified as critical for color stability upon 32 tive potential followed by the mutant lacking catalase exposure to light (Møller et al. 2000, 2003b). 33 but having SOD activity (Barriere et al. 2001). In order to control spoilage and pathogenic bacte- 34 Certain starter cultures seem to possess antioxida- ria in dry-cured ham and other products, high- 35 tive capacity, which has been proven for strains of pressure treatment has been introduced for sliced 36 staphylococci and lactic acid bacteria when growing products. Oxidation of MbFeIINO becomes slower 37 on media containing various fatty acids (Talon et al. under pressure, and high pressure is accordingly not 38 2000). This antioxidative effect is improved for most expected to cause discoloration (Bruun-Jensen and 39 strains when the media is supplemented with man- Skibsted 1996). At pressure treatment under 4000 40 ganese, suggesting a mechanism involving SOD atmospheres, little if any lipid oxidation was found 41 activity confirming the results obtained in model sys- to be induced in Iberian ham (Andres et al. 2004); 42 tems for the relative importance of catalase and SOD. thus, high hydrostatic pressures is a promising new 43 The effect of microbial activity on lipid oxidation technology also in relation to the zero-Listeria toler- 44 in dry-fermented sausages is still uncertain, but ance enforced in some countries. 45 studies are emerging that point toward an overall 46 antioxidative effect of specific starter cultures. Free CONCLUSION 47 fatty acids in dry-fermented sausage was found to 48 increase significantly during 50 days independent of In fermented meat, color formation depends on two 49 the type of starter cultures added (Kenneally et al. mechanisms. For nitrate-cured products, nitrate 50 1998). The role of free fatty acids is interesting reduction mediated through microbial enzyme activ- 51 because free fatty acids inactivate heme compounds ity seems to be the bottleneck. In contrast, color for- 52 as prooxidants toward unsaturated lipids (Møller mation in nitrite-cured meat is less dependent on 53S et al. 2002). It can be speculated whether a similar microbial activity and may be characterized as 54N mechanism prevails in traditionally processed meat “purely chemical.” For both of these categories of 40454 20 203-216 r2.qxd 7/20/07 1:43 PM Page 213

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products, the most important pigment is nitrosylmyo- K Arihara, H Kushida, Y Kondo, M Itoh, JB Luchansky, 1 globin, which is very sensitive to the combined action RG Cassens. 1993. Conversion of metmyoglobin to 2 of light and oxygen. For certain other products, where bright red myoglobin derivatives by Chromobac- 3 nitrate and nitrite are not used in curing, other pig- terium violaceum, Kurthia sp, and Lactobacillus fer- 4 ments determine the product color. Zinc porphyrin, mentum JCM1173. J Food Sci 58:38–42. 5 which is less light-sensitive, is formed in a much C Barriere, R Bruckner, R Talon. 2001. Characterization 6 slower process requiring longer product maturation. of the single superoxide dismutase of Staphylococcus 7 Future research should be directed toward a better xylosus. Appl Environ Microbiol 67:4096–4104. 8 understanding of nitrate reduction by microbial C Barriere, D Centeno, A Lebert, S Leroy-Setrin, 9 nitrate reductases and especially their expression of JL Berdague, R Talon. 2001. Roles of superoxide dis- 10 relevant genes under the conditions prevailing in mutase and catalase of Staphylococcus xylosus in the 11 meat systems under fermentation. Although the inhibition of linoleic acid oxidation. FEMS Micro- 12 chemistry behind nitrite-induced color formation is biol Lett 201:181–185. 13 rather well documented, the kinetics and mechanism C Barriere, S Leroy-Setrin, R Talon. 2001. Characteri- 14 behind formation of zinc porphyrin is largely zation of catalase and superoxide dismutase in 15 unknown and deserves further attention also in rela- Staphylococcus carnosus 833 strain. J Appl Micro- 16 tion to development of new types of meat products. biol 91:514–519. 17 R Bonnett, S Chandra, AA Charalambides, KD Sales, 18 ACKNOWLEDGMENT PA Scourides. 1980. Nitrosation and nitrosylation of 19 hemoproteins and related Compounds 4. Pentacoor- 20 The Frode and Norma S. Jacobsens foundation is dinate nitrosylprotohem as the pigment of cooked 21 gratefully thanked for its continuing ﬁnancial sup- cured meat. Direct evidence from Electron Spin Res- 22 port to meat related research carried out within the onance spectroscopy. J Chem Soc—Perkin Trans 23 Food Chemistry group at Department of Food Sci- 18:1706–1710. 24 ence, Royal Veterinary and Agricultural University. L Bruun-Jensen, LH Skibsted. 1996. High-pressure 25 effects on oxidation of nitrosylmyoglobin. Meat Sci 26 REFERENCES 44:145–149. 27 R Cammack, CL Joannou, XY Cui, CT Martinez, 28 JC Acton, RL Dick. 1977. Cured color development SR Maraj, MN Hughes. 1999. Nitrite and nitrosyl 29 during fermented sausage processing. J Food Sci compounds in food preservation. Biochim Biophys 30 42:895–897. Acta Bioenerg 1411:475–488. 31 CE Adamsen, JKS Møller, K Laursen, K Olsen, LH CU Carlsen, JKS Møller, LH Skibsted. 2005. Heme- 32 Skibsted. 2005. Zn-porphyrin formation in cured iron in lipid oxidation. Coordination Chem Rev 249: 33 meat products. Effect of added salt and nitrite. Meat 485–498. 34 Sci 72:672–679. RG Cassens, ML Greaser, T Ito, M Lee. 1979. Reac- 35 G Alley, D Cours, D Demeyer. 1992. Effect of nitrate, tions of nitrite in meat. Food Technol 33:46–57. 36 nitrite and ascorbate on color and color stability of FJM Chartier, M Couture. 2004. Stability of the heme 37 dry, fermented sausage prepared using back slop- environment of the nitric oxide synthase from 38 ping. Meat Sci 32:279–287. Staphylococcus aureus in the absence of pterin 39 HJ Andersen, G Bertelsen, G Boegh-Soerensen, CK cofactor. Biophys J 87:1939–1950. 40 Shek, LH Skibsted. 1988. Effect of light and packag- J Chasco, G Lizaso, MJ Beriain. 1996. Cured colour 41 ing conditions on the color stability of sliced ham. development during sausage processing. Meat Sci 42 Meat Sci 22:283–292. 44:203–211. 43 AI Andres, JKS Møller, CE Adamsen, LH Skibsted. Y Chen, JPN Rosazza. 1994. A bacterial nitric-oxide 44 2004. High pressure treatment of dry-cured Iberian synthase from a Nocardia species. Biochem Biophys 45 ham. Effect on radical formation, lipid oxidation and Res Com 203:1251–1258. 46 colour. Eur Food Res Technol 219:205–210. ———. 1995. Puriﬁcation and characterization of 47

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Cloning, sequenc- national symposium on nitrite in meat products. 19 ing, and characterization of a gene (narT) encoding a Zeist, The Netherlands: Wageningen Center for Agri- 20 transport protein involved in dissimilatory nitrate cultural Publishing and Documentation, pp. 13–19. 21 reduction in Staphylococcus carnosus. Arch Micro- JKS Møller, CE Adamsen, LH Skibsted. 2003a. Spectral 22 biol 166:361–367. characterisation of red pigment in Italian-type dry- 23 I Fedtke, A Kamps, B Krismer, F Götz. 2002. The cured ham. Increasing lipophilicity during processing 24 nitrate reductase and nitrite reductase operons and and maturation. Eur Food Res Technol 216:290–296. 25 the narT gene of Staphylococcus carnosus are posi- JKS Møller, M Jakobsen, CJ Weber, T Martinussen, LH 26 tively controlled by the novel two-component system Skibsted, G Bertelsen. 2003b. Optimisation of colour 27 NreBC. J Bacteriol 184:6624–6634. stability of cured ham during packaging and retail dis- 28 DA Froehlich, EA Gullett, WR Usborne. 1983. Effect play by a multifactorial design. Meat Sci 63:169–175. 29 of nitrite and salt on the color, flavor and overall JKS Møller, JS Jensen, MB Olsen, LH Skibsted, 30 acceptability of ham. J Food Sci 48:152–154. G Bertelsen. 2000. Effect of residual oxygen on 31 M Garcia-Varona, EM Santos, I Jaime, J Rovira. 2000. colour stability during chill storage of sliced, pas- 32 Characterisation of Micrococcaceae isolated from teurised ham packaged in modified atmosphere. 33 different varieties of chorizo. Int J Food Microbiol Meat Sci 54:399–405. 34 54:189–195. JKS Møller, JS Jensen, LH Skibsted, S Knöchel. 2003. 35 B Gonzalez, V Diez. 2002. The effect of nitrite and starter Microbial formation of nitrite-cured pigment, nitro- 36 culture on microbiological quality of “chorizo”— sylmyoglobin, from metmyoglobin in model systems 37 A Spanish dry cured sausage. Meat Sci 60:295–298. and smoked fermented sausages by Lactobacillus 38 JH Houben, A Van Dijk. 2001. Effects of dietary vita- fermentum strains and a commercial starter culture. 39 min E supplementation and packaging on the colour Eur Food Res Technol 216:463–469. 40 stability of sliced pasteurized beef ham. Meat Sci JKS Møller, LH Skibsted. 2002. Nitric oxide and myo- 41 58:403–407. globins. Chemical Rev 102:1167–1178. 42 AG Karahan, ML Cakmakci, B Cicioglu-Aridogan, JKS Møller, L Sosniecki, LH Skibsted. 2002. Effect of 43 A Kart-Gundogdu. 2005. Nitric oxide (NO) and nitrosylmyoglobin and saturated fatty acid anions on 44 lactic acid bacteria—contributions to health, food metmyoglobin-catalyzed oxidation of aqueous methyl 45 quality, and safety. Food Rev Int 21:313–329. linoleate emulsions. Biochim Biophys Acta 1570: 46 PM Kenneally, G Schwarz, NG Fransen, EK Arendt. 129–134. 47 1998. Lipolytic starter culture effects on production MC Montel, F Masson, R Talon. 1998. Bacterial role in 48 of free fatty acids in fermented sausages. J Food Sci flavour development. Meat Sci 49:S111–S123. 49 63:538–543. H Morita, J Niu, R Sakata, Y Nagata. 1996. Red pig- 50 KB Killday, MS Tempesta, ME Bailey, CJ Metral. 1988. ment of Parma ham and bacterial influence on its for- 51 Structural characterization of nitrosylhemochromogen mation. J Food Sci 61:1021–1023. 52 of cooked cured meat: Implications in the meat-curing H Morita, R Sakata, Y Nagata. 1998. Nitric oxide 53S reaction. J Agric Food Chem 36:909–914. complex of iron(II) myoglobin converted from 54N 40454 20 203-216 r2.qxd 7/20/07 1:43 PM Page 215

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metmyoglobin by Staphylococcus xylosus. J Food Sci of the membrane–bound nitrate reductase from 1 63:352–355. Pseudomonas chlororaphis DSM 50135. Biochim 2 H Morita, R Sakata, S Sonoki, Y Nagata. 1994. Met- Biophys Acta Gen Subj 1723:151–162. 3 myoglobin conversion to red myoglobin derivatives SR Poock, ER Leach, JWB Moir, JA Cole, DJ Richard- 4 and citrate utilization by bacteria obtained from meat son. 2002. Respiratory detoxification of nitric oxide 5 products and pickles for curing. Anim Sci Technol by the cytochrome c nitrite reductase of Escherichia 6 (Jpn) 65:1026–1033. coli. J Biol Chem 277:23664–23669. 7 H Morita, H Yoshikawa, R Sakata, Y Nagata, H Tanaka. DJ Richardson. 2001. Introduction: Nitrate reduction 8 1997. Synthesis of nitric oxide from the two equiva- and the nitrogen cycle. Cell Mol Life Sci 58: 9 lent guanidino nitrogens of L-arginine by Lactobacil- 163–164. 10 lus fermentum. J Bacteriol 179:7812–7815. DJ Richardson, BC Berks, DA Russell, S Spiro, CJ Tay- 11 PA Morrissey, JZ Tichivangana. 1985. The antioxidant lor. 2001. Functional, biochemical and genetic diver- 12 activities of nitrite and nitrosylmyoglobin in cooked sity of prokaryotic nitrate reductases. Cell Mol Life 13 meats. Meat Sci 14:175–190. Sci 58:165–178. 14 SA Muller. 1990. Packaging and meat quality. Can Inst R Sakata. 2000. Studies on physicochemical character- 15 Food Sci Technol J 23:AT22–AT25. istics of red pigments in meat products. Anim Sci J 16 H Neubauer, F Götz. 1996. Physiology and interaction 71:1–16. 17 of nitrate and nitrite reduction in Staphylococcus JG Sebranek, JBJ Fox. 1985. A review of nitrite and 18 carnosus. J Bacteriol 178:2005–2009. chloride chemistry: Interactions and implications for 19 H Neubauer, I Pantel, F Götz. 1999. Molecular charac- cured meats. J Sci Food Agric 36:1169–1182. 20 terization of the nitrite-reducing system of Staphylo- F Shahidi, RB Pegg. 1995. Nitrite alternatives for 21 coccus carnosus. J Bacteriol 181:1481–1488. processed meats. 1223–1241. In: G Charalambous, 22 P Nioche, V Berka, J Vipond, N Minton, AL Tsai, ed. Food Flavors: Generation, Analysis and Pro- 23 CS Raman. 2004. Femtomolar sensitivity of a NO cess Influence. Amsterdam: Elsevier Science B.V., 24 sensor from Clostridium botulinum. Science 306: pp. 1223–1241. 25 1550–1553. LH Skibsted. 1992. Cured meat products and their oxida- 26 K Pant, AM Bilwes, S Adak, DJ Stuehr, BR Crane. 2002. tive stability. In: DA Ledward, DE Johnston, MK 27 Structure of nitric oxide synthase heme protein from Knight, eds. The Chemistry of Muscle Based Foods. 28 Bacillus subtilis. Biochemistry 41:11071–11079. London: Royal Society of Chemistry, pp. 266–286. 29 I Pantel, PE Lindgren, H Neubauer, F Götz. 1998. Iden- LH Stahnke. 1995. Dried sausages fermented with 30 tification and characterization of the Staphylococcus Staphylococcus xylosus at different temperatures and 31 carnosus nitrate reductase operon. Mol Genet Gen with different ingredient levels. 1. Chemical and 32 259:105–114. Bacteriological Data. Meat Sci 41:179–191. 33 G Parolari. 1996. Achievements, needs and perspec- R Talon, D Walter, S Chartier, C Barriere, MC Montel. 34 tives in dry-cured ham technology: The example of 1999. Effect of nitrate and incubation conditions on 35 Parma ham. Food Sci Technol Int 2:69–78. the production of catalase and nitrate reductase by 36 G Parolari, L Gabba, G Saccani. 2003. Extraction prop- staphylococci. Int J Food Microbiol 52:47–56. 37 erties and absorption spectra of dry cured hams made R Talon, D Walter, MC Montel. 2000. Growth and 38 with and without nitrate. Meat Sci 64:483–490. effect of staphylococci and lactic acid bacteria on 39 RB Pegg, F Shahidi. 1997. Unravelling the chemical iden- unsaturated free fatty acids. Meat Sci 54:41–47. 40 tity of meat pigments. Crit Rev Food Nutr 37:561–589. BG Tarladgis. 1962. Interpretation of the spectra of 41 RB Pegg, F Shahidi, NJ Gogan, SI DeSilva. 1996. meat pigments. II. Cured meats. The mechanism of 42 Elucidation of the chemical structure of preformed color fading. J Sci Food Agric 13:485–491. 43 cooked cured-meat pigment by electron paramag- A Uren, D Babayigit. 1997. Colour parameters of Turk- 44 netic resonance spectroscopy. J Agric Food Chem ish-type fermented sausage during fermentation and 45 44:416–421. ripening. Meat Sci 45:539–549. 46 JA Perez-Alvarez, ME Sayas-Barbera, J Fernandez- J Wakamatsu, T Nishimura, A Hattori. 2004. A Zn- 47 Lopez, MA Gago-Gago, MJ Pagan-Moreno, V porphyrin complex contributes to bright red color in 48 Aranda-Catala. 1999. Chemical and color character- Parma ham. Meat Sci 67:95–100. 49 istics of Spanish dry-cured ham at the end of the NJ Watmough, G Butland, MR Cheesman, JWB Moir, 50 aging process. J Muscle Foods 10:195–201. DJ Richardson, S Spiro. 1999. Nitric oxide in bacte- 51 D Pinho, S Besson, PJ Silva, B de Castro, I Moura. ria: Synthesis and consumption. Biochim Biophys 52 2005. Isolation and spectroscopic characterization Acta 1411:456–474. S53 N54 40454 20 203-216 r2.qxd 7/20/07 1:43 PM Page 216

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1 G Wolf, EK Arendt, U Pfahle, WP Hammes. 1990. G Wolf, A Strahl, J Meisel, WP Hammes. 1991. Heme- 2 Heme-dependent and heme-independent nitrite dependent catalase activity of lactobacilli. Int J Food 3 reduction by lactic-acid bacteria results in different Microbiol 12:133–140. 4 n-containing products. Int J Food Microbiol 10: JR Yen, RB Brown, RL Dick, JC Acton. 1988. Oxygen 5 323–329. transmission rate of packaging ﬁlms and light expo- 6 G Wolf, WP Hammes. 1988. Effect of hematin on the sure effects on the color stability of vacuum-packaged 7 activities of nitrite reductase and catalase in lacto- dry salami. J Food Sci 53:1043–1046. 8 bacilli. Arch Microbiol 149:220–244. 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53S 54N 40454 21 217-226 r1.qxd 7/20/07 1:44 PM Page 217

1 2 3 21 4 5 6 Texture 7 8 Shai Barbut 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION Spain. Overall, the authors concluded that there were 22 only small differences in the textural parameters of 23 The development of acceptable texture in fermented the products (Gimeno et al. 2000). Some of the 24 sausages is very important for the successful market- observed differences, especially in cohesiveness, 25 ing of high-quality products. Obtaining the desired were significantly correlated to the pH of the product. 26 texture is a multiphase process, which is complex and It should be noted that a greater variation should be 27 takes anywhere from a few days to several months. expected when such a Chorizo de Pamplona is pro- 28 Many factors affect the final texture of fermented duced outside of Spain by non-Spanish or Spanish 29 meat products. They include the ingredients used, pro- sausage makers (e.g., immigrants to other countries). 30 cessing parameters, acidification method, possible Table 21.2 shows a range of values obtained for 31 application of a low/medium heat treatment, drying 29 commercially produced Italian Felino Salamis, 32 and ripening conditions, as well as the interactions which were produced in the Parma surroundings and 33 taking place among these factors over an extended marketed by some of the largest Italian manufactur- 34 period of time. Overall, texture development takes ers (products obtained from eight different produc- 35 place in three major steps: extraction of proteins by ers at different times and places). This is a typical 36 salt during grinding and mincing, formation of a pro- dry-cured product with a mean marketing process- 37 tein gel during acidification, and strengthening of the ing time of 73 days; however, as indicated by the 38 protein gel by further denaturation during cooking authors, time can vary greatly from 17 to 160 days, 39 and/or drying. As shown in other chapters, there are depending on the product’s diameter, moisture con- 40 many types of fermented sausages on the market. tent, etc. In this case there were great differences in 41 Describing the unique texture of each one is beyond texture; modulus, elasticity index, and sensory 42 the scope of this chapter. However, the main factors hardness, which varied from 6.27–66.4 N/cm2, 43 contributing to texture development will be discussed 0.07–0.53, and 2.17–6.51, respectively, for the 29 44 along with examples of representative products. different products (Dellaglio et al. 1996). 45 46 TEXTURE OF COMMERCIAL TEXTURE DEVELOPMENT 47 PRODUCTS DURING FERMENTATION 48 49 Despite the large number of fermented products on Reducing the pH of meat is done for a variety of 50 the market, there are very few scientific papers reasons. Historically, the most important reason 51 describing commercial products. Table 21.1 shows was to extend the storage life by producing an 52 texture variations among five commercial dry- acidic environment unfavorable for pathogenic and 53 fermented Chorizo de Pamplona produced in Northern spoilage microorganisms. Other reasons include the S54 N55 217 40454 21 217-226 r1.qxd 7/20/07 1:44 PM Page 218

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1 Table 21.1. Composition, texture, and color (lightness-L* and redness-a*) parameters of 2 five commercial Chorizo de Pamplona dry-fermented Spanish sausages. 3 4 Brand 1 Brand 2 Brand 3 Brand 4 Brand 5 5 pH 4.65ab 4.62ab 4.73b 4.89c 4.55a 6 b b c a b 7 Moisture (%) 31.62 31.69 33.82 30.02 31.56 8 Hardness (g) 7154c 5438ab 5475ab 5170a 6264bc 9 Springiness (mm) 0.57ab 0.61b 0.59b 0.57ab 0.51a 10 Cohesiveness 0.50b 0.51b 0.49b 0.45a 0.50b 11 b ab ab a ab 12 Gumminess (g) 2072 1695 1640 1436 1873 13 L* 46.8a 51.8bc 54.3c 52.3bc 49.2ab 14 a* 20.4a 25.9c 26.1c 25.8c 23.5b 15 16 Source: Gimeno et al. (2000). 17 abc Means followed by a different superscript are significantly different (P Ͻ 0.05). 18 19 20 Table 21.2. Range of chemical, physical, and sensory values obtained for 29 commercially produced Italian 21 Felino Salamis. 22 Variable Mean Minimum Maximum Standard Deviation CV (%) 23 24 Moisture (%) 38.0 28.0 48.9 5.4 14.3 25 Protein (%) 29.7 23.5 36.7 3.3 11.2 26 27 pH 5.92 5.40 6.36 0.26 4.4 28 Fat (%) 28.0 21.0 34.3 3.3 11.8 29 NaCl (%) 4.32 3.35 5.20 0.48 11.0 30 Modulus (N/cm2) 25.7 6.27 66.4 17.3 67.4 31 32 Elasticity index 0.35 0.07 0.53 0.10 28.6 33 Sensory*-color 5.03 2.89 6.15 0.60 11.9 34 Saltiness 4.23 3.71 4.91 0.32 7.6 35 Acidity 4.79 4.05 5.57 0.42 8.8 36 37 Hardness 3.76 2.17 6.51 1.06 28.2 38 Elasticity 4.62 3.16 5.87 0.82 17.8 39 Overall accept 4.28 2.47 5.56 0.79 18.5 40 41 Source: Dellaglio et al. (1996). 42 * Sensory analysis—13 semitrained panelists, using a 1–7 scale for each attribute (anchor points not mentioned). 43 44 45 production of unique acid/tangy flavors and acidifi- backslopping), inoculating the product with selec- 46 cation to assist speeding up the drying process by ted microorganisms, to using glucono-delta-lacton 47 bringing the pH closer to the iso-electric point of the (GDL) or encapsulated acids. Depending on the 48 meat proteins where the binding of water is signifi- method, the time of the acidification process can take 49 cantly reduced. a few minutes, using encapsulated acids, to a few days 50 As described in previous chapters, there are differ- or even weeks, using natural microbial fermentation. 51 ent ways to achieve acidification and pH reduction. The most popular acidification method is microbial 52 They can vary from relying on the natural lactic acid fermentation because GDL and encapsulated acids 53S bacteria found in the raw meat, inoculation with do not produce the unique flavor notes derived from 54N meat from a previous successful batch (also called enzymatic activity during microbial fermentation. 40454 21 217-226 r1.qxd 7/20/07 1:44 PM Page 219

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Heating Phase 1 5.5E+04 2 Control 5.0E+04 Encp. citric 3 Encap. Lactic 4 4.5E+04 Encp. GDL 5 ) a Liq Lactic 6 4.0E+04 Form 7 (Gí, kP 8 us

l 3.5E+04 u 9 3.0E+04 10

ge mod 11 a 2.5E+04

tor 12 S 13 2.0E+04 14 1.5E+04 15 16 1.0E+04 17 30 3540455055 60 65 70 75 18 ° Temperature ( C) 19 Figure 21.1. Effect of acidification method on storage modulus (G', kPa) of meat batters during heating 20 (1.5ûC/min) (from Barbut 2005). 21 22 23 24 However, various processors around the world do dialyzed against pH 4.0 buffers in 0.25 M KCl at 25 use the fairly fast acidifying GDL and the very fast 4°C. A fine strand gel was formed, which actually 26 acidifying encapsulated acids. Inoculation with a required a lower protein concentration to form the 27 specially prepared starter culture is the preferred gel than for heated myosin at pH 5.5 or 6.0. Electron 28 microbial method now because it minimizes unex- microscopy revealed a network formation by parallel 29 pected results, such as failure due to slow/no activity alignment of filaments in pairs and end-to-side inter- 30 of the lactic acid bacteria, and also help in achieving actions forming the so-called Y-junctions at low salt 31 the safety margins required in terms of reducing the level, and a more right-angled network at higher salt 32 chance of E. coli 0157:H7 survival in the product level. 33 (Pond et al. 2001). Ngapo et al. (1996) showed gel formation, meas- 34 Texture development during the acidification ured as an increase in Young’s modulus, due to grad- 35 process is primarily the result of meat protein denat- ual pH reduction by GDL, of a bovine myofibrillar 36 uration and coagulation. During the grinding and solution kept at 4°C. Increasing GDL concentration 37 mincing procedures, the added salt promotes the resulted in faster and more extensive disruption of 38 solubilization and extraction of proteins (primarily myofibrillar protein, leading to greater stiffness than 39 myosin and actin) from the myofibrils, forming a at high pH values. A similar gelation can be 40 sticky protein film around the minced particles. Dur- observed in Figure 21.1, which shows the initial 41 ing the succeeding acidification process, the solubi- storage modulus (G') values of a nonfermented con- 42 lized proteins coagulate and form a strong gel that trol beef meat batter (pH 5.60), fermented batter (by 43 binds the fat and meat closely together. Fretheim lactic acid bacteria; pH 4.60), and chemically acidi- 44 et al. (1985) reported that a 1% myosin solution, fied batter (by liquid lactic acid; pH 4.62) (Barbut 45 kept at 5°C, could spontaneously form a gel if the 2005). The G' values of the three treatments at 30°C 46 pH was slowly lowered to below 5.5. The acid- were significantly different (13.1, 41.0, and 26.7 47 induced denaturation was the basis of the gel formed kPa, respectively; P Ͻ 0.05). The fermented meat 48 where the stiffness depended on the pH, with a max- batter represents a typical process where the pH is 49 imum at pH 4.5 in the presence of 0.6 M KCl. At pH gradually being lowered by lactic acid bacteria. 50 above 5.0, part of the myosin remained dissolved, Sausage makers often use such gel formation, or 51 suggesting an incomplete protein denaturation. rather the increase in stiffness, to get a rough estimate 52 Hermansson et al. (1986) have also demonstrated of the development of the fermentation process; they S53 spontaneous gel formation of myosin solutions often squeeze the product and feel the change in N54 40454 21 217-226 r1.qxd 7/20/07 1:44 PM Page 220

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1 texture. The direct addition of liquid lactic acid was matrix. The coagulation of proteins that takes place 2 used only to demonstrate the devastating effect of during the fermentation phase is associated with the 3 such a practice (it is not used in commercial produc- release of water, and this water is continuously 4 tion) due to a very fast protein denaturation. Overall, removed during the drying process. As the drying 5 a sudden acidification results in clumping of the meat process continues, the more tightly bound water will 6 particles and a sudden loss of the smooth texture of be released as well, though at a slower rate, and cre- 7 the raw meat batter. Quinton et al. (1997) tried to add ate a more dense and chewy structure due to further 8 vinegar and liquid citric acid to a meat and vegetable- protein denaturation and degradation of protein by 9 stick product and made a similar observation. They endogenous or microbial enzymes. Processors 10 reported that direct addition of acids “resulted in flac- sometimes, describe the whole process as the devel- 11 cid, noncohesive raw mix that could not be extruded opment of a body or a typical bite. The net result is 12 into sticks, especially at pH 4.6.” When they used usually an increase in firmness, but a fine balance is 13 encapsulated citric and lactic acids, no such problems required between forces contributing to hardening 14 were encountered because there was no immediate and forces contributing to softening or protein break- 15 pH drop. Overall, the introduction of microencapsu- down by proteolytic enzymes. 16 lation in the late 1960s helped address the problem of Warnants et al. (1998) followed the development 17 controlled release of acids in meat acidification. of stiffness in fermented salami dried for 11 weeks. 18 Roughly, the acidification process used by the meat Although the product diameter is not mentioned, the 19 industry can be compared to other fermented food trend reported is typical for dried sausages. Their first 20 systems that are produced by lowering the pH. One measurement of the penetration force after 3 days 21 such popular example is yogurt, where G' increases (the end of fermentation) was 5 Ϯ 1 N (Table 21.3). It 22 as fluid milk is fermented to give a gel-like character- more than doubled after 3 weeks of drying at 16°C 23 istic (Haque et al. 2001). This transformation is and 83% relative humidity. Further drying showed a 24 attributed to pH reduction (to around 4.5), which numerical but not statistical increase in the penetra- 25 results in less suppression of electrostatic repulsion tion force. Benito et al. (2005) evaluated the changes 26 between casein micelles and agglomeration into a in myofibrillar proteins, sarcoplasmic proteins, non- 27 three-dimensional casein network structure. protein nitrogen and amino acid nitrogen in dry- 28 fermented Spanish Salchichon sausage dried for 29 20 weeks. They also evaluated the effect of adding a 30 TEXTURE DEVELOPMENT pure proteolytic enzyme (EPg 222 from Penicillium 31 DURING RIPENING chrysogenum) to one of the treatments. The results 32 showed a progressive reduction in myofibrillar 33 During the ripening process of fermented sausages, proteins in both the control and enzyme-added treat- 34 which can last from a couple of weeks to a few ment (Table 21.4), with a faster reduction in the EPg 35 months (depending on the product, diameter, etc.), 222 sausage. Sarcoplasmic proteins also decreased, 36 there are considerable changes in the meat batter’s and, as expected, nonprotein nitrogen significantly 37 38 39 Table 21.3. Effect of fatty acid composition on oxidation (TBA values), texture, and color of dry salami. 40 Ripening Time Dietary Treatment 41 42 Day 3 Week 3 Week 5 Week 7 Week 9 Week 11 Diet 1 Diet 2 Diet 3 Diet 4 43 44 PUFA:SFA* 0.40 0.42 0.39 0.37 0.40 0.41 0.34 0.38 0.45 0.45 45 FFA 2.3a 5.5b 6.5bc 8.0d 8.4d 7.2cd 6.3 5.9 6.6 6.6 46 TBA – 79 93 207 320 406 134 137 184 429 47 a b b b b b a a b b 48 Penetration 5 13 16 18 17 19 22 17 12 12 49 L* 55.2a 50.8b 48.5b 48.1b 50.6b 50.7b 49.5 50.4 50.2 50.8 50 a* 13.0 14.1 14.4 14.4 14.0 13.3 13.9 13.8 14.0 14.2 51 52 Source: Warnants et al. (1998). 53S * PUFA = polyunsaturated fatty acids; SFA ϭ saturated fatty acids; FFA ϭ free fatty acids (in g oleic acid equivalents/100 g 54N fat); TBA ϭ thiobarbituric acid (ug); penetration force to puncture a 1 cm thick slice (N); color L* ϭ lightness value and a* ϭ redness value of the product. 40454 21 217-226 r1.qxd 7/20/07 1:44 PM Page 221

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increased over the 20-week period. At the end of dry- TEXTURE DEVELOPMENT 1 ing, the texture profile analysis hardness value was DURING COOKING 2 almost twice as high for the control as compared to (NONDRIED/SEMIDRIED) 3 the EPg 222 treatment (2,925 Ϯ 331 versus 1,593 Ϯ 4 257 N, respectively; samples: 16 mm thick cm com- Some fermented/acidified meat products receive a 5 pressed to 8 mm). Other significant differences were heat treatment during manufacturing. This includes 6 reported for gumminess (1,000 Ϯ 299 versus 442 Ϯ products such as cooked salami, which is typically 7 72 N, respectively) and chewiness (610 Ϯ 261 versus not dried, and some products that are only semidried. 8 294 Ϯ 70N cm, respectively), but not for springiness It is interesting to note that after the mid-1990s, with 9 (about 0.62) and cohesiveness (about 0.31, ratio). the introduction of the regulation regarding E. coli 10 Severini et al. (2003) reported on the effect of a 0157:H7 reduction/elimination in dry-fermented 11 55-day drying period (20°C at 80% relative humid- sausages in North America, a few medium- and large- 12 ity) of a dry Italian salami and showed firmness val- size processors decided to add a heating step to the 13 ues (13 mm plunger used for penetration) starting at production of their traditionally dried noncooked 14 7 N for the sausage mixture, 8 N for the sausage after products. The regulation requires demonstrating a 15 16 days, 7.8 after 25 days, 4.8 after 42 days (called 2 or 5 log reduction in E. coli 0157:H7, depending 16 half ripening) and 4.7 N at the end of ripening at 55 on the one of the five options that can be selected by 17 days. The product tested was produced by natural USA and Canadian processors (Pond et al. 2001). 18 fermentation (no started culture added), so a pH of This cooking step helps meet the regulation and/or 19 5.0 was not reached until after 15 days of ripening. add another level of security; however, it is impor- 20 The pH stayed at this level until the end, while water tant to note that a heating step may cause some 21 activity went down to 0.89. The authors compared changes to the traditional products’ characteristics. 22 the treatment mentioned above to products where 33, When a meat product is cooked, the heat causes the 23 50, and 66% of the pork backfat was replaced with proteins to unfold, denature, and form a gel (Barbut 24 extra virgin olive oil. The replacement resulted in 2005). A typical gelation curve of a meat batter is 25 some differences in texture along the ripening period seen in Figure 21.1 (see control treatment), where the 26 but at the end of the 55-day period the differences beginning of denaturation starts at 45°C (i.e., the 27 were very small. slight elevation in G' value), corresponding to 28 myofibrillar proteins denaturation), and the beginning 29 of protein gelation at about 52°C. At this point a stiff 30 Table 21.4. Effect of using a proteolytic enzyme protein matrix starts to form and G' values increase by 31 (EPg 222) on myofibrillar protein and texture of a dry- a factor of about four once the temperature reaches 32 fermented sausage. 70°C. This basic behavior of a meat protein system 33 Ripening Enzyme has also been shown in beef, pork, and chicken pro- 34 Days Control Treated teins (Montejano et al. 1984). As mentioned above, 35 pH reduction also causes a certain degree of protein 36 Myofibrillar 0 123 Ϯ 41 128 Ϯ 31 denaturation and gelation. However, during heating, 37 protein 3 123 Ϯ 1a1 117 Ϯ 2b2 preacidified meat batters exhibit different gelation 38 (mg/g DM) 20 45 Ϯ 3a2 35 Ϯ 3b3 patterns (or G' value curve, Figure 21.1, than the fer- 39 35 35 Ϯ 23 32 Ϯ 23 mented and lactic acid treatments) because of the par- 40 50 27 Ϯ 2a4 20 Ϯ 2b4 tial pre-denaturation that occurs prior to cooking. The 41 75 18 Ϯ 15 19 Ϯ 24 microbial fermented product (pH 4.60 prior to cook- 42 145 17 Ϯ 2a5 9 Ϯ 2b5 ing) had a stiff and coherent texture prior to heating 43 Hardness (N) 145 2925 Ϯ 331a 1593 Ϯ 257b and could be thinly sliced without any problem main- 44 taining its shape. The initial G' value was actually 45 Ϯ a Ϯ a Cohesiveness 145 0.35 0.12 0.28 0.05 similar to the control treatment after cooking to 70°C 46 Gumminess (N) 145 1000 Ϯ 299a 442 Ϯ 72b (Figure 21.1). During heating of the fermented prod- 47 Chewiness 145 610 Ϯ 261a 294 Ϯ 70b uct a reduction in G' value between 30 and 40°C was 48 (Ncm) probably attributed to fat softening. Later the G' value 49 stayed the same up to 55°C, and then an additional 50 Source: Benito et al. (2005). gelation of myofibrillar proteins took place. The final 51 For a given row, values followed by a different letter (a,b) G' value (46 kPa) was significantly higher (P Ͻ 0.05) 52 are significantly different (P < 0.05). For a given ripening than that of the control (42 KPa). The addition of liq- S53 time (column), protein values with different numbers (1–5) uid lactic acid (immediate pH drop to 4.60) resulted N54 are significantly different. in very fast protein denaturation, crumbling of the 40454 21 217-226 r1.qxd 7/20/07 1:44 PM Page 222

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1 meat particles, and water release. This is clearly seen the liquid lactic acid treatment also indicates that pro- 2 in the light micrograph (Figure 21.2e), where the tein denaturation took place prior to cooking, but the 3 denaturation of the meat particles caused an incoher- texture was different from the LAB fermented treat- 4 ent microstructure of the cooked product compared to ment. This was reﬂected in the dense microstructure 5 the more dense structure of the control and the encap- of the microbial treatment (Figure 21.2f; see also the 6 sulated citric and GDL acids treatments (Figure many tiny black dots of the stained fermenting bacte- 7 21.2a,b,d, respectively). The initial high G' value of ria). The encapsulated citric and GDL acids (coated 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 Figure 21.2. Light micrographs of cooked meat batters: (a) control, (b) acidiﬁed with encapsulated citric acid, 53S (c) with encapsulated lactic acid, (d) with encapsulated glucono-delta lacton, (e) with liquid lactic acid, (f) fer- 54N mented with lactic acid bacteria. Bar 0.2 mm (from Barbut 2006). 40454 21 217-226 r1.qxd 7/20/07 1:44 PM Page 223

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with hydrogenated vegetable oil, which according to EFFECTS OF PROCESSING 1 the manufacturer is designed to melt and release the PARAMETERS 2 acids at 57–61, and 60–64°C, respectively) showed 3 G' value curves fairly similar to the control, but with a Processing parameters such as particle size of lean 4 higher final G' values. This was the result of the addi- meat and fat, temperature of added fat, degree of 5 tional pH reduction and protein denaturation. The mixing, etc., have a significant effect on the texture 6 deviation, in G' value during cooking, from the con- and sensory attributes of any meat product—and 7 trol were seen at 62°C for the citric acid and 66°C for fermented products are no exception. However, not 8 the GDL (Figure 21.1). Overall, the advantage of much has been reported in the literature in the area 9 using encapsulated acids is the removal of the adverse of fermented products. van’t Hooft (1999) conducted 10 effect of crumbling and water release prior to cook- a study in which various factors were compared 11 ing. The microstructures of these two treatments (Table 21.5). Mathematical models were constructed 12 (Figure 21.2b,d) show less dense structure compared to investigate the means and variance of the drying 13 to the control, but are fairly similar to the LAB treat- rate, true fracture stress and strain, Young’s modulus, 14 ment in terms of spacing among meat particles. The wall shear stress, firmness, and hardness of the final 15 encapsulated lactic acid addition showed some water product (drying to 80% of the original weight). The 16 release prior to cooking, and stiffening of the struc- main factors that were found to influence the binding 17 ture prior to cooking (higher initial G', Figure 21.1), and texture of the final product were 18 probably due to some acid release resulting from 19 damaged/weak capsules prior to heating. In terms of • Chopping time with salt 20 the microstructure, this also resulted in gaps among • Lean meat temperature 21 the meat particles (Figure 21.2c) compared to the • Fat temperature 22 control and the other two encapsulated acids. Large • Sharpness of the chopping knives 23 deformation testing (fracture force) of the above- • Application of vacuum mixing to the raw meat 24 mentioned products was also preformed (data not batter prior to stuffing 25 shown), but not always correlated with the small 26 strain values (G'). Other research groups have also Overall, the first four factors significantly affected 27 reported this phenomenon in different meat systems most of the responses to the variables studies, except 28 (Montejano et al. 1984). drying time. However, as indicated by the author, the 29 30 31 Table 21.5. Factors evaluated during the production of semidry-fermented sausage*. 32 Class Parameter Low Level High Level 33 34 Ingredients Fat hardness (iodine value) 65 70 35 Skin, precook time (min) 10 12 36 Skin type Cheek Back 37 Starter culture (addition) Dry Suspension 38 Processing Fat freezing temp (°C) –18 –30 39 Meat temp (°C) –2 7 40 Knife sharpness Sharp Blunt 41 Fat adding temp (°C) –18 –10 42 Chopping time (sec) 30 60 43 Chopping with salt (sec) 30 60 44 Casings restrain Slack Tight 45 Stuffing rate** 35 60 46 Batter vacuum time (sec) 0 90 47 Distance knife to wall (mm) 1 2 48 49 Source: van’t Hooft (1999). 50 * Batches made from 5 kg pork, 4.4 kg beef, 4.4 kg pork fat, 1.25 kg cooked pork skin, salt, and nitrite. Using a Laska 60 L 51 bowl chopper with six knives. Casing 58 mm cellulose. Fermentation at 25°C and later 18°C for 4 days, cold smoke, and 52 drying at 16°C, 82% relative humidity to 80% of original stuffed weight. S53 ** Machine setting scale 1–100. N54 40454 21 217-226 r1.qxd 7/20/07 1:44 PM Page 224

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1 relations of the factors to the binding and structure These comments illustrate the possible interactions 2 of the semidried products produced are complex among the different factors, where changing one 3 because of interactions and because the models used parameter can influence other parameters. The 4 were not decisive enough to optimize all the parame- reader should also keep in mind that processing 5 ters. In any case, as will be discussed below, these parameters not found to have a major effect in that 6 parameters can be used to provide certain guidelines. study (distance of the knives to the chopping bowl, 7 For example, salt chopping time, which had the skin cooking time, and the type of skin used) might 8 greatest effect on the variance of structure parame- be influenced by the type of equipment used in other 9 ters, is very important in providing a homogeneous processing plants (e.g., different ratio of chopping 10 batter. Mixing for 20 seconds was insufficient to knives speed to bowl chopper speed). 11 obtain a good distribution of the salt and resulted in a 12 large variation in the Young’s modulus compared to 13 mixing for 60 seconds. A higher lean meat tempera- EFFECTS OF PRODUCT 14 ture (−2°C), a higher initial fat temperature (−10°C), MODIFICATION WITH NONMEAT 15 blunt knives, longer chopping time with salt, and 16 vacuum mixing after chopping (to remove trapped INGREDIENTS 17 air bubbles) resulted in improved binding leading to Several modifications, such as changing the fatty acid 18 generally higher fracture stress, Young’s modulus, composition, type of salt, and supplementary dietary 19 and hardness. However, sharp knives resulted in a fiber, have been reported. The area of substituting/ 20 better visual appearance, and because this is a very reducing fat and increasing the level of unsaturated 21 important factor in marketing such a product, sharp fatty acids to obtain a more favorable nutritional 22 knives would be preferred. In fact, the parameters profile of fermented sausage commonly produced 23 leading to improved binding could induce a water- with pork and/or beef fat has received most attention. 24 binding capacity of the mince proteins that is too Such modifications usually also affect the texture of 25 high, impeding the water release during the drying the final product because of differences in the viscos- 26 of the product. ity of the fats/oils, as well as different interactions 27 In a further study, van’t Hooft (1999) concentrated with the meat proteins. Warnants et al. (1998) manip- 28 more on the five major factors listed above and ulated the diet fed to pigs to increase the level of 29 added a few more suggestions: polyunsaturated fat. When the backfat was used to 30 produce dry salamis (drying up to 11 weeks), they 31 • Batter chopping time with salt: 60 to 80 seconds reported significant differences in the penetration 32 to obtain a sufficient protein exudate to bind lean force and sensory analysis (Table 21.3). The penetra- 33 meat particles. But avoid too much chopping tion force values of the first two diets were statistically 34 because this may cause high adhesiveness, lower than the last two diets, which contained a higher 35 which leads to greasiness and subsequently a level of unsaturated fat. The treatments of diets 3 and 36 weaker structure as well as too strong a water 4 were rejected by the sensory panel because of an 37 binding oily texture, and diet 4 was also reported to have a 38 • Vacuum mixing: apply to batters chopped for a noticeable fishy off-odor due to higher-than-normal 39 short time where it can help binding. However, in oxidation by products (see TBA values; Table 21.3). 40 appropriately chopped/prolonged mixed batters, Muguerza et al. (2001), replaced pork backfat 41 such vacuum mixing can result in greasiness and with 0, 10, 12, 20, 25, and 30% preemulsified olive 42 low firmness of the final product oil (oil:water:soy protein isolate 10:8:1) in a Chorizo 43 • Meat temperature: influenced by the chopping de Pamplona fermented dried Spanish sausage. In 44 process and is affecting the batter wall shear terms of nutritional content, cholesterol was lowered 45 stress. However, the relationships to the studied by 12–13% in the 20–25% replacement treatments, 46 processing parameters were not completely and about 22% in the 30% treatment. Oleic acid 47 clarified (18:1) was significantly higher (P Ͻ0.05) in the 48 • Fat temperature: above −7.5°C resulted in more 15–30% replacement treatments, and linoleic (18:2) 49 melted fat and required more salt-soluble proteins in the 10–25% replacements. All sausages with 50 exudate to stabilize the fat. Applying a longer 10–25% replacements were acceptable by a sensory 51 chopping time and/or salt chopping time will taste panel. The texture and color were comparable 52 raise the amount of exudate, but increase water with that of a commercial formulation. However, it 53S binding and impede the drying process. should be noted that the hardness, gumminess, and 54N 40454 21 217-226 r1.qxd 7/20/07 1:44 PM Page 225

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chewiness of the control product (no fat substitution) The sensory quality of sausages with mixed salt 1 were higher than in the replacement treatments. The treatment was lower, but were classified acceptable. 2 authors concluded that up to 25% replacement with The authors indicated that the lower pH value of the 3 preemulsified olive oil was possible, but a higher sausage with the salt mixture (pH 4.86 versus 5.04 4 level was unacceptable due to considerable fat drip- for the control) could have resulted in greater protein 5 ping during ripening. denaturation and decreased binding capacity of the 6 In a fermented and cooked summer sausage, meat proteins, which would explain the results of the 7 Shackelford et al. (1990) reported that high content salt mixture. 8 of oleic acid in the pork fat (achieved by replacing 9 the corn and soy bean diet with 10% safflower oil, SUMMARY 10 sunflower oil, or canola oil) resulted in lower sensory 11 panel scores for springiness, cohesiveness, texture, The complex and long processing procedure of fer- 12 and overall palatability (P Ͻ 0.05) of the 25% fat mented meat products, compared to products such as 13 level treatment. When 15% fat level was used, the bologna and cooked ham, requires close attention 14 high oleic acid treatments were comparable to the to details. The steps in the manufacturing of fer- 15 control for all sensory properties. The authors con- mented product involve acidification, drying, and 16 cluded that using high levels of mono unsaturated fat ripening that may take anywhere from a few days to 17 was less desirable in the conventional summer a few weeks and even months. The texture of the 18 sausage with 25% fat, but produced a highly palat- final products is therefore a combined result of the 19 able reduced fat (15%) sausage, which provided a ingredients and various processing parameters used, 20 reduction of 13% in the saturated fatty acids. as described in this chapter. Furthermore, the inter- 21 The use of soluble dietary fibers (inulin; a blend of action among the different ingredients (meat and 22 fructose polymers extracted from plant material) as a nonmeat), product’s dimension (e.g., stuffing diame- 23 fat substitute has also been tried in dry-fermented ter), and processing conditions after stuffing (e.g., 24 sausages (Mendoza et al. 2001). The idea was to add drying temperature, relative humidify) also play an 25 an ingredient that can help counteract the increase in important role in the texture and acceptability of the 26 hardness due to fat reduction and also help raise final product. Manufacturing high-quality fermented 27 dietary fibers in the diet. Adding inulin at 6, 7, 10, product requires good understanding of such factors 28 and 11.5% to a low-fat (13.5% as compared to a and their interactions. 29 high-fat with 31% fat) naturally fermented sausage 30 (i.e., without a starter culture), resulted in an overall REFERENCES 31 improvement in sensory properties due to the soft 32 texture and tenderness; values were similar to the S Barbut. 2005. Effects of chemical acidification and 33 conventional high-fat control product. Texture profile microbial fermentation on the rheological properties 34 analysis showed a similar trend. On the other hand, a of meat products. Meat Sci 71:397–401. 35 fat reduction by itself (31.0 to 13.5%) resulted in ———. 2006. Fermentation and chemical acidification 36 higher hardness as well as lower springiness and of salami-type products—Effect on yield, texture and 37 cohesiveness values. Overall, inulin addition helped microstructure. J Muscle Foods 17:34–42. 38 maintain a softer texture, which is more difficult to MJ Benito, M Rodriquez, MG Cordoba, MJ Andrade, JJ 39 maintain in low-fat dried products than in nondried Corboda. 2005. Effect of the fungal protease Epg222 40 products because of the moisture loss during the on proteolysis and texture in the dry fermented 41 long ripening process. sausage “Salchichon.” Sci Food Agric 85: 273–280. 42 Gimeno et al. (1999) reported on the effect of sub- S Dellaglio, E Casiraghi, C Pompei. 1996. Chemical, 43 stituting some of the 2.6% common salt (NaCl) in physical and sensory attributes for the characteriza- 44 Chorizo de Pamplona sausage with a mixture of tion of an Italian dry-cured sausage. Meat Sci 45 1.0% NaCl, 0.55% potassium chloride, and 0.74% 42:25–35. 46 calcium chloride in order to reach a mixture with the K Fretheim, B Egelandsdal, O Harbitz, K Samejima. 47 same ionic strength, but with 40% less sodium. They 1985. Slow lowering of pH induces gel formation of 48 reported that the salt mixture affected the texture myosin. Food Chem 18:169–178. 49 parameters, showing significantly lower hardness O Gimeno, D Ansorena, I Astiasarán, J Bello. 2000. 50 values (71 versus 62 N for the control and mixture, Characterization of Chorizo de Pamplona: Instru- 51 respectively), cohesiveness (0.54 vs. 0.44), gummi- mental measurements of colour and texture. Food 52 ness (38 vs. 27 N), and chewiness (25 vs. 17 Ncm). Chem 69:195–200. S53 N54 40454 21 217-226 r1.qxd 7/20/07 1:44 PM Page 226

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1 O Gimeno, I Astiasarán, J Bello. 1999. Inﬂuence of par- ﬁbrillar protein at chilled temperatures. Meat Sci

2 tial replacement of NaCl with KCl and CaCl2 on tex- 42:3–13. 3 ture and color of dry fermented sausages. J Agric TJ Pond, DS Wood, IM Mumin, S Barbut, MW 4 Food Chem 47:873–977. Griffiths. 2001. Modeling the survival of Escherichia 5 A Haque, FK Richardson, ER Morris. 2001. Effect of coli O157:H7 in uncooked, semi-dry, fermented 6 fermentation temperature on the rheology of set and sausages. J Food Prot 64:759–766. 7 stirred yogurt. Food Hydrocol 15:593–602. RD Quinton, DP Cornforth, DG Hendricks, CP Bren- 8 AM Hermanson, O Harbitz, M Langton. 1986. Forma- nand, YK Su. 1997. Acceptability and composition of 9 tion of two types of gels from bovine myosin. J Sci some acidified meat and vegetable stick products. 10 Food Agric 37:69–84. J Food Sci 62:1250–1254. 11 E Mendoza, ML Garcia, C Casas, MD Selgas. 2001. C Severini, T De Pilli, A Baiano. 2003. Partial substitu- 12 Inulin as fat substitute in low fat, dry fermented tion of pork backfat with extra-virgin olive oil in 13 sausages. Meat Sci 57:387–393. “salami” products: Effects on chemical, physical and 14 JG Montejano, DD Hamann, TC Lanier. 1984. Ther- sensorial quality. Meat Sci 64:323–331. 15 mally induced gelation of selected comminuted SD Shackelford, MF Miller, KD Haydon, JO Reagan. 16 muscle systems—Rheological changes during pro- 1990. Evaluation of the physical, chemical and sen- 17 cessing; final strengths and microstructures. J Food sory properties of fermented summer sausage made 18 Sci 49:146–152. from high-oleate pork. J Food Sci 55:937–941. 19 E Muguerza, O Gimeno, D Ansoreno, JG Bloukas, B-J van’t Hooft. Development of binding and structure 20 I Astiasarán. 2001. Effect of replacing pork backfat in semi-dry fermented sausages. 1999. PhD Thesis, 21 with pre-emulsified olive oil on lipid fraction and sen- Utrecht University, Utrecht, The Netherlands. 22 sory quality of Chorizo de Pamplona—A traditional N Warnants, MJ Van Oeckel, Ch V Boucque. 1998. 23 Spanish fermented sausage. Meat Sci 59: 251–258. Effect on incorporation of dietary polyunsaturated 24 TM Ngapo, BHP Wilkinson, F Chong. 1996. 1, fatty acids in pork backfat on the quality of salami. 25 5-Glucono-delta-lactone induced gelation of myo- Meat Sci 49:435–445. 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53S 54N 40454 22 227-240 r1.qxd 7/20/07 1:46 PM Page 227

1 2 3 22 4 5 6 Flavor 7 8 Karsten Tjener and Louise H. Stahnke 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION sensory methods. Gas chromatography-olfactometry 22 (GC-O), combining classical chromatography with 23 This chapter deals with flavor and flavor formation the detection system of the human nose, is one of the 24 in fermented sausage and dry-cured ham: the preferred approaches. The other prevailing approach 25 description of flavor compounds of relevance to the is correlation of sensory profile and amount of spe- 26 overall sensation of fermented sausage and dry- cific volatiles. In principle, it is also possible to com- 27 cured ham, the pathways through which the com- pare the amount of a given volatile with the sensory 28 pounds are formed, and finally how to impact the threshold value in a similar matrix, but the approach 29 flavor formation processes in relation to dry-cured suffers from the lack of threshold values obtained in 30 ham and fermented sausage production. relevant matrices as well as quantitative determina- 31 Flavor is defined as the total impression of taste, tion of volatiles in fermented sausages and dry- 32 odor, tactile, kinesthetic, temperature, and pain cured hams. 33 sensations perceived through tasting (Rothe 1972; 34 ISO 5492:1992). However, only taste compounds, Dry-cured Ham 35 defined as molecules with the ability to trigger a 36 In a series of studies, GC-O was applied to identify response in the taste buds, and aroma (odor) com- 37 the odor-active compounds of Italian and Spanish pounds, defined as volatiles with the ability to reach 38 dry-cured hams (Flores et al. 1997; Blank et al. and generate response in the receptors of the olfac- 39 2001; Carrapiso et al. 2002a,b; Carrapiso and Garcia tory epithelium, will be treated. 40 2004). The studies report from 15 to 44 odorous 41 volatiles belonging to a variety of chemical classes, 42 IMPORTANT AROMA such as alcohols, aldehydes, acids, esters, ketones, 43 COMPOUNDS aromatic, sulfur and nitrogen compounds. Table 22.1 44 summarizes the odorous volatile compounds identi- IDENTIFICATION 45 fied in those five studies. 46 Approximately 400 volatiles have been identified in In Parma ham methional (potatolike), (E)-2- 47 fermented sausage (Stahnke 2002); from dry-cured nonenal (fatty, leatherlike), and sotolone (seasoning- 48 ham the number is approximately 200 (Ruiz et al. like) were reported to be the most potent odorants, 49 2002). However, many of those volatiles do not con- followed by 3-methylbutanoic acid (sweaty, musty), 50 tribute to dry-cured ham or fermented sausage p-cresol (phenolic, musty) and phenylacetic acid 51 aroma because of high sensory threshold values. (honeylike, spicy) (Blank et al. 2001). The most 52 Identification of the more important aroma com- potent odorants found in three studies of Iberian 53 pounds requires combination of analytical and ham were hexanal (green), (Z)-3-hexenal (acornlike), S54 N55 227 40454 22 227-240 r1.qxd 7/20/07 1:46 PM Page 228

1 Table 22.1. Odor-active aroma compounds in Italian and Spanish dry-cured hams. 2 3 Compound Descriptor Source 4 Alcohols 5 1-Penten-3-ol Onion, toasted 1 6 1-Octen-3-ol Mushroom, rustlike 3,4,5 7 3-Methyl-1-butanol Green 1 8 3-Methyl-2-hexanol Potato, wheat 1 9 10 Aldehydes 11 Propanal Almondlike, green 4 12 Pentanal Nutty, toasted, fruity 5 13 Hexanal Green 1,3,4,5 14 Heptanal Fruity, fatty 4,5 15 Octanal Green-fresh 1,3,4,5 16 Nonanal Green 1 17 (E)-2-Hexenal Olive oil-like 3,4,5 18 (Z)-3-Hexenal Acornlike 3,5 19 (E)-2-Heptenal Almondlike, fruity, fried foodlike 5 20 (E)-2-Octenal Fruity, rancid 4,5 21 (E)-2-Nonenal Fatty, leatherlike 2 22 2-Methylpropanal Nutty, toasted, pungent 3,4,5 23 3-Methylbutanal Cheesy-green, malty, nutty, toasted 1,3,4,5 24 2-Methylbutanal Rancid, almondlike, toasted 5 25 Acids 26 Acetic acid Acidic, vinegar 1,2 27 Butanoic acid Sweaty 2 28 3-Methylbutanoic acid Sweaty, musty 2 29 30 Esters 31 Methyl butanoate Sweet-caramel 1 32 Methyl hexanoate Boiled meat 1 33 Methyl 2-methylpropanoate Fruity 1 34 Ethyl butanoate Fruity 1 35 Ethyl 2-methylpropanoate Fruity 4,5 36 Ethyl 2-methylbutanoate Fruity 1,3,5 37 Ethyl 3-methylbutanoate Fruity 2 38 Ketones 39 2-Pentanone Green, fruity, tropical 4,5 40 2-Hexanone Floral-apple 1 41 2-Heptanone 4,5 42 6-Methyl-5-heptan-2-one Citrus-candy 1 43 1-Penten-3-one Rotten, sewerlike, fruity 3,5 44 1-Octen-3-one Mushroomlike 2,3,4,5 45 3-Hydroxy-2-butanone Fruit-red Jell-o 1 46 2,3-Butanedione Buttery, caramellike 1,4,5 47 Aromatic Compounds 48 Phenylacetaldehyde Honeylike 2 49 Phenylacetic acid Honeylike, spicy 2 50 p-Cresol Phenolic, musty 2 51 o-Xylene Sweet-fruit candy 1 52 53S 54N

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Table 22.1. (Continued) 1 2 Compound Descriptor Source 3 4 m-Xylene Smoked-phenolic 1 5 p-Xylene Smoked-phenolic 1 6 Sulfur Compounds 7 Hydrogen sulfide Boiled or rotten eggs, sewagelike 3,4,5 8 Methanethiol Rotten eggs or meat, sewagelike, cheesy 3,4,5 9 Dimethyldisulfide Dirty socks 1 10 Dimethyltrisulfide Rotten egglike, burnt 5 11 Methional Potatolike 2,5 12 3-Mercapto-2-pentanone Fruity, sewagelike, fatty, cured hamlike 4,5 13 2-Methyl-3-furanthiol Cured hamlike, toasted, nutty 3,4,5 14 2-Furfurylthiol Boiled meatlike, cured hamlike 5 15 Nitrogen Compounds 16 2-Acetyl-1-pyrroline Roasty, overheated meatlike, cured hamlike 2,3,4,5 17 2-Propionyl-1-pyrroline Stewlike, boiled meatlike, rancid 3,4,5 18 Methylpyrazine Nutty 1 19 2,6-Dimethylpyrazine Toasted nut 1 20 1(H)-Pyrrole Meaty 1 21 Miscellaneous 22 4-Hydroxy-2,5-dimethyl-3(2H)-furanone Caramellike, sweet 2 23 2-Butoxyethanol Dark, toast-meaty 1 24 2-Pentylfuran Hamlike 1 25 Sotolone Seasoninglike 2 26 27 Sources: (1) Flores et al. (1997); (2) Blank et al. (2001); (3) Carrapiso et al. (2002a); (4) Carrapiso and Garcia 28 (2004); (5) Carrapiso et al. (2002b). 29 30 31 32 3-methylbutanal (malty, nutty, toasted), 1-octen-3- sensory profiling, cured flavor was correlated to 33 one (mushroom), 1-octen-3-ol (mushroom, rustlike), 2-methylbutanal (Ruiz et al. 1999), methyl ketones, 34 hydrogen sulfide (boiled or rotten eggs, sewagelike), and straight-chain alcohols (Buscailhon et al. 1994), 35 methanethiol (rotten eggs or meat, sewagelike) and respectively. The authors Hinrichsen and Pedersen 36 2-methyl-3-furanthiol (nutty, dry-cured hamlike, (1995) found a correlation between nutty, cheesy, and 37 toasted) (Carrapiso et al. 2002a,b; Carrapiso and salty flavor notes and the methyl-branched aldehydes, 38 Garcia 2004). The meaty-smelling compounds secondary alcohols, methyl ketones, ethyl esters, 39 2-methyl-3-furanthiol, 2-furfurylthiol, 3-mercapto-2- and dimethyltrisulfide. Finally, aged odor was 40 pentanone, 2-acetyl-1-pyrroline, and 2-propionyl-1- correlated to methyl-2-methylpropanoate, ethyl-2- 41 pyrroline were all detected by Carrapiso et al. methylbutanoate, 2-methyl-3-buten-2-ol, 3-methyl- 42 (2002b). They all have very low odor thresholds 1-butanol, and 3-hydroxy-2-butanone (Careri et al. 43 (Rychlik et al. 1998) and may contribute signifi- 1993). 44 cantly to dry-cured ham aroma although present in 45 Fermented Sausage low amounts. Meaty aroma compounds found in Ser- 46 rano dry-cured ham were identified as 2-pentylfuran Table 22.2 compiles the odorous volatiles detected 47 (hamlike), 1-H-pyrrole (meaty) and 2-butoxyethanol by GC-O in fermented sausages. Compared to 48 (dark toast, meaty), and the latter two further dry-cured ham (Table 22.1), more acids, sulfides, 49 correlated with the pork flavor detected by sensory terpenes, and phenolic compounds seem to be 50 evaluation (Flores et al. 1997). In studies where important in sausages. The presence of sulfides is 51 instrumental flavor analysis was combined with closely linked to garlic (Mateo and Zumalacárregui 52 S53 N54 40454 22 227-240 r1.qxd 7/20/07 1:46 PM Page 230

1 Table 22.2. Odor-active aroma compounds in European dry-fermented sausages, with or without mold 2 coverage, smoked or nonsmoked. 3 Compound Descriptor Source 4 5 Alcohols 6 1-Octene-3-ol Mushroom 1,3 7 8 Aldehydes 9 Acetaldehyde White glue, green 1,5,7 10 Pentanal Synthetic, green leaves 4 11 Hexanal Green 1,3,4,5,6,7,8 12 Heptanal Pelargonium, potatoes, green leaves 1,3,5 13 Octanal Orange, spicy, pelargonium 1,4,5,7 14 Nonanal Herb, geranium, dry, spicy, citrus 4,5,8 15 Decanal Cucumber, cardboard, dry grass 4 16 (Z)-x-Hexenal Old cooked potatoes, nauseous 5 17 (Z)-4-Heptenal Unpleasant bread 5 18 (E)-2-Octenal Deep-fried, musty 5,8 19 (E)-2-Nonenal Fresh, cucumber 4,5 20 (E)-2-Decenal Deep-fried 1 21 (E,E)-2,4-Nonadienal Fatty 8 22 (E,E)-2,4-Decadienal Deep-fried fat 4 23 (E,Z)-2,4-Decadienal Rancid, fatty, tallowy 6,8 24 2-Methylbutanal Butter, caramel, rancid 3,4,5 25 3-Methylbutanal Sour cheese 4,5 26 3-Methylhexanal Sour, green, acetone 4,5 27 Acids 28 Acetic acid Vinegar 1,4,5,6,7 29 Propanoic acid Sour, sweaty 5,6 30 Butanoic acid Cheesy, sweaty 1,5,6,7 31 Pentanoic acid Sweaty 6 32 Hexanoic acid Sweaty, putrid 6 33 Decanoic acid Pungent, repellent 6 34 2-Methylpropanoic acid Cheesy, sweaty 5,6,7 35 3-Methylbutanoic acid Cheesy, sweaty socks 1,4,5,6,7 36 Esters 37 Methylbutanoate Fruity, ﬂowery 6 38 2-Methylpropylacetate Candy, fruit 4,5 39 Ethylacetate Artiﬁcial fruity 6 40 Ethylpropanoate Fruity 6 41 Ethylbutanoate Fruity, candylike 2,4,5,6,7 42 Ethylpentanoate Fruit 4,5 43 Ethyloctanoate Fatty, green 8 44 Ethyl-2-methylpropanoate Fruity, candylike, pineapple 2,4,5,7,8 45 Ethyl-2-methylbutanoate Fruity, candylike, pineapple 2,4,5 46 Ethyl-3-methylbutanoate Fruity, candylike, chutney 2,5,6,8 47 Ethyl lactate (ethyl-2-hydroxypropanoate) Chutney, sweet 1 48 Propylacetate Sourish apple, candy 4 49 50 Ketones 51 2-Heptanone Pelargonium, nettle, fruity 5 52 2-Nonanone Plant, herb, weak fruity 4,6 53S 3-Octen-2-one Hot soil, mushroom 5 54N

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Table 22.2. (Continued) 1 2 Compound Descriptor Source 3 1-Octene-3-one Mushroom 7,8 4 2,3-Butadione Butter strong 1,2,4,5,6,7 5 2,3-Pentadione Butter weak 5 6 3-Hydroxy-2-butanone Butter 6 7 8 Aromatic Compounds 9 Phenylacetaldehyde Honey 1 10 2-Phenylethanol Flowery, rose 6 11 2-Methoxyphenol (guiaicol) Smoked, bandage, phenolic 1,5,7 12 2-Methoxy-4-methylphenol (methyl guiaicol) Smoked, car tire 1 13 p-Cresol Musty, smoky 6 14 Naphthalene Roses, soap 1 15 Eugenol Cloves, spicy, smoky 6 16 Sulfur Compounds 17 Methanethiol Cabbage, feces, putrid 1,2,7 18 Allyl-1-thiol Salami, onion, cured meat 1,2,7 19 Furfuryl thiol Burned coffee 2 20 Methylthiirane Salami, onion 1,2,7 21 3-Methylthio-1-propene Gas, onion, unpleasant 3 22 Methional Cooked potato 1,7,8 23 Dimethylsulfide Sulfur, putrid 7 24 Dimethyldisulfide Garlic 6 25 Dimethyltrisulfide Cabbage, metallic 1,7 26 Methylallylsulfide Strong garlic 1,2,6,8 27 Methylallyldisulfide Garlic 6 28 Diallylsulfide Garlic 6 29 30 Nitrogen Compounds 31 x-Pyridineamine Cooked meat 1 32 2-Acetyl-1-pyrroline Popcorn 2,7,8 33 Dimethylpyrazine Coffee, toasted 5 34 Terpenes 35 β-Myrcene Herbal 6 36 Limonene Fresh 6 37 γ-Terpinene Herbal 6 38 α-Pinene Peppery 6,8 39 β-Pinene Sharp, dry 6 40 4-Terpineol Greenish 6 41 1,8-Cineol Menthol 6 42 Terpinolene Fruity, eucalyptus 3 43 Linalool Flowery, heavy 6,8 44 45 Sources: (1) Stahnke (1998); (2) Stahnke (2000); (3) Meynier et al. (1999); (4) Stahnke (1994); (5) Stahnke (1995a); 46 (6) Schmidt and Berger (1998); (7) Stahnke et al. (1999); (8) Blank et al. (2001). 47 48 1996), terpenes to pepper (Berger et al. 1990), and or microbiological processes occurring during 49 the phenolic compounds to smoke (Hollenbeck ripening. 50 1994). Thus, many of the compounds detected in In a study of French, Italian, Spanish, and German 51 fermented sausage but not in dry-cured ham were sausages, Schmidt and Berger (1998) found dial- 52 “added” to the sausage and not the result of chemical lyldisulfide (garlic), eugenol (cloves, spicy, smoky), S53 N54 40454 22 227-240 r1.qxd 7/20/07 1:46 PM Page 232

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1 3-methyl butanoic acid (sweaty), acetic acid (vinegar), mature flavor with methyl ketones, methyl-branched 2 linalool (flowery, heavy), methylallyl sulfide (garlic), aldehydes and some allylic sulfur compounds 3 and diallyl sulfide (garlic) to be the most important (Stahnke et al. 2002). Volatile analysis combined 4 odor compounds. In Italian-type salami hexanal with sensory assessment was also used to identify 5 (green), methional (cooked potato) and 2-acetyl-1- volatiles linked to other odor notes. Buttery odor 6 pyrroline (roasty, popcorn) were identified as the was correlated to the presence of diacetyl (Berdagué 7 most potent odorants followed by ethyl-2-methyl- et al. 1993b; Viallon et al. 1996), cheesy odor with 8 propanoate (fruity), ethyl-3-methylbutanoate (fruity, 2-methyl propanoic acid and 2- and 3-methylbu- 9 sweet), α-pinene (pepperlike), 1-octen-3-one (mush- tanoic acid (Stahnke 1995a; Viallon et al. 1996), ran- 10 room), and (E)-2-octenal (musty) (Blank et al. 2001). cid flavor to straight-chain aldehydes (Stahnke 1994, 11 Based on GC-O studies of two Northern (smoked) 1995a; Stahnke et al. 1999), and finally sourish and 12 and two Mediterranean (nonsmoked, mold- acid notes to acetic, butanoic, and hexanoic acids 13 fermented) fermented sausages, Stahnke et al. (1999) (Stahnke 1995a, 1999). The authors Stahnke et al. 14 concluded that the basic dried sausage odor was (1999) also correlated garlic flavor with allylic sulfur 15 composed of the following components: methanethiol compounds, smoked flavor with aromatic and O- 16 (cabbage, putrid), methional (cooked potato), heterocyclic compounds, and spicy/piquant flavors 17 dimethylsulfide (sulfur, putrid), dimethyltrisulfide with terpenes. 18 (cabbage, metallic), diacetyl (butter), ethylbutanoate In summary, the main constituents of dry-cured 19 (fruit), ethyl-2-methylpropanoate (fruit), acetalde- ham and fermented sausage odor are straight- and 20 hyde (glue, yogurt), acetic acid (vinegar), butanoic branched-chain aldehydes; acids; their methyl- and 21 acid (cheese), 2-methylpropanoic acid (cheese), ethyl esters; aromatic, heterocyclic, and sulfur com- 22 3-methylbutanoic acid (sweaty socks), guaiacol (phe- pounds; methyl ketones; and terpenes. 23 nolic), hexanal (green), octanal (orange), 1-octen-3- 24 one (mushroom), and several unidentified compounds FORMATION 25 (pelargonium, cooked meat, sulfur, onion, dry, rub- 26 ber, earthy, deep-fried, cress, cucumber). In the same This section shortly describes the origin of some of 27 study, the Northern European sausages differed from the most important groups of aroma compounds. 28 the Mediterranean molded sausage types by a pop- Detailed reaction schemes are outside the scope of 29 corn note in the latter and coffee/roasted, phenolic, this chapter and will not be shown. 30 sulfur, and vinegar notes in the former. The Straight-chain alcohols, aldehydes, and to some 31 coffee/roasted note was ascribed to 2-furfuryl thiol, extent also the acids are formed by autoxidation of 32 the phenolic note to guaiacol, the vinegar note unsaturated fatty acids. The complex chemistry of 33 to acetic acid, and the popcorn note to 2-acetyl-1- the various autoxidation processes is thoroughly 34 pyrroline. The sulfur note remained unidentified. reviewed by several researchers (Love and Pearson 35 Another study on 10 different Northern and Southern 1971; Frankel 1980; Ladikos and Lougovois 1990) 36 European sausages confirmed the differences and will not be treated further here. 37 found by Stahnke et al. (1999) and additionally Methyl ketones can be formed by autoxidation of 38 showed that 2-acetyl-pyrroline was mainly unsaturated fatty acids, but microbial β-oxidation is 39 present close to the edge of the molded sausages also possible and would explain the correlation 40 (Stahnke 2000). between the presence of staphylococci and elevated 41 The GC-O studies from which Table 22.2 was put levels of methyl ketones observed in several studies 42 together revealed only two components with odor (Berdagué et al. 1993b; Stahnke 1995a; Montel et al. 43 descriptors directly linked to fermented sausage, 1996). Furthermore, Engelvin et al. (2000) discov- 44 namely allyl-1-thiol (salami, cured meat) and its ring ered the β-oxidation pathway in S. carnosus. 45 closure methyl-thiirane (salami, onion) (Stahnke As mentioned above, the straight-chain acids are 46 1998, 2000; Stahnke et al. 1999). However, by com- to some extent formed by autoxidation of unsatu- 47 bination of volatile and sensory analyses it was rated fatty acids. However, the C2–C6 acids are also 48 shown that salami odor was correlated with the pres- derived from microbial degradation of pyruvate. 49 ence of ethyl esters and methyl ketones (Stahnke Lactic acid bacteria, staphylococci, and yeasts are all 50 1994, 1995a; Stahnke et al. 1999) as well as the capable of producing short straight-chain acids 51 methyl-branched aldehydes 2-methyl propanal and (Gottschalk 1986; Westall 1998; Olesen and Stahnke 52 2- and 3-methylbutanal (Stahnke 1994, 1995a; 2000; Beck et al. 2002). 53S Hagen et al. 1996). Likewise, cured odor correlated Esters are generated by reaction of an alcohol and 54N with methyl ketones (Berdagué et al. 1993b) and a carboxylic acid (or acyl-CoA). The amount of ester 40454 22 227-240 r1.qxd 7/20/07 1:46 PM Page 233

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present in fermented meat is strongly correlated to SUGARS 1 the amount of precursor alcohol, precursor acid, or 2 Glucose, sucrose or more complex sugars are typi- both (Stahnke 1994, 1995b, 1999a; Montel et al. 3 cally added to facilitate acid production by lactic 1996; Tjener et al. 2004c). The origin of esters is not 4 acid bacteria during fermented sausage processing. well established. LAB, Staphylococcus, yeast, and 5 When sugars are added in excess, the residual mold can form esters (Stahnke 2002), but chemical 6 amount will contribute to sweet taste in the final formation is also possible, in particular when acyl- 7 sausage, given that they are present in levels above CoAs are involved (Yvon and Rijnen 2001). 8 their sensory threshold. Threshold values of glucose The methyl-branched aldehydes 2-methylpropanal, 9 and sucrose are 1.4 and 0.6%w/ , respectively (Plattig 2-methylbutanal, and 3-methylbutanal and the corre- w 10 1984). Traditional manufacture of dry-cured hams sponding acids are primarily formed by degradation 11 does not include any addition of sugars, and any of the amino acids valine, isoleucine, and leucine, 12 sweet taste perceived from this product type is most respectively. In fermented sausage the formation of 13 likely caused by other compounds. the methyl-branched aldehydes and acids is ascribed 14 to staphylococci. By inactivation of the aminotrans- 15 ferase gene of S. carnosus Madsen, Beck et al. ATP DERIVATIVES 16 (2002) showed that transamination is the first step in Among the ATP derivatives, IMP (inosine monophos- 17 the degradation of the branched-chain amino acids. phate) is the dominant component in meat postrigor 18 The second step is presumably a decarboxylation (Lawrie 1995), but it is gradually converted into ino- 19 reaction whereby the methyl-branched aldehyde is sine and further to hypoxanthine during ripening of 20 formed, as showed by Beck et al. (2002). Subse- fermented sausage and dry-cured ham (Dierick et al. 21 quently, the aldehyde may be either reduced or, as in 1974; Kohata et al. 1992; Mateo et al. 1996). In ready- 22 the study by Beck et al. (2002), oxidized into the cor- to-eat Chorizo, the level of IMP (0.007–0.13 ␮mol/g) 23 responding acid. was below the umami taste threshold, and the level of 24 The allylic sulfur compounds listed in Table 22.2 its predominant derivative, hypoxanthine (6.9–8.7 25 most likely originate from garlic (Schmidt and ␮mol/g), was considered being negligible to the taste 26 Berger 1998), whereas methional, methanethiol, of Chorizo (Mateo et al. 1996). However, in another 27 dimethyldisulfide, and dimethyltrisulfide are proba- study, lower levels of hypoxanthine (0.5–1.5 ␮mol/g) 28 bly derived from methionine through the same path- were found to correlate with bitter taste of aged pork 29 ways as demonstrated in cheese ripening cultures meat (Tikk et al. 2005). 30 (Stahnke 2002). 31 The aromatic compounds phenylacetaldehyde, 32 ORGANIC ACIDS phenylacetic acid, p-cresol, guiaicol, and methyl- 33 guiaicol are possibly of microbial origin, although L- and D-lactic and acetic acids are the predominant 34 the pathways are unrevealed in meat cultures acids contributing to the sour taste of fermented 35 (Stahnke 2002). However, the phenolic compounds sausages (Montel et al. 1998; Stahnke 2002). The 36 listed in Table 22.2 may also arise from smoke. total lactic acid level varies from 0.4–3.1 g/100 g dry 37 matter among a wide range of Northern European 38 and Mediterranean sausage types (Vandekerckhove 39 IMPORTANT TASTE COMPOUNDS and Demeyer 1975; Johansson et al. 1994; Dellaglio 40 The basic taste sensations salt, sweet, sour, bitter, et al. 1996; Mateo et al. 1996; Demeyer et al. 2000; 41 and umami are triggered by a diverse group of mole- Bruna et al. 2000a). Typically, the level of acetic acid 42 cules. This group includes inorganic salts, sugars, is ten- to twentyfold lower than the level of lactic 43 ATP (adenosine triphosphate) derivatives, organic acid (Johansson et al. 1994; Mateo et al. 1996; 44 acids (of which some are volatile), free amino acids, Demeyer et al. 2000). The presence of C3–C6 45 and smaller peptides (Mateo et al. 1996). straight-chain acids is sometimes reported, but they 46 are unlikely to contribute significantly to the sour 47 taste due to low abundance (100–1000-fold less than 48 SALT acetic acid [Dainty and Blom 1995]). However, the 49 The salty taste in dry-cured ham and fermented studies by Bruna et al. (2000a, 2001, 2003) showed 50 sausage is mainly caused by sodium chloride. Typi- that propionic acid level of the same magnitude as 51 w cal levels in final products are 4.7–8.7% /w and lactic acid is obtainable by surface inoculation 52 w 3.5–4.6% /w for dry-cured ham and fermented with Penicillium aurantiogriseum or Penicillium S53 sausage, respectively (Wirth 1989; Toldrá 2002). camemberti. N54 40454 22 227-240 r1.qxd 7/20/07 1:46 PM Page 234

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1 FREE AMINO ACIDS AND PEPTIDES Isabel et al. 1999; Cava et al. 1999, 2000; Cava and 2 Ventanas 1999; Timon et al. 2002; Virgili and The degradation of protein into smaller peptides and 3 Schivazappa 2002) influence those measures. The free amino acids has been extensively studied in fer- 4 combination of the level of curing salts and the mented sausages as well as dry-cured hams, and 5 process parameters time, temperature, and humidity excellent reviews exist for both product types 6 is decisive for the chemical, enzymatic, and micro- (Toldrá et al. 1997; Toldrá and Flores 1998; Ordonez 7 biological processes leading to flavor formation et al. 1999; Sanz et al. 2002). 8 in dry-cured ham. The effect of salt, temperature, The free amino acids and smaller peptides may 9 etc., on enzymatic activity is extensively covered trigger sweet, sour, bitter, and umami taste when sur- 10 (Sarraga et al. 1989; Toldrá 1992, 2002; Motilva and passing their respective taste threshold values. 11 Toldrá 1993; Toldrá et al. 1997; Toldrá and Flores Mateo et al. (1996) determined the content of the 12 1998), and also the effect on chemical processes individual free amino acids in Chorizo and predicted 13 such as autoxidation is described in some detail glutamic acid to produce umami response, leucine a 14 (MacDonald et al. 1980; Coutron-Gambotti et al. bitter response, alanine a sweet response, and valine 15 1999; Andres et al. 2004). However, microbial activ- and lysine both sweet and bitter responses in this 16 ity in dry-cured ham as affected by processing product. Henriksen and Stahnke (1997) studied 17 parameters is not well studied. Although meat starter water-soluble fractions from different sausage types 18 cultures are applied in the dry-cured ham processing and found correlation between sour taste and glu- 19 industry, the effect of such starter cultures on flavor tamic acid; between bitterness and lysine, valine, 20 formation is scarcely researched. leucine, isoleucine, and proline; and between bouil- 21 In summary, the desired flavor of dry-cured ham is lon taste and a mixture of different amino acids and 22 obtained by choosing the right meat quality (feed, smaller peptides with high content of methionine, 23 breed, age, sex), the right processing conditions, and phenylalanine, alanine, glycine, glutamic acid, argi- 24 curing ingredients to control the enzymatic and nine, and histidine residues. 25 microbial activity, and finally to choose the right It is well established that glutamate acts as a flavor 26 (if any) adjunct culture. enhancer giving umami taste (Kato et al. 1989). In 27 Iberian ham, sodium glutamate together with small, 28 bitter peptides are thought to be the main nonvolatile FERMENTED SAUSAGE 29 contributors to the strong characteristic flavor of this 30 A fundamental difference between fermented saus- product (Ruiz et al. 2002). In other dry-cured hams, 31 ages and dry-cured ham is that sausage processing the bitter taste was related to high amounts of 32 allows the distribution of ingredients, including lipophilic amino acids and lipophilic oligopeptides 33 starter cultures, enzymes, flavor precursors, and (Sforza et al. 2001) and to elevated levels of methion- 34 cofactors, inside the product and not only on the sur- ine, asparagine, and isoleucine (Virgili et al. 1998). 35 face. Consequently, the microflora of fermented Based on peptide composition, it was predicted that 36 sausages has much better opportunities to influence various dipeptides could contribute to sour, bitter, 37 flavor formation compared to the microflora of dry- and umami taste of Serrano ham (Sentandreu et al. 38 cured ham. Fermented sausage flavor is probably 2003). However, in the same study it was also 39 affected by meat quality in a similar way as des- stressed that the free amino acids were more impor- 40 cribed for dry-cured ham. Likewise, the processing tant to dry-cured ham flavor as compared to peptides. 41 conditions (temperature, time, relative humidity) 42 influence the endogenous meat enzyme activities 43 HOW TO CHANGE FLAVOR and chemical reactions by the same mechanisms as 44 in hams. However, the processing conditions do, as DRY-CURED HAM 45 other environmental factors, also influence micro- 46 In principle, dry-cured ham flavor is determined bial activity and thus microbial flavor formation. 47 by meat quality, processing conditions, curing Before changing process parameters one should 48 ingredients, and adjuncts. The fatty acid composi- therefore consider the effect on chemical, enzy- 49 tion and the ratio of various proteolytic, peptidolytic, matic, as well as microbiological processes. 50 and lipolytic enzymes in muscle and adipose tissue The impact of growth parameters on the activity of 51 are some of the important measures of meat quality, lactic acid bacteria is described in Chapter 18, “Influ- 52 and numerous studies report that breed (Berdagué ence of Processing Parameters on Cultures Perfor- 53S et al. 1993a; Antequera et al. 1994; Gou et al. 1995; mance,” and will not be treated further here. However, 54N Cava et al. 2004) and feed (Manfredini et al. 1992; several studies focus on the aroma formation by 40454 22 227-240 r1.qxd 7/20/07 1:46 PM Page 235

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staphylococci as affected by ripening time (Mateo affected the staphylococci’s ability to degrade 1 and Zumalacárregui 1996; Sunesen et al. 2001; methyl-branched amino acids in a reaction medium. 2 Ansorena et al. 2000; Misharina et al. 2001; Tjener et The authors studied the effect of growth phase, tem- 3 al. 2003, 2004b; Olesen et al. 2004) and various envi- perature, NaCl concentration, and concentration of 4 ronmental factors, such as temperature (Stahnke leucine, isoleucine and valine and found that all 5 1995b, 1999b; Tjener et al. 2004c), salt (Stahnke parameters had a significant influence on the produc- 6 1995b; Olesen et al. 2004; Tjener et al. 2004c), tion of methyl-branched acids. Optimal production 7 nitrite, nitrate, and ascorbate (Stahnke 1995b, 1999b; of starter cultures, such as the staphylococci, may 8 Olesen et al. 2004), and pH (Stahnke 1995b; Tjener thus lead to enhanced flavor formation. 9 et al. 2004c). Finally, it is possible to change the flavor profile 10 The change of growth parameters and ripening by adding substrates or cofactors of specific path- 11 time, as described above, is one way to change flavor ways. Some examples are the addition of free amino 12 formation. Another strategy is to change microbial acids (Herranz et al. 2005) and α-ketoglutarate 13 composition within the sausage, e.g., from S. carno- (Herranz et al. 2003; Tjener et al. 2004a). The addi- 14 sus to S. xylosus. Generally, S. carnosus produces tion of proteolytic or lipolytic enzymes with the 15 more of the methyl-branched aldehydes and acids objective of generating flavor precursors is in this 16 than S. xylosus, S. warneri, S. saprophyticus, S. equo- context the same as adding the precursors directly. 17 rum, and K. varians (Berdagué et al. 1993b; Montel The addition of enzymes with the purpose of accel- 18 et al. 1996; Stahnke 1999a; Søndergaard and Stahnke erating flavor formation has been extensively stud- 19 2002; Olesen et al. 2004; Tjener et al. 2004c), whereas ied, and most of the work was reviewed by 20 S. xylosus normally produces more diacetyl than Fernandez et al. (2000). The authors concluded that 21 S. carnosus (Berdagué et al. 1993b; Montel et al. the level of flavor precursors by addition of prote- 22 1996; Stahnke 1999a; Søndergaard and Stahnke olytic or lipolytic enzymes did not necessarily lead 23 2002; Olesen et al. 2004; Tjener et al. 2004c). to increased flavor formation unless the formation of 24 The use of yeast, streptomyces, or molds is yet flavor compounds from their precursors is acceler- 25 another approach reported to influence sausage fla- ated as well. 26 vor (Eilberg and Liepe 1977; Bruna et al. 2000b, In summary, fermented sausage flavor depends on 27 2003; Olesen and Stahnke 2000; Lopez et al. 2001; raw materials, processing parameters, type and 28 Flores et al. 2004; Hierro et al. 2005). amount of starter culture, the conditions at which the 29 Another strategy is to change the inoculation level starter culture is produced, the use of adjunct cul- 30 of, e.g., staphylococci. In one study, low inoculum of tures as yeast, mold and streptomyces, and addition 31 S. carnosus resulted in a low level of, e.g., methyl- of substrates or cofactors of specific pathways. 32 branched aldehydes, whereas high inoculum resulted 33 in a high level of methyl-branched aldehydes. The PERSPECTIVES 34 effect was more pronounced in the beginning of 35 the ripening phase and was almost leveled out after As shown in the previous sections, considerable 36 3 weeks of ripening. Thus, the strategy seems most knowledge on the identity and the formation of flavor 37 valuable for fast-ripened sausages (Tjener et al. compounds in fermented and dry-cured meat prod- 38 2004b). ucts has been gathered. However, a number of topics 39 Often staphylococci grow very little or not at all still remain to be solved in order to completely under- 40 during sausage processing (Lücke 1998). The initial stand and control the flavor forming processes. Fur- 41 state of a culture, and thereby also the preinoculation ther research should be focused on two subjects 42 conditions, may therefore be very important for the primarily. First of all, after many years of research it is 43 following activity of this culture. Møller et al. (1998) still impossible to make a synthetic dry-cured ham or 44 showed that agitation during precultivation increased fermented sausage flavor by mixing the pure compo- 45 3-methyl butanol and 3-methyl butanal formation by nents, since all of the important components (volatiles 46 S. xylosus in minimal medium, and Masson et al. as well as nonvolatiles) and the concentrations at 47 (1999) demonstrated an increase in 3-methyl but- which they occur are still not known. Certainly more 48 anal and 3-methyl butanoic acid production by research is needed within this area in order to know 49 S. carnosus, when grown in complex compared to exactly which compounds to focus on. Second, in 50 defined medium prior to inoculation in reaction order to actively manipulate the concentrations of rel- 51 medium with leucine. Olesen and Stahnke (2003) evant flavor compounds, in this way controlling the 52 cultivated S. carnosus and S. xylosus under various flavor development, it is crucial to know their origin S53 conditions and investigated how this precultivation and pathway of formation. For several of the already N54 40454 22 227-240 r1.qxd 7/20/07 1:46 PM Page 236

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1 known compounds, the origin is speculated but still of Penicillium auratiogriseum to the volatile 2 not confirmed. Knowing exactly which pathways to composition and sensory quality of dry fermented 3 target is an excellent starting point for efficient devel- sausages. Meat Sci 59:97–107. 4 opment of new starter cultures or other ingredients ———. 2003. Changes in selected biochemical and 5 with improved flavor-forming capabilities. sensory parameters as affected by superficial inocu- 6 lation of Penicillium camemberti on dry fermented 7 REFERENCES sausages. Int J Food Microbiol 85:111–125. 8 S Buscailhon, JL Berdagué, J Bousset, M Cornet, 9 AI Andres, R Cava, J Ventanas, E Muriel, J Ruiz. 2004. G Gandemer, C Touraille, G Monin. 1994. Relations 10 Lipid oxidative changes throughout the ripening of between compositional traits and sensory qualities of 11 dry-cured Iberian hams with different salt contents French dry-cured ham. Meat Sci 37:229–243. 12 and processing conditions. 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1 SM Madsen, HC Beck, P Ravn, A Vrang, AM Hansen, JA Ordonez, EM Hierro, JM Bruna, L de la Hoz. 1999. 2 H Israelsen. 2002. Cloning and inactivation of a Changes in the components of dry-fermented sausages 3 branched-chain-amino-acid aminotransferase gene during ripening. Crit Rev Food Sci Nutr 39:329–367. 4 from Staphylococcus carnosus and characterization of KH Plattig. 1984. The sense of taste. In: JR Piggott, ed. 5 the enzyme. Appl Environ Microbiol 68: 4007–4014. Sensory Analysis of Foods. London: Elsevier 6 M Manfredini, A Badiani, N Nanni. 1992. Relationship Applied Science Publishers, pp. 1–22. 7 between dietary fat and quality characteristics of fat M Rothe. 1972. Zur terminologie der grundbegriffe der 8 and Parma-type ham in heavy pigs. Ital J Food Sci aromaforschung. Nahrung 16:473–481. 9 25–32. J Ruiz, E Muriel, J Ventanas. 2002. The flavour of Iber- 10 F Masson, L Hinrichsen, R Talon, MC Montel. 1999. ian ham. In: F Toldrá et al., ed. 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Meat Sci 25:241–249. 31 vica and Staphylococcus xylosus in cured meat model S Schmidt, RG Berger. 1998. Aroma compounds in fer- 32 systems. Int J Food Microbiol 42:101–117. mented sausages of different origins. Lebensm— 33 MC Montel, F Masson, R Talon. 1998. Bacterial role in Wiss Technol 31:559–567. 34 flavour development. Meat Sci 49:S111–S123. MA Sentandreu, S Stoeva, MC Aristoy, K Laib, 35 MC Montel, J Reitz, R Talon, JL Berdagué, AS Rousset. W Voelter, F Toldrá. 2003. Identification of small 36 1996. Biochemical activities of Micrococcaceae and peptides generated in Spanish dry-cured ham. J Food 37 their effects on the aromatic profiles and odours of a Sci 68:64–69. 38 dry sausage model. Food Microbiol 13:489–499. S Sforza, A Pigazzani, M Motti, C Porta, R Virgili, 39 MJ Motilva, F Toldrá. 1993. Effect of curing agents and G Galaverna, A Dossena, R Marchelli. 2001. 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different ingredient levels. Part II. Volatile compo- carnosus during sausage fermentation. Meat Sci 1 nents. Meat Sci 41:193–209. 67:711–719. 2 ———. 1998. Character impact aroma compounds in ———. 2004b. Growth and production of volatiles by 3 fermented sausages. Proceedings 44th ICoMST, Staphylococcus carnosus in dry sausages: Influence 4 Barcelona, Spain, 2, 786–787. of inoculation level and ripening time. Meat Sci 5 ———. 1999a. Volatiles produced by Staphylococcus 67:447–452. 6 xylosus and Staphylococcus carnosus during growth ———. 2004c. The pH-unrelated influence of salt, 7 in sausage minces. Part I. Collection and identifica- temperature and manganese on aroma formation by 8 tion. Lebensm—Wiss Technol 32:357–364. Staphylococcus xylosus and Staphylococcus 9 ———. 1999b. Volatiles produced by Staphylococcus carnosus in a fermented meat model system. Int J 10 xylosus and Staphylococcus carnosus during growth Food Microbiol 97:31–42. 11 in sausage minces. Part II. The influence of growth F Toldrá. 1992. The enzymology of dry-curing of 12 parameters. Lebensm—Wiss Technol 32:365-371. meat products. In: FJM Smulders, F Toldrá, J Flores, M 13 ———. 2000. 2-Acetyl-1-pyrroline—Key aroma com- Prieto, eds. New technologies for meat and meat prod- 14 pound in Mediterranean dried sausages. In: P ucts. Nijmegen, The Netherlands: Audet, pp. 209–231. 15 Schieberle, K-H Engel, eds. Frontiers of Flavour Sci- ———. 2002a. Manufacturing of dry-cured ham. In: F 16 ence. Garching, Germany: Deutsche Forschungsanstalt Toldrá, ed. Dry-Cured Meat Products. Trumbull, 17 für Lebensmittelchemie, pp. 361–365. Connecticut: Food & Nutrition Press, Inc., pp. 27–62. 18 ———. 2002. Flavour formation in fermented sausage. ———. 2002b. Effect of raw materials and processing 19 In: F Toldrá, ed. Research Advances in the Quality of quality. In: F Toldrá, ed. Dry-Cured Meat Products. 20 Meat and Meat Products. India: Research Signpost, Trumbull, Connecticut: Food & Nutrition Press, Inc., 21 pp. 193–223. pp. 189–210. 22 LH Stahnke, A Holck, A Jensen, A Nilsen, E Zanardi. F Toldrá, M Flores. 1998. The role of muscle proteases 23 2002. Maturity acceleration of Italian dried sausage and lipases in flavor development during the process- 24 by Staphylococcus carnosus—Relationship between ing of dry-cured ham. CRC Crit Rev Food Sci Nutr 25 maturity and flavor compounds. J Food Sci 67: 38:331–352. 26 1914–1921. F Toldrá, M Flores, Y Sanz. 1997. Dry-cured ham fla- 27 LH Stahnke, LO Sunesen, AD Smedt. 1999. Sensory vor: Enzymatic generation and process influence. 28 characteristics of European, dried, fermented Food Chem 4:523–530. 29 sausages and the correlation to volatile profile. Pro- P Vandekerckhove, D Demeyer. 1975. The composition 30 ceedings of the 13th Forum for Applied Biotechnol- of Belgian dry sausage (salami). Fleischwirtsch 31 ogy, pp. 559–566. 55:680–682. 32 LO Sunesen, V Dorigoni, E Zanardi, LH Stahnke. C Viallon, JL Berdagué, MC Montel, R Talon, JF 33 2001. Volatile compounds released during ripening Martin, N Kondjoyan, C Denoyer. 1996. The effect 34 in Italian dried sausage. Meat Sci 58:93–97. of stage of ripening and packaging on volatile con- 35 M Tikk, K Tikk, MA Tørngren, MD Aaslyng, AH tent and flavor of dry sausages. Food Res Int 29: 36 Karlsson, HJ Andersen. 2005. The development of 667–674. 37 inosine monophosphate and its degradation products R Virgili, C Schivazappa. 2002. Muscle traits for long 38 during aging of pork of different qualities in relation matured dried meats. Meat Sci 62:331–343. 39 to basic taste—And retro nasal flavor perception of R Virgili, C Schivazappa, G Parolari, CS Bordini, 40 the meat. Proceedings of 51st ICoMST, Baltimore, M Degni. 1998. Proteases in fresh pork muscle and 41 USA:97. their influence on bitter taste formation in dry-cured 42 ML Timon, L Martin, MJ Petron, A Jurado, C Garcia. ham. J Food Biochem 22:53–63. 43 2002. Composition of subcutaneous fat from dry- S Westall. 1998. Characterization of yeast species by 44 cured Iberian hams as influenced by pig feeding. their production of volatile metabolites. J Food 45 J Sci Food Agric 82:186–191. Mycol 1:187–201. 46 K Tjener, LH Stahnke, L Andersen, J Martinussen. F Wirth. 1989. Reducing the common salt content of 47 2003. A fermented meat model system for studies of meat products. Possible methods and their limita- 48 microbial aroma formation. Meat Sci 66:211–218. tions. Fleischwirtsch 69:589–593. 49 ———. 2004a. Addition of α-ketoglutarate M Yvon, L Rijnen. 2001. Cheese flavour formation by 50 enhances formation of volatiles by Staphylococcus amino acid catabolism. Int Dairy J 11:185–201. 51 52 S53 N54 40454 23 241-256 r1.qxd 7/21/07 9:57 AM Page 241

1 2 3 4 5 6 7 8 9 10 11 12 13 14 Part V 15 16 17 18 19 Product Categories: 20 21 22 General Considerations 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 S54 N55 40454 23 241-256 r1.qxd 7/21/07 9:57 AM Page 243

1 2 3 23 4 5 6 Composition and Nutrition 7 8 Daniel Demeyer 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION: FOODS FOR in developing countries, whereas the lack of con- 22 NUTRIENT SUPPLY AND/OR formity of meat composition with the goals recently 23 HEALTH set for healthy nutrition (Table 23.2) (WHO 2003) 24 receive most attention in industrialized countries. 25 The emphasis in any discussion of food composition 26 in relation to nutrient content differs considerably 27 with the overall level of food supply. This is especially 28 true when meat and meat products are discussed in the THE NUTRIENT SUPPLY FROM 29 environment of either developing or industrialized MEAT AND MEAT PRODUCTS 30 countries. The latter remain characterized by an exces- The term meat covers all edible tissue of slaughter 31 sive average per capita supply of food energy, mainly animals. The live weight of pork and beef, the major 32 as meat and milk, amounting to 127%, 346%, and species involved in industrial production of meat 33 476%, respectively, of the corresponding low values products, contains on average about 85% of such tis- 34 in the former (WHO 2003) (Table 23.1). sues, involving about 35% red muscle and between 35 Nutritional problems in developing countries there- 10% (beef) and 20% (pork) adipose tissue (Table 36 fore continue to relate to the coping of nutrient supply 23.3). Pork adipose tissue is almost exclusively used 37 with food shortages and malnutrition, whereas in in the manufacture of comminuted meat products, 38 industrialized countries they mainly reﬂect the effects together with predominantly pork muscle tissue, but 39 of overnutrition. These effects relate to a number of also involving beef muscle, depending on the type 40 complex societal disturbances, including a convincing of product. 41 and causal relationship between the excessive con- 42 sumption of energy-dense, micronutrient-poor foods 43 and the incidence of the so-called noncommunicable 44 PROTEIN AND FAT chronic diseases. The latter involve obesity and the 45 associated risks of type 2 diabetes, cardiovascular dis- The largest amounts of macronutrients supplied by 46 eases (CVD), and cancer as well as dental disease and meat and meat products are by far the protein and fat 47 osteoporosis. Besides sugars and starch, the amounts of animal muscle (19% and 2.5%, respectively) and 48 and types of fatty acids and cholesterol present in ani- adipose tissue (90% fat). Actual protein and fat con- 49 mal products are seen as the main culprits in this tents, as well as the corresponding amino and fatty 50 respect, whereas salt is seen as a main causative factor acid composition, vary considerably depending on 51 for CVD (WHO 2003). several endogenous (e.g., anatomic location, breeds, 52 For these reasons, the nutrient content of meat is sex, species) and exogenous (mainly feeding regime) 53 of primary importance in relation to nutrient supply factors. S54 N55 243 40454 23 241-256 r1.qxd 7/21/07 9:57 AM Page 244

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1 Table 23.1. Vegetable and animal sources of dietary Table 23.3. Estimated distribution of edible parts of 2 energy 1997Ð1999. meat animals (% of live weight). 3 Industrialized Developing Beef Pig 4 Countries Countries 5 Red muscle tissue 33 44 6 kcal/c/d Removable adipose tissue 11 24 7 Total 3380 2681 —Fat 21 8 Vegetable 2437 2344 —Rind 3a 9 Animal 943 337 10 Blood 6 4 kg/c/y 11 Organs 5 4 Meat 88.2 25.5 12 Milk 212.2 44.6 Stomach and intestines 5 4 13 Skin 10 – 14 Source: WHO (2003). 15 kcal/c/d = kcal per capita per day. Gelatin—derived 3 3 b 16 kg/c/y = kg per capita per year. from bones 17 All 73 83 18 19 Source: from Demeyer (1981). 20 Table 23.2. Ranges of population nutrient intake a About 35% in back fat. goals. 21 b 25% in skeleton. 22 Dietary Factor Goala 23 24 Total fat 15–30% 25 Saturated fatty acids 10% about 24% in low-value meat cuts such as those 26 Polyunsaturated fatty 6–10% derived from the neck, the head, and the lower limbs 27 acids (PUFAs) (Bender and Zia 1976). The collagen content of the 28 n-6 PUFAs 5–8% skin covering subcutaneous fat (rind) can be esti- 29 n-3 PUFAs 1–2% mated at about 50% (Wood 1990) and, taking into 30 Trans-fatty acids 1% account potential gelatin production from the skele- 31 Total carbohydrate 55–75% ton, it can be estimated from the data in Table 23.2 32 Free sugars (Mono- and 10% that close to 45% of total pork protein consists of col- 33 disaccharides) lagen. Together with low value meat cuts, rind is used 34 to a variable extent in the production of comminuted Protein 10–15% 35 meat products. Lee et al. (1978) showed that 95% of 36 Cholesterol 300 mg/d the variation in PER (protein efficiency ratio) of meat 37 Sodium chloride (sodium) 2 g/d products is explained by their collagen content, a 38 Fruits and vegetables 400 g/d finding confirmed by Pelczynska and Libelt (1999). 39 In Demeyer (1981), the author could relate the essen- Dietary fiber and nonstarch From foodsb 40 tial amino acid content of meat products to the colla- polysaccharides (NSP) 41 gen content (%) in crude protein following y 0.28; 42 x 40.06 (n 54; R2 0.90) with y molar Source: WHO (2003). 43 a % of [ileu leu lys phe( tyr) met( cys) thr % = % of total dietary energy. 44 b 25 and 20 g per day provided by fruits and vegetables. val] and x 100 (8.OH-proline)/crude protein. In 45 view of the high level of protein consumption in 46 industrialized countries, protein quality was already pronounced as being “of no importance whatsoever” 47 Protein 48 by Bender and Zia (1976). According to Lee et al. 49 Muscle protein quality is determined by its content (1978), an acceptable upper limit for protein quality 50 of essential amino acids, a value inversely related corresponded to about 30% of collagen. to its connective tissue content. Collagen, the major 51 Fat 52 connective tissue protein, is almost completely 53S devoid of methionine and tryptophane, and its pro- It is well known that in contrast to beef adipose tis- 54N portion in muscle protein may reach values up to sue, the PUFA content of pork backfat can easily be 40454 23 241-256 r1.qxd 7/21/07 9:57 AM Page 245

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Table 23.4. Range of polyunsaturated fatty acid levels in meat (% of total fatty acids). 1 2 b Intramuscular Fat 3 Pork Backfata Beef Pork 4 5 n-6 PUFAs 6 Linoleic acid 12–20 12–14 1.5–18 7 Long chain fatty acids 0.5–1.5 1–3 1–4 8 n-3 PUFAs 1–3 0.5–4 9 Linolenic acid 2–11 1–2 1 10 Long chain fatty acids – 1 1 11 12 cis9trans11 CLA 4–8 1–6 13 n-6/n-3 1–10 14 Total fat (%) 70–82c 1–10bc 1–6 15 16 a From Warnants et al. (1998). 17 b From Raes et al. (2004). 18 c From Wood (1990). 19 20 21 22 23 24 increased by dietary means (see, e.g., Warnants et al. inclusion of CLA-rich oils in the diet. Table 23.4 25 1998) (Table 23.4). As summarized by Raes et al. illustrates the type of variability observed for PUFA 26 (2004) and De Smet et al. (2004), the intramuscular levels in meat. 27 fat content may vary between about 1% (high value In comminuted meat products, protein contents 28 cuts of very lean pork and beef breeds) and 6% and may decrease to values as low as 10% and fat con- 29 such variation is also affecting fatty acid composi- tent may increase to levels as high as 40%, as illus- 30 tion. The contents of saturated (SFA) and monoun- trated in Food Composition tables (see, e.g., 31 saturated (MUFA) fatty acids increase faster with Anonymous 2005). 32 increasing fatness than does the content of polyun- Within a total diet, the amounts of protein and fat 33 saturated (PUFA) fatty acids, resulting in a decrease supplied by meat and meat products are determined 34 in the relative proportion of PUFA. In beef, fat level by the level of meat consumption within that diet. 35 and fat composition are to a large extent determined For years, average per capita consumption of meat 36 by breed, whereas in pork, nutrition will have a has been estimated using carcass production data 37 larger impact. The fat level also influences the n-6/ yielding values in the neighborhood of 90 kg per 38 n-3 PUFA ratio, due to the difference of this ratio in capita per year (kg/c/y) for the affluent societies of 39 polar and neutral lipids. However, these effects are Western Europe and North America. Such values, 40 much smaller than the effects that can be achieved by however, neglect organs but include bone. Correc- 41 dietary means. Although linseed or linseed oil inclu- tion for the latter lowers such values by about 20%. 42 sion in animal diet supplies α-linolenic acid (LNA), The removal of other nonedible parts before con- 43 the conversion of LNA to its longer chain metabolite sumption can again be estimated at 20% (Honikel 44 eicosapentanoic acid (EPA) seems to be limited, 1992; Chizzolini et al. 1999). Actual meat consump- 45 resulting in only a small increase in their intramuscu- tion can thus be estimated as 0.90 0.8 0.8 46 lar fat. For both ruminants and monogastrics, the 57.6 kg/c/y or 158 g per capita per day (g/c/d). Such 47 only effective way to increase docosahexanoic acid value is within the range of data collected for 48 (DHA) levels in intramuscular fat seems to be the Western Europe in dietary survey type of studies, 49 feeding of fish oil or fish meal. A desirable increase reporting mean value extremes of 85 (low fat con- 50 of c9t11CLA (conjugated linoleic acid) in lamb or sumers in Spain) and 184 (high fat consumers in 51 beef meat can be achieved to some extent by feeding Belgium) g/c/d for adult males (Williams et al. 52 strategies affecting rumen metabolism. In pigs, how- 1999). More recent data, derived from household S53 ever, CLA incorporation is mainly achieved by the budget surveys without correction for inedible parts N54 40454 23 241-256 r1.qxd 7/21/07 9:57 AM Page 246

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1 and other losses (Naska et al. 2006) report an overall It has been reported that within meat and meat 2 average of 191 g/c/d for total meat, covering 63 g/c/d products, pork and poultry are the main sources 3 of meat products. Such data are subject to very high of niacin and selenium, respectively (Lombardi- 4 variability, with variation coefﬁcients often exceed- Boccia et al. 2004), the former being very low in 5 ing 100%. Nevertheless, they suggest that for years beef because of the speciﬁcity of ruminant metabo- 6 now meat and meat products contribute between 34 lism. Animal diets are often supplemented with 7 and 42% to dietary protein and between 13 and 25% α-tocopherol to control oxidation and color stability 8 to dietary fat supply, respectively (Hermus and of both pork and beef. The amounts clearly exceed 9 Albers 1986; D’Amicis and Turrini 2002; Valsta that of its use as a food additive and improve the 10 et al. 2005). In the same period, the percentage con- vitamin E status of the meat (McCarthy et al. 2001). 11 tribution of fat to total energy intake has deviated As summarized earlier (Demeyer 1981), it has been 12 little from 40%, in line with an estimated 10–20% recognized for years that heme iron is a much more 13 contribution of meat and meat products to total available iron source than vegetable iron and that 14 energy intake (Chizzolini et al. 1999). High-fat com- meat and meat products are major providers of vita-

15 minuted meat products such as salami contribute min B12. On the other hand, the very low manganese 16 more to fat consumption than fresh meat (D’Amicis levels in meat (0.4 ppm) hamper meat fermenta- 17 and Turrini 2002), whereas the contribution of tion (Hagen et al. 2000), whereas low values for cal- 18 processed meats in total meat consumption is higher cium content (100 ppm) are reflected in a Ca/P 19 in high-fat than in low-fat consumers (De Henauw ratio 0.1 and contribute to calcium intakes below 20 and De Backer 1999; Chizzolini et al. 1999). recommended levels (Lombardi-Boccia et al. 2003). 21 Introduction of even low amounts of meat in the 22 diet improves development and/or prevention of ane- OTHER NUTRIENTS 23 mia, as repeatedly observed in infants (Hallberg 24 Meat and meat products should not be considered et al. 2003; Hadler et al. 2004; Taylor et al. 2004; 25 “empty calories” because they are a more important Tympa-Psirropoulou et al. 2005; Krebs et al. 2006; 26 source for several other nutrients than for fat. A selec- Lachat et al. 2006). Improvement of available iron 27 tion of data calculated from food composition and and zinc as well as protein supply has been proposed 28 food consumption surveys for The Netherlands and as mechanisms for this finding. The need for dietary 29 Italy, reported between 1986 and 2003 (Table 23.5), heme iron is also suggested by the findings that a 30 illustrates that meat and meat products cover about high proportion of women of child-bearing age are 31 20% and between 30 and 40% of dietary fat and iron deficient (Hill 2002) and that iron intake of ado- 32 protein intake, respectively. Corresponding values lescent girls was considerably lower than recent rec- 33 for several vitamins (niacin, pyridoxin, thiamine, ommendations in Belgium, a typical industrialized 34 and riboflavin) and trace elements (zinc, iron, and country (Pynaert et al. 2005). Recent work from Bel- 35 selenium) exceed 20%, indicating a high energetic gium estimated that beef and pork supplied about 36 density for these nutrients in meat and meat products 20% of the dietary supply of long chain n-3 PUFA 37 (Leveille and Cloutier 1986). for adolescent boys and girls (De Henauw et al. 38 39 Table 23.5. Contribution (%) of meat and meat products to daily nutrient intake. 40 41 Referencea (5) (2) (4) (1) (2) (3) 42 43 Protein 27 34 34 27 42 44 Thiamine 21 34 Iron 38 20 26 45 Niacin 40 46 Zinc 24 40 38 46 Pyridoxin 39 26 Selenium 23 21 47 Riboflavin 21 24 48 Fat 23 20 49 Calcium 2 Sodium 6 50 Magnesium 5 Potassium 4 51 Phosphorous 7 Copper 12 52 53S a (1) Van Dokkum (1987); (2) Lombardi-Boccia et al. (2003); (3) D’Amicis and Turrini (2002); (4) Hermus and Albers 54N (1986); (5) Lombardi-Boccia et al. (2002). 40454 23 241-256 r1.qxd 7/21/07 9:57 AM Page 247

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2006). The same authors stated that n-3 PUFA they clearly indicate a shift from animal fat to sugar 1 enrichment at the level of animal production would and vegetable oil intake in most industrialized coun- 2 result in a substantial increase of that supply. Recent tries. The data also show a total energy intake well 3 work from Australia also pointed out that meat above the requirement explaining the failure of popu- 4 and meat products account for more than 40% of lation reductions in obesity (Willett 2003). This is 5 long chain n-3 PUFA intake, when docosapen- illustrated for Belgium in Table 23.6 and also high- 6 tanoic acid (DPA) is included (Howe et al. 2006) lights the limited importance of meat and meat prod- 7 and cover about 25–30% of CLA intake in Western ucts in energy supply. 8 populations (Schmid et al. 2006). As pointed out It is therefore not surprising that in a recent 9 by Razanamahefa et al. (2005) however, more pre- approach, emphasis is shifted from fat intake to intake 10 cise analytical data on fatty acid composition of of “energy-dense foods” as a “convincing increasing 11 meat and meat products are required to evaluate such risk” of obesity (WHO 2003), in line with the recent 12 ﬁndings. suggestion that fat intake has a less important role as 13 a determinant of obesity (Willett 2003). Excessive 14 energy intake is probably associated with excessive 15 MEAT AND MEAT PRODUCTS IN generation of ATP by mitochondrial oxidative phos- 16 HEALTHY NUTRITION phorylation known to result in free radical produc- 17 tion. This then leads to oxidative damage to the lipid, 18 Food consumption is clearly related to the rapidly protein, and nucleic acid building blocks of the cell 19 increasing incidence of chronic noncommunicable membranes, enzymes, and genes, processes proba- 20 disease, in both industrialized and developing bly involved in the generation of chronic disease and 21 countries (WHO 2003). Over the past 50 years this aging (Weidruch 1996; Demeyer et al. 2004). Con- 22 association has been related to a steadily increasing siderable attention in this respect is directed toward 23 consumption of animal fat and, more speciﬁcally, to cholesterol. Although dietary goals recommend 24 the increased consumption of saturated fat present in cholesterol intakes less than 300 mg/d, and meat and 25 dairy products and beef. This has resulted in nutri- meat product intake may provide up to half of that 26 tional guidelines advocating a decrease in fat con- amount, cholesterol intake and serum cholesterol 27 sumption and in various proposals to decrease fat levels are recently considered of less importance for 28 content and alter fat composition in animal production the incidence of CVD (Chizzolini et al. 1999). Other 29 (Demeyer and Doreau 1999; Givens 2005). However, factors relating to genetics and dietary antioxidant 30 although available statistics are based on production intake appear to be very important. The latter could 31 and trade rather than on actual consumption of foods, be involved in the protection against oxidation of 32 33 34 35 Table 23.6. Evolution of food energy intake and distribution in Belgiuma. 36 % of 1974 Values 37 38 1974 1984 1994 2000 39 40 Total (kcal/c/d) 3242 (2300)b 106 110 111 41 Contribution (%) of 42 Cereals 21.6 110 97 100 43 Sugars 11.0 94 133 130 44 Vegetable oils 10.9 102 127 138 45 Animal fats 15.4 92 79 75 46 Meat 8.5 104 97 87 47 Beef 2.8 78 61 57 48 Pork 4.5 113 100 84 49 Poultry 1.2 125 167 167 50 51 a Source: calculated from Anonymous (2006). 52 b ( ) = average energy requirement calculated from the population composition (Anonymous 2006a) and a physical activity S53 level (PAL) = 1.70 (Anonymous 2005). N54 40454 23 241-256 r1.qxd 7/21/07 9:57 AM Page 248

248 Product Categories: General Considerations

1 both dietary and endogenous cholesterol. Cholesterol ing health, beyond the supply of nutrients, having a 2 oxides or oxysterols group about 70–80 compounds, clearly identified beneficial effect on a target func- 3 and a number of those have been identified in meats tion of the human body and/or lowering the risk of 4 and in atherosclerotic lesions. Their toxicity proba- disease (Diplock et al. 1998). Functional food com- 5 bly involves interference with the gene expression ponent supply from meat may cover creatine (Pegg 6 related to cell viability (Chizzolini et al. 1999). et al. 2006) and carnosine (Park et al. 2005) as 7 Another shift in emphasis concerns the substitu- well as angiotensin converting enzyme inhibitors 8 tion of food based for nutrient-based nutritional (Arihara et al. 1999). 9 guidelines. Based on a “probable increasing risk” of As a final note, the role of meat products in 10 colon cancer incidence, “preserved meat (e.g., saus- sodium intake should be considered. Meat as such is 11 ages, salami, bacon, ham)” is one of the rare food relatively poor in sodium; meat products, however, 12 groups for which a moderate consumption is recom- contain about 2% added salt, values increasing up to 13 mended (WHO 2003). Since 1975, meat consump- 6% in dried products. It can be estimated that meat 14 tion has indeed been associated with colorectal and meat products provide 20–30% of salt (NaCl) 15 cancer reflected in recommendations for healthy eat- intake (Jiménez-Colmenero et al. 2001). The latter 16 ing from the American Cancer Association and the value ranges between 9–12 g/d (3.5 and 5 g/d Na) 17 U.S. National Cancer Institute. These involved less for industrialized countries, a value well above 18 frequent eating of red meat to be substituted with the dietary goal of 5 g/d (2 g/d Na) (Table 23.2). 19 chicken or fish and moderation in the eating of salt- The recent finding of a close relationship between 20 cured, smoked, and nitrite-cured foods (Kim and consumption of cured meat products and the inci- 21 Mason 1996). Recent European work (Norat et al. dence of hypertension (Paik et al. 2005) high- 22 2005) has confirmed the association of red and lights the importance of meat processing in sodium 23 processed meat consumption with colorectal cancer overconsumption. 24 risk. The hazard ratio significantly increased from 25 0.96 to 1.35 when increasing average consumption RECOMMENDED MEAT INTAKES 26 from 20 g/d to >160 g/d. The association was 27 stronger for processed than for unprocessed red General approaches to healthy nutrition also recom- 28 meat, and risks were reduced by increased fiber mend reductions in meat consumption (Srinivasan 29 intake. The mechanism underlying the association et al. 2006; Emmons et al. 2005; Walker et al. 2005). 30 may relate to endogenous nitrosation of heme iron or From the arguments presented above and the general 31 colonic formation of toxic heme metabolites (Sesink agreement on the place of (red) meat as an important 32 et al. 1999) and/or to heterocyclic amines (HCAs) food and a major supplier of essential nutrients, it is 33 and/or polycyclic aromatic hydrocarbons (PAHs) difficult to rationalize recommended daily meat 34 produced during the preparation of meat or produc- intakes. In one discussion, Murphy and Allen (2003) 35 tion of meat products. The first mechanism is con- recommend a daily consumption between 140 and 36 sidered to be more likely because chicken is a major 200 g of lean meat or meat substitutes for adults. It is 37 contributor to HCA intake and no association was evident that a distinction should be made between 38 observed between poultry intake and colorectal red (beef, sheep, and pork) and white (poultry) meat 39 cancer risk in the study (Norat et al. 2005). (Willett and Stampfer 2003), whereas most modera- 40 Evidence has been presented for the multifactorial tion should be associated with meat products. In a 41 causes for colon cancer (Emmons et al. 2005a) as recent description of a “Mediterranean diet,” a mod- 42 well as for genetic differences in its association with erate meat consumption of a maximum of 100 g/d is 43 meat consumption (Chan et al. 2005; Luchtenborg proposed (Coene 2001), whereas according to 44 et al. 2005; Brink et al. 2005). Recent reports also Weisburger (2002), total protein intake can be as low 45 highlight the association of red meat and meat prod- as 10 g/d, equivalent to about 50 g of meat daily. 46 ucts both with the incidence of various diseases as According to the COMA (1998) report, health risks 47 well as with the dietary supply of functional food were present only for those consuming over 140 g 48 components. The former suggest association of red of red meat daily, whereas Norat et al. (2002) 49 meat and/or meat products consumption with type 2 derived the zero-risk level for red meat consumption in 50 diabetes (Song et al. 2004; Fung et al. 2004), ovarian relation to colorectal cancer risk at 70 g per week. 51 cancer (Kiani et al. 2006), prostate cancer (Wolk Bingham (1999) refers to the protective effects of bran, 52 2005), and degenerative arthritis (Hailu et al. 2006). resistant starch, and vegetables, which may not apply, 53S The latter concerned compounds defined as improv- however, at high meat intakes (300–600 g/d). 54N 40454 23 241-256 r1.qxd 7/21/07 9:57 AM Page 249

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THE EFFECTS OF the amount of collagen in the sausage protein may 1 FERMENTATION ON THE reach 35% (Reuterswärd et al. 1985). German legis- 2 NUTRITIONAL AND HEALTH lation differentiates sausages according to collagen 3 content (Anonymous 2006b) (Table 23.7), and this 4 PROPERTIES OF MEAT affects price and protein quality but not digestibility 5 Both the nutrient content and health properties of as measured in humans (Reuterswärd et al. 1985). 6 fermented meat products (sausages) are affected by Improvement of the health value of meat products 7 has been tried for years, e.g., by replacement or low- 8 • The mixing of raw materials and additives used. ering of fat using collagen (Arganosa et al. 1987) 9 • The metabolic and chemical changes of the and of salt content using alginates (Schmidt et al. 10 sausage mix components during processing. 1986). Fermented sausages with fat contents below 11 2–3% or where sugar substitutes for salt exist, such 12 as the Thai Nham (Visessanguan et al. 2006) and 13 EFFECTS OF NATURE OF RAW MATERIALS AND Chinese products (Savic et al. 1988), respectively. In 14 ADDITIVES general, however, sausage levels of fat and salt con- 15 The many types of fermented sausages have been tinue to be considered undesirable in the light of the 16 narrowed down by industrial production and a major recent goals for healthy nutrition. Proposals for par- 17 distinction can be made between those produced in tial substitution of pork backfat (Mendoza et al. 18 shorter and longer ripening periods (Demeyer and 2001; Muguerza et al. 2004; Valencia et al. 2006), as 19 Stahnke 2002). In both, pork and/or beef are mixed well as of NaCl (Gelabert et al. 2003) continue to be 20 and comminuted (cuttered) with pork backfat and published. Table 23.8 illustrates to what extent the 21 salt, processes allowing for the incorporation of substitution of pork backfat by soy or fish oil or by 22 large amounts of pork fat and low-value meat cuts, inulin can improve the nutrient content and health 23 including rind. For most fermented sausage types, value of fermented sausages, as reflected in improved 24 such practice is reflected in a high calorific value, a PUFA contents, n-6/n-3 ratios, and even dietary fiber 25 high fat content, a high salt content, and a limited content. The interventions referred to affect sensory 26 nutritional quality of the sausage crude protein. A evaluation to an acceptable extent, provided addi- 27 major action, characteristic for the manufacture of tional precautions are taken, such as the use of extra 28 meat products in general, is the addition of 2–3% antioxidants with fish oil (Valencia et al. 2006). 29

salt (NaCl) containing about 0.5% of NaNO2. For the A wider application and development of such tech- 30 longer ripened products, nitrate rather than nitrite nologies involving other meat products is recom- 31 may be used. Table 23.7 illustrates the extreme vari- mended (Fernández-López et al. 2004). 32 ability of protein quality as well as of fat and salt con- Substitution of salt is the more delicate interven- 33 tent for European sausages. It should be noted that tion because of its universal role in meat technology 34 35 36 Table 23.7. Variability in proximate composition of fermented sausages. 37 38 Demeyer et al. (2000) Salgado et al. (2005) 39 Short Ripening Long Ripening Home-Made Industrial 40 41 Dry matter (DM) (%) 57 (4)c 67(3) 51–82d 46–82 42 % in DM 43 Crude protein (CP) 31 (7)d 28 (9) 8–32 9–29 44 Crude fat 61 (7) 61 (8) 52–85 58–80 45 NaCl 5.3 (12) 6.1 (11) 0.6–12.2 1.7–3.9 46 Carbohydrates 0.56 (23) 0.40 (18) 0.3– 3.0 0.05–2.2 47 Collagen 15–35a 1.6– 6.3b 2.1–15.9b 48 49 a % of CP: extreme values for types of fermented sausages distinguished in the German legislation (Anonymous 2006b). 50 b % of DM. 51 c ( ) = coefﬁcient of variation. 52 d Minimum-maximum values. S53 N54 40454 23 241-256 r1.qxd 7/21/07 9:57 AM Page 250

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1 Table 23.8. Effects of fat substitution on nutrient content and health value of fermented sausage. 2 b 3 Reference (1) (2) (3) 4 % Pork fat Inulin Fish Oil Soy Oil 5 Reduction by 0 → 75c 0 → 25 0 → 25 6 a → → → 7 Crude fat 49 17 Cholesterol (mg/100 g) 102 134 34 33 8 Dietary fibera 0 → 24 % of total fatty acids 9 kcal/100 g 392 → 251 Long chain n-3 PUFA 0.02 → 0.82 0.17 → 0.22 10 n-6 PUFA 4.33 → 4.20 14 → 22 11 → → 12 PUFA/SFA 0.46 0.56 0.40 0.73 13 n-6/n-3 13.9 → 3.0 11.0 → 11.5 14 15 a % of dry matter. 16 b (1) Mendoza et al. (2001); (2) Valencia et al. (2006); (3) Muguerza et al. (2004). 17 c → control → modified formulations. 18 19 to improve texture and taste as well as providing a given to the production of Zn-porphyrin, the red pig- 20 hurdle for bacterial development (Ruusunen and ment in Mediterranean products such as Parma ham, 21 Puolanne 2005). Nevertheless, partial substitution whose formation is inhibited by nitrite addition 22 using calcium ascorbate has lowered sausage salt (Adamsen et al. 2006). The color of some types of 23 level from 2.3% down to 1.4%, with limited damage oriental and Mediterranean products, is often also 24 to sensory quality while ensuring a simultaneous determined by the addition of vegetable components 25 increase of the sausage calcium and ascorbate con- and/or spices such as chili peppers added to Spanish 26 tent, nutrients known to be very low and absent in chorizo (Fernández-Fernández et al. 1998). Addition 27 meat, respectively (Gimeno et al. 2001). of spices and plant material such as herbs or their 28 Arguments for the omission of nitrate and nitrite extracts does not only affect flavor and color but also 29 in meat processing have been formulated for about may have positive antimicrobial and/or antioxidant 30 20 years now (Wirth 1991), mainly based on the effects. This was recently shown for tea catechins 31 cytotoxic effects of N-nitroso compounds, such as (Yilmaz 2006), pepper (Martinez et al. 2006), a 32 nitrosamines formed by the colon flora and/or during lemon peel preparation (Aleson-Carbonell et al. 33 meat processing from meat precursors (Mirvish 2004), extracts of grape seed and pine bark (Grün 34 et al. 2002). Older (Mahoney et al. 1979), although et al. 2006), and paprika (Revilla and Quintana 35 contradictory, results (Lee et al. 1984; Greger et al. 2005). Furthermore, end products are enriched in 36 1984) suggest a possible negative effect of nitrite on desirable plant secondary compounds such as 37 the bioavailability of iron, and fermented sausages lycopene (Osterlie and Lerfall 2005) and trace ele- 38 produced without nitrite/nitrate were shown to be ments such as manganese, lacking in meat and essen- 39 acceptable to consumers (Skjelkvåle et al. 1974). tial for microbial fermentation (Demeyer and Stahnke 40 It is known now that the need for nitrate/nitrite in 2002). The “functional compounds” involved may be 41 color development, flavor, and microbiological added to the sausage mix, or raw materials may be 42 safety can be alleviated by use of a purified natural used derived from animals fed these compounds. 43 cooked cured-meat pigment (Pegg et al. 2000) The latter was shown, e.g., to be more effective for 44 and/or an adapted hurdle technology. The latter is the improvement of color stability using vitamin E 45 mainly based on water activities less than 0.85 (Mitsumoto et al. 1993), a finding suggesting pre- 46 (Chawla and Chandler 2004). In the EU, the impor- ferential use of raw materials derived from animals 47 tance of the cured meat color has, of course, further fed diets enriched in polyphenols (Lopez-Bote 2000; 48 been diminished by legislation, allowing the use of Tang et al. 2000). 49 coloring agents such as Monascus red (Angkak), 50 cochineal, and betanin, derived from yeast, a scale EFFECTS OF PROCESSING 51 insect, and red beet, respectively (Finkgremmels 52 et al. 1991). These arguments should stimulate both The main processes occurring after cuttering and 53S use and further development of alternatives for affecting the nutritional and health value of fer- 54N nitrite. Special attention in this regard should be mented sausages are the metabolism of protein, lipid, 40454 23 241-256 r1.qxd 7/21/07 9:57 AM Page 251

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Table 23.9. End products of sausage metabolism derived from carbohydrates, protein, and lipid. 1 2 Sausage Type 3 Short Ripening Long Ripening 4 5 DM (%) 57 (4)a 67 (3) 6 From Carbohydrate Metabolism (g/kg DM) 7 Lactic acid 19 (12) 15 (17) 8 Acetic acid 0.6 (14) 0.5 (19) 9 Residual sugar as glucose 1.0 (23) 0.7 (18) 10 11 From Protein Metabolism (% of TN) 12 Peptide α-NH -N 3.0 (13) 2.7 (16) 2 13 Free α-NH -N 2.3 (10) 3.7 (13) 2 14 Ammonia-N 0.3 (22) 1.0 (18) 15 Lipid Metabolism 16 Free fatty acids (% of total FA) 2.7 (4) 3.7 (11) 17 18 Source: calculated from Demeyer et al. (2000). 19 a ( ) coefficient of variation; DM dry matter; TN total nitrogen; FA fatty acids. 20 21 22 23 and carbohydrate by both bacterial and meat enzy- of the salt-solubilized myofibrillar protein can explain 24 matic activity and the physicochemical processes of the improvement of protein digestibility and protein- 25 oxidation affecting both the lipid and protein fraction. efficiency ratio after fermentation of sausage mix 26 (Eskeland and Nordal 1980). It was clearly demon- Sausage Metabolism 27 strated that endogenous lipases are by far the main 28 Bacterial fermentation mainly involves the produc- responsible enzymes for the liberation of free fatty 29 tion of lactic acid from carbohydrates added or pres- acids during ripening. This lipolysis involves prefer- 30 ent. As shown in Table 23.9, the final amount of ential release of polyunsaturated fatty acids, both 31 lactic acid represents about 1–2% of the sausage, because of the more important phopholipase activity 32 being present in about equal amounts as the L- and on muscle membrane phospholipids and the speci- 33 D-isomer (Demeyer et al. 2000). At these levels, the ficity of fat cell lipases (Molly et al. 1996). Increasing 34 physiological effects of the slower metabolism of the with ripening time, up to 5% of the total fatty acids 35 latter isomer are minor (Ewaschuk et al. 2005). present in the sausage may be present as free fatty 36 Besides bacterial fermentation of carbohydrates, acids. The nutritional consequences of this change 37 however, important hydrolytic changes brought about are not obvious but it may increase fatty acid suscep- 38 by the joint action of bacterial and meat enzymes tibility to oxidation (Ansorena et al. 1998), although 39 affect the sausage protein and lipid composition. Summo et al. (2005) found evidence for a high 40 Subject to considerable variation, myofibrillar pro- degree of primary oxidation forming oxidized tria- 41 teins representing about 5% of the sausage crude pro- cylglycerols as well as peroxides early in the sausage 42 tein are hydrolyzed, yielding about equal amounts of ripening. 43 shorter peptides and free amino acids, the values 44 Lipid and Protein Oxidation increasing with ripening time. Further deamination 45 and decarboxylation yield small amounts of ammo- Apart from the prooxidant effects of sodium chloride, 46 nia and amines, respectively. Provided starter organ- the PUFAs in sausage lipids are highly susceptible to 47 isms do not produce amines, amine production autooxidation because of the increase in oxygen con- 48 mainly reflects initial bacterial contamination of the tent due to the high degree of comminution. Also, 49 raw materials used (Ansorena et al. 2002). Small the added nitrate/nitrite may initially promote oxida- 50 amounts of the branched-chain amino acids, particu- tion although the later produced nitric oxide may 51 larly leucine, are metabolized with production of act as an antioxidant. Both lipid autooxidation and 52 major aroma compounds. The proteolytic protein microbial betaoxidation of saturated fatty acids S53 changes, together with the acid-induced denaturation are desirable reactions to some extent because the N54 40454 23 241-256 r1.qxd 7/21/07 9:57 AM Page 252

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1 compounds formed are important contributors to fla- heating, it was shown that the process of curing 2 vor. Excessive autooxidation however leads to color lowered available methionine content from 2.3% 3 defects and ultimately rancidity rendering the prod- to 1.8% in total amino acids. Such change is impor- 4 uct unacceptable for consumption. A concentration tant because methionine was recognized early on as 5 of thiobarbituric reactive substances (TBARS) the limiting amino acid in meat protein (Dvoràk 6 expressed as malondialdehyde 1 à 2mg/kg pro- 1972). 7 vides a limit for acceptability (Zanardi et al. 2004). Smoking, a technology mainly limited to the 8 However, before such condition is reached, lipid shorter and faster production types, merits a final 9 autooxidation and, in particular, cholesterol oxida- consideration in relation to oxidation. The process is, 10 tion, could generate toxic molecules lowering the of course, of paramount importance in relation to 11 health value of the product (Zanardi et al. 2004). For both sensory and health properties of the product, as 12 a fast fermented industrial sausage type, the latter discussed elsewhere. It was also shown, however, 13 authors reported that neither nitrites/nitrates nor that it has antioxidant effects, restoring methionine 14 ascorbate, added for enhancement of color develop- availability as well as lowering peroxide content 15 ment, nor added spices significantly affected oxida- (Strange et al. 1980). 16 tion evaluated by measurement of TBARS. The 17 extent of cholesterol oxidation (%) was very low in REFERENCES 18 all cases, but values were lowered even more by use 19 of the additives, except for spices. The data suggest CE Adamsen, JKS Moller, K Laursen, K Olsen, LH 20 that final levels of lipid oxidation are determined to Skibsted. 2006. Zn-porphyrin formation in cured 21 a large extent by the initial level of oxidation in meat products: Effect of added salt and nitrite. Meat 22 the raw materials. Sammet et al. (2006) recently Sci 72:672–679. 23 confirmed the absence of an antioxidant effect of L Aleson-Carbonell, J Fernandez-Lopez, E Sendra, 24 nitrite, but found in contrast that the use of meat E Sayas-Barbera, JA Perez-Alvarez. 2004. Quality 25 from pigs fed α-tocopherol–enriched diets produced characteristics of a non-fermented dry-cured sausage 26 fermented sausages clearly less oxidized than those formulated with lemon albedo. J Sci Food Agric 27 obtained from pigs fed a control diet. Although 84:2077–2084. 28 showing higher contents of PUFAs, sausages pre- Anonymous. 2005. Belgische Voedingsmiddelentabel 29 pared from extensively reared pigs were not subject 4th ed. Brussels: Nubel, 92 pp. 30 to more oxidation than those prepared from inten- Anonymous. 2006. 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rectal cancer: A prospective cohort study. The Sanchez, G Berglund, I Mattisson, G Hallmans, 1 Netherlands. Canc Causes & Contr 16:1041–1054. R Palmqvist, NE Day, K-T Khaw, TJ Key, M San 2 AW Mahoney, DG Hendrickx, TAG Gillett, DR Buck, Joaquin, B Hémon, R Saracci, R Kaaks, E Riboli. 3 CG Miller. 1979. Effect of sodium nitrite on the bio- 2005. Meat, Fish, and Colorectal Cancer Risk: The 4 availability of meat iron for the anaemic rat. J Nutr European Prospective Investigation into Cancer and 5 109:2182–2189. Nutrition. J Nat Cancer Inst 97:906–16. 6 L Martinez, I Cilla, JA Beltran, P Roncales. 2006. T Norat, A Lukanova, P Ferrari, E Riboli. 2002. Meat 7 Effect of Capsicum annuum (red sweet and cayenne) consumption and colorectal cancer risk: Dose- 8 and Piper nigrum (black and white) pepper powders response meta-analysis of epidemiological studies. 9 on the shelf life of fresh pork sausages packaged in Int J Cancer 98:241–256. 10 modiﬁed atmosphere J Food Sci 71:S48–S53. 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Total N-nitroso com- characteristics of emu Dromaius novae hollandiae 21 pounds and their precursors in hot dogs and in the meat and its value-added products. Food Chem 22 gastrointestinal tract and feces of rats and mice: Pos- 97:193–202. 23 sible etiologic agents for colon cancer. J Nutr RB Pegg, KM Fisch, F Shahidi, F Title. 2000. The 24 132:3526S–3529S. replacement of conventional meat curing with nitrite- 25 M Mitsumoto, RN Arnold, DM Schaefer, RG Cassens. free curing systems. Fleischw 80 (5):86–89. 26 1993. Dietary versus postmortem supplementation of E Pelczynska, K Libelt. 1999. Nutritional value of 27 vitamin E on pigment and lipid stability in ground sausages as a factor of their connective tissue con- 28 beef. J Anim Sci 71:1812–1816. tent. Fleischw 79(7):86–88. 29 K Molly, D Demeyer, T Civera, A Verplaetse. 1996. I Pynaert, C Matthys, M Bellemans, M De Maeyer, 30 Lipolysis in a Belgian sausage: Relative importance S De Henauw, G De Backer. 2005. Iron intake and 31 of endogenous and bacterial enzymes. Meat Sci dietary sources of iron in Flemish adolescents. Eur J 32 43:235–244. Clin Nutr 59:826–834. 33 E Muguerza, O Gimeno, D Ansorena, I Astiasarán. K Raes, S De Smet, D Demeyer. 2004. Effect of dietary 34 2004. New formulations for healthier dry fermented fatty acids on incorporation of long chain polyunsat- 35 sausages: A review. Trends Food Sci & Technol urated fatty acids and conjugated linoleic acid in 36 15:452–457. lamb, beef and pork meat: A review. Anim Feed Sci 37 SP Murphy, LH Allen. 2003. Nutritional importance of Technol 113:199–221. 38 animal source foods. J Nutr 133:3932S–3935S. L Razanamahefa, L Lafay, M Oseredczuk, A Thiebaut, 39 A Naska, D Fouskakis, E Oikonomou, MDV Almeida, L Laloux, M Gerber, P Astorg, JL Berta. 2005. 40 MA Berg, K Gedrich, O Moreiras, M Nelson, Dietary fat consumption within French population 41 K Trygg, A Turrini, AM Remaut, JL Volatier, A and quality data on major food contributors composi- 42 Trichopoulou. Group Authors.: DAFNE participants. tion Bull du Cancer 92(7–8):647–657. 43 2006. Dietary patterns and their socio-demographic AL Reuterswärd, H Andersson, NG Asp. 1985. 44 determinants in 10 European countries: Data from Digestibility of collagenous fermented sausage in 45 the DAFNE databank. Eur J Clin Nutr 60:181–190. man. Meat Sci 14:105–121. 46 T Norat, S Bingham, P Ferrari, N Slimani, M Jenab, I Revilla, AMV Quintana. 2005. The effect of dif- 47 M Mazuir, K Overvad, A Olsen, A Tjønneland, F ferent paprika types on the ripening process and 48 Clavel, M-C Boutron-Ruault, E Kesse, H Boeing, MM quality of dry sausages. 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1 ripening of Chorizo de cebolla, a Spanish traditional I Valencia, D Ansorena, I Astiasarán. 2006. Nutritional 2 sausage. Effect of the system of manufacture: Home- and sensory properties of dry fermented sausages 3 made or industrial. Food Chem 92:413–424. enriched with n-3 PUFAs. Meat Sci 72:727–733. 4 K Sammet, R Duehlmeier, HP Sallmann, C Von LM Valsta, H Tapanainen, S Mannisto. 2005. Meat fats 5 Canstein, T Von Mueffling, B Nowak. 2006. Assess- in Nutrition. Meat Sci 70:525–530 Sp. Iss. 6 ment of the antioxidative potential of dietary sup- W Van Dokkum. 1987. Vlees en Gezondheid. Proc 7 plementation with alpha-tocopherol in low-nitrite Symposium “Het Vleesvraagstuk,” Utrecht, Septem- 8 salami-type sausages. Meat Sci 72:270–279. ber 1987. 9 Z Savic, ZK Sheng, I Savic. 1988. Wurste nach Chine- W Visessanguan, S Benjakul, S Riebroy, M Yarchai, 10 sischer art. Fleischw 68.5:570–580. W Tapingkae. 2006. Changes in lipid composition 11 A Schmid, M Collomb, R Sieber, G Bee. 2006. Conju- and fatty acid profile of Nham, a Thia fermented pork 12 gated linoleic acid in meat and meat products: sausage, during fermentation. Food Chem 94: 13 A review. Meat Sci 73:29–41. 580–588. 14 GR Schmidt, WJ Means, AD Clarke. 1986. Using P Walker, P Rhubart-Berg, S McKenzie, K Kelling, 15 restructuring technology to increase red meat value. RS Lawrence. 2005. Public health implications of 16 J Anim Sci 62:1458–1462. meat production and consumption. Public Health 17 ALA Sesink, DSML Termont, JH Kleibeuker, R Van Nutr 8:348–356. 18 der Meer. 1999. Red meat and colon cancer: The N Warnants, MJ Van Oeckel, CV Boucque. 1998. 19 cytotoxic and hyperproliferative effects of dietary Effect of incorporation of dietary polyunsaturated 20 heme. Cancer Res 59:5704–5709. fatty acids in pork backfat on the quality of salami. 21 R Skjelkvåle, TB Tjaberg, M Valland. 1974. Compari- Meat Sci 49:435–445. 22 son of salami sausage produced with and without R Weidruch. 1996. Caloric Restriction and aging. Sci 23 addition of sodium nitrite and sodium nitrate. J Food Amer 274(1):32–38. 24 Sci 39:520–524. JH Weisburger. 2002. Lifestyle, health and disease pre- 25 Y Song, JE Buring, JE Manson, SM Liu. 2004. A vention: The underlying mechanisms. Eur J Cancer 26 prospective study of red meat consumption and type Prev 11(suppl 2):S1–S7. 27 2 diabetes in middle-aged and elderly women. Dia- WHO. 2003. Diet, Nutrition and the Prevention of 28 betes Care 27:2108–2115. Chronic Diseases. Report of a joint WHO/FAO expert 29 CS Srinivasan, X Irz, B Shankar. 2006. An assessment consultation, WHO Technical Report Series 919; 30 of the potential consumption impacts of WHO 148 pp. http://www.who.int/dietphysicalactivity/ 31 dietary norms in OECD countries. Food Policy publications/trs916/summary/en/print.html. 32 31:53–77. WC Willett. 2003. Dietary fat and obesity: Lack of an 33 ED Strange, RC Benedict, AJ Miller. 1980. Effect of important role. Scandin J Nutr 47:58–67. 34 processing variables on the methionine content of WC Willett, MJ Stampfer. 2003. Rebuilding the food 35 frankfurters. J Food Sci 45:632–634. pyramid. Sci Amer 288.1:64–71. 36 C Summo, F Caponio, MT Bilancia. 2005. The oxida- C Williams, M Wiseman, J Buttriss. 1999. Supplement 37 tive degradation of the lipid fraction of ripened eds. Food-based Dietary Guidelines—A Staged 38 sausages as influenced by the raw material. J Sci Approach. Br J Nutr 81(Suppl.2):S29–S153. 39 Food Agric 85:1171–1176. F Wirth. 1991. Restricting and dispensing with curing 40 SZ Tang, JP Kerry, D Sheehan, DJ Buckley, PA agents in meat-products. Fleischw 71 (9):1051–1054. 41 Morrissey. 2000. Dietary tea catechins and iron- A Wolk. 2005. Diet, lifestyle and risk of prostate 42 induced lipid oxidation in chicken meat, liver and cancer. Acta Oncologica 44:277–281. 43 heart. Meat Sci 56:285–290. JD Wood. 1990. Consequences for meat quality of 44 A Taylor, EW Redworth, JB Morgan. 2004. Influence reducing carcass fatness. In: JD Wood, AV Fisher, 45 of diet on iron, copper, and zinc status in children eds. Reducing fat in meat animals. New York: Else- 46 under 24 months of age. Biol Trace Elem Res vier Applied Science, pp 344–397. 47 97:97–214. Y Yilmaz. 2006. Novel uses of catechins in foods. 48 E Tympa-Psirropoulou, C Vagenas, D Psirropoulos, Trends Food Sci & Technol 17:64–71. 49 O Dafni, A Matala, F Skopouli. 2005. Nutritional E Zanardi, S Ghidini, A Battaglia, R Chizzolini. 2004. 50 risk factors for iron-deficiency anaemia in children Lipolysis and lipid oxidation in fermented sausages 51 12–24 months old in the area of Thess alia in Greece. depending on different processing conditions and 52 Intern J Food Sci Nutr 56:1–12. different antioxidants. Meat Sci 66:415–423. 53S 54N 40454 24 257-266 r1.qxd 7/20/07 3:49 PM Page 257

1 2 3 24 4 5 6 Functional Meat Products 7 8 Diana Ansorena and Iciar Astiasarán 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION (Higgs 2000). The contribution of meat to vitamin B 22 dietary intake is also relevant. However, from a 23 There is a growing consumer awareness of the rela- healthy point of view, an excessive presence of these 24 tionship between nutrition and health, leading to products in the diet can not be recommended, espe- 25 the relatively new concept of optimal nutrition, cially for certain population groups, because they 26 by which food components have the potential to are also sources of saturated fatty acids and choles- 27 improve the health and well-being of individuals and terol, and in some particular cases, such as occurs 28 maybe to reduce the risk from, or delay the develop- with dry-fermented sausages, of significant salt con- 29 ment of, major diseases (Diplock et al. 1999). tent. These components are known to be implicated 30 Advances in food science and technology are now in the development in some of the most prevalent 31 providing the food industry with increasingly diseases today (cardiovascular diseases, hyperten- 32 sophisticated methods to control and alter the physi- sion, atherosclerosis, etc.). Health organizations all 33 cal structure and the chemical composition of food over the world have promoted the choice of a diet 34 products. Consumers are also aware of the market low in saturated fat and cholesterol and moderated in 35 potential for functional foods, based on the principle total fat as a mean of preventing cardiovascular heart 36 of added value linked to health benefit. In this con- disease (AHA 2000; USDA 2000). Furthermore, it is 37 text, the meat industry is now making a great effort known that n-3 fatty acids have important roles in the 38 to try to fulfill the expectations of consumers and to modulation and prevention of human diseases, par- 39 offer a wide variety of healthier products, by means ticularly coronary heart disease, showing also other 40 of avoiding undesidered substances, reducing them well-recognized positive health effects (antitrombo- 41 to appropriate limits, and/or increasing the levels of genic, antiinflammatory, hypotrigliceridemic, pre- 42 other substances with beneficial properties (Jiménez- vention of arrhythmias, etc.) (Connor 2000). Besides 43 Colmenero et al. 2001). this, hypertension, which is one of the main risk fac- 44 Meat and meat products are significant sources of tors of cardiovascular disease, is known to be corre- 45 high biological value protein and they also supply a lated with an excessive sodium intake from the diet 46 wide range and amount of micronutrients. Red meat (Law et al. 1991; Truswell 1994). 47 and, consequently, red meat derivatives, are excellent Those data show the possible target goals for the 48 sources of iron, having 50–60% in the heme form, meat industry to improve their products and supply 49 which is absorbed by a more efficient mechanism arguments for the development of healthier dry- 50 than the nonheme iron form found in plant foods. fermented sausages that could contribute to minimize 51 Meat is also the major contributor to zinc intakes in the negative effects of some of their constituents. 52 developed countries and is one of the richest natural On the other hand, the technological process of 53 sources of an important reducing agent, glutation elaboration of this type of meat derivatives enables S54 N55 257 40454 24 257-266 r1.qxd 7/20/07 3:49 PM Page 258

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1 the addition of healthy ingredients, and even the fer- NaCl and 1% KCl. The sodium reduction in these 2 mentation process that takes place at the beginning products was about 25%, in comparison with the 3 of the process leads to study of the potential health traditional formulation, and the Na/K ratio 4 benefits of microbial starter cultures selection. In decreased from 4.38 to 0.87 (Ibañez et al. 1996). In 5 consequence, improvement of nutritional and func- another work, the same authors found that the nitro- 6 tional properties of dry-fermented sausages can be sation process and carbohydrate heterofermentative 7 tackled by trying different approaches. activity of the starter cultures was favoured in dry- 8 fermented sausages manufactured with a mixture 9 containing 1.37% NaCl and 0.92% KCl in compari- 10 MODIFICATION OF THE son with sausages manufactured with 2.73% NaCl 11 MINERAL CONTENT IN (Ibañez et al. 1995). 12 DRY-FERMENTED SAUSAGES Other strategies try to formulate healthier dry- 13 fermented sausages not only by the NaCl reduction, 14 NaCl has an important influence on the final taste but also by the simultaneous addition of interes- 15 and texture of dry-fermented sausages, and it also ting minerals such as magnesium and/or calcium, 16 plays an important role in the guaranty of the micro- both required for maintaining an adequate bone 17 biological stability, making it difficult to achieve a health. Results of some experiments are shown in 18 significant reduction without affecting the final qual- Table 24.1. Gimeno et al. (1998), when using 19 ity of these products. Gelabert et al. (1995) found a 22.5 g/kg of a mixture of 1% NaCl, 0.55% KCl,

20 decrease in the overall acceptability of fuet when 0.23% MgCl2, and 0.46% CaCl2 to replace the tradi- 21 potassium lactate, glycine, and KCl were used at tional 26 g/kg salt content in chorizo (16 mm particle 22 higher levels than 30%, 20%, and 40%, respectively. size), found that the sodium content decreased from 23 These salts were also used by Gou et al. (1996) as 1.88% in the control sausage to 0.91% in the modi- 24 sodium chloride substitutes in salami, finding impor- fied product. However, sensorial acceptability was 25 tant flavor defects with substitutions of more than scored lower, mainly due to the lower salty taste. In 26 40% for the three substitutes. Metallic taste limits chorizo de Pamplona, a traditional Spanish dry- 27 the use of KCl, an excessive sweetness is given by fermented sausage of 3 mm particle size, a mixture

28 high concentrations of glycine, and an abnormal of 1% NaCl, 0.55% KCl, and 0.74% CaCl2 (total 29 taste is detected when using K-lactate. Levels of sub- amount of salts of 22.9 g/kg) was used, decreasing 30 stitution higher than 30% for potassium lactate or the sodium content from 1.35% in the control to 31 higher than 50% for glycine also caused some tex- 0.82% in the modified sausage (Gimeno et al. 1999). 32 ture defects. Mixtures of these salts (KCl/glycine Significant increases of potassium and calcium 33 and K-lactate/glycine) at levels of 40–70% offered (from 0.21 to 0.60% and from 154 mg/100 g to 34 no advantages over those found in the individual 320 mg/100 g, respectively) were observed in those 35 components (Gelabert et al. 2003). Ibañez et al. products, which could be of interest from the nutri- 36 (1997) did not find significant differences between tional point of view (although no bioavailability 37 products manufactured with 3% of NaCl (control) studies had been carried out). Concerning sensorial 38 and modified products manufactured with 1.5% quality aspects, instrumental measures of texture 39 40 41 Table 24.1. Content of Na, K, and Ca2 of different dry-fermented sausages. 42 43 Chorizo Chorizo de Pamplona 44 Control A(*) Control B(**) Control C(***) 45 46 Na (g/100 g sausage) 1.88 0.91 1.35 0.82 1.98 1.07 47 K (g/100 g sausage) 0.55 1.11 0.21 0.60 – – 48 2 49 Ca (mg/100 g sausage) 84.60 182.90 154.50 319.9 130 400 50 Mg2 (mg/100 g sausage) 25.6 182.00 41.64 43.16 – – 51 52 A(*) 1% NaCl, 0.55% KCl, 0.23% MgCl2, 0.46% CaCl2. Gimeno et al. (1998). 53S B(**) 1% NaCl, 0.55% KCl, 0.74% CaCl2. Gimeno et al. (1999). 54N C(***) 1.4% NaCl, 2.9% sodium ascorbate. Gimeno et al. (2001a). 40454 24 257-266 r1.qxd 7/20/07 3:49 PM Page 259

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(TPA analysis) and color (CIEL*a*b*) showed some the diet (CAST 1991; Myer et al. 1992; Morgan 1 slight differences with regard to traditional products. 1992; Irie and Sakimoto 1992; Cherian and Sim 2 The applied mixture of salts did not affect the devel- 1995; Romans et al. 1995; Mitsuharu et al. 1997; 3 opment of the starter culture and guaranteed the Cava et al. 1997, 1999; de la Hoz et al. 1996; Enser 4 hygienic quality of the products (Gimeno et al. et al. 2000). Concerning dry-fermented sausages 5 2001a). Hoz et al. (2004) carried out an experiment in which 6 Signiﬁcant reduction in the sodium content has also salchichón, a Spanish dry-fermented sausage, was 7 been achieved through a partial substitution of NaCl manufactured using different combinations of back- 8 by different percentages of calcium ascorbate. The fat and meat obtained from animals fed on diets rich 9 sodium percentage decreased from 1.98% in the con- in polyunsaturated n-3 fatty acids (linseed oil) 10 trol sausages to 1.07% in the highest ascorbate exper- and/or olive oil and α-tocopherol. Enrichment of the 11 imental batch (29.17 g ascorbate/kg and 14 g sodium animal diets with linseed or linseed and olive oils, 12 chloride/kg) (Gimeno et al. 2001b). Calcium content signiﬁcantly increased (P 0.05) the percentage of 13 consequently increased from 130 mg/100 g (control) linolenic acid (5–8 fold), arachidonic acid (3–5fold), 14 to 400 mg/100 g, which is 50% of the RDAs for this EPA (2-fold) and DHA (1.5-fold) in sausages. Prod- 15 mineral. However, some small differences in the ucts from raw material from pigs fed on diets 16 instrumental measures of color and texture were again enriched only with linseed oil (no α-tocopherol) 17 noticed. The taste of these dry-fermented sausages showed high lipid oxidation susceptibility, develop- 18 was generally described as less salty, which is some- ing rancid taste. 19 times positively evaluated by consumers. Other approaches for improving the lipid fraction 20

Finally, addition of CaCl2 at two different con- of dry-fermented sausages introduce modifications 21 centrations (0.5% and 0.05%) has been assayed in in the formulation of products. First trials assayed 22 order to formulate calcium-enriched dry-fermented the incorporation of olive oil, alone or preemulsified 23 sausages (Flores et al. 2005). The sensory analysis with soy protein, as partial substitutes for pork back- 24 showed that the panel preferred in color (P 0.05), fat, with the aim of increasing the MUFA content. 25 aroma (P 0.001), taste (P 0.001), and overall Bloukas et al. (1997) in Greek dry-fermented saus- 26 quality (P 0.001) the control sausage over the Ca ages and Muguerza et al. (2001) in chorizo de Pam- 27 0.5% batch, and no significant differences were plona, concluded that it was possible to incorporate 28 detected between the control and Ca 0.05% batch. up to 20% and 25%, respectively, of preemulsified 29 These differences were associated to the high degree olive oil, developing technologically and sensory 30

of oxidation suffered by the 0.5% CaCl2 batch, which acceptable products. When a higher percentage of 31 showed higher TBA values and higher content of typ- substitution was used, drip fat was observed. These 32 ical volatile compounds derived from lipid oxidation. products showed increments in the MUFA and 33 An opposite effect was observed with the 0.05% PUFA fractions, and reduction in the cholesterol 34

CaCl2 addition, which showed an important effect on content, improving consequently their nutritional 35 volatile generation during ripening by inhibiting the quality. A simultaneous study of reduction and 36 generation of lipid oxidation products and promoting incorporation of olive oil was further done in Greek 37 the generation of methyl branched alcohols and ethyl dry-fermented sausages. Reduced- and low-fat dry- 38 esters that contribute to proper sausage aroma. fermented sausages can be prepared with 20% and 39 10% fat in the meat mixture, showing a 37% and 40 FAT MODIFICATIONS IN 53% lower fat content, respectively, than traditional 41 DRY-FERMENTED SAUSAGES sausages (30% fat). The reduction in the fat level led 42 to an increase in the weight loss, hardness, and firm- 43 Modification of the lipid fraction in meat products ness of the modified sausages, which were also 44 can be done by the reduction of the fat content darker and with a higher red intensity color (a* 45 and/or the simultaneous addition of nonlipid fat value) than control. However, the substitution of 46 replacers or substitutes in order to minimize texture 20% of the pork backfat by preemulsified olive oil 47 defects (Jiménez-Colmenero et al. 2001). Recent did not affect weight loss, obtaining lighter products 48 studies pointed out the interest of a change in the and with a higher yellow intensity (b* value). 49 dietary lipid profile of foodstuffs into a healthier pat- Muguerza et al. (2002). 50 tern, instead of a reduction in the total dietary fat by Different concentrations of extra-virgin olive oil 51 using other substances. As a way to reach this objec- have also been tested in salami (conventional fat 52 tive, different studies try to modify the composition content of 15%). The formulation with 5% olive oil, S53 of the animal raw matter (pigs) through a change in corresponding to 33.3% substitution of pork backfat N54 40454 24 257-266 r1.qxd 7/20/07 3:49 PM Page 260

260 Product Categories: General Considerations

1 was sensorial judged best of all formulations, and it and 75% and 59% of the AI in the case of batch B, 2 was found to be stable during the shelf life (Severini contributing in a significant way to the dietary intake 3 et al. 2003). Kayaardi and Gök (2004), in a similar of EPA and DHA. However, lipid oxidation analysis 4 fat content fermented product (sucuk), achieved sig- revealed potential degradation problems in the rich- 5 nificant cholesterol reductions and increments of est n-3 batch. No sensory analysis was done in these 6 oleic and linoleic acid contents in products with a products due to detected off-odor. An experiment 7 partial substitution of 40% and 60% of beef fat by carried out by partial substitution of pork backfat by 8 preemulsified olive oil. Sensorial analysis showed deodorized oil (25% substitution) overcame this 9 that sucuk prepared by replacing 40% were rated inconvenience. The developed products supplied 10 higher in terms of taste, appearance, and texture than 0.64 g EPA/100 g product and 0.46 g DHA/100 g 11 the other sausages. product, which could mean 85% of the adequate 12 Some other studies modify the lipid fraction by intake in these acids if a portion of 50 g sausage 13 incorporating high PUFA oils from vegetable and is considered. No signs of oxidation were found by 14 animal origin into the dry-fermented sausages for- TBA analysis, and none of the dienals and trienals 15 mulations. Table 24.2 reports the fatty acids modifi- reported as secondary lipid oxidation products typi- 16 cations caused in these cases. cal from fish oil were detected in the modified 17 Concerning vegetable oils, a 25% substitution of sausages. A sensory evaluation panel did not find 18 pork backfat by preemulsified soy and linseed oils differences in general acceptability, concluding that 19 significantly reduced the content of saturated fatty the modified products can be considered a techno- 20 acids, increasing the PUFA content, mainly at the logically viable functional food. 21 expense of linoleic acid and α-linolenic acid, res- One of the potential problems derived from these 22 pectively (Muguerza et al. 2003; Ansorena and modifications could be an acceleration of the oxida- 23 Astiasarán 2004a). The α-linolenic acid content in tive processes due to the increment in unsaturated 24 modified sausages elaborated with linseed oil nearly fatty acids, particularly polyunsaturated ones, which 25 reached the possibility of including the nutritional are more prone to oxidation. Stability of olive oil or 26 claim, still to be approved, “high in omega-3,” linseed-containing sausages, both products includ- 27 because it reached 2.5 g/100 g, and 3 g/100 g are ing BHT and BHA as antioxidants, was evaluated 28 needed for that purpose. A significant and positive after storage during 2 and 5 months under different 29 reduction in the n-6/n-3 ratio was observed when packaging conditions (aerobic, vacuum, and/or 30 using linseed oil, decreasing from 13.4 in control modified atmosphere) (Ansorena and Astiasarán 31 sausages to 1.9 in modified ones, attributed also to 2004b; Valencia et al. 2006b). No signs of lipid 32 this α-linolenic acid increment. Interesting incre- oxidation measured by TBA, peroxides, and volatile 33 ments in the PUFA/SFA ratio were also detected in aldehydes were detected for modified sausages, 34 both experiments, from 0.4 to 0.7. The relevance of a regardless of the packaging system used, and only 35 high PUFA/SAT ratio was shown in the epidemio- slight changes in the fatty acid profiles were noticed 36 logical Nurses Health Study, in which it was proved in comparison to those products analyzed just at the 37 that this ratio was strongly associated with a lower end of the ripening process. Control sausages were 38 risk of CHD (Hu et al. 1999). Modified sausages affected by oxidation when stored aerobically, 39 were similar to their respective control in sensory whereas they were well preserved under vacuum or 40 properties, and no lipid oxidation was detected in MAP conditions. These results confirmed the stabil- 41 any case. ity of the new formulations and the maintenance of 42 Concerning animal origin high PUFA oils, a fish the health benefits during their storage period. 43 oil extract (omega-3 700 Solgar Vitamins) and a 44 deodorised fish oil (Lysi) have been tested, by addi- 45 tion of two different amounts to the formulation INCORPORATION OF FIBER INTO 46 (5.3 g/kg-batch A and 10.7 g/kg-batch B) in the first DRY-FERMENTED SAUSAGE 47 case (Muguerza et al. 2004), and by partial substitu- FORMULATION 48 tion of pork backfat (25%) in the second experiment 49 (Valencia et al. 2006a). It has been postulated that an Well-established physiological properties and health 50 adequate intake (AI) for adults is 0.22 g/day for both benefits of dietary fiber have been described. It has 51 EPA and DHA in 2000 kcal/day (Simopoulos 2002). uniquely significant physical effects in the gut and in 52 According to this, 50 g of modified sausages would addition, through fermentation, is a major determi- 53S make up approximately 34% and 30% of the AI for nant of large bowel function (Cummings et al. 2004). 54N DHA and EPA, respectively, in the case of batch A, Its physical properties in the small bowel affect lipid 40454 24 257-266 r1.qxd 7/20/07 3:49 PM Page 261

1 2 3 4 5 6 7

Deodorized 8 9 10 11 12 13

10.7 g/kg) Control Substitution 14 15 16 17 18 19 not determined. 20 21 22 23 24 25 26 27 28 29

polyunsaturated fatty acids. nd polyunsaturated 30

31 32 33 34 35 36 37 38 39 40 41 monounsaturated fatty acids; PUFA monounsaturated fatty acids; PUFA 25% 25% Modiﬁed A Modiﬁed B 25% 42

43 44 Soy Oil (1)Soy Linseed +A (2) Oil (3) Fish Oil +A (4) Fish 45 46 BHA 200 ppm). 47 48

Cholesterol and other lipid-related parameters obtained in dry-fermented sausages elaborated with high PUFA content oils. obtained in dry-fermentedCholesterol and other lipid-related parameters with high PUFA sausages elaborated 49 50

PUFA/SFA 1.6 2.0 1.4 1.8 1.851 1.8 1.8 1.7 1.7 saturated fatty acids; MUFA saturated fatty acids; MUFA 52 (mg/g product) product) (g/100 g product) (g/100 g product) product) product) SFA (g/100 g SFA 12.82 MUFA 5.16PUFA 10.79 15.51n-3 (g/100 g 7.88 14.05n-6 (g/100 g 0.41 13.85 5.16 4.75 10.33 14.25 0.66 7.22 7.22 11.59 11.73 0.36 10.36 6.28 4.78 14.51 2.57 11.57 4.96 13.28 5.20 0.36 10.08 6.77 14.60 5.89 0.59 9.99 12.30 4.61 4.65S53 1.07 11.98 5.62 5.70 0.31 4.34 1.41 4.22 1: Muguerza et al. (2003). 2: Ansorena and Astiasarán (2004b). 3: Muguerza et al. (2004). et al. (2006a). Valencia 4: Table 24.2. Table ParameterCholesterol 92.96 Control∑ 87.71 Substitution∑ Control∑ nd Substitution∑ Control∑ nd (+5.3 g/kg)n-6/n-3 ( MUFA PUFA/SFA 94 (BHT A: antioxidants 11.5 0.4 90 11.0 0.7 91 13.4 0.4 102 1.9 0.7 134 16.1 0.5 7.8 0.5 5.3 0.6 13.9 0.5 3.0 0.6 SFA SFA N54

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262 Product Categories: General Considerations

1 absorption and the glycemic response. Epidemiolog- Concerning dry-fermented sausages, different 2 ical studies point out that many of the diseases of types of fiber have been added alone or combined 3 public health significance—obesity, cardiovascular with some other ingredients into various types of 4 disease, type 2 diabetes—can be prevented or treated formulations (Table 24.3). Mendoza et al. (2001) 5 by increasing the amounts and varieties of fiber- manufactured low-fat content dry-fermented saus- 6 containing foods. According to the American ages with different percentages of powdered inulin, 7 Dietetic Association, recommendations are estab- which impacts a softer texture and a tenderness, 8 lished in 20–35 g/day for healthy adults and age plus springiness, and adhesiveness very similar to that of 9 5 g/day for children, and in addition, a higher fiber conventional sausages. A concentration of 11.5% 10 intake provided by foods is likely to be less calori- gave products with the lowest calorific value, and 11 cally dense and lower in fat and added sugar (Marlett they showed better sensorial results than control 12 et al. 2002). However, current intakes of fiber do not low-fat products (no inulin added, 6.3% pork 13 cover the recommended values, and incorporating it backfat) but still statistically lower than control 14 into frequently consumed foods would be an inter- high-fat products (no inulin added, 25% pork back- 15 esting strategy to reach those values. fat). Also Garcia et al. (2002) manufactured 16 17 18 Table 24.3. Results for % fat reduction, calorific value, and acceptability in relation to the use of inulin, cereals, 19 and fruit fibers as texture modifiers in low-fat dry-fermented sausages. 20 21 % Fat Caloric Value 22 Reduction (kcal/100 g) Acceptability 23 (1) Experiment with inulin Control high fat 392.2 7a 24 Control low fat 48.5 305.7 5.9b 25 R1 (7% inulin) 65.3 257.1 5.5b 26 R2 (6%inulin) 55.9 271.0 5.4b 27 R3 (11.5% inulin) 64.6 242.9 6.1b 28 R4 (10% inulin) 60.1 251.5 5.4b 29 30 (2.1) Experiment with wheat Control 25% fat 435.9 6.48 31 and oat fibers Control 6% fat 60 275.2 5.71a 32 Wheat 3% 65 281.9 4.04b 33 Wheat 1.5% 61 300.6 6.10a 34 Oat 3% 66 270.8 4.42b 35 Oat 1.5% 68 264.6 6.12a 36 Control 10% fat 62 287.9 5.99a 37 Wheat 1.5% 61 292.3 5.09a 38 Oat 1.5% 63 289 5.57a 39 (2.2) Experiment with fruit fibers Control 25% fat 435.9 6.48 40 Control 6% fat 60 275.2 5.71ab 41 Peach 3% 63 277 4.88b 42 Peach 1.5% 57 290 6.08a 43 Apple 1.5% 59 289.5 5.39ab 44 Orange 1.5% 62 287.7 5.94ab 45 Control 10% fat 62 287.9 5.99a 46 Peach 1.5% 59 277.5 5.93a 47 Apple 1.5% 61 269.9 6.00a 48 Orange 1.5% 57 278.9 6.33a 49 50 (1) Experiment with inulin. Source: Mendoza et al. (2001). Data with the same superscripts did not show significant 51 differences between them at level of significance of P 0.05. 52 (2) Experiment with dietary fibers (cereal 2.1 and fruit 2.2). Source: García et al. (2002). Data with the same superscripts 53S did not show significant differences between them at level of significance of P 0.05. Statistical treatment applied 54N independently. 40454 24 257-266 r1.qxd 7/20/07 3:49 PM Page 263

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dry-fermented sausages with 6% and 10% pork prebiotics are “nondigestible food components that 1 backfat with addition of cereal (wheat and oat) and beneficially affect the host by selectively stimulating 2 fruit (peach, apple and orange) dietary fibers, at the growth and/or activity of one or a limited number 3 1.5% and 3% concentration. These authors found of bacteria in the colon, which have the potential to 4 that sensorial and textural properties of batches with improve host health.” Some of the claimed benefits 5 3% dietary fiber were the worst, due to their hard- for probiotics are to alleviate lactose maldigestion, 6 ness and cohesiveness. The best results were increase resistance to invasion by pathogenic species 7 obtained with sausages containing 10% pork backfat of bacteria in the gut, shorten of rotavirus diarrhoea, 8 and 1.5% fruit fiber especially those with orange stimulate the immune system, and possibly protect 9 fiber. An appreciable decrease of the energetic against cancer. These microorganisms should fulfill 10 value was also achieved in these products. Aleson- other properties to be considered suitable as prebi- 11 Carbonell et al. (2003) formulated dry-fermented otics: resistance to gastric acidity and the action of 12 sausages with the addition of raw and cooked albedo bile salts, the adhesion to intestinal epithelial cells, 13 at five concentrations, evaluating the effect on the and the growth capability in the presence of prebi- 14 final chemical composition and on the sensorial otic carbohydrates (Bezkorovainy 2001; Ouwehand 15 properties. Products with 2.5% raw albedo and with et al. 2002). 16 2.5%, 5%, and 7.5% cooked albedo were those Most probiotic bacteria are lactic acid bacteria, 17 with sensory properties similar to control and with mainly lactobacilli and bifidobacteria, although ente- 18 improved nutritional benefits due to the fiber incre- rococci have also been used in the past as probiotics 19 ment and to the presence of active biocompounds (Foulquié Moreno et al. 2006). In consequence, food 20 from albedo, which also contributed to produce a products fermented by lactic acid bacteria and not 21 significant (P 0.05) reduction in the residual receiving heating treatment have been investigated in 22 nitrite levels and to delay oxidation development. order to evaluate their potential health benefits. In 23 The use of by-products of citrus fruits such as this context, the meat industry has made an effort to 24 albedo is also an environmentally friendly strategy find novel starter cultures with additional value. 25 (Fernández-López et al. 2004). Concerning meat products, different probiotic and 26 Sugar beet fiber, at a concentration of 2%, has also potential probiotic cultures isolated from other 27 been used in the production of Turkish-type salami, sources than meat have been applied as starter cul- 28 in combination with different concentrations of three tures for the production of dry-fermented sausages. 29 interesterified vegetable oils prepared from either However, more interesting strategies that combine 30 palm, cottonseed, or olive oil, leading to products commercial probiotic culture and meat starter cul- 31 similar to traditional ones regarding appearance, tures have been also successfully used. Andersen 32 color, texture and sensory scores, and with a signifi- (1998) used Bifidobacterium lactis Bb-12, Staphylo- 33 cantly higher fiber content (Javidipour et al. 2005). coccus xylosus DD-34, and Pediococcus pentosaceus 34 PC-1 to produce dry-fermented sausages. Even the 35 possibility of using only starter cultures with probi- 36 USE OF DRY-FERMENTED otic properties has been assayed. Erkkilä et al. (2001) 37 SAUSAGES AS PROBIOTICS examined the technological properties of three 38 selected probiotic Lactobacillus rhamnosus strains 39 According to the conclusions of the EC Concerted concluding that particularly Lactobacillus rhamno- 40 Action on functional foods, the gut is an obvious tar- sus GG, LC-705, and E-97800 seem to be suitable 41 get for the development of functional foods because for the production of dry sausages, whereas L. rham- 42 it acts as an interface between the diet and the events nosus LC-705 was not highly adapted to the meat 43 that sustain life. Among the functions related to the environment, resulting in slower growth rates and 44 physiology of the gut are those that involve the acidification of the dry sausage. 45 colonic microflora, the optimal intestinal function Other approaches in this field identify different 46 and stool formation, those that are associated with starter cultures or isolated strains from different 47 the gut-associated lymphoid tissue (GALT), and types of dry-fermented sausages, with the aim of 48 those that depend on the products of nutrient fer- exploring their probiotic potential by means of in 49 mentation. All these functions can be modulated by vitro and in vivo tests. For that purpose, Klingberg 50 different food components, such as probiotics, prebi- et al. (2005) isolated 22 strains of nonstarter Lacto- 51 otics, and symbiotics (Bellisle et al. 1998). As bacillus from 15 Scandinavian-type fermented 52 already known, probiotics are “foods that contain sausages. Nine of them were able to grow at 37°C S53 live bacteria which are beneficial to health,” whereas and lowered pH below 5.1 in a meat model. These N54 40454 24 257-266 r1.qxd 7/20/07 3:49 PM Page 264

264 Product Categories: General Considerations

1 strains and another 19 from a culture collection were • Partial substitution of NaCl in dry-fermented 2 evaluated by in vivo methods, including survival sausages by other salts as calcium ascorbate could 3 upon exposure to pH 2.5, bile tolerance, adhesion imply interesting health benefits, because of both 4 capacity to the human colon adenocarcinoma cell the sodium reduction and calcium supply. 5 lineCaco-2, and antimicrobial activity against poten- • The use of highly unsaturated oils as partial 6 tial pathogens. Results obtained in this study con- substitutes for pork backfat is an interesting way 7 firmed that 3 strains isolated from sausages, 2 to change the fatty acid profile of dry-fermented 8 identified as Lactobacillus plantarum and another as sausages. Reduction of saturated fatty acids and 9 Lactobacillus pentosus, were successfully applied as better PUFA/SFA and n-6/n-3 ratios can be 10 starter cultures for production of fermented sausage. achieved by using olive oil, linseed oil, and 11 No sensorial differences were detected with regard deodorized fish oil, among others. Addition of 12 to commercial sausages, and good technological antioxidants and proper packaging conditions are 13 properties were described for those products, effective against potential lipid oxidation 14 reaching high cell numbers (4.7*107—2.9*108 processes. 15 cfu/g) and good pH decrease (to 4.8–4.9) at the end • Partial substitution for pork backfat by inulin and 16 of ripening. other dietary fibers such as albedo, seems to be a 17 In a similar work, Pennacchia et al. (2004) per- viable strategy to develop low-fat dry-fermented 18 formed a screening procedure, by isolation at low pH sausages and to incorporate active biocompounds 19 and in presence of bile salts, to select potentially pro- into formulations. 20 biotic Lactobacillus strains directly from 10 fer- • Research into functional properties of starter 21 mented meat products. Twenty-eight Lactobacillus cultures point out the possible development of 22 strains, from the initially 150 isolated ones, were able novel dry-fermented sausages with health 23 to resist low pH and to grow in conditions simulating benefits. 24 the intestinal environment, suggesting their value as 25 probiotics. Twenty-five of them, identified by 16S REFERENCES 26 rDNA sequencing as 13 L. plantarum, 7 L. brevis, 27 and 6 L. paracasei, were selected for a further study, AHA. 2000. Dietary guidelines revision. A statement 28 in which a complete characterization was carried out, for Healthcare Professionals from the Nutrition 29 together with adhesion tests and analysis of growth Committee of the American Heart Association. Cir- 30 capability in the presence of different carbohydrates culation 102, pp. 2284–2299. 31 (Pennacchia et al. 2006). The results of this paper L Aleson-Carbonell, J Fernández-López, E Sayas- 32 demonstrated that several Lactobacillus strains iso- Barberá, E Sendra, JA Pérez-Alvarez. 2003. Utiliza- 33 lated from sausages possess these two properties, tion of lemon albedo in dry-cured sausages. J Food 34 pointing at the potential in future development of Sci 68(5):1826–1830. 35 novel dry-fermented sausages by using potential L Andersen. 1998. Fermented sausages produced with 36 functional starter cultures. They also show that it the admixture of probiotic cultures. 44th Inter- 37 would be useful to take into account the specific national Congress of Meat Science and Technology, 38 utilization patterns of a probiotic strain before incor- Barcelona, pp. 826–827. 39 porating it into a product containing prebiotic sub- D Ansorena, I Astiasarán. 2004a. The use of linseed oil 40 stances, such as novel symbiotic fermented sausages. improves nutritional quality of the lipid fraction of 41 Nevertheless, these authors also conclude that the dry-fermented sausages. Food Chem 87:69–74. 42 particular probiotic effect on human health can be ———. 2004b. Effect of storage and packaging on 43 given based only on in vivo experiments. fatty acid composition and oxidation in dry fer- 44 This is in agreement with the European Consen- mented sausages made with added olive oil and 45 sus Document on functional foods (Diplock et al. antioxidants. Meat Sci 67:237–244. 46 1999), where it was concluded that the most perti- F Bellisle, AT Diplock, G Hornstra, B Koletzko, 47 nent aspect in communicating health-related benefits M Roberfroid, S Salminen, WHM Saris. 1998. 48 of functional foods is that any claim of their func- Functional Food Science in Europe. Br J Nutr 49 tionality must be scientifically based. 80(Suppl 1):S1–S193. 50 A Bezkorovainy. 2001. Probiotics: Determinants of sur- 51 CONCLUSIONS vival and growth in the gut. Am J Clin Nutr 73(2): 52 399S–405S. 53S The following are conclusions based on the research JG Bloukas, ED Paneras, GC Fournitzis. 1997a. Effect 54N cited by this chapter: of replacing pork backfat with olive oil on processing 40454 24 257-266 r1.qxd 7/20/07 3:49 PM Page 265

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and quality characteristics of fermented sausages. cocci in food and health. Int J Food Microbiol 1 Meat Sci 45:133–144. 106(1):1–24. 2 CAST (Council for Agricultural Science and Technol- ML García, R Domínguez, MD Gálvez, C Casas, MD 3 ogy). 1991. Foods, Fats and Health. Task Force Selgas. 2002. Utilization of cereal and fruit fibres in 4 Report No. 118. low fat dry fermented sausages. Meat Sci 60: 5 R Cava, J Ruiz, C López-Bote, L Martín, C García, 227–236. 6 J Ventanas, T Antequera. 1997. Influence of finishing J Gelabert, P Gou, L Guerrero, J Arnau. 1995. Efecto 7 diet on fatty acid profiles of intramuscular lipids, de la sustitución del NaCl por Lactato Potásico, Glic- 8 triglycerides and phospholipids in muscles of the ina o KCl sobre las características del “fuet.” AIDA, 9 Iberian pig. Meat Sci 45:263–270. Zaragoza. 10 R Cava, J Ruiz, J Ventanas, T Antequera. 1999. Oxida- ———. 2003. Effect of sodium chloride replacement 11 tive and lipolytic changes during ripening of Iberian on some characteristics of fermented sausages. Meat 12 hams as affected by feeding regime: Extensive feed- Sci 65:833–839. 13 ing and alpha-tocopheryl acetate supplementation. O Gimeno, I Astiasarán, J Bello. 1998. A mixture of 14 Meat Sci 52:165–172. potassium, magnesium and calcium chlorides as a 15 G Cherian, JS Sim. 1995. Dietary α-linolenic partial replacement of sodium chloride in dry fer- 16 acid alters the fatty acid composition of lipid mented sausages. J Agric Food Chem 46:4372–4375. 17 classes in swine tissues. J Agric Food Chem ———. 1999. Influence of partial replacement of NaCl 18

43:2911–2916. with KCl and CaCl2 on texture and colour of dry fer- 19 WE Connor. 2000. Importance of n-3 fatty acids in mented sausages. J Agric Food Chem 47:873–877. 20 health and disease. Am J Clin Nutr 71(1 SUPPL): ———. 2001a. Inﬂuence of partial replacement of 21

171S–175S. NaCl with KCl and CaCl2 on microbiological evolu- 22 JH Cummings, LM Edmond, EA Magee. 2004. Dietary tion of dry fermented sausages. Food Microbiol 18: 23 carbohydrates and health: Do we still need the ﬁbre 329–334. 24 concept? Clin Nutr Suppl 1(2):5–17. ———. 2001b. Calcium ascorbate as a potential par- 25 L De la Hoz, MO López, E Hierro, JA Ordoñez, MI tial substitute for NaCl in dry fermented sausages: 26 Cambero. 1996. Effect of diet on the fatty acid com- Effect on colour, texture and hygienic quality at dif- 27 position of intramuscular and intermuscular fat in ferent concentrations. Meat Sci 57:23–29. 28 Iberian pig cured hams. Food SciTech Int P Gou, L Guerrero, J Gelabert, J Arnau. 1996. Potas- 29 2(6):391–397. sium chloride, potassium lactate and glycine as 30 AT Diplock, PJ Agget, M Ashwell, F Bornet, EB Fern, sodium chloride substitutes in fermented sausages 31 MB Roberfroid. 1999. Scientiﬁc concepts of and in dry-cured pork loin. Meat Sci 42:37–48. 32 functional foods in Europe: Consensus document. JD Higgs. 2000. The changing nature of redmeat: 20 33 Br J Nutr 81: Supp1. years of improving nutritional quality. Trends Food 34 M Enser, RI Richardson, JD Wood, BP Gill, PR Sheard. Sci Tech 11:85–95. 35 2000. Feeding linseed to increase the n-3 polyunsatu- L Hoz, M D’Arrigo, I Cambero, JA Ordóñez. 2004. 36 rated fatty acids of pork: Fatty acid composition of Development of an n-3 fatty acid and α-tocopherol 37 muscle, adipose tissue, liver and sausages. Meat Sci enriched dry fermented sausage. Meat Sci 67(3): 38 55:201–212. 485–495. 39 S Erkkilä, ML Suihko, S Eerola, E Petäjä, T Mattila- FB Hu, MJ Stampfer, JE Manson, A Ascherio, GA 40 Sandholm. 2001. Dry sausage fermented by Lacto- Colditz, FE Speizer, CH Hennekens, WC Willett. 41 bacillus rhamnosus strains. Int J Food Microbiol 1999. Dietary saturated fats and their food sources in 42 64(1–2):205–210. relation to the risk of coronary heart disease in 43 J Fernández-López, JM Fernández-Ginés, L Aleson- women. Am J Clin Nutr 70(6):1001–1008. 44 Carbonell, E Sendra, E Sayas-Barberá, JA Pérez- C Ibáñez, L Quintanilla, I Astiasarán, J Bello. 1997. 45 Alvarez. 2004. Application of functional citrus Dry fermented sausages elaborated with Lactobacil- 46 by-products to meat products. Trends Food Sci Tech lus plantarum-Staphylococcus carnosus. Part II. 47 15(3–4):176–185. Effect of partial replacement of NaCl with KCl on 48 M Flores, P Nieto, JM Ferrer, J Flores. 2005. Effect of the proteolytic and insolubilization processes. Meat 49 calcium chloride on the volatile pattern and sensory Sci 46:277–284. 50 acceptance of dry-fermented sausages. Eur Food Res C Ibáñez, L Quintanilla, C Cid, I Astiasarán, J Bello. 51 Tech 221(5):624–630. 1996. Part I. Effect of partial replacement of NaCl 52 MR Foulquié Moreno, P Sarantinopoulos, E Tsakalidou, with KCl on the stability and the nitrosation process. S53 L De Vuyst. 2006. The role and application of entero- Meat Sci 44:227–234. N54 40454 24 257-266 r1.qxd 7/20/07 3:49 PM Page 266

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1 C Ibáñez, L Quintanilla, A Irigoyen, I García-Jalón, processing and quality characteristics of fermented 2 C Cid, I Astiasarán, J Bello. 1995. Partial replacement sausages. Meat Sci 6:397–404. 3 of sodium chloride with potassium chloride in dry fer- E Muguerza, O Gimeno, D Ansorena, JG Bloukas, 4 mented sausages: Influence on carbohydrate fermenta- I Astiasarán. 2001. Effect of replacing pork backfat 5 tion and the nitrosation process. Meat Sci 40:45–53. with pre-emulsified olive oil on lipid fraction and 6 M Irie, M Sakimoto. 1992. Fat characteristics of pigs sensory quality of chorizo de Pamplona, a traditional 7 fed fish oil containing eicosapentaenoic and docosa- Spanish fermented sausage. Meat Sci 59:251–258. 8 hexanoic acids. J Animal Sci 70:470–477. RO Myer, JW Lamkey, WR Walker, JH Brendemuhl, 9 I Javidipour, H Vural, OO Özbas, A Tekin. 2005. Effects GE Combs. 1992. Performance and carcass charac- 10 of interesterified vegetable oils and sugar beet fibre teristics of swine when fed diets containing canola oil 11 on the quality of Turkish-type salami. Int J Food Sci and added copper to alter the unsaturated:saturated 12 Tech 40(2):177–185. ratio of pork fat. J Anim Sci 70:1147–1423. 13 F Jiménez-Colmenero, J Carballo, S Cofrades. 2001. AC Ouwehand, S Salminen, E Isolauri. 2002. Probi- 14 Healthier meat and meat products: Their role as func- otics: an overview of beneficial effects. Antonie Van 15 tional foods. Meat Sci 59:5–13. Leeuwenhoek 82(1–4):279–89. 16 S Kayaardõ, V Gök. 2004. Effect of replacing beef fat C Pennacchia, D Ercolini, G Blaiotta, O Pepe, 17 with olive oil on quality characteristics of Turkish G Mauriello, F Villani. 2004. Selection of Lacto- 18 soudjouk (sucuk). Meat Sci 66(1):249–257. bacillus strains from fermented sausages for their 19 TD Klingberg, L Axelsson, K Naterstad, D Elsser, BB potential use as probiotics. Meat Sci 67:309–317. 20 Budde. 2005. Identification of potential probiotic C Pennacchia, EE Vaughan, F. Villani. Potential probi- 21 starter cultures for Scandinavian-type fermented otic Lactobacillus strains from fermented sausages: 22 sausages. Int J Food Microbiol 105(3):419–431. Further investigations on their probiotic properties. 23 MR Law, CD Frost, NJ Wald. 1991. By how much does Meat Sci 73:90–91. 24 dietary salt reduction lower blood presure?. I-Analysis JR Romans, DM Wulf, RC Johnson, GW Libal, WJ 25 of observational data among population. BMJ 302: Costello. 1995. Effects of ground flaxseed in swine 26 811–815. diets on pig performance and on physical and sen- 27 JA Marlett, MI McBurney, JL Slavin. 2002. Position of sory characteristics and omega-3 fatty acid content 28 the American Dietetic Association Health Implications of pork II. Duration of 15% dietary flaxseed. J Anim 29 of Dietary Fiber. J Am Diet Assoc 102 (7):993–1000. Sci 73:1987–1999. 30 E Mendoza, ML García, C Casas, MD Selgas. 2001. C Severini, T De Pilli, A Baiano. 2003. Partial substitu- 31 Inulin as fat substitute in low fat, dry fermented tion of pork backfat with extra-virgin olive oil in 32 sausages. Meat Sci 57:387–393. “salami” products: Effects on chemical, physical and 33 I Mitsuharu, K Yoshihiro, S Keiichi, O Yuuko, sensorial quality. Meat Sci 64(3):323–331. 34 A Hiroyuki. 1997. The effects of fish oil enriched AP Simopoulos. 2002. The importance of the ratio o 35 with n-3 polyunsaturated fatty acids on lipids and omega-6/omega-3 essential fatty acids. Biomed 36 tasty compounds of pork loin. Nippon Shokuhin Pharmacother 56:365–379. 37 Kagaku Kogaku Kaishi 43:1219–1226. AS Truswell. 1994. The evolution of diets for western 38 CA Morgan. 1992. Manipulation of the fatty acid com- diseases. In: Food. Multidisciplinary Perspectives. 39 position of pig meat lipids by dietary means. J Sci B Harris-While, R Hoffenderg, eds. Oxford, UK: 40 Food Agric 58:357–368. Basil Blackwell. 41 E Muguerza, D Ansorena, I Astiasarán. 2004. Functional USDA. 2000. US Department of Agriculture-US 42 dry fermented sausages manufactured with high levels Department of Health and Human Services: “Nutri- 43 of n-3 fatty acids: Nutritional benefits and evaluation tion and your health: Dietary guidelines for 44 of oxidation. J Sci Food Agric 84(9): 1061–1068. Americans.” Homes and Garden Bulletin No. 232. 45 E Muguerza, D Ansorena, JG Bloukas, I Astiasarán. Washington, D.C. U.S. Government Printing Office. 46 2003. Effect of fat level and partial replacement of I Valencia, D Ansorena, I Astiasarán. 2006a. Nutritional 47 pork backfat with olive oil on the lipid oxidation and and sensory properties of dry fermented sausages 48 volatile compounds of Greek dry fermented enriched with n-3 PUFAs. Meat Sci 72:727–733. 49 sausages. J Food Sci 68(4):1531–1536. ———. Stability of linseed oil and antioxidants con- 50 E Muguerza, G Fista, D Ansorena, I Astiasarán, JG taining dry fermented sausages: A study of the lipid 51 Bloukas. 2002. Effect of fat level and partial fraction during different storage conditions. Meat Sci 52 replacement of pork backfat with olive oil on 73:269–277. 53S 54N 40454 25 267-272 r1.qxd 7/21/07 9:58 AM Page 267

1 2 3 25 4 5 6 International Standards: USA 7 8 Melvin C. Hunt and Elizabeth Boyle 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION primary means for delivering rules, policies, and 22 guidelines to processors. 23 Fermented products made from muscle foods have 24 been produced for centuries. Demeyer and Toldrá 25 FEDERAL REGISTER PUBLICATIONS (2004), Incze (2004), and Toldrá (2004) provide 26 AND RELATED DOCUMENTS excellent descriptions of the art and science of the 27 manufacture of these products. Globally, consider- All official FSIS policy documents are published in 28 able variation exists in the sensory, chemical, and the Federal Register, including notices and proposed 29 physical properties of fermented (and often dried) rules, and interim and final rules. Final rules are 30 meat products. Because of this variation, it is chal- incorporated into the U.S. Code of Federal Regula- 31 lenging for regulatory agencies to categorize and tions, commonly referred to as the CFR, the year 32 create standards for these unique products. The pur- after a rule becomes final. Definitions and standards 33 pose of this chapter is to present the U.S. “standards” of identity or composition are found in 9 CFR, with 34 for these products. Although there is no one docu- Subparts I and J reserved for semidry-fermented 35 ment that describes in detail the regulatory issues for sausage and dry-fermented sausage, respectively. At 36 fermented meat and poultry products, a description this time, there are no standards of identity mandated 37 of United States Department of Agriculture (USDA) for these products. There are, however, other regula- 38 regulatory documents that relate to this product cate- tions that pertain to these product categories that are 39 gory are presented below. described later in this chapter. 40 41 42 FSIS DIRECTIVES:DIRECTIVES 43 U.S. REGULATORY PROCESS Directives contain instructions of an indefinite dura- 44 In the U.S., a total of nine government agencies are tion. Table 25.1 lists the series of directives that pro- 45 involved with ensuring that meat and poultry are vide instructions for processors. 46 safe and wholesome (Hale 1994). The USDA Food 47 Safety and Inspection Service (FSIS) is the primary 48 FSIS NOTICES government agency that develops and administers 49 regulations, standards, instructions, and guidelines Notices are temporary instructions that are sched- 50 for the meat and poultry industry. The following is uled to expire no later than 1 year from the issuance 51 a description of official documents that are the date. 52 53 S54 N55 267 40454 25 267-272 r1.qxd 7/21/07 9:58 AM Page 268

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1 Table 25.1. Series of FSIS directives providing CFR and the Meat and Poultry Inspection 2 instructions to processors. Regulations, Directives, Notices, and Guidelines. 3 Navigating these documents can be challenging Series 4 and requires some diligence; nevertheless, becoming Number Category 5 familiar with their contents is essential for inspec- 6 5000 Program services tion and product approval. 7 6000 Slaughter inspection 8 9 7000 Processed products REGULATORY DEFINITIONS AND 10 8000 Compliance evaluation and enforcement SPECIFICATIONS 11 9000 Exports and imports USDA FSIS requires that shelf-stable sausages 12 meet specific moisture protein ratio and pH require- 13 10000 Laboratory services ments. The following is a description of selected 14 11000 Facilities, equipment, and sanitation items that pertain to fermented meat and poultry 15 12000 Voluntary inspection products. 16 17 18 MOISTURE PROTEIN RATIO (MPR) AND PH 19 Nonrefrigerated or shelf-stable sausages must 20 have an MPR of 3.1:1 or less and a pH of 5.0 or less, 21 PROPOSED OR FINAL RULE unless commercially sterilized. This does not apply 22 A proposed or final rule typically includes the to products containing more than 3.5% binders or 23 following: 2% isolated soy protein. Specific MPR for dry, 24 semidry, and other sausages are shown in Table 25.2. 25 1. Preamble discussion of the provisions of the rule, 26 and the need for and the statutory basis for the 27 provisions. SHELF-STABLE SAUSAGE 28 2. A regulatory impact analysis estimating the future Dry sausage must have an MPR of 1.9:1, unless there 29 costs and benefits of the rule and discussion of is a specified MPR. Semidry, shelf-stable sausage 30 regulatory alternatives considered. If the rule is must have an MPR of 3.1:1 or less and pH of 5.0 or 31 designated “nonsignificant” or “exempt,” a less, unless commercially sterilized or unless MPR is 32 detailed regulatory impact analysis is not specified for a particular type of sausage. Alternatively, 33 required. nonrefrigerated, semidry, shelf-stable sausages are 34 3. A discussion of the estimated economic impact those that meet the following description: 35 of the regulations on small businesses. 36 4. Statements addressing relevant Executive orders. 1. Are fermented to a pH of 4.5 or lower (or the pH 37 5. Proposed regulatory provisions or final regulatory may be as high as 4.6 if combined with product 38 provisions that will be codified in the CFR. water activity no higher than 0.91). 39 6. A request for comment on a proposed rule, or, 2. Are in an intact form or, if sliced, are vacuum 40 if a final rule, a summary of comments received packed. 41 to the proposed rule and FSIS response to the 3. Have internal brine concentration no less than 5%. 42 comments. 4. Are cured with nitrite or nitrate. 43 7. A risk assessment of the rule for a “major” rule 5. Are smoked with wood. 44 that affects human health, human safety, or the 45 environment. DRY AND SEMIDRY SAUSAGES 46 47 Dry sausages may or may not be characterized by a THE FOOD STANDARDS AND LABELING POLICY 48 bacterial fermentation. When fermented, the inten- BOOK (USDA 2005) 49 tional encouragement of a lactic acid bacterial 50 This book provides definitions and descriptions that growth is useful as a meat preservative, as well as 51 aid processors with preparation of product labels. producing the typical tangy flavor. They may be 52 To be in compliance with all regulatory issues, the smoked, unsmoked, or cooked. Dry sausages that are 53S Policy Book must be used in conjunction with other “medium” dry generally lose about 70% of their 54N regulatory documents, including those found in the original weight, but the sausages typically range 40454 25 267-272 r1.qxd 7/21/07 9:58 AM Page 269

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Table 25.2. Speciﬁc moisture protein ratio (MPR) for dry, semidry, and other sausages. 1 2 Type Classiﬁcation Maximum MPR 3 Blockwurst Semidry 3.7:1 4 5 Calabrese Salami 6 Cervelat Semidry or dry No MPR requirement 7 Chorizo Semidry or dry 8 9 Easter Nola Salami 10 Farmer Sausage Cervelat Semidry 11 Farmer Summer Sausage Dry 1.9:1 12 Frizzes Dry 1.6:1 13 14 Genoa or Genoa Salami Dry 2.3:1 15 Goteborg Dry 16 Gothaer Cervelat Semidry 17 Landjaeger Cervelat Semidry 18 19 Lebanon Bologna Semidry 3.1:1 or less and pH 5 or less 20 Lyons Sausage Dry 21 Metz Sausage Semidry 22 23 Milan or Milano Salami Dry 1.9:1 24 Mortadella Semidry or dry 25 Pepperoni Dry 1.6:1 26 Poultry Salami Dry 1.9:1 27 28 Salami Dry 1.9:1 29 Sicilian Sausage Dry 2.3:1 30 Hard Salami Dry 1.9:1 31 Italian Sausage Dry 1.9:1 32 33 Sarno Dry 34 Soppresate Dry 1.9:1 35 Soujouk Dry 2.04:1 36 37 Summer Sausage Semidry 38 Thuringer Semidry 3.7:1 39 Touristen Wurst Semidry 3.7:1 40 Ukranian Sausage Dry 2.0:1 41 42 Adapted from USDA (2005). 43 44 45 from 60–80% of the original weight. Semidry saus- is applicable to using this species. Products that con- 46 ages are usually partially dried and fully cooked in a tain pork muscle tissue (not including pork hearts, 47 smokehouse. They are semisoft sausages with good pork stomachs, and pork livers) must be effectively 48 keeping qualities due to their lower pH. heated, refrigerated, or cured to destroy any possible 49 live trichinae. USDA FSIS describes in 9 CFR 50 318.10 various methods for control of trichinae in 51 TRICHINAE fermented meat and poultry products (CFR 2006). 52 Because fermented sausages (with no or minimal Many other terms listed in the Policy Book S53 thermal processing) often include pork, the following (USDA 2005) may be essential knowledge for N54 40454 25 267-272 r1.qxd 7/21/07 9:58 AM Page 270

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1 meeting USDA regulations, depending on the prod- • Alternative 2: Processors employ either a 2 uct, claims of regionality, or labeling terms that may postlethality treatment or a growth inhibitor for 3 be used with the product. Listeria on RTE products. Establishments opting 4 for this alternative are subject to more frequent 5 HACCP OPTIONS FSIS verification activity than for Alternative 1. 6 • Alternative 3: Processors employ sanitation 7 On July 25, 1996, the USDA FSIS mandated require- measures only. Establishments opting for this 8 ments designed to reduce the occurrence and num- alternative are targeted with the most frequent 9 bers of pathogens on meat and poultry products, level of FSIS verification activity. Within this 10 reduce the incidence of foodborne illness associ- alternative, FSIS places increased scrutiny on 11 ated with consuming these products, and provide a operations that produce hot dogs and deli meats. 12 framework for modernization of the meat and poultry In a 2001 risk ranking, FSIS and the Food and 13 inspection system. The new regulations required Drug Administration identified these products as 14 establishment of four new programs, three as pathogen posing relative high-risk for illness and death. 15 reduction measures and one for HACCP. Published guidelines (FSIS 2003) will assist pro- 16 USDA FSIS requires that a written HACCP plan cessors with implementing alternatives 1, 2, or 3. 17 include all decision-making documents and support- 18 ing documentation used in the development of an 19 HACCP plan. HACCP records for slaughter activi- VALIDATION 20 ties and refrigerated product must be maintained for 21 1 year; records for frozen, preserved, or shelf-stable From a regulatory standpoint, USDA FSIS requires 22 products must be stored for 2 years. All HACCP that manufacturers of dry and semidry sausage prod- 23 records must be maintained on-site for a minimum ucts either: (1) demonstrate, through a process vali- 24 of 6 months. After 6 months, records may be stored dation study, the ability to reduce a 7 log inoculum Ն 25 off-site, providing they can be retrieved within 24 of E. coli O157:H7 by 5 logs, or (2) institute a sta- 26 hours if requested by inspection authorities. tistically based sampling program for finished prod- 27 Fermented meat and poultry products are shelf- uct that affirms the absence of E. coli O157:H7 28 stable and ready-to-eat and they must meet USDA (Appendix B). In 1997, the American Meat Institute 29 HACCP regulations. Products such as dry salami, released a white paper titled “Good Manufactur- 30 summer sausage, and pepperoni would be consid- ing Practices for Fermented Dry & Semi-Dry 31 ered to fall under the HACCP category of not heat- Sausage Products” (AMIF 1997). This paper pres- 32 treated, shelf-stable meat and poultry products. If a ents pathogens of concern in fermented products and 33 fermented sausage was fully cooked, it would fall discusses strategies for controlling pathogens, includ- 34 into the heat-treated, shelf-stable meat and poultry ing Staphylococcus aureus, E. coli O157:H7, Listeria 35 product category. monocytogenes, Salmonella, and Trichinella spiralis. 36 USDA FSIS has mandated a rule that requires pro- In addition, good manufacturing practices for differ- 37 ducers to employ effective measures to control Liste- ent stages throughout production, cleaning, and san- 38 ria monocytogenes in ready-to-eat products. Most itation are discussed. 39 FSIS verification activities are concentrated in those 40 establishments that rely solely on sanitation for pre- ACKNOWLEDGMENT 41 venting Listeria contamination, and in which there is 42 no limitation on the growth of the pathogen if present Contribution No. 07-9-B from the Kansas Agricul- 43 in the product. Establishments would need to select tural Experiment Station, Manhattan, KS 66506 44 the most effective strategies to control Listeria and U.S.A. 45 would select one of the three following alternatives: 46 REFERENCES 47 • Alternative 1: Processors employ both a 48 postlethality treatment and a growth inhibitor for AMIF (American Meat Institute Foundation). 1997. 49 Listeria on RTE products. Establishments opting Good Manufacturing Processes for Fermented Dry & 50 for this alternative are subject to FSIS verification Semi-dry Sausage Products. http://www.amif.org/ 51 activities that focus on the postlethality treatment FactsandFigures/SAUSAGE.pdf. 52 effectiveness. Sanitation is important, but it is CFR (Code of Federal Regulations). 2006. Code of 53S built into the degree of lethality necessary for Federal Regulations (CFR): Main Page. http://www. 54N safety as delivered by the postlethality treatment. gpoaccess.gov/cfr/index.html. 40454 25 267-272 r1.qxd 7/21/07 9:58 AM Page 271

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Demeyer D and F Toldrá. 2004. Fermentation. In: Jensen, Sciences. Oxford: Elsevier, Academic Press, pp. 1 Devine, Dikeman, eds. Encyclopedia of Meat Sci- 1207–1216. 2 ences. Oxford: Elsevier, Academic Press, pp. 467–474. F Toldrá. 2004. Dry Curing. In: Jensen, Devine, Dike- 3 FSIS (Food Safety and Inspection Service). 2003. FSIS man, eds. Encyclopedia of Meat Sciences. Oxford: 4 Rule Designed to Reduce Listeria monocytogenes in Elsevier, Academic Press, pp. 360–366. 5 Ready-to-Eat Meat & Poultry. http://www.fsis.usda. USDA (United States Department of Agriculture). 6 gov/Fact_Sheets/FSIS_Rule_Designed_to_Reduce_ 2005. Food Standards and Labeling Policy Book. http:// 7 Listeria/index.asp. www.fsis.usda.gov/OPPDE/larc/Policies/Labeling_ 8 DS Hale. 1994. Inspection. In: Kinsman, Kotula, Policy_Book_082005.pdf. 9 Breidenstein, eds. Muscle Foods: Meat, Poultry and USDA-FSIS. 1999. Compliance Guidelines for Cool- 10 Seafood Technology. New York: Chapman and Hall, ing Heat-Treated Meat and Poultry Products (Sta- 11 pp. 163–185. bilization). Appendix B. http://www.fsis.usda.gov/ 12 K Incze. 2004. Dry and semi-dry sausage. In: OPPDE/rdad/frpubs/95-033F/95-033F_Appendix% 13 Jensen, Devine Dikeman, eds. Encyclopedia of Meat 20B.htm. 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 S53 N54 40454 26 273-288 r2.qxd 7/20/07 3:52 PM Page 273

1 2 3 26 4 5 6 International Standards: Europe 7 8 Reinhard Fries 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION Food Chain versus End Product 22 23 For appropriate testing, sometimes the chain is Food technology has grown historically, reflecting 24 observed, sometimes the final product should be summarized experience from the past, giving also 25 observed. The end product with its chemical compo- tribute to changes of nutritional habits of a popula- 26 sition is defined by recipes characterizing raw incom- tion. Today’s scientific insight (technological as well 27 ing materials as well as the manufacturing procedure. as from the hygienic point of view) potentially 28 This is true also for meat quality factors (e.g., tender- provides a high level of food technology and 29 ness). Meanwhile, some factors can be determined hygiene. 30 only in the food chain itself, i.e., ecological balance 31 in the food chain, or the observation of bioethical fac- 32 tors during the keeping of food animals. THE TERM OF QUALITY 33 Disciplines, Parameters, and Methods 34 GENERAL REMARKS 35 For comparison of results, an agreement on the tech- A food chain consists of several different operators— 36 nical performance (the method that is finally used to sellers, customers, and finally consumers—all pay- 37 characterize the quality factor) is necessary. Disci- ing attention to different aspects of quality. So, a 38 plines and parameters of analysis may be: general agreement on quality does not exist: Quality 39 is dependent on the point of view—legal or internal • Biochemistry (glycogen, lactate, content of 40 use, producer’s or customer’s—as well as the given ATP/ADP/AMP, IMP or Inosine and further 41 circumstances. Factors of quality also reflect the decomposition products thereof). 42 period of history or a particular geography and the • Chemistry (representing the basic composition of 43 sociocultural situation. a sample, e.g., protein or adipose-tissue content). 44 In Figure 26.1, each circle represents the view of • Physical examination (using pH, water binding 45 quality of particular groups, with one point of view capacity, or temperature). 46 being separate from the others, but overlapping, and • Bacteriology, representing hygiene relevant 47 simultaneously covering a different scope of the total agents (spoilage), agents of technological 48 of quality factors. However, there are some common relevance (lactobacilli), or zoonotic agents (health 49 and basic items: In each food commodity, main factors). The bacteriological composition is 50 issues are the health factor of the product as well as commanded by the ecology (intrinsic and 51 its nutritional and technical value (composition and extrinsic factors) of a food as well as the initial 52 shelf life). status of incoming raw materials. 53 S54 N55 273 40454 26 273-288 r2.qxd 7/20/07 3:52 PM Page 274

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1 • Organoleptic (e.g., pH, color, structure, consistency Regarding the nutrition value, the best food conver- 2 of the sample). Spoilage, i.e., decomposition, would sion rate of the food animal species provides a good 3 be recognized at a given time. So the sensory of relationship of effort and efficacy. 4 foods or esthetical quality (Latin qualitas: “how 5 is it”) is an important issue, too (Figure 26.2). Changing Ideas of Quality in the Past 6 In the EU, the concern with poultry meat was differ- 7 POULTRY MEAT ent at different times (Petersen 1997), beginning 8 with the need for supply of high-quality animal pro- 9 Poultry meat is accepted worldwide without any tein, followed by the technical ability to stock high 10 religious or sociocultural restrictions. Accordingly, numbers of birds independent of the climate in com- 11 consumption has greatly increased in the last decades. bination with keeping the animals healthy in large 12 numbers. Consecutively, human concerns (consumer 13 protection, amount of workload) were raised, and 14 finally the environmental impact and animal well 15 being was a concern. 16 17 Food Chain Technology and Hygienic 18 Consequences 19 In low-cost countries, one will find abattoirs with a 20 lower grade of mechanization, whereas in the EU, 21 poultry meat production is characterized by high 22 technological standards in slaughter and processing, 23 including further processing. More sophistication is 24 to be expected. 25 To run an abattoir at high speed (up to 12,000 26 birds/h) requires sophisticated technology. Accord- 27 Figure 26.1. Overlapping circles of quality adoption. ingly, in primary production, large herds supply 28 29 30 31 Primary 32 Production Animal health, animal welfare Meat Production 33 (diseases, technopathy) Line (Abattoir) 34 Technology of the line 35 36 37 38 39 Final status of Meat for further processing 40 fresh poultry meat water binding capacity 41 pH/ conductivity 42 consistency 43 44 45 46 47 Health status Welfare Shelf life Nutritive value Sensory 48 status status 49 Zoonotic agents keeping APC weight smell 50 Residues technique temperature % muscles color 51 Resistance age of the % adipose tissue consistency 52 product % bones taste % process structure 53S 54N Figure 26.2. Different quality aspects are caused from different stages. 40454 26 273-288 r2.qxd 7/20/07 3:52 PM Page 275

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these abattoirs, with vertical flux from breeding Table 26.1. Isolates found during poultry meat 1 via several steps of husbandry, transport, lairage to processing: Carcasses and water (Fries 1988, 2005). 2 slaughter and processing, with a transport-technology 3 Micrococcaceae 463 already fitting into the slaughter line. 4 In such chains exists a permanent transfer of Gram-positive irregular rods 395 5 (zoonotic) agents from primary production into the Streptococcus and related 158 6 abattoir. Identical strains of salmonellae or Campy- Enterobacteriaceae 87 7 lobacter might be found over days and throughout 8 the line, vertically as well as horizontally, and at sev- Bacillus 159 9 eral positions, the bird brings in its burden from Lactobacillus 99 10 prior stages. Such events are—at present—either an Listeria 45 11 unavoidable part of the technology, or they happen 12 Flavobacterium 11 because of accidents. 13 So, even an elaborate and reliable hygiene pro- Acinetobacter 7 14 gram in the abattoir cannot prevent that transfer, Moraxella 5 15 because effective barriers, which could be able to Others 11 16 control zoonotic agents, do not exist at this stage of 17 Total 1,440 isolates the line (Fries 2002). 18 Over the years, Salmonella was found most fre- 19 quently in low numbers. Recently in the Netherlands, 20 Dufrenne et al. (2001) detected in most samples Sal- quality of waste) also are of growing importance 21 monella in numbers of <10/carcass, which is still (Table 26.1). 22 comparable to the quantitative data from broiler car- 23 casses (end product) some 20 years ago, where most Legal Requirements 24 frequently positive cases did not exceed a contamina- Poultry meat processing has to observe human and 25 tion rate of <10/total skin (Fries 2002). animal health and must be aware of animal well being. 26 27 Animal Welfare and Well Being Worker’s protection might be an additional task. So, in primary production, foreign substances, 28 Animal welfare in the holdings is an issue in inten- zoonotic agents and resistance, zoonoses, other dis- 29 sive keeping. Circumstances of holding technology eases, as well as technical performance of the proce- 30 and management lead to losses because of condem- dures (reflecting animal welfare) are important 31 nation and downgrading, which touches the ethics of factors of the process. In the end, a wholesome meat 32 animal keeping, another and only recently raised or meat product with acceptable quality and shelf 33 term of quality. These days, such animal rights stability should be available. 34 would be discussed. In 2002, Germany was the first For that, the animals must remain under control 35 country to grant the animals rights in the rank of the during their whole lifespan (“from stable to table”). 36 constitution (Cherney 2004). It might be done on a mandatory basis (“control of 37 38 Poultry Meat as Such control” or direct supervision) as well as based on an internal control, which is a question of organization, 39 Organoleptic aspects (taste, smell, consistency, personnel, and political decisions, and which is not 40 appearance) or the usability of the food (shelf life, to be discussed here. 41 storability under extreme climates, or the meat, from According to Regulation (EC) 178/2002, the 42 its composition, fitting into further processing lines) farmer as a food entrepreneur bears responsibility for 43 are important. With regard to shelf stability, the gen- the animal intended to produce food for human con- 44 eral spoilage microflora and their pattern throughout sumption (product responsibility). Consequently, the 45 the processing line should be considered, too. Hav- food chain character of animal production has been 46 ing used nonselective media for isolation, Table 26.1 brought into Regulation (EC) 854/2004 on the sur- 47 gives a picture of microbiological colonization of veillance of food animals. Moreover, for poultry, 48 poultry and (scalding- and chilling-) water samples Regulation (EC) 2160/2003 must be followed, requir- 49 during processing. ing freedom of Salmonella, e.g., for layers from 50 November 17, 2007, for broilers from November 17, 51 Environment 2008, or for turkeys from November 17, 2009. 52 Finally, impact on the environment (release of In anticipation of the philosophy of the food S53 N54 NH3, energy costs of processing, quantity, and chain, earlier legislation of the EU (by then EEC) 40454 26 273-288 r2.qxd 7/20/07 3:52 PM Page 276

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1 reflects particular details of the whole production • Organic acids and related compounds 2 line: • Anorganic acids and related compounds 3 • Phosphorus (sodium phosphates, • Regulation (EEC) 1538/91 (“quality”): 4 trisodiumphosphate TSP, tripotassiumphos- Classification of carcasses (carcass weight, skin 5 phate TPP, sodiumhypophosphite) condition, fat content), state of evisceration (e.g., 6 • Sulphur (sulfur dioxide, sodium disulfite) New York dressed), trade conditions 7 • Anorganic nitrogen compounds such as • Directive 92/117/EEC for “Zoonoses”: 8 nitrite. Concealment of zoonotic agents 9 • Physical factors • Directive 71/118/EEC for Poultry Meat Hygiene 10 • Application of different wave lengths and Inspection Procedures: Flock health, 11 • X-rays residues, antemortem inspection, post mortem 12 • Ultraviolet inspection with macroscopic lesions to be 13 • Microwaves observed, processing procedures (water uptake, 14 • Ultrasonic evisceration efficacy). 15 • Application of temperature 16 • High temperature 17 FERMENTED MEAT PRODUCTS • Low temperature 18 The term of (poultry) meat products implies that the • Use of mechanical devices 19 material has been processed beyond the point of • Washing and sanitizing 20 fresh meat. Such lines are located at the very end of • Desiccation of surfaces 21 the food chain. Here, characteristics of the incoming • Pressure 22 raw material are needed, because it is the initial • Prohibition of atmosphere (vacuum, 23 “raw” material for the next, and qualitatively differ- modified atmosphere) 24 ent, processing line. • Biological factors 25 • Competing flora (starters, protective cultures) 26 Food Preservation as Such • Metabolites (bacteriocines, colicine, 27 For food technology in general, one should expect natamycine) 28 the product to cover specific requirements: • Enzymes (lysozyme, lactoperoxidase) 29 30 • No health impacts to consumers (safety) In the end, factors affecting microbial associations 31 • Storable within a certain and product-typical time and metabolism are (Mossel 1979, modified). 32 frame (shelf stability) 33 • Acceptable in the sense of sensory value • Intrinsic Factors: Nutrient composition, 34 • No loss of nutrients or losses remaining in an structural barriers, antimicrobial constituents unavoidable low range (nutritive value) 35 (naturally occurring or added), pH, aW, Eh, 36 • Production costs in an acceptable range amount of initial bacteria 37 • No rendering problems or remaining residues • Extrinsic Factors: Temperature, type of 38 from the applied technology packaging, duration of exposure 39 • No risk in the application of the technique for • Implicit Factors: Antagonisms and synergisms, 40 workers generation time of bacteria. 41 Ingredients, recipes, and technology result in a par- 42 ticular commodity with a given composition and From their microbiological background, particular 43 with given quality items as well as a product-specific technological procedures can correspond with a par- 44 microbiological composition—i.e., the respective ticular impact on microbiological colonization of the 45 technical sequence has a specific impact on microor- material (Table 26.2). 46 ganisms, leading to no restriction, to the cease of 47 metabolism, or even to the destruction of the biolog- The Product 48 ical agent. Such a preservation potential of actions is In Europe, many fermented meats are cured using 49 based on chemical, physical, or biological action; nitrites or nitrates. In that case, preservative mea- 50 these three main disciplines convey different ways of sures and factors are chemical (nitrite or nitrate), 51 antimicrobiological pressure: physical (a ), and (micro-) biological (lactobacilli, 52 W 53S • Chemical factors as well as micrococci, if nitrate is used as a preserva- 54N • Alcohols tive), which results in the characteristic sensory of • Aldehydes (glutare-di-aldehyde) that product. 40454 26 273-288 r2.qxd 7/20/07 3:52 PM Page 277

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Table 26.2. Impact of technological commodity on microbiological background. 1 2 Technology Microbiological Background Commodity 3 Organic acids, lactobacilli Impact on pH (lower) Fermented sausage 4 5 Wrapping (folia), MAP E (atmosphere change) All products h 6

Drying, salting, sugar treatment aw (lowering) Fermented sausage, dried products 7 Chilling/deep freezing Slowdown/cease of metabolism Fresh meat, convenience products 8 9 Preserves/pasteurization Heat/pressure (destroying Canned foods, milk, egg products 10 of the structure) 11 Starter cultures Microbiological competition Fermented sausage 12 Nitrite/nitrate, smoking Adverse (toxic) chemical agents Salami-type sausage 13 Combinations pH/a /nitrite Shelf-stable products 14 W 15 16 17 Fermented products need some time to reach the Table 26.3. Comparison of examination results 18 final pH. In some areas such as Germany, some between fast-ripening and slow-ripening products. 19 products are marketed within 3 to 5 days, after which 20 the product would be sold as a fresh fermented Fast-ripening Slow-ripening 21 sausage. That environment would allow salmonellae Products Products 22 23 to survive. Consequently, “fresh” fermented prod- pH 4.8–5.2 5.3–.8 24 ucts must be considered still to harbor still salmonel- (use of nitrite) (use of nitrate) lae, if such organisms have been initially present. 25 a <0.90 >0.90 Recently a new type of product has emerged (“meat W 26 preparations”), in which meat is only salted and some lactobacilli ϩϩϩ ϩϩϩ 27 spices are added. Products like this do not belong to micrococci ϩϩϩ 28 the category of meat products, because they are not 29 APC log 8 log 8 30 preserved by means of reduced aW/pH, and because they are only refrigerated. Therefore the risk of salmo- 31 nellae in such “meat preparations” remains high, 32 For health-related microbiology, the presence/ especially in products stemming from raw material 33 absence of zoonotic agents, in particular in very with high prevalence of salmonellae, such as poultry. 34 recently prepared “fresh” fermented products, are These products have not been fermented. 35 important items. However, the risk of a positive For a fermented product, preservation techniques 36 Salmonella result is low because of the low pH and and sensory state are typical. A product of European 37 low a . origin (e.g., salami) would be characterized as w 38 follows: 39 MICROBIOLOGICAL 40 • Chemical composition: In Germany, the German SAFEGUARDING IN FOOD 41 Food Collection (Deutsches Lebensmittelbuch, 42 N.N. 2002) refers to the percentage of animal- CHAINS 43 originated (muscle) protein as well as a product of RISK ANALYSIS (RA) 44 typical sensory and appearance. 45 • Product typical sensory: The characteristic taste Risk analysis comprises the scientific assessment of 46 and odor, consistency, and mincing state depends hazards in a given line as well as—separately— 47 on the product characteristics. So, some drawing consequences as managerial steps (Figure 48 fermented sausages have been minced very 26.3). So, RA is an analytical instrument directed 49 intensively, and others only to a lesser extent (the backward toward the given line, first to find out pos- 50 original product of “salami”) or not at all (cured sible hazards and second to determine possible risks. 51 and fermented “raw ham”). The risk profile shortens this procedure in a more 52 practical way. However, the approach is basically the S53 Besides that, important results of examination same. A risk profile provides an initial evaluation N54 depend on the technology used (Table 26.3). of the food safety issue in relation to the scope of 40454 26 273-288 r2.qxd 7/20/07 3:52 PM Page 278

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1 Food 2 in case of 3 occurrence of pathogenic 4 organisms (hazard identification) 5 6 a) Risk Assessment: 7 * hazard identification 8 * hazard characterization 9 * exposure assessment 10 in order to prevent human harm caused by food 11 12 establishing of guidelines 13 (voluntary, targeting hygiene) 14 * GMP, GHP * Specifications 15 result: 16 * Indicators 17 18 no risk (of human harm) risk (to consumer’s health) 19 20 21 no particular control 22 b) Risk Management: 23 development of (microbial) control options 24 a) in the framework of general terms 25 b) in the case of a particular plant: 26 using principles of (preventive) HACCP: 27 * setting criteria: preferably instantly meaningful methods 28 (i.e., mostly physical criteria: pH, time, temperature) 29 30 * establishing monitoring systems incl. corrective actions procedures for regularly verification (microbiological 31 methods). Limits serving as verification of the system 32 being under control 33 * documentation 34 35 c) Risk Communication: 36 to consumers 37 to the industry 38 to the trade to the authorities 39 40 Figure 26.3. Interrelations between Risk Assessment and Risk Management, as well as HACCP (based on 41 European Commission 1998; Lammerding 1997). 42 43 44 public health concerns, based on available informa- systems. ALOP is derived from a risk assessment 45 tion and surveying available control measures. and is expressed as, e.g., the likelihood to suffer a 46 The terms Appropriate Level of Protection food-related illness from a food serving, or the num- 47 (ALOP) and Food Safety Objectives (FSO) may ber of cases per 100,000 consumer years. The setting 48 serve as sort of practical and achievable blueprints of an ALOP is a risk management decision. 49 for what could be realistically reached at the present Food Safety Objectives (FSO) may be an impor- 50 state. The SCVPH (2003 I) stated: tant element in guidance on options to be taken for 51 the future safety of foods. The concept is still evolv- 52 Appropriate Level of Protection (ALOP) is a quan- ing and no deﬁnition has yet been agreed upon. The 53S tiﬁcation of the disease burden within a country SCVPH (2003 III) quotes Codex document CX/FH 54N linked to the implementation of food safety (2001) as follows: “The maximum frequency and/or 40454 26 273-288 r2.qxd 7/20/07 3:52 PM Page 279

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concentration of a microbiological hazard in a food From its origin, HACCP is an instrument for 1 at the time of consumption that provides the appro- aspects of human health concern, having been devel- 2 priate level of health protection (ALOP).” oped for safeguarding the manufacturing of space 3 food. Using the term hazard (and HACCP), this 4 approach is limited to human safety (NACMCF 5 FOOD SAFETY CONCEPTS IN PRACTICE 1998). 6 After a period of end product control, today the From the legal point of view, control measures, 7 whole sequence of the technical procedure gets based on the principles of HACCP, must be imple- 8 under control, using hygienic elements (e.g., GHP) mented in the food chain, with the exception of pri- 9 or recording methods (e.g., observation sheets). mary production (Reg. 852/2004, Art. 5 and Annex 1) 10 Such elements are needed for the construction of (see also Figure 26.3). 11 more sophisticated practical solutions, such as 12 HACCP. In that sense, Good Hygiene Practice (GHP 13 in primary as well as secondary production) or Good CRITERIA—REGULATION (EC) 14 Management Practice (GMP) are prerequisites for 2073/2005 15 the final HACCP performance. 16 The SCVPH (1999 I) stated that the use of GHP The implementation of limits into food legislation 17 and other elements and the implementation of Haz- has been frequently discussed between the member 18 ard Analysis Critical Control Point (HACCP) sys- states of the EEC or later on of the EU. Already years 19 tems ensure that undesirable microorganisms are ago, in the case of special foods being estimated as 20 eliminated or minimized to an extent that they can- important, legal quality parameters and microbio- 21 not cause harm to human health. logical limits were laid down. Generally, in cases of 22 HACCP means knowledge of a line and regular legal background, the estimation of results of an 23 checking and recording its control. The concept analysis is easily done. 24 looks forward and determines Critical Control As an example, with Decision 2001/471/EC, the 25 Points in that line and, as far as possible, their elimi- examination of carcasses (not for poultry) was based 26 nation (Figure 26.3). If the critical point cannot be on legal parameters; however, with limited legal con- 27 deleted, at least it must be kept under technical consequences (improvement of hygiene in an abattoir 28 trol by regular checking and recording. after checking the procedure and appropriate mea- 29 HACCP systems need also to be rapid. So, fast and sures to be taken). 30 reliable results are requisites. In most cases, instru- Moreover, in several other legislative documents we 31 ments based on physical factors such as time, temper- find already microbiological criteria, which have been 32 ature, volume (e.g., water consumption), weight, or recently repealed with the Directive 2004/ 41/EC. 33 also magnetism (metal detectors) or pH would be Most of such criteria had a national template. As 34 used. In general, results of direct measurements can an example, from the German Society of Hygiene 35 be data from the technical procedure; observations and Microbiology, criteria expressed as guidelines 36 provided by human beings such as macroscopic and as limits have been published, but not on a legal 37 parameters during meat inspection; other processing base (N.N. 2006). These criteria serve as guidance 38 records (observation sheets); or results based on (m) values or warning limits (M). For finally fer- 39 microbiological tests such as recording, cleaning, mented meat products (fully fermented and shelf- 40 and disinfection efficacy in the premises. stable), the values read as shown in Table 26.4). 41 42 Table 26.4. Guidance (m) values or warning limits (M) for finally fermented meat products. 43 Product m M 44 45 Enterobacteriaceae For spreading log 2/g log 3/g 46 For slicing (firm) log 3/g log 4/g 47 48 Coagulase-pos.staphylococci log 3/g log 4/g 49 E. coli log 1/g log 2/g 50 Salmonella n.d. in 25 g 51 L. monocytogenes log 2/g 52 S53 n.d.: not detectable. N54 40454 26 273-288 r2.qxd 7/20/07 3:52 PM Page 280

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1 Since the days of the Fresh Meat Directive In the end, scientific information from these opinions 2 64/433/EEC from the 1960s, community food legisla- served as a template for Regulation (EC) 2073/2005, 3 tion has proceeded very far. Having been confronted which has been in force in all member states since 4 with the BSE disaster in the EU, and triggered also by January 1, 2006. With this Regulation, food safety 5 the recognition of a need for a basic readjustment of and process hygiene criteria were introduced into EU 6 legislation philosophy, EU hygiene legislation experi- food business, simultaneously repealing Decision 7 enced a new arrangement. Parallel to, and after the 93/51/EEC. Moreover, Decision 2001/471/EC as 8 implementation of the European Food Safety Admin- amended had also been taken out of force. In particu- 9 istration (EFSA), now located in Parma, Italy, the lar, the regulation contains (see Annexes 2 and 3): 10 Regulations of the Hygiene Package went into force. • Food safety criteria and process hygiene criteria 11 Among them, Regulation (EC) 2073/2005 is based on • The commodity of concern 12 several opinions in particular from the Scientific • The parameter for each commodity to be tested for 13 Committee on Foods (SCF) and the Scientific Com- • The number to be sampled 14 mittee on Veterinary Measures Related to Public • The limit for c (accepted number of n exceeding 15 Health (SCVPH), later also from the newly founded the limit m) 16 scientific panel on biological hazards (BIOHAZ). • Limits for m and M 17 Opinions are scientific-based statements answering to • Amount of sample material to be taken and to be 18 questions from the commission (Terms of Reference). given into analysis 19 Here, opinions were produced on: • Sampling days shall be changed each week to 20 ensure coverage of each working day 21 • The setting of criteria as such (result: Criteria 22 should be relevant and effective in relation to From its basic philosophy, the Regulation states the 23 consumer health protection): SCVPH (1999I) following: 24 • Listeria monocytogenes in foodstuffs (result: Foods should not contain microorganisms/ 25 Concentration in foodstuffs should be Ͻ100 toxins/metabolites in quantities that present an 26 cfu/g): SCVPH (1999 II) unacceptable risk for human health. 27 • Vibrio vulnificus and Vibrio parahaemolyticus With this, the idea of zero tolerance was aban- 28 in seafood (result: Codes of practice should be doned. On the contrary, here the idea of ALOP and 29 implemented): SCVPH (2001) of FSO is taken over as a schedule for practice, 30 • Norwalklike viruses (result: No use of fecal which has already been mentioned in the opinion of 31 bacteria as indicators, but E. coli should be used the SCVPH on Salmonella. 32 in case of application of bacteriological 33 indicators): SCVPH (2002) Food business operators have an obligation to 34 • Gelatin specifications (result: In terms of withdraw unsafe food from the market. 35 consumer health, the Salmonella criterium would Here, the idea of responsibility, of transparency, 36 be sufficient): SCF (2002) and of the obligation for action from the basic 37 • Verotoxigenic E. coli in foodstuffs (result: Hygiene Regulation (Reg. (EC) 178/2002) has been 38 Guidelines to reduce fecal contamination along taken over. 39 the food chain should be established for special The producer of the food decides on the commod- 40 food category lines): SCVPH (2003 I) ity (ready to be consumed or with a need for 41 • Staphylococcus enterotoxins in milk and cheeses cooking or other procedure in order to provide its 42 (result: The amount of staphylococcal safety and compliance with the said criteria, 43 enterotoxins should be laid down in particular which should be labelled). 44 specific milk products): SCVPH (2003 II) Here, the responsibility of the food producer is 45 • Salmonella in foodstuffs (result: Criteria for stated again, but also the freedom to decide upon the 46 Salmonella should be set only if they are practical product category. However, the operator remains 47 and able to protect consumers; otherwise, responsible, in particular, in case of an accident. 48 guidelines should be established): SCVPH 49 (2003 III) Sampling plans, analytic methods should be com- 50 • Salmonella and Enterobacter sakazakii in infant parable for the comparability of results. However, 51 formula (result: E. sakazakii seems to be more methods other than reference methods should be 52 difficult to control than Salmonella; the family allowed. 53S of Enterobacteriaceae should be used as an Here, the choice of the method has been left to the 54N indicator): SP BIOHAZ (2004) analyst. However, analysts should be able to make 40454 26 273-288 r2.qxd 7/20/07 3:52 PM Page 281

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their applied method transparent and they should be environment of a holding or a factory with a given 1 able to make it comparable with the reference machinery and processing line. It should also be 2 method. considered whether the sampling should be done 3 prior to or after packaging the original bulk. 4 For a certain transitorial time, a derogation 5 should be granted. In such a case, the product Number of samples to be taken in relation to the lot 6 should be legal only on the domestic market. for sampling: 7 With this, a transitorial time for the adoption was In larger bulks, the level of aggregation of bacteria 8 granted out of practical reasons; however, with the is of importance for the probability of recovery, and 9 product to be marketed only on the limited domestic the sensitivity of a result depends (beside others) on 10 market, a fast implementation of the content of this the amount of samples taken. The more samples 11 regulation should be expected. examined, the higher the probability of finding, e.g., 12 Salmonella. If the prevalence of an environmental 13 Finally, studies may be performed on compliance pathogen such as Salmonella in food commodities is with the criteria during the shelf life of a product. 14 regarded as low, the probability of detecting the 15 Here, and with other comments also, the content is pathogen might also be low, resulting in a perhaps open for continuous further development, which 16 false feeling of security when the samples are to be 17 should enable the commission to keep in contact taken. with the users of such a regulation, to improve it and 18 However, for purposes of international trade, the 19 to find other solutions in case of impracticability. It sampling plan has to be standardized, and two- or should be kept in mind that in the EU, a historically 20 three-class plans are widely in use. The following are 21 grown and different picture of nutritional records their advantages: and traditions exists. 22 • Small and fixed amounts of samples (e.g., n ϭ 5) 23 • One or two limits for the specific parameter to be 24 GENERATING examined (e.g., m ϭ lg 3; M ϭ lg 5) 25 26 MICROBIOLOGICAL DATA • Fixing the number of samples allowed to exceed the limit of m (e.g., c ϭ 2). 27 IN PRACTICE 28 Two-class plans are performed as presence-absence 29 Basically, criteria must not be understood exclu- tests. sively in the sense of the microbiological discipline. 30 There exist also technical (e.g., temperature or time) The amount of materials to be sampled: 31 or chemical criteria (e.g., residue limits or lower or Especially in large bulks such as powders, the 32 upper limits of other—spoilage—substances). In amount of material for the actual analysis must be 33 any case, if using criteria, they should be clearly considered. Here, different directives have requested 34 elaborated and located at the appropriate place in the different sampling size. Simultaneously, the fre- 35 line. More decisions should be made: quency of sampling is a point of concern. In the Reg- 36 ulation (EC) 2073/2005, different frequencies have 37 • The character of the criterion been established for specific parameters. 38 • The stage of setting the criterion 39 • The laboratory aspects (prelaboratory, 40 IN-LABORATORY in-laboratory, postlaboratory) 41 Qualitative (yes/no) approach versus quantitative Several aspects influence the outcome of a test. 42 testing: Much work has already been done on comparison of 43 Testing for Salmonella is hampered by the most methods (in-laboratory), however, in particular pre- 44 frequently used yes/no procedure. In these cases, analytic decisions have a huge influence on the prac- 45 only the percentage of infected animals or contami- tical use of an analytic result. 46 nated samples can be recovered. Quantitative data 47 are lacking as background information for the 48 severeness of Salmonella as a prevalence in process- 49 PRELABORATORY ing lines. Using a qualitative approach, pooling of 50 Stage of processing, respectively, points of sampling: samples is another option. Regulation (EC) 2073/ 51 Samples might be taken from very different places 2005 refers to this technique with fishery products 52 and stages: within/during the food chain or produc- (food safety criteria) and with poultry carcasses S53 tion line, at its end (end-product-control), or in the (process hygiene criteria), too. N54 40454 26 273-288 r2.qxd 7/20/07 3:52 PM Page 282

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1 The microbiological nature of a criterion as such: plans, the number of c determines the number of 2 The use of a criterion depends on the quality of batches to fall within the unwanted range. So, crite- 3 information that it is intended to obtain. So, in ria can be set as a quantitative limit or as a yes/no 4 the case of Salmonella, the particular serotype result. This question remains to be solved with 5 and phagetype should be determined. Moreover, respect to each food commodity. For food categories 6 molecular-biological criteria are appropriate in order referred to in this Regulation, this has been done. 7 to find out sources of a contamination and the epi- It is also of importance to know which outcome is 8 demiological transfer pathways. expected from this testing. The SCVPH (1999 I) as a 9 scientific body of the EU has offered as characteriza- 10 Methods and internal laboratory quality assurance: tion: “A microbiological criterion for foodstuffs 11 Methods should follow particular criteria, which defines the acceptability of a process, product or food 12 reflect the “truth” of a result, i.e., specificity, accu- lot based on the absence or presence, or number of 13 racy, precision, lower limit of detection, or sensitiv- microorganisms and/or quantity of their toxins/ 14 ity. Practicability and applicability also may be of metabolites, per unit of mass, volume or area.” 15 interest (see also Figure 26.4): As food safety items, the purpose of microbiologi- 16 • Accuracy (N.N. 1982): Ability of a test to give a cal criteria is to protect the health of the consumer by 17 true measure of the item being tested. providing safe, sound, and wholesome products, and 18 • Repeatability (Precision) (N.N. 1982): Ability of to meet the requirements of fair practices in trade. 19 a test to give consistent results in repeated tests. The mere existence of criteria does not protect 20 • Sensitivity (N.N. 1982): Ability of a test to give consumer health per se. So, such criteria should be 21 a positive result when an animal is diseased established and applied only where there is a need 22 (or a sample is positive). It is measured as the and where they can be shown to be effective, practi- 23 proportion of samples (animals) that gives a cal and realistic (SCVPH 1999 I). 24 positive test result. Too-low sensitivity may lead 25 to false negative results. The Character of Microbiological Criteria 26 • Specificity (N.N. 1982): Grade of exclusiveness of 27 a reaction. The ability of a test to give a negative Microbiological criteria currently used can be under- 28 result when the sample is negative. Lack of stood differently. Basically, we separate the terms of 29 specificity may lead to false positive results. standard, guideline, and specification. 30 Use in the sense of standard: 31 Methods applicable uniformly to various groups of The term microbiological standard has a legal 32 foods should be given preference over methods that meaning; it is based on legislation and a failure to 33 apply only to individual commodities. Methods to be comply with the limits, which results in actions such 34 used for legal purposes require validated and/or ISO as rejection, withdrawal, or other. 35 or EN methods. National reference laboratories have Standards should be used only if the aim can be 36 been established for cases of specific problems. achieved. Otherwise, the tool becomes weak and 37 counterproductive. 38 POSTLABORATORY 39 Use in the sense of guideline: Interpretation of the Test Results 40 Microbiological guidelines are used to assess 41 For the assessment of a result, limits necessarily whether GMP has been followed in a food processing 42 should be set. As can be demonstrated with the class line. They may represent limits, which are possible 43 44 45 Type of Mistake Accidental Systematic Bad 46 Mistake Mistake Mistake 47 48 No Result 49 50 51 Precision Optimal Poor Good - 52 Accuracy Optimal Good Poor - 53S 54N Figure 26.4. Precision and accuracy (Henniger 1980). 40454 26 273-288 r2.qxd 7/20/07 3:52 PM Page 283

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from the technological capacity, and they should not of certain products of animal origin intended for 1 be exceeded. human consumption and amending Council Direc- 2 In this sense, a test result reflects the appropriate- tives 89/662/EEC and 92/118/EEC and Council 3 ness of a procedure within a given line. Data beyond Decision 95/408/EC. Off. J. EC L195/12 of 2.6.2004. 4 the limit indicate possible failures, a need for cor- Directive 71/118/EEC of 15 February 1971. Off. J. EC 5 recting actions to be taken in technology in order to L055/23 of 8.3.1971. 6 improve the hygiene at that specific point of the line. Directive 91/497/EEC of 29 July 1991 Amending and 7 Such criteria are intended to guide the manufacturer consolidating Directive 64/433/EEC. Off. J. EC 8 and help ensure good hygiene practice. L268/69 of 24.9.1991. 9 In such a case, only the collecting of data is Directive 92/117/EEC of 17 December 1992. Off. J. EC 10 mandatory: For statistical purposes or in order to L62/38 of 15.3.1993. 11 obtain more information about suspect circum- J Dufrenne, W Ritmeester, E Delfgou-Van Asch, F Van 12 stances and environments—at present without mar- Leusden, R de Jorge. 2001. Quantification of the con- 13 ket or any other legal consequences—data are to be tamination of chicken and chicken products in The 14 gathered (in the sense of a continuous sampling in a Netherlands with Salmonella and Campylobacter. 15 given lot) on a mandatory basis in order to get the J Food Prot 64:538–541. 16 requested information. European Commission. 1998. Microbiological criteria: 17 For poultry farming, for example, it would make Collation of scientific and methodological informa- 18 sense to identify holdings with a high prevalence of tion with a view to the assessment of microbiological 19 Salmonella in order to focus on such holdings for risk for certain foodstuffs. Food—Science and tech- 20 reduction measures. Such a system has been shown niques; Directorate—Gen. Industry, Brussels, EUR 21 to be useful over the years in pig production. This 17638 EN, 107 pp. 22 way the situation might improve stepwise, using Sal- R Fries. 1988. Bakteriologische Proze␤kontrolle in der 23 monella testing before setting a criterion in the sense Geflügelfleischgewinnung.Habilitationsschrift, 24 of a standard. Tierärztliche Hochschule Hannover. 25 ———. 2002. Reducing Salmonella transfer during 26 Use in the sense of specifications: industrial poultry meat production. World’s Poult Sci 27 Such criteria are used only for contract purposes J 58:527–540. 28 between producer and customer. Such particular ———. 2005. Spoilage Microorganisms in the Course 29 specifications are not binding for other contractors. of Poultry Processing. Feedinfo News Scientific 30 Reviews. October 2005. Available from URL. 31 REFERENCES http://www.feedinfo.com. 32 G Henniger. 1980. Enzymatische Analyse: Empfind- 33 DJR Cherney. 2004. Western Coordinating Committee— lichkeit, Richtigkeit, Präzision, Fehlerquellen. Vor- 34 204 Goals and why they are important to the future of trag GDCh—Kursus: “Enzymatische Analyse” am 35 animal production systems. Poult Sci 83:307–309. 16.9.1980 in München. 36 Decision 2001/471/EC of 8 June 2001 laying down AM Lammerding. 1997. An overview of microbial food 37 rules for the regular checks on the general hygiene safety risk assessment. J Food Prot 60:1420–1425. 38 carried out by the operators in establishments accord- DAA Mossel. 1979. The microbial associations of 39 ing to Directive 64/433/EEC on health conditions for foods of animal origin. Archiv für Lebensmittelhy- 40 the production and marketing of fresh meat and giene 30:82–83. 41 Directive 71/118/EEC on health problems affecting N.N. 1982. Livestock Disease Surveys: A Field Manual 42 the production and placing on the market of fresh for Veterinarians. Australian Bureau of Animal 43 poultry meat. Off. J. EC L165/48 of 21.6.2001. Health, 35 pp. 44 Decision 2004/379/EC of 26 April 2004 amending Deci- ———. 2002. Deutsches Lebensmittelbuch—Leitsätze 45 sion 2001/471/EC . Off. J. EC L144/1 of 30.4.2004. 2002, Verkehrsbezeichnung, Qualität und Zusam- 46 Decision 93/51/EEC of 15 December 1992 on the mensetzung. Bundesanzeiger Verlagsges. mbH, 47 microbiological criteria applicable to the production Köln, 398 S. 48 of cooked crustaceans and molluscan shellfish. Off. ———. 2006. Veröffentlichte mikrobiologische Richt 49 J. EC, L13/11 of 21.1.1993. und Warnwerte zur Beurteilung von Lebensmitteln 50 Directive 2004/41/EC of the European Parliament and (Stand Juni 2005).(http: www.lm-mibi.uni-bonn.de/ 51 of the Council of 21 April 2004 repealing certain DGHM.html). 52 Directives concerning food hygiene and health con- National Advisory Committee on Microbiological Cri- S53 ditions for the production and placing on the market teria for Foods (NACMCF). 1998. Hazard Analysis N54 40454 26 273-288 r2.qxd 7/20/07 3:52 PM Page 284

284 Product Categories: General Considerations

1 and Critical Control Point Principles and Application Scientific Committee on Veterinary Measures relating to 2 Guidelines. J Food Prot 61:762–775. Public Health (SCVPH). 1999 II. Opinion of the 3 J Petersen. 1997. Trends und Entwicklungen in der Zucht SCVPH on Listeria monocytogenes (adopted 4 und haltung von Geflügel und Kaninchen. Vorträge 23.9.1999). http://europa.eu.int/comm/food/fs/sc/ 5 der 49. Hochschultagung der Landwirtschaftlichen scv/out25_en.pdf. 6 Fakultät der Universitüt Bonn vom 18.2.1997 in Bonn, Scientific Committee on Veterinary Measures relating 7 pp 165–75. Landwirtschaftsverlag Münster-Hiltrup. to Public Health (SCVPH). 2001. Opinion of the 8 Regulation (EC) No 2073/2005 of 15 November 2005 SCVPH on Vibrio vulnificus and Vibrio para- 9 on Microbiological Criteria for Foodstuffs. Off. J. EU haemolyticus (in raw and undercooked seafood) 10 L338/1 of 22.12.2005. (adopted 19–20 September 2001). http://europa. 11 Regulation (EC) No 854/2004 of the European Parlia- eu.int/comm/food/fs/sc/scv/out45_en.pdf. 12 ment and of the Council of 29 April 2004. Off. J. EC Scientific Committee on Veterinary Measures relating 13 L226/83 of 25.6.2004. to Public Health (SCVPH). 2002. Opinion of the 14 Regulation (EC) No. 178/2002 of the European Parlia- SCVPH on Norwalk-like Viruses (adopted 30–31 15 ment and the Council of 28 January 2002. Off. J. EC January 2002). http://europa.eu.int/comm/food/fs/sc/ 16 L31/1 of 1.2.2002. scv/out49_en.pdf. 17 Regulation (EC) No. 2160/2003 of the European Par- Scientific Committee on Veterinary Measures relating 18 liament and the Council of 17 November 2003. Off. to Public Health (SCVPH). 2003 I. Opinion of the 19 J. EC L325/1 of 12.12.2003. SCVPH on Verotoxogenic E. coli (VTEC) in food- 20 Regulation (EC) No. 852/2004 of the European Parlia- stuffs (adopted 21–22 January 2003). http:// 21 ment and the Council of 29 April 2004. Off. J. EC europa.eu.int/comm/food/fs/sc/scv/out58_en.pdf. 22 L226/3 of 25.6.2004. Scientific Committee on Veterinary Measures relating 23 Regulation (EEC) No 1538/91 of 5 June 1991 introduc- to Public Health (SCVPH). 2003 II. Opinion of the 24 ing detailed rules for implementing Regulation SCVPH on Staphylococcal enterotoxins in milk 25 (EEC) No 1906/90 on certain marketing standards products, particularly cheeses (adopted 26–27 March 26 for poultry. Off. J. EC L143/11 of 7.6.1991. 2003). http://europa.eu.int/comm/food/fs/sc/scv/ 27 Scientific Committee on Foods (SCF). 2002. Opinion out61_en.pdf. 28 of the SCF on specifications for gelatine in terms of Scientific Committee on Veterinary Measures relating 29 consumer health. Adopted 27.2.2002. http://europa. to Public Health (SCVPH). 2003 III. Opinion of the 30 eu.int/comm/food/fc/sc/scf/out122_en.pdf. SCVPH on Salmonellae in Foodstuffs (adopted on 31 Scientific Committee on Veterinary Measures relating 14–15 April 2003). http://europa.eu.int/comm/food/ 32 to Public Health (SCVPH). 1999 I. Opinion of the fs/sc/scv/onl66_en.pdf. 33 SCVPH on “The evaluation of microbiological crite- Scientific Panel on Biological Hazards. 2004. Opinion 34 ria for food products of animal origin for human con- of the SPBIOHAZ on a request from the commission 35 sumption.” (http://europa.eu.int/comm/food/fs/sc/scv/ related to the microbiological risks in infant formu- 36 outcome_en. html# opinions). lae and follow-on formulae. EFSA J 113:1–35. 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53S 54N 40454 26 273-288 r2.qxd 7/20/07 3:52 PM Page 285

ANNEXES 1 2 3 Annex 1: Microbiological Criteria for Foodstuffs in the Reg. (EC) 2073/2005 4 5 Food Safety Criteria 6 26 food categories 7 6 parameters (microorganisms/toxins/metabolites) 8 Salmonella 9 Listeria monocytogenes 10 Staphylococcal enterotoxins 11 Enterobacter sakazakii 12 E. coli 13 Histamines 14 Different limits 15 Different sample numbers 16 Different stages of sampling 17 Different interpretation support 18 Process Hygiene Criteria 19 Meat and meat products 20 Milk and dairy products 21 Egg products 22 Fishery products 23 Vegetables, fruits, products thereof 24 Rules for sampling 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 S54 N55 285 40454 26 273-288 r2.qxd 7/20/07 3:52 PM Page 286

1 2 3 4 5 6 7 market during their market shelf-life during their market shelf-life 8 control of the food who business operator, has produced it 9

10 ) Products placed on the ) Products placed on the 6 6 11 12 13 ) applies 14 3 15 16 17 18 19 20 ) reference Analytical the criterion Stage where 2 ) EN/ISO 11290-2 ( ) left the immediate 5 21 7 22 23 24 25 Absence in 25 g 26 27 ) Limits ( 28 1 29 5 0 1.1.2006 From 5 EN/ISO 6579 0 Absence in Products placed on the EN/ISO 6579 Products placed on the 5 0 100 cfu/g EN/ISO 11290-2 ( 5 0 100 cfu/g ( 5 0 1.1.2006 From EN/ISO 6579 Products placed on the 30 Sampling- plan ( 31 32 33 34 35 36 37 38

39 Microorganisms/their Salmonella Salmonella Listeria monocytogenes Listeria monocytogenes Salmonella 40 41 42 43

44 I of Reg.Annex 2 (Chapter 1 of Annex (EC) 2073/2005): safety criteria Food 45 , other 46 , other than 47 48 49 50 51 support of the growth support of the growth preparations made from poultry meat intended to be eaten cooked Absence in 10 g 1.1.2010 From Absence in 25 g during their market shelf-life meat intended to be eaten cooked Absence in 10 g 1.1.2010 From during their market shelf-life L. monocytogenes than those intended for infants and for L.monocytogenes those intended for infants and for special medical purposes 5 0 Absence in 25 g ( EN/ISO 1129-0-1 Before the food has special medical purposes 52 products excluding eaten raw, the manufacturing where process or the composition of the product will eliminate the salmonella risk 25 g during shelf-life market 53S categoryFood metabolites toxins, n c m M method ( 54N 1.8. Meat products intended to be 1.5. Minced meat and 1.3. to Ready-to-eat foods unable 1.2. to Ready-to-eat foods able 1.9. Meat products made from poultry

286 40454 26 273-288 r2.qxd 7/20/07 3:52 PM Page 287

1 2 3 4 5 6 7 8 9

is not able to demonstrate, is not able 10 tee that the limit of 100 cfu/g the effectiveness of the HACCP of the HACCP the effectiveness lf-life: 11 duct will not exceed the limit 100 duct will not exceed

fe. 12 13 14 15 16 17 18 19 20 21 22 23

before the food has left immediate control of producing 24 25 26 27 28 0.94, products with a shelf-life of less than 5 days are automatically considered are automatically 0.94, products with a shelf-life of less than 5 days 29 Յ L. monocytogenes

w 30 31 32 33 5.0 and a 34 Յ 35 36 number of sample units giving values over m or between m and M. m or between over values number of sample units giving 37 ϭ 38 39 40 41 42 0.92, products with pH

Յ 43

w 44

M. 45

ϭ 46 4.4 or a 47 Յ in ready-to-eat foods able to supportof in ready-to-eat foods able the growth in other ready-to-eat foods: 48 49

in different food categories: in different 50 51

number of units comprising the sample; c 52

ϭ S53 n points 1.1–1.24 m For The most recent edition of the standard shall be used. that the pro to demonstrate, the satisfaction This criterion applies if the manufacturerof competent authority, is able dishes of 90 mm diameter. dish of 140 mm diameter or on three Petri 1 ml of inoculum is plated on a Petri he when left the immediate control of producing food business operator, have This criterion applies to products before they Products with pH to belong to this category. Other categories of products can also belong to this category, subject to scientific justification. of products can also belong to this category, Other categories to belong this category. or good hygiene practice of the process. or good hygiene L. monocytogenes • observed indicate the absence of bacterium, if all the values satisfactory, • of the sample units. if the presence of bacterium is detected in any unsatisfactory, L. monocytogenes • observed are < the limit, if all the values satisfactory, • are > the limit. of the values if any unsatisfactory, Salmonella • observed indicate the absence of bacterium. if all the values Satisfactorily, • of the sample units. if the presence of bacterium is detected in any Unsatisfactorily, 1 2 3 5 enough to guaran fix intermediate limits during the process that should be low The operator may cfu/g throughout the shelf-life. at the end of shelf-life. is not exceeded 6 7 the limit of 100 cfu/g throughout shelf-li that the product will not exceed to the satisfaction of the competent authority, 8 of the test results Interpretation refer to each sample unit tested. The limits given can be used also for demonstrating they quality of the batch tested. However, The test results demonstrate the microbiological the limit of 100 cfu/g throughout she to demonstrate that the product will not exceed he is not able business operator when N54

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288 Annex 3 (Chapter 2 of Annex I of Reg. (EC) 2073/2005): Process hygiene criteria 2.1. Meat and products thereof

Sampling- Analytical Stage where Action in case of plan (1) Limits (2) reference the criterion unsatisfactory Food category Microorganisms n c m M method (3) applies results 2.1.5. Poultry carcases of broilers Salmonella 50 (5)7 (6) Absence EN/ISO 6579 carcases after Improvements in and turkeys in 25 g of chilling slaughter a pooled hygiene and sample review of of neck process skin controls, origin of animals and biosecurity measures in the farm of origin 1n ϭ number of units comprising the sample; c ϭ number of sample units giving values between m and M. 2For points 2.1.3–2.1.5 m ϭ M. 3The most recent edition of the standard shall be used. 5The 50 samples are derived from 10 consecutive sampling sessions in accordance with the sampling rules and frequencies laid down in Regulation (EC) 2073/2005. 6The number of samples where the presence of salmonella is detected. The c value is subject to review in order to take into account the progress made in reducing the salmonella prevalence. Member States or regions having low salmonella prevalence may use lower c values even before the review.

Interpretation of the test results The limits given refer to each sample unit tested, excluding testing of carcases where the limits refer to pooled samples. The test results demonstrate the microbiological quality of the process tested. Salmonella in carcases: • satisfactory, if the presence of Salmonella is detected in a maximum of c/n samples, • unsatisfactory, if the presence of Salmonella is detected in more than c/n samples. After each sampling session, the results of the last ten sampling sessions are assessed in order to obtain the n number of samples. 40454 27 289-300 r1.qxd 7/20/07 3:54 PM Page 289

1 2 3 27 4 5 6 Packaging and Storage 7 8 Dong U. Ahn and Byungrok Min 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION pallet with stretched wrap. This chapter focuses on 22 the primary packaging. 23 The primary function of food packaging is to pre- Most fermented meat products consumed today 24 vent or retard the deterioration of nutritional, are processed sausage-type products. They undergo 25 organoleptic, and aesthetic quality in foods and to several key processing steps, such as fermentation, 26 provide the protection against environmental con- drying, smoking, and cooking after the meat batter is 27 tamination. Packaging of meat products is defined as stuffed into casings. At the end of the process, the 28 . . . the enclosure of products in a wrap, pouch, whole sausages with casing or sliced products are 29 bag, box, cup, tray, can, tube, bottle or other packaged for storage and commercial distribution. 30 container form to perform one or more of the The characteristics of final products and their distri- 31 following functions: 1) containment for han- bution conditions for retail are very important factors 32 dling, transportation and use; 2) preservation that should be considered for the choice of packag- 33 and protection of the contents for required shelf ing material and systems. This chapter describes the 34 and use life; 3) identification of contents, quan- function of packaging, packaging materials, and 35 tity, quality and manufacturer; 4) facilitate dis- packaging systems that can be applied to fermented 36 pensing and use [FSIS 2000]. meat products. 37 38 Packaging of meat products can be practically 39 classified into three levels: primary, secondary, and FUNCTIONS OF FOOD 40 tertiary level (Dawson 2001). The primary level is the PACKAGING 41 package that directly contacts with meat products, 42 and consumers usually purchase the products with The functions of food packaging can be categorized 43 the primary package at retail. Therefore, it should into four areas: containment, protection, conve- 44 provide most of the functions of food packaging, nience, and communication (Robertson 1993). 45 such as protection, information, etc. The most com- 46 mon packaging material for the primary packages of CONTAINMENT 47 meat products is plastic film. The secondary level of 48 packaging contains several single primary packages Containment is the basic function of packaging. The 49 and protects the primary packages from environmen- products must be contained in appropriate packages 50 tal hazards during distribution. The tertiary packages before they are transferred. The products should be 51 hold many secondary packages for loading, unload- well contained in order for the packages to achieve 52 ing, and shipping. The most common example is a their functions successfully. 53 S54 N55 289 40454 27 289-300 r1.qxd 7/20/07 3:54 PM Page 290

290 Product Categories: General Considerations

1 PROTECTION reusing by consumers but also improves the effi- 2 ciency in making secondary and tertiary packages Protection is considered as the major function of 3 for easy handling, shipping, and storing, which are food packaging. Moreover, the packaging is regarded 4 directly related to cost. as one of the major preservation methods of foods 5 and retards or prevents quality deterioration during 6 handling, transportation, and storage. Packages COMMUNICATION 7 should: 8 The package carries information that is required by 9 • Protect food products from the contamination of law, wants to be known by consumers, and is 10 microorganisms and vermin to ensure safety and claimed by the manufacturer. It provides an interface 11 prevent spoilage, which is one of the hottest for the communications between consumers and 12 issues in foods. manufacturers. The information that must be shown 13 • Minimize the transfer of heat and light energy, on the label of meat products is product name, 14 which can activate or accelerate chemical USDA inspection legend, net weight, handling state- 15 reactions such as lipid oxidation and discoloration ment, ingredient statement, nutrition facts, date 16 and microbiological process. (related to shelf life), name and address of manufac- 17 • Prevent gas transfer from outside to inside and turer, and safe handling instructions. In addition, 18 vice versa. Therefore, packages should serve as a packaging can serve as an excellent marketing tool 19 barrier against oxygen, water vapor, and volatile and is called the “silent salesman.” The purchasing 20 compounds. The permeability of packages to desire of consumers can be boosted by package 21 oxygen is very important because oxygen is the design, information provided, etc. For example, the 22 most important factor that causes oxidative use of transparent packaging enables consumers to 23 changes in meat products during storage. observe the whole product and improve their sense 24 Packaging as a barrier for water and volatiles of reliability about the product. Unique shape, col- 25 transfer prevents dehydration and weight loss, ors, and graphics of packaging help consumers spot 26 loss of good flavor from food products, and and identify the products at retail. In addition, pro- 27 contamination of volatile compounds that viding information such as instructions for use, 28 influence the flavor of food products negatively. nutrients content, ingredients list, and expiration 29 • Prevent the physical damage by external force date is critical for customers purchasing the product. 30 or pressure and the contamination of dust and 31 foreign elements. However, it should be reminded PACKAGING MATERIALS 32 that packaging cannot improve the original 33 quality but retard the rate of quality loss. The most important factors that should be consid- 34 ered for the selection of suitable packaging materials 35 are the identification of the most limiting factors for CONVENIENCE 36 the preservation of food quality and safety, such as 37 Packaging should be designed for the convenient use sensitivity to oxygen and water, shelf life, potential 38 of products not only from the consumer’s standpoint of microbial contamination, storage conditions, and 39 but also from the handler’s. One of the consumer’s development of requirements for the packaging 40 great needs in a modern lifestyle is to eat and handle materials on the basis of limiting factors. Other 41 foods conveniently. Two concepts, the apportion- packaging features are strength factors such as ten- 42 ment of products and the shape of the primary pack- sile, elongation, tear strength, and resistance to 43 aging in package design, have contributed to the puncture; permeability to gas, moisture, grease, and 44 improvement of the convenience in food packaging volatiles; machinability factors such as stiffness, 45 (Robertson 1993). The apportionment function of static accumulation, and slip; marketing factors such 46 food packaging allows us to apportion industrial- as transparency, gloss, and film color; anticlouding 47 sized products into serving-sized products, which characteristics; and cost (Kropf 2004). 48 can be consumed conveniently. The effective methods The packaging materials can be classified into 49 and package design for apportioning should be three categories on the basis of their properties: bar- 50 considered for individual food products because rier, strength, and sealing properties (Dawson 2001; 51 consumers’ needs and manufacturing conditions for Lundquist 1987). For example, aluminum foil is usu- 52 each food product vary. In addition, the proper shape ally used as a layer because of its barrier properties 53S of the primary package can improve not only for light and gases. Polyester (polyethylene tereph- 54N the convenience in opening, storing, reclosing, and thalate, PET) is added for strength, and polyethylene 40454 27 289-300 r1.qxd 7/20/07 3:54 PM Page 291

Packaging and Storage 291

as a sealing agent. The classification of major pack- discoloration in the presence of oxygen. In the 1 aging materials for each property is as follows: absence of oxygen or under anaerobic conditions, 2 nitric oxide separated from the heme group is not 3 • Barriers: aluminum foil, ethylene vinyl alcohol oxidized and can be reattached to the heme. Also, 4 (EVOH), polyvinyl chloride (PVC), polyvinyli- oxygen can accelerate lipid oxidation, which is 5 dene dichloride (PVDC, Saran®), and closely related to the flavor deterioration and discol- 6 acrilonitriles (Barex®) oration in meat. In addition, microorganisms are 7 • Strength properties: polyester (PET), nylon, and directly and indirectly involved in discoloration. 8 polypropylene (PP) Under aerobic conditions, some aerobes can produce 9 • Sealing properties: polyethylene (PE), ionomers hydrogen peroxide, which can oxidize heme pig- 10 (Surlyn®), and polystyrene (PS) ments. Also, because most of the fermented meat 11 No individual packaging material can satisfy all products undergo a drying process, rehydration by 12 desired requirements for packaging materials. Thus, residual moisture or inflow of outside water vapor 13 in most cases, a number of individual packaging can provide favorable environments for microbial 14 materials are combined and arranged as a layer to growth, resulting in microbial spoilage. Therefore, 15 produce a multilayer-structured material called a the presence of moisture, oxygen, and/or air is the 16 film that has the desired characteristics (Dawson primary quality limiting factor for the fermented 17 2000). The selection of specific individuals for a meat products. In addition, loss of moisture, loss of 18 multilayer film to use for the packaging depends on flavor compounds, and influx of undesirable 19 the specific requirements of package for a specific volatiles can be other quality limiting factors during 20 product. The thickness of a film commonly ranges storage. Therefore, low permeability to oxygen, 21 from 0.0254 mm (1 mil or 0.001 in) to 0.3054 mm water vapor, and other volatiles; strength and dura- 22 (12 mil or 0.012 in) (Kropf 2004). Generally, the bility throughout the distribution; machinability fac- 23 film consists of three or more layers. Each layer, tors such as flexibility, heat sealability, shrinkability, 24 arranged from outside to inside, contributes specific and printability; marketing factors; and cost are 25 properties to overall structure: the outside layer important in selecting packaging materials for fer- 26 should provide scuff/abrasion-resistance ability and mented meat products. Oxygen transmission rate 27 printability. The middle layer(s) generally has (have) (OTR) and water vapor permeability (WVP) are 28 barrier properties, especially to oxygen and water defined as “gas or vapor exchange rates, at a relative 29 vapor, and mechanical structure. The inner layer next humidity and temperature conditions stipulated for a 30 to the product (called the sealant layer) should have one mil thick (0.0254 mm or 0.001 in) film at one 31 heat sealability and compatibility with the product. atmospheric pressure” (Dawson 2000). 32 Another important consideration in the selection of Numerous packaging materials are available for 33 packaging materials, especially for the inner layer of fermented meat products: paper and coated-paper 34 a film, is the migration, which refers to the transfer products; cellulose products; cellophane; metals 35 of compounds from the packaging materials to food such as tin, aluminum, and stainless steel; ceramics; 36 products by leaching or diffusion, resulting in food glass; chemically treated rubber; and plastics. How- 37 safety issues and flavor deterioration. Slip and ever, multilayer films made of various plastics are 38 antifogging agents, heat-degradable products from the most common packaging materials for the fer- 39 film, and ink components are considered major mented meat products because of its versatile func- 40 causes for the migration problem in plastic package tions, cost, and convenience. 41 (Driscoll and Paterson 1999). Therefore, those The following section is a brief description of 42 should be chemically inert. individual packaging materials (Table 27.1) that can 43 Major quality deteriorations in fermented meat be used as a part of the multilayer film for the pack- 44 products are discoloration; development of lipid oxi- aging of fermented meats. 45 dation, which causes flavor deterioration; weight 46 loss; and microbial spoilage. The pigment com- BARRIER PROPERTIES 47 pounds that are responsible for the color of fer- 48 Aluminum Foil mented meat products are nitric oxide myoglobin for 49 uncooked and nitrosyl hemochromogen for cooked Aluminum foil provides an ultimate barrier for oxy- 50 meat (Aberle et al. 2001). The color stability in fer- gen, water vapor, and light, and is heat- and cold- 51 mented meat products is considerably influenced by resistant. In addition, it offers a great surface for the 52 light, oxygen, and microbial spoilage. Nitrosyl application of graphics on the package. However, S53 hemochromogen is very sensitive to light-induced aluminum foil should be used with plastic polymers N54 40454 27 289-300 r1.qxd 7/20/07 3:54 PM Page 292

1 2 3 4 5 Polyethylene; 6 ϭ References

7 (1995) Day (1995) Day 8 9 10 11 Polypropylene; PE Polypropylene; 3

12 ϭ 13

14 4 15 5 200 (1995) Day 16 200 17 24–120 18 19 Rate Transmission 20 21

22 terephthalate; PP Polyethylene

23 ϭ 24 2 25 5 4

26 4 27 28 2000–2500 7 (1995) Day 29

Oxygen Water Vapor Vapor 30 Oxygen Water 31

32 chloride; PET polyvinylidene

33 ϭ 34 35 36 37 38 39 40

41 chloride; PVDC polyvinyl thermoformability cold stability vapor permeability; high vapor good heat ﬂexibility; sealability thermoformability barrier heat resistance barrier; rigidity barrier 42 ϭ Oxygen barrier 0.2–1.6 Heat sealing agentHeat sealing agent; 2100–7100 2500–5000 7–24 170 (1995) Day (1995) Day 43 barrier; vapor Strength; water

44 <50, barrier; 50–200, semi-; 200–5000, medium-; >5000, high-. at 23°C, 0% RH unless noted. ORT: 1 45 Ϫ

46 atm • ) vapor Oxygen and water 15.5 77.5 Soroka (1999) 1 ®

47 WVTR: <10, barrier;at 38°C, 90% RH. 10–30, semi-; 30–100, medium; >100, high-. Ϫ 1 ) Excellent heat sealing agent 4650–6975 at 25°C 23–31 (2001) Dawson 48 Ϫ ® day ) vapor Oxygen and water 0.8–9.2 0.3–3.2 (1995) Day • ® 49 day 2 Properties of plastics for packaging of fermented meat products. of fermented Properties packaging of plastics for • Ϫ

50 2 m Ϫ ethylene vinyl alcohol; PVC vinyl ethylene •

51 m (Saran ϭ • 1 1 (unplasticized) (plasticized) vapor Oxygen and water and water High oxygen 120–160 2000–10000 22–35 (1995) Day mil (Polyester) Strength; good heat and 50–100 20–30 (1995) Day

52 Polystyrene. 1 1 • 1 (Oriented) mil 3 ϭ 1 1 1

53S • EVOH EVOH cm g Depending on moisture. of plasticity. Depending on moisture and level Barrier Properties EVOH PS Table 27.1. Table Plastics Primary Functions Rate Transmission 1 2 3 4 5 PS PP Nylon (nylon6) Strength, thermostability; 80 PVC PVDC Ionomer (Surlyn Acrilonitriles (Barex Sealing Properties PE 54N PVC Properties Strength PET

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with sealing properties, such as PE, because the foil it is printable and heat-resistant. PVDC has been 1 is not heat-sealable. Its common thickness in the commonly used as a layer of multilayer pouches, 2 multilayer film is 0.0003 to 0.0005 in. Although alu- bags, and thermoformed packages for various meat 3 minum foil has been primarily used in flexible retort products such as frankfurters and ham. Furthermore, 4 pouches, it is applicable for the packaging of fer- PVDC is preferably used for modified atmosphere 5 mented meat products because of its great barrier packaging (MAP). 6 properties for oxygen, water vapor, and light. 7 ® Acrilonitriles (Barex ) 8 Ethylene Vinyl Alcohol (EVOH) Acrilonitriles provides excellent barrier properties to 9 Ethylene vinyl alcohol is produced by the saponifi- water vapor, oxygen, and other gases; good clarity; 10 cation of ethylene, vinyl alcohol, and vinyl acetate good strength; and good chemical resistance to most 11 polymers. The superior properties of EVOH are its organic solvents. Acrilonitriles are commonly used 12 impermeability to oxygen and other gases such as for thermoformed plastic containers due to its rigidity. 13 CO2, N2, and other volatiles. However, EVOH is 14 hydrophilic and can absorb moisture due to the pres- 15 ence of hydroxy group in its backbone structure, and STRENGTH PROPERTIES 16 thus adversely affects oxygen barrier properties of Polyester (Polyethylene Terephthalate, PET) 17 EVOH under high humidity conditions. The gas bar- 18 rier properties of EVOH are determined by the ratio Polyester is produced by condensation of polyhydric 19 between vinyl alcohol, which has gas barrier proper- alcohol such as ethyleneglycol and polyfunctional 20 ties, and ethylene polymer, which has water-resisting acid such as dimethyl terephthalic acid. PET pro- 21 properties. EVOH is often used as a core layer in vides the excellent tensile strength, great stability 22 − multilayer film between layers of other polymers over a wide range of temperature ( 60 to 220°C), 23 such as PE, PP, and PET with high moisture barrier good chemical resistance, outstanding mechanical 24 properties. In addition, EVOH has high mechanical properties, good clarity, excellent printability, and 25 strength, elasticity and surface hardness, trans- moderate barrier properties to oxygen, water vapor, 26 parency, high gloss, good abrasion resistance, great and other gases for the food packaging film. PET 27 barrier to oil and organic solvents, and heat stability. is applicable to vacuum packaging, sterilizable 28 EVOH has been extensively used for the packages of pouches, and cook-in-bag applications for meat 29 various meat products, which need protection from products because of its strength and heat stability. 30 oxygen. Nylon 31 32 Polyvinyl Chloride (PVC) Nylon, polyamide, is produced by the condensation of 33 Polyvinyl chloride is produced by the polymeriza- diamines and diacids. Nylon provides excellent heat 34 tion of vinyl chloride monomers, and is simply resistance, thermal stability, thermoformability, and 35 called vinyl. Two major forms of PVC are used in low temperature flexibility; it is strong and abrasion-, 36 food packages: unplasticized and plasticized. The alkali-, acid-resistant, and offers good barrier proper- 37 unplasticized PVC is very clear and glossy, and it ties to oxygen and other gaseous phases. However, 38 has high tensile strength and rigidity, shows low nylon can absorb moisture and is well permeated by 39 water vapor and oxygen permeability, and is resist- water vapor, resulting in weakening tensile strength 40 ant to oil and grease as well as acid and alkali. The and lowering oxygen barrier properties. Therefore, it 41 plasticized PVC has excellent transparency and is often used as a layer of multiple layer film with 42 gloss, high flexibility, good heat sealability and high other layers such as PE and PP, which can provide 43 oxygen and water vapor permeability, and has been moisture barrier properties and heat sealability. It can 44 widely used for wrapping fresh red meat cuts at be used for vacuum packaging, pouches, cook-in-bag, 45 supermarkets in order to keep fresh meat color. and ovenproof trays for meat products because of its 46 strength, thermal resistance, and stability. 47 Polyvinylidene Dichloride (PVDC, Saran®) 48 Polypropylene (PP) Polyvinylidene dichloride is produced by the copoly- 49 merization of polyvinylidene and polyvinyl chloride. Polypropylene is a polymerized product from propy- 50 The characteristics of PVDC include excellent bar- lene monomers. Two types of PP depending on its 51 rier properties to water vapor, oxygen, and other manufacturing processes have been generally used 52 gases; good clarity; good resistance to grease; and as packaging materials: disoriented (or cast, CPP) S53 great abrasion-resistance. It can be heat-sealed, and and oriented (OPP). CPP is very weak, so that it is N54 40454 27 289-300 r1.qxd 7/20/07 3:54 PM Page 294

294 Product Categories: General Considerations

1 not recommended for the layer with strong proper- adhesion capability to other packaging materials 2 ties. CPP is primarily used as a sealing layer in retort including aluminum foil. In addition, ionomer has 3 pouches due to its relatively high temperature- good oil and grease resistance, good clarity, and 4 resistance. OPP has very good tensile strength, great resistance to stress cracking. It is commonly used as 5 water vapor barrier properties, resistance to high a sealing agent of vacuum packages for processed 6 temperature, clarity, and rigidity. For meat products, meat products, but is expensive. 7 OPP can be applied to cook-in-steam or boil processed Polystyrene (PS) 8 products and cook-in-bag products because of its 9 high heat resistance and water vapor impermeability Polystyrene (PS) is produced by the polymerization 10 in boiling or steam cooking. of styrene. PS is transparent, hard, fragile, and low- 11 strength material, which can be thermoformed into 12 disposable containers or clear trays. PS can be 13 SEALING PROPERTIES foamed to form expanded polystyrene (EPS) (Styro- 14 Polyethylene (PE) foam), which is thermoformed into trays for fresh 15 meats. Both PS and EPS have poor oxygen and water 16 Polyethylene is polymerized from polyethylene vapor barrier properties as well as good resistance to 17 monomers, and is generally divided into three major acids and alkalis. 18 types with different molecular structure, properties, and manufacturing processes: low density (LDPE), 19 PACKAGING SYSTEMS 20 linear low density (LLDPE), and high density PE 21 (HDPE). LDPE is a good sealing agent at relatively The major quality changes that limit the shelf life of 22 low temperature, which can be fused into itself in the fermented meat products include discoloration, lipid 23 opposite layer to make good liquid-tight seals. oxidation, rehydration, dehydration, and microbial 24 LDPE is slightly semitransparent; has good tensile, spoilage. In addition to selecting suitable packaging 25 impact, and tear strength. It also has excellent resis- materials, appropriate packaging systems should be 26 tance to acids, alkalis, and inorganic solvents, but is applied in order to retard or prevent those unfavor- 27 sensitive to oil, grease, and organic solvents. In addi- able quality changes in the products during storage 28 tion, LDPE provides good barrier properties to water and distribution. Because barrier properties to oxy- 29 vapor, but poor to oxygen and other gases, which gen, water vapor, and other gases are crucial for 30 makes it appropriate for packaging of fresh fruits packaging materials, the capability of removing or 31 and vegetables. LLDPE has greater chemical resis- minimizing headspace inside the packages or chang- 32 tance, higher strength, and higher resistance to stress ing headspace to low oxygen conditions is important 33 cracking than LDPE. HDPE shows greater heat for packaging systems. Among available packaging 34 resistance, tensile strength, rigidity, and chemical systems, vacuum packaging and modified atmo- 35 resistance than LDPE. In addition, HDPE provides sphere packaging (MAP) have been widely used 36 improved barrier properties to water vapor as well as for fermented meat products. In recent years, active 37 oxygen and other gases. However, HDPE has less packaging has been introduced and intensively 38 translucence, lower impact and tear strength, and developed in response to consumer and market 39 considerably worse sealability than LDPE. PE is a demands. Major packaging systems including vac- 40 relatively low-cost packaging material. uum packaging, modified atmosphere packaging 41 (MAP), active packaging, and aseptic packaging are Ionomer (Surlyn®) 42 described below. 43 Ionomer (Surlyn®) is produced by polymerizing eth- 44 ylene with a small amount of unsaturated carboxylic VACUUM PACKAGING 45 acid, such as methacrylic acid, followed by neutral- 46 ization of the polymers with derivatives of metals, Vacuum packaging is the primary packaging system 47 such as sodium or zinc, which results in the poly- used in fermented meat products to delay or prevent 48 mers with low levels of covalently attached sodium the quality deterioration by oxygen. Vacuum pack- 49 or metal ions that form ionic cross-links to provide aging is generally described as a packaging system 50 enhanced rigidity. Ionomer is an excellent sealing in which all air in a gas-impermeable container is 51 agent and is used over a wide range of conditions. substantially evacuated by vacuum pump before 52 Ionomer is primarily used for the meat-contact and sealing the container. Vacuum packaging provides 53S heat-sealing layer in multiple layer films because of oxygen-free or, strictly speaking, extremely low 54N its broad range of heat-sealing temperature and great levels of oxygen environment for the products to 40454 27 289-300 r1.qxd 7/20/07 3:54 PM Page 295

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prevent or delay quality deterioration such as discol- this is followed by vacuuming out. The open end of 1 oration, lipid oxydation ﬂavor deterioration, weight the bag is heat-sealed, and then the chamber is 2 loss, rehydration, and microbial spoilage in pack- exposed to atmosphere so that the package is col- 3 aged products. The extremely low level of oxygen as lapsed onto products by higher external pressure. 4

well as production of carbon dioxide (CO2) from the The package collapse may cause deformation if the 5 products in the container not only prevents the product does not have certain extent of resistance. 6 growth of aerobic microorganisms but also provides Therefore, vacuum packaging may not be appropri- 7 a favorable condition for facultative and/or anaero- ate for less-firm products. Use of a thermoformed 8 bic microorganisms, such as lactic acid bacteria, plastic tray as a container can be a good approach for 9 which can produce lactic acids (Kropf 2004). These soft products. PP, PS, nylon, or PET can be used to 10 conditions are also very helpful for protecting color provide structural properties in laminates for ther- 11 fading in cured meat products because oxygen plays moformed base trays, where those are combined 12 a critical role in the oxidation of heme pigments such with an EVOH as a gas barrier and PE as a heat 13 as nitrosyl hemochromogen, the major heme pig- sealing agent (e.g., PP/EVOH/PE, PS/EVOH/PE, 14 ments in cooked, cured meat products. nylon/EVOH/PE, or PET/EVOH/PE structures) 15 The most critical properties of packaging materi- (Toldrá et al. 2004). 16 als required for vacuum packaging are excellent bar- 17 rier properties to gases, especially oxygen and water 18 MODIFIED ATMOSPHERE PACKAGING vapor, in order to maintain vacuum condition during 19 storage and distribution. The flexibility of material Modified atmosphere packaging (MAP) is one of the 20 to be tightly attached to the entire surface of prod- most common preservative technologies wherein air 21 ucts and good puncture resistance are also important in the package is replaced by a single gas or a mix- 22 properties. Because no plastic material can meet all ture of inert gases, such as carbon dioxide and nitro- 23 requirements, a multilayer film of several plastics gen to prevent quality deterioration and microbial 24 with different properties has been usually used for spoilage of food products. The gas composition of 25 vacuum packaging. Among the materials, nylon and within-package atmosphere is not controlled after 26 PE have been commonly used for vacuum packaging incorporation of gas mixtures and sealing, but can be 27 (Toldrá et al. 2004). Nylon (polyamide) is an ideal changed by metabolism within the products itself 28 material for vacuum packaging because of its high and/or microorganisms present, diffusion of certain 29 tensile strength, good puncture and abrasive resis- gases into or out of the products, and transfer of 30 tance, and good barrier properties to oxygen and gases through the packaging film into or out of the 31 other gases. However, nylon is hydrophilic and has package (Kropf 2004). 32 poor heat sealability, and is generally laminated with In practice, the products are retained within a flex- 33 PE. In nylon/PE multilayer film, nylon serves as a ible pouch or bag, or on the thermoformed base tray, 34 barrier to oxygen and other gases and a mechanical and then a single gas or gas mixtures are flushed to 35 supporter, and PE is a barrier to water vapor and a modify the atmosphere, followed by sealing. There 36 heat sealing agent. The number of layers in a multi- are two methods to modify the atmosphere within 37 layer film is dependent upon designated shelf life the package: gas flushing and compensated vacuum 38 and storage and distribution conditions. For the gas flushing (Mullan and McDowell 2003). The gas 39 products that need long shelf life and are stored and flushing method is a simple method wherein air 40 distributed at room temperature, as in most of the within the package is removed by flushing with a 41 fermented meat products, plastic layers with more continuous gas stream before sealing. It is less effec- 42 powerful barrier properties to oxygen and other tive for removing air and consumes large amounts of 43 gases such as EVOH and PVDC should be added to flushing gas although the operation rate is very fast. 44 the film (Toldrá et al. 2004). However, products with Thus, the gas flushing method is not suitable for 45 short shelf life and stored at refrigerated temperature oxygen-sensitive products. On the other hand, the 46 can be packaged with a film with reduced barrier compensated vacuum gas flushing method consists 47 properties or even single-layer HDPE. of two steps: evacuation and gas flushing steps. The 48 Several vacuum systems have been developed on air within a package is evacuated to achieve a vac- 49 the basis of how to make air-deficient environment uum state, and, subsequently, the package is flushed 50 within the package: vacuum clip, snorkel, Pi-vac, with modified atmosphere. The consumption of 51 and vacuum chamber systems (Kropf 2004). In vac- flushing gas for this method is low, but the operation 52 uum chamber systems, a flexible pouch or bag con- rate is slow. This is a more efficient method in S53 taining product is placed in the vacuum chamber; decreasing residual air (especially oxygen) than the N54 40454 27 289-300 r1.qxd 7/20/07 3:54 PM Page 296

296 Product Categories: General Considerations

1 gas flushing method, and thus is a better method for a minimal level, resulting in prevention or delay of 2 oxygen-sensitive products such as fermented meat oxidative changes in products. Nitrogen does not 3 products. Zanardi et al. (2002) reported that MAP have antimicrobial effects, but provides an anaerobic 4 filled with 100% nitrogen is more efficient than vac- condition where the growth of aerobic microorgan- 5 uum packaging in preventing lipid oxidation and dis- isms responsible for microbial spoilage is inhibited. 6 coloration in Milano-type fermented sausage under Various types of containers (e.g., flexible pouches 7 commercial conditions because of lower residual or bags and thermoformed trays) can be used for 8 oxygen content in MAP. MAP. Use of flexible pouches is cost effective 9 Three major gases have been commonly used in because they can be applied for fast gas-flushing 10 MAP: oxygen, carbon dioxide, and nitrogen. Others, methods. However, without adequate information, 11 such as carbon monoxide, sulfur dioxide, nitrous consumers may consider the MAP flexible pouches 12 oxide, ozone, chlorine, and argon also have been as vacuum-failed, impaired products because of the 13 investigated for use in MAP (Davies 2003). Gas baggy appearance, resulting in rejection of the prod- 14 mixtures with various compositions can be used in ucts (Toldrá et al. 2004). Therefore, semirigid or 15 MAP depending on the features of products. As rigid thermoformed trays have been generally uti- 16 mentioned previously, it is essential to minimize the lized as the main container for MAP in fermented 17 amount of residual oxygen within a package atmo- meat products. The requirements of packaging mate- 18 sphere for fermented meat products. Therefore, rials for MAP are similar to those of vacuum pack- 19 modified atmosphere within a package is generally aging, and thus the same approaches can be used for 20 composed of carbon dioxide and nitrogen: com- the selection of packaging materials and design of 21 monly, 20–30% carbon dioxide and 70–80% nitro- multilayer films for MAP. 22 gen (Toldrá et al. 2004). Carbon dioxide is well 23 known to have inhibitory effects on the growth of ACTIVE PACKAGING 24 aerobic microorganisms, especially gram-negative 25 aerobes such as pseudomonads, resulting in delaying In recent decades, huge attention has been paid to 26 the initiation of microbial spoilage. It also has shown active packaging as a promising technology, capable 27 to have inhibitory effect on enzymatic activities. of satisfying increased consumer demand for safer,

28 Increasing the proportion of CO2 within the atmo- more convenient, and better quality foods as well as 29 sphere of a package up to about 25% improves the changes in business environments such as diversiﬁ- 30 antimicrobial effect (Kropf 2004). In addition, the cation of markets (globalization, internet-shopping,

31 antimicrobial effect of CO2 is significantly greater at etc.), increased complexity of logistics and distribu- 32 temperatures below 10°C than at 15°C or higher tion, and more tightened law. Active packaging is 33 (Mullan and McDowell 2003). On the other hand, defined as an innovative technology where certain 34 carbon dioxide promotes the growth of psychotropic additives are introduced into a packaging film or 35 lactic acid bacteria and increases the production of within a packaging container and provide desired 36 lactic acid (Kropf 2004). Carbon dioxide is soluble roles, by the interaction with headspace or foods, to 37 in water, lipids, and some other organic compounds. extend shelf life and maintain and/or improve nutri- 38 Carbon dioxide, therefore, can be dissolved in meat tional and organoleptic quality and microbial safety 39 products, and its solubility increases with decreasing of foods (Labuza and Breene 1989; Day 2003). 40 temperature. Due to its solubility in meat products Packaging may be termed active when it provides

41 and loss during storage, CO2 should be sufﬁciently desired roles for food preservation other than an 42 introduced into the package atmosphere to maintain inert barrier to external environments (Day 2003).

43 its antimicrobial effect. In addition, the loss of CO2 The prerequisites for the development and applica- 44 can cause reduction of headspace volume in the tion of active packaging for specific food products 45 package, which results in package collapse onto are to understand the mechanisms of quality deterio- 46 products. Another important gas consisting of modi- ration and identify the key factors limiting shelf life. 47 fied atmosphere for fermented meat products is The development and application of an active pack- 48 nitrogen. Nitrogen is relatively inert and has a low aging system is based on a fine match of packaging 49 solubility in water and other food components. properties to the requirements of packaging for spe- 50 Therefore, it is generally used as a filler gas (about cific food products (Rooney 1995). As shown in 51 70–80% for meat products) to avoid the possibility Table 27.2, various types of active packaging tech- 52 of package collapse when a high concentration of niques have been introduced and developed: oxygen 53S carbon dioxide is used. Nitrogen as a filler gas plays scavengers, carbon dioxide scavengers/emitters, 54N a significant role in the dilution of residual oxygen to moisture absorbers, ethanol emitters, antimicrobial 40454 27 289-300 r1.qxd 7/20/07 3:54 PM Page 297

Table 27.2. Various active packaging systems applicable for fermented meat products.1 1 2 Type of Active Packaging System Core Substances Applied and Mechanisms 3 4 Oxygen scavengers Chemical system 5 Oxidation of iron-based powder with catalysts 6 Sulﬁte salt/copper sulfate 7 Oxidation of ascorbic acid 8 Oxidation of photosensitive dyes/singlet oxygen acceptor 9 Oxidation of oxidizable polymers 10 Oxidation of unsaturated fatty acids 11 Consumption by immobilized yeast on solid materials 12 Conversion of oxygen to water vapor by platinum catalysts 13 Enzymatic system 14 Reaction of glucose oxidase and alcohol oxidase with substrates 15 Carbon dioxide Ferrous carbonate/metal halide 16 Scavengers/emitters Ascorbic acid/sodium bicarbonate 17 Iron powder-calcium hydroxide 18 Calcium oxide/hydrating agents (e.g., silica gel, activated charcoal) 19 Ethanol emitters Absorbed or encapsulated ethanol 20 Ethanol spraying 21 22 Antimicrobial releasers Organic acids (e.g., acetic acid, citric acid, benzoic acid, sorbic acid) 23 Salts and acid anhydrides of organic acids 24 Bacteriocins (e.g., nisin, pediosin, lacticin) 25 Enzymes (e.g., lysozyme, lactoperoxidase, chitinase) 26 Metals (e.g., silver zeolite, copper) 27 Antibiotics (e.g., natamycin) 28 Fungicides (e.g., sulfur dioxide, benomyl, imazail) 29 Metal chelators (e.g., EDTA, lactoferrin, conalbumin) 30 Natural phenols (e.g., catechin, hydroquinone) 31 Oligo and polysaccharides (e.g., chitosan and its oligomer) 32 Plant volatile compounds (e.g., allylisothiocyanate) 33 Antimicrobial peptides (e.g., attacin, cecropin, magainin) 34 Extracts (e.g., grapefruit seed extract) and essential oils 35 Amines and ammonium compounds 36 Antioxidant releasers BHA, BHT, TBHQ, tocopherol 37 Moisture absorbers Silica gel, natural clays (e.g., montmorilonite), calcium oxide, 38 molecular sieves 39 Superabsorbent polymers (e.g., polyacrylate salts, graft copolymers of 40 starch, propylene glycol) 41 Flavor/odor absorbers Baking soda, activated carbon/clays, active cellulose acetate, citric 42 acid, ferrous salt/organic acids 43 44 Flavor/odor releasers Desired food ﬂavors 45 Light absorbers/regulators UV blocking agents, hydroxybenzophenone 46 Time-temperature indicators Color change or development on packages 47 48 1 Sources: Vermeiren et al. (1999); Suppakul et al. (2003); Day (2003); Ozdemir and Floros (2004). 49 50 51 52 S53 N54

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1 and antioxidant releasers, ﬂavor/odor absorbers/ CO2 emitters on the basis of either ferrous carbonate 2 releasers, temperature control packaging, and selec- or a mixture of ascorbic acid and sodium bicarbon-

3 tive gas permeable films (Ozdemir and Floros 2004). ate can dually function as both CO2 emitter and pos- 4 The major factor for quality deterioration in fer- sible oxygen scavenger (Vermeiren et al. 1999). The 5 mented meat products is presence of oxygen, which use of this dual-functioned system in MAP with car- 6 facilitates oxidative rancidity, flavor deterioration, bon dioxide, which is soluble to water and lipid, is 7 discoloration, loss of nutrition, and growth of aerobic highly useful in extending shelf life. 8 bacteria, mold, and fungi. Therefore, oxygen scav- Other useful packaging for fermented meat prod- 9 engers are highly applicable to fermented meat ucts is antimicrobial and antioxidant active packag- 10 products, which are sensitive to oxygen. Various ing systems. Ethanol is widely recognized as a 11 techniques are currently available for oxygen scav- microbial growth suppressor. Ethanol emitters are 12 enging systems. The majority of commercial oxygen generally used in sachets where ethanol is absorbed 13 scavenging systems is based on the oxidation of or encapsulated in carrier materials and is released 14 iron powder with catalysts. The most well-known by exchange with water vapor (Ozdemir and Floros 15 technique is incorporation of a small size of sachet 2004). Therefore, the rate of ethanol release from 16 containing oxygen scavengers such as iron-based sachet to head space within a package depends upon 17 powders along with catalysts into the package the permeability of sachet barrier to water vapor. 18 (Ozdemir and Floros 2004). The major disadvantage Ethano-emitting films with an adhesive-backed film 19 of this method is the possibility of accidental inges- that can be attached on the inside of a package is also 20 tion of the contents in the sachet by consumers. available (Suppakul et al. 2003). 21 Thus, as alternatives to sachet, other new approaches In recent years, however, most attention has been 22 have been developed: incorporation of oxygen scav- paid to the development of antimicrobial active 23 enging layers into laminated trays and plastic films packaging systems because of food safety concerns. 24 or inserts in the form of flat packets, cards, or sheets; In general, the package for antimicrobial active 25 and oxygen scavenging adhesive labels, which can packaging systems is made by incorporating and 26 be applied to the inside of packages (Vermeiren et al. immobilizing antimicrobial agents into a film or by 27 1999). An enzymatic oxygen scavenging system, surface medication and surface coating of the agents 28 which uses oxidation enzymes such as glucose oxi- (Suppakul et al. 2003). The mode of actions of 29 dase and alcohol oxidase, is also available, either in antimicrobial agents in or on a film can be divided 30 packaging structure–incorporated or sachet-inserted into three categories: (1) direct diffusion to the prod- 31 form (Vermeiren et al. 1999). In addition, several ucts in the packaging system without headspace (e.g., 32 other technologies such as oxidation of ascorbic vacuum packaging); (2) in addition to (1), equilib- 33 acid, photosensitive dyes coupled with singlet oxy- rium evaporation from the film into the headspace 34 gen acceptors, oxidizable polymers and unsaturated followed by equilibrium sorption into the products; 35 fatty acids, immobilized yeast on solid materials, and (3) surface inhibition of microbials by surface- 36 and catalytic conversion of oxygen to water vapor by immobilized antimicrobial agents without migration 37 platinum in the presence of hydrogen gas are also (Quintavalla and Vicini 2002). Antimicrobial agents 38 available as oxygen scavenging systems (Labuza and that can be applied to active packaging are countless: 39 Breene 1989; Vermeiren et al. 1999; Ozdemir and for example, organic acids (e.g., acetate, citrate, 40 Floros 2004). Oxygen scavengers are commonly sodium benzoate, and sodium sorbate and their acid 41 used in association with vacuum packaging or MAP anhydrides), bacteriocins (e.g., nisin, pediosin, lac- 42 to remove residual oxygen within a food package. ticin), enzymes (e.g., lysozymes), metals (e.g., silver 43 The use of oxygen scavengers in combination with a and copper), fungicides (e.g., sulfur dioxide, beno- 44 film that has intermediate oxygen barrier properties myl, and imazalil), oligo and polysaccharides (e.g., 45 is more cost effective than using a film with very chitosan), antimicrobial peptides, amines, plant 46 high oxygen barrier properties alone (Vermeiren volatiles (e.g., allylisothiocyanate), metal chelators 47 et al. 1999). (e.g., ethylenediamine tetra acetic acid (EDTA), 48 Carbon dioxide emitters are useful active packag- lactoferrin, and conalbumin), and natural phenols 49 ing materials for fermented meat products because (e.g., catechin, extracts of grapefruit seed, essential

50 CO2 is effective in preventing the growth of surface oils) (Suppakul 2003; Cha and Chinnan 2004). 51 microorganisms. Because CO2 is more permeable Factors that should be taken into consideration in 52 than oxygen through most films used in food pack- designing and manufacturing antimicrobial films 53S aging, it is very difficult to control the concentration include the chemical nature of films/coatings, casting

54N of CO2 by ﬁlm alone (Labuza and Breene 1989). The process conditions, residual antimicrobial activity, 40454 27 289-300 r1.qxd 7/20/07 3:54 PM Page 299

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chemical interaction of additives with film matrix, and mold-ripened sausages during storage in order 1 characteristics of antimicrobial agents and foods, to control the growth of major pathogens such as 2 storage temperature, mass transfer coefficient, phys- Salmonella enteritica, Staphylococcus aureus, and 3 ical properties of packaging materials, food contact Listeria monocytogenes (Lücke 1998). 4 approval of antimicrobial agents, and cost (Quin- For fermented meat products, chemical and physi- 5 tavalla and Vicini 2002; Suppakul 2003). cal changes during storage are more critical than 6 Antioxidants such as BHT and BHA can be incor- microbial concerns. The major quality deteriorations 7 porated into plastic films in order to stabilize poly- in fermented meat products during storage are discol- 8 mers and thereby prevent film degradation. These oration, development of off-odors and off-flavors, 9 antioxidants are able to migrate into headspace dehydration, and weight loss. Vacuum packaging or 10 or products, resulting in an extended shelf life MAP with a high barrier plastic film to oxygen and 11 (Vermeiren et al. 1999). Other active packaging moisture can prevent or retard those primary quality 12 materials such as moisture absorbers, flavor/odor deteriorations during storage. However, vacuum 13 absorbers/releasers, time-temperature indicators, packaging and MAP is not suitable for all fermented 14 and light absorbers/regulators are applicable for fer- meat products, especially mold-ripened sausages 15 mented meat products. (Incze 2004). The surface of mold-ripened sausages 16 should be dry to maintain ideal appearance, but no 17 film can meet the properties of high moisture and low 18 ASEPTIC PACKAGING oxygen permeability. Therefore, the quality of mold- 19 Aseptic packaging is defined as the processes ripened sausages is more affected by storage condi- 20 wherein products, which are commercially sterilized tions. In this case, MAP combined with an active 21 or not, are placed in sterile packages under aseptic packaging system such as moisture absorbers may be 22 conditions and then the packages are hermetically one of the solutions to maintain the product’s appear- 23 sealed (Robertson 1993). The main objective of ance and prevent oxidative reactions and weight loss 24 aseptic packaging is to extend the shelf life of prod- at even room temperature during storage. In addition, 25 ucts at normal temperatures by reducing the initial during cold storage of mold-ripened sausages, phos- 26 microbial contamination and avoiding recontamina- phate crystals can appear on the products. 27 tion by microbes. There are two specific fields of An important food safety risk in fermented meat 28 application for aseptic packaging depending on products is production of biogenic amine, toxic com- 29 whether the products are sterilized or not before fill- pounds for human health. The amounts of biogenic 30 ing. The requirements for aseptic packaging are amines can be increased to hazardous levels during 31 eradication of microorganisms and protection of storage at room temperature (Komprda et al. 2001, 32 the packaging atmosphere for preventing any recont- 2004). Therefore, it is recommended that fermented 33 amination. In order to sterilize the food contact meat products be stored and distributed at refriger- 34 surface of packaging materials, numerous methods ated temperature—not only because the rates of 35 have been applied: irradiation, such as ultraviolet, chemical reactions such as lipid oxidation and 36 infrared, and ionizing-radiation; steam; hot air; discoloration, as well as transmission of oxygen, 37 extrusion; and chemical treatment with hydrogen through all plastic films are generally reduced as 38 peroxide, peracetic acid, ethylene oxide, and so on temperature is lowered (Romans et al. 2001), but 39 (Robertson 1993). also because the production rate of biogenic amines 40 during storage can be decreased at refrigerated con- 41 STORAGE ditions (Komprda et al. 2001, 2004). 42 43 The major characteristics of fermented meat prod- REFERENCES 44 ucts are low pH and water activity, which can pre- 45 vent the growth of food poisoning microbes during ED Aberle, JC Forrest, DE Gerrard, and EW Mills. 2001. 46 storage. The presence of salt, nitrite, and/or smoking Principle of Meat Science. 4th Ed. Dubuque, Iowa: 47 components added during processing inhibits bacte- Kendall/Hunt Publishing Co. DS Cha and MS 48 rial growth during storage. Therefore, if whole Chinnan. 2004. Biopolymer-based antimicrobial pack- 49 processes are well monitored by a well-designed aging: A review. Crit Rev Food Sci Nutr 44:223–237. 50 HACCP system, microbiological risk would not be AR Davies. 2003. Modified atmosphere and vacuum 51 the major concerns for fermented products. How- packaging. Ch. 11 in: Food Preservatives, 2nd ed. NJ 52 ever, it is recommended to keep the products below Russell and GW Gould, eds. New York: Kluwer S53 15°C for semidry sausages and below 25°C for dry Academic/Plenum Publishers, pp. 218–239. N54 40454 27 289-300 r1.qxd 7/20/07 3:54 PM Page 300

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1 PL Dawson. 2001. Packaging. Ch. 6 in: Poultry Meat BR Lundquist. 1987. Protective packaging of meat and 2 Processing. AR Sams, ed. New York: CRC Press, meat products. Ch. 14 in: The Science of Meat and 3 pp. 73–95. Meat Products, 3rd Ed. JF Price and BS Schweigert, 4 BPF Day. 1995. Active packaging for fresh produce. In: eds. Westport, Connecticut: Food & Nutrition Press, 5 Foods and Packaging Materials—Chemical Interac- Inc., pp. 487–505. 6 tions. P Ackermann, M Jägerstad, and T Ohlsson, M Mullan and D McDowell. 2003. Modified atmo- 7 eds. Cambridge, UK: The Royal Society of Chem- sphere packaging. Ch. 10 in: Food Packaging Tech- 8 istry, pp. 189–200. nology. R Coles, D McDowell, and MJ Kirwan, eds. 9 ———. 2003. Active packaging. Ch. 9 in: Food Pack- Boca Raton, Florida: CRC Press LLC., pp. 303–339. 10 aging Technology. R Coles, D McDowell, and MJ M Ozdemir and JD Floros. 2004. Active food packag- 11 Kirwan, eds. Boca Raton, Florida: CRC Press LLC., ing technologies. Crit Rev Food Sci Nutr. 44: 12 pp. 303–339. 185–193. 13 RH Driscoll and JL Paterson. 1999. Packaging and S Quintavalla and L Vicini. 2002. Antimicrobial food 14 food preservation. Ch. 23 in: Handbook of Food packaging in meat industry. Meat Sci 62:373–380. 15 Preservation. MS Rahman, ed. New York: Marcel G L Robertson. 1993. Food Packaging: Principle and 16 Dekker, Inc., pp. 687–733. Practice. New York: Marcel Dekker, Inc. 17 Food Safety and Inspection Service (FSIS). 2000. Meat JR Romans, WJ Costello, CW Carlson, ML Greaser, 18 packaging materials. http://www.fsis.usda.gov/OA/ and KW Jones. 2001. The Meat We Eat, 14th ed. 19 pubs/meatpack.htm. Danville, Illinois: Interstate Publishers, Inc. 20 K Incze. 2004. Mold-ripened sausages. Ch. 24 in: ML Rooney. 1995. Overview of active food packaging. 21 Handbook of Food and Beverage Fermentation Tech- Ch. 1 in: Active Food Packaging. ML Rooney, rd. New 22 nology. YH Hui, L Meunier-Goddik, AS Hansen, York: Blackie Academic & Professional, pp. 1–37. 23 J Josephsen, W Nip, PS Stanfield, and F Toldrá, eds. W Soroka. 1999. Fundamentals of Packaging Technol- 24 New York: Marcel Dekker, Inc., pp. 445–458. ogy. Herndon, Virginia: Institute of Packaging 25 T Komprda, J Neznalovà, S Standara, and S Bover-Cid. Professionals. 26 2001. Effect of starter culture and storage temperature P Suppakul, J Miltz, K Sonneveld and SW Bigger. 27 on the content of biogenic amines in dry fermented 2003. Active packaging technologies with an empha- 28 sausage poliˇcan. Meat Sci 59:267–276. sis on antimicrobial packaging and its applications. 29 T Komprda, D Smˇelá, P Pechová, L Kalhotka, J Stencl,ˇ J Food Sci 68:408–420. 30 and B Klejdus. 2004. Effect of starter culture, spice F Toldrá, R Gavara and JM Lagarón. 2004. Fermented 31 mix and storage time and temperature on biogenic and dry-cured meat: Packaging and quality control. 32 amine content of dry fermented sausages. Meat Sci Ch. 26 in: Handbook of Food and Beverage Fermen- 33 67:607–616. tation Technology. Y H Hui, L Meunier-Goddik, AS 34 DH Kropf. 2004. Packaging in Encyclopedia of meat Hansen, J Josephsen, W Nip, PS Stanfield, and 35 science, v. 3. WK Jensen, C Devine, and M Dikeman, F Toldrá, eds. New York: Marcel Dekker, Inc., 36 eds. New York: Elsevier Academic Press, pp. 943–976. pp. 445–458. 37 TP Labuza and WM Breene. 1989. Application of L Vermeiren, F Devlieghere, M van Beest, N de Kruijf, 38 “active packaging” for improvement of shelf-life and and J Debevere. 1999. Developments in the active 39 nutritional quality of fresh and extended shelf-life packaging in foods. Trends Food Sci Technol 40 foods. J Food Process Preserv 13:1–69. 10:77–86. 41 FK Lücke. 1998. Fermented sausages. Ch. 14 in: E Zanardi, V Dorigoni, A Badiani, and R Chizzolini. 42 Microbiology of Fermented Foods, vol. 2, 2nd Ed. 2002. Lipid and colour stability of Milano-type 43 BJB Wood, ed. New York: Blackie Academic & Pro- sausages: Effect of packing conditions. Meat Sci 44 fessional, pp. 441–483. 61:7–14. 45 46 47 48 49 50 51 52 53S 54N 40454 28 301-306 r1.qxd 7/20/07 3:55 PM Page 301

1 2 3 4 5 6 7 8 9 10 11 12 13 14 Part VI 15 16 17 18 19 Semidry-fermented Sausages 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 S54 N55 40454 28 301-306 r1.qxd 7/20/07 3:55 PM Page 303

1 2 3 28 4 5 6 U.S. Products 7 8 Robert E. Rust 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION CHEMICAL ACIDULANTS 22 23 Acidification is generally accomplished through Semidry sausages in the United States are generally 24 fermentation, although it can be also accomplished considered to be acidified processed meat products 25 through the use of chemical acidulants such as with a Moisture/Protein Ratio (MPR) of 3.7:1 or less. 26 encapsulated lactic or citric acid or glucono delta Once an MPR of 2.3:1 is reached, the product would 27 lactone (GDL). Although these chemical acidulants be considered a dry sausage. For the purposes of this 28 can achieve the desired acidity, there is some ques- discussion, we will concentrate on those products 29 tion as to whether the same subtleties of flavor can that fall into the MPR range of 3.7:1. With semidry 30 be achieved as with microbial fermentation. The sausages, shrinkage of about 15% would be 31 products containing encapsulated acidulants require expected during processing. The U.S. Department 32 heating to ϳ140°F (60°C) to release the acid from of Agriculture Food Safety Inspection Service 33 the vegetable fat coating. Acidulants are sometimes (USDA-FSIS) requires a “Keep Refrigerated” label 34 combined with lactic acid starter cultures to achieve on sausage products unless these are validated as 35 rapid pH drop. being shelf-stable. Previously they were considered 36 to be shelf-stable when they reached an MPR of 3.1:1 37 and a pH of 5.0 or less. Because the requirements for MICROBIAL FERMENTATION 38 a shelf-stable semidry sausage are constantly chang- 39 ing, it is recommended that the processor consult the For semidry sausages, a rapid fermentation cycle 40 current regulatory standards. is usually used. The fermenting organisms of pre- 41 ference are Pediococcus sp., often combined with 42 METHODS OF ACIDIFICATION Lactobacillus, which have an optimum growth tem- 43 perature of ϳ100°F (38°C). USDA-FSIS requires 44 Most U.S. semidry products are fermented to a pH of that “lactic acid starter culture” be included in the 45 approximately 4.8, although in some regions of the ingredient statement where a commercial starter 46 U.S. a pH of as low as 4.5 is considered desirable. culture is used. There are still some processors that 47 Rapid fermentation is considered desirable, not only depend on natural fermentation or the backslop 48 for safety considerations but for maximizing pro- method of inoculation. It is difficult to justify these 49 duction as well. It is important that the product reach practices in light of Hazard Analysis Critical Control 50 a pH of 5.3 or less before coagulase-positive Staphy- Point (HACCP) and Good Manufacturing Practice 51 lococcus can grow and produce toxin. (GMP) regulations. 52 53 S54 N55 303 40454 28 301-306 r1.qxd 7/20/07 3:55 PM Page 304

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1 FOOD SAFETY product where discrete particle distinction is impor- 2 tant. In the case of pork, meat from the shoulder 3 Because these products are considered “ready to eat” area carries a higher level of pigmentation and is 4 (RTE), several foodborne pathogens must be preferable. 5 addressed during manufacturing. USDA-FSIS con- Some processors like to include a limited amount 6 siders the presence of Salmonella, Listeria monocy- of high-collagen meats to provide the distinct colla- 7 togenes, and E. coli O157:H7 (products containing gen “eyes” that are often deemed desirable. For this 8 beef) in an RTE product as an adulterant. The reason beef or pork cheeks are often included in the 9 processor must be able to document, to the satisfac- meat ingredient list. These two meat ingredients, 10 tion of the regulatory agency (in this case USDA- however, often carry high microbial loads. Meats 11 FSIS), that the process used in production renders with high amounts of connective tissue—shank 12 the product safe from these foodborne pathogens. meat, for example—should be avoided because of 13 Appropriate HACCP plans and GMPs must be in the negative effect on texture. 14 place. Various researchers are working toward the In any case, meats with abnormal conditions such 15 development of computer models for the lethality of as soft oily fat or the PSE condition in pork and 16 foodborne pathogens. This computer modeling “dark cutting” beef should be avoided if a quality 17 might eventually supplant the necessity of lengthy end product is desired. 18 and expensive challenge studies. For most of the semidry sausage products, a meat 19 To detail the specific steps needed in an HACCP block with a fat content of 20–25% is typical. Leaner 20 plan and the processes needed to justify an acceptable products tend to be rubbery in texture unless a tex- 21 GMP for any specific semidry sausage would require ture modifier is added. To be labeled as a fat reduced 22 several lengthy chapters in themselves. Again, as pre- product, the fat content must be at least 25% less 23 viously mentioned, regulations are in a constant state than the manufacturer’s regular product. 24 of change, so a processor would be advised to consult 25 the up-to-date references on regulations. NONMEAT INGREDIENTS 26 Although Trichinae are no longer a serious threat 27 in the U.S., detailed regulations on the processing of Salt, NaCl, is normally added at levels of 2–3% 28 any RTE product containing pork are still parts of depending on market demands. Recently there has 29 the USDA-FSIS regulations in force and need to be been a trend toward reducing salt levels because 30 followed. consumer’s tastes have changed. 31 Sodium nitrite is added at 156 ppm. Traditionally, 32 MANUFACTURING PROCESSES sodium nitrate was used as the curing ingredient 33 because there was sufficient time for nitrate to be 34 In discussing manufacturing processes it is common reduced to nitrite by microbial action. With today’s 35 in the U.S. to express the level of ingredients added faster processing, the inclusion of nitrate has been 36 as a proportion of the meat block rather than as a dropped. The only exception might be Lebanon 37 proportion of the total formulation. Bologna, where a mixture of nitrate and nitrite is still 38 often used. 39 Sugar, particularly the simple sugar, dextrose, is MEAT SELECTION 40 critical to support fermentation. Dextrose is usually 41 Most of the sausages discussed here are either beef, added at levels of 0.5–0.75% depending on the pH 42 pork, or a mixture of the two. Poultry may some- drop required. Some products like Lebanon Bologna 43 times be included, but chicken or turkey semidry that are fermented to a very low pH may actually 44 sausages are rare. require levels of as high as 1.25%. Some of the 45 It is important to select meat ingredients that have more complex sugars may also be added, but gener- 46 minimum microbial loads to begin with. High levels ally these are limited unless a very low pH product 47 of bacteria can compete with the lactic acid starter is desired, because they may also support some 48 culture and often introduce unwanted flavors and fermentation. 49 textures as a result of their growth during the fer- Spices are what differentiate the semidry products. 50 mentation process. Obviously, selection of meat that Because of the long storage life, most processors pre- 51 is as free as possible from foodborne pathogens is fer the flavor of whole or ground spices over the spice 52 critical. extractives. With whole or ground spices, sterilized 53S Both lean and fat colors are important. Lean spices should be used so as not to introduce undesir- 54N should have a distinct red color and the fat should be able microorganisms into the product. Garlic, in the white. Yellow fat detracts from the appearance of a form of garlic powder, may or may not be added. 40454 28 301-306 r1.qxd 7/20/07 3:55 PM Page 305

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Natural smoke flavorings may be added to inten- collagen casings. Many of these have imbedded net- 1 sify smoke flavor. These flavorings can be added to ting or sting patterns for decorative purposes. There 2 the meat mixture in the form of an oil-based product, is also a variety of novelty casings made from 3 a maltodextrin-based product or a water-based solu- fibrous, collagen, or fabric materials. These come in 4 tion, provided the latter is appropriately buffered and a wide number of shapes and printed designs rang- 5 designed for direct inclusion in the product. ing from beer bottles to little pigs to pumpkins to 6 Christmas stockings, and they are useful when sup- 7 plying summer sausage to a niche market. 8 GRINDING/MIXING 9 10 Grinding and mixing are critical steps to ensure good STUFFING 11 particle distinction and to prevent fat smearing. The 12 A stuffer that will minimize smear is mandatory. meats should be kept as cold as possible and tem- 13 Most processors seem to lean toward a vane-type peratures of above 40°F (4°C) should be avoided. 14 vacuum stuffer. Keeping the meat below 40°F (4°C) Depending on the grinder, it is often preferable to 15 is critical to minimize smearing and prevent fatting- work with partially frozen meat to maintain good 16 out during the cooking step. The largest possible particle distinction. Close attention should be paid to 17 stuffing horn should be used, and this horn should be grinder maintenance. Knives and plates should be 18 free of nicks and dents. sharp and carefully matched. A bone removal system 19 should be used on the final grind. 20 A bowl chopper may be used for particle reduc- 21 FERMENTATION,SMOKING, AND COOKING tion. Although producing excellent particle dis- 22 tinction, it has the disadvantage of being highly Some type of conditioning step to bring the surface 23 operator-dependent for uniform particle size. Fur- of the product up to the initial fermentation tempera- 24 ther, it cannot be fitted with any type of bone removal ture in order to avoid moisture condensation on the 25 system as can a grinder. product as it is introduced to a high humidity atmo- 26 A coarse grind (1/2–3/8 in; 9–13 mm) of the meat sphere normally precedes the fermentation step in 27 ingredients is the first step. This may be followed by the thermal process. For these products, a culture 28 final grind to desired particle size or a mixing step that ferments at a temperature of about 100°F (38°C) 29 with the final grind following. The grind-grind-mix is the lactic acid culture of choice. The fermentation 30 is preferable because it reduces the chance of fat step is carried at 95% relative humidity (RH) and the 31 smearing and allows the use of a vacuum mixer prior desired pH can be attained in 24 hours or less. 32 to stuffing. It is also mandatory if whole spice or Following fermentation, a brief drying step pre- 33 other inclusions are to be added. In any event, a vac- cedes the smoking step. Smoke may be applied as a 34 uum mix prior to stuffing is desirable. vapor from a smoke generator or in the form of 35 For mixing, a paddle mixer with vacuum capabili- atomized liquid smoke. If liquid smoke is to be 36 ties is preferred. Mixing should be sufficient just to applied as a drench it can be done at this point. With 37 uniformly distribute the ingredients. Overmixing a liquid smoke drench, no predrying step is needed. 38 should be avoided. The accumulation of fat on the If liquid smoke is atomized, it is necessary to follow 39 paddles or sides of the mixer is an indication of the smoke supplier’s recommendation for applica- 40 overmixing. tion, which usually involves atomization followed by 41 a dwell step. 42 Because of microbial concerns, summer sausage 43 CASINGS and similar semidry products are usually fully 44 Fibrous, collagen, or natural casings are used for cooked, reaching a final internal temperature of 45 these semidry sausages as occasion demands. When 160°F (71°C). Lebanon Bologna is an exception to 46 using fibrous casings, if the casing is to shrink with this, but a lengthy process including cold smoking 47 the sausage as it loses moisture, a special coated cas- and holding at specified temperatures is involved. If 48 ing designed for dry and semidry sausage should be the product is not fully cooked, the process must be 49 used. For a product that will be peeled and sliced, a validated to ensure foodborne pathogen reduction. 50 regular fibrous casing would be the casing of choice. 51 As with all sausage casings, observing the Recom- 52 PACKAGING AND STORAGE mended Stuffing Diameter (RSD) is critical. S53 Laminated natural casings provide another alter- Vacuum packaging in a high-barrier plastic film is N54 native for summer sausage as do fabric-reinforced the most common packaging employed. With a sliced 40454 28 301-306 r1.qxd 7/20/07 3:55 PM Page 306

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1 product, if compaction of the slices is to be avoided, distinctly “tangy” and smoky flavor. In the traditional 2 an inert gas Modified Atmosphere package should process, the coarse ground meat is presalted and 3 be used. When held in inventory, even the shelf- held under refrigeration for a period of up to 1 week. 4 stable products should be stored under refrigeration 5 32–36°F (0–2°C) to obtain maximum storage life. SWEET BOLOGNA 6 7 Sweet bologna is a variation of Lebanon Bologna 8 DIFFERENT TYPES OF U.S. that is made with 10–12% sugar as opposed to 2–4% 9 SEMIDRY SAUSAGES in the Lebanon Bologna. 10 In this section the characteristics of some of the most 11 traditional products are explained. SNACK STICKS 12 13 This is a shelf-stable semidry sausage with a finished 14 SUMMER SAUSAGE diameter of approximately 19–15 mm. It is normally 15 The term summer sausage is applied to a loosely vacuum packaged in individual serving sizes and 16 defined variety of U.S. semidry sausages similar to used as a snack item. The formulation is similar to 17 some of the European cervelats. They generally have summer sausage. Sometimes jalapeno, chili, or other 18 a rather low pH, sometimes as low as pH 4.6. Sum- spicy peppers are added to produce a “hot” finished 19 mer sausage is usually a mixture of beef and pork, product. These are normally stuffed in an edible col- 20 although sausages made of beef alone are common. lagen casing that is designed specifically for this 21 The final grind size for this product ranges from type of product. 22 1/8–3/16 in (3–5 mm), and the predominant spice is 23 black pepper along with coriander and mustard. SAUSAGE FOR PIZZA 24 Sometimes whole peppercorns and whole mustard 25 seed are added. The product may contain garlic and This is a derivative of summer sausage with a spice 26 is smoked, or natural smoke flavoring is added. flavoring similar to the dry sausage, pepperoni. It 27 Some processors add nonmeat inclusions such as can be used as a substitute for true pepperoni on 28 diced cheese. When cheese is added, it is probably at price-competitive pizzas if appropriate labeling 29 the level of about 10% of the formulation. restrictions are observed. Sizing would be the same 30 Fibrous, collagen, natural, or laminated casings as for the true pepperoni. 31 are used and casing size ranges from 40–120 mm. 32 Some of these sausages may be stuffed in a variety REFERENCES 33 of novelty casings ranging from those shaped like 34 American footballs to beer bottles. Summer sausage S Barbut. 2006. Validating Lethality for Processing Dry 35 is often a component of food gift boxes in the U.S. Fermented Meat Products. Proc Thermal Processing 36 because of its shelf stability. A shelf life of up to a of RTE Meat Products. The Ohio State University, 37 year is not uncommon. March 28–30, 2006, The Ohio State University, 38 Columbus, Ohio. 39 JF De Holl. 1993. Encyclopedia of Labeling Meat and LEBANON BOLOGNA 40 Poultry Products. 10th Edition. St. Louis: Meat Busi- 41 This sausage is unique to the area around the town of ness Magazine. 42 Lebanon, Pennsylvania. It is an all-beef product with SA Palumbo, JL Smith, SA Ackerman. 1973. Lebanon 43 a final grind size of 3/32–3/16 in (2–5 mm). In addi- Bologna I. Manufacture and processing. J Milk and 44 tion to a prolonged cold smoking process, liquid Food Technol 36:497–503. 45 smoke flavoring may also be added. The lactic acid RE Rust. 2004. Ethnic Meat Products/North America. 46 starter culture used is often one that has been devel- Encyclopedia of Meat Sciences Vol. 1 Oxford, UK: 47 oped specifically for this product in order to enhance Elsevier Ltd, pp. 455–456. 48 the pathogen reduction in light of the fact that no ———. 2006. Good Manufacturing Processes Dry and 49 “cooking step” is involved. Casings would be the Semi Dry Sausages. Proc Dry and Semi Dry Sausage 50 same as those used for summer sausage. It is Short Course, Iowa State University April 11–13, 51 fermented to a very low pH (approximately 2006, Iowa State University, Ames, Iowa. 52 pH 4.2–4.4). Typically, it is not heated to a tempera- Standards and Labeling Policy, Book Change #10. 53S ture much above 135°F (57°C). The predominant 1995. United States Department of Agriculture 54N spice is black pepper along with cinnamon, cloves, (USDA)—Food Safety and Inspection Service allspice, and ginger. It is heavily smoked and has a Washington, D.C. 40454 29 307-318 r2.qxd 7/20/07 3:56 PM Page 307

1 2 3 29 4 5 6 European Products 7 8 Kálmán Incze 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION facturing was learned by other European butchers in 22 Germany, Hungary, France, etc. Its history goes back 23 When humans began to gather and hunt food in exces- close to 200 years in Hungary. 24 sive amounts, they must have experienced that, sooner This process of manufacturing salami was based 25 or later, food becomes inedible, either because of its on experience without knowing what microbial 26 offending taste or odor (spoilage) or because it activities take place, and the success of the manufac- 27 caused illness (toxicity). They must also have learned ture depended highly on the knowledge of the 28 that, with the help of methods that affect the food esteemed salami masters. Research and inventions 29 (cooking, frying, cooling, drying, salting), it remained of Louis Pasteur revolutionized the fermentation 30 edible for a longer time, with an added beneﬁt of industries; yet such a revolution in meat industry was 31 improvement in palatability. Drying food in the sun witnessed much later, when thanks to the works of 32 or above ﬁre in caves or in tents, where smoking con- Niven et al. (1959) and Niinivara (1993), strains with 33 tributed additionally to taste and length of storage, favorable effects were isolated, tested, and selected. 34 was later combined with salting and, if the salt was The results of this research were introduced in the 35 contaminated with nitrate, preservative and palatabil- meat industry about half a century ago and became 36 ity effect must have been experienced noticed too. step-by-step a generally accepted technology, yet 37 Because not only harmful microorganisms were pres- with basic differences among countries. In the United 38 ent in the ambient world of mankind, and because States, strong acidifying starter cultures (Pediococci) 39 salting and drying can cause a natural selection of the were used, whereas in Europe strains with good 40 useful microorganisms, this combination of methods nitrate reducing and aroma formation (Micrococci) 41 can be considered as an ancient way of fermentation. were introduced originally. Now, several different 42 (In this chapter, the word fermentation is used in a bacterial, yeast, and mold strains are available as 43 broader sense: metabolic activity of microorganisms starter cultures—less often as single strains, more 44 as well as enzymic activity of tissue enzymes, bring- often in combinations. Part III of this book, “Micro- 45 ing about all the chemical, biochemical, physical, biology and Starter Cultures for Meat Fermentation,” 46 and sensoric changes considered as fermentation.) deals with starter cultures in detail. 47 Fermentation of vegetables has been known for 48 many thousands of years; fermented meat products 49 have a much younger known history, although salt- SEMIDRY SAUSAGES, 50 ing and drying (not fermentation) of meat was also DEFINITION 51 known by Egyptians. Fermented salami (a dry 52 sausage type) was apparently “invented” 300 years The term semidry sausage is etimologically unequiv- 53 ago in Italy (Leistner 2005), and its method of manu- ocal; these products are dryer than water-added S54 N55 307 40454 29 307-318 r2.qxd 7/20/07 3:56 PM Page 308

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1 Table 29.1. Different types of semidry sausages. 2 Types Typical pH* Typical a * Storage Temperature 3 w 4 Raw fermented and dried Ͻ5.0–5.3 0.91–0.95 Ambient 5 Ͻ 6 Raw fermented and cooked 5.0–5.5 0.92–0.96 Ambient 7 Raw fermented cooked and dried 5.0–5.5 0.91–0.95 Ambient 8 Raw fermented dried and cooked 5.0–5.5 0.91–0.95 Ambient 9 Cooked and dried 5.6–5.7 0.93–0.96 Ͻ18°C** 10 Ͻ ** 11 Hot smoked and dried 5.6–5.7 0.93–0.97 18°C 12 Raw fermented, slightly dried (spreadable) Ͻ5.0–5.3 Ͻ0,97 Chilled** 13 *Serves only as guidance. 14 ** 15 Limited shelf life. 16 17 cooked meat products, but have higher moisture DIFFERENT TYPES OF SEMIDRY 18 content than dry sausages. According to the deﬁ- SAUSAGES 19 nition in the United States, semidry sausages are 20 fermented and cooked but usually not dried, with The most common semidry sausages in Europe are 21 the weight loss only during cooking (Sebranek the raw fermented and dried products. Although ear- 22 2004). lier natural microﬂora supported by carbohydrate 23 In Europe, the term semidry sausage is used in a addition had the task to complete fermentation in a 24 broader sense, because it may involve the types desired way, ensuring both good sensory value and 25 listed in Table 29.1. food safety, and backslopping was used only some- 26 As shown in the table, one may distinguish among times, real breakthrough came with the introduction 27 seven different types of semidry sausage with the of starter cultures or chemical acidulants or both. 28 following common features: This process contributed not only to safety and sen- 29 sory value but also to the more consistent quality. 30 • Most of these sausages are dried or at least lose The combination of lower pH value caused by lactic 31 weight after fermentation because of cooking acid fermentation or by direct addition of acidulant 32 or hot smoking; consequently they have a lower and the lower water activity ensures the storage at 33 aw value than cooked sausages or cooked hams room temperature without spoilage or growth of 34 to which water is added. pathogens. Some of these types are manufactured 35 • The typical aw values are higher than charac- with mold growth on the surface, but the majority 36 teristic aw values of dry sausages, which are are processed without it. Spontaneous mold growth 37 below 0.90. has been changed to mold starter application in most 38 • As a consequence of a higher aw value, texture cases. For safety reasons, molded semidry sausages 39 is softer than that of dry sausages. have to be dried to a lower aw value than the other 40 • There are also cooked or hot smoked and dried products because molds can metabolize lactic acid, 41 products without previous fermentation that can which causes elevated pH. This pH increase making 42 also be considered as semidry sausages. possible the growth of staphylococci, among other 43 • The taste of semidry sausages, with the exception undesired microorganisms. 44 of the only cooked and dried sausages (where It is valid for the raw fermented and dried 45 neither starter culture nor chemical acidulant sausages just as for the other groups that—within the

46 is used), is more acidic than that of dry sausages ranges given in Table 29.1—if aw is higher, a lower 47 because of the higher moisture content. pH value is needed to give a satisfactory protection 48 • Semidry sausages are sliceable due to coagulation against undesired growth of microorganisms and 49 caused by acidiﬁcation and/or heat treatment, vice versa. Final moisture content is in general 50 with the exception of spreadable sausages. below 40%. 51 • Spreadable semidry sausages are always raw and Raw fermented and cooked sausages were also 52 are dried to a lesser extent; for this reason, their manufactured earlier without starter culture or 53S shelf life is limited and chilled storage may be chemical acidulant, since the “age of starter cultures” 54N needed. now these products are cooked after a starter 40454 29 307-318 r2.qxd 7/20/07 3:56 PM Page 309

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culture–controlled fermentation. This heat treatment, safety; aw and pH values are more informative. Ini- 1 inactivating most vegetative forms of microbes, low- tial moisture content, initial salt (low molecular 2

ered pH, and aw values ensure a fair safety record weight substances in general) content, and extent of 3 even with a somewhat higher aw value. Final moisture drying, or initial moisture content, initial protein 4 content is, in general, above 40%, because usually content, and extent of drying define final aw and the 5 weight loss during cooking serves as the only drying final M:P ratio, respectively. As a matter of fact, the 6 effect. These types and spreadable sausages some- M:P ratio gives information on the extent of drying 7 times are called moist sausages, referring to the high of the lean meat part. It is worth mentioning that a 8 moisture content (Campbell-Platt 1995). borderline between dry and semidry sausages is not 9

Some raw fermented sausages are further dried always set similarly. In Europe, an aw value below 10 after cooking. This evidently makes them more stable 0.89–0.90, which equals an M:P ratio of about 11 and their sensory value is also upgraded. Heat treat- 1.2–1.3:1, is considered as a criterion for dry 12 ment is, of course, advantageous because pathogenic sausages (Incze 2004), whereas the U.S. uses an M:P 13 microorganisms (e.g., E. coli O157:H7, Listeria ratio of 1.9:1 for the same criterion (Sebranek 2004) 14

monocytogenes, etc.) are inactivated with much corresponding to an aw of cca 0.93. 15 higher probability than if the aw and pH were One of the main reasons of this difference may be 16 decreased only. It has to be mentioned, however, that that low acidic sausages are not uncommon in 17

raw semidry sausages in comparison with cooked Europe, where a lower aw value is needed for good 18 ones show a higher sensory value because bacterial shelf life and safety reasons. It is also interesting to 19 and tissue enzymes will contribute to the ﬂavor and note that the initial moisture content deﬁnes initial 20 aroma. protein content as well if the sausage is manufac- 21 Although not too common, there exists a technol- tured only of meat and fat without another protein 22

ogy in which cooking is applied only after fermenta- source; different aw values can belong to the same 23 tion and drying, with the aim of inactivating harmful M:P ratios (Table 29.2). 24 microbes and ensuring a better palatability than if 25 heat treatment is applied immediately after fermen- 26 tation, thus inhibiting extended metabolic activity of TECHNOLOGY OF SEMIDRY 27 microorganisms. SAUSAGES 28 There are also types of semidry sausages when 29 Unlike in the dairy industry where milk is (can be) neither spontaneous nor controlled (by starter cul- 30 pasteurized, thus the majority of pathogenic as well as ture) fermentation is applied, but the sausages are 31 spoilage microorganisms are inactivated before starter cooked and dried or hot smoked and dried, until the 32 cultures are added, there is practically no way in the a value is decreased. In these cases the a value 33 w w meat industry to support the growth of starter cultures itself is not inhibitory enough, but cooking or hot 34 by destroying competing natural microﬂora of meat smoking inactivates most vegetative forms and 35 batter. Consequently, good manufacturing practice is nitrite has also some inhibitory effect, so these prod- 36 even more important than with other types of meat ucts can be stored without refrigeration but usually 37 products in order that starter cultures can overcome for a shorter period of time (some weeks). 38 undesired microbes and become dominating. All the semidry sausages discussed until now are 39 Referring to Table 29.1, technologies differ from considered sliceable products, but a large group of 40 one another in fermentation, heat treatment, and dry- spreadable sausages should be listed among semi- 41 ing. Differences also can be in raw materials (pork, dry sausages, too. These types of products are manu- 42 beef, nonmeat proteins), in mincing, in smoking, and factured mostly in Germany and Austria. Spreadable 43 evidently in seasoning. sausages are raw and are dried only for some days 44 after smoking. 45 Semidry and dry sausages can be distinguished PRODUCTION OF MEAT BATTER 46 on the basis of water activity value (Incze 2004) or on 47 Selection of Ingredients and Additives the basis of moisture:protein (M:P) ratio (Sebranek 48 2004). According to Sebranek, the M:P ratio is Semidry sausages are manufactured mainly of pork 49 mainly used in the United States, whereas water or beef or a mixture of them, and less often from 50

activity (aw) is more common in Europe, even if the poultry and mostly with pork fat. (As ethnic food, 51 M:P is also given sometimes in recipes (Wahl 2004). pure beef or lamb with tallow is also a possible 52 Moisture content alone gives no sufﬁcient infor- choice for manufacturing semidry sausages.) Chilled S53 mation in terms of distinction or shelf life and meat and fat are used for grinding technology, frozen N54 40454 29 307-318 r2.qxd 7/20/07 3:56 PM Page 310

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1 Table 29.2. Differences between final moisture contents of dry and semidry sausages and their relation to 2 moisture:protein ratio depending on initial moisture content and drying loss. 3 Initial Moisture Drying Loss Moisture:Protein Ratio Moisture Content a 4 w 5 40% 30% 0.8:1 14.3% 0.836 6 50% 40% 0.7:1 16.7% 0.839 7 8 50% 38% 0.8:1 19.4% 0.864 9 40% 10% 2.5:1 33.3% 0.938 10 50% 15% 2.5:1 41.2% 0.946 11 12 60% 20% 2.5:1 50.0% 0.951 13 50% 30% 1.4:1 28.6% 0.913 14 60% 37% 1.4:1 36.5% 0.923 15 40% 5% 1.9:1 36.8% 0.946 16 17 50% 5% 3.2:1 47.4% 0.957 18 50% 10% 2.9:1 44.4% 0.951 19 50% 20% 2.1:1 37.5% 0.939 20 60% 5% 3.4:1 57.9% 0.962 21 22 60% 10% 3.1:1 55.6% 0.959 23 Data were calculated on the basis that 40%, 50%, or 60% initial moisture content was chosen; initial protein content was 24 accordingly 12%, 14%, or 16% respectively; initial salt content was 2.5% uniformly. 25 26 27 meat and fat (−4 to 8°C) are used for bowl chopper As a carbohydrate source for lactic acid forma- 28 technology. Firm fat is necessary instead of soft fat tion, dextrose is used the most, but other types of 29 for a nice, clear-cut surface. Nonmeat ingredients, sugars can also be used in a percentage of usually 30 plant proteins and skin (rind) can also be used. For between 0.3–0.7%. Dextrose supports the fastest, 31 safety reasons it is preferable to precook or to handle lactose slowest, acid formation that can be used, e.g., 32 the skin with acidic treatment; these treatments are in retarding pH drop and making metabolism of 33 advantageous not only from a hygienic but also from micrococci possible. Sodium or potassium ascorbate 34 a sensory point of view. improves color and retards oxidation of lipids 35 Semidry sausages usually are produced in a short (which acts also against nitrosamine formation); 36 time; therefore, chiefly nitrite curing salt is applied therefore, it is used extensively, being most effective 37 (0.4–0.6% NaNO2 blended with 99.4–99.6% NaCl). in the case of cooked semidry sausages. With these 38 Bacterial breakdown of nitrate to nitrite would need latter types of products, the addition of phosphates 39 longer time, and only nitrite is effective for color for- helps prevent fat separation during heat treatment. 40 mation and color stability, for microbial inhibition, For pH drop chemical acidulants also can be used 41 and as an antioxidant. alone or in combination with starter cultures. The 42 Finely or coarsely ground black pepper is the most most well-known substances are glucono-delta- 43 common spice used both in southern and northern lactone and encapsulated organic acids. The advan- 44 Europe, whereas red pepper and garlic are more typ- tage of their use is the almost immediate pH drop; 45 ical for Hungarian and southern European (Spanish, sausages with starter cultures are nevertheless of 46 Italian) sausages. Laurel is a typical seasoning for better quality and have longer shelf life. 47 several spreadable sausages, and common spices of 48 other meat products find their way to semidry COMMINUTION 49 sausage products, too. It is worth mentioning that the 50 relatively high carbohydrate content of red pepper Meat and fat are chilled (for grinding) or frozen 51 can cause an overacidification if not calculated with (bowl chopper technology); comminuted to the 52 added carbohydrate. Another extreme is the exces- desired particle size; and mixed with carbohydrate, 53S sive amount of red pepper, which makes good bind a starter culture if applicable, curing salt, and 54N and sliceability questionable. seasonings. A low temperature of meat and fat is 40454 29 307-318 r2.qxd 7/20/07 3:56 PM Page 311

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necessary for avoiding fat smearing, which causes praxis and in consumers’ preference can still be wit- 1 not only esthetic failure (individual meat and fat nessed within one country, e.g., Germany. Sausages 2 particles are not seen) but may retard or even inhibit in Northern Germany are more acidic than those 3 drying if a fat layer on the surface stops evaporation, produced in the southern part. 4 and as a result water activity is not reduced. Patho- Fast acidification and lower pH can be ensured by 5 genic and spoilage microorganisms have a good pediococci (P. acidilactici) that grow and metabolize 6 chance for growth when this happens, and safety carbohydrates at higher temperatures (37–40°C) or 7 cannot be ensured. by lactobacilli (L. sakei, L. curvatus) growing at 8 Comminution of ingredients and blending with lower temperatures (20–24°C). 9 additives takes place in one step in a bowl chopper, If higher final pH and more richness in aroma is 10 and yet extra blending is necessary if grinder required, staphylococci (S. carnosus, S. xylosus) or 11 technology is applied. In both cases, chopping, Kocuria varians are used alone or in combination 12 grinding, blending, and stuffing should be carried with lactobacilli. In the latter case, only slow acidifi- 13 out under vacuum for microbiological and color for- cation makes growth and the metabolic activity 14 mation purposes. of staphylococci—which do not tolerate low pH— 15 possible. This combination of starter cultures gives 16 Development of Binding, Structure, and Texture mutual benefit: better safety and more aroma rich- 17 Structure and texture of meat products are very ness. To enhance aroma formation, Debaryomyces 18 important characteristics perceived and judged by hansenii also is generally used together with Lacto- 19 consumers. The pH value and pH changes have a bacillus and Staphylococcus starter. Although these 20 definite influence on binding and structure. In high microorganisms are blended in the meat batter, 21 acidic sausages, major structural changes take place molds are inoculated onto the surface of sausages 22 during the first 48 hours (van’t Hooft 1999) when pH with the aim of meeting consumers’ demand, to ren- 23 decreases to 5.3 or below. The solubilized myofibril- der specific aroma and protect the sausage to some 24 lar proteins gelify and ensure the fixation of meat extent from the effect of light (rancidity) and from 25 and fat particles affecting binding. Myosin is the drying too rapidly. The most common mold starters 26 main gelating protein, but collagen fibrils also take are Penicillium nalgiovense, P. camemberti, and 27 part in cohesion and sliceability; at the same time P.chrysogenum. 28 sodium chloride plays an important role in binding. 29 van’t Hooft (1999) also quantified the amount of 30 CASINGS meat exudates necessary for good binding: exudates 31 below 15% gave poor cohesion, adhesion, and fat Several decades ago, natural casings (casing of pig, 32 binding. cattle, sheep, or horse) were used, and as a shortage 33 of natural casings occurred, research and develop- 34 ment intensified for replacing them with artificial 35 SELECTION OF STARTER CULTURES ones. All the artificial casings used for dried sausages 36 Northern European countries introduced starter cul- are common in their water vapor and gas permeabil- 37 tures earlier than the Mediterranean meat industry, ity, a necessity for making evaporation (drying) and 38 and semidry sausages are manufactured in most smoking possible. The main types are cellulose cas- 39 cases with starter cultures, but there are still some ings (nonfibrous and fibrous) and collagen casings. 40 products that are fermented and dried, cooked and These latter types are resistant to molds that can solu- 41 dried, or hot smoked and dried without starter bilize cellulose on molded sausages, causing plaques 42 cultures. and fattening, through. 43 One of the reasons that manufacture of high acidic Basically two types of cellulose casings exist, the 44

semidry sausages with higher aw value have been nonﬁbrous and the ﬁbrous types (Savic and Savic 45 characteristic in northern countries for long time 2002b). Both are permeable to water vapor and 46

while low acidic dry sausages with a low aw value gases; therefore, they can be used for raw fermented 47 were more common in southern European countries sausages, too. Nonﬁbrous cellulose casings have 48 is possibly because of safety reasons: Long-lasting excellent eye appeal with their bright surface and 49 drying at low temperature gives a product with a low transparency. In ﬁbrous casings, regenerated cellu- 50

aw value, ensuring good stability and safety at room lose ﬁbers reinforce a cellulose hydrate matrix; thus 51 temperature even without the acidiﬁcation that they possess higher strength and can be used for 52 would perform at higher temperature, a risky process manufacture of semidry sausages, both cooked and S53 if no starter culture is applied. This difference in raw. Depending on the end users’ need, the adhesion N54 40454 29 307-318 r2.qxd 7/20/07 3:56 PM Page 312

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1 properties of fibrous cellulose casings can be adjusted whereas in Mediterranean countries air drying instead 2 to facilitate casing removal (slicing operations) or to of smoking is the common technology for semidry 3 ensure a better adhesion. products. As mentioned, this brings about different 4 The other biodegradable polymer casing that is sensory values. 5 used in the meat industry is collagen casing. This For several decades, not only smoke directly gen- 6 type is also suitable for stuffing raw or cooked (hot erated in the meat plant by controlled combustion of 7 smoked) semidry sausages because of its good wooden chips or by friction of wooden logs has been 8 vapor, gas, and smoke permeability and sufficient applied, but liquid smoke also has been used, wherein 9 strength, as well as good adhesion (Savic and Savic an atomized liquid is deposited on the sausage. 10 2002a). The food laws of different countries regarding 11 The advantage of cellulose and collagen casing is liquid smoke regulate labeling differently. As an 12 the uniform caliber and, in the case of small-diameter advantage of liquid smoke, a low concentration of 13 collagen casings, there are edible varieties, too. The polycyclic hydrocarbons is claimed, which can also 14 mechanical strength of these artificial casings is be guaranteed by well-controlled smoke generation. 15 higher than that of natural casings (Effenberger A meeting of environmental requirements is more 16 1991). More on casings can be found in Part II, costly, however. More on smoking can be found in 17 “Raw Materials.” Part I, “Meat Fermentation Worldwide: History and 18 Principles.” 19 SMOKING 20 21 Smoking is also an ancient way of meat preserva- FERMENTATION, RIPENING, 22 tion; archeological findings prove that it was used AND DRYING 23 90,000 years ago (Sikorski 2004). 24 Depending on the temperature of smoke, we may In cases when a starter culture is applied, optimum 25 distinguish between cold smoking (15–25°C), warm incubation temperature for microorganisms sug- 26 smoking (approx. 30–50°C) and hot smoking gested by the supplier is adjusted so that starter 27 (approx. 60–90°C). For raw semidry products, cold microorganisms can perform expected metabolic 28 smoking is applied. Warm smoking is common with activity ensuring desired changes in pH drop, result- 29 cooked semidry sausages, but hot smoking also is ing in acid coagulation and texture formation, 30 used with some semidry products, when the internal enhancing microbial stability and safety, breaking 31 temperature (in the core) can reach a temperature of down of lipids and proteins, forming color and sta- 32 65°C or above. In order to produce a safe smoke low bility as well as safety, and forming good aroma. On 33 in polycyclic hydrocarbons, the combustion temper- these changes many papers are published where all 34 ature of the wood (mostly beech and other hard- the details can be found (Montel et al. 1998; Toldrá 35 woods) is strictly controlled. 2002; Demeyer and Toldrá 2004; Talon et al. 2004; 36 Smoking has several advantages in addition to Incze 2004; Part IV of this book). During incubation 37 sensory value (making air-dried and smoked prod- (fermentation), sausages can be smoked only if their 38 ucts entirely different in taste), both chemically and surface is dried. Depending on the type of starter, the 39 microbiologically. If applied sufficiently, possibly incubation temperature, and the technological aim, 40 more than once for several hours intensively, fermentation lasts shorter if product is cooked after- 41 smoking has a fair antioxidative as well as a ward, or longer if no heat treatment is applied. As 42 bacteriostatic and fungistatic effect. These latter mentioned earlier, some semidry sausages are dried 43 effects make protection against surface slime forma- after heat treatment, some are not. Fermentation 44 tion and mold growth easier and more reliable. Dif- with raw sliceable semidry products lasts longer 45 fusion of smoke constituents is restricted mainly than that of cooked, with reduced intensity parallel 46 to the outer layer of sausage and cannot be consid- with drying in order to increase salt concentration. 47 ered as an efficient antimicrobial factor inside the With products that are heat treated right after 48 sausage. stuffing and eventual smoking, practically no fer- 49 In order to avoid greyish discoloration, smoking is mentation takes place because bacterial and tissue

50 applied onto the dry surface of sausages and always enzymes are inactivated. As a result of drying, the aw 51 at lower relative humidity (70–80%). value decreases in all types of semidry sausages, 52 In south Austria, Germany, and Hungary, smoking resulting in an extended shelf life and safety. The 53S is not as typical as in these and in northern countries, initial moisture and salt content and the degree of 54N 40454 29 307-318 r2.qxd 7/20/07 3:56 PM Page 313

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drying determine the extent of shelf life and safety. SAFETY AND STABILITY OF 1 Sausages with lower pH and/or being heat treated SEMIDRY SAUSAGES 2 can lose moisture easier, i.e., the drying rate can be 3 higher than in the case of raw sausage with higher Safe and stable products can evidently be produced 4 pH (5.7 and above). Even then, sausages with lower only from ingredients and additives of high hygienic 5 pH can be dried at a rate not higher than the quality and applying GHP and GMP during process- 6 moisture-losing ability of the sausage. The driving ing. Combinations of (1) pH and aw drop; (2) pH 7 force of moisture diffusion (the difference between drop, heat treatment, and aw drop; or (3) heat treat- 8 the relative humidity of air and equilibrium relative ment and aw drop ensures safety and stability of such 9 humidity of product) has to be selected in such a way products. Although publications report on higher 10 that neither case hardening nor insufﬁcient drying resistance of Clostridium botulinum (Lund and Peck 11 will result. The range between 5–10% can be chosen 2000; Setlow and Johnson 2001; Austin 2001) 12 with acidic and/or heat treated sausages, but air against pH, aw, and other inhibiting factors, with 13 ϭ ϭ velocity must be taken into consideration. pH 4.6 and aw 0.94–0.96 as limiting values for 14 Reduction of ripening and drying time has always growth and similar growth-limiting values men- 15 been a thoroughly investigated research topic. The tioned concerning other pathogenic bacteria (E. coli, 16 main methods in achieving this objective were the EHEC, Listeria monocytogenes, Salmonella, 17 addition of enzymes such as proteinases (Blom et al. Staphylococcus, B. cereus); these are nevertheless 18 1996), reduction of initial moisture content by addi- measured under otherwise optimal conditions. In 19 tion of freeze dried meat or a remarkable volume of semidry sausages, a combination of several growth- 20 meat batter of already dried sausage (Ulmer et al. limiting factors act simultaneously, ensuring safety 21 2006). Although drying time could be reduced in at values that individually would not be sufﬁcient for 22 several cases, sensory characteristics were in most inhibition. This effect of hurdles (Leistner 2000) 23 Յ cases not equal to products manufactured, ripened, works well at pH values of 5.3 and parallel aw 24 and dried for the normal length of time. Sensory values at Յ0.95. A combination of reduced pH and 25 improvement has nevertheless been found (Bolumar aw as well as other hurdles ensure inhibition or inac- 26 et al. 2006) with the addition of cell-free extracts of tivation of not only bacteria (Salmonella, Staphylo- 27 Debaryomyces hansenii and L. sakei. coccus, Listeria, EHEC, etc.) but also parasites like 28 Toxoplasma (Anon 2006). Further safety-improving 29 factors include heat treatment, in special cases, heat 30 PHYSICAL CHANGES treatment in combination with aw reduction. More 31 During fermentation, ripening, smoking (if appli- details on the safety of fermented sausages, with 32 cable), cooking, or hot smoking (if applicable) and special regard to enterohaemorrhagic E. coli are dis- 33 drying, sausages lose weight. Because of lower pH cussed in publications (Leistner 1995; Incze 1998, 34 (near to the isoelectric point) or because of heat 2003; Toldrá 2002). On biogenic amines, polycyclic 35 treatment (coagulation), muscle protein loses mois- hydrocarbons, and nitrosamines, see publications of 36 ture more rapidly than traditional, low acidic Bauer and Paulsen (2001) and Bauer (2004). Myco- 37 sausages. As a result of this and because these prod- toxins are less a problem because semidry sausages 38

ucts are of higher aw than dry sausages, there are are in most cases produced without molds, and in the 39 huge differences between the whole processing time few cases when mold growth is desired, nontoxi- 40 of these two groups: 2–3 days versus 100 days or genic mold starters are used. 41 more. As for the drying rate of semidry sausages, Treatments ensuring safety are usually sufﬁcient 42 meat batter with higher fat content loses weight for stability of semidry sausages: growth of spoilage 43 slower and vice versa. Mainly muscle is capable of microorganisms is also suppressed or they are inacti- 44 losing water, because the moisture content of fat is vated. For this reason, shelf life is mainly limited 45 much lower and it takes more time to evaporating the by physical changes such as fat melting, rancidity, 46 same amount of moisture. In practice, it means that discoloration, extreme drying, and weight loss. Most 47

the lower initial aw (higher fat content) of a sausage of these changes can be prevented by MAP or vac- 48 does not result in a lower ﬁnal aw than a sausage of uum packaging. For more details on spoilage and 49 higher initial aw (lower fat content) after the same pathogenic microorganisms, see Kröckel (1995) 50 drying period, unless a signiﬁcantly higher driving and Chapters 41, “Spoilage Microorganisms: Risks 51 force of moisture is applied, which then means a and Control,” and 42, “Pathogens: Risks and Con- 52 high risk of case hardening. trols,” of this book. S53 N54 40454 29 307-318 r2.qxd 7/20/07 3:56 PM Page 314

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1 QUALITY DEFECTS OF SEMIDRY becomes a semidry product if dried for a shorter 2 SAUSAGES time and will be a dry sausage if dried for a longer 3 period. Evidently this is true only for high acidic 4 Some of the quality defects can be detected from sausages to avoid safety and/or spoilage risk. As a outside, others only after slicing. The reasons for 5 result of this different tenure, the aw values will dif- 6 defects can be manifold; the following are some fer from one another and the products will count, 7 more common failures: accordingly, as dry or semidry (see definitions ear- 8 • Contamination of raw materials (spoilage or lier in the section “Different Types of Semidry 9 safety risk) Sausages”). 10 • Poor technological quality of ingredients (soft fat, 11 offensive odor) 12 • Comminution of meat and fat not uniform TYPICAL SEMIDRY SAUSAGES OF DIFFERENT 13 • Temperature of fat during comminution not low COUNTRIES 14 enough (fat smearing, failing of structure) Austria 15 • Stuffing not suitable (fat smearing, air holes) 16 • Inadequate temperature during smoking, Manufacture and consumption of dry and semidry 17 ripening, and drying (too low: not suitable for sausages have a long tradition in Austria. Before 18 starters; too high: spoilage or safety risk; fat starter culture application, mainly dry sausages with 19 melting, not uniform in the room: remarkable or without a mold layer on the surface were pro- 20 differences among sausages) duced. But besides keeping the tradition of produc- 21 • Inadequate relative humidity and/or air velocity ing these types, the combination of milder drying 22 during smoking, ripening, and drying (too slow with the use of a starter culture made the production 23 drying: spoilage risk; undesired growth of of raw sausages with higher M:P ratio (semidry 24 Micrococci, yeasts, and molds on the surface; too sausages) possible, while ensuring microbial stabil- 25 rapid drying: case hardening risk; too high RH ity and safety. Known types are fresh salami (Frische 26 during smoking: color defects) Salami); Dekorsalami, which are peeled after dry- 27 • Inadequate pH drop (too low pH: sensory failure; ing, dipped in gelatin, and then covered with spices 28 too high pH: slow moisture loss; insufficient or grated cheese and called, accordingly, pepper, gar- 29 inhibition of microbial growth: spoilage and lic, and parmesan-decorated salami; Debreziner 30 safety risk, poor sliceability) Rohwurst (Debreziner raw sausage); Pusstawürstel 31 (Puszta sausage); Knoblauchwurst (garlic sausage); • Inadequate aw reduction (spoilage and safety risk) 32 • Selection of wrong packaging film or gaseous Haus würstel (home sausage); and frische Rohwurst 33 atmosphere (color defect, rancidity, eventual (fresh raw sausage). Garlic sausage and home sausage 34 mold growth if film has high oxygen permeability are eaten usually after cooking at home, because of 35 or residual oxygen percent is not sufficiently low their moderate drying (15%). All these are sliceable 36 (Ͻ1%) in modified atmosphere packages) products made of beef and/or pork, pork fat, starter 37 • Storage temperature is not suitable (too high: fat culture, spices, and curing salt, and dried to a weight 38 melting, separation, rancidity, spoilage loss of 15–30%. Paprika is not a typical spice of 39 possibility; too low: phosphate crystallization these products, but in some regions in Eastern Aus- 40 inside and outside the sausage) tria, paprika seasoning can also be characteristic 41 (Puszta sausage). The ratio of main ingredients is 42 approximately 70% meat and 30% fat, and the M:P 43 TYPES OF SEMIDRY SAUSAGES IN ratio varies between 1.6:1 and 3.0:1. 44 DIFFERENT COUNTRIES Production technology is similar to German pro- 45 cessing, and comminution and blending of raw 46 Although different types of semidry sausages are materials is done with a bowl chopper. Spreadable 47 characteristic for different countries, alterations can sausages are always raw fermented. “Officially” they 48 be detected between products under the same name are not dried, but during fermentation and smoking 49 even in the same country, depending on smaller or they lose weight about 5 max. 10%. As a result of pH 50 greater changes in technology. A difference can be drop and slight drying, these products are relatively

51 found in the aw value because sausages of the same stable and rather safe with a shelf life of 1–3 weeks 52 recipe and diameter can be dried for a shorter or depending on the storage temperature. Characteris- 53S longer period of time. Drafting somewhat arbitrarily, tic products are different types of Teewurst and Met- 54N we might say that a raw fermented dried sausage twurst, with M:P ratio ranging between 2.4:1 and 40454 29 307-318 r2.qxd 7/20/07 3:56 PM Page 315

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3.2:1; nevertheless, their share is definitely lower others are manufactured without it. Traditionally 1 than that of the sliceable products. Depending on these sausages are eaten raw, but sometimes heat 2 product and starter culture, fermentation (incuba- treated (cooked or fried) before consumption, which 3 tion) temperature can vary between 19 and 28°C contributes to safety. Some known sausages are 4 with 90–95% relative humidity (RH) that are saucisse de Montbeliard, smoked, also eaten raw, 5 decreased step by step to 14–18°C and 72–78% rela- produced along the Swiss border between Geneva 6 tive humidity. Smoking temperature is in general less and Basle; saucisse de Morteau, smoked and dried 7 than 20°C, and relative humidity during smoking is to some extent; and cabbage sausage, also smoked 8 less than 85%. Austrian semidry sausages are less and cooked as in Switzerland. 9 acidic (pH 5.2–5.3) than (North) German products. Further types are longeoles, pormoniers, and diots 10 (can be smoked), produced in the region of the French 11 Denmark Alps to Italy. The typical Corsican products with pork 12 There are two Danish dried products that fulfill the liver are figatelles-figatelli. Saucisson lorrain is the 13 definition of semidry sausages mentioned earlier: well-known, although produced only in small quan- 14 Danish salami (Dansk spegepølse) and South Jut- tity, smoked, dried (20–25% weight loss) sausage of 15 landic salami (Sønderjysk spegepølse). Northern France along the Belgian-German border. 16 Danish salami is produced from pork, and has a cal- Also along the German border a Teewurst-Mettwurst– 17

iber of 90–120 mm, a final pH of 4.7–4.9, and an aw of type smoked spreadable sausage is manufactured in 18 about 0.90. Seldom a starter culture, but more often Alsace with a starter culture. 19 glucono-delta-lactone, helps in pH drop. The salami 20 has a bright red color due to cochineal (E 120); nitrite Germany 21 salt is also used. Two production methods are known: 22 direct curing, wherein all curing additives containing Germany can be considered as “the home of 23 the meat-fat batter is stuffed in fibrous cellulose cas- sausages” in general, but also as “the home of semidry 24 ing, and tank curing, wherein sausages are put in a sausages.” Similarly to Austrian products, sliceable 25 tank with brine causing salt enrichment in the sausage and spreadable sausages are produced. A wide range 26 and moisture removal from it at the same time. In the of production includes manufacture both in small and 27 latter method, the original sausage mix contains less medium enterprises (SMEs) and at a large industrial 28 salt. Because of the high initial fat, high salt, and low scale, but in a huge variety. It is interesting to note that 29 moisture content (49%, 4.5% and 36%, respectively), in a book of recipes (Wahl 2004), analytical data, M:P 30 a rather moderate drying loss of 13% causes a low ratio, etc., are given for weight (drying) losses of 5, 10, 31 and 15% (spreadable) and 10, 20, and 30% (sliceable) final aw value, which in combination with a pH value 32 of 4.7–4.9 ensures a good microbial stability and to the same named products supporting the view men- 33 safety. tioned earlier concerning a wide range of semidry 34 South Jutlandic salami has a caliber of 55–60 mm, sausages depending only on the extent of drying. 35 Because of text restrictions, only some of the most the final pH is 4.7, the aw value is 0.90–0.95. Pro- 36 duced with pork, beef, and pork fat and 3–3.5% salt, typical products are discussed below. 37 sugar, spices, and nitrite, the meat is pre-cured for 24 38 hours in salt. Generally used starter cultures are Sliceable Sausages 39 L. plantarum, P. cerevisiae, and Micrococci, often in Schinkenplockwurst, coarse granulation, caliber 40 combination. Fermentation and drying starts simi- 70–90 mm, smoked, dried (M:P ratio if 20% drying 41 larly at 24°C and 94–95% RH, which are decreased loss ϭ 2.3:1, with 30%ϭ1.71). 42 gradually. After the pH drop, the products are Schlackwurst, finely chopped, stuffed in bung, 43 smoked. Being a traditional Danish product, this type smoked, dried (M:P ratio with 20% drying loss ϭ 44 is manufactured both by small and large enterprises. 2.2:1, with 30% ϭ 1.6:1). 45 Schinkenmettwurst, coarse granulation, pork meat 46 France only, natural casing, air-dried (M:P ratio with 20% 47 If we go southward, sausages with higher moisture drying loss ϭ 2.5:1, with 30% ϭ 1.9:1). 48 content (semidry products) become less and less Air-dried Mettwurst, coarse granulation (M:P 49 known and less popular; fully dried products (dry ratio with 20% drying loss ϭ 2.4:1, with 30% ϭ 50 sausages) on the other hand represent the definite 1.9:1). Mettwurst is a collective designation includ- 51 majority of such types. French semidry sausages are ing several types under different names. 52 only moderately dried, 10% weight loss being an Mettenden, medium-size granulation, pork intes- S53 average; in some of them a starter culture is used, tine or collagen casing smoked, dried (M:P ratio N54 40454 29 307-318 r2.qxd 7/20/07 3:56 PM Page 316

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1 with 10% drying loss ϭ 3:1, with 20% ϭ 2.5:1, with have been developed, practically all of them are raw 2 30% ϭ 1.9:1). fermented and dried. Semidry varieties fulfill the 3 safety requirements: a Ͻ 0.95 and pH Ͻ 5.3 at the In addition to a type-specific seasoning mixture, a w 4 starter culture, garlic, and MSG are added to practi- same time. Natural as well as artificial casings (small 5 cally all these sausages. intestine of pork, fibrous cellulose and collagen cas- 6 ing, respectively) are used with 30–65 mm. 7 Spreadable Sausages Black and white pepper, garlic, and caraway are 8 Teewurst, finely chopped, caliber up to 45 mm, common spices used. Most of these sausages contain 9 smoked, moderately dried (M:P ratio if 5% drying mild or hot or mixed paprika up to 1–2%. Starter 10 loss ϭ 3.2:1, with 10% ϭ 2.8:1). cultures and nitrite salt (2.4–2.5%) are added to the 11 Mettwurst, finely chopped, caliber same as above, meat batter, and all the sausages are smoked 2–4 12 smoked, moderately dried (M:P ratio similar to hrs/day after drying the surface for some days. Incu- 13 above). bation temperature: 20–22°C, RH starting with 14 Coarse Mettwurst, caliber same as above, smoked, 92–94%, decreasing parallel with temperature to 15 moderately dried, with garlic (M:P ratio if 5% drying about 80% and 13–14°C, respectively. Mold growth 16 loss ϭ 2.5:1, with 10% ϭ 2.3:1). on the surface is undesired. 17 Schmierwurst, finely chopped, caliber as above, Some typical products that are (were) produced: 18 smoked and moderately dried, with paprika (M:P ratio sausage of Kunság (kunsági kolbász), vine dresser 19 with 5% drying loss ϭ 3.5:1, with 10% ϭ 3.0:1). sausage (vincellér kolbász), Hercules salami, “thick” 20 All these spreadable sausages are produced with paprika sausage (paprikás vastagkolbász), Goliath 21 starter cultures in order to decrease the pH value, an sausage, Bajaer deli sausage (Bajai csemegekolbász), 22 absolute necessity because of a short drying period Bácskaer hot sausage (Bácskai csÌpös kolbász),

23 and relatively high aw. Spreadable sausages are Kulen, Stifolder, etc. All these are sliceable products 24 stored usually chilled in retail shops, though it is also in small intestine of pork, in cecum (Kulen), or in 25 suggested (Leistner, personal communication) that artiﬁcial casings of 30–80 mm with a ﬁnal moisture 26 storage at ambient temperature helps in reducing or of 32–38%. Spreadable sausage is not manufactured 27 eliminating pathogenic bacteria (Salmonella) in Hungary, even though earlier several trials with 28 through metabolic exhaustion, a phenomenon that good products have been launched; because of lack 29 does not work at low temperatures. of consumers’ acceptance they failed. 30 Hot smoked (lángolt kolbász) products, on the Hungary 31 other hand, are very popular and are produced with 32 Semidry sausages are manufactured mainly from nitrite curing salt and spices, paprika dominating. 33 pork and pork fat. The classic Hungarian semidry After hot smoking, they may be dried or not, and 34 sausage, summer tourist (nyári túrista), has been pro- because neither starter culture nor GDL is used, 35 duced for more than 60 years similarly to the original these products with 10–15% weight loss are usually 36 technology. The coarsely chopped pork and pork fat, consumed in cooked form. 37 blended with curing salt, now nitrite salt, and season- Italy 38 ings, is stuffed into collagen casings of 60–70 mm, 39 warm smoked and cooked right after smoking, and In the birthplace of dried sausages, classic dry 40 then dried to a weight loss of about 10–20%. Because sausages are still most popular, even if, unlike origi- 41 starter culture or chemical acidulant has never been nally, starter cultures now are extensively used inside 42 used, and the pH of the product (5.6–5.7) would have the meat batter and outside (mold starter). If the pH 43 no inhibitive effect against spoilage and pathogenic drops below 5.3, drying can be stopped earlier than

44 microbes, cooking ensures a fair microbial stability if dry sausage is aimed at, before the aw reaches 45 and safety even at aw values of 0.95–0.96 by inacti- 0.90: such can be the case, e.g., with Salame friu- 46 vating these microorganisms. A similar product, lano. In addition to salamis and raw dry sausages, 47 though with paprika and smaller diameter, Transyl- several of them with mold cover—and in addition to 48 vanian tourist (erdélyi túrista), was produced earlier raw sausages that are dried to less weight loss and 49 in larger volume. Farmer sausage (Farmer kolbász) meet the requirements of semidry products—a very 50 was originally manufactured for export purposes; it famous Italian sausage Mortadella can be consid- 51 is a cooked and dried product that now is also pro- ered as a semidry product that contains visible fat 52 duced for the domestic market. cubes in ﬁnely ground meat batter. 53S Three decades ago, starter culture technology was Stuffed in large diameter casing of several hun- 54N introduced in Hungary, too, and many different types dred mm, mortadella has to be heat-treated for many 40454 29 307-318 r2.qxd 7/20/07 3:56 PM Page 317

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hours, during which a remarkable cooking loss takes collagen casing, they are smoked and dried to a total 1 place. This way, Mortadella has a low initial micro- weight loss of 12–17%. The following are typical 2 bial count, having killed most undesired microorgan- products. 3 isms, and a good shelf life at ambient temperature, Polish smoked sausage contain spices black pep- 4 proven by the fact that these products are kept with- per, marjoram, and garlic. Different-quality pork is 5 out refrigeration in the shops. These sausages have ground, stuffed in pork small intestine, paired, 6 nothing to do with so-called “mortadella” that are smoked at 25–30°C to brown color, and dried at 7 produced in other countries and have a similar tech- 18–20°C, 70–80% RH until a 12% weight loss. 8 nology to Bologna-type sausages. Smoked frankfurters are made from pork and beef, 9 Italian dry and semidry sausages are made mostly nitrite salt, nutmeg and black pepper, and starter cul- 10 of pork, in some cases pork and beef (Salame Napoli), ture; stuffed in sheep or edible collagen casing (22 11 and pork fat with salt and black pepper—some with mm), and smoked and dried at 15–18°C, 80–85% 12 garlic, red pepper, or dill seed. They are stuffed in nat- RH until a 15% weight loss. 13 ural casings 40–70 mm, with some exceptions Semidry cervolat is made of pork and beef, paprika 14 (Napoli) not smoked, and ripened-dried for 25, 40, or in addition to black pepper, garlic, and coriander, and 15 45 days (Salciccia, Soppressata, and Friulano, curing salt; it is stuffed in 60 mm collagen casing, 16 respectively). If they are ripened longer, weight loss is and smoked and dried to a yield of 83%. 17 higher and they can be considered dry sausages. 18 19 The Netherlands ACKNOWLEDGEMENT 20 Dutch semidry sausages are manufactured from Sincere thanks are due to F. Bauer, L. Cocolin, B.-J. 21 pork and/or beef, and in some products with cooked van’t Hooft, W. K. Jensen, A. Pisula, E. Puolanne, 22 pork rind. Pork fat, starter culture, (mixed lacto- and J.-L. Vendeuvre for the information on national 23 bacilli and Micrococci) carbohydrate, nitrite salt, semidry product types. 24 and spices like pepper, cardamom, coriander, and 25 garlic are comminuted and blended in a bowl chop- REFERENCES 26 per at −1 to –5°C to a particle size between 1 and 10 27 mm, stuffed mostly in cellulose or collagen casing, Anonymous. 2006. Technologie spielt Schlüsselrolle. 28 incubated (fermented) until pH drop, smoked during Fleischwirtschaft 1:43–44. 29 fermentation and dried afterward. During fermenta- JW Austin. 2001. Clostridium botulinum. In: MP 30 tion, color formation, acidification, and gelation Doyle, LR Beuchat, TJ Montville, eds. Food Micro- 31 take place. During fermentation and drying tempera- biology. Fundamentals and Frontiers. Washington, 32 ture, RH and air velocity is controlled, initial tem- D.C.: ASM Press, pp. 329–349. 33 perature of 25°C is gradually decreased to 18°C, and F Bauer. 2004. Residues in meat and meat products. 34 RH is reduced from 95% to below 85%. A detailed Residues associated with meat production. In: WK 35 description of the technology is given by van’t Hooft Jensen, C Devine, M Dikeman, eds. Encyclopedia of 36 (1999). The most popular Dutch products are Meat Sciences. London: Elsevier Ltd. Academic 37 Salami, finely chopped; Cervelat, finely chopped, Press, pp. 1187–1192. 38 with higher fat content (35–40%); Snijworst, finely F Bauer, P Paulsen. 2001. Biogenic amines in meat and 39 chopped, with higher fat content (35–45%) and rind meat products. In: DML Morgan, V Milovic, M 40 added; Farmersmetwurst, coarsely chopped; and Krizek, A White, eds. COST 917—Biogenically 41 Chorizo (less spicy than the Spanish product). These Active Amines in Food. Luxembourg: Office for 42 products are ripened-dried for 2–3 weeks resulting Official Publications of the European Commission, 43 in a weight loss of about 12–20%. Water activity pp. 88–94. 44 drops to 0.92–0.95, pH drops to 4.5–5.2. H Blom, BF Hagen, BO Pedersen, AL Holck, L 45 Semidry sausages, also with reduced fat content Ayelsson, H Naes. 1996. Accelerated production of 46 (18% fat), are produced. Snack size Farmersmetwurst dry fermented sausage. Meat Sci 43:229–242. 47

is dried to a lower aw and therefore considered dry T Bolumar, Y Sanz, M Flores, M-C Aristoy, F Toldrá, 48 sausage. J Flores. 2006. Sensory improvement of dry- 49 fermented sausages by the addition of cell-free 50 Poland extracts from Debaryomyces hansenii and Lacto- 51 Semidry sausages are cooked and dried, made from bacillus sakei. Meat Sci 72:457–466. 52 pork or mixed with beef, with starter culture, carbo- G Campbell-Platt. 1995. Fermented meats—A world S53 hydrate, and nitrite salt. After stufﬁng in a natural or perspective. In: G Campbell–Platt, PE Cook, eds. N54 40454 29 307-318 r2.qxd 7/20/07 3:56 PM Page 318

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1 Fermented Meats. London: Blackie Academic & CF Niven, RH Deibel, GD Wilson. 1959. Production of 2 Professional, pp. 46–47. fermented sausages. US Patent 2, 907, 661. 3 D Demeyer, F Toldrá. 2004. Fermentation. In: WK Z Savic, I Savic. 2002a. Collagen casings. In: Sausage 4 Jensen, C Devine, M Dikeman, eds. Encyclopedia of Casings. Vienna: Victus Lebensmittelbedarf Ver- 5 Meat Sciences. London: Elsevier Ltd. Academic triebsgesellschaft, pp. 183–200. 6 Press. pp. 467–474. ———. 2002b. Cellulose casings. In: Sausage Casings. 7 G Effenberger. 1991. Wursthüllen. Kunstdarm. Bad Vienna: Victus Lebensmittelbedarf Vertriebsge- 8 Wörishofen: Hans Holzmann Verlag, pp. 33–34. sellschaft, pp. 215–238. 9 K Incze. 1998. Dry fermented sausages. Meat Sci JG Sebranek. 2004. Semidry fermented sausages. 10 49(Suppl 1):169–177. In: YH Hui, L Meunier-Goddik, ÅS Hansen, 11 ———. 2003. Ungarische Roh und Dauerwürste. J Josephsen, W-K Nip, PS Stanfield, F Toldrá, eds. 12 Fleischwirtschaft 11:30–34. Handbook of Food and Beverage Fermentation 13 ———. 2004. Dry and semi-dry sausages. In: WK Technology. New York: Marcel Dekker, Inc., pp. 14 Jensen, C Devine, M Dikeman, eds. Encyclopedia of 385–396. 15 Meat Sciences. London: Elsevier Ltd. Academic P Setlow, EA Johnson. 2001. Spores and their signifi- 16 Press, pp. 1207–1216. cance. In: MP Doyle, LR Beuchat, TJ Montville, eds. 17 L Kröckel. 1995. Bacterial fermentation of meats. Food Microbiology. Fundamentals and Frontiers. 18 In: G Campbell-Platt, PE Cook, eds. Fermented Washington, D.C.: ASM Press, pp. 33–70. 19 meats. London: Blackie Academic & Professional, ZE Sikorski. 2004. Traditional smoking. In: WK 20 pp. 69–109. Jensen, C Devine, M Dikeman, eds. Encyclopedia of 21 LF Leistner. 1995. Stable and safe fermented sausages Meat Sciences. London: Elsevier Ltd. Academic 22 world-wide. In: G Campbell–Platt, PE Cook, eds. Press, pp. 1265–1272. 23 Fermented Meats. London: Blackie Academic & R Talon, S Leroy-Sétrin, S Fadda. 2004. Dry fermented 24 Professional, pp. 160–175. sausages. In: YH Hui, L Meunier-Goddik, ÅS 25 ———. 2000. Basic aspects of food preservation by Hansen, J Josephsen, W-K Nip, PS Stanfield, 26 hurdle technology. Inter J Food Microbiol 55: F Toldrá, eds. Handbook of Food and Beverage Fer- 27 181–186. mentation Technology. New York: Marcel Dekker, 28 ———. 2005. European Raw Fermented Sausage: Inc., pp. 397–416. 29 Fundamental Principles of Technology and Latest F Toldrá. 2002. Dry-cured meat products. Trumbull, Con- 30 Developments. Proc 2005 International Symposium necticut: Food & Nutrition Press Inc., pp. 113–172, 31 on Fermented Foods. Chonbuk Natl Univ Jeonju, 221–231. 32 South Korea, pp. 29–39. K Ulmer, J Stankeviciute, M Gibis, A Fischer. 2006. 33 BM Lund, MW Peck. 2000. Clostridium botulinum. In: Reifezeitverkürzung bei schnittfester Rohwurst 2. 34 BM Lund, TC Baird-Parker, G Gould, eds. The Micro- Teil. Fleischwirtschaft 2:96–99. 35 biological Safety and Quality of Food. Gaithersburg B-J van’t Hooft. 1999. Development of binding 36 MD: Aspen Publishers, Inc., pp. 1057–1109. and structure in semi-dry fermented sausages. 37 MC Montel, F Masson, R Talon. 1998. Bacterial role in A multifactorial approach. Thesis. Universiteit 38 flavour development. Meat Sci 49(Suppl 1):111–123. Utrecht: Waageningen, Ponsen & Looyen, pp. 3–5, 39 FP Niinivaara. 1993. Geschichtliche Entwicklung des 109–132. 40 Einsatzes von Starterkulturen in der Fleischwirtschaft. W Wahl. 2004. Excellent German sausage recipes. 41 1. Stuttgarter Rohwurst Forum. Gewürzmüller, Bad Wörishofen: Holzmann Verlag GmbH & Co 42 pp. 9–20. KG, pp. 1.1–1.18, 2.1–2.10. 43 44 45 46 47 48 49 50 51 52 53S 54N 40454 30 319-326 r1.qxd 7/20/07 4:00 PM Page 319

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1 2 3 30 4 5 6 Dry-fermented Sausages:An Overview 7 8 Fidel Toldrá, Wai-Kit Nip, and Y. H. Hui 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION details on sausages produced in North America, the 22 Mediterranean area, and Northern Europe are pro- 23 Historically, the manufacturing procedures for fer- vided in three separate chapters in this part. Space 24 mented sausages have been adapted to the cli- limitation prohibits an in-depth discussion of fer- 25 matic conditions of the production area. For instance, mented sausages from other parts of the world. If 26 Mediterranean sausages have dried to low water interested, one can refer to the references provided at 27 activity values, taking advantage of long dry and the end of the chapter. 28 sunny days, whereas in Northern Europe fermented 29 sausages require smoking for further preservation 30 (Toldrá 2006). Summer sausage is another example FERMENTED SAUSAGES 31 traditionally produced in the summer and heated for WORLDWIDE 32 safety reasons (Zeuthen 1995). NORTH AMERICAN 33 Preservation is the result of a successive and 34 specific series of factors known as hurdle effects Manufacturing practices were brought to the United 35 (Leistner 1992). These hurdles follow a chain of States by the first European settlers. Today, many 36 events: typical European fermented sausages can be found 37 • Addition of nitrite, salt, and/or sugar in northern states, such as Wisconsin (Toldrá and 38 • Reduction of the redox potential Reig 2006). Lebanon bologna is a semidry-fermented 39 • Introduction of lactic acid bacteria sausage originally from Lebanon (Pennsylvania). 40 • Lowering pH It is produced entirely from beef, with the addition 41 • Decreasing water activity of black pepper, fermented to a very high pH and 42 • Smoking heavily smoked (Rust 2004). Pepperoni is produced 43 from pork and/or beef and seasoned with red pepper, 44 The sausage remains stable after this sequence of ground cayenne pepper, pimento, anise seed, and 45 hurdles (Leistner 1995). Lactic acid bacteria play garlic. This sausage has a small diameter and is 46 important roles in safety, nutrition, and sensory smoked. 47 quality (Toldrá et al. 2001). These microorganisms 48 develop important reactions essential for the devel- SOUTH AND CENTRAL AMERICAN 49 opment of adequate color, texture, and flavor 50 (Demeyer and Toldrá 2004). Details have been pre- There is a general Spanish and Italian influence on 51 sented in previous chapters. different typical fermented meat products in many 52 Some of the most important fermented sausages Latin American countries. This is the case of Italian 53 produced worldwide are listed in this chapter, with a Milano and Cacciaturi, which are consumed in many S54 short description for each type. However, more countries, such as Uruguay, Brazil, and Mexico. N55 321 40454 30 319-326 r1.qxd 7/20/07 4:00 PM Page 322

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1 MEDITERRANEAN juniper. It is dry-cured, smoked, cooked, and dried 2 for 3–5 days (Pisula 2004). There are many types of dry-fermented sausages 3 that have been produced for centuries in the Mediter- 4 ranean area. They are usually dried due to the partic- EASTERN EUROPEAN 5 ular climate and are rarely smoked. These types of 6 The Hungarian salami is a good example of a typical sausages depend on the diameter, shape, size, spices 7 salami, which is intensively smoked and then the and seasonings, and sensory characteristics. They 8 surface is inoculated with mold starters or sponta- receive different names according to the geographic 9 neous mold growth covering the surface (Incze origin; sometimes the names differ even between 10 2004). This salami is seasoned with white pepper, very close and small areas (Toldrá 2006). Pork is the 11 garlic, red wine, and paprika. Similar sausages main meat used, and fungi starters may be used for 12 are Winter salami, also produced in Hungary, and development on the external surface (Talon et al. 13 Hermannstädler produced in Romania (Roca and 2004). Salamis of medium diameter (around 6 cm) 14 Incze 1990). Russian salami and Moscow salami include French Menage, French Saucisson d’Alsace, 15 are produced in Russia from pork, and, optionally, Italian Turista, and Spanish Salchichón; those of 16 some beef. larger diameter such as French Varzi, Italian Milano, 17 and Italian Crespone may be ripened for more than 18 60 days (Toldrá 2004). Spanish chorizo, which has a MIDDLE EASTERN 19 strong red color, is seasoned with garlic, pepper, and 20 Fermented sausages are produced from many vari- oregano (Toldrá 2002). 21 eties of animals, such as beef, buffalo, mutton, lamb, 22 goat, camel, and horse meat in Middle Eastern coun- 23 tries. Pork meat is not used because of religious pro- NORTHERN EUROPEAN 24 hibition. Sausages, which can contain rice, wheat, 25 Many fermented meats are produced in North corn, and rice flour, are cured and smoked. Different 26 Europe, for example, the Greuβner salami is pro- flavors are given depending on the addition of olive 27 duced in Thuringia (Germany). It is a sliceable oil, garlic, cinnamon, onion, paprika, black pepper, 28 sausage produced from beef and some pork, and fat rosemary, etc. Fermented and strongly smoked beef 29 and flavored with garlic, pepper, and other spices. sausages were produced in Lebanon and expanded to 30 Production includes a long-term fermentation other countries (El Magoli and Abd-Allach 2004). 31 process and cold smoking. Another example is the Soudjouk sausages, using only beef, buffalo, and/or 32 Rügenwalder teewurst, a semidry-fermented sausage, mutton and fat-tailed sheep, are produced in Turkey 33 also produced from beef and pork, which is fer- (Gökalp and Ockerman 1985). These sausages may 34 mented and cold-smoked (Gibis and Fischer 2004). be heavily seasoned with spices and other ingredi- 35 The Austrian katwurst is another example of long- ents, including garlic, red and black pepper, cumin, 36 dried sausage. The Swedish metwursk contains some pimento, and olive oil. 37 potato in addition to spices and seasonings. Other 38 meats can be added to the formulation of sausages AFRICAN 39 in Scandinavian countries: for instance, horse meat 40 in farepolse, toppen, trondermorr, stabbur, and Biltong is a typical South African meat product. It is 41 sognekorr in Norway and kotimainen meetwurst in produced from young and lean carcasses from either 42 Finland; lamb meat in lambaspaeipylsa in Iceland; cattle or game, especially from areas like the round, 43 reindeer meat in poro meetwurst in Finland and loin, or tenderloin. The meat is ripened and dried 44 rallersnabb gilde in Norway (Campbell-Platt 1995). until losses exceed 50%. Salt, sugar, pepper, and 45 Several fermented sausages are produced in roasted ground coriander are added. Vinegar and salt- 46 Poland from pork and some beef, game, or poultry peter can also be used (Strydom 2004). Other typical 47 (Pisula 2004). Krakowscha sucha is produced from fermented and sun-dried products have been summa- 48 pork and beef with the addition of black pepper, rized by Campbell-Platt (1995). Most of them are 49 nutmeg, and garlic. These sausages are dry-cured, produced in Northeastern Africa. Miriss and Mussran 50 smoked, cooked, and dried for about 3 weeks. are made from fat surrounding a lamb’s stomach and 51 Kabanosy is produced from pork with addition of small intestine, respectively. Other similar products 52 black pepper, nutmeg, and caraway. It is smoked and are Twini-digla and Um-tibay. Beirta is made in the 53S dried for 3–5 days. Jalowcowa is produced from Sudan from goat meat with offal; Kaidu-digla is 54N pork and a little beef with the addition of pepper and made from chopped bones; and Dodery, Mulaa 40454 30 319-326 r1.qxd 7/20/07 4:00 PM Page 323

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el-sebit, and Aki-el-Muluk are made from crushed quality all year round. However, the traditional pro- 1 bones, marrow, and fat. cedure is still practiced in the rural areas of China. 2 Aap Gon Cheong: Aap (duck) Gon (liver) Cheong 3 (sausage) is a specialty Cantonese product made in a 4 EAST ASIAN similar manner as the Lap Cheong, with the cut-up 5 Most information on fermented sausages for East duck liver replacing the pork. The amount produced 6 Asian countries has been derived from recipe books is small because of less availability of the duck liver. 7 in both Chinese and English. The English references Traditionally, soy sauce is one of the main ingredi- 8 include Aidells et al. (2000), Campbell-Platt (1995), ents in the making of duck liver sausage and con- 9 Inglis et al. (1998), Leistner (1995), Rogers (2003), tributes to a special flavor. 10 Solomon (2002), and Trang (2006). Although their Gam Ngan Cheong: Gam (gold) Ngan (silver) 11 descriptions here do not detail the fermentation stage, Cheong (sausage) is a very special Cantonese prod- 12 most East Asian sausages require short- or long-term uct, which does not use intestine or casing as the 13 fermentation during the manufacturing processes. “container.” A chunk of pork fat is cut into a wedge 14 shape and wrapped with thin slices of pork liver, 15 Chinese Sausage which has been marinated with salt and sugar. The 16 Lap Cheong (La chang): The name Lap Cheong is a Gam Ngan Cheong is then dried by the natural 17 general term for Chinese sausages, but it can also be process the same way Lap Cheong is cured. Because 18 used to describe Chinese pork sausages. Literally, it of concern about cholesterol and fat intakes, this 19 means winter (lap) intestines (cheong or sausage), product is less popular today. 20 which can be interpreted as intestines stuffed in the Chicken liver sausage w/wo pig liver: This is a 21 winter. Traditionally, Lap Cheong is made in the modification of the Aap Gon Cheong, with chicken 22 winter months to take advantage of the lower tem- and/or pig liver replacing the duck liver. 23 peratures, which reduce the chance of spoilage dur- 24 Singaporean Sausage ing curing after the sausages are stuffed. The 25 ingredients used to make Lap Cheong in China vary Singaporean sausages are similar to Chinese 26 from place to place, but basically they are cut-up sausages. This is understandable because the major- 27 pork and pork fat (now they are ground-up pork and ity of the population in Singapore is of Chinese eth- 28 fat), sugar, and salt, with optional ingredients such nic origin. They produce sausages such as Special 29 as soy sauce, alcoholic beverages, spices, and others. grade (less fat) pork sausage, Chicken sausage, and 30 The amount of pork fat used also varies depending Pig liver sausage. 31 on the types of Lap Cheong, with regular and low-fat 32 Thai Sausage types more common now. In the old days, when the 33 intestines or casings were stuffed at the beginning of Sai Ua (a dried Northern Thai sausage): This Thai 34 the winter season, they were hung with a string in a sausage is made by stuffing pork with Thai curry 35 ventilated area to gradually dry up the ingredients paste (onion, galangal, lemon grass, parsley’s root, 36 and the surface, and also to produce the typical fla- curcuma, chili, and salt mashed in shrimp paste) into 37 vor (odor, color) and texture. With the decrease in a pork casing. It is dried and roasted before con- 38 water activity and moisture content, the product sumption. 39 finally hardened and could be kept edible (after Northeastern sour Thai-style sausage: This sour 40 cooking) for the winter and spring months. It was Northeastern Thai sausage is made with ground 41 not uncommon to have mold and yeast development pork, cooked rice, nitrite, erythorbate, pepper, salt, 42 on the surface of the dried product when the relative and sugar. After stuffing the mixture into a pork cas- 43 humidity was high. To extend the shelf life, some ing, it is kept at room temperature for about 24 hours 44 people also stored small amounts of the dried prod- to allow lactic acid fermentation. The sour sausage 45 ucts in oil in a sealed container in a cool place. This requires thorough cooking, such as roasting or fry- 46 practice could keep the product available for the ing before consumption. 47 summer and even the autumn months. For several Nham (Thai fermented sausage): This Thai 48 decades, industrial production of Lap Cheong has sausage is made similarly to the making of North- 49 been modified by curing or drying the green sausage eastern Thai sour sausage except that chili and pork 50 in temperature- and humidity-controlled dryers to skin are also added and they are packed in a bamboo 51 speed up the process. The products are also now leaf or plastic film. After keeping (fermenting) for 52 packed in vacuum-sealed pouches with recommen- 3–4 days at room temperature, they are then ready S53 dations to store them in the refrigerators to maintain for cooking and consumption. Currently, some N54 40454 30 319-326 r1.qxd 7/20/07 4:00 PM Page 324

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1 manufacturers apply irradiation treatment to kill par- IMPORTANCE OF FERMENTED 2 asites and ensure safety. SAUSAGES 3 Goon Chiang: This Thai sausage is made first by 4 marinating the pork with nitrite at refrigerating tem- Fermented sausages are very popular with most pop- 5 perature for 24 hours, followed by grinding and mix- ulation groups—such as American, Chinese, Span- 6 ing with sugar and erythrobate before stuffing into a ish, German, and so on—who consume meat. 7 pork casing. It is dried at 60°C to appropriate dryness. Science and technology have played an important 8 This sausage requires cooking before consumption. role in better quality and storage time for fermented 9 sausages, especially in Europe, and Central, South, Filipino Sausage 10 and North America. However, the development and 11 Longamisa: Longamisa is a sweet sour sausage production processes for fermented sausages in 12 made at the rural level using lean pork, pork fat, countries such as China, Hong Kong, and Taiwan are 13 white vinegar, soy sauce, and sugar as ingredients. still an art more than a science. 14 After stuffing, it can be smoked or cooked fresh. Most of us like fermented sausages, and this type 15 of processed meat products has been and will con- Korean Sausage 16 tinue to be significant in our diets. 17 Sundae: This popular street-vendor Korean stuffed 18 sausage is made with pig’s blood, rice, green onions, 19 garlic, minced pork, and sweet potato vermicelli, ACKNOWLEDGMENT 20 stuffed into small and large pig’s intestines. It is The authors thank their personal friends and col- 21 steamed before consumption. leagues in East Asian countries for contributing sig- 22 Soonday: This is also a popular Korean stuffed nificant information on sausages associated with 23 sausage sold at public markets. The stuffing consists their native countries. 24 of firmly cooked rice, crushed garlic and cloves, 25 crushed fresh ginger, black or white pepper, Korean 26 sesame oil, crushed sesame seeds, crushed scallions, REFERENCES 27 and either beef or pork blood. The mixture is stuffed 28 into small beef intestines. The sausages are cooked B Aidells. 2000. Bruce Aidells’s Complete Sausage 29 in water before consumption. Book. Berkeley, California: Ten Speed Press, 314 pp. 30 G Campbell-Platt. 1995. Fermented meats—A world Nepalese Sausage 31 perspective. In: Fermented Meats. G Campbell-Platt, 32 Nepalese sausage is similar to German sausage PE Cook, eds. London: Blackie Academic & Profes- 33 except chicken is used as the main meat ingredient. sional, pp. 39–51. 34 D Demeyer, F Toldrá. 2004. Fermentation. In: Encyclo- Sri Lankan Sausage 35 pedia of Meat Sciences. W Jensen, C Devine, M Dike- 36 Sri Lankan sausage is made with lean pork, pork fat, mann, eds. London: Elsevier Science, pp. 467–474. 37 toasted and ground coriander seeds, ground cinna- SB El-Magoli, MA Abd-Allach. 2004. Ethnic meat 38 mon, ground clove, ground black pepper, finely products: Middle East. In: Encyclopedia of Meat 39 grated nutmeg, salt, and vinegar. After stuffing, they Sciences. W Jensen, C Devine, M Dikemann, eds. 40 are cold smoked at temperature not higher than 30°C London: Elsevier Science, pp. 453–455. 41 for a few hours to appropriate dryness. They require M Gibis, A Fischer. 2004. Ethnic meat products: 42 proper cooking before consumption. Germany. In: Encyclopedia of Meat Sciences. 43 Countries such as India, Indonesia, Japan, and W Jensen, C Devine, M Dikemann, eds. London: 44 Malaysia do not have specialties such as fermented Elsevier Science, pp. 444–451. 45 sausages, although ethnic groups within these coun- HY Gökalp, HW Ockerman. 1985. Turkish-style fer- 46 tries have their ethnic heritage sausages, such as mented sausage (soudjouk) manufactured by adding 47 Chinese, Thais, and so on. However, European-style different starter cultures and using different ripening 48 sausages are now produced in Japan and India. temperatures. Fleischwirtschaft 65:1235–1240. 49 HF Ho. 2004. Ethnic meat products: China. In: Ency- 50 clopedia of Meat Sciences. W Jensen, C Devine, PACIFIC RIM 51 M Dikemann, eds. London: Elsevier Science, 52 Pepperoni is produced in Australia. Vento salami is pp. 441–444. 53S also produced in Australia from beef with pepper- K Incze. 2004. Mold-ripened sausages. In: Handbook 54N corns and red wine (Campbell-Platt 1995). of Food and Beverage Fermentation Technology. 40454 30 319-326 r1.qxd 7/20/07 4:00 PM Page 325

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YH Hui, LM Goddik, J Josephsen, PS Stanﬁeld, AS C Devine, M Dikemann, eds. London: Elsevier Sci- 1 Hansen, WK Nip, F Toldrá, eds.New York: Marcel ence, pp. 440–441. 2 Dekker Inc., pp. 417–428. R Talon, S Leroy-Satrin, S Fadda. 2004. Dry fermented 3 K Inglis, G Francione, L Invernizzi. 1998.Tropical sausages. In: Handbook of Food and Beverage 4 Asian Style. North Clarendon, Vermont: Periplus Fermentation Technology. YH Hui, LM Goddik, 5 Editions, 223 pages. J Josephsen, PS Stanﬁeld, AS Hansen, WK Nip, 6 L Leistner. 1992. The essentials of producing stable and F Toldrá, eds. New York: Marcel Dekker, Inc., 7 safe raw fermented sausages. In: New Technologies pp. 397–416. 8 for Meat and Meat Products. JM Smulders, F Toldrá, F Toldrá. 2002. Dry-cured Meat Products. Trumbull, 9 J Flores, M Prieto, eds. Nijmegen, The Netherlands: Connecticut: Food & Nutrition Press, pp. 63–88. 10 Audet, pp. 1–19. ———. 2004. Ethnic meat products: Mediterranean. 11 ———. 1995. Stable and safe sausages world-wide. In: Encyclopedia of Meat Sciences. W Jensen, 12 In: Fermented Meats. G Campbell-Platt, PE Cook, C Devine, M Dikemann, eds. London: Elsevier Sci- 13 eds. London: Blackie Academic & Professional, ence, pp. 451–453. 14 pp. 161–175. ———. 2006. Meat fermentation. In: Handbook of 15 FK Lücke. 1985. Fermented sausages. In: Microbiol- Food Science, Technology and Engineering. YH Hui, 16 ogy of Fermented Foods. BJB Wood, ed. London: E Castell-Perez, LM Cunha, I Guerrero-Legarreta, 17 Elsevier Applied Science, pp. 41–83. HH Liang, YM Lo, DL Marshall, WK Nip, 18 A Pisula. 2004. Ethnic meat products: Poland. In: F Shahidi, F Sherkat, RJ Winger, KL Yam, eds. Boca 19 Encyclopedia of Meat Sciences. W Jensen, C Raton, Florida: CRC Press, volume 4, pp. 181-1– 20 Devine, M Dikemann, eds. London: Elsevier Sci- 181-12. 21 ence, pp. 456–458. F Toldrá, M Reig. 2006. Sausages. In: Handbook of 22 M Roca, K Incze. 1990. Fermented sausages. Food Food Product Manufacturing. YH Hui, ed. In press. 23 Reviews International 6:91–118. John Wiley & Sons. 24 J Rogers. The Essential Asian Cookbook, 2003. Berke- F Toldrá, Y Sanz, M Flores. 2001. Meat fermentation 25 ley, California: Thunder Bay Press, 304 pp. technology. In: Meat Science and Applications. YH 26 RE Rust. 2004. Ethnic meat products: North America. Hui, WK Nip, RW Rogers, OA Young, eds. New 27 In: Encyclopedia of Meat Sciences. W Jensen, C York: Marcel Dekker, Inc., pp. 537–561. 28 Devine, M Dikemann, eds.. London: Elsevier Sci- C Trang. The Asian Grill: Great Recipes, Bold Flavors. 29 ence, pp. 455–456. 2006. San Francisco: Chronicle Books, 168 pp. 30 C Solomon. The Complete Asian Cookbook. 2002. P Zeuthen 1995. Historical aspects of meat fermenta- 31 North Clarendon, Vermont: Tuttle Publishing, 512 pp. tions. In: Fermented Meats. G Campbell-Platt, PE 32 PE Strydom. 2004. Ethnic meat products: Africa. Cook, eds. London: Blackie Academic & Profes- 33 In: Encyclopedia of Meat Sciences. W Jensen, sional, pp. 53–67. 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 S53 N54 40454 31 327-332 r1.qxd 7/20/07 4:04 PM Page 327

1 2 3 31 4 5 6 U.S. Products 7 8 Robert Maddock 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION DEFINITIONS 22 23 As with most meat processing endeavors, United In order to understand dry sausage production in the 24 States dry sausage production borrows heavily from United States, it is important to know the definitions 25 European knowledge and background, especially associated with American dry sausage production. 26 German and Italian (Pearson and Gillet 1996). In The United States Department of Agriculture 27 addition, true dried sausages are not especially com- (USDA) is responsible for the regulatory naming 28 mon in the United States marketplace, and are gener- and labeling of dried sausage. Some commonly used 29 ally produced by smaller, specialized manufacturers terms in United States sausage production and some 30 for specific ethnic markets (Price and Schweigert of the actual USDA definitions of all sausage types 31 1987)—for example, Italian markets in the North- follow (in alphabetical order): 32 east and German in the Midwest. The annual produc- Acidulation: The process of reducing the pH by 33 tion of dried sausage in the United States is hard to the direct addition of organic acids. The majority of 34 estimate, but is likely less than 5% of total sausage acidulants in common use require the protection of 35 production. Essentially no large United States an encapsulation process. Because many different 36 sausage manufacturers produce a true dried sausage. encapsulating agents are in use, adherence to the 37 In fact, in the United States it is uncommon to find supplier’s specific recommendations is required. 38 dried sausage in a typical supermarket or grocery Approved source: A source that has been deter- 39 store. Some sausages commonly thought of as dried, mined to conform to principles, practices, and stan- 40 such as pepperoni or salami, are easy to find, but the dards that protect public health. 41 typical United States manufacturing process for Casings: The natural animal stomachs, intestines, 42 these sausages involves a cooking and smoking step, or bladders, or manufactured casings of cellulose or 43 making them more similar to a semidried sausage collagen, which are used to contain comminuted 44 than to a dried. Adding a cooking step to dried meat or poultry mixtures for sausages. 45 sausage manufacturing has been done in response to Cooked sausages and/or smoked sausages: Saus- 46 the many United States government regulations and ages that are chopped or ground, seasoned, cooked, 47 definitions associated with dried sausage produc- and/or smoked and come in various shapes and sizes. 48 tion. These regulations have resulted in many The most common of all United States sausage prod- 49 processors either discontinuing their production of ucts, they include sausages such as cotto salami, liver 50 dried sausages or adding a cooking step to their tra- sausage, wieners, bologna, knockwurst, and others. 51 ditional sausage production, resulting in semidried Degree-hours: Degrees are measured as the 52 sausage rather than a true dried sausage. excess over 60°F (15.6°C) (the critical temperature 53 S54 N55 327 40454 31 327-332 r1.qxd 7/20/07 4:04 PM Page 328

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1 at which staphylococcal growth begins). Degree- mixture to lactic acid, and thus lower the pH. Once 2 hours are the product of time at a particular tempera- the pH reaches 5.3 or less, the environment for 3 ture and the “degrees.” Degree-hours are calculated Staphylococcus aureus and other pathogenic 4 for each temperature used during fermentation. The microbes growth is effectively controlled if the 5 limitation of the number of degree-hours depends process continues lowering the pH to a more stable 6 upon the highest temperature in the fermentation value or the process begins drying the product at a 7 process prior to the time that a pH of 5.3 or less is low temperature. However, reducing pH to 5.3 is 8 attained. Processes attaining less than 90°F (32.2°C) insufficient to destroy Escherichia coli O157:H7 9 prior to reaching pH 5.3 are limited to 1200 degree- without a cooking step in the sausage-making 10 hours; processes exceeding 89°F (31.7°C) prior to process (Muthukumarasamy and Holley 2007). Fer- 11 reaching pH 5.3 are limited to 1000 degree-hours, mentation to a pH of Յ4.6 at 90°F (32.2°C) or 110°F 12 and processes exceeding 100°F (37.8°C) prior to (43.3°C) requires further holding and potentially a 13 reaching pH 5.3 are limited to 900 degree-hours. heat process in order to destroy E. coli O157:H7 if it 14 Dry sausage: A sausage made of chopped or were present. During fermentation of sausages to a 15 ground meat that may or may not be characterized by pH of 5.3, it is necessary to limit the time during 16 a bacterial fermentation. Dried sausages are always which the sausage meat is exposed to temperatures 17 stuffed into casings and started on a carefully con- exceeding 60°F (15.6°C). 18 trolled air-drying process. Some dry sausage is Fermentation culture: An active culture of one or 19 given a light preliminary smoke, but the key produc- more bacteria, which affects the rapid development of 20 tion step is a relatively long, continuous air-drying a lower pH in dry and semidry-fermented sausages. 21 process. When fermented, the intentional encourage- Fresh sausage: A sausage made of fresh, uncured 22 ment of a lactic acid bacteria growth is useful as a meat, generally cuts of fresh pork, and sometimes 23 meat preservative as well as producing the typical beef. When ice or water is used to facilitate chopping 24 tangy flavor. The meat ingredients, after being mixed and mixing, it is limited to a maximum of 3% of the 25 with spices and curing materials, are generally held total formula. Product must be kept refrigerated and 26 for several days in a curing cooler. As a result of bac- is cooked before consumption. Bratwurst, “Italian” 27 terial action, a pH of 5.3 or less is obtained and the sausage, whole-hog, and most pork sausages, are 28 sausage is then dried to remove 25–50% of the mois- common examples. 29 ture to yield a moisture/protein ratio in compliance Identifiable source: Can include the name and 30 with USDA Food Safety Inspection Service (FSIS) address of the immediate supplier and the actual 31 requirements. Medium-dry sausage is about 70% of source or location of the supplies. 32 its “green” weight when sold. Less dry and fully Moisture/Protein Ratio (MPR): The ratio of mois- 33 dried sausage ranges from 80–60% of original ture content to protein content in the sausage. 34 weight at completion. According to the USDA, dry Potentially hazardous food (PHF): A food that is 35 sausage must have a Moisture Protein Ratio (MPR) natural or synthetic and that requires temperature 36 of 1.9:1 or less. Nonrefrigerated, semidry, shelf- control because it is in a form capable of supporting 37 stable sausage must have an MPR of 3.1:1 or less the rapid and progressive growth of infectious or 38 and a pH of 5.0 or less, unless commercially steril- toxigenic microorganisms; the growth and toxin pro- 39 ized. Some of the dry sausages produced in the duction of Clostridium botulinum. 40 United States are chorizo (Spanish, smoked, highly Semidry-fermented sausage: A sausage made of 41 spiced), frizzes (similar to pepperoni but not chopped or ground meat that, as a result of fermenta- 42 smoked), pepperoni (not cooked, air-dried), Lola or tion, reaches a pH of 5.3 or less and undergoes up to 43 Lolita and Lyons sausage (mildly seasoned pork 15% removal of moisture during the fermentation/ 44 with garlic), salami (Italian; many different types of heating process. Examples of semidry-fermented 45 “salami” are made; most are usually made from pork sausages made in the United States include summer 46 but may have a small amount of beef and are sea- sausage, thuringer, cervelat, and Lebanon bologna. 47 soned with garlic), summer sausages including Trichina treatment: A defined method to render 48 cervelats (may be fully cooked and dried after cook- food products, i.e., pork and game meat products, 49 ing or fermented and dried), and landjäger (Ameri- free of the Trichinella spiralis parasite. 50 can landjäger is often fully cooked). Uncooked smoked sausage: A unique product that 51 Fermentation: That part of the process in which has characteristics of fresh sausage, but is smoked 52 lactic acid producing bacteria (from the starter without the addition of heat (cooking), resulting in a 53S culture or “mother” culture) convert fermentable product with a different flavor and color. 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and requires prominent labeling for consumers, modern collagen and fibrous casing will provide simi- 1 because the product can look fully cooked. “Smoked lar performance as natural casing. The shelf life of dry 2 Pork Sausage” is included in this class. sausages is excellent, attributed to the high salt: mois- 3 ture ratio and a lower than optimal pH for bacterial 4 growth. Dried sausages can be kept without refrigera- 5 UNITED STATES tion, but are commonly refrigerated by consumers 6 MANUFACTURING PROCESSES after purchase. Raw sausages, which are not submitted 7 FOR DRIED SAUSAGES to the smoking process, are known as air-dried 8 sausages and often have a highly attractive appearance 9 The properties of most American dry sausages and a yeasty-cheesy flavor. Air-dried sausages are 10 depend upon the products of bacterial fermentation marked with or without mold overlay and are more 11 and by biochemical and physical changes occurring commonly produced in the northeastern United States. 12 during the long drying or aging process. Many pro- American dry sausages can be sold as new dry 13 ducers will use starter cultures in the manufacturing sausages (about 20% weight loss from original 14 of dry sausages; however, it is still common to use weight), moderately dry sausages (about 30% weight 15 bacteria existing in the meat as a source for fermenta- loss) and dry sausages (about 40% weight loss), 16 tion, even though this practice is discouraged by many depending upon the market. Drying or aging is a key 17 experts and inspectors. Production time depends upon operation, especially in dry sausage production. The 18 many factors including possible smoking, drying most critical point in drying is to avoid the pro- 19 period, diameter and physical properties of casings, nounced surface coagulation of proteins and the 20 sausage formulation, choice and methods of prepar- formation of a surface skin. If the sausages lose 21 ing meat, conditions of drying, etc. Often, overall moisture too rapidly during the initial stages of the 22 processing time may require up to 90 days. Also, the drying period, the surface becomes hardened and a 23 final pH of dry sausages is usually somewhat higher crust or ring develops immediately adjacent to the 24 (5.0–5.5) than that of semidry sausages, and can casing. This hardened ring inhibits further loss of 25 increase during the aging process. moisture and the sausage has an excessively moist 26 American dry sausages are mainly coarsely or center. Only a sufficiently wet and soft casing, a high 27 moderately chopped (the majority of small-diameter relative humidity at the outset of the drying opera- 28 dry sausages) and very occasionally finely chopped. tion, and the use of a lower relative humidity in the 29 Some varieties of dry sausages are cold smoked advanced stages of the process will permit moisture 30 (54–64°F, 12–18°C) but sometimes they are not; in to migrate from the interior of the sausage into the 31 some countries, dried sausage may be heavily outer layer. Thus, the sausage should dry from the 32 spiced, however, United States dried sausages tend inside outward. If the outer layers of the sausage 33 to be milder, and they usually have less smoked fla- become hard, the diffusion of water will be inhibited 34 vor and salt than found in European sausages. In and the sausage tends to spoil. 35 principle, American dried sausages are processed by Unlike much American sausage manufacturing, 36 long, continuous air-drying, sometimes after a com- the production of dried sausage is still somewhat 37 paratively short period of smoking. It is important to “low tech.” It is not uncommon for much variation in 38 note that processing of dried sausages can be as var- the steps, conditions, and drying of the sausage. At 39 ied as the production of any other sausage type. the start of the drying or the drying and smoking 40 Some production systems utilize a long period of process, relative humidity can be as high as 98%, but 41 moderate temperatures (90–120°F, 32–49°C) and can vary depending upon the sausage being pro- 42 light smoke to promote fermentation and flavor duced. In the following 2–4 days the relative humid- 43 development. These extremely smoky sausages are ity must gradually but slowly be reduced. Too much 44 more common in the upper Midwest of the United humidity in the drying room favors the development 45 States and are usually marketed as “summer of mold and sliming of products. Dried sausage from 46 sausages” versus the heavier-spiced dried sausages the midwestern and western United States will usu- 47 found in many ethnic communities that are often ally not be displayed with visible mold. However, 48 referred to collectively as “salamis.” other areas do not object to a light white surface 49 American dry sausages are stuffed in both natural mold at the beginning of the drying process. It is 50 and artificial casings of varying diameters, with 2.5 believed that mold contributes to the specific flavor 51 in (60 mm) and larger being common. Natural cas- of some products. 52 ings are preferred by many because they adhere In the traditional production process of dried S53 closely to the sausages as sausages shrink. 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1 flora or a starter culture. In order to achieve safe fer- Table 31.1. Formulation for U.S. hard salami, 2 mentation of the raw sausage, it is important to give naturally fermented. 3 the microflora the proper growth conditions. Types Ingredients kg g 4 of bacteria used can sometimes be controlled by 5 the incubation times and temperatures used. For Beef chuck 18 6 example, the raw sausage mixture, containing meats, Pork jowl 18 7 curing salts, and sugar, can be placed in 15–18 cm 8 deep pans and kept for 2–4 days at 37–39°F (3–4°C). Pork trimmings 9 9 After remixing, the mix is stuffed into casings and Salt 1.5 10 the drying process continued at 54–59°F (12–15°C) Sugar 0.7 11 with or without simultaneous smoking. A number of 12 alternative procedures are found in practice. White pepper 85 13 American dried sausage makers will use frozen Sodium nitrate 60 14 concentrated starter cultures rather than natural Garlic powder 10 15 microflora to improve the speed and consistency of 16 production. The addition of starter culture allows for 17 a shorter “greening” or microbial growth time. Table 31.2. Formulation for U.S. pepperoni, starter 18 When using starter cultures in current production culture added. 19 practices, desired acidity can be achieved within 24 20 hours at a high (95–106°F, 35–41°C) incubation Ingredients kg g 21 temperature. This is in contrast to traditional manu- 22 facturing processes, which often require up to 72 Beef chuck 14 23 hours to achieve the required microbial growth to Pork heart 7 24 lower pH. Pork trimmings 20 25 In general, American dried sausage manufacturers 26 will add sugar from 0.3–2.0% of the weight of the Pork cheek 4.5 27 raw product. If dextrose or other easily degradable Salt 1.5 28 sugars are used, acidification is fast and the amount Sugar 0.5 29 of sugar added should be somewhat lower. In opposi- Sweet paprika 340 30 tion, corn syrup solids, which are common in many 31 sausage recipes, must be added at higher levels. Ground pepper 170 32 Whole fennel 100 33 BASIC FORMULATIONS AND PROCESSES FOR Red pepper 100 34 SELECTED LARGE-DIAMETER DRIED SAUSAGES Sodium nitrite 7 35 36 Table 31.1 is an example of a formulation for a hard Commercial starter 37 salami made in the United States, naturally fer- culture 38 mented (no starter culture). The procedure follows. 39 5 minutes. Stuff into suitable sized casings (around 40 Procedure: 40 mm for natural, or No. 1 fibrous). Hold for 9–11 41 Grind lean meat through 30 mm plate and fat pork days at 39–43°F (4–6°C) and 60% relative humidity. 42 through a 60 mm plate. Mix all ingredients for about Transfer to green room at 50°F (10°C) and 70% rela- 43 5 minutes. Store in 25 cm deep trays for 48–96 tive humidity for 48 hours. Smoke for 24–60 hours 44 hours. Stuff into suitable-sized casings (sewed bungs at 100°F (38°C). Hold for approximately 21 days at 45 for natural, or No. 5 fibrous). Hold for 9–11 days at 54°F (12°C) and 70% relative humidity. 46 39–43°F (4–6°C) and 60% relative humidity. Allow 47 to dry until desired MPR is reached. 48 Table 31.2 shows an example of a formulation for SAFE PRODUCTION OF DRIED 49 a pepperoni made in the United States, starter cul- SAUSAGES IN THE UNITED 50 ture added. The procedure follows: STATES 51 52 Procedure: Dried sausages were generally produced without any 53S Grind lean meat through 30 mm plate and fat pork special USDA inspection protocols prior to 1994, 54N through a 60 mm plate. Mix all ingredients for about when an outbreak of E. coli O157:H7 poisoning was 40454 31 327-332 r1.qxd 7/20/07 4:04 PM Page 331

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attributed to dry sausages. According to reports from bacteria used for fermentation may be incorporated 1 USDA, from November 16 through December 21, into the chopped or ground meat to produce fer- 2 1994, a total of 20 laboratory-confirmed cases of mented products safely: 3 diarrhea caused by E. coli O157:H7 were reported to 4 1. The preferred and most reliable method is to use a the Seattle-King County Department of Public 5 commercially prepared culture, which is handled Health, in the Pacific Northwest of the United States. 6 and used as prescribed by the manufacturer. Epidemiologic investigation linked E. coli O157:H7 7 2. A less acceptable procedure is the use of a portion infection with consumption of a commercial dry- 8 of a previously fermented and controlled cured salami product distributed in several western 9 “mother” batch. Because this is less precise than states. Three additional cases subsequently were 10 using a commercial culture, it is important that identified in northern California. These illnesses 11 the inoculum derived from the mother batch be raised questions about the effectiveness of control- 12 composed of a vigorous culture capable of ling E. coli during the production of dry-fermented 13 producing a rapid pH decline. sausage. In response, the USDA’s Food Safety and 14 Inspection Service (FSIS) developed specific proto- A third method, used historically, relied on lactic 15 cols to identify problems, which encompass options acid bacteria, which naturally occur in fresh meat, to 16 to correct them. These protocols must be followed or initiate the fermentation. Although this practice had 17 the product must be heat treated to control potential been used in the past and was the original art of mak- 18 E. coli contamination. Specifically, USDA requires ing fermented sausage, the method is highly unreli- 19 commercial producers of dry and semidry-fermented able and should be used by only experienced sausage 20 sausages in the United States to follow 1 of 5 safety manufacturers and may require more rigorous exam- 21 options: ination of the safety of the final product. 22 Acidulation: An alternative to commercial starter 23 • Achieve a 5-log kill using a heat process (145°F, cultures for reducing pH in sausage batters is direct 24 63°C for 4 minutes). acidulation by USDA-approved acidulants such as 25 • Develop and validate individual 5-log citric acid, lactic acid or glucono delta lactone 26 inactivation treatment plans. (GDL). These ingredients should be incorporated 27 • Conduct a hold-and-test program for finished into the sausage batter following procedures recom- 28 product. Depending on type of product, 15–30 mended by the manufacturer. 29 individual chubs must be subsampled per lot. Because dry sausages are not usually cooked, 30 • Propose combinations that demonstrate a people “at risk” (the elderly, very young children, 31 collective 5-log kill. pregnant women, and those with weakened immune 32 • Initiate a hazard analysis critical-control point systems) might want to avoid eating them. The 33 system that includes raw batter testing and a following are recommendations made for the safe 34 2-log inactivation in fermentation and drying. consumption of dried sausages (MacDonald et al. 35 2001): 36 37 PROCESS CONTROL POINTS FOR DRIED SAUSAGE • Dry-fermented sausages produced should be 38 MANUFACTURING subject to heat treatment or equivalent processes 39 pH Control: Fermented and acidulated sausages that will result in acceptable reductions of 40 shall attain a pH of 5.3 or lower through the action of pathogenic E. coli and other pathogens. 41 lactic acid forming bacteria or by direct acidulation • The effectiveness of the hold-and-test option 42 within the time frame defined in the GMP. It is should be reevaluated. 43 important to reach a pH below 5.3 to control the • Individuals who are at high risk of serious 44 growth of pathogenic microorganisms, including outcomes following infection with E. coli, 45 staphylococci and pathogenic E. coli. To assure that including children, the elderly, and individuals 46 the pH decreases normally, pH readings must be with immuno-compromising conditions, should 47 taken from each lot. It is important to record all pH not consume uncooked, dry-fermented sausage 48 measurements before the product surface tempera- products. 49 ture reaches 110°F (43°C) or before the degree-hour • This warning should be noted in the HACCP plan 50 limitation has been reached and before any final heat and on the label. 51 treatment, if used, is initiated. • Questions about exposure to dry-fermented 52 Fermentation: There are two general means rec- sausages should routinely be asked of all reported S53 ommended for use by which lactic acid–forming cases of E. coli O157:H7 and other infections. N54 40454 31 327-332 r1.qxd 7/20/07 4:04 PM Page 332

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1 REFERENCES containing micro-encapsulated probiotic lactic acid 2 bacteria. Food Microb 24:82–88. 3 D MacDonald, M Fyfe, A Paccagnella, J Fung, J Harb, AM Pearson, TA Gillet. 1996. Processed Meats, 3rd ed. 4 K Louie. 2001. Escherichia coli O157:H7 outbreak New York: Chapman & Hall. 5 linked to salami, British Columbia, Canada, 1999. JF Price, BS Schweigert. 1987. The Science of Meat 6 Health Canada Field Epidemiology Training Pro- and Meat Products, 3rd ed. Westport, Connecticut: 7 gram Abstracts. Food & Nutrition Press. 8 P Muthukumarasamy, RA Holley. 2007. Survival of 9 Escherichia coli O157:H7 in dry fermented sausages 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53S 54N 40454 32 333-348 r3.qxd 7/20/07 4:12 PM Page 333

1 2 3 32 4 5 6 Mediterranean Products 7 8 Juan A. Ordóñez and Lorenzo de la Hoz 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION referred to “goats casings filled with blood and fat” 22 (Liepe 1982). The Romans inherited the custom of 23 The production of foods using microorganisms is eating these food products from the Greeks, perfect- 24 one of humanity’s most ancient activities. Through- ing the techniques for their preparation and adding 25 out history, man soon realized the beneficial effects other ingredients. In pre-Christian Rome, during the 26 of some fermentations and, in fact, archaeological flower festivals and lupercalias, large amounts of fer- 27 remains and historical documents show that man mented sausages were eaten (“termicina,” “circeli,” 28 produced wine, beer, bread, cheeses, and sausages, and “botuli”), made from pork, pork fat, pepper, and 29 among other products, many years ago. All these cumin seeds. Given the pagan nature of these festi- 30 products are the result of fermentations, some vals, during the first few centuries A.D. the church 31 accompanied by ripening phenomena, which are, banned the festivals and also prohibited the con- 32 essentially, processes of breakdown and synthesis sumption of fermented sausages because of its close 33 due to the action of enzymes of different origin: associations with these celebrations. This prohibition 34 some naturally present in the raw material, others was maintained until 494 A.D., when these festivities 35 intentionally added for this purpose, and those pro- were Christianized (Sanz 1988). Since the Roman 36 vided by the microorganism. The result is a product Empire, these products have spread worldwide. 37 with a longer shelf life and the accumulation in it of Today, the importance of curing as a method of 38 aromatic and sapid compounds responsible for the preserving meat has declined owing to the develop- 39 taste and odor of the final product. ment of other preservation systems (refrigeration, 40 Drying and fermentation of meat is, probably, the freezing, heat treatments, etc). However, in contrast, 41 oldest form of meat conservation (Roca and Incze other aspects of the curing process have become 42 1990). The two processes are mentioned together more important, such as the flavor and color of the 43 because, in practice, they cannot be separated. Cur- foods, and today, fermented sausage production can 44 ing of meat probably first occurred by chance as a be considered more as a method of transformation 45 way to conserve meat by adding salt in countries and diversification than preservation. In any case, 46 of the Mediterranean area (Adams 1986). Around the Mediterranean dry-fermented sausages have an 47 1500 B.C., it was found that the shelf life of meat was exceptional hygienic background, which was guar- 48 considerably extended if, after mincing finely, it was anteed by both low pH (Ͻ4.5–5) and low water 49 mixed with salt and aromatic herbs, introduced into activity (Ͻ0.90) of the end product, when it is deliv- 50 casings and then dried (Palumbo and Smith 1977), ered to the market. 51 which also resulted in a product highly appreciated Raw fermented sausages, in general, can be 52 for its pleasant taste. The first documented evidence defined as products made by selecting, chopping, 53 for this process was found in book XVIII of The and mincing meat and fat, with or without offal, S54 Odyssey, written by Homer 900 years B.C., which mixed with seasoning, spices, and authorized N55 333 40454 32 333-348 r3.qxd 7/20/07 4:12 PM Page 334

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1 additives, that are then ripened and dried (cured) The meat also has a variable composition, and its fat 2 and, sometimes, smoked. However, regional cus- content depends on the species used and the anatom- 3 toms, environmental variations, family recipes and ical region of the animal from which it is taken. Gen- 4 other factors have given rise to a wide range of fer- erally, meat from adult animals is preferred, owing to 5 mented sausages, and it can be said that there are its higher myoglobin content, which favors a more 6 almost as many types of sausages as there are geo- appropriate color (Lücke 1998). Likewise, the fat 7 graphical regions or even manufacturers, although should be firm, with a high melting point and a low 8 their production process always requires the combi- content of polyunsaturated fatty acids, because this 9 nation of fermentation and dehydration. In the spe- causes the fermented sausage to turn rancid more 10 cific case of Mediterranean countries, and also quickly and to exude fats (Frey 1985). One excep- 11 Portugal, air-dried sausages made with spices are the tion to this is found in Spain, where some regions 12 most common type, with relatively long ripening manufacture sausages with adipogenic pork (Iberian 13 times compared to central and northern European pig), which contains a high level of unsaturated fats 14 countries, where fermentation is usually accompa- (Ordoñez et al. 1996), giving rise to products highly 15 nied by a smoking process or a less intense drying appreciated by the consumer. 16 process (Flores 1997). In the United States, the most Mincing of the meat and fat is done at low temper- 17 common fermented sausages are semicured, fer- atures (between −5 and 0°C) to achieved a clean cut 18 mented rapidly at relatively high temperatures, with and to avoid the release of intramuscular fat from 19 a short drying period (Lücke 1998). fatty meats, which could cause changes in the color 20 Dry-fermented sausages can be classified accord- and the drying process during ripening (Frey 1985). 21 ing to a range of criteria, such as the acidity, the minc- Once the meat and fat have been comminuted, the 22 ing size of the ingredients, the addition or absence of starter culture (lactic acid bacteria, belonging to the 23 starter cultures, the presence or absence of molds on Lactobacillus genus and the nitrate and nitrite 24 the surface, the addition of some ingredients and of reducer bacteria Micrococcaceae), curing salts (salt, 25 seasoning, the diameter and type of casing used, etc. nitrates, and nitrites), additives (colorants, ascorbic 26 Mediterranean sausages can be classified as fer- acid, etc.), and other ingredients (sugars glutamate, 27 mented meat products with a high acidity because, aromatic herbs, and spices) are added. All the ingre- 28 usually, the fermentation and ripening temperature, dients are placed in a kneader, after which the 29 concentration of curing salts used, pH, and addition of sausages are made by filling natural or synthetic cas- 30 carbohydrates most commonly used in these products ings. This operation is carried out in a vacuum to 31 are similar to those described by Incze (1992) for that exclude, as far as possible, oxygen from the matrix 32 type of fermented sausage. and to prevent abnormal fermentations and the 33 development of undesirable colors and flavors. 34 Once the casings have been filled, the sausages are 35 PRODUCTION OF placed in ripening rooms under conditions of con- 36 MEDITERRANEAN trolled temperature (18–24°C), relative humidity ϳ 37 DRY-FERMENTED SAUSAGES ( 90%) and air movement (0.5–0.8 m/s). The 38 sausages are kept here for 1 to 2 days during which the 39 There is a very wide range of Mediterranean fer- fermentation is produced. In this phase, two basic 40 mented sausages because, as mentioned above, it microbiological reactions proceed simultaneously, 41 depends on several factors, although its manufactur- and they influence one another. These consist of the 42 ing always is based on a combination of fermenta- formation of nitric oxide by nitrate and nitrite- 43 tion and drying. For example, in Spain, more than reducing bacteria (Micrococcaceae) and the genera- 44 50 varieties have been described, although many are tion of lactic acid by lactic acid bacteria (mainly 45 handmade on a small-scale (MAPA 1997). Table 32.1 Lactobacillus spp. in Mediterranean sausages). These 46 shows the ingredients for quite a few Mediterranean two phenomena are well known, and several authori- 47 fermented sausages from several countries. The tative reviews have been published in which the mech- 48 manufacture of dry-fermented sausages consists, anisms and interaction of the reactions taking place 49 essentially, of a number of sequential steps that are are thoroughly discussed (Palumbo and Smith 1977; 50 shown in the flow chart in Figure 32.1. The meat Liepe 1982; Lücke 1998; Nychas and Arkoudelos 51 used depends on eating habits, customs, and the 1990; Demeyer et al. 1992; Ordoñez et al. 1999; 52 preferences availing in the geographical region Toldrá 2002). Although the nitrite reactions occurring 53S where the fermented sausage is produced. This is during curing have been studied extensively, the actual 54N usually pork, sometimes mixed with beef, although, chemical reactions are not conclusive due to the for religious reasons, in Turkey lamb or cow is used. high reactivity of nitrite, the complexity of the meat 40454 32 333-348 r3.qxd 7/20/07 4:12 PM Page 335

Table 32.1. Typical formulation (g/100 g) of selected Mediterranean dry-fermented sausages. 1 2 Salchichón/Saucisson Salami Fuet Chorizo Lukanka 3 (Spain/France) (Italy) (Spain) (Spain, Portugal) (Bulgaria) 4 (1) (1) (1) (1) (2) 5 6 Lean pork 35–70 45–84 60–70 80–65 25 7 Pork fat 10–25 14–25 30–40 20–40 20.0 8 9 Lean beef 0–50 0–37 0–20 0–20 55.0 10 Fermentable sugars* 0.2–0.5 0.3–0.7 0.1–0.4 0.6–0.8 – 11 Curing salts** 2.0–2.4 1.8–2.5 2.0–2.4 1.8–2.1 2.24 12 13 Flavoring or spices: 14 —Whole/ground black 0/0.2–0.2/0.4 0/0.2–0.1/0.14 0/0.3–0/0.2 0.30 15 — pepper 16 —White pepper – 0–0.2 0–0.3 – 17 —Red pepper – – – – 0.20 18 —Sweet/hot paprika – – – 1.5/0–2.5/1.5 – 19 —Cumin – – – – 0.20 20 —Red pepper paste – – – – – 21 —Garlic – 0–0.2 – 0.2–1.2 – 22 —Other spices *** *** *** – – 23 **** Others : 24 —Sodium glutamate 0.25 – 0–0.15 – – 25 —Powdered milk 0–0.6 0–2.5 – 0–2.5 – 26 —Caseinate 0–0.6 – 0–1.0 – – 27 —White wine – – – – – 28 —Liquid smoke – – – 0.20 29 30 Sources: (1) Several sources; (2) Balev et al. (2005). * 31 Glucose is commonly used and, sometimes, glucose plus lactose. 32 **Curing salts are composed by NaCl, nitrate, nitrite, and ascorbic acid. A typical mix is, respectively, NaCl/NO Na/NO Na/ 3 2 33 ascorbic acid. 2.5/0.095/0.0065/0.094)(w/w/w/w) (Díaz et al. 1997). 34 ***The spices, ﬂavorings, and aromatic herbs used are very variable cinnamon, oregano, aniseseed, dill, posemary, etc. **** 35 Coloring is used in some products, (e.g., cochineal, ponceau 4R). 36 37 38 Selection of meat Meat (deboning and mincing) 39 (pork and/or beef) 40 41 Addition of starter culture, additives and curing agents 42 (salt, sugar, nitrate, nitrite, spices, others) 43 44 Stuffing into casings Mixing to form the paste 45 46 47 Fermentation (18–24ºC, RH ~ 90%, 24–48 hours) 48 49 50 Ripening 1st phase: 10–14ºC, decreasing RH to ~ 85% in about 4 days 51 52 Ripening 2nd phase: 10–14ºC, RH 85–87%, var. time (e.g., 1–5 months) S53 N54 Figure 32.1. Steps in the manufacture of dry-fermented sausages.

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1 substrate and the different processing types of the 1986) approximately 1.8 mol of lactic acid per mole 2 product (Cassens 1995). Nevertheless, several func- of metabolized hexose and around 10% of other 3 tions have been attributed to nitrites: it is involved in substances (formic and acetic acids and ethanol) 4 both color (Eakes et al. 1975; Giddings 1977) and ready to act as substrates for the synthesis of some 5 odor (Cross and Ziegler 1965; Bailey and Swain 1973) flavor compounds. This phenomenon produces a 6 development; it prevents the growth of several undesir- decrease in the pH from an initial value of 5.8–6.2 7 able microorganisms (Hauschild et al. 1982; Pierson close to 5.0 or below (Figure 32.2), which has several 8 and Smoot, 1982) and it has an antioxidant activity beneficial effects on both the manufacturing process 9 (Sato and Hegarty 1971; MacDonald et al. 1980). and the shelf life of the product, such as the control of 10 The sausage ingredients play an important role in microbiota and enzymic reactions, the reduction of 11 the selection of the proper microbiota of the sausages. water retention capacity of proteins favoring the dry- 12 The initial microbial population of meat is always ing, acceleration of the gelation of myofibrillar pro- 13 varied and is similar to that found in fresh meat. It teins, and regulation of color formation reactions. 14 includes gram-negative (Pseudomonas spp., Achro- The presence of molds in Mediterranean fermented 15 mobacter spp., Moraxella spp., Enterobacteriaceae, sausages is usually attractive for the consumers and, 16 etc.) and gram-positive (Bacillus spp., Lactobacillus in fact, spores now are often inoculated to enhance 17 spp., Enterococcus spp., Brochotrix thermosphacta, growth of a given species. Owing to its aerobic 18 Micrococcaceae, etc.) bacteria (Gill 1986; Gill and character, its growth is limited to the surface of the

19 Newton 1977). On the contrary, the water activity (aw) sausage (Geisen 1993). The count is very slow in the 20 has passed from 0.98 (in fresh meat) to 0.96 (in fresh first few days (on the order of 102–103 cfu/cm2) but 21 sausage) due to the addition of curing salts and other rapidly increases, reaching values of 106–107 cfu/cm2 22 solutes (e.g., sugars), which, together with the specific in around 25 days (Roncales et al. 1991). The molds 23 inhibitory effects of nitrate and nitrite and the low most frequently isolated belong to the genera Penicil- 24 oxygen tension, favors the selection of both lactic acid lium and Aspergillus, although sometimes molds of 25 and Micrococcaceae bacteria. These circumstances the genera Cladosporium, Scopularopsis, Alternaria, 26 are normally brought about in naturally produced dry- and Rhizopus (Leistner and Eckardt 1979; Pestka 27 fermented sausages (without starters), which causes 1986) are identified. The presence of molds on the 28 the inhibition of organisms responsible for the sausage surface can lead to both desirable and detri- 29 spoilage of fresh meat (gram-negative bacteria, espe- mental effects. The main positive actions are the 30 cially pseudomonads) and allows the rapid growth of antioxidant effect by oxygen consumption, peroxide 31 the former lactic acid bacteria and Micrococcaceae, degradation, and protection against light, which leads 32 which are the dominant bacteria until the end of to the inhibition of oxidative phenomena and color 33 the ripening period (Figure 32.2). Similarly, these stabilization (Bruna et al. 2001; Sunesen and Stanhke 34 conditions stimulate the growth of the starter cultures 2003); the protection against spontaneous coloniza- 35 when these are added. The sugar metabolism of lactic tion with undesirable molds, including toxinogenic 36 acid bacteria naturally present, or those included in ones (Lücke and Hechelmann 1987); the favorable 37 the starter, in the Mediterranean dry-fermented saus- characteristic white or greyish appearance of the 38 ages is mainly homolactic, yielding (Gottschalk mycelium and conidia (Lücke 1998); and the contri- 39 bution to the development of flavor compounds, 40 aw pH which has been recently reviewed by Sunensen and 9 6.5 41 0.96 Stahnke (2003). The adverse effects are usually related 42 7 6.0 to the growth of unwanted species, and the main 43 0.94 effects correspond to the production of mycotoxins 5.5 44 5 0.92 and antibiotics. The inoculation of nontoxinogenic 45 5.0 strains is a very useful approach to avoid colonization 46 3 0.90 by undesirable molds. Penicillium nalgiovense is a 47 4.5 much-used strain with this goal because it is nontox- Log viable count (cfu/g) 0.88 48 inogenic, easily establishes on the surface, and pro- 49 5 10 15 20 25 30 35 vides an attractive appearance. 50 Ripening time (days) After fermentation, the temperature and relative 51 Figure 32.2. Microbial (total viable count [ _____ ]), humidity are reduced to reach values of 10–14°C 52 lactobacilli ( ------) Micrococcaceae ( ...... )] and 85–87%, respectively, in 4–6 days, which are ...... 53S and chemical [pH ( ------) and aw ( ------)] maintained until the end of the ripening period, dur- 54N changes during ripening of dry fermented sausages. ing which many flavor compounds develop. 40454 32 333-348 r3.qxd 7/20/07 4:12 PM Page 337

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RIPENING OF MEDITERRANEAN processes of degradation or synthesis. The former 1 DRY-FERMENTED SAUSAGES are metabolic and chemical transformations of 2 major compounds in the raw material (proteins and 3 A ripening sausage is a highly complex biochemical triglycerides and other less-abundant lipids) and the 4 system in which numerous reactions are taking added compounds (sugars). The metabolic reactions 5 place, many equilibria are established, and several are catalyzed by enzymes of different origin: some 6 metabolic and chemical pathways cross. The result- proceed from the microorganisms which, at one time 7 ing product of these reactions often becomes the or another, participate in the ripening process; and 8 substrate for other ones. The composition of a fer- others come to the sausage directly from the meat 9 mented sausage, therefore, is constantly changing and fat that form part of its normal composition. 10 during the ripening process. Small extrinsic or endo- Undoubtedly, the first reactions to come into play are 11 genous modifications can produce significant shifts degradative phenomena: hydrolytic reactions (gly- 12 in the equilibrium of the reactions involved, which colysis, proteolysis and lipolysis) catalyzed by micro- 13 gives rise to some of the actions taking place during bial or endogenous enzymes, and chemical reactions 14 the manufacturing process. One example, is the (mainly lipid autooxidation) catalyzed by the oxygen 15 repercussions of the casing diameter of salchichon from the air that penetrates the fermented sausage. 16 (Ͼ3 cm) versus fuet (Ͻ3 cm); in the first case, the The first phases of the hydrolytic and chemical 17 volatile fraction is dominated by compounds derived processes are well known and, essentially, consist, 18 from microbial activities (e.g., esters, alcohols, etc.) respectively, of the degradation of sugar by lactic acid 19 and, in the second case, volatile compounds pro- bacteria during the aforementioned process of fer- 20 duced by autooxidative phenomena are more preva- mentation; the rupture of the triglyceride-ester bond 21 lent (e.g., aldehydes and ketones) due, in this case, to by the action of lipases, with the subsequent forma- 22 the mixture inside being more exposed to oxygen tion of free fatty acids and diglycerides, monoglyc- 23 (Edwards et al. 1999). In any case, during ripening a erides, or glycerol, depending on the extent and 24 wide range of compounds are generated (peptides, position of the bond cleavage in the lipid molecule; 25 free amino acids, free fatty acids, amines, ketones, the fragmentation of proteins due to the action of pro- 26 aldehydes, etc). Some of these are accumulated in teinases, yielding polypeptides of different molecular 27 the matrix, contributing directly to the taste (e.g., size that are, later, attacked by other proteases pro- 28 amino acids and amines and long chain fatty acids, ducing nonprotein nitrogen fractions (small peptides, 29 which are nonvolatile sapid compounds) and/or odor amino acids, amines); and, finally, the formation of 30 (e.g., short chain fatty acids released by the action hydroperoxides from unsaturated fatty acids, which, 31 of esterases) and others that become aromatic sub- in the more advanced stages, result in the appearance 32 stances (e.g., amino acids and amines, which are of low molecular weight organic compounds. All 33 transformed into ketoacids, aldehydes, etc., by these reactions involved in the mentioned degrada- 34 oxidative deaminations or free fatty acids, which, tive phenomena are well known and have been ana- 35 mainly unsaturated, can undergo chemical oxidations lyzed in detail in several articles (Dainty and Blom 36 and yield, among other compounds, ketones or fun- 1995; Ordoñez et al. 1999; Toldrá 2002) and an 37 gal catabolism that give rise to the generation of update of chemistry of reactive oxygen in food is 38 methyl ketones and secondary alcohols). All these described by Choe and Min (2006). 39 compounds accumulate in the medium at different In this context, it is well known that the enzymatic 40 concentrations, and they are responsible for the fla- luggage of the LAB is responsible for the metabolism 41 vor and odor of the final product. These compounds of sugars but, perhaps, the origin of enzymes pre- 42 can be of diverse origin: some are the result of micro- dominant in the lipolytic and proteolytic processes 43 bial phenomena (glycolysis and numerous other occurring in the sausage mixture is less clear. 44 reactions derived from the enzymatic luggage of the Traditionally, the microbiota of fermented sausages 45 microorganisms present); some arise from biochem- has been considered to be responsible for the break- 46 ical reactions of endogenous origin (lipases and pro- down of lipids that occurs during ripening (Alford 47 teases) and others from chemical phenomena (mainly et al. 1971; Cantoni et al. 1967; Demeyer et al. 1974; 48 nitric oxide reactions and lipid autooxidation); and, Selgas et al. 1988; Talon et al. 1992). In fact, many 49 finally, others accumulate from the addition of certain studies in vitro have been focused on the extracellu- 50 ingredients (e.g., spices and seasonings). The phe- lar and intracellular activity of lactic acid bacteria 51 nomena that produce these low molecular weight (Nordal and Slinde 1980; Sanz et al. 1988; Papon 52 compounds, generally absent or only present in and Talon 1988, 1989; Samelis and Metaxopoulus S53 small amounts in the initial substrate, correspond to 1997) and Micrococcaceae (Selgas et al. 1988, 1993; N54 40454 32 333-348 r3.qxd 7/20/07 4:12 PM Page 338

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1 Talon et al. 1992; Coppola et al. 1997) isolated from period (Toldrá et al. 1991; Motilva et al. 1993). Taking 2 Mediterranean fermented sausages (Italian, French, into account that the pH of the fermented sausages 3 Greek, and Spanish). In general, all these authors varies from 4.8 to 6.0 along the ripening, acid lipase 4 have detected some activity and, in accordance with could develop an important activity in these products. 5 the results, speculations have been made about their Also, in studies conducted on dry hams, lipases of 6 participation in lipolytic phenomena during ripening. subcutaneous adipose tissue presented less stability 7 However, Garcia et al. (1992) observed a similar con- than muscular lipases (Toldrá et al. 1991). The neu- 8 tent of free fatty acids in fermented sausages manu- tral lipase would be the main agent responsible for 9 factured aseptically and in others inoculated with lipolysis in adipose tissue because it would not be 10 lactobacilli and/or micrococci. They concluded that affected by variations in the NaCl concentration 11 the release of fatty acids could be due, at least in part, between 1 and 10 g/l and would, also, be slightly

12 to the endogenous lipases in the meat. Montel et al. reduced as the aw dropped from 0.98 to 0.62 (Motilva 13 (1993) also studied the lipolysis of dry-fermented and Toldrá 1993). The role played by this enzyme in 14 sausages. They demonstrated that noninoculated the lipolysis of fermented sausages during ripening 15 sausages presented slightly lower levels of fatty acids may not be highly important, because its optimum 16 than batches inoculated with different starter cul- pH is almost neutral and, as mentioned previously, 17 tures, and, therefore, also considered the endogenous fermented sausages have an acid pH. Moreover, the 18 lipases as main responsible of lipolysis. Also, Hierro NaCl concentration of these products is approxi- 19 et al. (1997), with aseptic fermented sausages, con- mately 22–25 g/Kg (Toldrá et al. 1992), which could 20 cluded that more than 60% of the free fatty acids also adversely affect the activity of neutral lipase. 21 released proceeded from the action of these enzymes. Finally, there is some doubt about the possible par- 22 They also described a greater release of linoleic ticipation of muscular and adipose esterases in the 23 and oleic fatty acids than in batches inoculated with ripening process of fermented sausages and cured 24 starter cultures. Kenneally et al. (1998) manufactured hams. Esterases are more sensitive to variations in

25 batches of fermented sausages with different starter NaCl concentrations and aw than lipases (Motilva and 26 cultures using, as a control, a batch to which glu- Toldrá 1993). However, in experiments carried out in 27 cone-δ-lactone and several antibiotics (penicillin, dry hams, both muscle tissue and adipose tissue 28 streptomycin, and amphotericin) were added to pre- esterases presented a good activity during the whole 29 vent microbial growth. The results showed that there process (Toldrá et al. 1991). Nonetheless, it seems 30 were no signiﬁcant differences in the levels of free that they do not play an important role in lipolytic 31 fatty acids among the batches made with or without phenomena, due, among other reasons, to the lack of 32 the starter culture. From this, they also concluded a suitable substrate (Motilva et al. 1993). 33 that lipolysis is mainly due to endogenous enzymes. Similarly, the fragmentation of protein macromole- 34 It, therefore, seems that endogenous lipases play an cules in dry-fermented sausages has been traditionally 35 important role in the breakdown of lipids in fer- attributed to the activity of major microorganisms 36 mented sausages. present during the ripening process. Many studies 37 Cured ham is a good model in which to study the have been conducted in the past to investigate the 38 activity of endogenous enzymes because it usually proteolytic activity of Micrococcaceae and lactic 39 presents bacterial loads below 104 cfu/g inside it acid bacteria in isolated strains from fermented 40 (Molina and Toldrá 1992). Hence, Motilva and Toldrá sausages. In the case of Micrococcaceae, several 41 (1993), in samples of Biceps femoris of pork, studied authors have reported that these bacteria actively

42 the effect of curing salts and aw on muscular and adi- participate in the proteolytic phenomena that take 43 pose lipase activity and they reported that acid lipase place during the ripening of fermented sausages 44 could participate actively in muscular lipolysis (Bacus 1986; Selgas et al. 1993), causing an impor- 45 throughout the curing process because it is strongly tant increase in the free amino acid content (Bacus 46 activated as the concentration of NaCl is increased 1986). In fact, endopeptidase, aminopeptidase, and

47 and aw is reduced. However, the basic and neutral dipeptidase activities have been detected in these 48 lipases are strongly inactivated as the aw diminishes. It bacteria (Bhowmik and Marth 1989). However, 49 was confirmed in dry hams, where the activity of studies conducted on Staphylococcus sp. (Montel 50 these two enzymes increases during the first 2 months et al. 1992), S. carnosus (Hammes et al. 1995), and 51 of curing to diminish rapidly afterward, at the start of S. xylosus (Bermell et al. 1992) show that Micrococ- 52 the drying phase, maintaining some degree of activ- caceae do not present a very important proteolytic 53S ity until the end of the process. On the other hand, activity. Hierro et al. (1999) did not found differ- 54N acid lipase remains active during the whole ripening ences in myofibrillar proteins or in the amino acid 40454 32 333-348 r3.qxd 7/20/07 4:12 PM Page 339

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profile after inoculating sausages with a strain of processes. With regard to catepsins, research has 1 Staphylococcus sp. been carried out in model systems that simulate the 2 Similar considerations can be made about the lac- conditions of the ripening of fermented sausages 3 tic acid bacteria. Hence, some authors (Cantoni et al. (Toldrá et al. 1992), to determine which of these 4 1975; Montel et al. 1992; Fadda et al. 1998) have could be the most relevant in proteolytic phenom- 5 observed in vitro proteolytic activity of lactic acid ena. They concluded that catepsins B, L, and D are 6 bacteria strains isolated from fermented sausages, mainly active in the first stages of the production 7 and they concluded that these bacteria played a very process (mixing and fermentation), whereas only 8 important role in the protein breakdown. However, catepsin L could present a significant activity during 9 other authors (Nordal and Slinde 1980; Lücke and the drying step. On the other hand, they also agreed 10 Hechelmann 1987; Bermell et al. 1992; Hierro et al. with Demeyer et al. (1992) that muscular proteins 11 1999) did not detect proteolytic activity, or only (endopeptidases) of the type of catepsin D, are 12 negligible activity, in these bacteria, and Law and mainly active during the fermentation phase, 13 Kolstad (1983) indicated that there was no direct evi- whereas bacterial exopeptidases are mainly active in 14 dence that these microorganisms contribute signifi- the drying phase. Nonetheless, other muscular 15 cantly to the flavor and odor of fermented meat enzymes, such as type II dipeptidyl aminopeptidases 16 products, and that the role of their enzymes in meat would collaborate with the latter because the pH of 17 proteolysis is uncertain. Later, Kröckel (1995) con- the dry-fermented sausages is adequate for their 18 sidered that the extent to which lactic acid bacteria activity (Bird and Carter 1980) and also that of the 19 and/or Micrococcaceae participate in the proteolytic carboxypeptidases (Toldrá et al. 1992). 20 changes during the ripening of dry-fermented saus- 21 ages and cured hams is unknown. 22 Data supporting the proteolytic activity due to FLAVOR DEVELOPMENT 23 endogenous enzymes, however, are more consis- IN MEDITERRANEAN 24 tent. To demonstrate their importance in sausage DRY-FERMENTED SAUSAGES 25 ripening, some authors conducted experiments in 26 CONVERSION OF FREE AMINO ACIDS which microbial growth was inhibited by making the 27 AND FATTY ACIDS sausages in aseptic conditions (Garcia et al. 1992; 28 Montel et al. 1993; Hierro et al. 1999) or by adding One of the most important phenomena that occurs 29 antibiotics (Verplaetse et al. 1992; Molly et al. during the ripening of fermented sausages is the 30 1996). No differences were observed between the transformation of amino acids that appear as a con- 31 nitrogen fractions in batches without microorgan- sequence of the breakdown of proteins. These amino 32 isms and batches to which different starter cultures acids can accumulate in the matrix, contributing to 33 were added. Moreover, the addition of pepsatin and the overall taste of the product, but play a much 34 leupeptin to inhibit the proteinases produced a sharp more important role as a substrate for other reactions 35 drop in the rate of actin and myosin degradation that can produce a wide range of aromatic and 36 (Verplaetse et al. 1992). On the basis of these results, sapid compounds, some of which are shown in 37 these authors deduced that endogenous proteases Figure 32.3. As in cheeses (Engels and Visser 1996; 38 play a more important role than bacterial ones in the Yvon et al. 1997), these transformations are mainly 39 development of proteolytic phenomena, and Hierro due to the activity of microbial enzymes (Hinrichsen 40 et al. (1999) even express that these bacteria have a and Pedersen 1995) although some chemical reac- 41 very low participation, if any, in the process. tions (Strecker degradation) could also occur (Yvon 42 The importance of muscular proteases in protein et al. 1997; Ordoñez et al. 1999). Among these 43 fragmentation is also supported, as in the case of reactions, transamination is a very important event 44 lipolysis, by studies conducted in cured ham (Toldrá because it is the first step in amino acid catabolism, 45 et al. 1993; Buscailhon et al. 1994; Martin et al. which requires the presence of an α-keto acid accep- 46 1998). Catepsins are the proteases that develop most tor for the amino group, commonly α-ketoglutarate 47 activity during ripening, whereas the calpains have a (Yvon et al. 1998). This reaction is catalyzed by 48 neglible action because they are relatively sensitive aminotransferases and these enzymes have been 49 to acidity because calpain I loses 82% of its maxi- demonstrated to initiate the catabolism of aromatic, 50 mum activity at pH 5.5, and calpain II is even more branched-chain, and sulphur amino acids (Dias and 51 sensitive (Dransfield 1993). This, together with the Weimer 1998; Yvon et al. 1997; Engels et al. 2000), 52 addition of curing salts (Sarraga et al. 1989), makes yielding, among other products, branched aldehy- S53 it highly unlikely that calpains participate in these des, which play a central role in the flavor of the final N54 40454 32 333-348 r3.qxd 7/20/07 4:12 PM Page 340

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1 Meat proteins 2 Proteolysis 3 4 Lower nitrogen fractions, polypeptides, peptides 5 Proteolysis 6 7 Free amino acids Oxidative 8 Transamination Decarboxilation Sulphur amino 9 Deamination acids Degradation 10 Amino acids NH3 11 Amines Phenol, indol α-ketoacids Sulfur compounds 12 CO2 NH3 13 Alcohols Reduction Aldehydes Oxidation Acids 14 15 Esters 16 17 Figure 32.3. Formation of aromatic and sapid compounds from the protein degradation. 18 19 20 product because they have been associated with the synthesis and, similarly, butanal and 2-methylbutanal 21 ripened flavor of both dry ham (Careri et al. 1993; are produced during isoleucine formation. 22 Hinrichsen and Pedersen 1995) and dry-fermented Like amino acids, free fatty acids are the source of 23 sausages (Stahnke et al. 2000). In fact, they have many volatile compounds, which are integrated in 24 been detected in several cured products, such as the flavor compounds. It has been mentioned above 25 bacon (Andersen and Hinrichsen 1995); Italian dry that lipolytic activity has been demonstrated in 26 ham (Barbieri et al. 1992; Hinrichsen and Pedersen several lactic acid bacteria, but these organisms are 27 1995); Iberian dry ham (Garcia et al. 1991; Lopez not able to degrade the resultant free fatty acids. 28 et al. 1992); and Italian (Stahnke and Zeuthen 1992), Therefore, the contribution of these substances to 29 French (Berdague et al. 1991), and Spanish (Edwards the flavor directly derives from their accumulation 30 et al. 1999; Mateo and Zumalacarregui 1996) dry- in the sausage. However, the molds may play an 31 fermented sausages. These compounds may be gen- important role in lipid degradation, because these 32 erated by certain microorganisms from amino acids microorganisms can degrade, via partial β-oxidation, 33 (Hinrichsen and Pedersen 1995) via transamination- the free fatty acids released by lipases, yielding 34 decarboxylation, as demonstrated in Streptococcus methyl-ketones, which may be reduced to secondary 35 lactis, var. maltigenesis for leucin, which is con- alcohols, as it has been described in cheeses for 36 verted in 3-methylbutanal (MacLeod and Morgan Penicillium camemberti and Penicillium roqueforti 37 1955) and several other lactic acid bacteria, mainly (McSweeney 2004). 38 from cheeses (Alting et al. 1995; Christensen et al. 39 1999; Smit et al. 2000; Yvon et al. 1998). Other ROLE OF MOLDS IN THE FLAVOR COMPOUNDS 40 pathways may also proceed, such as the deamination- GENERATION 41 decarboxylation of amino acids by Strecker degra- 42 dation (Ordoñez et al. 1999), which has been Given the aerobic character of molds, these cannot 43 demonstrated in dry-cured Iberian ham (Garcia et al. be present in the fermented sausage interior, but are 44 1991; Barbieri et al. 1992; Ventanas et al. 1992). This often found on its surface. Maybe this is why little 45 reaction involves the interaction of α-dicarbonyl attention has been paid to their potential role in the 46 compounds and α-amino acids, yielding an aldehyde flavor and odor of fermented sausages. Several 47 with one less carbon atom than the initial amino acid, authors (Mateo and Zumalacarregui 1996; Meynier 48 which results in the formation of 3-methylbutanal, et al. 1999; Schmidt and Berger 1998; Stahnke et al. 49 2-methylbutanal, and phenylacetaldehyde from, 2002) have detected a number of volatiles com- 50 respectively, leucine, isoleucine, and phenylalanine. A pounds in dry-fermented sausages with molds in the 51 third possibility for the generation of branched alde- fungal casing coat, but few authors have analyzed 52 hydes is through the biosynthesis of amino acids. In the origin of these substances and the effect of dif- 53S this pathway, 2-methylpropanal and 3-methylbutanal ferent molds on the overall quality and volatile com- 54N are formed as by-products of valine and leucine position in Mediterranean dry-fermented sausages. 40454 32 333-348 r3.qxd 7/20/07 4:12 PM Page 341

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Singh and Dincho (1994) studied the protective authors analyzed Spanish commercial dry sausages 1 effect of molds in Bulgarian dry sausages Sredna with different times of ripening, diameters (salchi- 2 Gora and observed, in relation to the flavor, that the chon versus fuet), and spices (salchichon versus 3 three strains (two belonging to Penicillium camem- chorizo), which had different mincing degrees 4 berti and one to P. nalgiovensis) used slightly (whole versus ground). Sulphurous compounds and 5 improved the overall acceptability compared to the 3-hexanol were detected only in chorizo, derived, 6 controls (without molds), although there were not respectively, from garlic and paprika. In sausages 7 differences between the strains. Garcia et al. (2001) made with ground pepper, the terpenes were the 8 compared the sensory properties of Spanish dry- major components and reached much higher levels 9 fermented sausages (salchichon), using one strain of than in dry-fermented sausages that were manufac- 10 Mucor spp., a commercial strain of P. nalgiovensis, tured with whole peppercorns. The narrower the 11 and two of Penicillium spp. A correlation between diameter of sausages, the higher the levels of com- 12 proteolytic and lipolytic activities of molds and over- pounds from lipid oxidation, and, finally, the longer 13 all acceptability was observed. P. nalgiovensis had a the ripening times, the greater the ester contents. 14 better external aspect than the other strains, but it pre- 15 sented the lowest enzyme activity, giving rise to the 16 lowest sensory parameters. A recent study (Hierro ATTEMPTS TO ACCELERATE THE 17 et al. 2005) deals with volatile generation in dry- RIPENING OF MEDITERRANEAN 18 fermented sausages by three mold species (Mucor DRY-FERMENTED SAUSAGES 19 racemosus, Penicillium aurantiogriseum, and Peni- 20 cillium camemberti) inoculated onto the surface of Since, on the one hand, in the Mediterranean regions 21 sausages. A starter culture composed of Lactobacillus a longer ripening time is preferred to produce dry 22 plantarum, Staphylococcus carnosus, and Staphylo- meat products (Flores 1997) and, also, since protein 23 coccus xylosus was used. As a control, a noninocu- and fat degradation are two central phenomena dur- 24 lated batch was prepared, which was sprayed on the ing dry-fermented sausage ripening, several attempts 25 surface with a 25% potassium sorbate solution. Four have been made to either accelerate the ripening 26 patterns were distinguished. Each one was dominated period or enhance the flavor. This would permit stor- 27 by different volatile classes, i.e., aldehydes from lipid age time to be shortened and would increase the 28 degradation, esters, branched aldehydes from amino profit margin and competitiveness of the final prod- 29 acid catabolism and alcohol, and branched aldehy- uct. To do this, enzymes of different origins have 30 des, which, respectively, corresponded to control, been used: bacterial (Næs et al. 1992, 1994; Hagen 31 M. racemosus, P. camemberti, and P. aurantiogriseum et al. 1996; Zapelena et al. 1997, 1998), fungal (Diaz 32 batches. The four batches were perfectly discrimi- et al. 1992, 1997; Zapalena et al. 1998, 1999; 33 nated by a principal component analysis. The authors Ansorena et al. 1998b, 2000) and vegetal (Diaz et al. 34 even concluded that “it is possible to generate aroma 1996, 1997; Melendo et al. 1996) proteinases, mix- 35 profiles in mold-ripened sausages depending on the ture of bacterial proteases (Diaz et al. 1993, 1997), 36 fungal strain used”...and, at the same time,... and bacterial (Næs et al. 1992), fungal (Zalacain 37 “control the level of lipid autooxidation.” et al. 1997a,b,c; Ansorena et al. 1998a,b, 2000) yeast 38 (Zalacain et al. 1995, 1996), and animal (Paleari 39 1991; Fernandez et al. 1991, 1995a,b) lipases. All 40 ROLE OF HERBS AND SPICES IN THE FLAVOR these authors obtained similar results, i.e., an increase 41 COMPOUNDS FORMATION in the amounts of substances derived from proteoly- 42 Other factors providing volatiles substances to dry- sis (nonprotein nitrogen and free amino acids) and 43 fermented sausages are the herbs and spices and the lipolysis (free fatty acids) was observed, but no, or 44 physical state (whole or ground) in which these are few, effects were obtained in relation to an improve- 45 added to the sausage. Black pepper, for example, ment in the sensory properties and overall accept- 46 provides terpenes, and garlic and paprika provide ability. Probably the most reasonable conclusion has 47 sulphurous compounds and 3-hexenol, respectively been described by Diaz et al. (1997). In brief, when 48 (Edwards et al. 1999). proteinases are added to the sausages, proteolysis is 49 In summary, flavor development is a very complex enhanced, with an increase in the different nitrogen 50 phenomenon, in which many degradation and syn- fractions (water soluble, nonprotein, and total volatile 51 thesis reactions take place. These are modulated by basic nitrogens) and quantitative and qualitative 52 different internal and external agents. The work of changes in free amino acids. Similarly, the addition S53 Edwards et al. (1999) clearly demonstrates this. These of lipases accentuates the hydrolysis of triglycerides, N54 40454 32 333-348 r3.qxd 7/20/07 4:12 PM Page 342

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1 yielding variable amounts of free fatty acids. How- means that valine and leucine were the donors of the 2 ever, the sensory analysis showed that solely, and amino group. However, the best results were obtained 3 only in some cases, a slight increase in the flavor when addition of α-ketoglutarate was combined with 4 may be obtained. Therefore, the addition of lipases a lactic acid bacteria strain of cheese origin (Lacto- 5 and proteinases alone is not useful for shortening the coccus lactis, subsp. cremoris NCDO 763) highly 6 ripening period, but this approach may be used to active in glutamic acid metabolism (Yvon et al. 7 provide precursors (free amino acids or fatty acids) 2000). It has also been described (Tjener et al. 2004) 8 to be transformed into flavor compounds (aldehydes, that addition of α-ketoglutarate enhances the for- 9 ketones, alcohols, etc.). After this statement, the mation of volatile compounds by Staphylococcus 10 same authors (Diaz et al. 1997; Ordoñez et al. 1999; carnosus during the fermentation of sausages. 11 Fernandez et al. 2000) hypothesized that to shorten 12 the ripening, or to improve the sensory quality of fer- CONCLUSIONS 13 mented sausages, in addition to adding proteinases or 14 lipases, it is necessary to create conditions or to add Mediterranean dry-fermented sausages have been 15 either an efficient starter or another kind of enzyme, to produced and consumed for many centuries. Tradi- 16 convert the free amino acid and fatty acids into tionally, they were made by artisanal methods in both 17 volatile flavor compounds in a shorter time than usual. industrial and craft level. However, the developments 18 According to the former hypothesis, several in both food engineering and meat product technol- 19 attempts have been made to increase the transami- ogy, the consumer demands, and the globalization 20 nase and oxidative deaminase activities during pro- trend in the latter twentieth century have pushed the 21 duction of dry-fermented sausages, by either surface meat industry to produce dry sausages on a grand 22 inoculation of molds or by adding microbial (fungal scale. As a consequence, it has been necessary to 23 or bacterial) crude extracts as an ingredient, together manufacture a normalized end product. To reach this 24 with proteases, to provide increased amounts of goal, the conditions of fermentation/ripening (tem- 25 amino acids. These experiments showed that the perature, humidity, and air flow) are narrowly con- 26 addition of mold intracellular extracts from Mucor trolled and starter cultures are generally used. The 27 racemosus (Bruna et al. 1999, 2000a), Penicillium production of Mediterranean dry-fermented sausages 28 aurantiogriseum (Bruna et al. 2000b, 2001a,b), and is based, as in other varieties from Northern and Cen- 29 Penicillium camemberti (Bruna et al. 2002) produce, tral Europe, on a combination of fermentation and 30 in comparison with the control, an increase in both drying, but in Mediterranean countries the smoking 31 the free amino acid and ammoniacal fractions. Like- treatment is not generally applied, and air-drying and 32 wise, higher levels of branched aldehydes were the addition of spices are prevalent circumstances, 33 observed, which led to an improvement in the flavor. together with a longer ripening time than that of 34 The fungal superficial inoculation of these species Northern and Central European products. 35 produced similar results (Bruna et al. 2000a, 2001a,b, In relation to the scientific advances, many studies 36 2003). However, the combination of both appro- have been made to clear up both the source of agents 37 aches was much more effective (Bruna et al. 2000a, responsible for the phenomena occurring during 38 2001a,b). In similar experiments with bacterial ripening and the precursors and reactions giving rise 39 extract, the same trend was observed but a weaker to flavor compounds. Likewise, several attempts 40 effect, with negligible effects on the sensory quality have been made to either accelerate the ripening or 41 (Herranz et al. 2006). When commercial free amino enhance the flavor of dry-fermented sausages. For 42 acids were added during the mixing of ingredients, that, the addition of exogenous enzymes (lipases 43 both a higher total volatile content and a slight and proteinases) from bacterial, fungal, vegetal, or 44 improvement in flavor was observed, without addi- animal origin has been assayed. The results showed 45 tion of any enzyme extract (Herranz et al. 2005). that higher amounts of compounds from hydrolytic 46 Another approach to enhance the flavor of dry saus- processes (e.g., amino acids and free fatty acids) 47 ages has been based on the fact that α-ketoglutarate were formed, but sensory analysis demonstrated that 48 is, as mentioned previously, the first step in amino solely, and only in some cases, a slight increase in 49 acid catabolism. In this sense, it has been described flavor was obtained. Accordingly, an approach based 50 (Herranz et al. 2004) as the effect of the addition of on the amino acid transformation has been also 51 α-ketoglutarate on both volatile substance generation investigated, which involves the enrichment of 52 and flavor. An increase in the glutamic acid content the matrix with aminotransferase and deaminase 53S was observed and, at the same time, an increase in activities by adding bacterial or fungal extracts or 54N 2-methyl propanal and 3-methyl butanal, which inoculating molds at the surface. It is expected that 40454 32 333-348 r3.qxd 7/20/07 4:12 PM Page 343

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Meat Sci C Garcia, JJ Berdague, T Antequera, C Lopez-Bote, JJ 51 34:205–216. Cordoba, J Ventanas. 1991. Volatile components of 52 ———. 1996. Effect of the addition of papain on the dry cured Iberian ham. Food Chem 41:23–32. 53S dry fermented sausage proteolysis. J Sci Food Agric ML Garcia, C Casas, VM Toledo, MD Selgas. 2001. 54N 71:13–21. Effect of selected mould strains on the sensory 40454 32 333-348 r3.qxd 7/20/07 4:12 PM Page 345

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properties of dry fermented sausages. Eur Food Res dry fermented sausages by the surface inoculation of 1 Technol 212:287–291. selected mould species. Eur Food Res Technol 220: 2 ML Garcia, MD Selgas, M Fernandez, JA Ordoñez. 494–501. 3 1992. Microorganisms and lipolysis in the ripening LL Hinrichsen, SB Pedersen. 1995. Relationship among 4 of dry fermented sausages. Inter J Food Sci Technol flavor, volatile compounds, chemical changes, and 5 27:675–682. microflora in Italian type dry-cured ham during 6 R Geisen. 1993. Fungal starter cultures for fermented processing. J Agric Food Chem 43:2932–2940. 7 foods: Molecular aspects. Trend Food Sci Technol K Incze. 1992. Raw fermented and dried meat products. 8 4:251–256. Fleischwirtschaft 72:1–5. 9 GG Giddings. 1977. Basis of color in muscle foods. PM Kenneally, G Schwarz, NG Fransen, EK Arendt. 10 CRC Crit Rev Food Sci Nutr 9:81–114. 1998. Lipolytic starter culture effects on production 11 CO Gill. 1986. The control of microbial spoilage in of free fatty acids in fermented sausages. J Food Sci 12 fresh meats. In: AM Pearson and TR Dutson, eds. 63:538–543. 13 Advances in Meat Research. Meat and Poultry L Kröckel. 1995. Bacterial fermentation of meat. 14 Microbiology. Westport, Connecticut: AVI Publish- In: G Campbell-Platt and PE Cook, eds. Fermented 15 ing Co., pp. 49–88. Meats. Glasgow: Blackie Academic and Profes- 16 CO Gill, KG Newton. 1977. The development of aero- sional, pp. 69–109. 17 bic spoilage flora on meat stored at chill tempera- BA Law, J Kolstad. 1983. Proteolytic systems in lactic- 18 tures. J Appl Bacteriol 43:189–195. acid bacteria. Antonie Van Leeuwenhoek. J Micro- 19 G Gottschalk. 1986. Bacterial Metabolism. New York: biol 49:225–245. 20 Springer-Verlag, pp. 214–224. L Leistner, C Eckardt. 1979. Occurrence of toxinogenic 21 BF Hagen, JL Berdague, AL Holck, H Næs, H Blom. Penicillia in meat-products. Fleischwirtschaft 59: 22 1996. Bacterial proteinase reduces maturation time 1892–1896. 23 of dry fermented sausages. J Food Sci 61:1024–1029. HU Liepe. 1982. Starter cultures in meat production. 24 WP Hammes, I Bosch, G Wolf. 1995. Contribution of In: HJ Rhem and G Reed, eds. Biotechnology. 25 Staphylococcus carnosus and Staphylococcus pis- Basilea: Verlag Chemie, pp. 169–174. 26 cifermentans to the fermentation of protein foods. MO López, L Hoz, MI Cambero, E Gallardo, G Reglero, 27 J Appl Bacteriol 79:S76–S83. JA Ordoñez. 1992. Volatile compounds of dry hams 28 AHW Hauschild, R Hilsheimer, G Jarvis, DP Raymond. from Iberian pig. Meat Sci 31:267–277. 29 1982. Contribution of nitrite to the control of FK Lücke. 1985. The microbiology of fermented 30 Clostridium botulinum in liver sausage. J Food Prot meats. J Sci Food Agric 36:1342–1343. 31 45:500–506. FK Lücke. 1998. Fermented sausages. In: BJB Wood, 32 B Herranz, M Fernandez, L de la Hoz, JA Ordoñez. ed. Microbiology of Food Fermentation. London: 33 2006. Use of bacterial extracts to enhance amino acid Appl Sci Publ pp. 441–483. 34 breakdown in dry fermented sausages. Meat Sci FK Lücke, H Hechelmann. 1987. Starter cultures for 35 72:318–325. dry sausages and raw ham composition and effect. 36 B Herranz, M Fernandez, E Hierro, JM Bruna, JA Fleischwirtschaft 67:307–314. 37 Ordoñez, L de la Hoz. 2004. Use of Lactococcus lactis B MacDonald, JI Gray, LN Gibbins. 1980. Role of 38 subsp cremoris NCDO 763 and alpha-ketoglutarate to nitrite in cured meat flavor. Antioxidant role of 39 improve the sensory quality of dry fermented saus- nitrite. J Food Sci 45:893–897. 40 ages. Meat Sci 66:151–163. P MacLeod, ME Morgan. 1955. Leucine metabolism 41 B Herranz, L de la Hoz, E Hierro, M Fernandez, JA of Streptococcus lactis var maltigenes .1. Conversion 42 Ordoñez. 2005. Improvement of the sensory proper- of alpha-ketoisocaproic acid to leucine and 43 ties of dry-fermented sausages by the addition of free 3-methylbutanal. J Dairy Sci 38:1208–1214. 44 amino acids. Food Chem 91:673–682. MAPA (Ministerio de Agricultura, Pesca y Alimentación). 45 E Hierro, L de la Hoz, JA Ordoñez. 1999. Contribution 1997. Inventario Español de productos tradicionales. 46 of the microbial and meat endogenous enzymes to Madrid: Servicio de Publicaciones del MAPA. 47 the free amino acid and amine contents of dry fer- L Martin, JJ Cordoba, T Antequera, ML Timon, J Ven- 48 mented sausages. J Agric Food Chem 47:1156–1161. tanas. 1998. Effects of salt and temperature on prote- 49 ———. 1997. Contribution of microbial and meat olysis during ripening of Iberian ham. Meat Sci 50 endogenous enzymes to the lipolysis of dry fer- 49:145–153. 51 mented sausages. J Agric Food Chem 45:2989–2995. J Mateo, JM Zumalacarregui. 1996. Volatile com- 52 E Hierro, J Ordoñez, JM Bruna, C Pin, M Fernandez, pounds in chorizo and their changes during ripening. S53 L de la Hoz. 2005. Volatile compound generation in Meat Sci 44:255–273. N54 40454 32 333-348 r3.qxd 7/20/07 4:12 PM Page 346

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1 PLH McSweeney. 2004. Biochemistry of cheese ripen- MA Paleari, L Piantoni, F Caloni. 1991. Prove sull 2 ing. Inter J Dairy Technol 57:127–144. impiego della lipasi nella preparazione dei salumi. 3 JA Melendo, JA Beltran, I Jaime, R Sancho, P Ind Alim 30:1072–1074. 4 Roncales. 1996. Limited proteolysis of myofibrillar SA Palumbo, JL Smith. 1977. Chemical and microbio- 5 proteins by bromelain decreases toughness of coarse logical changes during sausage fermentation and 6 dry sausage. Food Chem 57:429–433. ripening. In: L Orly and J St. Angelo, eds. ACS Sym- 7 A Meynier, E Novelli, R Chizzolini, E Zanardi, posium Series no. 47, pp. 279–294. 8 G Gandemer. 1999. Volatile compounds of commer- M Papon, R Talon. 1988. Factors affecting growth and 9 cial Milano salami. Meat Sci 51:175–183. lipase production by meat lactobacilli strains and 10 I Molina, F Toldrá. 1992. Detection of proteolytic activ- Brochothrix thermosphacta. J Appl Bacteriol 64: 11 ity in microorganisms isolated from dry-cured ham. 107–115. 12 J Food Sci 57:1308–1310. ———. M Papon, R Talon. 1989. Cell location and 13 K Molly, D Demeyer, T Civera, A Verplaetse. 1996. partial characterization of Brochothrix thermo- 14 Lipolysis in a Belgian sausage: Relative importance sphacta and Lactobacillus curvatus lipases. J Appl 15 of endogenous and bacterial enzymes. Meat Sci Bacteriol 66:235–242. 16 43:235–240. JJ Pestka. 1986. Fungi and mycotoxins in meats. In: 17 MC Montel, R Talon, JL Berdague, M Cantonnet. 1993. AM Pearson and TR Dutson, eds. G Advances in 18 Effects of starter cultures on the biochemical char- Meat Research. Meat and Poultry Microbiology. 19 acteristics of French dry sausages. Meat Sci 35: Westport: AVI Publishing, Vol 2, pp. 277–309. 20 229–240. MD Pierson, LA Smoot. 1982. Nitrite, nitrite alterna- 21 MC Montel, R Talon, M Cantonnet, J Fournaud. 1992. tives, and the control of Clostridium botulinum in 22 Identification of Staphylococcus from French dry cured meats. CRC Crit Rev Food Sci Nutr 17: 23 sausage. Lett Appl Microbiol 15:73–77. 141–187. 24 MJ Motilva, F Toldrá. 1993. Effect of curing agents and M Roca, K Incze. 1990. Fermented sausages. Food Rev 25 water activity on pork muscle and adipose subcuta- Inter 6:91–118. 26 neous tissue lipolytic activity. Z Lebens Unte Forsch J Samelis, J Metaxopoulus. 1997. Lipolytic activity of 27 196:228–232. meat lactobacilli isolated from naturally fermented 28 MJ Motilva, F Toldrá, P Nieto, J Flores. 1993. Muscle Greek dry sausage. Fleischwirtschaft 77:165–168. 29 lipolysis phenomena in the processing of dry cured B Sanz. 1988. El ayer, hoy y mañana de la Broma- 30 ham. Food Chem 48:121–125. tología. Madrid: Instituto de España. Real Academia 31 H Næs, AL Holck, L Axelsson, HJ Andersen, H Blom. de Farmacia, pp. 1–92. 32 1994. Accelerated ripening of dry fermented sausage B Sanz, D Selgas, I Parejo, JA Ordoñez. 1988. Charac- 33 by addition of a Lactobacillus proteinase. Inter J teristics of Lactobacilli isolated from dry fermented 34 Food Sci Technol 29:651–659. sausages. Inter J Food Microbiol 6:199–205. 35 H Næs, BO Pedersen, AL Holck, L Axelsson, V Holten, C Sarraga, M Gil, J Arnau, JM Monfort, R Cusso. 1989. 36 H Blom. 1992. Fermentation of dry sausage. The Effect of curing salt and phosphate on the activity of 37 effect of added proteinase and lipase from lacto- porcine muscle proteases. Meat Sci 25:241–249. 38 bacilli. Proc 38th Inter Cong Meat Sci Technol, Cler- K Sato, GR Hegarty. 1971. Warmed-over flavor in 39 mond Ferrand, pp. 815–818. cooked meat. J Food Sci 36:1098–1102. 40 J Nordal, E Slinde. 1980. Characteristics of some S Schmidt, RG Berger. 1998. Aroma compounds in fer- 41 lactic acid bacteria used as starter cultures in dry mented sausages of different origins. Food Sci Tech- 42 sausage production. Appl Environ Microbiol nol Lebens Wissen Technol 31:559–567. 43 40:472–475. MD Selgas, ML Garcia, GD Garcia de Fernando, JA 44 GJE Nychas, JS Arkoudelos. 1990. Staphylococci. Ordoñez. 1993. Lipolytic and proteolytic activities 45 Their role in fermented sausages. J Appl Bacteriol of micrococci isolated from dry sausages. Fleis- 46 69:S167–S188. chwirtschaft 73:1175–1176, 1179. 47 JA Ordoñez, EM Hierro, JM Bruna, L de la Hoz. 1999. MD Selgas, B Sanz, JA Ordoñez. 1988. Selected char- 48 Changes in the components of dry-fermented acteristics of micrococci isolated from Spanish dry 49 sausages during ripening. CRC Crit Rev Food Sci fermented sausages. Food Microbiol 5:185–193. 50 Nutr 39:329–367. BJ Singh, D Dincho. 1994. Molds as protective cultures 51 JA Ordoñez, MO Lopez, E Hierro, MI Cambero, for raw dry sausages. J Food Prot 57:928–930. 52 L de la Hoz. 1996. Efecto de la dieta de cerdos G Smit, A Verheul, R van Kranenburg, E Ayad, R 53S ibéricos en la composición en ácidos grasos del Siezen, W Engels. 2000. Cheese flavour development 54N tejido adiposo y muscular. Food Sci Technol Inter by enzymatic conversions of peptides and amino 2:383–390. acids. Food Res Inter 33:153–160. 40454 32 333-348 r3.qxd 7/20/07 4:12 PM Page 347

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LH Stahnke, A Holck, A Jensen, A Nilsen, E Zanardi. M Yvon, S Berthelot, JC Gripon. 1998. Adding alpha- 1 2000. Flavour compounds related to maturity of ketoglutarate to semi-hard cheese curd highly 2 dried fermented sausage. Proc 46th Inter Cong Meat enhances the conversion of amino acids to aroma 3 Sci Technol, Buenos Aires, pp. 236–237. compounds. Inter Dairy J 8:889–898. 4 ———. 2002. Maturity acceleration of Italian dried M Yvon, E Chambellon, A Bolotin, F Roudot-Algaron. 5 sausage by Staphylococcus carnosus. Relationship 2000. Characterization and role of the branched 6 between maturity and flavor compounds. J Food Sci chain aminotransferase (BcaT) isolated from Lacto- 7 67:1914–1921. coccus lactis subsp. cremoris NCDO 763. Appl Env- 8 LH Stahnke, P Zeuthen, 1992. Identification of volatiles iron Microbiol 66:571–577. 9 from Italian dried salami. Proc 38th Inter Cong Meat M Yvon, S Thirouin, L Rijnen, D Fromentier, JC Gripon. 10 Sci Technol, Clermond Ferrand, pp. 835–838. 1997. An aminotransferase from Lactococcus lactis 11 LO Sunesen, LH Stahnke. 2003. Mould starter cultures initiates conversion of amino acids to cheese flavor 12 for dry sausages—Selection, application and effects. compounds. Appl Environ Microbiol 63:414–419. 13 Meat Sci 65:935–948. I Zalacain, MJ Zapelena, I Astiasarán, J Bello. 1995. 14 R Talon, MC Montel, G Gandemer, M Viau, Dry fermented sausages elaborated with Lipase from 15 M Cantonnet. 1992. Lipolysis of pork fat by Staphy- Candida cylindracea. Comparison with traditional 16 lococcus warneri, S. saprophyticus and Micrococcus formulations. Meat Sci 40:55–61. 17 varians. Appl Microbiol Biotechnol 38:606–609. ———. 1996. Addition of lipase from Candida cylin- 18 K Tjener, LH Stahnke, L Andersen, J Martinussen. dracea to a traditional formulation of a dry fer- 19 2004. Addition of alpha-ketoglutarate enhances for- mented sausage. Meat Sci 42:155–163. 20 mation of volatiles by Staphylococcus carnosus dur- I Zalacain, MJ Zapelena, MP de Peña, I Astiasarán, 21 ing sausage fermentation. Meat Sci 67:711–719. J Bello. 1997a. Application of lipozyme 10,000 L 22 F Toldrá. 2002. Dry-cured Meat Products. Trumbull, (from Rhizomucor miehei) in dry fermented sausage 23 Co: Food & Nutr Press, Inc., pp. 89–148. technology: Study in a pilot plant and at the industrial 24 F Toldrá, MJ Motilva, E Rico, J Flores. 1991. Enzyme level. J Agric Food Chem 45:1972–1976. 25 activities in the processing of dry-cured ham. Proc ———. 1997b. Lipid fractions of dry fermented 26 37th Inter Cong Meat Sci Technol. 1–6. Kulmbach, sausages change when starter culture and/or Asper- 27 Germany, pp. 954–957. gillus lipase are added. J Food Sci 62:1076–1079. 28 F Toldrá, E Rico, J Flores. 1992. Activities of pork ———. 1997c. Use of lipase from Rhizomucor miehei 29 muscle proteases in model cured meat systems. in dry fermented sausages elaboration: Microbial, 30 Biochimie 74:291–296. chemical and sensory analysis. Meat Sci 5:99–105. 31 ———. 1993. Cathepsin-B, cathepsin-D, cathepsin-H MJ Zapelena, D Ansorena, I Zalacain, I Astiasarán, 32 and cathepsin-L activities in the processing of dry J Bello. 1998. Dry fermented sausages manufactured 33 cured ham. J Sci Food Agric 62:157–161. with different amounts of commercial proteinases: 34 SO Tomek, A Bulgay. 1991. Influence of environmental Evolution of total free alpha-NH2-N groups and 35 RH on some physical and chemical properties of sensory evaluation of the texture. Meat Sci 36 Turkish sukuk. Proc 37th Inter Cong Meat Sci Tech- 49:213–221. 37 nol 1–6. Kulmbach, Germany, pp. 958–961. MJ Zapelena, I Astiasarán, J Bello. 1999. Dry fer- 38 J Ventanas, JJ Cordoba, T Antequera, C Garcia, C mented sausages made with a protease from 39 Lopezbote, MA Asensio. 1992. Hydrolysis and mail- Aspergillus oryzae and or a starter culture. Meat Sci 40 lard reactions during ripening of Iberian ham. J Food 52:403–409. 41 Sci 57:813–815. MJ Zapelena, I Zalacain, MP de Peña, I Astiasarán, 42 A Verplaetse, D Demeyer, S Gerard, E Buys. Endoge- J Bello. 1997. Effect of the addition of a neutral pro- 43 nous and bacterial proteolysis in dry sausage fermen- teinase from Bacillus subtilis (Neutrase) on nitrogen 44 tation. Pro 38th Inter Cong Meat Sci Technol, fractions and texture of Spanish fermented sausage. 45 Clermond Ferrand, 1992, pp. 851–854. J Agric Food Chem 45:2798–2801. 46 47 48 49 50 51 52 S53 N54 40454 33 349-358 r2.qxd 7/20/07 4:14 PM Page 349

1 2 3 33 4 5 6 North European Products 7 8 Jürgen Schwing and Ralf Neidhardt 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION a relatively short time and which leads to a distinct 22 acidic flavor, though astringency and stingy mouth- 23 This chapter covers the dry sausage traditions of feel should be avoided. This meat batter resembles 24 northern Europe; the quality aspects of dry-fermented the meat batter of Milan salami very much, with its 25 sausages, the ingredients and processing technolo- very fine grain size (Löbel 1989), hence the habit of 26 gies commonly used, and the characteristic recipes the English-speaking world to call all fermented 27 of various countries. Of course, the great number of sausages “salami.” The same is done in this chapter. 28 products makes it impossible to give an exhaustive Molded sausages are still of relatively little impor- 29 overview, also it is generally acknowledged that a tance in northern Europe, even if the market trend 30 great number of names and designation very often seems to point toward a milder, more rounded 31 cover the same type of product or a very similar prod- Mediterranean sausage flavor. 32 uct. This makes it particularly difficult to describe 33 differences. 34 QUALITY CRITERIA Northern Europe represents a huge number of 35 sausages and production traditions in the following The definition of quality is quite difficult, because it 36 countries: Benelux, Scandinavia, Germany, Switzer- depends not only on objective criteria laid down in a 37 land, Hungary, Poland, Russia, Baltic countries, written form and measurable, but depends as well 38 Bulgaria, Romania, Czech Republic, Slovakia, on subjective perceptions linked to cultural back- 39 Austria, Slovenia, Ukraine, Serbia, Croatia, England, ground and history. Hofmann (1974) defined quality 40 Ireland, etc. Some of these countries produce fer- as the summary of all quality-relevant parameters. 41 mented sausages, which can be classified either as For administrative safety, measurable parameters 42 north European or as south European depending on like pH, water activity, and microbiology aspects are 43 the consumers’ appreciation of the quality character- relevant quality parameters. In addition, protein/fat 44 istics: however, this chapter will deal with only the ratio, connective tissue, water binding capacity, and 45 typical northern types. type of fat of the raw materials are important for 46 processors. Of course the consumer expects good 47 CHARACTERISTICS OF NORTH flavor, good mouth-feel, and consistency on top of 48 EUROPEAN SAUSAGES the safety aspects. 49 A long tradition for producing dry-fermented 50 In general, north European sausages are smoked and sausages in northern Europe exists with a great 51 lower in pH than south European sausages. The number of product names. Some are very similar in 52 majority of the sausages are prepared with a finely appearance; others are very different in their features 53 minced meat batter, which is fermented and dried in (BAFF 1985). The following characteristics are used S54 N55 349 40454 33 349-358 r2.qxd 7/20/07 4:14 PM Page 350

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1 in order to describe the quality of a dry-fermented Dry Ingredients 2 sausage: The addition of nitrite is important for the color for- 3 • General appearance (color, molded, smoked, mation and for the microbiological stability, espe- 4 coated, etc.) cially in the first days of ripening (Leistner and Gould 5 • Caliber of casing/type of casing 2002). Furthermore, nitrite participates in the flavor 6 • Grain size of meat and fat particles formation. Nitrite is added to nearly all north Euro- 7 • Meat composition (lean, fat, connective tissue, pean fermented sausages in a mixture with common 8 others) salt; only a few specialties are made with nitrate. 9 • Degree of fattiness and quality of fat (color, taste) To achieve a proper acidification and an excellent 10 • Salt contents (type of salt, level, and health flavor and to improve and stabilize color, the use of 11 aspects) bacterial starter cultures is widely spread in northern 12 • Color (added colorant, natural color formation) Europe (Hansen 2003). As compared to southern 13 • Aroma (smell of smoke, molds, yeasts, Europe, processors in northern Europe mostly use 14 fermentation, etc.) starter cultures, which results in a fast pH drop. Due 15 • Taste features (acidity level, fermentation flavor, to the fast starter cultures, the use of GDL (glucono- 16 maturity) delta-lactone) is becoming less common. 17 • Texture (firmness, elasticity, crumbliness, etc.) Sugar is primarily added as an energy source for 18 the starter cultures. Glucose, maltose, sucrose, and 19 derivatives from starch hydrolysis are predominantly 20 SAUSAGE INGREDIENTS used. In the past, sucrose was the most commonly 21 Raw Materials used sugar because it was easy to get hold of. Now it 22 is mostly substituted by glucose to obtain a fast pH 23 Dry, red meat from older animals is preferred, but drop. Lactose becomes more and more unpopular 24 sometimes cooked pork skin is used as well. The raw for allergenic reasons, in particular in Germany. It is 25 material is usually used fresh and/or frozen. Pork important that the sugar type corresponds to the 26 meat must fulfill the following criteria: pH >5.8 and 1 needs of the specific starter culture, especially 27 pH Ͻ5.8; if chilled at 0°C, meat must be used 24 when fast fermentation is targeted (Hansen 2003). 28 within 3 to 5 days postmortem; if frozen at −30°C Carbohydrates like potatoes, potato starch, or potato 29 to 18°C, meat must be stored for a maximum of flakes are added in specific sausage types (see 30 90 days. For beef, the following criteria must be met: Swedish Mettwurst) and are responsible for the char- 31 pH Ͻ5.8; if chilled at 0°C, meat must be used 24 acteristic acidification profile, flavor, and texture of 32 within 3 to 14 days postmortem; if frozen at −30°C those products. 33 to 18°C, frozen storage should be a maximum of The most important spice for north European 34 180 days (Fischer 1988). salami is pepper (Piper Nigrum L.). White pepper is 35 The fatty part is usually from pork, which has to usually used as fine ground pepper and black as 36 be fresh, white, and firm to avoid strong “oiling out” coarsely ground pepper. Other spices are ginger, 37 and rancidity. The shelf life of a fermented sausage cardamom, garlic, coriander, cumin, mace, paprika, 38 is often limited by changes in the fat. The best fat is rosemary, nutmeg, and clove (especially in northern 39 from neck or back of the pork carcass. The amount Holland); sweet peppers or mustard seeds are used in 40 of polyunsaturated fatty acids should be below 12%. Hungarian products (see below). 41 The fat must fulfill the following: well chilled before 42 freezing; chilled Ͻ2°C, stored for a maximum of 43 3 days; frozen at −30°C to −18°C, storage for a PROCESSING TECHNOLOGY 44 maximum of 90 days (Fischer 1988). 45 Meat from poultry should be dry and dark; there- The microbiological stability of a north European 46 fore, meat from the leg is preferred. Poultry fat is sausage is based on low pH, low water activity, and 47 problematic for its softness and may lead to strong smoke application externally (Potthast and Eigner 48 oiling out. Horse, reindeer, and venison meat is used 1988). Traditionally, fermented sausage processors 49 for specialties. These meats need, in general, an did not use cultures, and they produced under cli- 50 addition of backfat in order to obtain good finished matic conditions, which were strongly influenced by 51 fermented products. The fermentation profile has to the weather of the moment. Therefore, sausages 52 be adjusted as compared to products with pork and were often dried extensively to make the sausages 53S beef because these meats have initially high pH well safe. Now the use of starter cultures and controlled 54N over pH 6.2. fermentation and drying conditions result in a 40454 33 349-358 r2.qxd 7/20/07 4:14 PM Page 351

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controlled pH drop and an optimization of water • Fast fermentation: fermentation temperature 1 loss. The hurdle principle ensures that a sausage 22–26°C, less than 30 hours to archive pH 5.3, 2 technology can be considered safe even when drying final pH usually 4.5–4.9, final water activity 3 is less extensive than in the past (Stiebing 1993). Ͼ0.90, total production time 2–3 weeks 4 (Neuhäuser 1988). 5 Meat Batter Preparation • Very fast American-style fermentation: 6 The most important comminution technology in fermentation temperature 32–43°C, less than 7 15 hours to archive pH 5.3, final pH Ͻ4.8, final 8 northern Europe in fermented sausage production is Ͼ the combined chopping and mixing technology of water activity 0.90, total production time 9 the bowl chopper. The standard procedure is the 1–3 weeks, cooking often applied to stop 10 following: Frozen lean meat is chopped coarsely in fermentation and/or increase safety (Bacus 1984). 11 12 the bowl chopper. All ingredients except salt are The boundaries between the three technologies over- 13 added. Chopping continues to desired particle size lap, but in northern Europe the fast fermentation is 14 (1–4 mm), frozen fat (−18°C, 0°F) is added, chop- the most used technology now. Due to less technical 15 ping continues to the final particle size of the end possibilities and a smaller range of available starter 16 product, and fresh, minced meat is supplied and cultures, the slower traditional fermentation was 17 mixed in homogeneously. At the very end, salt is used in the past. Other technologies, such as ripen- 18 added and minimal blending is applied to ensure a ing in brine or ripening under vacuum and/or pulsat- 19 homogeneous distribution. The final batter must ing atmospheric pressure, disappeared mostly when 20 have a temperature not higher than about −4°C the use of industrial fermentation equipment became 21 (25°F). Smearing has to be avoided as much as more widespread (Neuhäuser 1988). In fact, the 22 possible during the subsequent stuffing procedure machinery plays a crucial role in modern sausage 23 (Stiebing 1993). making, and this role is very often underestimated. 24 This is especially true in the area of fermentation 25 Fermentation and Drying equipment (Stiebing 1993). The ripening scheme in 26 Table 33.1 shows a standard process for fermenta- After stuffing, the sausages are fermented and 27 tion and drying in northern Europe. dried/ripened by applying the appropriate tempera- 28 ture, humidity, and speed of air (circulation) with 29 respect to the wanted end product (Stiebing and SAUSAGE TYPES OF CENTRAL 30 Rödel 1987). Roughly, the fermentation pattern may EUROPE 31 be divided into three schemes: 32 GERMANY • Traditional fermentation: fermentation temp- 33 erature usually 18–24°C, more than 40 hours to German sausage quality is mainly characterized by 34 achieve pH 5.3, final pH often >5.0, final water the usage of valuable parts of the carcass (ham, belly, 35 activity Ͻ0.90, total production time >3 weeks trimmings, etc.); hence, the consumer does not con- 36 (BAFF 1990). sider the stage of drying as an obligingly important 37 38 39 Table 33.1. Standard ripening program for north European technology. 40 41 Time (Hours) Temperature (°C/°F) RH* (%) Smoking 42 4 25/77 – 43 44 24 24/75 94 45 24 22/72 92 Moderate 1–2 h 46 24 20/68 90 47 48 24 20/68 88 Moderate 1–2 h 49 24 18/64 86 50 24 18/64 84 Moderate 1–2 h 51 14–16/57–61 78–82 Postripening 52 S53 * RH ϭ relative humidity. N54 40454 33 349-358 r2.qxd 7/20/07 4:14 PM Page 352

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1 feature of quality assessment. Yet in some areas and The following recipe represents the standard recipe. 2 in some products, this is the case, e.g., for landjäger The meat block: 40% frozen, lean sow meat, 40% 3 sausage in south Germany. Traditionally, the Ger- chilled, lean pork meat, and 20% frozen backfat. 4 man butcher chooses good raw materials as a basis Further ingredients are nitrite salt, pepper, garlic, 5 for successful production. Many recipes are based sugars, starter cultures, and sometimes, mustard 6 on pork, beef, and backfat of excellent quality seeds. The standard production procedure is as fol- 7 (Löbel 1989). The typical characteristics are: lows: Sow meat is chopped coarsely in the bowl 8 chopper and dry ingredients except salt are added. • Northwestern Germany: strongly smoked, 9 Further comminution continues to a particle size of relatively soft sliceable or spreadable products 10 approximately 2 mm. Fat is put in and minced to a having soft fats and a “fresh meat” characteristic 11 particle size of 3–4 mm. Pork meat, processed with mild, low acid features. 12 through a 2–3 mm plate, is added, and the whole • Westphalia: smoked, firm, and sliceable products 13 mixture is chopped to the desired particle size. Dur- with a distinct sour feature; ideally there is a 14 ing the last rounds of chopping, nitrite salt is added distinct fermentation flavor; very often a fast 15 and mixed in homogenously. The mince is stuffed technology is used and sausages are stuffed in big 16 into casings and ripened at 24°C at 95% relative diameter casings with limited drying. 17 humidity (RH) in the beginning. The temperature is • Southern Germany: mildly smoked, well- 18 lowered to 12–14°C and the humidity to about dried products with only very mild acidic 19 80% (Ϯ2) step by step, until a water loss of approxi- characteristics; sausages may be molded 20 mately 25% is obtained. instead of smoked. 21 Schlackwurst has a very fine particle size and its • Eastern Germany: mildly smoked, well-dried 22 name originates from the casing type used (colon or products with low acidic features and special 23 Schlacke in German). The basic meat block is 24% characteristic spice combinations (cumin). 24 lean beef, 10% lean pork, and 66% pork belly. Typi- 25 German specialties are Teewurst, Thuringer Bratwurst cal ingredients are nitrite salt, pepper, and honey. 26 (raw fermented type of semidry coarse grained There are several regional differences. Braun- 27 sausage), Cervelatwurst, Feldkieker, Eichsfelder schweiger Schlackwurst contains only pork meat 28 (based on freshly slaughtered meat), Katenrauch- and backfat instead of pork belly. The spices are 29 wurst, etc. pepper, ginger, and red wine. Thüringer Schlack- 30 The most-often sold German salami product is a wurst contains pepper and cardamom. Westfälische 31 sliced large salami type called Stapelpack salami, Schlackwurst is made of 20% beef, 20% pork belly, 32 which is based on Westphalian salami (Figure 33.1). 20% backfat, and 40% very lean pork. The season- 33 Both the meat block and the seasoning of the stan- ings are based on pepper, ginger, raspberry syrup, 34 dard Westphalian salami vary between producers. rum, and mustard seeds. 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53S 54N Figure 33.1. Stapelpack salami from Germany. 40454 33 349-358 r2.qxd 7/20/07 4:14 PM Page 353

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Cervelatwurst has as fine a particle size as smoke flavor. A typical ripening scheme is the fol- 1 Schlackwurst or even finer, but it is stuffed in lowing: Press in a rectangular shape for 24–48 hours 2 different casings. The origin of Cervelatwurst is a at 24°C, 94% RH, the meat hanging on sticks, fol- 3 cooked Milan-type of salami from Italy. The meat lowed by continued ripening for 24 hours at 20°C, 4 block for this high-quality product is 30% very lean 88% RH and 48 hours at 18°C, 82% RH with occa- 5 beef, 46% lean pork, and 24% backfat. Typical sea- sional smoking; and further dry at 14–16°C, 6 sonings are ground pepper, pepper seeds, and 76–78% RH to a weight loss of 35%. 7 juniper brandy, but different recipes exist in each Teewurst is a high-quality, spreadable raw 8 region of Germany, such as Westphalia, Thüringen, sausage (Figure 33.2). It can be divided into two 9 or Holstein. One main recipe variation stems from groups: coarsely grained Teewurst (3–4 mm particle 10 the use of different brandy types like rum, whiskey, size) and finely minced Teewurst (no visual par- 11 or Steinhäger, which is based on herbal extracts. ticles, creamy appearance). Teewurst must not have 12 Feldkieker is a specialty from a region around a firm structure, and therefore the meat particles 13 the town of Göttingen. The special feature of this should be surrounded by fat particles or an oily fat 14 product is the use of just-slaughtered warm meat phase to avoid protein coagulation. In addition, 15 (derived from the traditional sausage making tech- pH should not be lowered below 5.3. The use of 16 nique when cold stores were not available and meat about 3% sunflower oil helps maintain the soft 17 had to be preserved immediately after slaughtering). texture. The water loss is usually 5–10%. A typical 18 The 70% lean pork and 30% backfat are minced processing scheme is the following: The meat block 19 through a 2 mm plate and mixed with salt, pepper, (2 mm) contains 20% lean beef, 60% lean pork, 20 raspberry syrup, honey, and red wine or rum. The 17% backfat, and 3% sunflower oil; meat tem- 21 mince is stuffed into a calf’s bladder and ripened for perature is −2°C to ϩ2°C. Backfat is chopped to a 22 8–12 months. The sausage is either air-dried or cream in the bowl chopper. Meat, sunflower oil, and 23 smoked. all other ingredients except salt are added. The 24 Landjäger is a sausage from the south of blend is chopped with high speed until 15°C to 25 Germany. The origin is from the region of Voralberg desired consistency. Salt is added while chopping 26 in Switzerland, Elsaβ in France, and Schwarzwald in at low speed in order to reach a homogenous dis- 27 Germany. Landjäger is cased in small pork intestine tribution of salt. The end temperature should be 28 and gets its characteristic rectangular shape from a approximately 18°C. After filling, the ripening 29 special pressing step during fermentation. The meat scheme is the following: 2 days at 20°C/85–90% 30 block is based on beef rich in collagen tissue, pork, RH; smoking at 20°C, 85% RH until the desired 31 and backfat. Typical spices are pepper and cumin. In golden color is obtained, followed by 6 h at 26°C, 32 some recipes, parts of the beef are substituted with 80% RH to obtain a slight film of fat and further 33 smoked cured ham, which results in a very strong ripening at 14°C, 78–80% RH. 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 S53 Figure 33.2. German Teewurst. N54 40454 33 349-358 r2.qxd 7/20/07 4:14 PM Page 354

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1 AUSTRIA fresh. The frozen meat is ground to 3 mm in the bowl 2 chopper. Halfway through this process, the backfat Austrian sausages are very similar to the German 3 is added and while comminuting further the fresh salamis; however, a typical Austrian product is 4 meat part is put on top of the blend. Salt is added Kantwurst. Kantwurst gets its characteristic square 5 close to the end. The final mince has a meat particle shape and unique taste from a special pressing and 6 size of about 3 mm with larger particles of backfat curing process close to traditional Bavarian technol- 7 (3–5 mm). The mince is stuffed in a natural pork ogy. Kantwurst is cured for 7 weeks and reaches a 8 casing formed into a ring of approximately 400 g. weight loss of about 35%. Traditionally, Kantwurst 9 Drying normally takes 15–20 days and results in a could be kept for a long time when stored in a cool 10 weight loss of 25–30%. The standard product is and dry place. 11 treated with smoke. Seasonings are pepper, corian- 12 der, mustard, and salt. In the northern part of the 13 THE NETHERLANDS (AND FLEMISH-SPEAKING Netherlands, ginger and clove are added as a spe- 14 BELGIUM) cialty and the product is dried to about 40%. 15 The Dutch salami always has added garlic. The Dutch dry sausages can be divided into two major 16 meat block contains 10% lean beef, 40% pork meat, groups: spreadable and nonspreadable sausages, 17 10% belly, and 30% backfat. Normally, the meat is the latter having about two-thirds of the market. The 18 80% frozen and 20% fresh. Sometimes about 10% traditional process is slow, aiming at a very aromatic 19 ground pork skin is added for a softer texture. The fermentation flavor. Typical Dutch dry sausages 20 frozen meat is ground to 3 mm in the bowl chopper. are Boerenmetworst, salami and cervelat (Dutch: 21 Halfway through this process, the backfat is added snijworst). 22 and while comminuting further the fresh meat part is The most common product of Boerenmetworst is 23 put on top of the blend. Salt is added close to the end. without added garlic (Figure 33.3). The meat block 24 The final mince has a uniform particle size of 3–5 contains 20% lean beef, 50% pork meat, and 30% 25 mm. The grain size could be smaller in the western backfat. Normally, the meat is 80% frozen and 20% 26 part of the country. Sometimes the particle size is as 27 fine as 0.5–1 mm. The sausages are normally stuffed 28 into a beef casing of 90–110 mm diameter to a total 29 sausage weight of 4.5–5 kg. The drying and ripening 30 takes around 30 days, and a weight loss of 25–30% 31 is common. The normal product is nonsmoked. 32 Seasonings are pepper, garlic, red wine, and green 33 pepper (sometimes visible). In the southern part 34 of the Netherlands, the sausages are more of the 35 French type with respect to spices and application 36 of molds. 37 Snijworst (Dutch cervelat) is always smoked and 38 very close to some German products. The spices are 39 pepper, nutmeg, mace, and coriander. In the south of 40 the country, the product is more coarse-grained and 41 beef replaces part of the pork. The meat block con- 42 tains 10% lean beef, 40% pork meat, 10% belly, and 43 30% backfat. Normally, the meat and fat is added 44 frozen to the bowl chopper and ground to a uniform 45 size of 3–5 mm. The sausages are normally stuffed 46 into a beef casing of 90–110 mm diameter to a total 47 sausage weight of 4.5–5 kg. The drying and ripening 48 takes around 30 days, and a weight loss of 25–30% is 49 common. 50 51 BELGIUM AND LUXEMBOURG 52 53S Belgium salami is a smoked sausage similar to 54N Figure 33.3. Dutch Boerenmetworst. the Dutch salami. The special Baguette salami in 40454 33 349-358 r2.qxd 7/20/07 4:14 PM Page 355

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Belgium is a nonsmoked sausage, which resembles a and it is a typical feature of the product that droplets 1 Mediterranean product. of free oil form on the slice surface during slicing 2 (Incze 1986). 3 Szegediner salami is made of beef, pork, and back- 4 IRELAND AND ENGLAND fat, but sometimes the recipe can be on a pork-only 5 Ireland and England do not have a real fermented basis. The traditional casing was the big horse intes- 6 sausage tradition, but today American-style pepper- tine; today the stuffing is done in a 60 mm fibrous 7 oni and continental-style products are produced in casing. After the classical cold smoking period for 8 both countries in small amounts. 1 day, the sausages are fermented for 3–4 days. The 9 target of 30% weight loss is reached in 3–4 months. 10 The salamis acquire a nice whitish grey mold cover 11 SAUSAGE TYPES OF EASTERN over time and have a firm to very firm texture and 12 EUROPE excellent keeping qualities. Usually, processors inoc- 13 ulate only the ripening chamber with molds and not 14 HUNGARY the sausages directly (dipping or spraying). 15 Hungarian salami is one of the two world trade- Budapest salami is a sausage based on 66% lean 16 names for salami (Figure 33.4). The other is Milano well-trimmed pork and 34% backfat. Its production 17 salami. Hungarian company tradenames like Pick is very similar to Szegediner salami. 18 from Szeged or Herz from Budapest have carried the Hot Kolbász is a pure pork or a pork/beef product 19 fame of Hungarian sausage making all over the with hot spices, coarsely ground, stuffed into 28–30 20 world. The Hungarian salami is one of the rare prod- mm casings, fermented for 3–4 days, smoked for 1 21 ucts combining smoke and mold application. In day, and dried afterward according to desired weight 22 original Hungarian salami, sugars and starter cul- loss. This can take up to 6 weeks; the normal time is 23 tures are still not allowed. The traditional technology 2–3 weeks. The end products weigh 200 to 250 g. 24 was based on the Italian predrying technique: After a Typical spices used in Kolbász are white pepper, 25 coarse grinding (6 mm), the meat was stored at low hot and sweet paprika, cumin, coriander, and garlic. 26 temperature for 1 day, followed by a second and final Kolbász is not mold covered. 27 grinding (2.6–3.2 mm). The sausages were stuffed 28 and hung for smoking over open fires. The pH did 29 BULGARIA not drop below 5.5 during processing (Incze 1986). 30 The seasonings used for Hungarian salami comprise Bulgaria has a long-standing tradition for making 31 white pepper, hot and sweet peppers, Tokay wine, fermented meat products under naturally well-suited 32 garlic, etc. The flavor profile of traditional Hungar- climate conditions. One of the outstanding products 33 ian salami should not contain any acidic notes at all is Loukanka, with its traditionally flat shape, very 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 S53 Figure 33.4. Hungarian salami. N54 40454 33 349-358 r2.qxd 7/20/07 4:14 PM Page 356

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1 dry and slightly crumbly texture, and extremely mild often cooked to an internal temperature of 68–70°C 2 fermentation flavor (total absence of acidic note). after production. A typical Polish sausage type is 3 Loukanka is a finely grained lean sausage with good Polska. Traditionally, this sausage was not fer- 4 definition of the particles and with a typical cumin- mented, but now manufacturers use starter cultures 5 pepper spice background in addition to the normally to achieve a pH drop. Pork meat is minced through 6 strong garlic flavor. It is mainly made from pork only an 8 mm plate and mixed with all other ingredients. 7 and stuffed into casings of 28–30 mm. The flat shape Afterward, the mince is stuffed into natural casing 8 is obtained by pressing the sausage during the initial (30–32 mm) longer than 35 cm, ripened at 6°C for 9 mild fermentation cycle; a typical pH curve should 2 days and smoked at 22°C for about 2 days. The 10 not go below 5.3. When pressing is done too late, the finished Polska is still quite soft due to the limited 11 sausage forms crevices inside and the end product drying. 12 is very often spoiled. Its strong drying leads to 13 extremely good keeping. Other Bulgarian specialties RUSSIA 14 are Ambaritsa, Babek, and Kalofer. 15 Typical Russian fermented sausages with a long 16 CZECH REPUBLIC AND SLOVAKIA tradition are Salami Moscow-type and Salami 17 Russian-type. In Russia, the product must lose In Slovakia, German-type salamis with mild acidity 18 40% weight, and the pH must be 5.0 (Ϯ0.1). Salami and good fermentation flavor are common. A lightly 19 Moscow-type is made of 25% backfat and 75% lean smoked, well-rounded flavor profile is typical. On 20 beef. The particularity is the big size of the fat the other hand, in the Czech Republic, sausages 21 (7–8 mm). The mince is stuffed in 45 mm casings, are traditionally German-type salamis with very 22 and the product is finished when a weight loss of strong smoke features and relatively fatty meat bat- 23 45% is obtained. The surface of the sausage is quite ters (Figure 33.5). Furthermore, the use of clove is 24 rough due to the big fat pieces, which have less very typical for Czech salamis. Hercules salami, 25 shrinkage than the lean part. Polican, and Paprikas, are the most important ones. 26 Russian Salami is made in two different qualities. A specialty is Lovecky salami, which has a charac- 27 The lean type is made of 30% backfat and 70% lean teristic rectangular shape from a special pressing. 28 meat, which can be pork, beef, or a combination of 29 the two (50/50). The standard type is made of 50% POLAND 30 backfat, 25% lean pork, and 25% lean beef. Both 31 Most Polish salamis are more or less German-type types have a particle size of 3 mm, are stuffed in 45 32 salamis, but for safety reasons the sausages are very mm casings, and have a weight loss of 40%. 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53S 54N Figure 33.5. Czech dry sausages. 40454 33 349-358 r2.qxd 7/20/07 4:14 PM Page 357

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SAUSAGE TYPES OF mutton, lamb, goat, horse, offal (liver, heart), blood, 1 SCANDINAVIA reindeer, moose, etc. A large range of so-called Morr 2 sausages are produced based on old traditional 3 DENMARK recipes. As an example Fårepølse contains mutton 4 Danish dry-fermented sausages basically cover two meat, goat, and beef, and Stabbur contains different 5 sausage types: the red-colored salami and the meats that could be pork, beef, horse, mutton, etc. 6 spegepølse that exist in many different qualities. Traditionally dry sausages from sheep and salamis 7 The typical Danish salami sausage is smoked and with meat from wild animals are produced all over 8 is characterized by added color and finely minced Scandinavia. The use of meat from free-ranging ani- 9 meat with a high fat content of up to 55% fat in the mals and the addition sometimes of blood to the 10 finished product. The meat block contains 55% sausages gives a special strong flavor to the end 11 pork/beef and 45% backfat, and typical ingredients products. Variation in quality is quite frequent 12 are nitrite salt, potato starch, maltodextrin, glucose, because there is a natural spread in the quality of raw 13 white pepper, garlic powder, sodium ascorbate, materials under these conditions. 14 carmine, and starter culture. The meat is chopped 15 16 coarsely in a bowl chopper, dry ingredients except FINLAND salt are mixed thoroughly into the minced meat, and 17 the coarsely chopped fat, nitrite salt, and salt are The two famous and most important Finish fer- 18 mixed in at the end. The mince is stuffed into 60–120 mented sausages are Kotimainen and Venäläinen. In 19 mm collagen casings and the sausages fermented general, these salamis are quite sour and acidic 20 and dried until the desired water loss is achieved. (pH 4.6–4.9), and they have a strong smoke taste. 21 The second type of Danish fermented sausage is The particle size and the fat contents are of major 22 similar to German Westphalian salami in production importance. Products with a lot of visual fat are 23 technology, but is often more fatty, more acidic, and unpopular in Finland. The fat contents of salamis are 24 less aromatic. The sausages are smoked and the usually lower than 20% and the particle size is about 25 degree of comminution may vary from coarse (6 mm) 2 mm. Technology and ripening is according to the 26 to fine (2 mm). Often, juniper berries are used for German procedure. Dominant spices are pepper and 27 smoking to obtain a special flavor. A special smoked garlic. 28 taste called eel-smoked is obtained when the backfat 29 is smoked before freezing. The meat block contains, 30 typically, 1/3 pork, 1/3 beef, and 1/3 backfat. REFERENCES 31 32 J Bacus. 1984. Utilization of microorganisms in meat 33 SWEDEN processing. A handbook for meat plant operators. 34 Swedish Medwurst is characterized by its content of Letchworth, UK: Research Studies Press Ltd, 35 boiled potatoes (usually 10–20%) or potato flakes. pp. 1–170. 36 Quite often a high fermentation temperature, such as BAFF. 1985. Kulmbacher Reihe Band 5. Mikobiologie 37 30–35°C, is used, and sometimes the Medwurst is und Qualität von Rohwurst und Rohschinken. Kulm- 38 also heat-treated after fermentation. The meat block bach, DE: Bundesanstalt für Fleischforschung, 39 contains 50% pork, 22% fat, 20% boiled potatoes, pp. 2–15, 34–39. 40 and 3% water; other ingredients are salt, saccharose, ———. 1990. Kulmbacher Reihe Band 10. Sichere 41 pepper, onion, ascorbate, and starter culture. Meat Produkte bei Fleisch und fleischerzeugnissen. Kulm- 42 and fat are chopped in the bowl chopper. The dry bach, DE: Bundesanstalt für Fleischforschung, 43 ingredients are mixed thoroughly into the chopped pp. 70–80, 96–97, 102–103. 44 TM meat and the mince stuffed into casings. The Chr. Hansen. 2003. Bactoferm Meat Manual, vol 1. 45 sausages are allowed to equilibrate to room tempera- Production of Fermented Sausages with Chr. Hansen 46 ture over night. The sausages are fermented at 29°C, Starter Cultures, 1st ed. Hørsholm, DK: Chr. Hansen 47 80% RH for 5 days and smoked shortly each day. A/S, pp. 1–31. 48 A Fischer. 1988. Handbuch der Lebensmitteltechnolo- 49 gie—Fleisch, Produktbezogene Technologie, 50 NORWAY Rohwurst. Stuttgart, DE: Ulmer Verlag, pp. 518–546. 51 Many Norwegian salamis are similar to the German- K Hofmann. 1974. Notwendigkeit und Vorschlag einer 52 style sausages, but typical for Norwegian fermented einheitlichen Definition des Begriffes “Fleischqual- S53 sausages is the use of “unusual” meats, such as ität.” Fleischwirtsch 54:1607–1609. N54 40454 33 349-358 r2.qxd 7/20/07 4:14 PM Page 358

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1 K Incze. 1986. Technologie und Mikrobiologie derUn- K Potthast, G Eigner. 1988. Neuere Ergebnisse über die 2 garisches Salami. Fleischwirtsch 66:1305–1311. Zusammensetzung von Räucherrauch. Fleischwirtsch 3 L Leistner, GW Gould. 2002. Hurdle Technologies: 68:651–655. 4 Combination Treatments for Food Stability, Safety A Stiebing. 1993. Vorlesungsscripe im Fach— 5 and Quality. New York: Kluwer Academic/Plenum Technologie der fermentierten Fleischwaren, Studi- 6 Publishers, pp. 1–208. engang Fleischtechnologie. Lemgo, DE: University 7 J Löbel. 1989. Mailänder salami & Co. Düsseldorf, DE: of Applied Sciences. 8 Econ Taschenbuchverlag, pp. 50–80. A Stiebing, W Rödel. 1987. Einﬂuβ der Luft- 9 S Neuhäuser. 1988. Fleischwaren-Handbuch, Technik geschwindigkeit auf den Reifungsverlauf von 10 und technologie. Hamburg, DE: Behr’s Verlag, Rohwurst. Fleischwirtsch 67:236–240, 1020–1030. 11 pp. 129–197. 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53S 54N 40454 34 359-368 r1.qxd 7/21/07 12:07 PM Page 359

1 2 3 4 5 6 7 8 9 10 11 12 13 14 Part VIII 15 16 17 18 19 Other Fermented Meats 20 21 22 and Poultry 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 S54 N55 40454 34 359-368 r1.qxd 7/21/07 12:07 PM Page 361

1 2 3 34 4 5 6 Fermented Poultry Sausages 7 8 Sunita J. Santchurn and Antoine Collignan 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION has been a renewed interest in poultry and poultry 22 products, especially 100% pure poultry products 23 Poultry meat is very popular among Western con- including sausages. This can be explained by several 24 sumers (Bruhn 1994), while also offering an excel- factors. First, the increasing price of red meat and 25 lent source of animal protein for consumers in the facility to set up poultry production (compared 26 developing countries (Gascoyne 1989). According to with cattle or pigs), particularly in developing coun- 27 FAO (FAOSTAT data 2004), poultry meat ranks sec- tries, have played a determining role in the world- 28 ond in meat produced worldwide, after pork, and wide spread of the poultry industry. Poultry meat 29 represents more than 30% of the total world meat also benefits from a healthier image than red meats 30 production with 77 million tons/year. It also ranks and, as such, appeals to the ever-increasing health- 31 first in exported meat with 8 million tons/year. Raw- conscious consumers. Last but not least, poultry and 32 poultry-meat-based production (i.e., salted, dried, its derived products are not snubbed by any religious 33 smoked, etc,) is fairly recent (20 years old) and, in or cultural group (Santos and Booth 1996; Jackson 34 today’s world market, remains very marginal com- 2000; Kubberod et al. 2002), contrary to pork and 35 pared to pork or beef delicatessen meat. This beef. In fact, it is highly appreciated by Muslims, 36 explains the poor indexing of these products. Hindus, and Tamils. Moreover, halal practice is com- 37 As a matter of fact, traditionally, poultry animals monly implemented in poultry processing plants in 38 are consumed just after slaughtering. This is due to countries where a Muslim community forms a non- 39 the family size of the animals, which allowed total negligible part of the population demographics. 40 consumption of the animal without the need to pre- Demand from these groups for new poultry products 41 serve parts of its carcass. In contrast, other farm ani- has led to the development of poultry bologna, franks, 42 mals (pig, cattle), once slaughtered, could not be burgers, nuggets, and sausages. 43 eaten at once and a large proportion of the carcass In the particular case of fermented poultry prod- 44 needed to be preserved for later use. In this way, ucts, there is no real tradition of consumption as with 45 people started to salt, dry, and, unknowingly, ferment red meat. Nevertheless, some raw cured poultry- 46 pork or beef portions. based products have been developed, and a few are 47 Nevertheless, a narrow range of poultry deli- even available on the market, such as smoked or 48 catessen meats have appeared on the market, chiefly dried duck fillet (Lesimple et al. 1995), poultry jerky 49 cooked products such as cured thigh meat (Bater (Carr et al. 1997), and dry sausage with partial or 50 et al. 1993), patties (Reddy and Rao 1997), sausages complete poultry meat content (Holley et al. 1988). 51 (Beggs et al. 1997), and raw products to be cooked, These fermented delicatessen products and, in par- 52 e.g. bacon (Walters et al. 1992). More recently, there ticular, fermented sausages do not yet have a broad 53 S54 N55 361 40454 34 359-368 r1.qxd 7/21/07 12:07 PM Page 362

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1 market distribution. This may be explained by prob- FERMENTED POULTRY SAUSAGE 2 lems associated with the raw material characteris- 3 tics. Indeed, the first development of fermented SPECIFIC CHARACTERISTICS OF RAW POULTRY 4 poultry sausages, dating back to the 1970s, met with MEAT AND FAT IN RELATION TO PROCESS 5 two main obstacles. The first one concerns sanitary AND QUALITY 6 hazards arising from the presence of pathogens on The characteristics of poultry meat and fat, together 7 the poultry carcass. In fact, Salmonella spp. and with those of other meats and fats, are presented in 8 Campylobacter jejuni are commonly present in poul- Tables 34.1 and 34.2, respectively. 9 try meats (Bryan and Doyle 1995). These pathogens Apart from the fact that poultry meat is generally 10 constitute a serious public health hazard accentuated lower in fat than pork or beef, it is also less fibrous 11 by the high raw poultry meat pH. Moreover, preva- and more tender. Moreover, its ultimate pH tends 12 lent contamination by Listeria monocytogenes in to be higher than that of pork and beef, with high- 13 slaughterhouses, poultry processing plants (Franco activity muscles (thigh and leg) generally showing 14 et al. 1995), and all raw poultry meats (Glass and a higher ultimate pH than low-activity muscles 15 Doyle 1989) has also limited the development of (breast, wing) (Table 34.1) (Jones and Grey 1989). 16 these products. In addition, poultry meat has on average, a lower 17 The second limitation is linked to the poor myoglobin content than beef. This explains the 18 technological properties of poultry fat tissue. The lighter color of poultry compared to beef. Further- 19 latter is weakly structured and is characterized by more, color varies with the type of muscle within a 20 a low melting point of fat leading to greasiness carcass: in poultry, thigh meat is darker than breast 21 defects (Arnaud et al. 2004). As such, this fat can- meat (Miller 2002). 22 not be used as lard and is not suitable for pro- The composition of poultry fat (abdominal and 23 ducts like dry-fermented sausages requiring a skin) is significantly different from that of beef and 24 structured fat. pork fats (Table 34.2). First, its water content is 25 This chapter sums up the different existing poul- higher and fat content lower, with chicken skin 26 try fermented products with particular reference to showing more marked differences. Like beef and 27 fermented sausages, the technological constraints pork fat, chicken abdominal fat is an association of 28 associated to their sanitary and sensorial qualities, fat cells held in a loose framework of connective 29 and promising alternatives to overcome them. tissue. However, it contains more water and less 30 31 32 33 34 35 Table 34.1. Characteristics of poultry, beef, and pork lean. 36 Poultry Beef Pork References 37 38 Postrigor pH Breast: 5.6–5.8 5.5–5.7 5.7–5.9 Jones and Grey 39 Thigh: 6.1–6.4 1989; Varnam and 40 Sutherland 1995; 41 Ricke et al. 2001 42 Myoglobin content 8-week-old poultry: 3-year-old: 5-month-old: Miller 2002 43 (mg/g); visual color dark meat 0.40, 4.60; bright 0.30; grayish 44 dull red; white meat red pink 45 0.01, grayish white 46 14-week-old turkey: 47 dark meat 0.37, dull red; 48 white meat 0.12, dull red 49 50 Water content (wt %) 75.4 74.0 71.5 Kirk and 51 Sawyer 1991 52 Protein (wt %) 23.4 20.3 20.7 53S Fat (wt %) 1.0 4.6 7.1 54N 40454 34 359-368 r1.qxd 7/21/07 12:07 PM Page 363

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Table 34.2. Characteristics of poultry, beef, and pork fat. 1 2 Poultry Fat 3 Abdominal Skin Beef Fat Pork Fat References 4 5 Water (wt %) 33.8 57.1 24.0 21.1 Kirk and Sawyer 1991 6 Protein (wt %) 5.4 14.0 8.8 6.8 7 8 Fat (wt %) 63.3 31.2 66.9 71.4 9 Cholesterol (mg/100 g) 40–63 75–140 50–120 Viau and Gandemer 1991; 10 Foures 1992 11 Fat melting point (°C) 26.6 0.2 42–45 32–38 Foures 1992; Arnaud 12 et al. 2004 13 Consistency at room temperature semi liquid solid solid 14 15 * MUFA (g/100 g) 44.0 46.6 16 Oleic acid C18:1(g/100 g) 38.2 36.5–43.0 39.0–45.0 17 PUFA* (g/100 g fat) 25.7 11.4 18 19 Linoleic acid C18:2 (g/100 g) 23.8 1.4–3.9 8.5–12.0 20 SFA* (g/100 g) 30.3 42.0 21 Stearic acid C18:0 (g/100 g) 5.8 15.5–23.0 12.8–17.7 22 23 *MUFA monounsaturated fatty acids; PUFA polyunsaturated fatty acids; SFA saturated fatty acids. 24 25 26 collagen, accounting for its lower protein content 70% of SFA are in the external position (positions 1 27 and very weak structure. In contrast, chicken skin is and 3) (compared to 30% in lard) (Arnaud et al. 28 composed of cellular layers (a superficial epidermal 2004). According to Entressangles (1992), this is of 29 and inner dermal layers) that hold the subcutaneous significance to intestinal absorption of fatty acids 30 adipose tissue (Dingle 2001). Hence its higher pro- because most of the unsaturated fatty acids will be 31 tein content and greater level of structure compared assimilated by the organism, and most of the SFA will 32 to pork or beef fat. Poultry fat also contains less sat- be eliminated. Considering that SFA, unlike MUFA 33 urated fatty acids and more monounsaturated and and PUFA, raise blood cholesterol levels and cause 34 polyunsaturated fatty acids than pork or beef fat. atherosclerosis (Higgs 2002), chicken fat thus repre- 35 This justifies the lower melting point of chicken fat sents a nutritionally better fat than pork or beef fats. 36 and its semiliquid consistency at ambient tempera- 37 ture (Foures 1992; Arnaud et al. 2004). 38 The distinctive features of poultry lean and fat have THE FERMENTED POULTRY SAUSAGE-MAKING 39 a direct bearing on formulations, processes, and end- PROCESS 40 product organoleptic quality, as will be discussed later The fermented poultry sausage making process fol- 41 in the chapter. Not to be neglected are their repercus- lows the same basic technology used for the process- 42 sions on the nutritional quality of processed products. ing of fermented red meat sausages (Figure 34.1). 43 Indeed, from a nutritional standpoint, poultry The main process steps are grinding/blending of 44 meat is leaner than red meats and thus constitutes an ingredients, stuffing into casings, fermentation, 45 excellent source of high-quality proteins for weight and drying. Nevertheless, due consideration must 46 control diets (Mountney and Parkhurst 1995). Fur- be given to the specific nature of poultry raw materi- 47 thermore, compared to pork and beef fat, poultry als when designing formulations and monitoring 48 fat contains higher levels of mono- (MUFA) and processes. 49 polyunsaturated (PUFA) fatty acids and lower levels 50 of saturated fatty acids (SFA) (Foures 1992; Arnaud Grinding/Blending of Ingredients 51 et al. 2004). More than 82% of fatty acids of 52 chicken fat in the internal position (position 2) on Formulations include standard ingredients, namely S53 the triglyceride (TGA) are unsaturated fatty acids and meat (80–90%), fat (7–15%), curing agents (sodium N54 40454 34 359-368 r1.qxd 7/21/07 12:07 PM Page 364

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1 Raw material meat block, beyond which excessive softening of the 2 (deboned chicken thigh with skin) final sausage is noted. Use of chilled material and 3 sharp knives ensures a cleaner cut and better particle 4 definition. Once the meat is chopped, the other 5 ingredients are mixed in. It is to be borne in mind 6 Grinding/blending that comminution and any other mechanical working 7 (curing salts, seasonings, and tend to cause more physical disruption to poultry 8 starter culture) meat that red meat, poultry being less fibrous and 9 more tender. In addition, the presence of NaCl, at 10 ionic strength 0.6 or higher, generally causes extrac- Sausage mix 11 tion of myofibrils from meat fibers into exudate. (pH: 6.3, water activity: 0.97) 12 Chicken, and especially its white muscle, produces 13 exudates easily (Varnam and Sutherland 1995). 14 Although exudate is important for binding and 15 Stuffing maintenance of product structure, high levels of 16 (artificial collagen casing) myofibrillar protein solubilization into exudates lead 17 to a decrease in fibrous texture, hence less chewiness 18 in the final sausage. Furthermore, the water-holding 19 capacity of solubilized myofibrillar proteins is 20 Fermentation higher, which tends to retard subsequent drying 21 (6 phases: 6°C/24h/90%RH; 14°C/6h/90%RH; rate. Use of dark meat (thigh and leg), coarse grind- 22 18°C/6h/85%RH; 22°C/6h/85%RH; ing of lean, and addition of salt in the last instance 23 18°C/6h/85%RH; 14°C/24h/85%RH) are measures that minimize the above-mentioned 24 problems. 25 Another point important to consider is the nature 26 of the fat component. Fat sources in pure poultry 27 Drying sausages are either abdominal or skin fat. As has 28 (14°C/18 days/75%RH) been pointed out earlier, poultry fat is more fluid at 29 room temperature. As a result, during ingredient 30 blending, and contrary to pork and beef fats, which 31 Fermented dry sausage remain discrete particles, poultry fat liquefies easily 32 (pH: 4.8-5.7 depending on starter and tends to coat the lean meat particles. In the case 33 used; water activity: 0.82) of dry sausages, this fat layer constitutes a barrier to 34 moisture removal during the drying stage. Conse- 35 Figure 34.1. Fermented dry poultry sausage- quently, the amount of poultry fat or skin is usually 36 making process. restricted to 15%, which is low compared to red 37 meat formulations (23–32%) (Bacus 1986). Also, fat 38 is better incorporated toward the end of the blending 39 nitrite 0.008%, sodium nitrate 0.008%, salt 1.5–3%), cycle. In addition, and of particular relevance for 40 seasonings (e.g., ground pepper, ground mustard, pure poultry sausage, mixing time should be just 41 paprika, garlic powder, allspice, and/or coriander), sufficient for the purpose and temperature main- 42 and carbohydrates (e.g., glucose, sucrose, lactose, tained low (e.g., use of frozen ingredients, cooling of 43 0.7–1.5%) (Keller and Acton 1974; Deumier and mixer/blender, cooling of processing room). 44 Collignan 2003). A grinder equipped with a metal Traditionally, fermented red meat sausages have a 45 plate bearing holes of specific diameter (3–8 mm) or typical red color arising from the reactions between 46 a bowl cutter may be used. Although skin-on meat is myoglobin and added nitrite. Poultry meat, espe- 47 more conveniently ground as such, it is advisable to cially white muscle, is poorly pigmented and devel- 48 grind/chop lean and fat separately for better particle ops a less intense red color than pork or beef. The 49 definition in the end product. Mechanically deboned incorporation of poultry fat in formulations tends to 50 poultry meat (MDPM), still cheaper than hand- further lighten the overall sausage color due to fat- 51 deboned poultry meat, has also been successfully coating of the lean. However, processors may have 52 incorporated in sausage formulations (Holley et al. recourse to permitted colorings, such as Monascus 53S 1988; Ricke et al. 2001). However, the maximum red (Angkak), cochineal, and betanin, to achieve 54N proportion of MDPM should stand at 15% of the specified color intensity (Miller 2002). 40454 34 359-368 r1.qxd 7/21/07 12:07 PM Page 365

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As mentioned before, poultry meat and fat have a metabolism of the lactic acid-producing bacteria. 1 high average moisture content. Furthermore, the This is of particular relevance to poultry sausage 2 amount of fat in poultry sausage formulation is gener- mix, which presents a high initial pH. Rapid pH 3 ally low. As a result, the sausage mix has a relatively decrease is essential to curb the growth of patho- 4 high initial moisture content and water activity genic bacteria that may be present in poultry mate- 5 (0.97–0.98) (Deumier and Collignan 2003; Alter et al. rial (meat and skin). At the end of the fermentation 6 2006). In addition, its pH value is also fairly high, cycle, the pH value should decrease to at least 5.3 to 7 ranging from 5.9 to 6.53 (Holley et al. 1988; Deumier prevent staphylococcal toxin production (Bacus 8 and Collignan 2003; Alter et al. 2006). These two fac- 1986). Besides, so long as the pH remains above the 9 tors make the sausage mix very favorable to microbial isoelectric point of meat proteins (pH 5) (Keller and 10 growth. Use of poultry skin, a major source of micro- Acton 1974), the water-holding capacity of poultry 11 biological contamination, may further magnify the lean stays high, which in turn may retard the sub- 12 hazard. The presence of nitrite and salt is important to sequent rate of drying. Instead of relying solely 13 inhibit the growth of Clostridium botulinum and upon fermentation-produced acidulants (lactic acid), 14 gram-negative pathogens such as Pseudomonas, processors may employ chemical acidulants, in the 15 Escherichia coli, and Salmonella (Bacus 1986; Ricke form of gluco-delta-lactone or organic acids, to 16 et al. 2001). Still, the high level of raw poultry meat stimulate pH decrease (Bacus 1986). 17 and a high salt content may selectively promote the 18 growth of Staphylococcus aureus. In this regard, it is 19 DRYING essential to limit the time the sausage mix is left at 20 temperatures above 15.6°C in the initial stages of pro- Depending on the extent of drying, two types of 21 cessing while the pH is still above 5.3 (limit value for sausages may be distinguished: semidry and dry 22 toxin production by S. aureus) (Bacus 1986). sausages, showing a product water activity of 23 As a pH-control measure, commercially available 0.92–0.94 and 0.85–0.86, respectively (Roca and 24 starter cultures, most commonly composed of lactic Incze 1990). As mentioned earlier, poultry sausage 25 acid bacteria (LAB), are used to rapidly acidify the formulations are generally high in moisture. Conse- 26 sausage mix (Baran et al. 1973; Acton and Dick quently, measures must be taken to prevent too rapid 27 1975; Deumier and Collignan 2003). When nitrates release of surface moisture and to ensure uniform 28 are used instead of nitrites, starter cultures contain- drying and product integrity (Bacus 1986). This 29 ing nitrate-reducing strains (Kocuria or Staphylo- involves, in the initial stages of processing, the con- 30 coccus) are used. The inoculum is more often and trol of the degree of meat and fat comminution, tem- 31 conveniently added at the end of the blending cycle, perature to limit fat liquefaction, level and time of 32 although it may sometimes be manually mixed after salting, and the rate of acidification. Uneven drying 33 a short holding time to allow proper binding of the may also be minimized through the use of casings of 34 meat (Baran et al. 1973). suitable water permeability and appropriate time/ 35 temperature/relative humidity drying schedules. Dry- 36 Stuffing ing process temperatures must also be controlled to 37 minimize fat loss, thus maximizing process yield. 38 Following inoculation, the mix is stuffed into a cas- 39 ing using a mechanical stuffer/clipper. The only 40 appropriate option for pure poultry sausages is artifi- END PRODUCT QUALITY 41 cial casing such as fibrous collagen casings. Again, The most noticeable defect in fermented pure poul- 42 care must be taken to minimize temperature increase try sausages is associated with the nature of poultry 43 to prevent fat smearing (Varnam and Sutherland fat. Despite the fact that pure poultry sausages are 44 1995). In some cases, namely for dry sausages, lower in fat than pork or beef sausages, an unappeal- 45 sausages are then dipped in a suspension of mold ing oily surface invariably appears due to fat exuda- 46 culture (Penicillium candidum and P. nalgiovencis), tion even at room temperature (Keller and Acton 47 which eventually grows into a white or gray coat on 1974; Deumier and Collignan 2002), thus giving a 48 the outer surface of the casing (Deumier and Collig- false impression of a high-fat product to consumers. 49 nan 2003). Using beef or pork fat in place of poultry fat usually 50 corrects the greasiness problem (Acton and Dick 51 Fermentation 1975; Bacus 1986). However, sausages so produced 52 Fermentation is the next stage, during which incuba- are not acceptable for no-pork and no-beef consumer S53 tion conditions are set to favor active growth and groups. N54 40454 34 359-368 r1.qxd 7/21/07 12:07 PM Page 366

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1 In addition, the higher level of unsaturated fatty slaughtered at 63 days (Viénot 2003). The chicken fat 2 acids in poultry fat makes sausages more susceptible has a higher melting point and therefore does not 3 to rapid fat oxidation, thereby increasing the risks cause any of the previously mentioned problems 4 of rancidity during drying and storage. The use of associated with standard poultry fat. However, sani- 5 permitted antioxidants may, however, inhibit such tary hazards associated with poultry skin still persist. 6 oxidative defects. Another promising avenue for pure poultry deli- 7 Moreover, abdominal fat is soft and lacks struc- catessen meats, including fermented sausages, rests 8 ture. This leads to poor particle definition in the on the improvement of the technological properties 9 finished product, which significantly reduces its of poultry fat. Recently, pilot-scale dry fractionation 10 marketability (Deumier and Collignan 2002). For of chicken fat succeeded in producing a fat fraction, 11 this reason, poultry skin fat is preferred because it stearin, with a higher melting point (Arnaud et al. 12 confers more chewiness and may simulate the white 2004) and with good sanitary quality due to the heat 13 flecks in salami-like products (Bacus 1986). How- treatment of fat before fractionation. Stearin is solid 14 ever, fat exudation still prevails. In addition, poultry at room temperature and closely resembles lard, and 15 skin is a notorious source of microbiological con- as such would eliminate greasiness defects in all- 16 taminants and its incorporation into sausage formu- poultry sausages. However, unlike pork or beef fat, 17 lations, especially raw sausages, implies accrued the stearin fraction is not sufficiently structured for 18 risks of pathogens. use in poultry formulations. And ongoing research 19 Indeed, poultry and poultry products have been the by the same authors aims at constructing a stearin 20 cause of many foodborne disease outbreaks most fre- with sufficient body for good particle definition in 21 quently involving Salmonella spp., Clostridium per- the end product. 22 fringens, and Staphylococcus aureus (Mead 1989). 23 Campylobacter jejuni is another pathogen associated 24 mainly with poultry-meat consumption (Bryan and OTHER FERMENTED PRODUCTS 25 Doyle 1995). The high incidence of Listeria monocy- 26 togenes in raw and even processed poultry products Although dry sausage is the most common fer- 27 is also a major concern (Franco et al. 1995). To coun- mented poultry meat, a few other fermented poultry 28 teract these sanitary hazards, at the end of the fer- products may also be found. 29 mentation cycle and prior to drying of fermented Among these, the French dry-cured magret is 30 poultry sausages, a mild heating treatment may be probably the most famous. It is made with the breast 31 applied to kill heat-sensitive pathogenic bacteria of Moulard duck, a breed used for foie gras (fattened 32 (Keller and Acton 1974; Raccach and Baker 1979). duck liver). The magret refers specifically to the lobe, 33 Such types of sausages fall into the semidry group or half breast of the Moulard duck, well known for 34 and have a significantly different organoleptic profile its excellent quality. In particular, the flavor of this 35 from that of dry sausages. In the case of more tradi- processed fillet is related to exogenous and endoge- 36 tionally processed dry sausage, no thermal treatment nous compounds and their distribution between the 37 for preservation or safety purposes is practiced. As a fatty panicle and the lean (Lesimple et al. 1995). The 38 result, these sausages are in essence raw meats that process is well described by Poma (1998). After cut- 39 have been salt-cured, incubated, and dried. Moreover, ting carcasses, raw duck fillets (with subcutaneous 40 dry sausage is usually eaten by the consumer without fatty panicle) are subjected to salting and drying. 41 further cooking. For these reasons, in addition to Salting consists of a dry rub of the fillet with salt 42 good manufacturing practices during production and (20 g/kg) and pepper (2 g/kg) followed by curing at 43 slaughtering, and better sanitization of carcasses at 4°C for 12 h. After curing, the magret is hung in a 44 the primary levels of production, poultry sausage thermostated chamber at 25°C and 80% of relative 45 processors must also abide by strict hygiene regula- humidity during 48 h for fermentation. Next, the 46 tions (sanitation, employee hygiene, and good manu- magret is dried in the same chamber with an initial 47 facturing practices). step at 16–18°C for 24 h followed by a second one 48 at 12–13°C for 1–2 weeks. The dry-cured fillets are 49 commercialized either entire or thinly sliced. Often RECENT ADVANCES 50 vacuum-packed, they keep for 2 months under refrig- 51 Successful commercialization of fermented dry pure eration. Dry magret makes a great appetizer. It may 52 chicken sausage was recently made possible through be served with fruit slices (pear, melon, or fig 53S the use of thigh meat and skin fat originating from a depending on the season) as part of an antipasto plat- 54N selected slow-growing breed of chicken (Guignol) ter, or sliced thinly on top of a green salad. 40454 34 359-368 r1.qxd 7/21/07 12:07 PM Page 367

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Other dried and cured duck meats that are encoun- KLH Beggs, JA Bowers, D Brown. 1997. Sensory and 1 tered may be classified as fermented products. It is the physical characteristics of reduced-fat turkey frank- 2 case of NAN-AN, a traditional dried duck meat (more furters with modified corn starch and water. J Food 3 than 100 years old) from China (Jiangxi province) Sci 62:1240–1244. 4 processed from Big Gunny ducks. The process mainly CM Bruhn. 1994. Consumer perceptions of quality. In: 5 includes cutting of the carcass into five pieces, salting Minimal Processing of Foods and Process Optimiza- 6 in brine and sun drying (Yongchang 1989). tion: An Interface RP Singh, FAR Oliveira, eds. 7 Prosciutto is originally a traditional Italian cured London: CRC Press, pp. 493–504. 8 pork meat which is cured and further matured by air FL Bryan, MP Doyle. 1995. Health risks and conse- 9 drying. However, it can also be made with magret, quences of Salmonella and Campylobacter jejuni in 10 Moulard duck breast (Smith 2005). In the particular raw poultry. J Food Protect 58:326–344. 11 case of Moulard magret prosciutto, a mixture of salt MA Carr, MF Miller, DR Daniel, CE Yarbrough, JD 12 and spices (juniper berries, bay leaf, coriander, pep- Petrosky, LD Thompson. 1997. Evaluation of the 13 percorns, and garlic) is used for curing under refrig- physical, chemical and sensory properties of jerky 14 eration (4°C) for 24 hours. Without rinsing, the processed from emu, beef, and turkey. J Food Qual 15 magret is then hung in a dry chamber at 10°C for 20:419–425. 16 2 weeks. F Deumier, A Collignan. 2002. Evaluation des risques 17 For many years, jerky and biltong meats have been sanitaires au cours du procédé de fabrication d’un 18 favorite snack foods for Americans and southern saucisson sec de volaille. 14èmes Rencontres Scien- 19 Africans, respectively. They are produced by using tifiques et Technologiques des Industries Alimentaires, 20 a combination of curing, drying, and sometimes AGORAL 2002, Nancy, France, 2002, pp. 443–449. 21 smoking procedures. People generally associate ———. 2003. The effects of sodium lactate and starter 22 jerky and biltong with beef. However, the meat cultures on pH, lactic acid bacteria, Listeria monocy- 23 sources are unlimited and it is not uncommon to use togenes and Salmonella spp. levels in pure chicken 24 meat from turkey, ostrich, and emu (Carr et al. dry fermented sausage. Meat Sci 65:1165–1174. 25 1997). Depending on the drying and/or storage con- J Dingle. 2001. Manufacturing leather from chicken 26 ditions, spontaneous lactic acid fermentation may be skin. Kingston, Australia: Rural Industries Research 27 favored, thus leading to a fermented end-product and Development Corporation 1–16. 28 (unpublished work). B Entressangles. 1992. Digestion et absorption des lipi- 29 des. In: Les Lipides. Institut Français Pour la Nutri- 30 tion, ed. Paris, France, pp. 17–33. 31 REFERENCES FAOSTAT data. 2004. http://faostat.fao.org/faostat 32 (24 May 2006). 33 JC Acton, RL Dick. 1975. Improved characteristics for C Foures. 1992. Tissus animaux. In: Manuel des Corps 34 dry, fermented turkey sausage. Food Prod Devel Gras. I. A Karleskind, ed. Paris: Lavoisier Tec & Doc, 35 9:91–94. pp. 242–260. 36 T Alter, A Bori, A Hamedi, L Ellerbroek, K Fehlhaber. CM Franco, EJ Quinto, C Fente, L Rodriguez-Otero, 37 2006. Influence of inoculation levels and processing A Cepeda. 1995. Determination of the principal 38 parameters on the survival of Campylobacter jejuni sources of Listeria spp. contamination in poultry meat 39 in German style fermented turkey sausages. Food and a poultry processing plant. J Food Protect 58: 40 Microbiol 23:701–707. 1320–1325. 41 E Arnaud, P Relkin, M Pina, A Collignan. 2004. Charac- J Gascoyne. 1989. The world turkey industry, structure 42 terisation of chicken fat dry fractionation at the pilot and production. In: Recent Advances in Turkey Sci- 43 scale. European J Lipid Sci Technol 106:591–598. ence. C Nixey, TC Grey, eds. London: Butterworths, 44 JN Bacus. 1986. Fermented meat and poultry products. pp. 3–9. 45 In: Advances in Meat Research, vol. 2. Meat and KA Glass, MP Doyle. 1989. Fate of Listeria monocyto- 46 poultry microbiology, AM Pearson, ed. Westport: Avi genes in processed meat products during refrigerated 47 Publishing Company, pp. 123–164. storage. Appl Envir Microbiol 55:1565–1569. 48 WL Baran, LE Dawson, KE Stevenson. 1973. Produc- J Higgs. 2002. The nutritional quality of meat. In: Meat 49 tion of a dry fermented turkey sausage. Poultry Sci Processing—Improving Quality. J Kerry, J Kerry, 50 52:2358–2359. DA Ledward DA, eds. Cambridge, England: Wood- 51 B Bater, O Descamps, AJ Maurer. 1993. Quality char- head Publishing Ltd., pp. 64–104. 52 acteristics of cured turkey thigh meat with added AR Holley, PA Jui, M Wittman, P Kwan. 1988. Survival S53 hydrocolloids. Poult Sci 72:349–354. of S. aureus and S. typhimurium in raw ripened dry N54 40454 34 359-368 r1.qxd 7/21/07 12:07 PM Page 368

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1 sausages formulated with mechanically separated M Raccach, RC Baker. 1979. Fermented mechanically 2 chicken meat. Fleischwirtschaft 68:194–201. deboned poultry meat and survival of Staphylococ- 3 MA Jackson. 2000. Getting religion—For your prod- cus aureus. J Food Protect 42:214–217. 4 ucts that is. Food Technol 54:60–66. KP Reddy, BE Rao. 1997. Influence of binders and 5 JM Jones, TC Grey. 1989. Influence of processing on refrigerated storage on certain quality characteristics 6 product quality and yield. In: Processing of Poultry. of chicken and duck meat patties. J Food Sci Technol 7 GC Mead, ed. Essex, England: Elsevier Science Pub- 34:446–449. 8 lishers Ltd., pp. 127–181. SC Ricke, I Zabala Diaz, JT Keeton. 2001. Fermented 9 JE Keller, JC Acton. 1974. Properties of a fermented, meat, poultry, and fish products. In: Food Microbiol- 10 semidry turkey sausage during production with ogy: Fundamentals and Frontiers MP Doyle, LR 11 lyophilized and frozen concentrates of Pediococcus Beuchat, TJ Montville, eds. Washington D.C.: ASM 12 cerevisiae. J Food Sci 39:836–840. Press, pp. 681–700. 13 RS Kirk, R Sawyer. 1991. Pearson’s Composition and M Roca, K Incze. 1990. Fermented sausages. Food Rev 14 Analysis of Foods. Essex, England: Longman Scien- Inter 6:91–118. 15 tific & Technical. MLS Santos, DA Booth. 1996. Influences on meat 16 E Kubberod, O Ueland, A Tronstad, E Risvik. 2002. avoidance among British students. Appetite 27: 17 Attitudes towards meat and meat-eating among ado- 197–205. 18 lescents in Norway: A qualitative study. Appetite P Smith. 2005. Duck Breast Prosciutto. www. 19 38:53–62. articledashboard.com (30/05/06). 20 S Lesimple, L Torres, S Mitjavila, Y Fernandez, L AH Varnam, JP Sutherland. 1995. Meat and Meat 21 Durand. 1995. Volatile compounds in processed duck Products—Technology, Chemistry and Microbiol- 22 fillet. J Food Sci 60:615–618. ogy. London, UK: Chapman & Hall. 23 GC Mead. 1989. Hygiene problems and control of M Viau, G Gandemer. 1991. Principales caractéris- 24 process contamination. In: Processing of Poultry. tiques de composition des graisses de volaille. Revue 25 GC Mead, ed. Essex, England: Elsevier Science Pub- française des corps gras 38:171–177. 26 lishers Ltd., pp. 183–220. E Viénot. 2003. Saroja volailles lance le saucisson sec 27 RK Miller. 2002. Factors affecting the quality of raw “pur poulet.” Filières Avicoles 43. 28 meat. In: Meat Processing. J Kerry, J Kerry, DA BS Walters, MM Lourigan, SR Racek, JA Schwartz, 29 Ledward, eds. Cambridge: Woodhead Publishing AJ Maurer. 1992. The fabrication of turkey bacon. 30 Ltd., pp. 27–63. Poultry Sci 71:383–387. 31 GJ Mountney, CR Parkhurst. 1995. Chemical and nutri- Z Yongchang. 1989. Preservation of the NAN-AN 32 tive characteristics. In: Poultry Products Technology. pressed salted ducks. International Congress of Meat 33 New York: Food Products Press, pp. 67–82. Science and Technology, Copenhagen, Denmark, 34 JP Poma. 1998. Le jambon sec et les petites salaisons. 1989, pp. 507–510. 35 Paris: Editions Erti. 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53S 54N 40454 35 369-374 r1.qxd 7/21/07 2:23 PM Page 369

1 2 3 35 4 5 6 Fermented Sausages from Other Meats 7 8 Halil Vural and Emin Burçin Özvural 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION has been achieved by decreasing mutton fat to a level 22 of 10% or less in meat products (Bartholomew and 23 In fermented sausage production, utilization of other Osuala 1986; Beriain et al. 1997). Also, undesirable 24 animals’ meat rather than pork, beef, and poultry is mutton characteristics may be prevented by applica- 25 not widely spread. Nevertheless, there are several tion of spices, curing, fermentation, and smoking 26 studies investigating the use of mutton, lamb, camel, (Bartholomew and Osuala 1986; Wu et al. 1991; 27 ostrich, horse, buffalo, and game meats for sausage Beriain et al. 1997). Limited work has been done on 28 production. In this chapter, each section includes fermented mutton sausages. 29 information on the raw materials from the men- Bartholomew and Osuala (1986) used mutton in 30 tioned animals, related studies on their manufacture the manufacture of summer sausages by incorporat- 31 into fermented sausages, and some details on local ing lean beef (10% fat), beef fat (87% fat), lean mut- 32 products. ton (5.5% fat), untrimmed mutton (16.5% fat), or fat 33 mutton trims (45% fat) into the formulation. They 34 MUTTON AND LAMB showed that mutton products containing 10% or less 35 mutton fat with added high levels of spices were 36 Mutton and lamb are consumed in almost all acceptable. Pepperoni is a heavily seasoned, smoked, 37 countries around the world. Moslem, Jewish, and dried fermented sausage generally made from pork 38 Hindu people do not eat pork and/or beef due to reli- and/or beef and sometimes from other meats such as 39 gious and cultural taboos; however, there is not any lamb. It is produced in central, northern, and eastern 40 handicap for mutton and lamb. Nevertheless, the Europe; North America; and Australia (Campbell- 41 production and consumption of sheep meat is lower Platt 1995). Bushway et al. (1988) examined chemi- 42 than pork and beef because of its odor, flavor, and cal, physical, and sensory properties of pepperoni 43 difficulty in deboning (Baliga and Madaiah 1971; produced with mutton (50%), fowl meat (50%), or 44 Anderson and Gillett 1974; Shaikh et al. 1991; beef fat. Their work indicated that spice mix, beef 45 Young et al. 1994). fat, and fermentation at 28°C for 48 hours, or until 46 Fat in mutton and lamb contains higher proportions the pH was reduced to 5.0 or less, prevented mutton 47 of saturated fatty acids than that of pork, and supplies flavor in the product. Wu et al. (1991) produced 48 a number of flavor components (Bartholomew and fermented mutton sausages with incorporation 49 Osuala 1986; Beriain et al. 1997). Lamb meat flavor of Pediococcus acidilactici H, Lactobacillus plan- 50 depends on age, diet, sex, breed, and body weight of tarum 27, and Lactacel 75 as a starter culture. The 51 the animal (Bartholomew and Osuala 1986; Beriain mince was stuffed into 20 mm collagen casings and 52 et al. 1997; Rousset-Akrim et al. 1997; Young et al. fermented at 30°C and 90–92% relative humidity. 53 1997; Sanudo et al. 2000). Reduction of mutton flavor When the pH was 4.9–5.0, the temperature was S54 N55 369 40454 35 369-374 r1.qxd 7/21/07 2:23 PM Page 370

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1 raised to 68°C for cooking. After cooking, the Fermentation was carried out for 36 hours at 26°C, 2 sausages were dried until achieving a moisture loss 96% relative humidity, and 1 m/s air velocity and 3 of 40–45%. According to the results, P. acidilactici then heated for 30 min at 55°C, followed by 70°C. 4 H and Lb. plantarum 27 were the more suitable cul- The products were cooled to 20°C and placed in a 5 tures for fermented mutton sausage. Cured dry mut- drying room at 15°C (relative humidity 59%) for 6 ton sausages formulated with 76% mutton and 24% 3 days. Vural (1998) reported that sausages with 7 fat were compared to pork sausages prepared with P. acidilactici had the best color, appearance, flavor, 8 the same meat and fat ratio. Sausages were fer- and general acceptability. 9 mented at 24°C with 60% and 90% relative humidity Sheep tail fat is an important constituent of Turkish 10 until pH was 4.9, and then dried in a drying chamber fermented sausage due to its strong influence on the 11 at 12–15°C while reducing the relative humidity sensory characteristics. Gökalp and Ockerman 12 gradually from 85% to 75%. It was reported that (1985) and Gökalp (1986) used 10% tail fat and 13 both mutton and pork sausages showed similar tech- Ayhan et al. (1999) added 15% tallow for the soud- 14 nological characteristics during processing, whereas jouk manufacture. Soyer (2005) and Soyer et al. 15 the flavor and texture of mutton sausages were not (2005) used 10, 20, and 30% sheep tail fat content 16 desirable (Beriain et al. 1997). and ripened the products at 20–22°C and 24–26°C. 17 In Norway, fermented lamb sausage is a common Soyer et al. (2005) showed that fat level affected 18 sausage type that is prepared differently in various water activity, color, and total viable and lactic acid 19 parts of the country (Helgesen et al. 1998). Fjellmorr bacteria counts of the sausages. Soyer (2005) found 20 in Norway and lambaspaeipylsa in Iceland are dried that total acidity, free fatty acids, and thiobarbituric 21 fermented sausage types that contain lamb in addi- acid (TBA) values were affected by fat level, ripen- 22 tion to beef and pork. In warmer countries of south- ing temperature, and time. It was shown that high 23 ern Europe (France) and North Africa, such as fat level and temperature increased the rancid flavor 24 Algeria, Tunisia, and Morocco, Merguez-type fer- and decreased the overall acceptability. In another 25 mented sausages, which contain pork, beef, lamb, or study, Vural (2003) manufactured Turkish semidry- 26 mixed meats, are consumed. Those sausage types are fermented sausages by substituting 0, 20, 60 or 27 usually stuffed in medium-large casings (50–70 mm), 100% of the beef fat and tail fat with interesterified 28 fermented at 30–35°C for 3–6 days and dried for a palm and cottonseed oils. Control groups had the 29 couple of days (Campbell-Platt 1995). Utilization of highest sensory scores, whereas vegetable oils reduced 30 mutton has also been tested in South African dry the ratio of saturated to unsaturated fatty acids and 31 sausages (Mellett 1991). increased the ratio of polyunsaturated to saturated 32 Turkish fermented sausage, which is called soud- fatty acids. 33 jouk or sucuk, is a popular fermented meat product 34 in Turkey. This product is produced and consumed GOAT 35 all over the country. Soudjouk or sucuk is made from 36 beef and/or water buffalo meat and/or mutton. Soud- Goat meat consumption varies globally due to cul- 37 jouk mixture also contains beef fat and sheep tail fat. tural, social, and economic conditions of consumers 38 The mixture is stuffed into air-dried bovine small (Norman 1991; Webb et al. 2005). Goat meat is gen- 39 intestines (Gökalp and Ockerman 1985; Gökalp erally preferred by people of local regions and sold 40 et al. 1988; Vural 1998; Bozkurt and Erkmen 2002; for domestic consumption. The world production of 41 Soyer et al. 2005). In traditional soudjouk produc- goat meat was 1.1 million tons in 1961 and 3.7 mil- 42 tion, starter cultures are not added and the fermenta- lion tons in 2001. However, the increase in goat meat 43 tion and drying stages are applied under natural production was less than other meats (Dubeuf et al. 44 climatic conditions. The ripening periods, tempera- 2004). 45 ture, and relative humidity applied in the production Goat meat is also called chevon; Henry Wallace 46 vary depending upon the traditional method used. gave this name in 1924 (Norman 1991). Although 47 Due to variable conditions and long processing time, goat meat (chevon) contains lower fat than other 48 manufacturers have started to use starter cultures types of red meats, its intense and natural flavor 49 (Gökalp 1986; Soyer et al. 2005). is undesirable to most consumers (Cosenza et al. 50 Vural (1998) produced Turkish semidry-fermented 2003; Kannan et al. 2006). However, Nassu et al. 51 sausages using P. acidilactici, Staphylococcus xylo- (2000) claimed that fermented goat meat sausages 52 sus plus P. pentosaceus and S. carnosus plus Lb. pen- could be a valuable alternative for fresh goat meat 53S tosus. The sausages were prepared with 74.5% beef, because fermented sausages produced with goat 54N 9.1% mutton, 10% beef fat, and 6.4% tail fat. meat had good consumer acceptability and suitable 40454 35 369-374 r1.qxd 7/21/07 2:23 PM Page 371

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physicochemical and microbiological characteristics (Dawood and Alkanhal 1995). El-Faer et al. (1991) 1 during storage. The sausages were fermented at examined the minerals and proximate composition of 2 23–12°C and 95–70% relative humidity during muscle tissues from shoulders, thighs, ribs, necks, and 3 14 days with the addition of the starter cultures humps of seven young (1–3 years) male camels. The 4 Lb. farciminis, S. xylosus, and S. carnosus. In a simi- mineral elements, protein, and ash contents in the var- 5 lar study it was shown that rosemary extract ious muscle tissues of the camels were generally simi- 6 (0.050%) gave an effective protection against oxida- lar to those in the same tissues of beef, but camel meat 7 tion in fermented goat meat sausages (Nassu et contained significantly less lipids (1.2–1.8% versus 8 al.2003). In a different study, Nassu et al. (2002) 4.0–8.0%) and 5–8% higher moisture content than 9 investigated the effect of different fat contents (5, 10, beef. Dawood and Alkanhal (1995) determined the 10 and 20%) in the processing, chemical characteris- proximate composition of some Najdi-male camels. 11 tics, and sensory acceptance of fermented goat meat They found that skeletal muscles had 68.8–76% mois- 12 sausages. To the sausages were added 0.02% S. xylo- ture, 19.4–20.5% protein, 4.1–10.6% fat, and 13 sus and P. pentosaceus, and they were matured 1.0–1.1% ash. 14 according to Nassu et al. (2003). The study indicated Limited work has been done on the use of camel 15 that the fat level did not significantly affect the pro- meat in fermented sausages. Kalalou et al. (2004) 16 cessing and sensory acceptance of the sausages. The produced fermented camel meat sausages with 17 authors also reported that a fat level of 10–20% was added Lb. plantarum to extend the shelf life of 18 considered the most adequate due to the low fat con- ground camel meat. They mixed minced camel lean 19 tent in goat meat. meat with hump fat in the proportion of 80/20, 20 The Angora goat originated in the district of stuffed the mixture into natural casings and dried the 21 Angora in Asia Minor (Anatolia) from where it sausages at 15–18°C at 70–80% relative humidity. 22 spread to the rest of the world. It is primary bred for The results showed that in 21 days pH dropped to 23 mohair (wool) production. Kolsarıcı et al. (1993) 4.5; water activity to 0.7; and coliforms, Entero- 24 investigated the utilization of Angora goat meat at cocci, and yeasts to nondetectable levels. However, 25 different ratios (25, 50, 75 and 100%) in soudjouk the authors concluded that the processing conditions 26 production. Physical, chemical, and sensory charac- should be better monitored for improving the 27 teristics of the soudjouks were examined during organoleptic quality of the product. 28 fermentation and storage periods that took place at 29 24 2°C, 90–95% relative humidity on the first day, 30 followed by 22 2°C, 80–85% relative humidity OSTRICH 31 for the last 6 days. The authors stated that goat meat 32 addition up to 50% level in soudjouk production The ostrich meat industry is largely developed in 33 did not cause any differences on physical, chemical, South Africa and has also been extended to Australia, 34 and sensory properties of soudjouks, in general, North and South America, Canada, Israel, and 35 when compared with the control (100% beef). recently to European countries such as Spain and 36 For sausages with 75% and 100% goat meat, physi- Italy (Kolsarıcı and Candog˘an 2002; Fernandez- 37 cal and chemical properties were good, but sensory Lopez et al. 2003). Ostrich meat can be used as a 38 properties not acceptable. Goat meat has also been healthy alternative to other meats (Fisher et al. 2000; 39 tested in fermented cabrito snack stick products Kolsarıcı and Candog˘an 2002; Fernandez-Lopez 40 (Cosenza et al. 2003). et al. 2003). It contains a favorable fatty acid profile 41 due to the high amount of polyunsaturated ω-3 fatty 42 CAMEL acids (Sales 1998; Fisher et al. 2000; Kolsarıcı and 43 Candog˘an 2002). In addition, ostrich meat has 44 Camel meat is not consumed all over the world, but it higher pH value, darker color, lower collagen solu- 45 is eaten in Sudan, Libya, Egypt, Saudi Arabia, and bility, and higher pigment content than beef. Its 46 India (El-Faer et al. 1991; FAO 2005). It is an alterna- cooking loss and cholesterol content are similar 47 tive product in arid areas where the climate conditions to those of beef (Sales et al. 1996; Kolsarıcı and 48 adversely affects other animals. In some societies, Candog˘an 2002; Fernandez-Lopez et al. 2003). 49 camel meat is the food of ritual celebrations (FAO The published data on fermented ostrich sausages 50 2005). Both young and mature animals are used in are very few. Böhme et al. (1996) produced Italian- 51 camel meat production. The carcass of animals type salami with ostrich meat by a combination of 52 younger than 1 year of age weighs about 100 kg and Lb. sake, Lb. curvatus, and Micrococcus spp. Sen- S53 the carcasses of the older animals, 200–300 kg sory characteristics of these products were compared N54 40454 35 369-374 r1.qxd 7/21/07 2:23 PM Page 372

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1 with the salami produced with glucono-delta-lactone cisely. The tenderness of buffalo meat resembles that 2 (GdL). Ostrich meat was fermented for 4 days at of beef. It has lower cholesterol content, and better 3 20–22°C, 97–99% relative humidity and ripened for hardness and resistance than beef (Paleari et al. 2000). 4 a further 11 days at 16–18°C, 40–60% relative Sachindra et al. (2005) claimed that buffalo meat 5 humidity. The study indicated that the combination is cheaper when compared to other meats and 6 of Lb. curvatus and Micrococcus spp. resulted in that products made from buffalo meat would be a 7 salami with acceptable texture and sensory quality. profitable foreign income. Buffalo meat produced 8 Dicks et al. (2004) produced ostrich meat salami by in India is largely exported in frozen condition. 9 using the cultures Lb. plantarum 423 and Lb. curva- Because there is not any taboo against the consump- 10 tus DF126 to prevent the growth of monocytogenes tion of buffalo meat in India, popularity of this meat 11 during production. The results indicated that bacteri- has been rising, and buffalo meat covers now 35.7% 12 ocins produced by the lactic acid bacteria, inhibited of the total meat consumption (Sekar et al. 2006). 13 the growth of monocytogenes in salami. In Turkey, water buffalo meat is generally used 14 together with beef and/or mutton in fermented 15 HORSE sausages (soudjouks) (Gökalp and Ockerman 1985; 16 Gökalp 1986; Gökalp et al. 1988). Gökalp and 17 The application of horsemeat in fermented sausages Ockerman (1985) manufactured Turkish-style fer- 18 is not common but is popular in Norway and Kaza- mented sausage (soudjouk) with 45% beef, 45% 19 khstan. Horsemeat consumption shows differences water buffalo meat, and 10% sheep tail fat and stud- 20 in historic times according to economy and cultural ied the effects of Lb. plantarum, Lb. plantarum M. 21 societies. Milk and meat of horse are still an impor- aurantiacus, Lb. plantarum M. aurantiacus 22 tant diet for the nomadic people of Eurasia. How- Debaryomyces hansenii as starter cultures at differ- 23 ever, in Western Europe, its consumption was partly ent ripening temperatures (12–14°C, 16–18°C, and 24 restricted because of its relation with pagan rites and 20–22°C). The authors showed that a mixed culture 25 festivals. There is a contamination risk of Salmo- of Lb. plantarum and M. aurantiacus should be used 26 nella and Yersinia enterocolitica and an infection and that the addition of D. hansenii had beneficial 27 possibility of Trichinella from horsemeat. However, effects on the decline of proteolytic and lipolytic 28 in some countries, particularly France, Italy, and microorganisms. It was reported that the lowest pro- 29 Japan, some people believe that raw consumption of teolytic and lipolytic counts were found by using the 30 horsemeat is safer than other meats and eat it raw or mixed culture of starters with ripening at 20–22°C. 31 after only minimal cooking (Gill 2005). Gökalp (1986) showed that using mixed cultures sig- 32 Kazy and Shuzhuk are popular sausages in Kaza- nificantly reduced the ripening period; dropped the 33 khstan. In Kazy production, raw sausage is dried, pH faster; and in addition to these effects, provided 34 boiled, or smoked. Hanging it outside for a week in better development of color, appearance, firm- 35 a sunny, aired place dries the sausage. Smoking ness, texture, flavor, odor and general acceptability. 36 is applied with dense smoke at 50–60°C for 12–18 D. hansenii in the starter mixture had positive effects 37 hours followed by drying at 12°C for 4–6 hours. in the development of soudjouk characteristics. 38 Shuzhuk is made from horsemeat and suet at equal Shehata (1997) prepared salamis with only buffalo 39 amounts. The meat is rubbed with salt and kept for meat and buffalo fat and evaluated the effects of Lb. 40 1–2 days in a cool place at 3–4°C. The meat and fat are plantarum and white mold coating on the quality of 41 cut into small pieces and stuffed into the gut with salt, the products. The white molds provided the best 42 pepper, and greens. The chubs are hung in a cool place organoleptic scores, whereas sausages with starter 43 for 3–4 hours, smoked at 50–60°C for 12–18 hours, cultures had the best color. In another study, Abou- 44 and dried for 2–3 days at 12°C (Chenciner 2005). Arab (2002) used buffalo and lamb meats in a 45 Trøndermorr, Fjellmorr, Sognemorr, and Stabbur sausage mixture and studied the effect of the fermen- 46 are dried fermented sausages produced in Norway. tation stage by a meat starter on the pesticides DDT 47 In some of these products, horsemeat is added into and lindane in fermented sausage. Fermentation was 48 the formulation. Horsemeat may also be used in Koti- accomplished at 30°C for 72 hours. The fermentation 49 mainen sausage in Finland (Campbell-Platt 1995). process decreased the pesticide residues in the prod- 50 ucts as a result of the activity of the meat starter. 51 BUFFALO 52 GAME MEAT 53S Because marketing of buffalo meat does not have 54N any specific regulations and the meat is generally Game meat includes deer, reindeer, wild boar, rabbit, sold as if beef, its consumption is not known pre- gazelle, and more. Generally, game meat has a 40454 35 369-374 r1.qxd 7/21/07 2:23 PM Page 373

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dark color, strong taste, and tough texture, but these ton and fowl. Can Inst Food Sci Technol J 21: 1 characteristics may vary according to the type and 415–418. 2 age of the animal (Soriano et al. 2006). Processing of G Campbell-Platt. 1995. Fermented meats—A world 3 game meat is not common due to the difﬁculties of its perspective. In: G Campbell-Platt, PE Cook, eds. 4 preparation and because most of the domestic con- Fermented Meats. London: Blackie Academic and 5 sumption is unprocessed. Also, the marketing of Professional, pp. 39–52. 6 game meat products is limited due to the hunting sea- R Chenciner. 2005. http://www.polosbastards.com/ 7 son (Soriano et al. 2006). However, today there is a artman/publish/horsemeat.shtml. 8 tendency for incorporating game meat to processed GH Cosenza, SK Williams, DD Johnson, C Sims, 9 meat products (Paleari et al. 2003; Soriano et al. CH McGowan. 2003. Development and evaluation of 10 2006). The literature on fermented sausages is still a fermented cabrito snack stick product. Meat Sci 11 limited, though. 64:51–57. 12 In Sudan, whole wild rabbit is skinned and then fer- AA Dawood, MA Alkanhal. 1995. Nutrient composi- 13 mented for 3 days. After fermentation, the carcass is tion of Najdi-camel meat. Meat Sci 39:71–78. 14 boiled in water and pounded to give a thick meaty LMT Dicks, FD Mellett, LC Hoffman. 2004. Use of 15 sauce (Campbell-Platt 1995). Reindeer meat is added bacteriocin-producing starter cultures of L. plan- 16 into some kinds of fermented sausages in Scandina- tarum and L. curvatus in production of ostrich meat 17 vian countries. In Finland Poro sausage and in Nor- salami. Meat Sci 66:703–708. 18 way Reinsdyr pølse include reindeer meat. Venison JP Dubeuf, P Morand-Fehr, R Rubino. 2004. Situation, 19 (deer meat) can also be utilized in summer sausage changes and future of goat industry around the world. 20 (semidried fermented sausage) production. 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Formulation and chase probability of dry fermented lamb sausages. N54 characteristics of a fermented pepperoni using mut- Food Qual Prefer 9:295–301. 40454 35 369-374 r1.qxd 7/21/07 2:23 PM Page 374

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1 I Kalalou, M Faid, TA Ahami. 2004. Improving the sensory characteristics of lamb carcasses from 2 quality of fermented camel sausage by controlling Britain and Spain. Meat Sci 54:339–346. 3 undesirable microorganisms with selected lactic acid A Sekar, K Dushyanthan, KT Radhakrishnan, R 4 bacteria. Int J Agri Biol 6:447–451. Narendra Babu. 2006. Effect of modified atmosphere 5 G Kannan, KM Gadiyaram, S Galipalli, A Carmichael, packaging on structural and physical changes in buf- 6 B Kouakou, TD Pringle, KW McMillen, S Gelaye. falo meat. Meat Sci 72:211–215. 7 2006. Meat quality in goats as influenced by dietary NS Shaikh, AT Sherikar, KN Bhilegaonkar, UD 8 protein and energy levels, and postmortem aging. Karkare. 1991. Studies on preparation of mutton 9 Small Ruminant Res 61:45–52. sausages. Journal Food Sci Technol 28:323–325. 10 N Kolsarıcı, K Candog˘an. 2002. Devekus¸u eti. Standart H Shehata. 1997. Production of buffalo salami. Effects 11 4:35–42. of bacterial starter cultures, gelatine coat with 12 N Kolsarıcı, A Soyer, K Turhan. 1993. Tiftik keçisi pasterma spices and mold coat on product quality. 13 etinin sucuk üretiminde kullanılabilme olanakları Fleischwirtschaft 77:1116–1118. 14 üzerine aras¸tırma, Ankara Üniversitesi Ziraat Fakül- A Soriano, B Cruz, L Gómez, C Mariscal, AG Ruiz. 15 tesi Yayınları, pp. 1–15. 2006. Proteolysis, physicochemical characteristics 16 FD Mellett. 1991. South African type dried sausage and free fatty acid composition of dry sausages 17 Addition of caudal fat from sheep carcasses. Fleis- made with deer (Cervus elaphus) or wild boar (Sus 18 chwirtschaft 71:680–681. scrofa) meat: A preliminary study. Food Chem 19 RT Nassu, FJ Beserra, LAG Gonçalves, T Feitosa. 2000. 96:173–184. 20 Manufacturing of a goat meat fermented sausage. A Soyer. 2005. Effect of fat level and ripening tempera- 21 7th International Conference on Goats, France, ture on biochemical and sensory characteristics of 22 pp. 672. naturally fermented Turkish sausages (sucuk). Eur 23 RT Nassu, LAG Gonçalves, FJ Beserra. 2002. Effect of Food Res Technol 221:412–415. 24 fat level in chemical and sensory characteristics of A Soyer, AH Ertas¸, Ü Üzümcüog˘lu. 2005. Effect of 25 goat meat fermented sausage. Pesq Agropec Bras processing conditions on the quality of naturally 26 37:1169–1173. fermented Turkish sausages (sucuks). Meat Sci 69: 27 RT Nassu, LAG Gonçalves, MAAP da Silva, FJ Baserra. 135–141. 28 2003. Oxidative stability of fermented goat meat VIC, Venison Information Centre. 2005. Available at 29 sausage with different levels of natural antioxidant. http://www.shopsaskatchewan.com/NorthBattleford/ 30 Meat Sci 63:43–49. venison_information_centre.htm. 31 GA Norman. 1991. The potential of meat from the goat. H Vural. 1998. The use of commercial starter 32 In: RA Lawrie, ed. Developments in Meat Science. cultures in the production of Turkish semi-dry fer- 33 Essex: Elsevier Science Publishers Ltd, pp. 89–157. mented sausages. Z Lebensm Unters Forsch A 34 MA Paleari, G Beretta, F Colombo, S Foschini, 207:410–412. 35 G Bertolo, S Camisasca. 2000. Buffalo meat as a ———. 2003. Effect of replacing beef fat and tail fat 36 salted and cured product. Meat Sci 54:365–367. with interesterified plant oil on quality characteristics 37 MA Paleari, VM Moretti, G Beretta, T Mentasti, of Turkish semi-dry fermented sausages. Eur Food 38 C Bersani. 2003. Cured products from different ani- Res Technol 217:100–103. 39 mal species. Meat Sci 63:485–489. EC Webb, NH Casey, L Simela. 2005. Goat meat qual- 40 S Rousset-Akrim, OA Young, J-L Berdague. 1997. Diet ity. Small Ruminant Res 60:153–166. 41 and growth effects in panel assessment of sheepmeat WH Wu, DC Rule, JR Busboom, RA Field, B Ray. 42 odour and flavor. Meat Sci 45:169–181. 1991. Starter culture and time/temperature of storage 43 NM Sachindra, PZ Sakhare, KP Yashoda, D Narasimha influences on quality of fermented mutton sausage 44 Rao. 2005. Microbial profile of buffalo sausage during J Food Sci 56:916–919, 925. 45 processing and storage. Food Control 16: 31–35. OA Young, J-L Berdague, C Viallon, S Rousset-Akrim, 46 J Sales. 1998. Fatty acid composition and cholesterol con- M Theriez. 1997. Fat-borne volatiles and sheepmeat 47 tent of different ostrich muscles. Meat Sci 49:489–492. odour. Meat Sci 45:183–200. 48 J Sales, D Marais, M Kruger. 1996. Fat content, caloric OA Young, DH Reid, ME Smith, TJ Braggins. 1994. 49 value, cholesterol content, and fatty acid composi- Sheepmeat odour and flavor. In: F Shahidi, ed. Flavor 50 tion of raw and cooked ostrich meat. J Food Comp of Meat and Meat Products. London: Blackie Aca- 51 Anal 9:85–89. demic & Professional, pp. 71–97. 52 C Sanudo, ME Enser, MM Campo, GR Nute, G Maria, 53S I Sierra, JD Wood. 2000. Fatty acid composition and 54N 40454 36 375-386 r1.qxd 7/21/07 12:38 PM Page 375

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1 2 3 36 4 5 6 U.S. Products 7 8 Kenneth J. Stalder, Nicholas L. Berry, Dana J. Hanson, and William Mikel 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION protect a market by not allowing a competing product 22 to use the same name—for example, country ham 23 There is no doubt that the dry-curing of meat, or versus a ham and water product. These are both ham, 24 as we in the United States call it, country curing, but they are produced in a very different manner. 25 specifically of pork, followed from European tradi- Title 9, Section 319.106 of the USDA-FSIS code 26 tions as the New World was beginning to be settled classifies Country Ham, Country Style Ham, Dry 27 as early as the 1500s (Voltz and Harvell 1999). Cured Ham, Country Pork Shoulder, Country Style 28 Explorers and settlers arrived in what eventually Pork Shoulder, and Dry Cured Pork Shoulder as 29 became the east coast of the United States of Amer- follows: 30 ica and often brought with them pigs to provide a 31 source of food. The pigs were preferred because they (a)–Country Ham, Country Style Ham, or Dry 32 had many offspring per female every year. Addition- Cured Ham, and Country Pork Shoulder, 33 ally, the pigs were allowed to roam freely and forage Country Style Pork Shoulder or Dry Cured 34 for their own food. As the pigs gained weight and Pork Shoulder are the uncooked, cured, dried, 35 became full grown, they were harvested. Settlers smoked or unsmoked meat food products made 36 found it desirable to preserve meat in order to provide respectively from a single piece of meat 37 food for their families over a longer period of time. Of conforming to the definition of ham, as 38 course, in those days there was no refrigeration and, specified in Sec. 317.8(b) (13), or from a single 39 hence, other methods of preservation were required. piece of meat from a pork shoulder. They are 40 The use of dry-cure methods had long been utilized in prepared in accordance with paragraph (c) of 41 Europe, and the early U.S. settlers applied the knowl- this section by the dry application of salt 42 edge they brought with them. Consequently, a new (NaCl), or by the dry application of salt (NaCl) 43 industry was born that still survives, mainly in the and one or more of the optional ingredients as 44 southeastern U.S., albeit dramatically changed. specified in paragraph (d) of this section. They 45 may not be injected with curing solutions nor 46 COUNTRY HAM STANDARDS placed in curing solutions. 47 (b)–The product must be treated for the destruction 48 The United States Department of Agriculture of possible live trichinae in accordance with 49 (USDA) Food Safety and Inspection Service (FSIS) such methods as may be approved by the 50 has developed standards (Code of Federal regula- Administrator upon request in specific instances 51 tions 2001) of identity in order to assure that con- and none of the provisions of this standard can 52 sumers are provided products they expect, based be interpreted as discharging trichinae treatment 53 on label claims. These standards can also serve to requirements. S54 N55 377 40454 36 375-386 r1.qxd 7/21/07 12:38 PM Page 378

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1 (c) —– 1. Nutritive sweeteners, spices, seasonings and 2 flavorings. —– 1. The entire exterior of the ham or pork 3 —– 2. Sodium or potassium nitrate and sodium or shoulder shall be coated by the dry 4 potassium nitrite if used as prescribed in this application of salt or by the dry application 5 section and in accordance with a regulation of salt combined with other ingredients as 6 permitting that use in this subchapter or 9 CFR permitted in paragraph (d) of this section. 7 Chapter 111, Subchapter E, or in 21 CFR —– 2. Additional salt, or salt mixed with other 8 Chapter 1, Subchapter A or Subchapter B. permitted ingredients, may be reapplied to 9 the product as necessary to insure complete 10 penetration. 11 —– 3. When sodium or potassium nitrate, or COMMERCIAL DRY-CURE HAM 12 sodium or potassium nitrite, or a combination PRODUCTION IN THE U.S. 13 thereof, is used, the application of salt shall 14 HISTORY be in sufficient quantity to insure that the 15 finished product has an internal salt content The country ham industry has its roots in the south- 16 of at least 4%. eastern U.S. Early U.S. colonial history suggests that 17 —– 4. When no sodium nitrate, potassium nitrate, the ham industry began around the time European 18 sodium nitrite, potassium nitrite or a settlers arrived in what is now Virginia. As early as 19 combination thereof is used, the application the 1600s, colonists realized that hogs were a critical 20 of salt shall be in sufficient quantity to insure valuable food resource if they were to survive in the 21 that the finished product has a brine New World. 22 concentration of not less than 10% and In 1779, Captain Mallory Todd of Smithfield, Vir- 23 a water activity of not more than 0.92. ginia, is often credited with starting the commercial 24 —– 5. For hams or pork shoulders labeled country or country ham industry in the United States, after he 25 country style, the combined period for curing shipped cured hams to the West Indies. The country 26 and salt equalization shall not be less than ham industry grew into a viable industry in this 27 45 days for hams, and shall not be less than region, and by the early 1900s Smithfield, Virginia 28 25 days for pork shoulders; the total time for boasted that it was the “ham capital of the world” 29 curing salt equalization, and drying shall not (Evans-Hylton 2004). 30 be less than 70 days for hams, and shall not be Another factor that contributed to the success of 31 less than 50 days for pork shoulders. During the industry was the fact that peanuts were a readily 32 the drying and smoking period, the internal available feed source for the hogs raised in the area. 33 temperature of the product must not exceed Peanut-fed hogs quickly became synonymous with a 34 95°F (35°C), provided that such temperature Smithfield Ham. In 1926, the Virginia General 35 requirement shall not apply to product dried Assembly passed legislation to protect the identity of 36 or smoked under natural climatic conditions. ham produced in Smithfield. A genuine Smithfield 37 —– 6. For hams or pork shoulders labeled dry Ham was defined as a dry-cured ham that was pro- 38 cured, the combined period for curing and duced within the city limits of Smithfield, Virginia 39 salt equalization shall not be less than from peanut-fed hogs from the “peanut belt” of 40 45 days for hams, and shall not be less than Virginia or North Carolina (Evans-Hylton 2004). This 41 25 days for pork shoulders; and the total time law was altered in 1966, when the peanut-fed hog dis- 42 for curing, salt equalization, and drying shall tinction was removed because peanut-fed hogs became 43 not be less than 55 days for hams and shall more difficult to procure (Evans-Hylton 2004). 44 not be less than 40 days for pork shoulder. Commercial country ham production has followed 45 —– 7. The weight of the finished hams and pork the evolution of the U.S. from a totally agrarian soci- 46 shoulders covered in this section shall be at ety to a more industrialized nation (D. Pilkington, 47 least 18% less than the fresh uncured weight personal communication, 2006). Beginning after 48 of the article. The total time for curing, salt World War II, many farmers or their children began 49 equalization, and drying shall not be less than to move to the city to look for work, and they desired 50 55 days for hams and shall not be less than familiar food including country ham. This started 51 40 days for pork shoulders. commercialization of the country ham industry in 52 (d)–The optional ingredients for products covered in the late 1940s and early 1950s (D. Pilkington, per- 53S this section are sonal communication, 2006). 54N 40454 36 375-386 r1.qxd 7/21/07 12:38 PM Page 379

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ORGANIZATION or would find difficult to do individually (National 1 Country Ham Association 2006). Some of the 2 In 1992, commercial dry ham producers in the U.S. defined responsibilities include “encourage the sale 3 organized themselves in order to have the interests of and use of country cured meats through cooperative 4 country ham producers represented nationwide methods of production, promotion, education, adver- 5 (National Country Ham Association 2006). The tisement, and trade shows” (National Country Ham 6 National Country Ham Association headquarters is Association 2006). Some of the issues addressed 7 located in the heart of country ham production in by country ham producers through actions of this 8 Conover, North Carolina. The majority of, but not organization include Hazard Analysis Critical Con- 9 all, dry-cure or country ham producers are members trol Points (HACCP), nutritional labeling, various 10 of this organization. Currently, there are 21 members research projects. Ultimately, the goals of the organi- 11 of the National country Ham Association (National zation are designed to increase consumer demand for 12 Country Ham Association 2006). The organization is country ham and other dry-cured meat products, 13 funded through a self-imposed check-off currently thereby improving the profitability of its members. 14 set at $0.0125 per ham, with minimum dues of Country ham enthusiasts can find out where to buy 15 $250.00 and a maximum of $6,250.00 annually product and find new and traditional country ham 16 (National Country Ham Association 2006). Addition- recipes at the www.countryham.org website. 17 ally, many producers have organized state county ham 18 associations. COUNTY HAM PRODUCTION 19 The organization maintains a website providing 20 contact information, products produced, and web- U.S. commercial country ham production is concen- 21 sites of its membership (National Country Ham trated in the southeastern portion of the country 22 Association 2006). Additionally, the association (Figure 36.1). This region of the country is where 23 holds an annual meeting for its membership. This country ham originated. This area also has the desir- 24 meeting not only includes an annual business meet- able climate for producing country hams utilizing 25 ing, but typically also includes educational seminars traditional methods. Today, country ham could be 26 that address the latest dry-cure research and regula- produced in any area of the U.S. because commercial 27 tory issues. The association also publishes a quar- ham processing facilities have the ability to control 28 terly newsletter addressing emerging issues. the curing environment. 29 The organization primarily functions to perform Members of the National Country Ham Associa- 30 duties that individual country ham producers cannot, tion cured approximately 3.4 million hams in 2004 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 Figure 36.1. U.S. states (Georgia, Kentucky, Missouri, Pennsylvania, North Carolina, Tennessee, and Virginia) S53 where country-cure meat products are produced. N54 40454 36 375-386 r1.qxd 7/21/07 12:38 PM Page 380

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1 (C. Cansler, personal communication, 2006). environment. Furthermore, country ham producers 2 Assuming a 9.1 kg average weight for each ham and are exploring a variety of marketing methods, rang- 3 a retail value of $11.00 per kg., the value of U.S. pro- ing from bubble packages of thinly sliced country 4 duced country hams produced by member of the ham, center-cut slices, biscuit-sized portions, etc., in 5 National Country Ham Association was approxi- addition to the traditional method of marketing 6 mately 340 million dollars in 2005. Nationwide, it is whole, bone-in cured hams. This is being done to 7 estimated that 6.5 million hams were produced in capture new markets and to serve more traditional 8 2004 (C. Cansler, personal communication, 2006) markets that demand product in a manner that meets 9 by both nonmembers and members of the National the consumer’s desire for meal components that are 10 Country Ham Association. These hams had a retail both easy and quick to prepare. 11 value in excess of 600 million dollars. Although this 12 represents a large quantity of hams, it is still a rela- COMMERCIAL CURING PROCESS 13 tively minor portion of pork meat produced in the 14 U.S annually. Approximately 110 million market pigs The curing process begins with the purchase of qual- 15 are processed annually in the U.S. These carcasses ity fresh or what the industry terms as green hams. 16 would produce 220 million hams annually (National Country ham processors begin by purchasing green 17 Pork Board 2005). The numbers of country hams hams from a harvest facility. These harvest facilities 18 processed annually represent approximately 3% of follow USDA guidelines for humanely and safely 19 the total fresh hams produced in the U.S. harvesting food animals. As the harvest process 20 The estimated production of country ham in the begins, the animal, and subsequently the carcass, and 21 U.S. by state is shown in Table 36.1. North Carolina all internal organs are thoroughly inspected. A vet- 22 led the U.S. in the estimated production of country erinarian or someone supervised by a veterinarian 23 ham, with over 2.7 million hams cured in 2004. closely examines every carcass for signs of disease 24 These hams had a retail value in excess of 273 mil- or abnormalities. After harvest, the carcass is chilled 25 lion dollars. North Carolina was followed by Ten- and then broken into primal cuts. One of these pri- 26 nessee, Missouri, Kentucky, Virginia, Georgia, and mal cuts, the ham, serves as the raw ingredient for 27 Pennsylvania in the production of country hams. As the country ham industry. In order for any portion of 28 more country ham producers and even pork produc- the harvested carcass to be sold commercially, it 29 ers recognize the opportunity to enter a value-added must have a stamp signifying that it has passed 30 market chain, country ham production is likely to required inspection. This includes all primal cuts 31 continue to rise. Additionally, country ham is mov- sold for further processing. Hence, all commercial 32 ing from a product that is regionally produced and country ham processors purchase their green hams 33 consumed to one that is being served in more areas from USDA-inspected harvest facilities so both the 34 of the country. This is particularly true of chain processor and later the consumer can be assured that 35 restaurants that have their origins in the southeastern the country curing process began with fresh hams 36 U.S. and have branched out into other areas of the that were wholesome and safe. 37 country. Moreover, country ham producers have rec- Once green hams have been purchased, the first 38 ognized an opportunity to add value when compet- step in the dry-curing process occurs. The hand appli- 39 ing against imported prosciutto and other types of cation of the dry-cure ingredients has not changed 40 ham produced in other areas of the world. Imported since the process was first identified. The ingredients 41 hams have traditionally demanded a relatively high in the curing mixture include (1) salt, (2) sodium 42 retail price, particularly on both the East and West nitrite/nitrate, (3) sugar, and (4) other spices. 43 Coasts of the U.S. U.S. country ham producers are Salt is really the only ingredient needed to per- 44 taking their product to these regions of the country form the dry-curing process. Salt provides flavor to 45 where opportunity exists to expand their market and dry-cured meat and acts as a preservative by inhibit- 46 sell product for premium prices. A good example is a ing growth and destroying microorganisms (Marriott 47 new dry-cure ham facility that was recently com- and Graham 2000). Additionally, it helps other dry- 48 pleted in Iowa, not considered the traditional dry- cure components migrate through the raw product 49 cure ham production area of the U.S. This new and dehydrates the meat, which also limits bacterial 50 facility is making a prosciutto-like dry-cured ham growth (Marriott and Graham 2000). 51 that is being marketed to high-end value-added Sodium nitrite/nitrate helps give the product the 52 markets on the east and west coasts of the U.S. The reddish color that country ham is known for. Addi- 53S company is using hams from pigs raised without the tionally, the sodium nitrate/nitrite acts as a pre- 54N use of antibiotics and in an outdoor or “natural” servative and inhibits the growth of Clostridium 40454 36 375-386 r1.qxd 7/21/07 12:38 PM Page 381

1 2 3 4 5 6 7 8 9 10 ciation, personal communication, weight of dry-cure hams is 9.1 kg weight 11 12 13 14 15

1 16 17 18 19 20 21 22 23 24 25

Produced, kgProduced, % Hams Produced, $ Value, Retail 26 27 28 29 30

2 31 32 33 34 35 36 37 38 39 40 41 42 43

Number of Estimated Number of of Hams Weight Total of Percentage 44 45 46 47 48 49 U.S. state. by country estimated production, percentage produced, and retail value ham producers, 50 51 52 S53 Data from members of National Country Ham Association only. Information was obtained from C. Cansler, National Country Ham Asso National Country Ham obtained from C. Cansler, Information was Association only. Data from members of National Country Ham Estimated 6.5 million hams produced by nonmembers and members of the National Country Ham Association in 2004. Assumes average Assumes average Association in 2004. nonmembers and members of the National Country Ham Estimated 6.5 million hams produced by 2006. and a retail value of $11.00 dollars per kg. and a retail value Table 36.1. Table State ProducersTotal1 Hams Produced 2 54–59 6,500,000 59,150,000 100 606,620,000 North CarolinaTennesseeMissouriKentuckyVirginia 20–25GeorgiaPennsylvania 10 5 10 2,730,000 7 2 2 1,690,000 24,843,000 910,000 780,000 260,000 15,379,000 65,000 65,000 8,281,000 42 7,098,000 2,366,000N54 26 591,500 591,500 273,270,000 14 12 4 125,170,000 1 1 91,100,000 78,050,000 26,030,000 6,500,000 6,500,000

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1 botulinum, the organism that causes botulism. this process the remainder of the dry-cure is applied 2 Nitrates and nitrites contribute the flavor distinctive to the partially cured meat. The ham is then refriger- 3 of cured meats and reduce the development of oxida- ated for an additional 35 days at approximately 4 tive rancidity and the associated rancid taste 2.5–3.0°C. 5 (Marriott and Graham 2000). After the initial curing process, excess salt is 6 Sugar is included to sweeten the taste of the ham washed from the surface of the ham. After washing, 7 and reduce the salty flavor of the ham to some the hams are placed in a mesh stockinette made of 8 degree. Sugar also provides an energy source for plastic or cotton with the hock, the small portion of 9 beneficial microorganisms that convert nitrate to the ham, down. The hams are hung on ham trees 10 nitrite during the curing process (Marriott and (Figure 36.3). The next step is called equalization or 11 Graham 2000). drying. In this process, the hams are placed in envi- 12 Other spices are added to provide flavor differ- ronmentally controlled chambers for a minimum of 13 ences, and their use varied from processor to proces- 13 days and up to 30 days. The temperature of the 14 sor. The amount of each of these ingredients varied chambers is maintained at 10–12.5°C, and the rela- 15 slightly from processor to processor and is what con- tive humidity is maintained between 55% and 70%. 16 tributes to flavor differences between individual This process dries the hams. Controlling the relative 17 processor’s products. Consumers usually favor the humidity helps prevent mold growth on the surface 18 flavor of ham from a given country ham processor. of the ham. Additionally, the temperature is used to 19 The slight differences in the curing ingredients con- kill Trichina spiralis. 20 tribute to these consumer flavor preferences. After equalization has occurred, hams can be 21 The dry-cure mixture is spread evenly or rubbed smoked for 7 to 8 days at a temperature of 41°C if 22 onto the ham, paying particular attention to the lean desired. Smoking of the hams is not necessary, but it 23 surfaces of the ham. Typically, the dry-cure mixture gives the surface of the hams a desirable deep golden 24 is packed into the hock to prevent spoilage that brown color (Figure 36.4). Smoking is not only for 25 occurs around the bone of the ham. Approximately, aesthetic purposes, but for purposes of adding flavor 26 one-half of the dry-cure mixture is applied in the ini- and aroma, which may be important when the final 27 tial application of the dry-cure ingredients. After the product is marketed as whole ham. Generally, smok- 28 application of the dry-cure mixture has occurred, the ing occurs by exposing the hams to smoke from saw- 29 hams are placed fat side down into large containers dust made from oak, hickory, apple, or some other 30 or plastic vats (Figure 36.2) and refrigerated. Plastic desirable wood material. Trichina control can occur 31 vats are used because they are easily cleaned. After by manipulating the temperature and duration during 32 5 to 7 days of refrigeration, the hams are resalted; the smoking and drying periods. Smoking is a 33 the industry calls this process overhauling. During process that is traditional to a Virginia Style Country 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53S 54N Figure 36.2. Salted hams in large plastic vats after dry-cure mixture has been placed on the surface. 40454 36 375-386 r1.qxd 7/21/07 12:38 PM Page 383

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Figure 36.3. Hams are placed in stockings and hung on “trees,” with the hock portion of the ham at the bottom. 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 Figure 36.4. Country-style hams that have been smoked have a golden brown color. 37 38 39 Ham. This processing step is applied to most hams HAM CURING AT HOME 40 produced in the U.S. The exception to this rule is 41 found in North Carolina, where almost all country Long before dry-cure ham production became com- 42 hams are not smoked. This unsmoked country mercialized or industrialized, dry-cure hams were 43 ham is often referred to as a North Carolina Style produced at home on the farm. The reason that coun- 44 Country Ham. try ham is produced and became popular in the 45 The next step in the process is referred to as aging. southeastern U.S. was that it had the climate required 46 The aging period can last 30 days or longer. Many to properly dry-cure ham. Settlers and farmers of 47 specialty hams and dry-cure hams from other areas the ham-producing states commonly experience 48 of the world are aged 18 to 24 months. The longer temperatures between 0° and 5.5°C during the win- 49 the ham is allowed to age, the bolder and more com- ter. These temperatures allowed farmers, curing their 50 plex the ﬂavor will become. Drying temperatures own hams, to avoid spoilage and freezing during 51 normally range from 21–25°C, with the relative these months (American Museum of Food 2006). 52 humidity (55–70% similar to that utilized during the The mild springs in these areas of the U.S. were ideal S53 equalization step. for equalization of the salt content in the ham N54 40454 36 375-386 r1.qxd 7/21/07 12:38 PM Page 384

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1 (American Museum of Food 2006). The hot and heavier hams produced from today’s market pig 2 humid summers experienced in this area were ideal make curing hams at home much different for cur- 3 for the aging process (American Museum of Food rent generations of curing enthusiasts when com- 4 2006). The hams were ready to eat around Thanks- pared with their predecessors. Heavy weight hams 5 giving time the following year. Thus, a tradition was can affect the rate of cure penetration, the amount of 6 born whereby country hams were produced and con- cure ingredients needed, and overall aging time. 7 sumed during the holiday time of the year (American Individuals that want to cure a smaller weight ham 8 Museum of Food 2006). may consider raising the pigs themselves, or make 9 Today, many country-cured hams are still pro- arrangements with a pork producer to purchase a 10 duced at home, passing the tradition of country ham pig(s) that is lighter than normal marketing weight. 11 production on to future generations. Many of the After the carcasses have gone through proper 12 home curers of country-cured ham remember days chilling, the hams are dissected (USDA 1967) from 13 gone by where many family members were involved the carcass and are ready to have the dry-cure mix- 14 with the entire process of slaughtering hogs on the ture applied or rubbed on the hams. Rather than 15 farm and producing country ham. These producers harvesting and processing the pigs at home, many 16 continuously try to keep the tradition alive for future people who want to produce their own country ham 17 generations. at home purchase fresh hams from a local supplier. 18 If producers want to raise and harvest their own High-quality fresh hams can be purchased from gro- 19 pigs on the farm, slaughtering should occur in the cery stores, local butcher shops, or locker plants. 20 winter months when the temperature drops below After the fresh hams have been prepared or 21 40°F (4.5°C) (Cole and Houston 1961). This is the purchased, the dry-cure mixture is applied to the 22 most extreme temperature that will still allow the hams. The dry-cure mixture ingredients include: 23 carcass to go through the rigor process without salt, sugar, and saltpeter. The exact mixture can vary 24 spoiling (USDA 1967). The average weight of mar- slightly. Table 36.2 shows examples of dry-cure mix- 25 ket hogs in 2004 was 121.1 kg (267 lb) in the U.S. tures commonly used for the home curing process. 26 (National Pork Board 2005). Carcasses produced Only salt is really needed to cure the ham. The other 27 from a pig weighing 121 kg will weigh approxi- ingredients provide the roles previously described. 28 mately 91 kg (200 lb). This large carcass will pro- After the dry-cure mixture has been applied to the 29 duce a raw ham in the 9 to 10 kg range. Both hams surface, the ham is placed in a container of some 30 represent approximately 25% (22.7 kg or 50 lbs.) of sort; a clean barrel or crock works well (USDA 31 the total carcass weight (National Pork Board 2005). 1967). If many hams are being cured, small wooden 32 Because average market weight continues to rise in boxes made of oak or other hardwood or a small 33 the U.S., ham weight will continue to increase. These building with shelving works well (Winfree and 34 35 36 37 Table 36.2. Dry-cure mixture ingredients commonly used when curing hams at home. 38 39 Formula Ingredient Amount, % Reference 40 1. Salt 79.0 Winfree (1975) 41 Sugar 19.8 United States Department of Agriculture (1967) 42 Saltpeter 1.2 Marriott and Ockerman (2004) 43 44 2. Salt 71.5 Cole and Houston (1961) 45 Sugar 27.2 46 Saltpeter 1.7 47 3. Salt 76.1 Skelley and Ackerman (1965) 48 Sugar 21.8 49 Saltpeter 2.1 50 4. Salt 83.0 Varney (1968) 51 Sugar 8.9 52 Saltpeter 1.8 53S 54N 40454 36 375-386 r1.qxd 7/21/07 12:38 PM Page 385

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Melton 1990). The temperature should be main- for ensuring food safety appropriately on the food 1 tained at 2–4°C (Cole and Houston 1961). The hams manufacturer or distributor, (5) helping food compa- 2 should remain in the cured mixture for a minimum nies compete more effectively in the world market, 3 of 30 days. After the salt curing, the hams should be and (6) reducing barriers to international trade. These 4 placed in cold water to remove excess salt and then factors go a long way toward ensuring that food, 5 allowed to dry (Cole and Houston 1961). The hams including country ham produced in the U.S., is safe 6 “equalize” approximately 30 days, which allows the and wholesome (USDA 2001). 7 salt to spread evenly throughout the meat. If smok- 8 ing is desired, a smoldering smoke made from hard- 9 woods (again oak, hickory, etc., are commonly used) COOKING 10 is utilized for a period of 2 days in order to obtain the 11 As with the European prosciutto or Serrano dry- desired color and flavor. 12 cured ham counterparts, properly cured country ham Cured meat should be stored in a cool, dry, well- 13 does not need to be cooked to be safely eaten. As ventilated area that is free of insects and rodents (Cole 14 more and more country ham is distributed around and Houston 1961). There are many things that have 15 the U.S., more of it is eaten in the more traditional been done in an attempt to control pests when storing 16 European way, uncooked. Some think that country whole cured hams. The first attempt is processing 17 ham or dry-cured ham has the best flavor this way hams in cooler weather when freezing temperatures 18 (Marriott and Graham 2000). will kill insects. Also, wrapping hams in meat wrap- 19 Traditions in the U.S. usually dictate that country ping paper ((USDA 1970) and inserting the wrapped 20 ham is cooked prior to consumption. One of the ham into a closely woven bag and hanging it in a cool 21 most common ways to cook country ham is by pan- dry place will help eliminate pest damage. Coating 22 frying it. Thin pieces of pan-fried country ham can the outside of a cured ham in lard or coating with a 23 be served with a variety of side dishes or put in the heavy layer of pepper is also thought to reduce the 24 middle of a homemade biscuit and eaten for break- incidence of insect infestation if curing at home (Cole 25 fast. Other methods of cooking country ham include and Houston 1961). A good summary of the required 26 oven-steaming, slow-cooking in the oven, and roast- curing times, temperatures, and relative humidity can 27 ing in an oven bag (Voltz and Harvell 1999; Marriott be found in a fact sheet by Varney (1968). 28 and Ockerman 2004). 29 Today’s consumer might feel that a large bone-in 30 SAFETY country ham is difficult to prepare. However, with 31 relative minimal effort a great meal can be prepared Country ham in the U.S. is produced commercially 32 with this American delicacy. The following steps are under very controlled conditions using only the safest 33 an easy way to cook a whole country ham: raw ingredients. A plant must adhere to many regula- 34 tions before USDA inspectors will allow a label 1. Remove the whole ham from its package. 35 (indicating inspected and passed) to be placed on the Country hams will often develop mold on the 36 product (Code of Federal Regulations 2001). Addi- surface of the skin during storage. This mold does 37 tionally, all country ham processors must have a not detract from the quality or safety and is easily 38 HACCP program in place. The HACCP program has removed by washing it off with tap water and a 39 seven principles including (1) conduct a hazard analy- stiff kitchen brush. In order to facilitate easier 40 sis, (2) determine the critical control points (CCPs), handling during cooking the shank may be 41 (3) establish critical limits, (4) establish monitoring removed at this time. 42 procedures, (5) establish corrective actions, (6) estab- 2. The harshness of the salt may be reduced by 43 lish verification procedures, and (7) establish record- soaking the ham for 24 to 48 hours prior to 44 keeping and documentation procedures (USDA 201). cooking. An easy way to accomplish this task 45 The HACCP program focuses on (1) identification is to soak the ham in a plastic cooler large enough 46 of hazards and preventing them from contaminating to hold the ham and 10 to 15 liters of water. For 47 the food supply, (2) basing the plan on sound science, best results, change the water 2 to 3 times during 48 (3) allowing more efficient and effective government the soaking process. 49 oversight (record-keeping allows inspectors to deter- 3. The ham now is ready to begin the cooking 50 mine whether a firm is complying with food safety process. Place the ham in a pot large enough that 51 laws over a period of time rather than how well it is it will hold the ham yet allow for the entire ham 52 doing on any given day), (4) placing responsibility to be covered with water. Simmer the ham over S53 N54 40454 36 375-386 r1.qxd 7/21/07 12:38 PM Page 386

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1 medium-low heat for 3–4 hours or until the bone NG Marriott, PP Graham. 2000. Some solutions to dif- 2 is nearly lose enough to remove by hand. ficulties of home-curing pork. Virginia Cooperative 3 4. Allow the ham to cool long enough that it may be Extension. Publication Number 458–872. Blacks- 4 safely removed from the cooking water. At this burg, Virginia: Virginia Polytechnic Institute and 5 time the ham can be transferred to an oven-safe State University. 6 cooking pan. Prior to oven-baking, the ham can NG Marriott, HW Ockerman. 2004. The ultimate guide 7 be dressed with pineapple rings, cherries, and to country ham an American delicacy. Radford, Vir- 8 clove or brushed with the cook’s favorite glaze. ginia: Brightside Press. 9 Cook the ham in a 175°C oven for 20 to 30 National Country Ham Association. 2006. National 10 minutes. This final cooking step is really needed Country Ham Association web site. Available at http:// 11 only to brown the outer surface of the ham and www.countryham.org. Accessed on April 30, 2006. 12 give it a pleasing appearance. National Pork Board. 2005. Quick Facts. Clive, Iowa: 13 5. Carve thin slices (5 to 6 mm) of the cooked ham National Pork Board. Available at http://www.pork. 14 prior to serving. org/NewsAndInformation/QuickFacts/porkFactPDFS/ 15 pg68-93.pdf. Accessed on April 20, 2006. 16 RESEARCH GC Skelley, Jr., CW Ackerman. 1965. Pork Processing 17 and Curing. Clemson Agriculture Extension Service. 18 Because the country ham industry represents such a Bulletin 77. Clemson, South Carolina: Clemson 19 small portion of the U.S. pork industry, relatively University. 20 little funding opportunities exist to evaluate ways to KJ Stalder, CC Melton, GE Conatser, SL Melton, MJ 21 improve it. Recent work has explored breed differ- Penfield, JR Mount, D Murphey, KJ Goddard. 2003. 22 ences (Stalder et al. 2003) and molecular marker Fresh and processed qualities of dry-cured hams 23 (Stalder et al. 2005) effects on various quality and from purebred Duroc and genetically undefined mar- 24 yield evaluations in country ham. These studies have ket hogs. J Muscle Foods 14:253–263. 25 reported that hams from purebred Duroc may have a KJ Stalder, MF Rothschild, SM Lonergan. 2005. Asso- 26 yield and quality advantage when compared to hams ciation between two gene markers and indicator traits 27 from pigs of unknown genetic background (Stalder affecting fresh and dry-cured ham processing quality. 28 et al. 2003). Additionally, two molecular markers, Meat Sci 69:451–457. 29 CAST and PRKAG3, have been shown to impact United States Department of Agriculture (USDA). 30 processing yield and moisture content of country- 1967. Slaughtering, cutting, and processing pork on 31 style cured hams. Other current studies under way the farm. Farmers’ Bulletin No. 2138. Washington, 32 include a project designed to identify methyl bro- D.C.: United States Department of Agriculture. 33 mide alternatives for use as pest infestation deter- ———. 1970. Protecting home-cured meat from insects. 34 rents. These studies are being conducted as a joint Home and Garden Bulletin No. 109. Washington, 35 project between Mississippi State, North Carolina D.C.: United States Department of Agriculture. 36 State, and the University of Kentucky in conjunction United States Food and Drug Administration. 2001. 37 with the National Country Ham Association. HACCP: A state-of-the-art approach to food safety. 38 Rockville, Maryland: U.S. Food and Drug Adminis- 39 REFERENCES tration. Available at http://www.cfsan.fda.gov/~lrd/ 40 bghaccp.html. Accessed on April 30, 2006. 41 American Museum of Food. 2006. Raw Talent: Amer- WY Varney. 1968. Curing and Aging Country-Style 42 ica’s ham artisans take on Europe’s masters. Available Hams Under Controlled Conditions. University of 43 at http://www.americanmuseumoffood.org/. Accessed Kentucky Cooperative Extension Service. Circular 44 on April 30, 2006. 617. Lexington, Kentucky: University of Kentucky. 45 Code of Federal Regulations. 2001. Title 9, Volume 2. J Voltz, EJ Harvell. 1999. The Country Ham Book. 46 Parts 200 to end. Revised as of January 1, 2001. Chapel Hill, North Carolina: The University of North 47 9CFR319.106, pp. 300–301. Carolina Press. 48 JW Cole, JW Houston. 1961. Country Style Pork. Uni- SK Winfree. 1975. Preserving Pork at Home. The Uni- 49 versity of Tennessee Agriculture Extension Service versity of Tennessee Agriculture Extension Service. 50 Publication 441. Knoxville, Tennessee: The Univer- Knoxville, Tennessee: The University of Tennessee. 51 sity of Tennessee. SK Winfree, CC Melton. 1990. Home Guide to Meat 52 P Evans-Hylton. 2004. Images of America: Smithfield, Curing. The University of Tennessee Agricultural 53S Ham Capital of the World. Charleston, South Car- Extension Service. Publication PB700-500-4/90. 54N olina: Arcadia Publishing. Knoxville, Tennessee: The University of Tennessee. 40454 37 387-392 r1.qxd 7/21/07 12:40 PM Page 387

1 2 3 37 4 5 6 Central and South American Products 7 8 Silvina Fadda and Graciela Vignolo 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION processed foods contributing to obesity, diabetes, 22 and other chronic conditions in this region (Romieu 23 Food habits in Central and South America are a blend et al. 1997). The balance between improving access 24 of cultural backgrounds, available foods, cooking to variety and maintaining dietary quality poses a 25 styles, and the foods of colonial Europeans. Although challenge for public health. In Mexico and Central 26 Spanish and Portuguese influence is largely docu- America, as elsewhere, this transition has been 27 mented in Latin American countries, eating habits fueled by globalization and urbanization. 28 could be divided into two main characteristic groups: The cuisine of South America differs among coun- 29 the highland foods of the Andes with a maize-based tries and regions. Some regions have a largely maize- 30 diet and the lowland dishes of southern countries that based diet, and others use rice as a meal base. Meat 31 are major beef producers. The cuisine in the moun- is also very popular and traditionally sides of beef, 32 tain areas is unique in South America, preserving hogs, lamb, and goats are grilled slowly for hours. 33 many dishes of the Incas. Another cooking method consists of steaming foods 34 in a pit oven, as with pachamanca in Perú, which 35 typically includes a young pig or goat (as well as 36 REGIONAL CHARACTERISTICS chicken, guinea pig, tamales, potatoes, and corn) 37 The diets of people in Mexico and Central America cooked under layers of hot stones, leaves, and herbs. 38 (Guatemala, Nicaragua, Honduras, El Salvador, Venezuelan and Colombian foods have strong Span- 39 Belize, and Costa Rica) have several common fea- ish influences, the food being prepared and cooked 40 tures, though within the region great differences in with olive oil, onions, parsley, cilantro, garlic, and 41 methods of preparation and local recipes exist. The cheese. Hot chili peppers as well as local fruits 42 basis of the traditional diet in this part of the world and vegetables are used as a garnish in most dishes. 43 is corn (maize), beans, and rice, with the addition of Chicken stew and sancocho (a meat stew with 44 meat, animal products, local fruits, and vegetables. starchy vegetables) are popular in Colombia; pabel- 45 Historically, major changes in the traditional diet lón caraqueño, consisting of flank steak served on 46 occurred during colonial times, when the Spaniards rice with black beans, topped with fried eggs and gar- 47 and other European settlers introduced the region to nished with plantain chips, is a typical meat dish in 48 wheat bread, dairy products, and sugar. The con- Venezuela. The cuisine of Guyana and Suriname is 49 sumption of meat and animal products (pork, beef, a culinary hybrid with African, Indian, Portuguese, 50 chicken, fish, and eggs), although popular, is often Dutch, Creole, and Chinese influences. A favorite 51 limited due to cost. However, an Americanized ver- meat dish is pepper pot, a stew made with bitter cas- 52 sion of popular foods as a result of culture intermin- sava juice, meat, hot pepper, and seasoning. In Brazil, 53 gling has been produced, with the increasing use of the Portuguese contributed with linguiça (Portuguese S54 N55 387 40454 37 387-392 r1.qxd 7/21/07 12:40 PM Page 388

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1 sausage) and spicy meat stews. The national dish of American poultry producers; broiler meat produc- 2 Brazil is feijoada, which consists of black beans tion is increasing steadily in response to strength- 3 cooked with smoked meats and sausages served with ened domestic demand and record exports. Due to 4 rice, sliced oranges, boiled greens, and hot sauce. In high beef price and consumer nutritional concerns, 5 Perú and Ecuador, food features an abundant use of the demand for poultry meat and poultry processed 6 potatoes and chili peppers. Due to the scarce avail- products has increased among consumers. Pork is 7 ability of other sources of meat as well as to geo- the world’s major supplier of animal proteins as the 8 graphical isolation, communities in the Andean principal meat staple in Central America. Domestic 9 plateaus consume meat from Camelidae (llama, gua- consumption of pork meat as well as processed pork 10 naco, and alpaca), whereas in the tropical coastal products is of great importance. The consumption of 11 regions ceviche, prepared from ﬁsh and seafood mar- meat and meat products in some Latin American 12 inated in sour lemon, is a typical dish. In Central and countries is summarized in Table 37.1. In this chap- 13 South America, along the Andes highland regions, ter, a contribution is presented on the meat foods 14 meat pies called empanadas as well as tamales con- currently prepared and consumed in Latin America 15 taining a mixture of chopped meat (beef and pork), as well as their safety status. 16 onions, potato, and different seasonings are tradition- 17 ally consumed. 18 The southern countries (Argentina, Chile, Uruguay, TYPICAL MEAT PRODUCTS, 19 and Paraguay), including Brazil, are major beef MICROBIAL ECOLOGY, AND 20 producers. Argentineans eat more beef per capita SAFETY RISKS 21 than people from any other country in the world PROCESSED MEAT PRODUCTS 22 (67 kg/person/year). Argentina is famous for asados, 23 grilled meat dishes, mainly beef, but also pork, Even though in Central America and the western 24 lamb, and chicken. Meat production in South Amer- areas of South America the consumption of meat and 25 ica is concentrated in Argentina, Uruguay, and animal products (pork, beef, chicken, ﬁsh, and eggs) 26 Brazil, where meat exportation constitutes an impor- is limited due to their high cost, a wide variety of 27 tant source of income. Nevertheless, a great diver- meat- and poultry-based products is consumed. 28 sity of meat products is produced and demanded by Meat is a perishable product unless some methods to 29 consumers. Poultry meat consumption has increased restrict bacterial proliferation are applied. Together 30 worldwide together with the increase in production. with drying and salting, fermentation is one of the 31 Brazil and Mexico are among the major Latin oldest forms of food preservation technology in the 32 33 34 35 Table 37.1. Meat and meat products consumption (kg/person/year) in Latin 36 America (2002Ð2005). 37 38 Processed Meat 39 Country Products Beef Pork Poultry 40 Argentina 8 67 5.7 23 41 42 Brazil 5.7 38 11 38 43 Chile 12 25.3 19.3 31.2 44 Uruguay 78 8.9 45 Mexico 4.4 15.4 17.7 26.6 46 47 Colombia 14.8 2.4 16 48 Costa Rica 6 16.4 3 21 49 Peru 5.5 5.2 23.9 50 51 Ecuador 14.9 16.4 52 Venezuela 4.6 17 4.3 31 53S Bolivia 19 8 16.24 54N Paraguay 37.6 28 17 40454 37 387-392 r1.qxd 7/21/07 12:40 PM Page 389

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world. For centuries, drying and salting have been poultry meat is strongly linked to culture and tradi- 1 used to keep meats available for consumption. tion, especially in rural households and village com- 2 Processed meat refers to those products that have munities, making a significant contribution to the 3 been subjected to a technological process such as diet of millions of individuals. Although wide local 4 heat treatment or decrease of water activity. Some variations between types of products and, indeed, 5 examples are cooked meat products such as hams, names were found, the differences usually amounted 6 pastrami, roast beef, corned beef, mortadella, wiener to little more than changes in the relative amounts of 7 sausages, turkey breast, and turkey sausages. the filling ingredients or in the type of pastry used. In 8 Mexico, Costa Rica, Brazil, Chile, and Argentina are addition, the bulk of such products is marketed on a 9 among the major processed meat producers. The national or at least a supraregional scale, although a 10 microbiological status of these products is highly relatively large number of small manufacturers dis- 11 dependent on raw materials and hygiene of handling tributing on a local basis or even from a single outlet 12 procedures. Cooked meat and poultry products are do exist. Raw comminuted meat products are those 13 generally either prepackaged in a permeable over- intended for sale in their natural raw state, such as 14 wrapping film or cut from a bulk block and may fresh sausages and burger products. These are made 15 become contaminated during production or process- from chopped (minced, ground) pork, beef, and 16 ing in homes or food establishments. Even when poultry meat and fat and usually include meat from 17 processing has been adequate and the product has various parts of the animal, including trimmings. In 18 been correctly handled after cooking, the most rele- specific products, offal is also used. A major type of 19 vant microorganisms of public health significance comminuted raw meat product is the burger-type 20 traditionally implicated are Staphylococcus aureus, product made from beef but also from poultry meats. 21 Listeria monocytogenes, Salmonellae, and shiga These, often circular in shape, may also contain filler 22 toxin-producing Escherichia coli. They are likely to and spices. Fresh pork sausages as well as blood 23 be present as a result of postprocessing contamina- sausages, which are very popular in Central and 24 tion due principally to poor cooling procedures and South American countries, are often implicated in 25 mishandling practices. Staphylococcal food poison- foodborne diseases. Even when it has been a long 26 ing is generally associated with highly manipulated tradition in these countries, the existence of street 27 foods, the etiological agent being S. aureus and its food sell-points constitutes a major health risk 28 related enterotoxins (Balaban and Rasooly 2000). because hygienic conditions for food preparation 29 Even when statistics for foodborne diseases due to cannot always be assured (Carrascal et al. 2002). The 30 contaminated meat products are scarcely available in raw comminuted meat products are among the foods 31 Latin American countries, contamination of proces- more commonly contaminated, and the strains iso- 32 sed meat and poultry products by L. monocytogenes lated from them have been frequently found to carry 33 has resulted in outbreaks of listeriosis. This pathogen virulence factors. Enterotoxin-producing E. coli, 34 can survive on processing equipment such as meat Yersinia enterocolitica, and Salmonellae associated 35 slicers as well as under different technological and with raw beef, pork, and poultry meat, respectively, 36 storage conditions (Lin et al. 2006). On the other are causative agents of food poisoning. Basic 37 hand, it is well known that cattle are a major reservoir hygienic precautions should be included during 38 of enterotoxigenic E. coli, an organism associated slaughtering, dressing, and conditioning of meat. On 39 with food illness that is often due to consumption of the other hand, comminuted meats are used as fill- 40 undercooked meat (Hussein and Bolliger 2005). ings in traditional Central and South American sta- 41 Although ground beef products are among the more ples such as tacos and enchiladas, generally made 42 commonly contaminated foods, the heat treatment with beef, pork, and poultry meat and served on corn 43 usually applied in processed meat products would tortillas or tamales, in which meat mixed with corn 44 decrease the prevalence of this meatborne pathogen. meal is wrapped in corn and banana leaves and 45 Other major types of spoilage of these products empanadas, a tasty meat filled pastry. Because these 46 results from the growth of yeast and, more com- meat fillings are cooked as a stew containing onions, 47 monly, molds on the surface of the product, which potatoes, and seasonings, the most important organ- 48 may be exacerbated by environmental conditions ism compromising public health that may be present 49 (type of packaging, temperature changes). is S. aureus, although Salmonella and coliforms (as 50 fecal contamination markers) could also be encoun- 51 tered (Carrascal et al. 2002). This is likely to occur 52 COMMINUTED MEAT PRODUCTS principally as a result of poor cooling procedures S53 In Latin America, the high consumption of food and contamination by handling. It is estimated that N54 specialties containing comminuted beef, pork, and 25% of the population carry S. aureus on their skin 40454 37 387-392 r1.qxd 7/21/07 12:40 PM Page 390

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1 and hands and, consequently, the organism may be LAB, in particular lactobacilli and gram-positive, 2 transferred to the cooked product. Due to the heat coagulase-negative cocci (Staphylococcus and Kocu- 3 stability of Staphylococcal enterotoxins, once formed ria), are the two major groups involved in the fer- 4 in the food they cannot be eliminated. Furthermore, mentation of dry-cured sausages; these bacterial 5 if the storage temperature exceeds 8°C, S. aureus groups account for texture, color, and sensorial 6 may then multiply to significant numbers. Slow changes. The ability of LAB to lower the pH and 7 cooling of these products also constitutes a serious produce bacteriocins prevents the growth of patho- 8 risk for heat-resistant spores of the genera Clostrid- genic and spoilage microorganisms, improving 9 ium and Bacillus, it being particularly favorable for hygienic safety. LAB also contribute to color and 10 Cl. perfringens. texture development, mainly through their acidifica- 11 tion capacity. On the other hand, Staphylococcus and 12 Kocuria reduce nitrate to nitrite, leading to the for- FERMENTED AND MEAT PRODUCTS 13 mation of the typical cured red color. By means of the 14 Fermentation is a relatively efficient, low-energy application of molecular techniques (RAPD analysis, 15 preservation process, which increases shelf life and DGGE and 16SrDNA sequencing), the identification 16 decreases the need for refrigeration or other forms and intraspecific differentiation of strains responsible 17 of food preservation technology. It is, therefore, a for artisanal Argentinean sausage fermentation was 18 highly appropriate technique for use in developing carried out (Fontana et al. 2005). Lactobacillus plan- 19 countries and remote areas where access to sophisti- tarum and Lactobacillus sakei were found to be the 20 cated equipment is limited. Fermented foods should dominant LAB during fermentation, and Staphylo- 21 be recognized as part of each country’s heritage and coccus saprophyticus represented the dominant 22 culture and efforts should be made to preserve the species among the Staphylococcus strains (Fontana 23 production methods. The most common types of et al. 2005). During sausage fermentation, a rapid 24 fermented meat products are salamis and salami- increase in LAB numbers within the first 5 days of 25 type sausages. These products are microbiologically ripening was observed, and these organisms were the 26 extremely stable by virtue of their low pH (usually dominant population at 14 days. As a result of acid 27 5.0) and water activity levels (0.7–0.8). Dry-cured production, total coliforms decreased after 5 days 28 sausages have traditionally been produced by fer- whereas yeast and molds were detected throughout 29 mentation combined with drying without heat the fermentation period. 30 treatment. Bacterial food-poisoning hazards are 31 minimal with dry sausages because the manufactur- DRIED,CURED, AND SALTED MEAT PRODUCTS 32 ing processes used usually inhibit pathogen growth. 33 Fermentation and drying have been reported to The preservation of foods in a sound and safe condi- 34 reduce the number of E. coli O157:H7 and L. mono- tion has long been an ongoing challenge for humans, 35 cytogenes mainly through lactic acid and bacteriocin drying and salting being ancient methods of keeping 36 production by lactic acid bacteria (LAB) naturally meat available for consumption when refrigeration 37 present in the meat batter or added as starter cultures was not yet available. It is well known that salted 38 (Lahti et al. 2001). Although fermented dry sausages beef was the very first “industry” in southern South 39 are produced in different Latin American countries, America, especially in Argentina and Uruguay, from 40 their manufacture has a long history in Argentina, colonial times to the advent of refrigeration around 41 mainly due to Italian and Spanish traditions as well the end of the 19th century. Beef from criollo cattle 42 as to the well-known quality of meat. Fermented dry naturally raised was salted and shipped to Europe as 43 sausages (salame) are produced in different regions tasajo. The major South American meat producers 44 of Argentina using local artisanal techniques for have also traditionally been important manufacturers 45 their preparation, which includes beef and/or pork of a wide variety of cured and salted meat products, 46 meat, pork fat, salt, and different spices. The meat, such as dry-cured ham and bondiola made from pork 47 fat and other ingredients are mixed together and hindlegs and neck muscle, respectively. In these 48 stuffed in pieces that can have different sizes accord- products, the increased concentration of salt and the 49 ing to the type of sausage to be produced in each progressive reduction in water activity are the most 50 region. In the northern region of Argentina, a tradi- important factors contributing to shelf stability. 51 tional fermented sausage is produced without micro- Even when molds can grow and develop on the outer 52 bial starters from fresh pork meat and lard that are surface of these meat products, they can be consid- 53S mixed with sugar, NaCl, nitrate/nitrite, and spices. ered as safe regarding public health concerns. Due to 54N 40454 37 387-392 r1.qxd 7/21/07 12:40 PM Page 391

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the higher cost of these cured and salted products individuals able to apply the basic microbiological 1 when compared with other processed ones, they are and technological principles to the production of tra- 2 not massively consumed. ditional foods. The implementation of the HACCP 3 Another dried meat product is jerky or charqui, system and Good Manufacturing Practices (GMPs) 4 which traditionally was made by exposure to sun, during food preparation is an essential complement 5 wind, and smoke as a way to preserve and extend the to improve hygienic quality and minimize safety 6 shelf life of meat. Jerky refers to dried meat strips risks of traditional meat products. 7 (beef, pork, lamb, venison, poultry) or ground and 8 formed meat. In South America, jerky is also made 9 from llama, alpaca, and guanaco, these being the REFERENCES 10 major protein source for many Amerindians groups N Balaban, A Rasooly. 2000. Staphylococcal enterotox- 11 in the Andes and in southern South America ins. Int J Food Microbiol 61:1–10. 12 (Franklin 1982). The fresh meat is cut into small A Carrascal, G Arrieta, S Máttar. 2002. Estudio prelim- 13 pieces, salted, pressed for several days, and dried, inar de la calidad microbiológica de los alimentos en 14 with the moisture allowed to drain freely from the la Costa Atlántica Colombiana. Inf Quinc Epidemiol 15 product. Because most of the water is removed, jerky Nac 7:161–176. 16 is shelf-stable and can be stored for months without C Fontana, P Cocconcelli, G Vignolo. 2005. Monitor- 17 refrigeration. The standards for jerky require a water ing the bacterial population dynamics during fermen- 18 content no higher than 45% and at least 15% salt in tation of artisanal Argentinean sausages. Int J Food 19 the lean. However, illnesses due to Salmonella and Microbiol 103:131–142. 20 E. coli O157:H7 from homemade jerky raise ques- C Fontana, G Vignolo, P Cocconcelli. 2005. PCR- 21 tions about the safety of traditional drying methods. DGGE analysis for the identiﬁcation of microbial 22 The USDA recommends meat to be heated at populations from Argentinean dry fermented 23 71–74°C before the dehydrating process in order to sausages. J Microbiol Meth 63:254–263. 24 destroy pathogenic organisms. After drying, bacteria W Franklin. 1982. Biology, ecology, and relationship to 25 become much more heat-resistant and likely to sur- man of the South American camelids. In: M Mares, 26 vive and, if pathogenic, they can cause foodborne H Genoways, eds. Mammalian Biology in South 27 illness. Comparison between heated and unheated America. University of Pittsburgh, pp. 457–489. 28 meat samples inoculated with E. coli O157:H7 J Harrison, M Harrison, B Rose. 1998. Survival of 29 before further processing into jerky strips and dehy- E. coli O157:H7 in ground beef jerky assessed on 30 dration showed a higher destruction rate of bacteria two plating media. J Food Prot 61:11–13. 31 when meat was precooked to 71°C prior to drying H Hussein, L Bolliger. 2005. Prevalence of Shiga toxin- 32 (Harrison et al. 1998; Kauer et al. 1998). Although producing Escherichia coli in beef cattle. J Food Prot 33 jerky has traditionally been a product eaten by low- 68:2224–2241. 34 income people, recently it has become fashionable to J Kauer, D Ledward, R Park, R Robson. 1998. Factors 35 use as snacks and ready-to-eat foods. affecting the heat resistance of Escherichia coli 36 O157:H7. Lett Appl Microbiol 26:325–330. 37 38 CONCLUSIONS E Lahti, T Johansson, T Honkanen-Buzalski, P Hill, E Nurmi. 2001. Survival and detection of Escheri- 39 With many of the traditional products in Africa, Asia, chia coli O157:H7 and Listeria monocytogenes 40 and Latin America, knowledge of the processes during the manufacture of dry sausage. Food Micro- 41 involved is poor and the production conditions vary biol 18, 21:75–85. 42 enormously from region to region, thus giving rise to C Lin, K Takeuchi, L Zhang, C Dohm, J Meyer, P Hall, 43 numerous variations of the basic product. The main M Doyle. 2006. Cross-contamination between pro- 44 idea is not to standardize the process and thereby lose cessing equipment and deli meats by Listeria mono- 45 this huge diversity, but to harness the tremendous cytogenes. J Food Prot 69:71–79. 46 potential of these methods have to increase not only I Romieu, M Hernández-Ávila, J Rivera, M Ruel, 47 the quantity but also the quality of food available to S Parra. 1997. Dietary studies in countries experienc- 48 the world’s population. Developing countries need ing a health transition: Mexico and Central America. 49 to build their resources of trained, knowledgeable Am J Clin Nut 65(Suppl):1159S–1165S. 50 51 52 S53 N54 40454 38 393-406 r2.qxd 7/21/07 1:00 PM Page 393

1 2 3 38 4 5 6 Mediterranean Products 7 8 Mario Estévez, David Morcuende, Jesús Ventanas, and Sonia Ventanas 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION The latter show, however, higher levels of fat than 22 industrial genotype pigs raised for other purposes 23 The necessity for meat preservation in the Mediter- (i.e., meat for fresh consumption) and are slaugh- 24 ranean countries has been fulfilled through the pro- tered at higher weights, because pigs reared for the 25 duction of ripened meats since ancient times. The production of Parma hams are slaughtered at 26 traditional ripening process includes the addition of 130–160 kg. The characteristics of the raw material 27 curing salt and the subsequent drying process aimed largely determine the procedure for the ham process- 28 at reducing the water activity of the product, which ing and the sensory features of the products. For 29 results in a shelf-stable muscle food. Among the instance, the former require a weak salting stage and 30 large variety of dry-cured meats produced in the a long ripening process (over 20 months), whereas 31 Mediterranean area, the dry-cured ham is the most the latter are moderately salted and ripened during a 32 outstanding example of a traditional ripened prod- shorter period of time (around 7–14 months). In gen- 33 uct, highly appreciated by European consumers. The eral, hams produced from free-range–reared rustic 34 manufacture of dry-cured meats using raw hams pigs are considerably more appreciated and reach 35 from autochthonous pigs has been done in these higher prices than those from intensively reared 36 countries throughout centuries. However, the intro- industrial genotypes. The great acceptability of these 37 duction of improved pig breeds during the 20th cen- products by consumers allows both ham versions to 38 tury led to a significant decrease of interest in these coexist in each country, keeping their peculiar char- 39 traditional products. Today, the production of dry- acteristics. For cultural and geographical reasons, 40 cured hams is mainly carried out in Spain, France, consumers prefer the appearance, taste, and flavor of 41 and Italy, in which two different types of dry-cured the dry-cured hams produced in their own countries 42 hams can be found, depending on the origin of raw according to the consumption data and the prefer- 43 material: ence and choice studies carried out. In the present 44 • Dry-cured hams from free-range–reared pigs chapter, the most relevant aspects regarding the pro- 45 derived from rustic genotypes, slaughtered at duction of dry-cured hams will be presented. After 46 high weights, and producing fatty carcasses that, the biological and technological factors deter- 47 (i.e., Iberian hams) mining the particular features of dry-cured hams 48 • Those hams from intensively reared pigs derived produced in the Mediterranean area will be described, 49 from industrial genotypes, with leaner carcasses, with special attention to those protected by the Euro- 50 and slaughtered at lower weights (Parma or pean policies 2081/92 and 2082/92 (P.D.O., P.G.I., 51 Serrano hams). T.S.G.). 52 53 S54 N55 393 40454 38 393-406 r2.qxd 7/21/07 1:00 PM Page 394

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1 PRODUCTION OF because these two factors have been highlighted as 2 DRY-CURED HAMS the more influential on the sensory quality of the 3 final product (Ventanas et al. 2005). Particularly in 4 Although the times and procedures for the manufac- hams from rustic breeds reared in traditional sys- 5 ture of dry-cured hams are highly variable depend- tems and protected by EU legislation, the origin of 6 ing on costumes and the type of product, there are the raw material is specially taken into considera- 7 common stages for all of them. In general, two dif- tion, because only certain pig breeds and finishing 8 ferent phases are clearly discerned: feeds are accepted in order to fit established quality 9 • The cold phase, which comprises the selection of policies. Among the most relevant quality traits, the 10 the raw material and the salting and postsalting fat content and fatty acid composition, myoglobin, 11 stages, aimed to reduce the water activity of the and antioxidants content decisively influence the 12 green ham down to 0.95 through the addition of appearance, flavor, and taste of the hams affect- 13 marine salt and the loss of water ing the consumer’s preference and acceptability 14 • The hot phase, which includes the drying and (Ventanas et al. 2005). From a technological point of 15 cellar stages, intended to smooth the progress of view, some other parameters such as the muscle pH, 16 the dehydration process through the increase of the temperature, and the weight of the green ham are 17 the temperature under certain relative humidity also considered. The preparation of the green hams 18 conditions and the development of the peculiar prior to the technological process includes the 19 sensory features of a ripened ham, caused by the removal of remaining blood from the core of the ham 20 onset of chemical and biochemical reactions and the adjustment of the shape and appearance of 21 the ham according to the quality policy of each type 22 Whereas the traditional procedures followed for the of product. 23 production of ripened hams in Mediterranean countries were perfectly adapted to the natural environ- 24 SALTING/CURING STAGE 25 mental conditions during the seasons, today, in some 26 cases, the development of artificial salting, drying, The raw ham is treated with marine salt leading to 27 and cellar rooms with controlled environmental con- the penetration of the sodium chloride into the core 28 ditions of temperature and relative humidity allow of the ham. Depending on the type of product, the 29 the development of standardized ripening processes. marine salt can be used together with potassium 30 Figure 38.1 summarizes the length of each phase for nitrate, sodium nitrite, and reducing sugars. For this 31 the production of the most representative dry-cured purpose, different techniques can be used: (1) direct 32 hams in the Mediterranean countries. salting in piles with alternative layers of hams and 33 salting mixture (i.e., Serrano and Iberian ham), and (2) mechanical or manual massage of the hams 34 SELECTION OF THE RAW MATERIAL 35 with the salting mixture at fixed periods of time 36 Green hams are selected principally by considering (i.e., Parma hams). The length of this stage gener- 37 the genetic and feeding backgrounds of the pigs ally depends on the weight of the ham, ranging from 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53S 54N Figure 38.1. Length of the main phases of the production process of the most representative European ripened hams. 40454 38 393-406 r2.qxd 7/21/07 1:00 PM Page 395

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17 hours to 48 hours per kg of weight. The salting ripening of dry-cured hams is dependent on the bal- 1 chamber is kept at refrigeration temperatures (below ance between anti- and prooxidant factors, mainly 2 5°C) in order to avoid microbial spoilage and at high the fatty acid composition and antioxidant status of 3 relative humidity (RH) (around 85–90%) to facili- the muscles (Cava et al. 2000), whereas the lipolytic 4 tate the penetration of the salt and to avoid an exces- changes are caused by endogenous (muscle) and 5 sive dehydration of the muscles’ surface. exogenous (microbial) enzymes (Toldrá 1998). 6 7 8 POSTSALTING STAGE CELLAR STAGE 9 The salt is homogeneously distributed throughout After the drying stage, the dry-cured hams are kept 10 the lean of the ham muscles according to osmosis in cellars under lower temperatures (12–20°C) dur- 11 and diffusion mechanisms. Consistent with the pre- ing a highly variable period of time, depending on 12 vious stage, the conditions of the environment are the type of product. The length of this stage is gener- 13 still controlled in terms of temperature (3–5°C) and ally related to the sensory quality of the final product 14 RH (85–95%). At the end of this stage (30–90 days), because the development of greatly appreciated sen- 15 the water activity of the lean tissues decreases sory features (bouquet) in dry-cured hams occurs 16 (below 0.96) as a result of the addition of salt and the during this last stage. The length of this stage is par- 17 dehydration of the hams, with weight shrinkages up ticularly remarkable for dry-cured hams derived 18 to 17–18%. The low water activity reached at the end from rustic breeds with high fat content, such as the 19 of this stage allows the increase of the temperature Iberian ham (up to 24 months). 20 and continues the gradual dehydration of the hams. 21 22 SPANISH DRY-CURED HAMS 23 DRYING STAGE The production of dry-cured meat products in Spain 24 The environmental conditions in the Mediterranean has been done since ancient times. Currently, Spain 25 area during late spring and the summer (relatively is the world’s largest producer of dry-cured ham, 26 high temperatures and decreasing relative humidity) with approximately 39 million cured hams per year. 27 allowed the dehydration of the hams following tradi- In the Iberian Peninsula, especially in mountainous 28 tional procedures in drying rooms in which the tem- regions, with climates that are mild and dry in the 29 perature and the RH were controlled by opening or summer and cold in the winter, the environmental 30 closing the windows. Nowadays, the drying process conditions throughout the year allowed the produc- 31 is carried out in artificial drying chambers, making tion of such shelf-stable meat products as sausages, 32 the production of dry-cured hams season-indepen- shoulders, loins, and especially dry-cured hams. 33 dent and a more homogeneous practice. The length Among dry-cured hams, the most representative 34 of the process, the temperature and relative humidity products are those coming from intensively reared 35 program, and the number of drying cycles are pig breeds such as the Serrano ham, and those 36 designed depending on the type of dry-cured ham. obtained from extensively reared rustic pigs, such as 37 Longer drying periods are commonly used for high- the Iberian pigs (Iberian dry-cured ham) and the 38 quality dry-cured hams from free-range–reared rus- Alentejano pigs (Barrancos ham in Portugal). 39 tic pigs (i.e., Iberian hams) following traditional 40 procedures, whereas a short drying process at higher 41 SERRANO HAM temperatures is usually chosen for hams from indus- 42 trial genotype pigs (i.e., Serrano hams). At the present time, Serrano ham is protected by the 43 The degradation of lipids and proteins mainly European Community as a Traditional Speciality 44 occur during this stage, with those having a decisive Guaranteed, and the production is carried out in mod- 45 effect on the sensory properties of the final product. ern installations called secaderos equipped with 46 The lipolytic degradation of muscle lipids leads to modern technology, keeping a constant high-quality 47 the generation of free fatty acids, which are sub- level. Serrano hams are produced using raw material 48 sequently degraded through oxidative reactions from intensively reared industrial genotype pigs 49 (Gandemer 1998; Toldrá 1998). Muscle proteins are (Landrance, Large White, and other commercial 50 denatured and degraded by proteases leading to the crossbred pigs including Duroc). Serrano ham is 51 generation of low-weight nitrogen containing mole- produced throughout the whole country and, there- 52 cules (amino acids and peptides) and causing loss of fore, certain differences or nuances regarding produc- S53 protein functionality and solubility (Toldrá 1998). tion procedures might exist depending on the zone N54 The intensity of the oxidative processes during the and according to the producers. The fundamental 40454 38 393-406 r2.qxd 7/21/07 1:00 PM Page 396

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1 stages in the Serrano ham manufacture are the protects the exceptional environmental system, 2 following: Dehesa, in which Iberian pigs have been tradition- 3 The ham is first salted for a period of between 0.8 ally free-range–reared. As a consequence of their 4 and 2 days per kg in order to avoid microbial exceptional characteristics, the European Union pro- 5 spoilage. It is then left to stand for at least 40 days to tects these traditional products with five Protected 6 allow the salt to diffuse through the ham. This is fol- Designations of Origin, one in Portugal—the Pre- 7 lowed by a period of maturing, at least 110 days, and, sunto de Barrancos in the Alentejo region—and four 8 finally, by a period of aging during the time neces- in Spain—Jamón de Guijuelo in Castilla y León 9 sary to complete a minimum of 7 months. During region, Dehesa de Extremadura in the homonymous 10 the last stage, biochemical processes caused by region, and Jamón de Huelva and Jamón Valle de los 11 oxidative reactions and enzymes from microorgan- Pedroches in the Andalucia region. The Iberian pig 12 isms and from porcine tissues take place leading to has been a constant inspiration source for researchers 13 the traditional quality and its peculiar taste and who have profusely studied, during more than 15 14 aroma. A Serrano ham slice has a characteristic pink years, different aspects related to their genetic traits, 15 to purple color, and the fat has a shiny appearance, their traditional rearing system, and the muscle 16 with an aromatic and pleasant taste. foods produced with their tissues, with special atten- 17 Serrano ham can be bought in three different tion to the Iberian dry-cured ham (Ventanas et al. 18 forms: (1) with bone, the way that it is usually eaten 2005). 19 in Spain, which requires a good knife in order to 20 obtain very thin, almost transparent, slices; (2) bone- Elaboration of the Iberian Dry-cured Ham 21 less hams, processed the same way as the Serrano The final product quality depends on the quality of 22 ham with a bone, although at the end of the maturing the raw matter, clearly influenced by the genotype 23 process and before packaging, the bone is removed; and the feeding background, which also affects the 24 and (3) sliced and vacuum packaged. technological process. 25 Particular Serrano-type Hams 26 Raw Matter 27 Protected Designation of Origin (PDO) Teruel ham: The Iberian pig is a rustic and highly lipogenic 28 The entire province of Teruel (northeast Spain) is breed. The lipogenic ability of Iberian pigs together 29 included in the production of Teruel ham, the first with the feeding strategy used during its fattening 30 Spanish P.O.D. for meat products. Teruel ham is period lead to green hams with a thick subcutaneous 31 obtained from 50% Duroc x (Landrace/Large White) fat layer (above 5 cm) and high intramuscular fat 32 pigs slaughtered with a live-weight of 120 to 130 kg. (8–10% in biceps femoris) content (Ruiz-Carrascal 33 Fresh ham with hoof and weight no less than 11.5 kg et al. 2000; Ventanas 2001; Carrapiso and García 34 are cured during 12 to 14 months in the highlands of 2005), which is appropriate from a technological 35 Teruel, more than 800 miles above sea level. point of view. In addition, the feeding background 36 Protected Geographical Indication Trevélez ham: influences the quality of the final product as it affects 37 Trevélez ham is elaborated in the rocky area of Alpu- the fatty acid composition and antioxidant level of 38 jarras (Granada, Andalucia region), in factories that the porcine tissues leading to Iberian dry-cured 39 are higher than 1200 meters above sea level. Cured hams with different nutritional and sensory charac- 40 hams from Trevélez have a rounded shape, keep their teristics. With the purpose of improving reproduc- 41 rind and hoof, and have a final weight of approxi- tive and productive parameters, the Iberian pig has 42 mately 8 kg. been commonly crossed with other breeds. Cur- 43 rently, only Iberian x Duroc (up to 50%) crosses are 44 IBERIAN HAM allowed for the production of Iberian dry-cured 45 hams, and in such case, only the pure Iberian breed 46 The Iberian dry-cured ham is the most representative is allowed as a maternal line (BOE 2001). The pro- 47 traditional Spanish meat product, and it represents ductive cycle of Iberian pigs involves an extensive 48 an outstanding example of a high-quality meat prod- system of breeding and comprises three stages: 49 uct, comparable to the most exquisite food products 50 in the world. Iberian ham production, which repre- • Weaning, for around 50 days 51 sents only about 8% of the total hams produced in • Growing, from postweaning until 90–100 kg 52 Spain, has largely contributed to the development • Fattening, the last stage, which has a great 53S of the regional meat industry in the southwest of importance regarding the classification of the 54N the Iberian Peninsula. Furthermore, it supports and animal and their products 40454 38 393-406 r2.qxd 7/21/07 1:00 PM Page 397

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There are three models of fattening allowed (López- southwestern Spain, with cold winters and warm and 1 Bote 1998): Montanera, Recebo, and Cebo. With dry summers. Although the basic steps followed for 2 Montanera, pigs are fed during the final fattening dry-cured ham production are similar in all types of 3 period ad libitum with natural resources (grass and ham, temperatures reached during ripening of Iber- 4 acorns) in the Dehesa, a sylvopastoral system with a ian hams are higher, and the length of the process is 5 variety of Quercus species and grass. The length of much longer than those used for the production of 6 this period is variable, but it is generally carried out other Mediterranean dry-cured hams. The following 7 from October to February, when maturation of are the fundamental stages in the Iberian ham pro- 8 acorns takes place. Feeding pigs on acorns and pas- duction (Ventanas 2001): 9 ture has two main consequences on the composi- 10 Green hams preparation: This step includes the tional traits of meat from Iberian pigs: high oleic 11 slaughtering operations, pig quartering, and V-shape acid and antioxidant (tocopherol) content (Cava 12 cutting. The hoofs are kept for the manufacture of et al. 2000; Daza et al. 2005). The high amount of 13 Iberian hams (see Figure 38.2). oleic acid in both subcutaneous and intramuscular 14 fat (IMF) leads to a very bright slice surface and Salting: This step is carried out by rubbing the legs 15 very soft fat. Moreover, together with the high oleic with salt containing nitrites and nitrates. Then the 16 acid content, the high amount of natural antioxidant pieces are piled up in salting chambers or in contain- 17 make muscles from Iberian pigs reared in the ers. The chambers must be at 0–4°C and 90–95% 18 Montanera system highly stable against lipid oxida- relative humidity. The pieces are buried in salt from 19 tion. When the fattening period finishes, the animals 0.70 to 2 days per kg (see Figure 38.3). The trend is 20 are slaughtered at around 160 kg live weight and are to employ shorter times than 1 day per kg. 21 14–18 months old. 22 Postsalting: After salting, hams are gently brushed in The Recebo fattening model is employed when 23 order to remove remaining salt from their surfaces the resources of the Dehesa are insufficient for the 24 and are transferred to the postsalting chamber. The pigs to reach the optimum slaughtering weight, so it 25 initial temperature is 5°C, and it is slowly increased is necessary to add a complementary diet based on 26 to 13°C; the relative humidity decreases from 90% commercial feeding. With the Pienso model, the pigs 27 to 80%. This step lasts around 80–100 days, longer are mainly fed commercial feed. 28 than in the processing of other hams. The high intra- 29 muscular fat content in Iberian hams hinders the 30 Processing water and salt diffusion through the whole piece, 31 which explains the length of the postsalting stage. The traditional manufacture process of Iberian hams 32 is perfectly adapted to the natural environmental Drying: In this step, the hams are exposed to higher 33 conditions of the mountainous areas located in temperatures and lower relative humidity, in order to 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 S53 Figure 38.2. Green Iberian hams. N54 40454 38 393-406 r2.qxd 7/21/07 1:00 PM Page 398

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Figure 38.3. Salting stage: piles of alternative layers 27 of hams and marine salt. Figure 38.4. Drying stage: dry-cured hams hanging 28 in the drying chambers. 29 30 31 accelerate the drying process. This phase is partially hams (see Figure 38.5). This feature, together with 32 carried out in summertime, and, thus, length and the high levels of oleic acid, supports the formation 33 temperature varies with the geographical location, of pleasant flavor compounds, affects in a positive 34 reaching almost 30°C in some areas (see Figure way the sensorial perception (e.g., juiciness), and 35 38.4). During this stage, the fat melts and it impreg- provides a healthy nutritional quality (Ventanas 36 nates the muscular fibers, acting as a real solvent of 2001). Iberian dry-cured ham shows a more intense 37 volatile compounds. color than other hams due to the high myoglobin concentration of the muscles of these pigs, as a con- 38 Cellar: Finally, hams are kept in a cellar for at least sequence of their age at slaughter and the exercise 39 12–24 additional months at a temperature of 10 to the pigs should accomplish to gain the food, espe- 40 20°C and relative humidity from 65% to 82%. The cially in free-range systems. 41 length of this step is critical for quality attributes to An intense and pleasant flavor perception has been 42 fully develop, especially the generation of volatile highlighted as one of the most peculiar features 43 compounds (Ruiz et al. 2002). 44 of Iberian dry-cured hams. The characterisation of certain odor-active compounds (Carrapiso et al. 45 Sensory Features of Iberian Dry-cured Hams 46 2002) among the total volatile components isolated 47 Sensory characteristics of Iberian dry-cured hams from the headspace (HS) of Iberian dry-cured hams 48 are greatly appreciated by consumers, explaining the (Andrés et al. 2002) has been one of the most out- 49 considerably high prices reached in the market. standing approaches to understanding the mechanisms 50 Among these features, marbling, color, and flavor of generation of volatile compounds in dry-cured 51 seem to be largely influential for Iberian ham accept- hams and the role of particular volatile compounds 52 ability (Ruiz-Carrascal et al. 2000). The marbling of on the aroma perception of such dry-cured products. 53S Iberian dry-cured ham is typically abundant and evi- The main routes involved in the formation of rele- 54N dent, much more intense than in other dry-cured vant volatile compounds include proteolysis, lipid 40454 38 393-406 r2.qxd 7/21/07 1:00 PM Page 399

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Figure 38.5. Surface of an Iberian dry-cured ham. 28 29 30 oxidation, and amino acid degradation through 900 m. Pigs are intensively reared and slaughtered in 31 Maillard reactions and Strecker degradation the Emilia-Romagna, Lombardy, Piedmont, Veneto, 32 (Ventanas et al. 1992; Ruiz et al. 2002). Table 38.1 Tuscany, Umbría, Marche, Abruzzi, Lazzio and 33 summarizes the major volatile compounds isolated Molise areas, under regulated conditions and ﬁxed 34 from the HS of dry-cured hams produced in the feeding arrangements. 35 Mediterranean area. For the manufacture of Parma hams, the green 36 hams are ﬁrst placed in special cold storage rooms 37 for 24 hours in order to reduce the leg temperature 38 ITALIAN DRY-CURED HAMS from 40°C to 0°C and harden the meat, thereby facil- 39 itating the trimming. During this phase, the weight 40 PARMA HAM losses are around 1%. The trimming of the green 41 The Parma hams are the most representative Italian hams involves removing external fat and rind from the 42 dry-cured hams because of their mass production leg. The green hams acquire their peculiar “chicken 43 and rate consumption. The Parma ham owns a Pro- drumstick” shape, with those showing the slightest 44 tected Designation of Origin (PDO) since 1970. imperfection being discarded. In addition to the aes- 45 These hams are produced using raw material from thetic purpose of the trimming phase, it is also aimed 46 Landrace and Large White pigs, although Duroc to facilitate the salting step. After trimming, hams lose 47 genotypes are accepted for crossing purposes. Ani- up to 24% of their weight in fat and muscle. 48 mals are usually slaughtered at 160 kg, and are at Parma hams are salted using wet and dry salt: wet 49 least 9 months of age. All Parma ham producers salt for treating the skin, and sprinkled dry salt for 50 must be located within the geographical boundaries the lean parts. Using any other type of additive such 51 of the Parma production area, 5 km south of the via as nitrates and nitrites is not allowed. Hams are 52 Emilia, limited to the east by the river Enza and on stored in cold storage rooms at a temperature rang- S53 the west by the river Stirone, and up to an altitude of ing between 1°C and 4°C at about 80% relative N54 40454 38 393-406 r2.qxd 7/21/07 1:00 PM Page 400

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1 Table 38.1. Major volatile components of different types of dry-cured hams. 2 3 Iberian Serrano Parma Bayonne 4 Aldehydes 5 2-Methylpropanal *** *** ** ** 6 3-Methylbutanal **** **** *** *** 7 2-Methylbutanal **** *** *** *** 8 Pentanal **** *** *** *** 9 Hexanal **** **** **** **** 10 Heptanal *** *** *** *** 11 2,4-Nonadienal ** ** ** ** 12 Octanal *** ** *** ** 13 Phenylacetaldehyde *** ** ** ** 14 Nonanal *** *** *** *** 15 Decanal ** * * * 16 17 Alcohols 18 2-Methylpropanol ** ** ** ** 19 1-Penten-3-ol *** *** *** *** 20 2-Pentanol *** *** * * 21 3-Methylbutanol *** *** ** ** 22 1-Pentanol *** *** *** *** 23 1-Octen-3-ol ** ** ** ** 24 Ketones 25 Butanone *** *** *** *** 26 2-Pentanone *** *** *** *** 27 2-Hexanone *** ** * * 28 2-Heptanone *** *** * * 29 2-Octanone *** ** * * 30 n-Alkanes 31 Hexane *** ** ** ** 32 Octane *** *** ** *** 33 Sulphur Compounds 34 Dimethyl disulphide *** *** ** ** 35 Dimethyl trisulphide ** ** * * 36 37 Adapted from Sabio et al. (1998). 38 Amount of volatile compound in Area Units (AU): * (AUϽ105), ** (105ϽAUϽ106), *** (106ϽAUϽ107), 39 **** (107ϽAUϽ108). 40 41 42 43 humidity. After 6–7 days stored in these rooms, After removing the residual salt, hams are stored 44 called preliminary salting rooms, the hams are in the so-called resting rooms for 60–90 days at 45 cleaned of residual salt and sprinkled with a second 1–5°C and 75% relative humidity (this is known as 46 thin coating of salt. Hams are then placed into a new the resting stage). During this stage, hams are 47 cold stored room, known as the ﬁnal salting room, allowed to “breathe” and should not become too dry 48 where they remain for 15–18 days depending on or too wet. The air in the resting rooms is changed at 49 their weight. A highly trained maestro salatore frequent intervals. The absorbed salt penetrates 50 ensures that the hams absorb just enough salt to deeply, becoming evenly distributed in the muscular 51 guarantee the safety and technological stability, pre- mass. During this phase, the ham weight reduces 52 serving simultaneously the peculiar “sweetness” of around 8–10%. Then, hams are washed with warm 53S Parma hams. At the end of this period, weight losses water and afterward the rind is brushed to remove 54N are around 3.5–4%. any residual salt or impurity. Hams are set out to dry 40454 38 393-406 r2.qxd 7/21/07 1:00 PM Page 401

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in the air when the weather conditions are favorable rated fatty acids (11%) are within the expected range 1 (dry, windy, and sunny) or in special drying rooms. for the fatty acid compositions of other commercial 2 The precuring phase is carried out in large rooms Italian, French, or Spanish dry-cured hams (Bosi 3 with windows where hams are hung on the tradi- et al. 2000; Gandemer 2002). Compared to the fatty 4 tional scalere (ladders). Windows are opened, con- acid profiles of dry-cured hams produced from free- 5 sidering the internal/external humidity as well as range–reared pigs fed on acorns or chestnuts (i.e., 6 that of the product. After precuring, hams are beaten Iberian ham, Corsican ham) Parma hams generally 7 to give them their typical rounded shape. The hollow contain lower levels of MUFA and higher of SFA 8 surrounding the best end is sprinkled with pepper to and PUFA (Gandemer 2002) (Table 38.2). Several 9 keep the contact dry. Between 8–10% of the ham recent studies have reported the possibilities of 10 weight is lost during this stage. enhancing the fatty acid composition, oxidative sta- 11 During the “greasing stage,” the hollow surround- bility, and aromatic profile of Parma hams through 12 ing the best end, exposed muscular parts, and any dietary means (Bosi et al. 2000; Pastorelli et al. 13 cracks are covered with a layer of ground pork fat, 2003). 14 mixed with a pinch of slay and ground pepper and, if 15 necessary, rice flour. The greasing has the purpose of 16 SAN DANIELE HAM (PROSCIUTTO DI SAN DANIELE) softening the superficial muscular layers, preventing 17 them from drying up too quickly compared to the This ham has been produced in the northeastern 18 inner layers, as well as causing additional moisture region of Friuli Venezia-Giulia for centuries, although 19 loss. According to the Italian legislation, this grease it has owned a Protected Designation of Origin 20 mixture is not considered an ingredient. (PDO) since 1996. As with the Parma hams, San 21 After greasing and upon reaching the seventh Daniele hams are another clear example of Italian 22 month, hams are moved to the cellars, which are hams produced using raw material from industrial 23 colder and less ventilated than precuring rooms. genotype pigs (Landrace and Large White) and 24 Before being removed, hams are subjected to sam- crossbred pigs using Duroc genotypes. San Daniele 25 pling, in which a special needle, made of horse bone hams are manufactured in the municipality of San 26 and having the feature of being able to quickly Daniele del Friuli, in the province of Udine (Italy). 27 absorb the aroma and subsequently lose it again, is Pig rearing and slaughter take place in Friuli- 28 inserted into various points of the muscular mass Venezia Giulia, Veneto, Lombardy, Piedmont, Emilia 29 and then sniffed by experts. During the curing phase, Romagna, Umbria, Tuscany, Marche, Abruzzi, and 30 chemical and biochemical reactions take place, lead- Lazio. At slaughter, pigs must have average weights 31 ing to the development on the particular scent and of no less than 160 kg and be at least 9 months 32 taste of Parma hams. Hams lose about 5% of their of age. 33 weight during this phase. The whole production process lasts 12 months at 34 After a period of 10 months for hams with a final least, of which a minimum of 8 months is used for 35 weight of 7–9 kg and 12 months for hams weighing air curing. Pig thighs are selected and suspended in 36 more than 9 kg, the Ducal Crown fire brand is affixed well-ventilated or refrigerated rooms for 24 to 36 37 as a sign of a Parma ham elaborated following the hours. The fat and hide is then trimmed, and the salt 38 established policies. curing process begins. The salt is applied through 39 The Parma ham is a seasoned dry-cured ham with a manual massage, repeating the procedure once 40 a curved shape, without the distal part of the ham a week for a month. The hams are then washed, 41 (the hoof is removed) or any other external blem- brushed, and dried either in sunlight or indoors, 42 ishes likely to impair the product’s image. The hams where temperatures never exceed 15°C. Once the 43 weigh between 8 kg and 10 kg, but never less than hams are dried, they are inspected for flaws and are 44 7 kg. The ham has a mild, delicate flavor, is slightly coated with a mixture of flour, lard, water, and pep- 45 salty, and has a fragrant and distinctive aroma. per. After that, San Daniele hams must be aged for at 46 Among the variety of factors influencing the sensory least 12 months, but some are aged for up to 2 years. 47 and technological quality of Parma dry-cured hams, During this time, the hams lose up to 30% of their 48 the compositional characteristics of the fat from the original weight. Whereas the ingredients and condi- 49 carcass has been highlighted as the most relevant tions involved in the production of San Daniele 50 (Bosi et al. 2000). Although the fatty acid composi- hams are identical to other types of Italian dry-cured 51 tion of Parma hams is highly dependent on the feed- hams, the unique climatic conditions of the Friuli 52 ing background of the pigs, the levels of saturated region, with its higher altitudes and drier air, has S53 (38%), monounsaturated (51%), and polyunsatu- been considered as one of the key factors influencing N54 40454 38 393-406 r2.qxd 7/21/07 1:00 PM Page 402

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1 Table 38.2. Intramuscular fat content and fatty acid composition of m. Biceps femoris from different types 2 of hams. 3 Iberian Serrano Parma Nero Siciliano Bayonne 4 5 IMF (g/100 g) 11.3 4.78 3.57 7.07 3.53 6 Fatty Acids (%) 7 C12:0 0.07 0.07 – 0.08 – 8 C14:0 1.27 1.37 1.18 1.09 1.08 9 C16:0 22.92 24.48 21.65 22.55 22.91 10 C18:0 7.45 10.98 12.67 11.08 12.53 11 C20:0 0.22 – 0.14 0.14 – 12 13 Saturated 31.93 37 35.99 34.94 36.52 14 C16:1 3.39 3.41 3.05 2.94 3.32 15 C18:1 54.51 47.99 49.99 39.53 43.6 16 C20:1 – 0.97 0.86 0.8 0.57 17 Monounsaturated 57.9 52.37 54.04 43.29 47.49 18 C18:2 9.41 9.62 7.77 16.75 11.7 19 C18:3 0.65 0.53 0.21 0.93 0.5 20 C20:4 0.11 0.97 0.61 2.35 3.1 21 Polyunsaturated 10.17 11.01 8.59 20.03 15.3 22 23 Adapted from Timón et al. (2001), Lo Fiego et al. (2005), and unpublished data from the authors. 24 25 26 27 the unique ﬂavor and texture of these hams. It is a regions of Emilia-Romagna, Lombardy, Marche, 28 matured, dry-cured ham with a guitar-shaped exte- Umbria, Latium, and Tuscany. 29 rior and intact distal part (the hoof remains in the These are salted, naturally cured, uncooked hams 30 ham). The most peculiar external feature of San with a rounded shape and bowed at the top. The 31 Daniele hams is the presence of the bottom part of hams normally weight around 8 to 9 kg and have a 32 the leg bone, providing a very rustic-looking appear- light to bright red color when sliced, with little inter- 33 ance. It is appreciated for its pink meat, its creamy, and intramuscular fat. 34 smooth texture, and salty-sweet ﬂavor. Whole San Recently, Pugliese et al. (2005) have studied the 35 Daniele hams weight from 8 to 10 kg, but never less fatty acid composition and sensory characteristics 36 than 7.5 kg. of Toscano dry-cured hams from the Cinta Senese 37 breed pig compared to those from crossbreeding 38 Cinta Senese with a commercial breed pig (Large CINTA SENESE HAM 39 White). Fat of hams from Cinta Senese purebred 40 These hams are known as Toscano dry-cured ham pigs had lower SFA and PUFA but higher MUFA 41 according to the policy of the Protected Designa- proportions than those from crossbred pigs. More- 42 tion of Origin. In contrast to the Parma and San over, Toscano dry-cured hams from Cinta Senese 43 Daniele hams, the Cinta Senese ham is manufac- purebred pigs scored higher for juiciness than hams 44 tured using green hams from an ancient pig breed from crossbred pigs. 45 very common in Tuscany since the twelfth century. 46 Pigs are characterized by a black skin and white NERO SICILIANO HAM 47 stripe all over the front legs, the chest and the shoul- 48 ders. Cinta Senese is one of the pig breeds allowed The Nero Siciliano pigs derive from a population of 49 by the PDO for the production of the Toscano dry- pigs whose presence in Sicily is well documented at 50 cured ham. least since the nineteenth century (Moretti et al. 51 Toscano ham processing takes place in the tradi- 2004). This breed, reared on wood pasture, is charac- 52 tional production area including the entire territory terized by high roughness and strength. The popula- 53S of the region of Tuscany. Production and slaughter of tion of Nero Siciliano pigs is currently declining. 54N animals for raw material are carried out in the The population size is probably below 900 sows, 40454 38 393-406 r2.qxd 7/21/07 1:00 PM Page 403

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distributed in a large number of small herds and The production of French dry-cured hams is 1 including a free-ranging population of about 200 clearly influenced by the procedures followed in 2 sows. However, there are conditions for developing Spain and Italy. Bayonne hams are nitrified, salted 3 successful conservation programs. Traditionally, using dry salt and then kept in postsalting chambers 4 Nero Siciliano pigs have used both for fresh meat around 10 weeks. The ripening process lasts between 5 consumption and for the production dry-cured 9–10 and 12 months with the temperatures going 6 sausages (Moretti et al. 2004) and most recently for from 20–22°C (at the first stages) to 14–18°C fol- 7 the production of dry-cured hams. According to lowing the procedures followed for the production of 8 recent studies (Pugliese et al. 2004) the fat and meat Parma hams. 9 compositional traits of extensively reared Nero Sicil- Dry-cured Bayonne hams exhibit a rounded shape 10 iano pigs are similar to those found in other free- and their external appearance reminds of that of 11 range–reared pigs (i.e., Iberian or Corsican pigs) fed Parma hams: the hoof is removed and the head of the 12 on natural resources such as acorns and chestnuts. femur can be superficially seen. Compared to other 13 types of dry-cured hams, Bayonne hams contain 14 relatively high moisture content and relatively low 15 FRENCH DRY-CURED HAMS pigment concentration. Buscailhon et al. (1994) 16 The tradition of producing dry-cured hams from reported the relationship between the concentration 17 free-range–reared autochthonous pigs fed on acorns of heme pigments in Bayonne hams, their appear- 18 and chestnuts in France arises from the Roman ance, and their quality, highlighting these parameters 19 Empire. The coins carved into a ham shape found as decisive for consumer acceptability. The lean of 20 in Nîmes in the first century B.C. support that Bayonne hams has a pink, mild-ripened appearance. 21 hypothesis. In order to improve the quality of Bayonne hams 22 However, the population of rustic breeds in France in terms of texture and flavor, the aforementioned 23 is now decreasing and the production of dry-cured authors suggested extending the duration of the 24 hams is relegated to southern France, whereas north- ripening process up to the length currently used. 25 ern producers are devoted to the production of cooked According to Buscailhon et al. (1994), a longer 26 (Paris) or smoked (Alsace) hams. Lately, several ini- ripening process would increase the generation of 27 tiatives aimed to retrieve particular rustic breeds proteolysis-derived compounds and aroma volatiles. 28 (Noir de Bigorre, Corsican, Vasque, Limousine) and Additionally, a higher drying course would lead to 29 traditional production systems have been started in more firm hams and an extended chewing quality, 30 order to fulfill increasing consumer demand. The which would increase the perception of taste and fla- 31 production of improved industrial genotype pigs vor compounds. 32 (Large White, Landrace, and Duroc) specifically for 33 34 the manufacture of certain dry-cured products such NOIR DE BIGORRE HAM as Bayonne and Lacaunne hams has been recently 35 increased as well. The Noir de Bigorre ham is produced using raw 36 material from the Noir de Bigorre (traditionally 37 called Gascon) rustic and vigorous pig breed tradi- 38 BAYONNE HAM tionally reared in southwest France. The Noir de 39 The Bayonne hams own a Protected Geographical Bigorre pigs are reared in a semiextensive system 40 Indication and are the most representative French with availability of natural resources (grass, acorns, 41 hams due to the large amount of hams produced per and chestnuts), and they are slaughtered at high 42 year (around 500,000). These hams are manufac- weights (around 160 kg) with at least 12 months of 43 tured using raw material from industrial genotype age. Currently, the Noir de Bigorre ham is in the 44 pigs slaughtered at around 120 kg. Green Bayonne process of obtaining a Protected Designation of 45 hams show intermediate characteristics between Origin. In opposition to the commercial pigs used 46 those from rustic pig breeds (i.e., Noir de Bigorre) for the production of Bayonne hams and similarly 47 and other commercial pigs in terms of subcutaneous to other rustic breeds (i.e., Iberian pigs), Noir de 48 fat thickness (around 1.4 cm) and intramuscular fat Bigorre pigs generally have thick subcutaneous fat 49 content (2.8%), which largely determine the process (above 5 cm), and their muscles contain high 50 followed for their ripening. Bayonne hams are amounts of IMF (around 4%) and tocopherols. There- 51 ripened during an intermediate long process (9–12 fore, green hams from Noir de Bigorre pigs show a 52 months) leading to dry-cured hams of commercial high suitability for being subjected to a lengthy S53 quality. ripening process. In fact, Noir de Bigorre hams are N54 40454 38 393-406 r2.qxd 7/21/07 1:00 PM Page 404

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1 processed following traditional procedures similar to option to French consumers looking for dry-cured 2 those carried out for the production of the Iberian hams of higher quality than that of Bayonne hams, 3 ham: a long drying course (above 18 months) under with the latter being, nevertheless, generally appreci- 4 natural environmental conditions with severe tem- ated by consumers. 5 perature changes. 6 The appearance of the Noir the Bigorre ham is 7 considerably different from that of Bayonne ham. REFERENCES 8 The coxal bone and the hoof are kept, giving to this 9 ham a similar appearance to that of the Spanish AI Andres, R Cava, J Ruiz. 2002. Monitoring volatile 10 Serrano-type ham. Compared to the lean of Bayonne compounds during dry-cured ham ripening by solid- 11 hams, that of Noir the Bigorre hams exhibits a redder phase microextraction coupled to a new direct- 12 and more intense color as a result of the higher con- extraction device. J Chrom A 963:83–88. 13 centration of heme pigments (0.32 versus 0.12 BOE. 2001. Real Decreto 1083/2001, de 5 de octubre, 14 mg/100 g) and a more severe drying process por el que se aprueba la norma de calidad para el 15 (Ventanas unpublished data; Robert et al. 2005). jamón ibérico, paleta ibérica y caña de lomo ibérico 16 Large differences between Bayonne and Noir de Big- elaborados en España. Boletín Oficial del Estado, 17 orre hams also have been described regarding other 247, 15 de octubre, 37830–37833. 18 chemical components. Compared to other commer- P Bosi, JA Cacciavillani, L Casini, DP Lo Fiego, 19 cial dry-cured hams (i.e., Parma or Bayonne hams), M Marchetti, S Mattuzzi. 2000. Effects of dietary 20 higher oleic acid (ϳ46%) and tocopherol (ϳ7 mg/kg) high-oleic acid sunflower oil, copper and vitamin 21 levels have been reported in Noir de Bigorre hams as E levels on the fatty acid composition and the quality 22 a likely consequence of the feeding background of of dry-cured Parma ham. Meat Sci 54:119–126. 23 the pigs (pasture, acorns) in outdoor systems S Buscailhon, JL Berdagué, J Bousset, M Cornet, 24 (Ventanas unpublished data). Recent sensory studies G Gandemer, C Touraille, G Monin. 1994. Relations 25 carried out in different French dry-cured hams estab- between compositional traits and sensory qualities of 26 lished a certain relationship between compositional French dry-cured ham. Meat Sci 37:229–243. 27 traits and sensory quality, revealing large differences S Buscailhon, G Gandemer, G Monin. 1994. Time- 28 between types of hams. As a likely consequence of related changes in intramuscular lipids of French 29 the higher levels of IMF, Noir de Bigorre hams dry-cured ham. Meat Sci 37:245–255. 30 showed a higher juiciness than Bayonne hams (Tovar AI Carrapiso, C García. 2005. Instrumental colour of 31 et al. 2005; Robert et al. 2005), with that parameter Iberian ham subcutaneous fat and lean (biceps 32 highlighted as decisive for the overall acceptability of femoris): Influence of crossbreeding and rearing sys- 33 the dry-cured products. Hedonic tests carried out by tem. Meat Sci 71:284–290. 34 Tovar et al. (2005) in collaboration with the Centre AI Carrapiso, A Jurado, M Timón, C García. 2002. 35 Technique de la Salaison, de la Charcuterie et des Odor-active compounds of Iberian hams with differ- 36 Conserves de Viandes (CTSCCV) revealed that ent aroma characteristics. J Agric Food Chem 50: 37 amongst French dry-cured hams Noir de Bigorre 6453–6458. 38 hams received higher scores by French consumers R Cava, J Ventanas, J F Tejeda, J Ruiz, T Antequera. 39 than Bayonne hams. The aroma perceived from 2000. Effect of free-range rearing and α-tocopherol 40 Bayonne hams was described as “mild and slightly and copper supplementation on fatty acid profiles 41 rancid,” whereas that from Noir de Bigorre hams and susceptibility to lipid oxidation of fresh meat 42 showed a more intense and complex aromatic profile. from Iberian pigs. Food Chem 68:51–59. 43 The authors considered that the high level of IMF and A Daza, AI Rey, J Ruiz, CJ Lopez-Bote. 2005. Effects 44 the high levels of oleic acid and tocopherols caused of feeding in free-range conditions or in confinement 45 the generation of high levels of certain lipid-derived with different dietary MUFA/PUFA ratios and 46 volatiles during the long ripening process of the Noir α-tocopheryl acetate, on antioxidants accumulation 47 de Bigorre hams, which likely contributed pleasant and oxidative stability in Iberian pigs. Meat Sci 48 aromatic notes and enhanced their flavor traits. 69:151–163. 49 Consistently, volatile compounds described as odor- G Gandemer. 1998. Lipids and meat quality, lipolisis, 50 active compounds in Iberian dry-cured hams (Car- oxidation and flavour. In: Proc of the 44th ICoMST, 51 rapiso et al. 2002) and contributors of pleasant flavor Barcelona, Spain, pp. 106–119. 52 notes, have been described as volatile components of ———. 2002. Lipids in muscles and adipose tissues, 53S Noir de Bigorre hams (Sans et al. 2004). The Noir de changes during processing and sensory properties of 54N Bigorre hams are currently considered a promising meat products. Meat Sci 62:309–321. 40454 38 393-406 r2.qxd 7/21/07 1:00 PM Page 405

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DP Lo Fiego, P Macchioni, P Santoro, G Pastorelli, J Ruiz-Carrascal, J Ventanas, R Cava, A I Andrés, 1 C Corino. 2005. Effect of dietary conjugated linoleic C García. 2000. Texture and appearance of dry cured 2 acid (CLA) supplementation on CLA isomers con- ham as affected by fat content and fatty acid compo- 3 tent and fatty acid composition of dry-cured Parma sition. Food Res Int 33:91–95. 4 ham. Meat Sci 70:285–291. E Sabio, MC Vidal-Aragón, MJ Bernalte, JL Gata. 5 C López-Bote. 1998. Sustained utilization of the Iber- 1998. Volatile compounds present in six types of dry- 6 ian pig breed. Meat Sci 49:S17–S27. cured ham from south European countries. Food 7 VM Moretti, G Madonia, C Diaferia, T Mentasti, MA Chem 61:493–503. 8 Paleari, S Panseri, G Pirone, G Gandini. 2004. P Sans, MJ Andrade, E Muriel, J Ruiz. 2004. Etude du 9 Chemical and microbiological parameters and sen- profil des composes volatils de jambons secs issus de 10 sory attributes of a typical Sicilian salami ripened in porcs de race Gasconne. Proceedings of the 5th Inter- 11 different conditions. Meat Sci 66:845–854. national Symposium of the Mediterranean Pig, 12 G Pastorelli, S Magni, R Rossi, E Pagliarini, P Baldini, Tarbes, France, 2004, 66–67. 13 P Dirinck, F Van Opstaele, C Corino. 2003. Influence ML Timón, J Ventanas, AI Carrapiso, A Jurado, 14 of dietary fat on fatty acid composition and sensory C García. 2001. Subcutaneous and intermuscular fat 15 properties of dry-cured Parma ham. Meat Sci 65: characterisation of dry-cured Iberian hams. Meat Sci 16 571–580. 58:85–91. 17 C Pugliese, R Bozzi, G Campodoni, A Acciaioli, F Toldrá. 1998. Proteolysis and lipolysis in flavour 18 O Franci, G Gandini. 2005. Performance of Cinta development of dry-cured meat products Meat Sci 19 Senese pigs reared outdoors and indoors: 1. Meat 49:S101–S110. 20 and subcutaneous fat characteristics Meat Sci 69: J Tovar, E Gilbert, C García, J Ventanas. 2005. Establec- 21 459–464. imiento de un perfil sensorial del jamón francés Noir 22 C Pugliese, G Calagna, V Chiofalo, VM Moretti, de Bigorre. Proceedings of the 3th Mondial Congress 23 S Margiotta, O Franci, G Gandini. 2004. Comparison of Dry-cured Ham, Teruel, 2005, pp. 419–420. 24 of the performances of Nero Siciliano pigs reared J Ventanas. 2001. Tecnología del Jamón Ibérico. 25 indoors and outdoors: 2. Joints composition, meat Madrid: Ediciones Mundi-Prensa, pp. 15–45. 26 and fat traits. Meat Sci 68:523–528. J Ventanas, JJ Cordoba, T Antequera, C García, 27 N Robert, S Basly, C Dutertre. 2005. Efecto del proceso C Lopez-Bote, MA Asensio. 1992. Hydrolysis and 28 de maduración en la textura del Jamón IGP Bayonne. Maillard reactions during ripening of Iberian ham. 29 Proceedings of the 3th Mondial Congress of Dry- J Food Sci 57:813–815. 30 cured Ham, Teruel, 2005, pp. 411–412. S Ventanas, J Ventanas, J Ruiz, M Estévez. 2005. 31 J Ruiz, E Muriel, CJ Lopez-Bote. 2002. The flavour of Iberian pigs for the development of high-quality 32 Iberian ham. In: F Toldrá, ed. Research Advances in cured products. In: SG Pandalai, ed. Recent Research 33 the Quality of Meat and Meat Products. Trivandrum, Developments in Agricultural and Food Chemistry. 34 India: Research Signpost, pp. 289–309. Trivandrum, India: Research Signpost (6), pp. 27–53. 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 S53 N54 40454 39 407-414 r2.qxd 7/20/07 4:47 PM Page 407

1 2 3 39 4 5 6 North European Products 7 8 Torunn T. Håseth, Gudjon Thorkelsson, and Maan S. Sidhu 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION FENALÅR:NORWEGIAN DRY-CURED LEG 22 OF LAMB OR MUTTON 23 Salting and drying are considered to be of the oldest 24 Fenalår is to a large extent a Norwegian specialty, and most important methods for preservation of meat 25 although there is a small production in some other (1). The main focus of this chapter is on the typical 26 European countries such as Iceland (2). The name and traditional dry-cured meat products from Norway, 27 fenalår comes from the word fenad, which is an old Iceland, and the Faeroe Islands. Among these dry- 28 Norwegian word for mutton, and the word lår mean- cured products, some are generally unknown and dif- 29 ing leg. Curing of mutton and lamb legs has a long ferent from dry-cured products in the rest of Europe. 30 tradition in Norway, back to the time of the Vikings 31 more than a thousand years ago. Today, fenalår is a NORWAY 32 popular dry-cured product. The classic way of eating 33 Several types of dry-cured meats are produced in Nor- it is with very thin and unfermented crisp bread 34 way, and the annual production is about 1,700 tons or with normal bread, followed by scrambled eggs 35 (ACNielsen Norway AS), in addition to an import of and beer. Traditionally, a sheath knife was used to 36 about 300 tons. The most important dry-cured prod- cut off slices of the dry-cured meat, and many 37 ucts of whole meat in Norway are fenalår (fenalaar), people still do it this way at home. Modern use 38 which is dry-cured leg of lamb or mutton, and speke- includes a variety of dishes where dry-cured ham 39 skinke, which is dry-cured ham. The processes can be used. 40 described in this chapter are for the dry-cured meat The most common raw material for fenalår is leg 41 products with bone, but processing of deboned of lamb or mutton. Norway has the largest sheep 42 tumble-salted products are widespread. Dry-cured production among the Nordic countries (3), mostly 43 meat has a long tradition in Norway, and the con- the long-tailed crossbreed Norwegian White Sheep 44 sumption is about 0.5 kg per capita. Sweden, Fin- (T. Kvame, personal communication, 2006). Norway 45 land, and Denmark do not have the same tradition in is a long country with differing climate and differing 46 producing and eating dry-cured meat products, and geography from the sea to the mountains; therefore 47 therefore the consumption here is much lower. The there is a wide variety in pasture, but as much as 48 industrial “bulk type” dry-cured products are low- 80% of the sheep graze in the highland or mountains 49 priced and have a high salt content. More expensive during the summer (Norwegian Sheep Recording 50 high-quality products with low salt content and more System 2005). In September the sheep and lambs are 51 pronounced dry-cured aroma and taste are produced brought to the farms a short time before slaughtering 52 at a small, but increasing, scale. for cultivated pasture and grain feeding if necessary 53 S54 N55 407 40454 39 407-414 r2.qxd 7/20/07 4:47 PM Page 408

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1 (S. Vatn, personal communication, 2006). The ani- ducers of higher-quality products. They also tend to 2 mal welfare is generally good and strongly regulated reduce the amount of salt in the product because the 3 by legislation. The animal transportation has a high salt amount in industrial bulk products tends to be 4 standard, and the drivers have comprehensive cours- very high, often as much as 7–10 %. Combinations 5 ing in animal welfare and transportation (E. Tolo, of finely ground vacuum salt, nitrite salt, and coarse 6 personal communication, 2006). marine salt has long been preferred, but coarse 7 Most sheep and lamb are slaughtered in the marine salt used alone gains ground as dry salting 8 autumn, and use of frozen and thawed legs is com- becomes more common. Some producers use herbs, 9 mon to ensure constant meat supply to the produc- spices, honey, syrup, or other natural ingredients 10 tion during all seasons. In addition, salting time is along with the salt, mainly for taste contribution. 11 reduced (4). The legs should preferably be at least Two important conditions separate the Norwegian 12 3 kg after cutting and covered by an evenly distrib- technology from the South European. First, the only 13 uted fat layer. The leg is traditionally cut similarly to climatic control of the salting rooms is temperature 14 Spanish hams, with the aitch bone (ischium) kept on, control. The relative humidity (RH) varies widely, 15 but the foot is removed. There is a new trend among depending on conditions such as the outdoor climate 16 some producers to remove the aitch bone and form and which other products are stored in the salting 17 the leg to a rounded shape, similar to Parma ham. room. Second, keeping the cured legs cold for salt 18 Traditionally dry-cured leg of mutton and lamb equalization (postsalting) was, until recently, rarely 19 were produced on farms holding sheep. The legs used in Norway. The extensive salting allows the salt 20 were pickle- or brine-cured in bins. Today, there are to reach the center of the leg during salting, and the 21 numerous small- and medium-size production plants temperature can safely be raised immediately after 22 in Norway, in addition to butchers, specialty shops, salting. Focus on salt reduction has led to extended 23 and some private production. Brine- or pickle-curing use of salt equalization after salting (5). A nonde- 24 is common (Figure 39.1), but there seems to be a structive method for measuring salt is a large advan- 25 trend to convert to dry salting, especially among pro- tage in both research and product development of 26 expensive dry-cured products, and computed tomog- 27 raphy (CT) is such a method (6). The CT scanner 28 measures density as the attenuation of X-rays sent 29 through an object and visualizes this in a gray-scale 30 image (7). The density is related to the salt content in 31 the meat. The distribution of salt through the meat is 32 also visualized (8). 33 Historically, farmers dried the salted hams in tra- 34 ditional Norwegian storehouses on pillars (stabbur), 35 each cured leg hanging in a separate bag of cloth. 36 A wire prevented contact between cloth and meat, 37 which was important to prevent flies from placing 38 their eggs in the meat. Today, modern production 39 plants have conditioned rooms where temperature, 40 relative humidity, and air velocity are controlled. 41 Small producers control only the temperature. Bulk 42 products are dried until a weight loss of 26–33%, 43 with little time for ripening processes, like proteoly- 44 sis and lipolysis, to occur. Fortunately, the industry 45 has increased its development activity the last sev- 46 eral years and an extended ripening time is increas- 47 ingly common. Smoking was traditionally used for 48 additional preservation in the western part of Nor- 49 way, where the climate is very humid. Today, many 50 producers smoke part of their production. The smok- 51 Figure 39.1. Salting Norwegian fenalår in a ing is very slight compared to the heavily smoked 52 Norwegian meat processing plant. The legs are German Schwarzwald hams. The purpose of the 53S soaked in saturated brine before being pickle-salted smoking is to add taste and flavor to the product and 54N (private photo, M. S. Sidhu). to prevent growth of mold. 40454 39 407-414 r2.qxd 7/20/07 4:47 PM Page 409

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SPEKESKINKE:DRY-CURED HAM WITH BONE rest of the country. The name pinnekjøtt comes from 1 pinne meaning wooden stick, and kjøtt, which is 2 Hams used for Norwegian bulk dry-cured hams are Norwegian for meat. The dry-cured meat is placed 3 9–10 kg, usually with less than 10 mm subcutaneous on wooden sticks during cooking. Pinnekjøtt is pro- 4 fat. The small size, leanness, and high enzymatic duced from the side of lamb or mutton. It is mainly 5 activity of these fresh hams make them best suited produced in the slaughtering season for lamb, and 6 for shorter production times (9). A common indus- for this reason, the raw material is mainly used fresh. 7 trial curing method for bulk bone-in ham is pickle- The meat is placed in bins and coarse salt between 8 curing, where dry salt and meat extract turns into the layers ensures that all meat surfaces are in con- 9 brine, but dry salting and brine salting are also used. tact with salt. The meat is dry-, pickle-, or brine- 10 Bulk hams are heavily salted for 2–4 weeks and cured, or combinations of these methods. There are 11 given none or a short salt equalization. Some hams two salting strategies, either restricted or complete 12 are smoked, but not all, in contrast to what may be salting. Restricted salting lasts from 1.5 days (15) 13 the general idea (10). The hams are dried for 2.5–3 and up to 3–4 days, whereas complete salting lasts 14 months at roughly 15°C and 75% RH. Bulk hams until the meat is more or less saturated with salt after 15 have a weight loss of 25–30% and the salt content 4–10 days. Some producers rinse the salted meat in 16 can be as high as 10%. Salt distribution, proteolytic fresh water for some hours to a couple of days, to 17 index, and protein degradation is shown to be vari- prevent salt precipitation during drying. 18 able among Norwegian dry-cured hams (11). The salted meat is dried at 10–15°C, usually in 19 High-quality hams are generally produced from rooms without humidity control, but often under 20 older, heavier, and fattier pigs. The hams are dry- high air circulation from fans. If controlled, the 21 salted with coarse marine salt, the curing time being humidity is usually quite low, about 65–75% RH. 22 considerably shorter than for bulk ham (12). The The drying time varies widely; 10–15 days is com- 23 equalization period lasts from a few weeks up to mon, but as much as 6–7 weeks can be found (15). 24 4 months. Climatic conditions during drying are simi- About 25% are cold-smoked at room temperature, 25 lar to those of bulk ham, but small scale producers either during or after drying. The variation in total 26 may also use stabbur for drying, where the climatic water loss is considerable. This is due to large differ- 27 conditions are dependent on the outdoor conditions. ences in processing conditions such as time and tem- 28 During drying, the meat surface is covered with a thin perature, and differences in the raw material, such as 29 layer of fat or a mixture of fat and rice flour to prevent the size of the meat pieces, bone mass, and amount 30 excessive drying and to keep the surface smooth. The of fat (T. C. Johannessen, personal communication, 31 total production time is from 7 to 24 months, depend- 2006). 32 ing upon initial size and weight of the fresh hams and 33 the desired degree of ripening. These hams have a 34 pronounced dry-cured ham flavor and aroma, due to ICELAND AND THE FAEROE 35 proteolysis and lipolysis (13, 14). The final salt con- ISLANDS 36 tent is usually between 5 and 7%. 37 Food safety is an increasingly important public Iceland and the Faeroe Islands were settled from 38 health issue. Dry-cured meat is more or less shelf- Norway during the 8th and 9th centuries. Sheep 39 stable, depending on the ingredients and process farming has been the most important part of agri- 40 used, but is frequently contaminated with molds. All culture, and the production systems were adapted 41 together, mold contamination on dry-cured meat is a according to local environmental conditions. The 42 significant quality problem leading to a major eco- sheep breeds are directly descended from the first 43 nomic impact for the producers due to cleaning and sheep brought to the islands, belonging to the North 44 loss of products. Molds may also produce mycotox- European short-tailed group of sheep (16). The 45 ins that are toxic compounds with diverse effects on sheep live outdoors, grazing on wild pasture and cul- 46 health. tivated hayfields, but in Iceland they are kept in sheds and fed on dried hay or silage during the cold- 47 est part of the winter. 48 49 PINNEKJØTT:NORWEGIAN DRY-CURED 50 SIDE OF LAMB OR MUTTON HANGIKJÖT:COLD-SMOKED ICELANDIC 51 LAMB MEAT The dry-cured specialty pinnekjøtt is the traditional 52 and popular Christmas Eve dinner in the western The traditional preservation methods of lamb meat S53 part of Norway, and its popularity is increasing in the in Iceland are drying, smoking, and whey pickle N54 40454 39 407-414 r2.qxd 7/20/07 4:47 PM Page 410

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1 fermentation (17). Hangikjöt is a traditional product shortened the smoking times, cut down on the use of 2 in Iceland, which is eaten as a main part of festive nitrates, and introduced ascorbates as brining aids in 3 dinners like Christmas and Easter, but also as cold order to reduce the formation of N-nitrosamines (20). 4 cut slices on bread and as a starter. 5 Hangikjöt was traditionally produced from fresh SKERPIKJØT:AIR-DRIED FAEROESE SHEEP MEAT 6 meat after slaughtering in the autumn. Today, 7 hangikjöt is produced throughout the year from The local sheep production system and the ancient 8 frozen lamb carcasses of 17–19 kg. The carcass is slaughtering and processing traditions for sheep 9 cut into legs, forequarters, rack, and flanks. The legs meat in the Faeroe Islands are unique, compared to 10 and forequarters are thawed overnight in cold run- both the Nordic countries and to Europe. Most 11 ning water before salting. Different salting methods lambs are home-slaughtered at the farms or in the 12 are used. At one time, nitrate salt was common for villages and used for production of the traditional 13 dry-curing, but it is now fully replaced by nitrite salt. air-dried lamb meat called skerpikjøt. The Faeroese 14 Dry-curing is also combined with, or replaced by, have preserved a Viking food culture and it is part of 15 brine immersion. Multineedle injection was intro- daily life in the islands, and skerpikjøt is a national 16 duced during the 1970s and is used by some factories dish. Skerpikjøt means meat sharpened by cold and 17 in combination with brine-curing. Brine strengths wind. It is cut into thin slices and eaten, or placed on 18 (9–15 %), salting times (1–6 days), and use of other bread—preferably buttered rye bread. 19 ingredients (ascorbates, phosphates) vary between Newly slaughtered, whole carcasses of lamb, ewe, 20 producers or brands of hangikjöt. and ram are cut directly after slaughtering. The meat 21 Until the late 19th century, smoking took place is neither salted nor smoked but dried directly. To 22 over open fireplaces (18) for 2–3 weeks before the facilitate and speed up the drying, the carcass is 23 meat was moved to other parts of the kitchen or in opened up and flattened by cutting through the ribs 24 storage huts (hjallur) to dry. The fuel in the old on one side close to the backbone. Skerpikjøt is 25 kitchens was dried peat, dried sheep dung, and in dried in hjallur, mostly placed along rivers or brooks 26 some places birchwood. Today, specially designed on low banks or at places with good air circulation, 27 cold smoke ovens are used in the meat processing where drying of meat, by experience, is good. 28 plants. Dried sheep dung is always needed because Drying takes place during winter from October to 29 the smoke from it gives the meat its distinct flavor. March. Early winter the temperature is around 30 The smoking time is 1–5 days, depending on how 5–10°C and the humidity is rather high; later, the 31 many times the fuel is lit. temperature and humidity decreases. A weight loss of 32 Hangikjöt is still an important product in Iceland. about 35% during drying has been reported (21). 33 In 1975, the consumption was about 4.5 kg/person Water activity decreases from 0.97% to 0.90% dur- 34 (17), but it has declined to about 2.5 kg/person in ing the first month but changes little after that. Pro- 35 2005, calculated as meat with bone. Traditionally tein degradation due to proteolysis leads to an 36 produced hangikjöt is eaten both raw and cooked. increase in the pH of the meat during the first 4 37 But today, most smoked lamb meat in Iceland can be months, before it decreases again—perhaps due to 38 consumed only if cooked thoroughly. Some products yeast fermentation of carbohydrates in the meat. The 39 are also produced in order to be eaten raw. They are chemical composition of three cuts of skerpikjøt is 40 always smoked with dried sheep dung and the total shown in Table 39.1 (22). 41 weight loss is 30–35%. There are different prefer- The quality of the final product is very much 42 ences in Iceland for the various types of hangikjöt. dependent on weather conditions, especially in the 43 Less salt and less smoke is the main trend. first period when temperature, humidity, and wind 44 The consumption of smoked foods in Iceland was have a great influence. The quality of the fresh 45 very high before the introduction of modern preser- meat—that is freshness, carcass conformation, fat 46 vation methods like freezing. It has been suggested condition, and fat composition—is also critical for 47 that it might have been a factor of importance in the processing and quality of the end product. Further 48 high incidence of stomach cancer in Iceland, among studies are needed to explain the influence of raw 49 other factors due to considerable amounts of poly- material and processing conditions on the quality of 50 cyclic aromatic hydrocarbons (17). A high amount skerpikjøt. But parallels can be drawn to studies on 51 of nitrosamines has been found; most are in home- dry-cured ham, e.g., the age at slaughter can affect 52 made products, but they are also in commercially the color, amount of intramuscular fat, and activity 53S produced products (19). The meat industry has of proteases (23), whereas raised temperature at the 54N 40454 39 407-414 r2.qxd 7/20/07 4:47 PM Page 411

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Table 39.1. Chemical composition (in percent) personal communication, 2006). The production is 1 of three cuts of Faeroese air-dried lamb meat, traditional and on a small scale. The meat is heavily 2 skerpikj¿t. salted and dried to give a long shelf life. The dry- 3 ing takes place outdoors during the winter and 4 Types Protein Fat Dry Matter Salt spring. 5 Shoulder 27.6 27.9 57.0 0.54 6 7 Leg 28.5 19.0 59.5 0.41 GERMANY 8 Flank 22.2 65.6 89.0 0.39 Various types of dry-cured meats are widely used in 9 Germany, such as smoked ham like Westphalia ham, 10 Adapted and modified from Marita Poulsen (22). air-dried ham, cured lard, and various cured muscles 11 (26). Smoked hams are usually pickle-cured, filling 12 end of the process can increase the risk of faults, as up with brine after some days. The meat is com- 13 with soft hams and color changes (24). monly left in slow running water for 12–24 hours 14 Health authorities have questioned the safety of and smoked for 4 to 20 days depending upon product 15 skerpikjøt because the slaughtering and drying, and the desired degree of smoking (27). Air-dried 16 which is the only hurdle, are not carried out under hams are produced from round-shaped cooled hams. 17 controlled conditions. Four outbreaks of botulism They are rubbed with nitrite salt and dry salted in sea 18 were reported in the Faeroe Islands in 1979, 1988, salt for 25–30 days at 0–2°C and 80% RH (27). The 19 and 1989 (25). Today, the health authorities in the hams are slightly dried at 20–23°C to get a dry rind, 20 islands are strict on accomplishing good hygienic before being kept at 8–12°C and 60–70% RH for salt 21 practices to avoid microbial contamination. equalization. A mixture of salt and pepper is rubbed 22 into the ham before drying, initially at 8–12°C at 23 LAPP TRADITIONS 60–70% RH and later at 12°C at 70–80% RH for 24 several months. 25 Lapp traditions of dry-curing reindeer meat are wide- Perhaps the most famous and exclusive dry-cured 26 spread in northernmost parts of Norway, Sweden, meat from Germany is Schwarzwälder Schinken, 27 and Finland. The production is generally for private Black Forest hams. These hams can be produced 28 consumption. Most parts of the carcass, such as side, only in Schwarzwald (the Black Forest) in the 29 ham, and shoulder, are dry-cured. In the Norwegian southwest of Germany following strict production 30 regions, there are two main types of products: tørka regulations, due to their EU origin protection and 31 (dried) meat and bokna meat (M. Trumf, personal production protection (28). The hams must come 32 communication, 2006). Both types are dry salted from specially raised pigs. To be accepted, the fresh 33 using coarse marine salt, but the amount and length hams must have the correct pH (usually below 6.0), 34 of salting varies. After salting, the meat is dried out- the right amount of fat, and a good hygienic quality 35 doors, hanging on wooden racks. The drying time including a sufficiently low core temperature (29, 30). 36 depends on both the climatic conditions and the type The hams are deboned and trimmed before salt- 37 of commodity produced. Tørka meat is dried for ing. A mixture of salt, herbs, and spices, such as gar- 38 roughly 1.5 months, sometimes 2 months, before it is lic, pepper, coriander, and juniper berries is rubbed 39 then ready for consumption. The final water content onto the hams by hand (31). This mixture is con- 40 is generally lower than in products such as dry-cured sidered important for the products’ final characteris- 41 ham. On the other hand, Bokna meat is dried for only tics, and the exact blend is each producer’s secret. 42 a couple of weeks. This product is roasted in the oven Brine is formed after 3–4 days in the salt/spice mix- 43 or in a pan before consumption. ture and the hams are left in the brine until they are 44 In northern Sweden, there is a long tradition for considered salty enough, approximately after 2 45 drying reindeer meat. The meat is sometimes weeks (32). Excessive salt is removed and the hams 46 smoked, and many different types and pieces of are left in cold rooms for salt equalization for 2–7 47 muscles are used, including tongue and heart. Pro- weeks (29, 33). Smoking is an important part of the 48 duction is usually on a small scale for home con- tradition. The hams are cold-smoked over branches 49 sumption or for local distribution and sale (S. Berg, and sawdust of pine or fir from the Schwarzwald 50 personal communication, 2006). district, at about 25°C for 2–3 weeks (33). The hams 51 Reindeer, mainly loin and steak, is also dry- are kept at about 15°C from 2 to several weeks for 52 cured in Lapland in the north of Finland (J. Hallikas, ripening. S53 N54 40454 39 407-414 r2.qxd 7/20/07 4:47 PM Page 412

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1 OTHER NORTH EUROPEAN Dry-cured meat is mainly sold presliced among the 2 COUNTRIES delicacies in larger stores (U. Væreth, personal com- 3 munication, 2006). 4 The production volume of whole dry-cured meat in 5 Denmark and Sweden is small, though that of dry- 6 fermented sausages is considerable. During the last ACKNOWLEDGMENTS 7 years, local production of dry-cured meat has been The authors wish to acknowledge the Norwegian 8 initiated in Sweden to compete with an increasing dry-cured meat producers who anonymously shared 9 import from Germany, Italy, Spain, and Norway. their production processes for the documentation 10 Whole hams are not produced, but, instead, cold- purpose of the chapter. We also wish to thank 11 smoked products of whole muscles are preferred. Mohamed Abdella and Terje Frøystein at the Norwe- 12 These products are sold in pieces or sliced. Cold- gian Meat Research Centre for useful comments. 13 smoked whole meat products are also produced at Special thanks to Per Berg at the Norwegian Meat 14 farms and small enterprises, but the drying pro- Research Centre for general discussion about the 15 cess and final water activity are hardly controlled production of fenalår in Norway and for valuable 16 (S. Berg, personal communication, 2006). comments. 17 Historically, dry-cured hams were produced at 18 Danish farms for home consumption, but there is no 19 industrial production of whole dry-cured meat in REFERENCES 20 Denmark to date (K. Teglmand, personal communi- 21 cation, 2006). However, fresh hams are exported to 1. A Riddervold. 2004. Matkonserveringsmetodene. 22 Italy for production of dry-cured ham, whereas Parma In: A Riddervold, P Berg, et al.: Spekemat. 2nd ed. 23 ham and other hams are imported to Denmark. Local Norway: H.W. Damm & Søn AS, pp. 35–65. 24 butchers and gourmet shops produce some dry- 2. P Berg. 2004. Fenalår—fra konserveringsmetode til 25 cured hams, the most famous being Skagenskinke smak av kultur. In: Kjøttets Tilstand 2004. Norwe- 26 (C. Vestergaard, personal communication, 2006). gian Meat Research Centre, pp. 4–8. 27 Finland has a small production of dry-cured reindeer 3. ÓR Dyrmundsson. ´ 2004. Sustainability of sheep 28 meat (J. Hallikas, personal communication, 2006). and goat production in North European countries— 29 The meat is salted and dried, but not as heavily as the From the Arctic to the Alps. 55th Annual Meeting of 30 traditional Lapp production. The products are usu- the European Association for Animal Production, 31 ally smoked. Bled, Slovenia, p. 4. 32 Poland has a tradition for dry-curing different 4. F-S Wang. 2001. Lipolytic and proteolytic proper- 33 whole muscles of pork (J. Mielnik, personal commu- ties of dry-cured boneless hams ripened in modified 34 nication, 2006). Typical muscles are loin, tender- atmospheres. Meat Sci 59:15–22. 35 loin/filet, neck, clod (outside chuck; Triceps brachii), 5. T Thauland. 2003. The effect of resting on the distri- 36 and ham muscles. The muscles are slightly salted, bution of salt during the production of dry-cured ham. 37 usually without any other additives, but lactic acid Proceedings of the 49th International Congress of 38 bacteria are sometimes used. Dry salting is most Meat Science and Technology, Brazil, pp. 479–480. 39 common, but brine injection is also commonly used 6. A Dobrowolski, R Romvári, P Allen, W Branscheid, 40 in larger muscles. The salted muscles are predried, P Horn. 2003. X-ray computed tomography as an 41 usually for about 10 days, before smoking for 4–12 objective method of measuring the lean content of a 42 hours. Predrying allows smoke to fasten to the meat pig carcass. Proceedings of the 49th International 43 surface. The drying is continued after smoking until a Congress of Meat Science and Technology, Brazil, 44 total weight loss of 20–30% is achieved. The final pp. 371–372. 45 salt content is usually about 3–3.5%. 7. CS Vestergaard, SG Erbou, T Thauland, J Adler- 46 Smoked meat products of pork and beef are popu- Nissen, P Berg. 2005. Salt distribution in dry-cured 47 lar in Lithuania, whereas sheep and game are hardly ham measured by computed tomography and image 48 used for dry-curing. People in cities use industrially analysis. Meat Sci 69:9–15. 49 produced dry-cured products. Small farms make dif- 8. T Thauland. 2005. Accuracy of CT scanning for pre- 50 ferent products for their own consumption, like diction of salt content in salted meat. Proceedings 51 smoked fat, ham, and sausages (S. Nominaitis, per- of the International Skjervold-symposium, Hamar, 52 sonal communication, 2006). The production of Norway, p. 44. 53S dry-cured meat in England is limited; bacon and 9. F Toldrá. 2002. Dry-cured meat products. Trumbull, 54N cooked cured ham are much more widespread. Connecticut: Food & Nutrition Press, pp. 193–194. 40454 39 407-414 r2.qxd 7/20/07 4:47 PM Page 413

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10. J Flores. 1997. Mediterranean vs. northern 20. G Thorkelsson. 1989. The effect of processing 1 European meat products. Processing technologies on the content of polycyclic aromatic hydro- 2 and main differences. Food Chem 59:505–510. carbons and volatile N-Nitrosoamines in cured and 3 11. MS Sidhu, K Hollung, P Berg. 2005. Proteolysis in smoked lamb meat. In: Bibliotheca “Nutritio et 4 Norwegian dry-cured hams: Preliminary results. Pro- Dieta.” JC Somogyi, HR Muller, eds. Basel: 5 ceedings of the 51st International Congress of Meat Karger, pp. 188–198. 6 Science and Technology, Maryland, 2005, pp. 50–51. 21. Annual Report of Heilsufrødiliga Starvstovan. 7 12. P Berg, T Thauland. 2004. Den norske, italienske 1990. 8 og spanske spekeskinken. In: A Riddervold, P 22. Marita Poulsen. 1995. Føroyskar Føð slutavlar. 9 Berg, et al.: Spekemat. 2nd ed. Norway: H.W. (Faeroese Food Composition tables). Heilsufrøð 10 Damm & Søn AS, pp. 96–133. iliga Starvsstovan, pp. 16–18. 11 13. F Toldrá. 2006. The role of muscle enzymes in dry- 23. C Sárraga, M Gil, JA Farcia-Regueiro.1993. Com- 12 cured meat products with different drying condi- parison of calpain and cathepsin (B, L and D) 13 tions. Trends Food Sci Technol 17:164–168. activities during dry-cured ham processing from 14 14. F Toldrá, M Flores. 1998. The role of muscle pro- heavy and light large white pigs. J Sci Food and 15 teases and lipases in ﬂavor development during the Agric 62:71–75. 16 processing of dry-cured ham. Crit Rev Food Sci 24. L Martin, JJ Córdoba, T Antequera, ML Timón, 17 Nutr 38:331–352. J Ventanas. 1998. Effects of salt and temperature 18 15. A Brimi, B Wilson. 1987. Frå Lom til Lyon. Koke- on proteolysis during ripening of Iberian ham. 19 bok frå naturens kjøken. 2nd ed. Norway: Univer- Meat Sci 49:45–153. 20 sitetsforlaget AS, p. 132. 25. EP Wandall, T Videro. 1976. Botulism in the 21 16. I Tapio, L Grigaliunaite, E Holm, S Jeppsson, Faeroe Islands. Ugeskr Laeger 153:833–835. 22 J Kantanen, I Miceikiene, I Olsaker, H Viinalass, 26. H Koch. 1966. Die Fabrikation feiner Fleisch— 23 E Eythorsdottir. 2002. Mitochondrial differentia- und Wurstwaren. 15th ed. Frankfurt am Main: Ver- 24 tion in Northern European sheep. The 7th World lagshaus Sponholz, pp. 124–130. 25 Congress on Genetics Applied to Livestock Pro- 27. H Koch, H Fuchs, Gemmer. 1992. Die Fabrikation 26 duction, Montpellier, France. Session 26. Manage- feiner Fleisch—und Wurstwaren. 20th ed. Frank- 27 ment of genetic diversity, pp. 26–38. furt am Main: Deutscher Fachverlag, pp. 69–76. 28 17. T Thorsteinsson, G Thordarsson, G Hallgrimsdot- 28. http://www.tannenhof-schinken.de/english/schutzver- 29 tir. 1976. The Development of Smoking of Food in band.html, printed 060214. 30 Iceland. In: Advances in Smoking of Foods. Inter- 29. http://www.adler-schinken.de/start.htm, printed 31 national Joint IUPAC/IUFoST Symposium in 60214. 32 Warszawa, Poland, pp. 185–189. 30. http://www.schinken-wein.de/content/index.html, 33 18. H Gísladóttir 2004. Hangikjöt í rót upp rís: um printed 060214. 34 reykhús og önnur reykingar´ymi. In: Árbók Hins 31. http://www.pfau-schinken.de/komplett-rahmen.html, 35 Islenzka Fornleifafélags 2002–2003, pp. 151–162. printed 060214. 36 19. MJ Dennis, GS Cripps, AR Tricker, RC Massey, DJ 32. http://www.tannenhof-schinken.de/english/produk- 37 McWeeny. 1984. N-nitroso compounds and polycyclic tion.html, printed 060214. 38 aromatic hydrocarbons in Icelandic smoked cured 33. http://www.schwarzwaelder-schinken-verband.de/ 39 mutton. Food Chem Toxicol 22:305–306. herstellung/index.html, printed 060214. 40 41 42 43 44 45 46 47 48 49 50 51 52 S53 N54 40454 40 415-418 r1.qxd 7/21/07 12:43 PM Page 415

1 2 3 40 4 5 6 Asian Products 7 8 Guang-Hong Zhou and Gai-Ming Zhao 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION autumn of the next year. After a long ripening 22 process, muscle protein and fat are hydrolyzed to 23 Asia has a long and glorious history with splendid some extent by internal enzymes, and many small 24 dietary culture. Many traditional ripened meat prod- peptides, free amino acids, free fatty acids, and 25 ucts were developed in Asia, among which Jinhua volatiles are produced, which eventually contribute to 26 ham is the most famous in the world. Jinhua ham has the unique flavor of Jinhua ham (Zhao et al. 27 attractive color, unique flavor, and bamboo-leaflike 2005a,b,c; Du and Ahn 2001; Huan et al. 2005). For 28 shape. Its roselike muscle, golden-yellow skin, and example, the content of the free amino acids in final 29 pure white fat make Jinhua ham one of the most Jinhua ham products is 14 to 16 times that in green 30 preferred items in Chinese cuisine. The processing ham, among which, Leu, Ile, Phe, Glu, Ser, and Lys 31 technology of Jinhua ham was introduced to Euro- are 30 times higher (Zhao et al. 2005a). There are 168 32 pean countries by the famous Italian traveler Marco volatile compounds identified in the biceps femoris 33 Polo in the 13th to 14th centuries and had an impor- from Jinhua ham using SPME coupled with GC-MS, 34 tant impact on dry-cured ham processing technology including hydrocarbons, aldehydes, ketones, esters, 35 in the world, especially on Parma ham of Italy. In alcohols, carboxylic acid, heterocyclic compounds, 36 fact, the processing technologies of most dry-cured lactones, and sulphur compounds, etc. (Zhao 2004; 37 hams in China are evolved from Jinhua ham. In this Huan 2005). 38 chapter, the traits and traditional processing technol- 39 ogy of Jinhua ham are introduced. 40 PROCESSING OF JINHUA HAM 41 HISTORY AND TRAITS OF Processing of Jinhua ham consists of six stages: 42 JINHUA HAM green ham preparing, salting, washing, sun drying 43 and shaping, ripening, and postripening. There are 44 Jinhua ham is formed and produced in Jinhua Dis- more than 90 steps. The flow route of Jinhua ham 45 trict, Zhejiang Province of China. The earliest legend processing is shown in Figure 40.1. 46 regarding the Jinhua ham processing method may be Jinhua ham is traditionally processed under natu- 47 traced back to the Tang Dynasty (A.D. 618–907). ral conditions. Its unique quality is due not only to 48 However, Jinhua ham was formally named by the elaborate processing technology, but also related to 49 first emperor of the South Song Dynasty about 800 the unique local geographic terrain and climate. 50 years ago (Wu and Shun 1959). About 70% of Jinhua District is a mountainous 51 Typical Jinhua ham processing generally takes region with four distinct seasons; air temperature 52 8–10 months, starting from winter and finishing in goes up and down regularly without tremendous 53 S54 N55 415 40454 40 415-418 r1.qxd 7/21/07 12:43 PM Page 416

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1 temperature, salt has difficulty penetrating meat 2 Green ham preparation when it is below 0°C, whereas the fast growth of 3 microbes will occur when temperature is above 4 Salting 15°C. According to the experience, undesirable 5 water loss will happen, which causes insufficient salt 6 penetration when the ambient humidity is below 7 Soaking and washing 70% RH. Otherwise, much of the salt will flow away 8 in the form of brine, causing pastiness on the ham 9 Sun drying and shaping surface when the humidity is above 90%. So, the 10 desirable ambient temperature and humidity for salt- 11 ing is 5–10°C and 75%–85% RH, respectively. 12 Ripening Average salting duration is about 30 days, varying 13 from 25 days for small hams (less than 5 kg) and 14 35 days for big hams (more than 8 kg), during which 15 Postripening each ham is salted 5–7 times. Usually no nitrate or 16 nitrite is used. 17 Grading and storage The first salting is also called drain salt. The aim 18 is to remove the remaining blood and superabundant 19 water from the muscle and restrain microbe intru- 20 Figure 40.1. Flow route of Jinhua ham processing. sion. The first salting requires the entire meat surface 21 to be covered with a thin layer of salt. Where bones 22 and main blood vessels are located, a little more salt 23 fluctuation, which is desirable for processing dry- should be used. As a result of salting, part of the 24 cured meat products. Usually, the room temperature water-soluble proteins, the remaining blood, and 25 in winter is from 0–10°C, favorable for ham salting. some water seep out of the meat surface, which is 26 In spring, air temperature goes up to 20°C or higher, favorable for growth of microorganisms. So the sec- 27 suitable for sun drying. The summer in Jinhua Dis- ond salting should start no later than 24 hours after 28 trict is usually very hot. The temperature can go up the first salting. It requires the entire meat surface to 29 to 40°C, ideal for ham ripening. Postripening begins be covered with a thick layer of salt. The quantity of 30 in autumn when temperature falls. The whole pro- salt used this time is the largest among all the salting 31 cessing of Jinhua ham, beginning in winter and fin- procedures. The following salting processes are car- 32 ishing in the next autumn, takes 8–10 months. ried out at 5-day intervals for the purpose of supple- 33 mentary salt in the ham. After 5–7 times of salting, 34 the muscle normally becomes firm and the color GREEN HAM PREPARATION 35 of the meat surface becomes dark red, indicating that 36 Traditionally, only hindlegs from the Jinhua “Liang- the ham has been well salted. 37 touwu” pig or its cross-offspring could be used for 38 producing Jinhua ham. Desirable legs should be SOAKING AND WASHING 39 fresh, with thin skin, slim shank-bone, and well- 40 developed muscle; and covered by a thin layer of The purpose of this procedure is to remove superflu- 41 white fat. Broken bone should be particularly ous salt and wash off dirty substances on the meat sur- 42 avoided. The exposed part of bones, as well as the face. Usually, hams are initially soaked in water for 43 fat, tendon, and muscle membrane on the meat sur- 4–6 hours and then washed with bamboo brushes. 44 face of selected legs are cut off and then trimmed Water in the pools is changed after washing, and hams 45 into a shape like a bamboo leaf. The remaining blood are soaked again in water for another 16–18 hours. 46 should be squeezed out. A leg weight between 5.5 47 and 7.5 kg after trimming is preferred. SUN DRYING AND SHAPING 48 49 The objective of sun drying is to achieve appropriate SALTING 50 dehydration because insufficient dehydration may 51 Salting is a critical stage in Jinhua ham processing, cause ham spoilage. To keep balance, a pair of hams 52 and inappropriate salting may cause ham spoilage. of similar weight are tied by a rope and hung from a 53S The ambient temperature and humidity have great rack. Positioning is such that each ham gets enough 54N effects on the salting process. With regard to the sunshine and sound ventilation. When hams are 40454 40 415-418 r1.qxd 7/21/07 12:43 PM Page 417

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hung, the husk of the hoof is removed, water and dirt Usually, the ripening room should be well ventilated, 1 on the skin is razed off, and the brand is sealed on the room temperature should increase from 15°C to 2 skin. Then hams can be dismounted from racks for 37°C gradually, and humidity should be controlled 3 shaping. First, the ankle joint is straightened, and within 55% and 75%. 4 then the claw is pressed to curve inside; finally, the During ripening, skin and muscle shrink to some 5 two sides of the ham are pressed toward the middle extent because of loss of water, and bones around 6 to make a bamboo leaflike shape. After shaping, joints protrude out of the meat surface. Therefore, 7 hams are hung up in racks again. Sun drying can be hams are usually dismounted from the centipede 8 terminated when hams start to drop oil. It generally rack and retrimmed in mid-April. This is normally 9 requires about 7 sunny days. If several continuous the last shaping, and it requires cutting off protrud- 10 rainy days are incurred during this period, a layer of ing bones, superfluous skin, and fat. After reshaping, 11 yellow and sticky substances formed on the meat sur- hams are retied to the centipede rack for ripening. 12 face should be washed off with water on a sunny day. Usually, the ripening process terminates in mid- 13 The remaining hair on the skin is usually burnt off August when the temperature begins to drop. 14 with flame before dismounting the hams from racks. 15 16 POSTRIPENING 17 RIPENING After ripening, the meat surface becomes very dry 18 Ripening is the key process for the generation of and often covered with a thin layer of mold spores and 19 ham flavor substances. During this period, muscle dirt. So hams are first brushed clean and daubed with 20 proteins and fat are hydrolyzed to some degree, a thin layer of vegetable oil to soften the muscle and 21 mainly by an endogenous enzyme, which results in a prevent excessive fat oxidation; then they are piled 22 great amount of peptides, free amino acids, and free with skin side up for postripening. The postripening 23 fatty acids. The products constitute the main part of process is a process to stabilize and intensify the ham 24 ham taste substances and may continue to react with flavor; it is carried out in a warehouse and usually 25 each other or be hydrolyzed to produce volatiles that takes 2 months. During postripening, ham piles need 26 contribute to the unique aroma of Jinhua ham. turning over from time to time in case of unexpected 27 Ripening rooms are usually on the second floor. fermentation that affects ham quality. 28 When transferred to the ripening room, ham pairs 29 are fastened to the centipede rack (named for its cen- GRADING AND STORAGE 30 tipedelike shape) with the meat surface toward the 31 windows. Normally, various molds begin to grow It’s a common practice to grade Jinhua ham into dif- 32 20 to 30 days later and the meat surfaces of the hams ferent categories according to its quality, mainly 33 are gradually covered by several dominant molds. It depending on its intensity of aroma. The government 34 was reported (Toldrá and Etherington 1988; Molina authorized panel judges a ham’s aroma by inserting a 35 and Toldrá 1992; Sánchez and Crespo 2000; Zhao bamboo prod into a ham and smelling it when it’s 36 2004; Huan et al. 2005) that mold growth is not the pulled out. There are three fixed locations: namely 37 main contributor to ham flavor. However, the growth the “up,” “middle,” and “lower” positions on a ham 38 of some dominant molds may reflect a ham-ripening for this special purpose. Jinhua ham can be stored 39 condition in Jinhua ham processing. Normally, when for years, and it has the best flavor when stored for 40 gray and greenish molds dominate, it indicates desir- around 12 months. 41

able muscle salt content and water activity (aw). 42 White mold domination indicates higher and unde- REFERENCES 43 sirable water content and/or insufficient salt concen- 44 tration in the muscle. No mold growth indicates too M Du, DU Ahn. 2001. Volatile substances of Chinese 45 much salt in the muscle, which will make it difficult traditional Jinhua ham and Cantonese sausage. 46 for the hams to develop sufficient aroma and easy to J Food Sci 66:827–831. 47 grow worms (Wu and Shun 1959; Gong 1987). YL Gong. 1987. Jinhua ham Processing Technology. 48 Ham quality is susceptible to ripening room 1st ed. Beijing: Popular Science Press, pp. 4–22, 49 microclimate. High temperature with low humidity 73–75. 50 stimulates weight loss and fat oxidation, and with YJ Huan. 2005. Studies on the forming mechanism of 51 high humidity may result in ham spoilage. On the flavor compounds originated from lipid during pro- 52 other hand, low temperature, especially in the later cessing of Jinhua ham. PhD dissertation, Nanjing S53 phase of ripening, slows aroma formation in hams. Agricultural University, Nanjing, China. N54 40454 40 415-418 r1.qxd 7/21/07 12:43 PM Page 418

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1 YJ Huan, GH Zhou, GM Zhao, XL Xu, ZQ Peng. 2005. GM Zhao, GH Zhou, W Tian, XL Xu, YL Wang, X Luo. 2 Changes in flavor compounds of dry-cured Jinhua 2005a. Changes of alanyl amino peptidase activity 3 ham during processing. Meat Sci 71:291–299. and free amino acid contents in biceps femoris 4 I Molina, F Toldrá. 1992. Detection of proteolytic activ- during processing of Jinhua ham. Meat Sci 71: 5 ity in microorganisms isolated from dry-cured ham. 612–619. 6 J Food Sci 57:1308–1310. GM Zhao, GH Zhou, YL Wang, XL Xu, YJ Huan, JQ 7 FJC Sánchez, FL Crespo. 2000. Influence of molds on Wu. 2005c. Time-related changes in cathepsin B and 8 flavor quality of Spanish ham. J Muscle Foods L activities during processing of Jinhua ham as a 9 11:247–259. function of pH, salt and temperature. Meat Sci 70: 10 F Toldrá, DJ Etherington. 1988. Examination of cathep- 381–388. 11 sins B, D, H and L activities in dry-cures hams. Meat GM Zhao, GH Zhou, XL Xu, ZQ Peng, YJ Huan, ZM 12 Sci 59:531–538. Jing, MW Chen. 2005b. Studies on time-related 13 AF Wu, CY Shun. 1959. Jinhua Ham. 1st ed. Beijing: changes of dipeptidyl peptidase during processing of 14 China Light Industry, pp. 3. Jinhua ham using response surface methodology. 15 GM Zhao. 2004. Studies on the effects of muscle prote- Meat Sci 69:165–174. 16 olytic enzymes in the processing of Traditional Jinhua SW Zhu. 1993. Study on Taste and Taste Substances of 17 ham. 2004. PhD dissertation, Nanjing Agricultural Jinhua Ham. Food Sci (Chinese) 159:8–11. 18 University, Nanjing, China. 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53S 54N 40454 41 419-426 r1.qxd 7/21/07 1:02 PM Page 419

1 2 3 4 5 6 7 8 9 10 11 12 13 14 Part X 15 16 17 18 19 Biological and Chemical Safety of 20 21 22 Fermented Meat Products 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 S54 N55 40454 41 419-426 r1.qxd 7/21/07 1:02 PM Page 421

1 2 3 41 4 5 6 Spoilage Microorganisms: 7 8 Risks and Control 9 10 Jean Labadie 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION defects, the spoilages, their consequences, and in each 22 case the solutions proposed to limit their risk. 23 In Europe, the most popular dry-cured sausages are 24 salamis and salami-type sausages. Although such 25 products are, according to the customs of the coun- STICKY OR “SLIMY” SAUSAGES 26 tries, highly variable in terms of flavors, texture, color 27 and acidity, they are always the result of fermentations These sausages result from an overgrowth of 28 by two groups of bacterial species, which are included microorganisms on casings. The surface microflora 29 in the Lactobacillus and Staphylococcus genera. is clearly visible very early, noticeably during the 30 These species are mainly Lactobacillus sakei, Lacto- course of initial fermentation when the relative 31 bacillus pantarum, Staphylococcus saprophyticus, humidity of the fermentation chamber is high and 32 Staphylococcus xylosus, Staphylococcus warneri, and favorable to the microbial growth. Two factors 33 Staphylococcus equorum (Gevers et al. 2003; Fontana explain the defect: the high temperature inside the 34 et al. 2005; Corbière et al. 2006). All these bacteria, fermentation room and a film of liquid water clearly 35 which are now well identified by many taxonomical visible on the surface of the sausages. The microor- 36 approaches, including the most up-to-date molecular ganisms involved include yeasts, molds, and many 37 techniques, principally degrade the sugars (glucose or bacteria that grow heavily and form thick and sticky 38 saccharose) added to the raw sausage mixture at the biofilms. Ammonia flavors are almost always thor- 39 beginning of the processing. Such fermentations oughly produced and easily noticed. The aspect 40 largely contribute to the pH shift observed early in the of the biofilms are reminiscent of those observed 41 batter and to the more-or-less acidic pH of the fin- inside water pipes; they are grey, slimy, and have a 42 ished products. In terms of microbiology, the conse- strong smell. Pseudomonas spp. constitute the domi- 43 quence of a rapid acidification inside a meat mixture nant part of the flora isolated from these biofilms 44 is the selection of a few microbial species well fitted (Fournaud 1976; Migaud and Frentz 1978). The 45 to such conditions that are able to survive and/or to thick biofilms grown on the surface of casings 46 grow. Additionally, because salamis are always pro- threaten sausage manufacturers because they slow 47 gressively dried during the course of their ripening, down and limit the dehydration inside the ripening 48 the water activities (a ) rapidly reach levels (0.7–0.8), w chambers. The color of these sausages rapidly turned 49 which constitute insuperable hurdles for most of to yellow, green, or a combination of these colors. 50 the microorganisms not involved in fermentations The formation of the biofilms could also be observed 51 (Leistner 2000). Hence, spoilages are fortunately rare when processing is correctly managed, but in this 52 and they are always the consequence of defective case it is observed later, noticeably during the ripen- 53 manufacturing practices. This chapter describes these ing phase. The defect is generally the result of a S54 N55 421 40454 41 419-426 r1.qxd 7/21/07 1:02 PM Page 422

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1 malfunctioning of the drying room, but in this condi- the use of another surface treatment, for instance by 2 tion the bioﬁlms are not so pronounced and more using ﬂour, to give the sausages a white color and a 3 progressively formed, because of a lower tempera- smoothness indicating the moldy surfaces of nor- 4 ture. The microorganisms involved are resistant to mally ripened products. Unfortunately, these treat-

5 lower aw values; they are different from those isolated ments are costly and they almost systematically 6 from biofilms observed at the beginning of fermenta- demand a reintroduction of the sausages in packages 7 tions. They are mostly yeasts and Staphylococci. that often favor the mold regrowth. 8 The odors of slimy sausages are so pronounced In fact, practically, the best solution is a combina- 9 that they could be compared to some French soft tion of different treatments. 10 cheese like Epoisse or Livarot. These odors result 11 from the activity of numerous microbial enzymes, 12 proteases, peptidases, and desaminases. Many bacte- INOCULATION OF THE CASINGS WITH 13 ria are involved, among which Pseudomonas spp., WHITE MOLDS AND YEASTS 14 Brevibacterium spp., and Corynebacterium spp. are This treatment can be efficient, but results are haz- 15 the most important. ardous because microbial growth can be long before 16 Slimy sausages are also produced when the fol- a visible, acceptable result. It means that sometimes 17 lowing occurs: other wild molds already present in the ripening 18 • Products are dried at too low temperatures. chambers are able to grow in place of the added 19 • Water pellicles are observed on casings. They strains. In other cases, no mold growth is observed 20 result of rapid temperature and RH (relative for unexplained reasons. 21 humidity) shifts. 22 • Water condensations exist inside packaging. 23 INCREASING THE AIR VELOCITY 24 Solutions that could be used to avoid the production This solution is difficult to use because intense dry- 25 of sticky sausage are easy to set up: drying tempera- ing often leads to “crusting” of the sausages. Addi- 26 ture higher than 12°C, increasing the air velocity tionally, air fluxes will have to be avoided from the 27 inside the drying chamber, and packaging the fin- brushing chamber to the fermentation chamber in 28 ished products in a room of similar temperature. order to limit the airborne contaminations. 29 30 MOLDY SAUSAGES 31 FUMIGATION OF THE WORKING PLANT Moldy sausages are not spoiled inside the meat and 32 This cleaning and disinfection approach is not easy fat mixture, because molds are present only on the 33 to set up (hazardous for humans) and often inef- casings. Moreover, as their growth on casings is 34 ficient because many places are not cleaned and demanded by consumers of Romanic (France, Italy) 35 disinfected, such as crevices, corners, pipes, wood type sausages (Rödel et al. 1994), it is a difficult task 36 surfaces, etc. (Migaud and Frentz 1978). It is impor- for the producers to avoid an overgrowth of molds on 37 tant to understand that disinfection is to be carried sausages that are stored for several weeks before 38 out in the first part of the plants, where RH (relative their sale. In France, to get this “moldy” aspect, the 39 humidity) is still high. The materials used in the dry- most often inoculated species is Penicillium nal- 40 ing room are to be cleaned by the same approach or giovense. This mold gives the sausages a white 41 another specific one. 42 attractive color. However, sometimes other mold and 43 yeast species, also present on casings, grow so heav- 44 ily that they give the sausages unwanted colors TREATMENT WITH NATAMYCIN (PIRAMICIN) (green or brown, or a mixture of both) which are a 45 This treatment inhibits the growth of any molds on 46 cause of rejection by the consumers. Several solutions could be applied to avoid or to many food products, including cheese and sausages. 47 It is not convenient for all kind of sausages. 48 limit the growth of molds. They are briefly listed in 49 the next sections (Sirami 1998). 50 CONTROLLING RELATIVE HUMIDITY ELIMINATION OF THE MOLDS BY CLEANING AND 51 As indicated above, stabilizing sausages with a mold BRUSHING 52 flora biofilm that remains acceptable is a difficult 53S Eliminating the molds is only a part of the solution task, because it is always impossible to give all 54N because the aspect of the sausages always demands sausages the same aspect—that is, controlling the 40454 41 419-426 r1.qxd 7/21/07 1:02 PM Page 423

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mold growth in the same way on every sausage ANTIMOLD PRODUCTS 1 (Leistner 1995; Rödel et al. 1994). This is mainly 2 Among the efficient products that could be used on the consequence of the conditions chosen during the 3 sausages, pimaricin, a natural product produced by ripening phase. These conditions are optimal for fer- 4 Streptomyces natalensis, is often recommended. Dur- mentations to get tasty, colorful products, but they 5 ing the first 2 weeks, the pimaricin inhibits the are also optimal for mold growth on casings. This 6 growth of molds on the casings, but the efficiency means that some sausages are inevitably covered 7 of this antimicrobial molecule decreases rapidly with a thick mold biofilm. 8 (Sirami 1998). This time is, however, sufficient for Water is the most important parameter for the 9 products with a short ripening period of not longer growth of bacterial biofilm (Migaud and Frentz 10 than 2 weeks. In case of longer ripening, generally 1978). It is also true for mold biofilms, the growth of 11 4 weeks, it seems impossible to avoid the growth of which is favored by increased water quantities in the 12 molds because dehydration of the casings consider- vicinity of the sausage surface during the course of 13 ably limits the action of the piramicin. On German drying. This water comes from the meat particles of 14 raw sausages, potassium sorbate solutions (20%) are the batter and goes through the air of the drying 15 recommended and approved. These solutions are room by passing across the casings. By speeding up 16 used to avoid the “sausage bloom” sometimes the air velocity inside fermentation rooms it is pos- 17 observed on some batches (Leistner 1985). sible to eliminate the surface water of sausages more 18 rapidly; however, crusting frequently results from 19 such operations. That is why other approaches are SOURING 20 preferable. This defect is generally detected in romanic (French, 21 Among the molds sometimes isolated, several Italian) salamis (Fournaud 1976; Flores and Bermell 22 Penicillium spp. strains producing toxins could grow 1996; Leistner 1985), but it could also be observed 23 on the casings (Leistner 1995). Their growth, how- sometimes in Hungarian and German salamis heavily 24 ever, never or rarely leads to toxin production, at contaminated by heterofermentative lactobacilli 25 least inside the sausages. Moreover, because gener- (Leistner 1995). Generally, it occurs at the end of 26 ally most of the sausages are peeled off before being ripening, and the problem is discovered mostly when 27 eaten, the intoxication hazard is limited. the sausages are eaten. Acidic sausages are often 28 As already outlined, inside the ripening chambers, observed in the largest pieces of the product and are 29 RH and temperatures are the most important factor practically not visible from the outside. Acidic 30 to be controlled and monitored (Sirami 1998). Dur- sausages display similar aspects of normal sausages, 31 ing the ripening time, RH must remain below 75% although some of them could be paler than the others. 32 on the sausage casings; molds grow slowly up to an Sometimes, the absence of molds, which are nor- 33 RH of 80%. Avoiding the growth of molds means mally present on casings, is an indication that the 34 that the air of the ripening chamber is to be renewed sausages could be soured, but many other causes 35 regularly, and that the RH value is below 80% any- could explain the less abundant growth of molds. 36 where in the ripening room. Getting homogeneous However, a combination of properties observed on 37 RH inside the chamber is a difficult task, and great big sausages, i.e, nude casings and soft texture at the 38 care in the renewing of the air is necessary. The end of ripening, is a good indication that the batters 39 growth of the molds on only one sausage is detri- of these big pieces are soured (Migaud and Frentz 40 mental to all the sausages present in the ripening 1978). After slicing, the batters of soured sausages 41 chamber. To get good results, manufacturers are gen- never show any transition to a coagulated state; 42 erally using very dry air fluxes, which are highly moreover, off-flavors reminiscent of lactic acid are 43 favorable to the production of crusty surfaces. detectable. The taste of these products is clearly 44 Sausages must also be well separated inside the acidic and even strongly acidic. The color is brown or 45 chamber to avoid air pockets where RH could pale pink on the outside of the sausage, close to the 46 remain high. casing. Surprisingly, the surface is a little “crusty,” 47 tough, and very dry. The casings are tightly attached 48 to the batter. When analyzed, the water content of 49 SMOKING these sausages is too high and the pH of the batter is 50 A slight smoking every day prevents, delays, or higher in the middle than on the outside of the 51 inhibits the mold flora; this explains why smoked sausages (Sirami 1998). Additionally, nitrate contents 52 sausages of northern Europe are contaminated by are high in the center of the sliced sausages, whereas S53 molds lately (Leistner 1987). no nitrite is observed in the same location. The acidic N54 40454 41 419-426 r1.qxd 7/21/07 1:02 PM Page 424

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1 taste, which is obvious in such products, is generally without favoring some spoilage microorganisms, for 2 well correlated to the pH. Because the D lactate is instance, Enterobacteriaceae. In this case, it is nec- 3 clearly involved in the acidic taste, it is likely that het- essary to work with meat of high microbiological 4 erofermentative bacteria, at least Leuconostoc spp. standards. 5 (L. mesenteroides, L. carnosum, L. gelidum), which 6 only produce D lactate from sugars, are involved in 7 the manufacturing defect. As a matter of fact, the ROTTEN SAUSAGES 8 defect is easily reproduced when these bacteria are This defect is fortunately rare in industrial produc- 9 experimentally introduced in batters (Sirami 1998). tions. It is, however, not so uncommon in small 10 Avoiding the defect is generally difficult because workshops of southern Europe, which are producing 11 it could arise for unpredictable reasons. However, it sausages without starters. In these workshops, the 12 is possible to limit the souring by different ways. lactic microflora responsible for fermentations are 13 One of the most efficient approaches is to slow down only those present inside the processed chopped 14 the fermentation process in order to get batters with meat and fat mixture and on the natural casings gen- 15 higher pH at the end of the ripening time. In order to erally used (Fournaud 1976; Migaud and Frentz 16 achieve such pH values, decreasing quantities of 1978; Sirami 1998). Two parameters are responsible 17 sugars should be introduced into the batter, and con- for the defect. First, acidification inside fermentation 18 ditions favoring rapid degradation of these carbohy- chambers is weak. The final pH of the finished prod- 19 drates must be favored. Moreover, because the final ucts is too high (pH 6.2). Second, the raw products 20 pH of the product is reached earlier in the fermenta- that are used to prepare the batters are sometimes of 21 tion process, a slight increase of its value is generally low microbiological quality—that is, high numbers 22 observed during the ripening phase. To be efficient, of Pseudomonas spp. and Enterobacteriaceae are 23 this approach demands the use of high microbial observed (>105/g) on meat and fat as well. The 24 quality batters in order to avoid the growth of Enter- sausages produced with such quantities of spoilage 25 obacteriaceae, which could be less inhibited by the organisms become rapidly full of crevices that con- 26 higher pH values achieved at the end of ripening. tain different glazes. They look like balloons. Chop- 27 ping liberates spoiled, putrid odors. These sausages 28 SELECTING THE STARTERS are obviously not consumable. As indicated above, 29 this defect is caused by the use of low-quality raw 30 Some selected strains degrade the sugars more rap- materials, but also by a lack of hygiene during the 31 idly than the heterofermentative Leuconostoc spp., course of processing. The solutions to this problem 32 which are thus inhibited. are clear, i.e., the choice of high-quality meat and 33 fat, and the respect of good manufacturing practices 34 INCREASING THE SALT CONTENT OF THE on the processing lines. 35 BATTER UP TO 35 G/KG 36 It is first necessary to predry the fats and chop up 37 meat at low temperatures. Such operations are car- DISCOLORED SAUSAGES 38 ried out in cold rooms at −1/–3°C for 24 hours. They 39 This problem is basically due to defective fermenta- are, however, responsible for weight losses that could 40 tion processes. Very often, the origin is a lack of be as large as 10% of the total weight of the products. 41 favorable bacterial species and particularly those 42 producing the microbial enzymes involved in color 43 DECREASING THE FERMENTATION TEMPERATURES formation (Hechelman 1985; Leistner 1985; Lücke 44 Low temperatures improve the biochemical activi- and Hechelman 1987; Sirami 1998). These enzymes 45 ties involved in aroma production, particularly in the are the nitrate reductase of Staphylococci or some- 46 Staphylococci (S. xylosus, S. warneri, S. carnosus). times Enterobacteriaceae. However, as Enterobacte- 47 These species thus produce less acid and more aro- riaceae are often involved in the production of 48 matic compounds. biogenic amines, meat containing too many of these 49 microorganisms should be eliminated. A selection of 50 starters with efficient nitrate-reductase activities, OPTIMIZING THE PROCESSING TO AVOID CRUSTING 51 among which staphylococci are the most interesting, AND BIOFILM GROWTH 52 is certainly a better solution. 53S It is difficult to decrease simultaneously the sugar No, or poor, reductase activities could also result 54N content of batters and the fermentation temperature in a very rapid acidification of the batter, which often 40454 41 419-426 r1.qxd 7/21/07 1:02 PM Page 425

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inhibits the growth of staphylococci. Lactic acid bac- microorganisms growing inside these sausages are 1 teria (mainly L sakei, L. curvatus, and L. plantarum) the same as observed in bigger pieces. 2 which are responsible of the pH reduction, are not This problem is difficult to detect, but it could be 3 inhibited and could also participate to the inhibition definitely eliminated by hanging the big sausages 4 observed by other mechanisms. The sausages with inside nets. 5 this defect are grey-brown or brown after chopping. 6 The discoloration is often observed when the quanti- 7 ties of sugars added to the raw sausage mix are large CRUSTY SAUSAGES 8 (15 g/kg or more). Generally, the sugars are thor- This defect is one of the most frequently observed in 9 oughly degraded so that the pH of the batter drops industrial workshops. The sliced sausages display 10 almost immediately; it reaches, generally within discolored zones. From the outside, close to the cas- 11 48 hours, values that are lower than pH 5. ings, a dark zone of several mm thickness is formed. 12 Discoloration of sausages could also be the result This crusty surface, which is generally very tough, 13 of peroxide production by greening lactic acid bacte- slows down (Rödel et al. 1994) the dehydration of 14 ria, almost always present in raw sausage mixes. Per- sausages during the ripening. The phenomenon 15 oxides, which are normally present in low quantities could have two main origins: 16 at the end of the ripening, are still present in these 17 sausages, because the Staphylococci which normally • Dehydration is too fast; the water that, from the 18 produce the enzymes (catalases and peroxydases) inner parts of the sausages reaches the surface 19 involved in their destruction are inhibited by the lac- and evaporates, remains inside the core of the 20 tic acid bacteria. Such fermentations are generally batter. 21 • An overgrowth of molds increases the speed of observed when high aw (0.96) values and high tem- 22 perature (25°C) are combined. The solution to this dehydration. As a result, a crust is formed under 23 problem is simple, although not always easy to the mold biofilm. 24

apply: decreasing the aw of the batter before stuffing Molds consume nutrients and water on the surface of 25 casings and decreasing the temperature inside fer- the sausages, which become crusty. These sausages 26 mentation chambers. remain soft, and they dry very slowly. Acidification 27 is slowed and the batter is destructured. As molds are 28 HOLLOW SAUSAGES consuming the lactic acid produced by fermentation, 29 the taste of these sausages is neutral and the slices 30 Hollow sausages are generally observed in large- rapidly lose their color in the air. The sausages are 31 diameter, heavy sausages, although the problem could circled by a dry surface. If the problem is identified 32 be observed sometimes inside small pieces (Fournaud during the course of ripening, it is possible to wash 33 1976; Migaud and Frentz 1978; Sirami 1998). The the surfaces with salted water in order to make pos- 34 hollows are located inside the upper part of the sible a further ripening. 35 sausage, close to the hanging string. The result of such 36 hollows is that fat rapidly becomes rancid, with fat CONCLUSION 37 and lean pieces of the batter not tightly cemented in 38 the same coagulated structure. The pieces of meat are Most of the batters used to process dry-cured 39 less colored and more-or-less large crevices are sausages contain high counts of microorganisms, 40 observed between fat and meat, inside the sausage which are numerous species of bacteria, molds, and 41 mix. Inside crevices, meat pieces appear dark-red yeasts. Fortunately in most cases, these organisms 42 or red-brown, and fats are yellow-orange. These are not harmful and they generally grow very slowly 43 crevices grow with time because fermentations due to or are progressively eliminated inside the sausages 44 spoilage bacteria inevitably occur in the increasing during fermentations and ripening. Thus, spoiled 45 hollows. Molds like Mucor or Penicillium, heterofer- fermented sausages are very uncommon, but they 46 mentative lactic acid bacteria like Leuconostoc spp., could be observed in peculiar conditions described 47 and spoilage bacteria such as Pseudomonas spp. and in this chapter. As shown, spoilage generally results 48 Enterobacteriaceae (Hechelman 1985) often form from processing defects in which the normally two 49 slimy biofilms inside the hollows. insuperable hurdles, i.e., acidification and drying, 50 Sometimes hollows are observed inside sausages are either too pronounced or insufficient. Finally, to 51 of small sizes. They are due to a defective coagula- avoid the defects due to spoilage in dry-fermented 52 tion of proteins by salt, or to the use of partly sausages, it is important to set up rapid and efficient S53 degraded muscle proteins in the sausage mix. The corrective measures during the processing and good N54 40454 41 419-426 r1.qxd 7/21/07 1:02 PM Page 426

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1 manufacturing practices along the fermentation and L Leistner. 1985. Empfehlungen fûr sichere produkte. 2 drying process. In: Mikrobiologie und Qualitât von Rohwurst und 3 Roschinken. Herausgegeben vom Institût fûr Mikro- 4 REFERENCES biologie, Toxikologie und Histologie der Bunde- 5 sanstadt fûr Fleishforshung, Kulmbach, pp. 219–244. 6 S Corbière Morot-Bizot, S Leroy, R Talon R. 2006. ———. 1987. Fermented meat products. Proc 33rd Int 7 Staphylococcal community of a small unit manufac- Cong Meat Sci Technol August 2–7, Helsinki. Fin- 8 turing traditional dry fermented sausages. Inter- land, pp. 323–326. 9 national J Food Microbiol 108(2):210–217. ———. 1995. Stable and safe fermented sausages 10 J Flores, S Bermell. 1996. Dry-cured sausages factors worldwide. In: Fermented Meats. G. Campbell-Platt, 11 inﬂuencing souring and their consequences. Fleis- P. Cook, eds. England: Blackie Academic and Profes- 12 chwirtschaft 76 (2):163–165. sional, pp. 160–175 13 C Fontana, P Sandro Coconcelli, G Vignolo. 2005. ———. 2000. Basic aspects of Food preservation by 14 Monitoring the bacterial population dynamics during hurdle technology. Inter J Food Microbiol 55: 15 fermentation of artisanal Argentinean sausages.Inter 181–186. 16 J Food Microbiol 103(2):131–42. FK Lücke, H Hechelman. 1987. Starter cultures for dry 17 J Fournaud. 1976. La microbiologie du saucisson sec. sausage and raw ham: Composition and effect. Fleis- 18 L’Alimentation et la Vie 64:2–3. chwirtschaft 67:307–314. 19 D Gevers, M Danielsen, G Huys, J Swings. 2003. M Migaud, JC Frentz. 1978. In: La Charcuterie Crue et 20 Molecular characterization of tet(M) genes in les Produits Saumurés, Ch 47, Les défauts de fabri- 21 Lactobacillus isolates from different types of fer- cation. Orly France: Soussana 12 rue des lances, 22 mented dry sausage. Appl Environ Microbiol 69(2): pp. 224–239. 23 1270–1275. W Rödel, A Stiebing, L Krockel. 1994. Ripening 24 H Hechelman. 1985. Mikrobell verursachte fehlfab- parameters for traditionnal dry sausages with a 25 rikate bei Rohwurst und rohschinken. In: Mikrobi- mould covering. Fleishwirtschaft 73:848–853. 26 ologie und Qualitât von Rohwurst und Roschinken. J Sirami. 1998. Saucisson sec: Défauts de fabrication. 27 Herausgegeben vom Institût fûr Mikrobiologie, In:L’Encyclopédie de la Charcuterie, Ch 28. Défauts 28 Toxikologie und Histologie der Bundesanstadt fûr de Fabrication. Frentz et al., eds. Orly, France: Sous- 29 Fleishforshung, Kulmbach, pp. 103–127. sana 12 rue des lances, pp. 1122–1124. 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53S 54N 40454 42 427-454 r1.qxd 7/20/07 5:11 PM Page 427

1 2 3 42 4 5 6 Pathogens: Risks and Control 7 8 Panagiotis Skandamis and George-John E. Nychas 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION Escherichia coli, Clostridium spp., and Staphylococ- 22 cus aureus not only in the final products, e.g., 23 Preservation of meat by fermentation and drying has sausages, but also in different production levels. This 24 been used for hundreds of years. For a long time, the can be due either to frequently contaminated raw 25 technology of fermentation has been considered an material, where there is a probability that some of 26 art; however, more recently the process has been the pathogenic organisms could cross the antimicro- 27 studied, and as a result, products of quality can now bial barriers imposed during processing, or to cross- 28 be repeatedly produced under controlled conditions. contamination at any stage of retailing. In both 29 Dry or semidry-fermented sausages are prepared by cases, they may be present in the final product, caus- 30 mixing ground meat with various combinations of ing a concern for the producers and for those respon- 31 spices, flavorings, salt, sugar, additives, and bacterial sible for public health, which has been the topic of 32 cultures. The mixtures, in bulk or after stuffing, are study of several research groups (Moore 2004). 33 allowed to ferment at different temperatures for The risk analysis assessment related to pathogenic 34 varying periods of time. Following fermentation, bacteria in sausages is based on the following steps: 35 the product may be smoked and/or dried under 36 controlled conditions of temperature and relative 1. Hazard identification, where all pathogenic 37 humidity. agents capable of causing adverse health effects 38 Today, these meat products are part of the daily and which may be present in sausages are 39 diet in many countries all over the world; recently identified. 40 they have become fashionable food products, and in 2. Hazard characterization, the qualitative and/or 41 urban centers their market has been increasing sig- quantitative evaluation of the nature of the adverse 42 nificantly. In general, fermented products are consid- health effects associated with the above-mentioned 43 ered safe foods due to the pH drop and, in the case of bacteria present in sausages. This step requires a 44 meat fermentation, simultaneous reduction of water dose-response assessment for chemical agents, and 45 activity during the transformation of raw tissue if the data are obtainable, this should also be 46 (muscle) to final (fermented) product. Although both attempted for biological or physical agents. 47 these factors are considered to restrict detrimental 3. Exposure assessment, the qualitative and/or 48 parameters that cause growth of pathogenics, their quantitative evaluation of the likely intake of 49 presence has been previously reported in different pathogenic agents via sausages, as well as 50 stages of production as well as at the retail level exposures to other sources if relevant. 51 (Ananou et al. 2005; Samelis and Metaxopoulos 4. Risk characterization, the qualitative and/or 52 1999; Thévenot et al. 2005a). Different surveys have quantitative estimation, including uncertainties 53 revealed the presence of Listeria monocytogenes, around the model parameters, of the probability S54 N55 427 40454 42 427-454 r1.qxd 7/20/07 5:11 PM Page 428

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1 of occurrence and severity of known or potential enterotoxins. However, a higher incidence of 2 adverse health effects in a given population based L. innocua in meat products, compared to L. mono- 3 on the previous three steps. cytogenes, was reported (Borges et al. 1999). In 4 salami, the occurrence of L. monocytogenes varies 5 from 5–23% (Borges et al. 1999). Salami does not 6 HAZARD IDENTIFICATION undergo heat treatment and are fermented under 7 It is generally accepted (Nychas et al. 2007) that, variable temperatures (mostly between 25–30°C, 8 with the exception of external surfaces of the animal according to the processing method adopted by the 9 and the gastrointestinal and respiratory tracts, the manufacturer), having a final pH between 4.8 and 10 tissue of healthy living food animals is essentially 5.2 and water activity around 0.85–0.90 (Borges 11 free of microorganisms—or at least they are unde- et al. 1999). The fermentation process is also variable 12 tectable or at extremely low populations (Mackey and can be conducted by the meat natural flora or by 13 and Derrick 1979). Once the animal is slaughtered, the addition of lactic acid starter cultures. The char- 14 however, the internal defense mechanisms (e.g., acteristics of these products make them susceptible to 15 lysozyme, antimicrobial peptides) that combat infec- the survival of L. monocytogenes and, therefore, there 16 tious agents in the living body are lost. Thus the is a potential risk to the consumer’s health. Despite 17 resulting meat becomes exposed to increasing levels the importance of L. monocytogenes in meat prod- 18 of contamination and, depending on various extrin- ucts, there are only a few studies on the occurrence of 19 sic parameters (temperature, packaging, processing this microorganism in fermented sausage. 20 method, etc.), may undergo rapid microbial decay. In addition to Salmonella, Staphylococcus aureus, 21 Unless effectively controlled, the slaughtering process and Clostridium botulinum, the microorganisms 22 may lead to extensive contamination of the exposed mainly studied in these products in the past decades 23 cut surfaces of muscle tissue with a vast array of (Tompkin et al. 1974), L. monocytogenes and 24 gram-negative and gram-positive bacteria as well Escherichia coli O157:H7, are the predominant 25 as fungi (Nychas et al. 2007). Sources of these pathogens currently under study (Hew et al. 2005; 26 microorganisms include the external surfaces of the Naim et al. 2004; Pond et al. 2001; Samelis et al. 27 animal, including the gastrointestinal tract, as well 2005). 28 as the environment (e.g., floors, contact surfaces, 29 knives, hands, etc.) with which the animal had con- HAZARD CHARACTERIZATION— 30 tact at some time before or during slaughter (Nychas DEFENSE MECHANISM 31 et al. 2007). Many slaughter procedures contribute 32 to the contamination as well, i.e., during removal of Although raw meat is an ideal medium for the growth 33 the hide/fleece of cattle/sheep, during dehairing of of many organisms due to richness in low molecular 34 pigskin, and during evisceration (Grau 1986). As a compounds—e.g., fermentable carbohydrates, pro- 35 consequence, pathogenic bacteria are present on the teins, peptides, amino acids, and other plentiful 36 meat products thereof. For example, testing of the growth factors such as vitamins and minerals—meat 37 microbiological quality of ready-to-eat dried and fermented products have a less favorable matrix for 38 fermented meat and meat products performed on bacterial growth, due to dynamic changes of pH and 39 2,981 samples sold at retail in the U.K., Wales, and aw as a consequence of the development of a micro- 40 Northern Ireland revealed that Staphylococcus bial association dominated by lactic acid bacteria. 41 aureus and Salmonella were present at 1.18% and Indeed, the pH drop, reduction in aw values, and bac- 42 0.06%, respectively (Little et al. 1998). In processed terial activity (e.g., competition and production of 43 meat products, including ready-to-eat food, the con- specific metabolites) are the important factors in 44 tamination ranges from 8–92% (Borges et al. 1999). the control of the development or die-off of patho- 45 In a similar survey in Italy, 5.3% of samples were genic organisms. However, many pathogens such as 46 found to be positive for Salmonella; in the U.S., it L. monocytogenes, E. coli O157:H7, Salmonella 47 was found that the prevalence in small-diameter spp., and Staphylococcus aureus have been reported 48 cooked sausages was 0.20% and the cumulative to survive or even grow in such unfavorable condi- 49 3-year Salmonella prevalence for dry and semidry- tions (Table 42.1). The ability of these pathogens to 50 fermented sausages was 1.43% (Levine et al. 2001). survive in acidic foods has been recently studied. It 51 For L. monocytogenes in dry and semidry- needs to be noted, however, that the effect of structure 52 fermented sausages, in the same study, the preva- and the physicochemical characteristics (buffering 53S lence was 3.25%; none of the RTE products tested capacity, local pH gradients, and nutrient availability 54N positive for E. coli O157:H7 or staphylococcal or diffusion etc.) of the fermented sausages have not 40454 42 427-454 r1.qxd 7/20/07 5:11 PM Page 429

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Table 42.1. Pathogenic bacteria commonly found environment. In humans, infections most often 1 on meats. affect the pregnant uterus, the central nervous sys- 2 tem, or the bloodstream. Symptoms vary, ranging 3 Type of Muscle Meat from mild flulike symptoms and diarrhea to life- 4 Bacteria Gram Rxn Fresh Processed threatening infections characterized by septicemia 5 and meningencephalitis. In pregnant women, the 6 ϩ Bacillus XXinfection spreads to the fetus, which will either be 7 Campylobacter –X born severely ill or die in the uterus, resulting in 8 ϩ Clostridium X abortion. Although human infections are rare, it is 9 Escherichia –X worth to note that is considered to be of the high 10 Klebsiella –X mortality type. 11 Kluyvera –X Listeriosis is an important disease in Europe due 12 ϩ Listeria XXto high morbidity and mortality in vulnerable popu- 13 Pseudomonas –XXXlations, although it remains a relatively rare disease 14 Salmonella –X in the EU. Meat and meat products (e.g., sausages) 15 ϩ Staphylococcus XXthat are contaminated with more than 100 L. mono- 16 Vibrio –X cytogenes bacteria per gram, and that are to be 17 Yersinia –X consumed without further heat treatment, are con- 18 X ϭ known to occur; XX ϭ most frequently isolated. sidered to form a direct risk to human health. These 19 Based on Nychas et al. 2007. food categories (ready-to-eat meat products) have 20 been typically identified as risk products for contam- 21 ination with Listeria. Member states in the European 22 Community have been instructed to report the 23 been taken into account in microbial safety risk presence of Listeria. So far comparison between 24 assessment of these products. data from different countries is difficult due to 25 differences of sample sizes and testing protocols. 26 However, it is generally considered that concentra- 27 ESCHERICHIA COLI tions of L. monocytogenes greater than 100 cfu/g are 28 Strains of the bacterium E. coli capable of producing required to cause human disease in healthy popula- 29 certain cytotoxins are reported as Verotoxigenic tions; therefore, qualitative results alone are not 30 Escherichia coli (VTEC). Enterohemorrhagic necessarily an indicator of risk (EU Regulation 31 E. coli, commonly referred to as EHEC, are a subset 2073/2005). 32 of the VTEC, harboring additional pathogenic fac- 33 tors. More than 150 different serotypes of VTEC 34 SALMONELLA SPP. have been associated with human illness; however, 35 the majority of reported outbreaks and sporadic Salmonella has long been recognized as the most 36 cases of VTEC infections have been attributed to important zoonotic pathogen of economic signifi- 37 serotype O157. There is a wide spectrum of symp- cance in animals and humans. 38 toms associated with VTEC infections, ranging from Human salmonellosis is usually characterized by 39 mild to bloody diarrhea, often accompanied by acute onset of fever, abdominal pain, nausea, and 40 severe abdominal cramps but usually without fever. sometimes vomiting. Symptoms are usually mild and 41 VTEC infection can also result in hemolytic uremic most infections are self-limiting, lasting a few days. 42 syndrome (HUS). HUS is characterized by acute However, in some patients, the infection may be more 43 renal failure, anemia, and lowered platelet counts. serious and the associated dehydration can become 44 HUS develops in up to 10% of patients infected with life threatening. In these cases, as well as when Sal- 45 VTEC O157 and is the leading cause of acute renal monella causes bloodstream infection, effective 46 failure in young children. antimicrobials are essential for treatment. Salmonel- 47 losis has also been associated with long-term and 48 sometimes chronic sequelae, e.g., reactive arthritis. 49 LISTERIA MONOCYTOGENES There are numerous foodborne sources of Salmo- 50 The genus Listeriae comprises six species, but nella, including a wide range of domestic and wild 51 human cases are almost exclusively caused by the animals and variety of foodstuffs (meat and meat 52 species Listeria monocytogenes. Listeriae are ubiq- products, including sausages). Transmission often S53 uitous organisms, which are widely distributed in the occurs when organisms are introduced in food N54 40454 42 427-454 r1.qxd 7/20/07 5:11 PM Page 430

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1 preparation areas and are allowed to multiply in inhibitory ecosystem for most bacterial pathogens, 2 food, e.g., due to inadequate storage temperatures or thereby transforming the perishable and hazardous 3 because of inadequate cooking or cross-contamination raw materials into products with extended keeping 4 of cooked food. The organism may also be transmitted quality and reduced risk of causing illness. The over- 5 through direct contact with infected animals and all antimicrobial effect elicited in fermented meats is 6 fecally contaminated environments. the sum of multiple hurdles of microbial (organic 7 acids, ethanol, and bacteriocins) or nonmicrobial 8 (reduced a , nitrites, nitrates, spices, phenolics, and STAPHYLOCOCCUS AUREUS w 9 heating temperature) origin. However, the manufac- 10 Staphylococcus aureus is of significant practical turing of traditional fermented products is character- 11 importance in meat. Not only can this bacterium ized by considerable variability in raw materials, 12 cause a variety of infections in meat animals, as well operation units, fermentation, and/or ripening condi- as in humans, but it is also the causative agent of a 13 tions. This in turn is reflected in the final pH and aw 14 major form of food poisoning. For example, in of the produced sausages, which have been reported 15 the U.S. during the period 1975–1979, 540 food- to vary from 4 to 7 and from <0.60 to >0.95, respec- 16 poisoning outbreaks were reported, with Staph. tively (Little et al. 1998). 17 aureus responsible for 28% (153 outbreaks). Misuse Starting in 1950, the outcome of numerous studies 18 of foods in food service operations seems to be the was the establishment of a new fermentation tech- 19 major cause of outbreaks, followed by mishandling nology involving the application of starter cultures 20 in the home. comprised of lactic acid bacteria (Incze 1998). Since 21 Bad practices during manufacturing, especially then, a variety of starter cultures has been evaluated 22 poor conditions of fermentation and poor conditions and used in practice, including acidifying cultures, 23 of storage, have led to food-poisoning outbreaks from aroma-forming cultures, nitrate-reducing starters 24 products, especially those that are consumed raw and even mold starters (Incze 1998). The application 25 (Nychas and Arkoudelos 1990; Pullen Genigeorgis of starter cultures was a “triumphal” step in the man- 26 1977). The occasional outbreaks of Staph. aureus ufacturing of fermented products, because it assured 27 food poisoning, which appear to be more common in safer products, especially those with short ripening 28 the USA and Canada than in Europe, have led to time. Among the antimicrobial factors associated 29 studies of the ability of Staph. aureus to grow in fer- with LAB, such as bacteriocins, hydrogen peroxide, mented sausages (Nychas and Arkoudelos 1990; 30 CO2, diacetyl, low redox potential, crowding low 31 Pullen and Genigeorgis 1977; Metaxopoulos et al. pH, and organic acids (Adams and Nicolaides 1997), 32 1981a,b). the main factors are organic acids and low pH. Com- 33 Illness results from the ingestion of water-soluble, monly, the level of organic acids, mainly lactic acid, 34 heat-stable enterotoxins (Minor and Marth 1972a,b) produced by lactic acid bacteria during fermentation 35 secreted by the staphylococcal cells. Secretion of exceeds the 100 mM (1% v/v), whereas the ultimate 36 these enterotoxins occurs at different periods of the pH is often in the range of 3.5–4.5. Another feature 37 growth phase, either as primary or secondary that is required so that LAB may elicit their antimi- 38 metabolites (Minor and Marth 1972a). crobial effectiveness is their numerical superiority 39 Among these many ubiquitous organisms, over any pathogens present. 40 Clostridium botulinum will inevitably be present However, despite the increase in safety level 41 from time to time (Table 42.1). However in this conferred by starter cultures and the manufacturing 42 overview the hazard of C. botulinum will not be process itself, there are still emerged or “developed” 43 discussed. pathogens that are capable of combating the inhibitory 44 environment of fermented meats ecosystems. The 45 EXPOSURE ASSESSMENT dominant pathogens that have been reported to be 46 presently capable of surviving during storage of fer- 47 Although it is well established that the majority of mented sausages include Salmonella, enterohem- 48 pathogens may gain access to the foods as a result of orrhagic Escherichia coli (EHEC), Staphylococcus 49 contamination during processing and storage, raw aureus, and Listeria monocytogenes (Incze 1998; 50 materials constitute the principal source of hazards Adams and Nikolaides 1997). Therefore, knowledge 51 (Adams and Mitchell 2002). Meat fermentation may of the ability of pathogens to survive and proliferate in 52 be viewed as a process that takes potentially haz- fermented meats is an important prerequisite in order 53S ardous raw materials and, through a two-stage to achieve the food safety objectives related to these 54N process of fermentation and ripening, develops an specific food ecosystems in a reliable and consistent 40454 42 427-454 r1.qxd 7/20/07 5:11 PM Page 431

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manner. Such knowledge may derive from challenge Hinkens et al. 1996), which were further established 1 tests of pre- and post-process inoculation of pathogens as lethality performance standards (USDA 2001). 2 in the meat batter and ﬁnal products, respectively. Following the release of these guidelines, research 3 These approaches will allow elucidation of the effect was focused on ﬁnding alternatives in the manufac- 4 of the manufacturing process as well as the combined turing process that will ensure 5-log reductions 5

effect of storage and product pH and aw on the inacti- (Freeman 1996). Among the predominant alterna- 6 vation of pathogens (Incze 1998). Moreover, these data tives was a heating step at 63°C for 4 minutes as well 7 allow for the development of predictive models that as combined antimicrobial treatments (Hinkens et al. 8 mathematically describe the inactivation of pathogens 1996). Ellajosyula et al. (1998) showed that fermen- 9 in fermented sausages (Pond et al. 2001). However, it tation of Lebanon bologna to pH 4.7 followed by 10 is expected that survival responses of pathogens will gradual heating to 48.9°C, in 10.5 hours is sufﬁcient 11

vary depending not only on the manufacturing process to destroy 5-log10 CFU of E. coli O157:H7. How- 12 and product characteristics, but also on whether inocu- ever, despite the indisputable effectiveness of cook- 13 lation is carried out in the meat batter or the ﬁnal ing in reducing pathogens, it is against the traditional 14 products, because the pathogens will be exposed to character of some dry-fermented sausages, which 15 different stresses. By way of example, Shadbolt et al. are known as raw products, whereas in some cases 16 (2001) demonstrated that exposure of E. coli to lethal cooking to an internal temperature of 63°C leads to 17 pH (pH 3.5) followed by exposure to lethal NaCl unacceptable products (Calicioglu et al. 1997, 2002). 18

(10%, aw 0.90), a sequence of stresses simulating the Thus, in order to evaluate the eligibility of the target 19 order of stresses encountered during fermentation of 5-log reduction, many challenge studies were per- 20 and ripening, is more deleterious than the reversed formed to validate various manufacturing processes, 21 sequence, or the simultaneous exposure to both as well as to ensure that storage of the ﬁnal products 22 stresses, a situation that simulates postfermentation may also contribute to the inactivation of a pre- or 23 inoculation. Thus, in order to conduct reliable expo- postprocess contamination (Table 42.2). Calicioglu 24 sure assessment reports related to fermented products, et al. (2001) evaluated the survival of E. coli 25 the need for challenge testing is intensive. A review 0157:H7 during manufacturing of semidry Turkish 26 of the reports on the survival/growth of the major soudjouk fermented with a commercial Pediococcus 27

pathogens in a variety of fermented dry or semidry and Lactobacillus starter culture (pH 4.48–4.55, aw 28 sausages is presented in the next paragraphs. It needs 0.88), or naturally fermented (pH 5.28–5.44, aw 29 to be noted that literature reports have focused consid- 0.90–0.91), without cooking or cooked at 54°C for 30 erably on EHEC and L. monocytogenes due to the 0–60 minutes and subsequently stored under vac- 31 severity of these pathogens and their ability to survive uum at 4 or 21°C. They found that cooking reduced 32 at extreme environmental conditions. E. coli O157:H7 to undetectable levels in sausages 33 fermented with starters, while it caused only 2–3 logs 34 in naturally fermented products. At both tempera- 35 ESCHERICHIA COLI O157:H7 tures, the population of E. coli O157:H7 that had 36 Enterohemorrhagic E. coli is considered highly survived the manufacturing process (without cook- 37

sensitive to the low pH and aw of dry-fermented ing) was further reduced by 1–4 logs within 28 days 38 sausages. Moreover, the presence of nitrite further of storage, while higher reduction was achieved at 39 contributes to the reduction of this bacterium. There- 21 compared to 4°C. Higher log reductions of E. coli 40 fore, once good manufacturing practices are in O157:H7 during storage occur in sausages fer- 41 effect, presence of E. coli O157:H7 in ﬁnal products mented with starter cultures compared to naturally 42 should be avoided (Incze 1998). However, E. coli fermented sausages, apparently due to the lower pH 43

O157:H7 was implicated in an outbreak involving and aw sustained with starters (Calicioglu et al. 2001, 44 consumption of presliced dry-fermented salami 2002). Note, however, that the potential of starters to 45 (CDC 1995a) with an estimated dose of less than 50 produce bacteriocin does not inﬂuence the gram- 46 cells (Tilden et al. 1996). A year later, another E. coli negative EHEC, as is the case with gram-positive 47 outbreak was linked to mettwurst, an uncooked pathogens, such as L. monocytogenes (Lahti et al. 48 semidry-fermented sausage in Australia (CDC 2001). The majority of studies on prefermentation 49 1995b). These two outbreaks prompted U.S. food inoculation of E. coli O157:H7 have concluded that 50 authorities to develop guidelines through which fer- the reduction of the pathogen caused by the manu- 51 mented sausage manufacturers were required to facturing process of fermented sausages, including 52 exhibit the 5-log unit reduction in the enterohemor- summer sausage, Turkish soudjouk, and local S53 rhagic E. coli during processing (Reed 1995; dry-fermented sausages, with pH 4.5–5.0 and N54 40454 42 427-454 r1.qxd 7/20/07 5:11 PM Page 432

1 2 10 3 10 10

4 Ϯ 1 Log = = = Ͻ 91.14– dependent, 17–21.9 min strain dependent, .18–2.21 min strain dependent reduction CFU/g reduction Log Log after 35d, LI: after 35d, not detected after 7 or 22d CFU/g after processing 116.93 min strain

5 50 55 60 CFU/g

6 D D 7 8 9 10 nd nd Present at nd nd 2.13 11 Conditions Ϯ 12 Ϯ

13 / T°C/ Pathogen w 14 0.90 15 16 17 Ϯ 18 19 20 21 22 23 24

25 tation Drying a 26 / Fermen- of Storage

27 Ϫ Ϫ 28 3 2 29

30 meat products. O157:H7 in traditional 31

32 E. coli 33 34 35 Process Conditions Product Final 36 nd nd nd nd ndY 4.7–5.1 nd N ndY nd D Y Y 24nd Y 14 nd 18–23 4.9 nd 15 nd 4.5–4.7 24.7 nd 4.5 nd 7,22 A HI: 2 37 10 10

38 6 39 10 –10 CFU/g 1.36 40 3 6–7 Log 41 Concen- Starter NO 42

43 ¥ nd nd 7.3 Log nd 6 Log nd 10 44 valence Data (Y/N) (Y/N) (Y/N) T°C T°C pH RH % Packaging Response 45 § 46 b a a b 47 48

49 of survival, and growth Data related to prevalence, 50 (matrix) CFU/g (sliced) 25 g sausages CFU/ 51 sausage CFU/g 0.06 0.01 0.08

52 of Type of Type Pre- tration Cultures Sugars NO 53S 2Dry C 2Dry 3 Salami C 4 C Fermented 1 Salami C

54N 42.2. Table a/a Sausages Study

432 40454 42 427-454 r1.qxd 7/20/07 5:11 PM Page 433

) 1

10 2 CFU/g CFU/g CFU/g CFU/g 3 10 10 10 10 10 = 4 Continues 13 = 21.5d, = ( reduction 12.05d 21.65d CFU/g depending on cooking Log reduction reduction depending on cooking reduction depending on starter culture, LI: 1 bellow Log 21d reduction after 14 or

3.6 3.6 5 6 7 8 9 13 D 10 4,21 V 4.0–7.0 4,22 V 1.7–4.7 11

Ϯ Ϯ 12 13

0.88 0.88 nd 4 V ~2 Log 14 15

Ϯ Ϯ 16 Ϯ 0.026 0.10.011 0.1 Log Log 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Y YN Y N 24Y/N Y 22 Y 24 4.55 Y 22 nd 5.48 nd 4.4 nd ndY nd ndY nd nd Y nd 37.7 4.4–4.6 Y nd nd 24 4.4–4.6 3.6, V nd D 17 3.6 V 5.0–5.2 0.90 D nd nd ~2.5–3.0 36 10 10 37

10 10 38 10 Ϯ Ϯ Ϯ 39 CFU/g CFU/g 40 41 42 43

nd 7 Log nd 7.5 Log nd 4.5 Log nd 7.65 nd 7.65 nd 4.68 nd 2.30– Y Y N 20–23 17 4.4–4.6 0.76–2.79– 44 15–17 A HI: 45 b a/b a a/b a/b a/b a/b 46 47 48 49 50 sausage CFU/gg slices luncheon bologna CFU/g bologna CFU/g bologna (model sausages) fermented 0.02 log log fermented 0.02 sausage 0.01 CFU/g CFU/g soudjouksoudjouk 0.04 Log 0.04 Log fermented 5.68 log 0.87 4.84 Log sausage CFU/g 51 52 S53 6 Lebanon C 7Dry C 7Dry 9 C Turkish 5 Lebanon C 8 C Turkish 10 Dry- C 11 Dry- C N54

433 40454 42 427-454 r1.qxd 7/20/07 5:11 PM Page 434

2 10 C,

3 °

4 C ° after 60, 90d detected ~3.5 Log 5 CFU/g reduction depending on inoculum history at 5 6 7 8 9 10 11 Conditions 12

13 / T°C/ Pathogen w 14 15 16 17 18 19 20 21 22 23 24

25 tation Drying a 26

27 / Fermen- of Storage Ϫ Ϫ 28 3 2 29 30 31 32 33 34

35 Process Conditions Product Final

36 N NY N Y Y 22–24 22–24 4.86–4.9 nd 31– 4 nd A/V Not 4.63– 0.90–Y 5,20 A Y Not detected nd 30,37 nd 4.0–4.2 nd nd nd Not 37 38 5

39 –10 5 2 5 40 Concen- Starter NO 41 42 43 nd 10 nd 10 nd 10 44 valence Data (Y/N) (Y/N) (Y/N) T°C T°C pH RH % Packaging Response

45 § ) 46 a/b b a 47 48

49 Continued ( 50 soudjouk(lumberjack CFU/ml CFU/gminced 4.8620 0.95 CFU/g at 32.5% detected meat, fermented meat cubes semidry in 25g25gsemidry in sausage fermented in sausage 0.94 0.94 51 beef roll)

52 of Type of Type Pre- tration Cultures Sugars NO 53S 13 Salami C Table 42.2. Table a/a Sausages Study 12 C Turkish 54N 14 Dry/15 P Spreadable P16 0% 0% C Fermented Absence Absence nd nd nd nd nd nd nd nd nd nd 4.0–6.0 4.0–6.0 0.85– 0.85– nd nd nd nd nd nd

434 40454 42 427-454 r1.qxd 7/20/07 5:11 PM Page 435

1 2 3

detected at 4°C, not detected at 20°C, LI: not detected at 4, 20°C 4 5 6 7 8 9 10 11

1); Erkkila et al. (available at 2000); 1); Erkkila et al. (available 12 13

: preprocessing inoculation (challenge) and 14 a/b 15 al. (1998); Sakhare and Rao (2003); Nissen 16 17 18 19 20 21 22 23 24 25 26 27

: postprocessing inoculation (challenge); C 28 b 29 30 31 32 33 34 35

Y nd nd 27 14 nd nd 4,20 V HI: 36 10 37

7 38 39 –10 1.25 Log 3 CFU/g 40 41 42 43

nd 10 44 45

a/b 46 : preprocessing inoculation (challenge) without storage; C a 47 48 49 50

sausage CFU/g sausage CFU/g– 51 52 S53 nd: no data, HI: high inoculum; LI: low inoculum. nd: no data, HI: high inoculum; LI: low P: prevalence study; C P: prevalence A: aerobically packaged; V: vacuum packaged. vacuum V: packaged; A: aerobically et al. (1999); Naim (2006); Samelis (2005); Uyttendaele (2001); Chikthimmah and Knabel (200 Based on Duffy Calicioglou et al. (2001); Glass et al. (1992); Lahti et al. (2001); Cosansu and Ayhan (2000); Rocelle et al. (1996); Little Ayhan et al. (2001); Glass (1992); Lahti Cosansu and Calicioglou Holck (1998); Rantsiou et al. (2005). 17 Dry C § ¥ 18 S-NP Fermented nd <10 N Y Y nd 0–14 5.62–5.70 nd ndCFU/g nd <10 storage; S-NP: survival study on naturally contaminated products. study on naturally storage; S-NP: survival N54

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436 Biological and Chemical Safety of Fermented Meat Products

1 aw 0.80–0.90 does not exceed the 2–3-log units sensitive than nonadapted cells when heated 2 (Glass et al. 1992; Faith et al. 1998a,b; Calicioglu (55–62°C) in pepperoni, whereas higher susceptibil- 3 et al. 1997, 2001, 2002; Nissen et al. 1998; Cosansu ity to heat was observed in postfermentation inocu- 4 and Ayhan 2000; Erkkilä et al. 2000; Chiktimmah lation (pH 4.8) compared to prefermentation 5 and Knabel 2001; Lahti et al. 2001; Naim et al. inoculation (pH 6.7). Clavero and Beuchat (1996) 6 2003). In contrast, all the above studies agree that investigated the survival of heat-stressed (52°C, 7 signiﬁcant reductions of E. coli O157:H7 are sus- 30 min) and nonstressed E. coli O157:H7 popula- 8 tained during subsequent storage of products for 1 to tions onto salami slices stored at 5 and 20°C for 9 6 months, especially at higher temperatures. In gen- 32 days. They found that heat stressed E. coli 10 eral, the reduction in pathogen numbers achieved by O157:H7 reduced by 4 logs after 8 days at 20°C and 11 storage at ambient temperatures is greater than that by 3 logs after 32 days at 5°C, while the nonstressed 12 achieved by storage at cooler temperatures. cells reduced by 4 and 2 logs at 20°C and 5°C, 13 Furthermore, given the high acid resistance of respectively, after 32 days. 14 some EHEC strains at pH 2.5–4.0 (Buchanan and Another useful insight was provided by Naim 15 Endelson 1996; Zhao et al. 1993), the survival of et al. (2004) who studied the survival of E. coli 16 E. coli inoculated postfermentation has been an O157:H7 during an in vitro challenge in a model 17 important area of investigation. Chiktimmah and system, which comprised of synthetic saliva (pH 6.7), 18 Knabel (2001) noted a 7-log reduction of E. coli synthetic gastric ﬂuid (pH 2.5), and synthetic 19 inoculated postfermentation on sliced vacuum- pancreatic juice (pH 8.0). E. coli O157:H7 was inoc- 20 packed Lebanon bologna (pH 5.0), fermented with ulated during meat batter preparation, survived 21 starters (pH 4.4–4.6), after 55 and 65 days of storage the manufacturing process, and then was exposed 22 at 13 and 3.6°C, respectively. Conversely, when to the GI tract model under static or dynamic pH 23 E. coli O157:H7 was inoculated in the meat batter conditions. It was found that the population of 24 (prefermentation inoculation) the total reductions pathogen reduced by 2 logs during fermentation and

25 obtained after fermentation and storage for 90 days ripening (final pH 5.0 and aw 0.90), it remained 26 did not exceed the 4-log units, suggesting that the stable throughout 120 minutes of exposure to gastric 27 gradual reduction of pH during fermentation acidity (pH 2.5), and showed hundred- or thousand- 28 induced acid resistance, which enhanced survival of fold increase during subsequent exposure to pancre- 29 pathogen during subsequent storage (Buchanan and atic juice (pH 8.0), depending on the duration of 30 Edelson 1998). Uyttendaele et al. (2001) illustrated previous exposure to gastric fluid. Finally, Duffy 31 that the lower are the levels of E. coli O157:H7 inoc- et al. (1999) evaluated the thermo-tolerance of three 32 ulated postfermentation on fermented sausage E. coli O157:H7 meat product isolates, injected 33 stored at 7 and 22°C the higher is the inactivation. (109 CFU/g) in the core of dry salami. The inocu- 34 This conclusion is also consistent with the findings lated salami samples were heated at 50, 55, and 35 of Nissen and Holck (1998) and Lahti et al. (2001). 60°C. The D values of the three isolates ranged from 36 It is notable however, that Nissen and Holck (1998) 91 to 110 minutes at 50°C, 17–22 minutes at 55°C, 37 observed an initial growth of the low inoculation and 1.1 to 2.2 minutes at 60°C. 38 level of E. coli O157:H7 at the early stages of 39 fermentation followed by rapid decrease to unde- LISTERIA MONOCYTOGENES 40 tectable levels. 41 Of high concern is also the survival of stress- Although L. monocytogenes is among the most fre- 42 adapted EHEC cultures on the final products, con- quently detected pathogens in fermented sausages 43 sidering potential cross-contamination scenarios by reaching prevalence levels of up to 30% (Levine 44 cells that are habituated in acidic or osmotically et al. 2001; Thévenot et al. 2005a), critical listeriosis 45 stressful niches within a plant producing dry- cases have yet to be linked to the consumption of fer- 46 fermented sausages. Leyer et al. (1995) inoculated mented sausages contaminated with L. monocyto- 47 (104–105 CFU/g) meat batter at pH 5.8 before fer- genes (Nightingale et al. 2006). So far, there is only 48 mentation for 15 hours at 32°C and shredded dry one epidemiological association of L. monocyto- 49 salami (pH 5.0) stored at 5°C for 5 days with a com- genes with salami, in Philadelphia, in the U.S, but no 50 posite of five acid-adapted or nonadapted E. coli confirmed outbreak due to the consumption of such 51 O157:H7 strains. Acid-adapted cultures were capable product with Listeria is reported (Moore 2004). 52 of surviving longer than nonadapted cultures in However, L. monocytogenes is ambiguous in nature 53S both foods. On the other hand, Riordan et al. (2000) (Tompkin 2002) and is thus considered a common 54N found that acid-adapted E. coli O157:H7 were more contaminant, which is highly difficult to control in 40454 42 427-454 r1.qxd 7/20/07 5:11 PM Page 437

Pathogens: Risks and Control 437

food-processing environments due to its high toler- sausages has been produced in the last decade 1 ance to low pH and high salt concentration (Farber (Table 42.3). The ﬁndings of these studies are 2 and Peterkin 1991). Therefore, it is likely that this reviewed in the next paragraphs. 3 bacterium may also survive the common manufac- Glass and Doyle (1989) reported a greater than 4 turing technologies of dry-fermented products, as 4 log units reduction in L. monocytogenes during fer- 5 has been the case with fermented dairy products mentation and drying of pepperoni fermented with 6 (Gahan et al. 1996). In this respect, legislation starter cultures. Farber et al. (1993) inoculated 7 authorities, such as the U.S. Department of Agricul- L. monocytogenes (approximately 104 CFU/g) in 8 ture (USDA) have published rules and guidelines to three types of sausages: German-style smoked 9 control the speciﬁc pathogen in fermented meats sausage fermented with Pediococcus pentocaceus 10

regarded as ready-to-eat foods (USDA 2003, 2004; starter (pH 4.8) and dried to aw 0.80, American-style 11 Ingham et al. 2004). By way of example, the reduc- smoked sausage fermented with Pediococcus acidi- 12

tion of pH due to the fermentation process, the lactici starter (pH 4.6–4.7) and dried to aw 0.87–0.89, 13 reduction of water activity via drying, and the depo- and naturally fermented Italian-style sausage 14

sition of inhibitory compounds via smoking are con- (pH 4.9–5.5) dried to aw 0.88–0.89. After drying, 15 sidered antimicrobial processes. These processes, which lasted 42 days for all three types of products, 16 according to the compliance guidelines (USDA all sausages were stored at 4°C for 70 days. In 17 2004), should not allow more than 1-log increase of sausages fermented with starters (i.e., American and 18 L. monocytogenes throughout the shelf life of the German-style), the population of L. monocytogenes 19 treated products. Moreover, these guidelines state reduced by 2–3 logarithms by the end of fermenta- 20

that aw <0.92, a relatively high water activity com- tion; smoking, drying, and storage caused another 21 pared to that of dry sausages, or pH <4.39, a mar- 1–2-log reduction, which dropped the pathogen close 22 ginal pH achieved after fermentation depending on to the detection limit. In contrast, in the Italian-style 23 the use of fermentable carbohydrates and starter sausage, L. monocytogenes showed slight growth 24 cultures, are the lowest levels permitting growth of during fermentation, remained constant during dry- 25 L. monocytogenes based on scientific evidence. ing, and reduced slightly during a 4-week holding 26 Moore (2004) summarized the guidelines for micro- period. Campanini et al. (1993) investigated the 27 biological quality of fermented meats and in the case behavior of L. monocytogenes (103–104 CFU/g) dur- 28 of L. monocytogenes, acceptable fermented meat ing maturation (49 days at 14–18°C) of Italian salami 29 products are considered those that contain equal to (pH after maturation 5.5–5.8) without starters, with a 30 or less than 100 CFU/g of L. monocytogenes, consis- bacteriocin-producing Lactobacillus plantarum, or 31 tent to some extent with the new EC regulation with a nonbacteriocin-producing variant of L. plan- 32 2073/2005 for microbiological criteria in foods. tarum. In sausages without starters, L. monocytogenes 33 However, in contrast to E. coli O157:H7, there are showed considerable growth throughout maturation 34 no lethality performance standards proposed for period. In sausages with bacteriocin-negative starter, 35 this bacterium in fermented meat products. In this survival without reduction of L. monocytogenes was 36 context, Ingham et al. (2004) screened 15 different observed, whereas significant decrease in numbers 37 fermented meats packed in vacuum or air for their of the pathogen were noticed in sausages with the 38 ability to inactivate postfermentation inocula of bacteriocin-positive starter. Similar findings in rela- 39 L. monocytogenes during storage at 21 or 4°C for tion to the effect of bacteriocin-producing starters on 40 11 weeks. The pH of the products ranged from 4.7 to prefermentation inoculation of L. monocytogenes in 41

5.6 and aw from 0.91 to 0.98; products with 50–60% dry sausage (pH 4.5, aw 0.75–0.87 after 49 days of 42 water phase salt (aw 0.27 and pH above 6.0) were also fermentation-drying) have been reported by Lahti 43 evaluated (Table 42.3). The results of the study et al. (2001), too. However, the inadequacy of Italian- 44 showed that inactivation of L. monocytogenes was style salami to reduce L. monocytogenes was also 45

higher on products with lower pH and aw as well as at confirmed by the challenge studies by Nightingale et 46 ambient temperatures. The authors recommended al. (2006). The application of bacteriocins as dipping 47 1-week postpackaging storage at room temperature solutions has also been evaluated as an alternative 48 prior to shipment to ensure elimination of the preservation and safety intervention following the 49 pathogen (Ingham et al. 2004) and challenge testing ripening and slicing of fermented sausages. Mattila 50 to validate various manufacturing processes for the et al. (2003) reported that immersion of sliced 51 lack of growth of L. monocytogenes. However, a sig- cooked sausages, bearing more than 2 logs of 52 nificant body of scientific evidence related to the sur- L. monocytogenes, into a pediocin preparation 0.9% S53 vival of L. monocytogenes in various dry-fermented reduced L. monocytogenes to undetectable levels N54 40454 42 427-454 r1.qxd 7/20/07 5:11 PM Page 438

1 2 3 10 10 10 10 2.90 Log 2.98 Log 1.54 Log 4 CFU/g CFU/g CFU/g CFU/g reduction depending on irradiation 5 6 7 8 9

10 Conditions nd nd 3.9–1.7 11

12 Ϯ 0.90 / T°C/ Pathogen

13 w 14 15

16 meat products. (7Ð52) in traditional 17 18 19 20 21 22 23 24 Listeria monocytogenes

25 tation Drying a 26 / Fermen- of Storage

27 Ϫ Ϫ 3 2 28 29 spp. and (1Ð6) 30 31

32 Listeria nd nd nd nd 4.9–6.6 nd nd nd nd 33 34 ¥ 35 Process Conditions Product Final N ndN nd nd ndN nd nd nd ndY 5.19 nd nd Y nd nd 5.10 Y nd nd nd nd 18–23 4.80 Present at 15 nd nd nd 4.5–4.6 Present at 24.7 nd nd Present at 36 37 38 10 10 10 39 10 in 25g in 25g 0.90 40 CFU/g 1.36 Log 41 Concen- Starter NO 42 43 nd 6 Log 44 valence Data (Y/N) (Y/N) (Y/N) T°C T°C pH RH % Packaging Response

45 § 46 a 47 48

49 of survival, and growth Data related to prevalence, 50 picantedulcedulce CFU/g CFU/g CFU/g sausage 51 salami in 25g 0.90

52 of Type of Type Pre- tration Cultures Sugars NO 53S 2 Salami3 Chorizo S-NP P ndLog 14.8% 3.36 Presence N nd nd nd nd 4.8–5.2 0.85– nd nd nd 4 Chorizo S-NP ndLog 5 3.57 Chorizo S-NP ndLog 6 1.17 Salami C 7 P Fermented 10%CFU/g <3 Y Y N 22–24 12 4.7–5.4 0.78– nd nd nd 1 Soudjouck P 9% Presence nd 8 Italian P 13.3% Presence nd nd nd nd nd 4.8–5.2 0.85– nd nd nd

54N 42.3. Table a/a Sausages Study

438 40454 42 427-454 r1.qxd 7/20/07 5:11 PM Page 439

) 1 2 3 4 Continues ( 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 in 25g in 25g in 25g CFU/g in 25g in 25g 40 41 42 43 44 45 46 47 48 49 50

sausages in 25g 25g sausages in 25g sausagessausages25g sausages in sausages in 25g in 25g in 25g fermented in sausage fermented in sausage sausage,sliced sausage in 25g in 25g 51 52 S53 9 Soudjouck P 7% Presence nd nd nd nd nd 4.9–6.7 nd nd nd nd

10 P Fermented 11 Fermented 3.25% P12 Presence Fermented nd P13 20% P Fermented nd Presence 14 19.05% Presence P Fermented nd 44% nd15 nd Presence Sausages 20% nd nd16 nd P nd Presence P Spreadable nd nd nd nd 25%17 nd 11.30% P Sliceable Presence nd nd Presence nd nd nd18 nd 4.8% nd nd nd Salami nd nd Presence19 nd nd nd nd P Raw P nd nd nd nd nd nd20 nd 1.59% 16.67% nd Salsiccia P nd nd nd <100 nd nd Presence21 nd nd nd nd P Fermented nd nd nd 11.54% nd nd nd22 Presence nd nd nd Salami nd 20% nd nd nd nd nd23 nd nd nd nd Presence Salami nd P nd nd nd nd nd nd nd nd nd P 10% nd nd nd nd nd nd nd nd Presence 16% ndN54 nd nd nd nd nd nd Presence nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd

439 40454 42 427-454 r1.qxd 7/20/07 5:11 PM Page 440

1 2 10

3 10 4 detected after 8d; LI: detected after 8d 5 reduction CFU/g reduction 6 7 8 9 10 11 Conditions 12 13 / T°C/ Pathogen

14 w 15 16 17 18 19 20 21 22 23 24

25 tation Drying a 26 / Fermen- of Storage

27 Ϫ Ϫ 28 3 2 29 30 31 32 33 34

35 Process Conditions Product Final 36 Y YN Y YY 30 Y Y 12 17–22Y Y 17–22 4.3–4.8 5.2–5.5 Y 20–22 0.87– 0.78– 15 nd nd Y nd 5.1–5.2 nd <2.0 Log nd nd Not nd nd nd 4.7 HI: 0.96 5 V 2.7 Log 37 10 10

38 4 5 39 –10 –10 3 3 sample 40 CFU/g in 25g in 25g 41 Concen- Starter NO 42 43 nd 7.4 Log nd 10 nd 10 nd 3.6 Log 44 valence Data (Y/N) (Y/N) (Y/N) T°C T°C pH RH % Packaging Response

45 § 46 ) 47 a a a b 48

49 Continued ( 50 sausage fermented CFU/gsausage fermented CFU/g 0.80 detected salami CFU/gsausage CFU/ 0.95 CFU/g 51 25g sausagesstyle in sausage sausage in 25g in 25g

52 of Type of Type Pre- tration Cultures Sugars NO 53S 32 Spanish-type C 33 Summer C 31 Naples-type C Table 42.3. Table a/a Sausages Study 54N 24 Salami25 Dry cured P26 P P Fermented 5%27 10% Salami 10% 20 nd28 Presence Presence Spanish- P nd nd nd P29 40% P nd Smoked nd nd30 3,70% Presence nd Presence nd Italian-style C 0,00% nd nd nd Presence nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd

440 40454 42 427-454 r1.qxd 7/20/07 5:11 PM Page 441

) 1

4 2

10 10 3 –10 = 10 10 2 = = 13 4 Continues ( 2 .97d CFU/g reduction Log CFU/g reduction Log CFU/g reduction CFU/g reduction CFU/g) after 28d 5.02d, 5.02d, D 5.02

3.6 3.6 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Y Y Y nd ndY 4.8 ndY 0.96 nd 5,21 Y 37.7 VY 2.5 Log nd Y Y 4.4–4.6 24N nd Y 16–18 Y 4.6–5.1 3.6,13 24 V 0.87– Y D 16–18 4 4.6–4.8 16–24 0.80 A 14 Detected 4 4.9–5.5 0.88– A 4 Detected A Detected 36 37 10 10

5 38 10 10 10 39 –10 CFU/g 4 3 5 sample sample sample 40 41 42 43

nd 3.9–4.2 Ynd Y 3.4 Log nd Y 3.1–3.7 ndnd N nd 3.8–4.0Y Y 4.9nd Y Y 6.7 Log 0.95 Ynd 5,21 nd nd V 10 nd ~3.0–3.3 ndnd 5.2 5.2 10 nd 0.95 0.95 5,21 10 Log 5,21 V 2.2 nd V 6.3 log 1.4–3.1 Y nd44 nd 37.7 nd 4.4–4.6 nd 3.6 V D 45 b b b b a/b a/b a/b a/b b 46 47 48 49 50 slices sausage CFU/gAmerican- style sausage CFU/gGerman- style sausages CFU/g28d 0.8828d –0.81 after 0.89 after (10 sausage CFU/ style sausagesummer Log sausage CFU/ CFU/ Italian- sausages) sausage Log summer Log bologna CFU/g bologna CFU/g bologna luncheon (model 51 52 S53 39 C Fermented 40 C Fermented 41 C Fermented 35 Summer C 37 Elk C 36 C Buffalo 38 Lebanon C 42 Lebanon C 34 Summer C N54

441 40454 42 427-454 r1.qxd 7/20/07 5:11 PM Page 442

1 2 10

3 10 10 10 4 CFU/g CFU/g CFU/g reduction CFU/g 5.33 Log Log Log Log Log 5 reduction depending on T°C reduction depending on T°C reduction depending on T°C depending on T°C reduction depending on T°C 6 7 8 9 10 11 Conditions 12 / T°C/ Pathogen

13 w 14 15 16 17 18 19 20 21 22 23 24 tation Drying a

25 / Fermen- of Storage Ϫ Ϫ 26 3 2 27 28 29 30 31 32 33 34 N nd ndN nd nd nd nd 4.9–5.1N 0.85 nd 7,25,30 nd A nd 2.77– nd 4.9–5.1N 0.90 nd 7,25,30 nd A nd 3.0–6.13 ndN 4.9–5.1 0.93 nd nd 7,25,30 A nd nd 2.38–4.62 4.9–5.1 nd 0.95 7,25,30 A nd 1.31–3.14 4.9–5.1 0.97 7,25,30 A 0.68–2.16

35 Process Conditions Product Final 36 10 10 10 10 10

37 Ϯ Ϯ Ϯ Ϯ Ϯ CFU/g CFU/g CFU/g CFU/g CFU/g Log Log Log Log 38 Log 39 Concen- Starter NO 40 41

42 nd 7.51 nd 7.51 nd 7.51 nd 7.51 nd 7.51 valence Data (Y/N) (Y/N) (Y/N) T°C T°C pH RH % Packaging Response 43

44 § 45 a/b a/b a/b a/b a/b 46 ) 47 48

49 Continued ( 50

51 (experimental)(experimental) 1.38 (experimental) 1.38 (experimental) 1.38 (experimental) 1.38 1.38

52 of Type of Type Pre- tration Cultures Sugars NO 53S 43 Chorizo C 44 Chorizo C 45 Chorizo C 46 Chorizo C 47 Chorizo C 54N 42.3. Table a/a Sausages Study

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1 2 3 after 46d after 60d detected after 35–49d; LI: not detected after processing 4 5 6 7 8 9 10 11 12 0.81 detected 13

: preprocessing inoculation (challenge) and 14 a/b

Mena et al. (2004); Villani etVillani Mena et al. (2004); al. (1997); Hugas 15 ); Nissen and Holck (1998); Samelis et al. 16 17 18 19 20 21 22 23 24 25 26 27

: postprocessing inoculation (challenge); C 28 b 29 30 31 32 33 34

Y Y Y 27 14 4.9 0.93 4,20 V Detected 35 36 37 7 10 10 38 –10 3 CFU/g Log 5.1–5.4 CFU/g 39 40 41 42

nd 2.92– Y Ynd Ynd 10 4.0–4.6 Y 20–24 17 Y 4.4–4.6 Y 0.76– 15–17 20–24 A 13–14 HI: 5.8–5.9 0.76– 10Not A 43 44 45

a/b a/b a/b 46 : preprocessing inoculation (challenge) without storage; C a 47 48 49 50 sausage CFU/g dry salami in 25g 30.3% detected sausage sausage 3.35, 0.88sausage detected Log 51 52 S53 nd: no data, HI: high inoculum; LI: low inoculum. nd: no data, HI: high inoculum; LI: low P: prevalence study; C P: prevalence 50 Dry C 51 Greek S-NP52 ndN S-NP Fermented nd Presence Y nd Y N 20–24 15–16 Y 5–5.2 Y 27.7–Not nd nd nd 14 5.62–5.7 nd nd nd Not A: aerobically packaged; V: vacuum packaged. vacuum V: packaged; A: aerobically et al. (1999); Encinas al Samelis (2005). et al. (2006); Borges 1–6: Based on Siriken (2003); et al. (2001); FDA/CFSAN et al. (2006); Levine et al. (1999); Siriken et al. (2005 a,b); Borges Thevenot 7–52: Based on et al. (2002); Ingham et al. (2004); Chikthimmah and Knabel (2001); Farber et al. (1993); Hew et al. (2005); Lahti (2001 et al. (1993); Hew et al. (2002); Ingham (2004); Chikthimmah and Knabel (2001); Farber Rantsiou et al. (2005). 49 Dry C 48 Dry-fermented C § ¥ storage; S-NP: survival study on naturally contaminated products. study on naturally storage; S-NP: survival N54

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444 Biological and Chemical Safety of Fermented Meat Products

1 within 21 days of storage of slices in vacuum packs. salami, Lebanon bologna sausages, and fresh sausages 2 In contrast, L. monocytogenes remained stable on the (Sauer et al. 1997; Nissen and Holck 1998; Moore 3 surface of nondipped samples. Nissen and Holck 2001; Giovannini et al. 2004). 4 (1998) monitored the survival of two inoculation Proposed lethality performance standards for RTE 5 levels of L. monocytogenes (103–104 and 105–107 products by the USDA require a 6.5-log CFU/g 6 CFU/g) during the fermentation, drying, and storage reduction of viable Salmonella populations for ﬁn-

7 of Norwegian fermented dry sausages (pH 4.8 and aw ished products (USDA 2001). However, concern has 8 0.89 after drying) at 4 and 20°C for 5.5 months. Inoc- been expressed for the survival of postprocessing 9 ulation was carried out in the center of stuffed Salmonella contamination, especially on sliced fer- 10 sausages. In accordance to the findings of Farber et al. mented meats, such as Lebanon bologna, as well as 11 (1993), the fermentation caused a limited reduction of in naturally fermented meat products. Thus, valida- 12 2–3-logs in L. monocytogenes numbers, whereas it tion of manufacturing processes and challenge test- 13 was the storage at the ambient temperature that totally ing on finished products for survival of Salmonella 14 eliminated the pathogen as opposed to cold storage. In is essential. For example, Escartin et al. (1999) 15 the study of Encinas et al. (1999), 21 lots of five vari- demonstrated the reduction of native salmonellas, 16 eties of naturally contaminated Spanish chorizo with present at levels up to 900 CFU/g in chorizos, to 17 (pH 4.27–4.57) or without (pH 4.8–5.20) starter cul- undetectable levels during storage at ambient and 18 tures were surveyed for presence and enumeration of cold temperatures. Detailed description of available 19 L. monocytogenes during maturation. The authors con- challenge studies is provided in Table 42.4.

20 cluded that in the naturally fermented chorizo vari- Ellajosyula et al. (1998) demonstrated a 5-log10 21 eties, the initial contamination of L. monocytogenes CFU reduction of S. Typhimurium in cooked Lebanon 22 survived at the same levels throughout maturation, in bologna sausages. In the same product, Chikthimmah 23 contrast to the chorizos fermented with starter cul- et al. (2001) showed that S. Typhimurium exhibited 24 tures, where L. monocytogenes reduced to unde- the most rapid reduction compared to E. coli 25 tectable levels. In another study, Hew et al. (2005) O157:H7 and L. monocytogenes inoculated both pre- 26 performed artiﬁcial contamination of laboratory-made and postfermentation. Likewise, in a comparative 6 27 chorizos of different aw (0.85–0.97) with 10 CFU/g evaluation of the survival of L. monocytogenes, 28 L. monocytogenes and stored them at ambient temper- E. coli O157:H7 and S. kentucky during manufacture 29 atures (21–30°C) and cold temperatures (6–8°C). The and storage of Norwegian dry salami, S. kentucky

30 pH of sausages was adjusted with vinegar and the aw was the most sensitive pathogen, which reduced to 31 by addition of NaCl. It was found that low water activ- undetectable levels after 46 days of storage both at 32 ity and ambient temperatures caused the highest ambient and cold temperatures. The same observa- 33 reductions of the pathogen. Chikthimmah and Knabel tion accounted for two inoculation levels, one low 34 (2001) demonstrated that prefermentation L. mono- (103 CFU/g) and one high (106 CFU/g). The higher 35 cytogenes contamination of Lebanon bologna batter sensitivity of Salmonella to the manufacturing 36 survives longer than postfermentation contamina- process of dry-fermented sausages, in comparison to 37 tion. This was also evident for E. coli O157:H7 (see other pathogens of concern, was also recently

38 above) and Salmonella Typhimurium. However, the observed in Italian-style salami of pH 4.4–4.8 and aw 39 survival potential of L. monocytogenes during fer- of 0.869–0.935 (Nightinghale et al. 2006). However, 40 mentation and drying has also been recognized as in the latter study, lower reductions in Salmonella 41 strain-dependent (Thévenot et al. 2005b). The out- serotype Enteritidis population were obtained in fer- 42 come of the aforementioned studies can be summa- mented nondried sausages than in fermented dried 43 rized from a safety standpoint as follows: the use of products, whereas drying to a 1.4:1 moisture protein 44 starters in combination with good manufacturing ratio (MPR) ensures higher reductions than drying 45 practices, drying to low water activity, and storage of to a 1.9:1 MPR. In contrast to the above studies, 46 sausages at room temperature minimize the risk S. Typhimurium and S. dublin have been reported to 47 of listeriosis by consumption of these products. survive the fermentation and drying process of

48 pepperoni at 5°C with starters (pH 4.5, aw 0.798 49 in the finished product) or without starters (pH 5.0, SALMONELLA SPP. 50 aw 0.805 in the finished product) (Smith et al. 1975). 51 In contrast to L. monocytogenes, Salmonella species A total of a 3-log reduction was achieved in the pres- 52 have been involved in outbreaks linked to the con- ence of starters (Lactobacillus and Pediococcus) 53S sumption of fermented meats and specifically of dry as opposed to only a 2-log reduction in naturally 54N 40454 42 427-454 r1.qxd 7/20/07 5:11 PM Page 445

) 1

(15), 2 3 after detected 42d– LI: not detected after 35d 25g 4 Continues ( 5 S. dublin 6 7 8 (12Ð14), 9

Conditions 10 11 12 30.3 in 25g / T°C/ Pathogen w 13 14 S. tymphimurium 15

(11) 16 17 18 19 20 21 22 23 Salmonella kentucky 24

tation Drying a 25 26 / Fermen- of Storage

Ϫ Ϫ 27 3 2 spp. (1Ð10), 28 29 30 31 Salmonella 32 33 34 Process Conditions Product Final 35 36 37 38 CFU/g Y Y Y 30, 37 nd 4.0–4.2 nd nd nd Not

in 25gCFU/g nd5 5.7039 22 detected detected Presence N Y Y nd nd 5.62– nd nd nd Not Concen- Starter NO 40 41 42 ¥ 43 nd 10 valence Data (Y/N) (Y/N) (Y/N) T°C T°C pH RH % Packaging Response 44

§ 45 46 a 47

(16) in traditional meat products. (16) in traditional 48

Data related to prevalence, survival, and growth of survival, and growth Data related to prevalence, 49 50 sausages in 25g 0.94 minced meat, fermented dry salami fermented sausage meat cubes 51

Type of Type of Type Pre- tration Cultures Sugars NO 52 S. Enteritidis S53 1 Dry/semidry P2 S-NP Fermented 0.08% nd Presence nd nd nd nd nd 4.0–6.0 0.85– nd nd nd 3 Chorizo4 Chorizo P S-NP nd 61.4% nd 0.04–900 nd nd nd nd nd nd nd nd nd nd nd nd nd18–nd nd 5, HI; nd nd nd 5 Soudjouck6 P Fermented P7 Greek S-NP8 nd 1.43% 7% P Spreadable nd nd nd 0% nd nd nd N nd nd Y nd nd nd Y nd nd nd nd 20–24 nd 15–16 nd nd 5.0–5.2 nd 27.7– 4.9–6.5 nd nd nd nd nd nd nd Absence nd nd nd nd nd nd nd ndin Absence 9 C Fermented and Table 42.4. Table a/a Sausages Study N54

445 40454 42 427-454 r1.qxd 7/20/07 5:11 PM Page 446

1 2

3 = = = 13

4 4.99d during production D 4.89d detected culture: not detected after 42d; With natural ﬂora: detected after 42d 5 3.6 3.6 6 7 8 9

10 Conditions 11 12 / T°C/ Pathogen

13 w 14 15 16 17 18 19 20 21 22 23 24

25 tation Drying a 26 / Fermen- of Storage

27 Ϫ Ϫ 3 2 28 29 30 31 32 33 34 Process Conditions Product Final 35 Ynd nd nd ndY nd 27 nd ndY/N 14 nd Y nd nd nd Y 4.4–4.6 nd nd nd 35 4,20 3.6– 4.4–4.6 V V 12 nd Not D 4.6–5.0 3.6 V 0.798– nd D A With 36

37 10 4

38 10 –10 39 3 4

40 Concen- Starter NO 41 42 43 valence Data (Y/N) (Y/N) (Y/N) T°C T°C pH RH % Packaging Response 44 ndnd 10 8 Log nd 7.5 Log nd 10

45 § 46 ) a/b b a/b a 47 48

Continued

49 ( 50 sausage in 25g bologna CFU/gbologna luncheon CFU/g bologna (model sausages) and 13 4.99d slices 51 sausage CFU/g pepperoni CFU/g 0.805 starter

52 of Type of Type Pre- tration Cultures Sugars NO 53S 10 Dry S-NP nd11 Dry C Presence N nd nd 12 12 nd nd nd nd Detected 14 Beef-pork C 13 Lebanon C Table 42.4. Table a/a Sausages Study 54N 12 Lebanon C

446 40454 42 427-454 r1.qxd 7/20/07 5:11 PM Page 447

1 2 10 10 3 CFU/- reduction depending on starter culture; Detected after 42d reduction CFU/g 4 5 6 7 8 9 10 11 12 0.96 Log 13

: preprocessing inoculation (challenge) and 14 a/b 15

); Samelis et al. (1998); Sakhare and Rao (2003); 16 17 18 19 20 21 22 23 24 25 26 27

: postprocessing inoculation (challenge); C 28 b 29 30 31 32 33 34 35 Y/N Y Y 35 12 4.6– 0.798– nd A ~2–3 36 37 10 38

4 39 40 41 42 43 nd 10 nd 7.4 Y Y Y 30 10~13 4.3–4.9 0.87– nd nd 5–6.9 44 45 46 : preprocessing inoculation (challenge) without storage; C a a a 47 48 49 50 style Log salami CFU/g pepperoni CFU/g 5.0 0.805 Log 51 52 S53 nd: no data, HI: high inoculum; LI: low inoculum. nd: no data, HI: high inoculum; LI: low P: prevalence study; C P: prevalence 16 Italian- C A: aerobically packaged; V: vacuum packaged. vacuum V: packaged; A: aerobically et al. (2001 1–10: Based on Little et al. (1998); Rantsiou (2005); Escartin et al. (2006); Levine et al. (1999); Siriken Chevallier et al. (2006). Chevallier 11: Based on Nissen and Holck (1998). 12–15: Based on Chikthimmah and Knabel(2001); Smith et al. (1975). 16: Based on Nightingale et al. (2006). storage; S-NP: survival study on naturally contaminated products. study on naturally storage; S-NP: survival 15 Beef-pork C § ¥ N54

447 40454 42 427-454 r1.qxd 7/20/07 5:11 PM Page 448

2 10 CFU/g

3 10

4 10 10 CFU/g detected reduction depending on formulation 2.51 Log CFU/g reduction depending on formulation 5 depending on the producer 6 7 8 9 10

11 Conditions 12

13 / T°C/ Pathogen 14 w 15 16 17 18 19 20 21 22 23

24 meat products. in traditional 25 26 tation Drying a

27 / Fermen- of Storage Ϫ Ϫ 28 3 2 29 30 31

32 aureus Staphylococcus 33 34 Process Conditions Product Final 35

36 Y/N Y YY/N Y 20,30 15 Y 4.6–4.8 20,30 0.82–nd nd 15 nd ndnd 4.6–4.8 2–3 Log 0.82– nd nd ndY/N nd nd 1.5–1.9 nd Y nd nd Y 4.0–6.0 35 nd 0.85– nd 4.0–6.0 nd nd 0.85– nd nd nd nd nd nd nd nd Not 37 38 10 10 1 2 CFU/g CFU/g 0.89 Log 6 CFU/g5 CFU/g 7 0.89 nd CFU/g 39 Log 10 Concen- Starter NO 10 40 ≥ 41 42

43 ¥ 44 valence Datand (Y/N) (Y/N) (Y/N)nd T°C 10 T°C pH RH % Packaging Response nd 10

45 § 46 a a a 47 48 49 Data related to prevalence, survival, and growth of survival, and growth Data related to prevalence, 50 drysaus-ages fermented CFU/gsausage sausagesausage 3.40 CFU/g CFU/g 0.95 0.94 Log 0.94 (salsiccia) Log 51 sausage 3.48 86.0 at >0.30–

52 of Type of Type Pre- tration Cultures Sugars NO 53S 5 P Spreadable 6 100% Beef C 7 <10 Fermented S-NP nd <2.0– nd nd nd nd nd 5.6–6.2 0.86– nd nd <2 or 4 3 Fermented S-NP nd 3.18– nd nd nd nd nd 6.2–6.5 76.5– nd nd Present 1 Chorizo C 2 Salchichon C 4 Dry/semi- P 1%

54N 42.5. Table a/a Sausages Study

448 40454 42 427-454 r1.qxd 7/20/07 5:11 PM Page 449

1 2 10 10

CFU/g 3

10 10 10 4 detected increase depending on pH, at 20°C: 0.3 Log CFU/g depending on the producer CFU/g 0.3–4.6 Log CFU/g reduction to 2.6 Log CFU/g increase, depending on pH 5 6 7 8 9 10 11 12 13 14 : preprocessing inoculation (challenge) and

a/b 15 16 ); Sameshima et al. (1998); Sakhare and Rao (2003); 17 18 19 20 21 22 23 24 25 26 27 28 : postprocessing inoculation (challenge); C b 29 30 31 32 33 34 35

Y Y nd 30,37 nd 4.0–4.2 nd nd nd36 Not 37 10 10 10 38 Log 5 CFU/g CFU/g 39 40 41 42 43 nd 4.1–4.2 Y Y Y 20,35nd nd 10 4.4–5.5 90–95 nd nd At °C: 44 45 46 a a : preprocessing inoculation (challenge) without storage; C a 47 48 49 50 mincedmeat, Fermented meat cubes CFU/g (soppres- Log sausage CFU/g 5.70 sausage 3.40 sata CFU/g lucana) salamisausage 3.0 0.95 Log Log 30.3%51 Log 52 S53 8 Fermented S-NP nd9 Greek dry <2.0– S-NP nd nd nd <2.0– N nd nd Y nd Y 5.2–6.4 20–24 0.89– 15–16 nd 5–5.2 nd 27.7– <2 or 4.16 nd nd <2 or 2.6 nd: no data. P: prevalence study; C P: prevalence 12 S-NP Fermented nd <100 N Y Y nd 0–14 5.62– nd nd ndCFU/g <100 Based on Gonzalez-Fandos et al. (1999); Little (1998); Smith (1978); Blaiotta (2004); Samelis (1998 Based on Gonzalez-Fandos Chevallier et al. (2006); Rantsiou (2005). Chevallier 11 C Fermented § ¥ storage; S-NP: survival study on naturally contaminated products. study on naturally storage; S-NP: survival 10 C Fermented N54

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450 Biological and Chemical Safety of Fermented Meat Products

1 fermented products. However, a cooking step at control measures at a single source. Thus, effective 2 60°C could eliminate the pathogen from the product. control strategies must consider the multiple points 3 at which pathogens can gain access to the human 4 food chain. The persistence and the ability of very STAPHYLOCOCCUS AUREUS 5 small numbers of these organisms to establish 6 Different types of fermented sausages have been life-threatening infections with serious long-term 7 implicated in staphylococcal food poisoning out- clinical consequences, particularly among at-risk 8 breaks (Lücke 1985). Staph. aureus may be com- sections of the human population, mean that many 9 monly found in industrial equipment surfaces and is elements of our food safety strategies have to be 10 salt- and nitrite-tolerant, thus being capable of grow- improved. 11 ing and producing enterotoxin during the initial Measures to control pathogens during food pro- 12 stage of fermentation (Gonzalez-Fandos 1999). duction, processing, and distribution, at retail level 13 Based on available reports, growth and enterotoxin and during commercial/domestic preparation should 14 production of Staph. aureus is highly dependent on be considered in detail. 15 the starter cultures used and the fermentation condi- Therefore, the best approach to control pathogens 16 tions, such as duration, temperature, and aeration is to implement HACCP principles into the food 17 (Smith and Palumbo 1978; Adams and Nicolaides safety management systems at all stages of food pro- 18 1997; Sameshima et al. 1998; Gonzalez-Fandos duction and distribution. However, due to their 19 1999). According to Gonzalez-Fandos (1999), growth unusual tolerance to low pH, of some pathogens, i.e., 20 and toxin production was not prevented at 20 and Listeria, E. coli, and Salmonella should be consid- 21 30°C in chorizos and salchichon, two traditional ered at higher risk than other pathogens (e.g., Staph.

22 Spanish sausages (pH 5.5–5.8 and aw 0.82–0.88), aureus) in meat products (sausages), which have a 23 regardless of the presence of starter cultures. The low pH and are minimally processed or are not 24 combination of lower fermentation temperature and cooked before consumption. 25 activity of starter cultures simply restricted growth The basic control measures should include the 26 and toxin production. However, Staph. aureus sig- following: 27 niﬁcantly reduced throughout ripening. Conversely, • A quantitative microbiological hazard analysis or 28 Sameshima et al. (1998) found that three starters, quantitative microbial risk assessment on current 29 added at an initial level of 107 CFU/g (some of them practices should be carried out for products in 30 being potential probiotics) were capable of inhibit- these categories. 31 ing both growth of 103 CFU/g Staph. aureus and • Training of personnel working in food 32 production of staphylococcal toxin in salami fer- preparation and food service industries coupled 33 mented at 20 or 30°C. Likewise, Smith and Palumbo with consumer education on hygienic handling 34 (1978) addressed the importance of starters and the and adequate cooking of food can play a role in 35 addition of glucose in meat fermentations in order to reducing the incidence of pathogenic infection. 36 enhance the injury of Staph. aureus by the metabolic 37 activity of starter cultures. Overall, a common fea- 38 ture of previous reports is that challenge tests with 39 St. aureus do not lead to standard conclusions REFERENCES 40 regarding the inhibition of growth and toxin produc- M Adams, R Mitchell. 2002. Fermentation and 41 tion (Adams and Nicolaides 1997), as is the case pathogen control: A risk assessment approach. Int J 42 with the other three pathogens. Thus, prevalence Food Microbiol 79:75–83. 43 studies and thorough validation of separate MR Adams, L Nicolaides. 1997. Review of the sensitiv- 44 processes are required to ensure minimization of risk ity of different foodborne pathogens to fermentation. 45 by staphylococcal food poisoning due to consump- Food Contr 8:227–239. 46 tion of fermented sausages (Table 42.5). S Ananou, M Garriga, M Hugas, M Maqueda, 47 M Martinez-Bueno, A Galvez, E Valdivia. 2005. Con- 48 CONTROL MEASURES trol of Listeria monocytogenes in model sausages by 49 enterocin AS-48 Int J Food Microbiol 103:179–190. 50 The ability of the above-mentioned pathogens to sur- G Blaiotta, C Pennacchia, F Villan, A Ricciardi, R Tofal, 51 vive in many low acid as well as low water activity E Parente. 2004. Diversity and dynamics of communi- 52 meat products makes it unlikely that complete sup- ties of coagulase-negative staphylococci in traditional 53S pression can be achieved by the application of fermented sausages. J Appl Microbiol 97:271–284. 54N 40454 42 427-454 r1.qxd 7/20/07 5:11 PM Page 451

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MF Borges, RS de Siqueira, AM Bittencourt, MCD G Duffy, DCR Riordan, JJ Sheridan, BS Eblen, 1 Vanetti, LAM Gomide. 1999. Occurrence of Listeria RC Whiting, IS Blair, DA McDowell. 1999. Differ- 2 monocytogenes in salami. Revista de microbiologia ences in thermotolerance of various Escherichia coli 3 30:362–364. 0157:H7 strains in a salami matrix. Food Microbiol 4 RL Buchanan, SG Edelson. 1996. Culturing enterohe- 16:83–91. 5 morrhagic Escherichia coli in the presence and KR Ellajosyula, S Doores, EW Mills, RA Wilson, 6 absence of glucose as a simple means of evaluating RC Anantheswaran, SJ Knabel. 1998. Destruction 7 the acid tolerance of stationary-phase cells. Appl of Escherichia coli O157:H7 and Salmonella 8 Environ Microbiol 62:4009–4013. typhimurium in Lebanon bologna by interaction of 9 M Calicioglu, NG Faith, DR Buege, JB Luchansky. fermentation pH, heating temperature and time. 10 1997. Viability of Escherichia coli O157:H7 in fer- J Food Prot 61:152–157. 11 mented semidry low-temperarure-cooked beef sum- JP Encinas, JJ Sanz, ML Garcia-Lopez, A Otero. 1999. 12 mer sausage. J Food Prot 60:1158–1162. Behaviour of Listeria spp. in naturally contaminated 13 ———. 2001. Validation of a manufacturing process chorizo (Spanish fermented sausage). Int J Food 14 for fermented, semidry Turkish soudjouk to control Microbiol 46:167–171. 15 Escherichia coli 0157:H7. J Food Prot 64: S Erkkila, M Venalainen, S Hielm, E Petaja, E Puolanne 16 1156–1161. T. Mattila-Sandholm. 2000. Survival of Escherichia 17 ———. 2002. Viability of Escherichia coli O157:H7 coli 0157:H7 in dry sausage fermented by probiotic 18 during manufacturing and storage of a fermented, lactic acid bacteria. J Sci Food Agric 80:2101–2104 19 semidry Soudjouk-style sausage. J Food Prot 65: (available online: 2000). 20 1541–1544. EF Escartin, A Castillo, A Hinojosa-Puga and 21 M Campanini, I. Pedrazzoni, S. Barbuti, P. Baldini. J Saldana-Lozano. 1999. Prevalence of Salmonella in 22 1993. Behaviour of Listeria monocytogenes during chorizo and its survival under different storage tem- 23 the maturation of naturally and artificially contami- peratures. Food Microbiol 16:479–486. 24 nated salami: Effect of lactic-acid bacteria starter European Union (EU). 2005. Commission Regulation 25 cultures. Int J Food Microbiol 20:169–175. 2073/2005/EC of 15 November 2005 on the micro- 26 Centers for Disease Control and Prevention (CDC). biological criteria for foodstuffs. Official Journal of 27 1994. Escherichia coli 0157:H7 outbreak linked the European Communities, 22.12.2005, L338/1–25. 28 to commercially distributed dry-cured salami— NG Faith, N Parniere, T Larson, TD Lorang, CW 29 Washington and California. Morbid Mortal Weekly Kaspar, JB Luchansky. 1998a. Viability of 30 Rep 44:157–160. Escherichia coli O157:H7 in salami following condi- 31 ———. 1995. Community outbreak of hemolytic ure- tioning of batter, fermentation and drying of sticks 32 mic syndrome attributable to Escherichia coli and storage of slices. J Food Prot 61:377–382. 33 0111:NM South Australia. Morbid Mortal Weekly NG Faith, RK Wierzba, AM Ihnot, AM Roering, TD 34 Rep 44:550–551,557. Lorang, CW Kaspar, JB Luchansky. 1998b. Survival 35 I Chevallier, S Ammor, A Laguet, S Labayle, V Castanet, of Escherichia coli O157:H7 in full- and reduced-fat 36 E Dufour, R Talon. 2006. Microbial ecology of a pepperoni after manufacture of sticks, storage of 37 small-scale producing traditional dry sausages. Food slices at 4°C or 21°C under air and vacuum, and bak- 38 Contr 17:446–453. ing slices on frozen pizza at 135, 191 and 246°C. 39 N Chikthimmah, SJ Knabel. 2001. Survival of J Food Prot 61:383–389. 40 Escherichia coli 0157:H7, Salmonella typhimurium, FAO/WHO. 2004. Risk Assessment; Appendix 41 and Listeria monocytogenes in and on vacuum pack- 4—Prevalence and incidence of L. monocytogenes in 42 aged Lebanon bologna stored at 3.6 and 13.0°C. fermented meat products (QMRA). 43 J ood Protect 64:958–963. JM Farber, E Daley, R Holley, WR Usborne. 1993. 44 MRS Clavero, LR Beuchat. 1996. Survival of Survival of Listeria monocytogenes during the 45 Escherichia coli O157:H7 in broth and processed production of uncooked German, American and 46 salami as influenced by pH, water activity and tem- Italian-style fermented sausages. Food Microbiol 47 perature and suitability of media for its recovery. 10:123–132. 48 Appl Environ Microbiol 62:2735–2740. JM Farber, PI Peterkin. 1991. Listeria monocytogenes, 49 S Cosansu, K Ayhan. 2000. Survival of enterohaemor- a food-borne pathogen. Microbiol Rev 55:476–511. 50 rhagic Escherichia coli O157:H7 strain in Turkish Food and Drug Administration, Center for Food Safety 51 soudjouck during fermentation, drying and storage and Applied Nutrition (FDA/CFSAN). 2003. Quanti- 52 periods. Meat Sci 54:407–411. tative assessment of the relative risk to public health S53 N54 40454 42 427-454 r1.qxd 7/20/07 5:11 PM Page 452

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1 from foodborne Listeria monocytogenes among manufacture of dry sausage using two different starter 2 selected food categories of ready-to-eat foods. Avail- cultures. Food Microbiol 18:75–85. 3 able at: http://www.fsis.usda.gov/OA/news/2003/ P Levine, B Rose, S Green, G Ransom W Hilli. 2001. 4 rtedata.htm. Pathogen testing of ready-to-eat meat and poultry 5 V Freeman. 1996. The E. coli equation. Meat products collected at federally inspected establish- 6 Poultry 44. ments in the United States, 1990–1999. J Food Pro- 7 CGM Gahan, B O’Driscoll, C Hill. 1996. Acid adapta- tect 64:1188–1193. 8 tion of Listeria monocytogenes can enhance survival GJ Leyer, L-L Wang, EA Johnson. 1995. Acid adaptation 9 in acidic foods and during milk fermentation. Appl of Escherichia coli O157:H7 increases survival in 10 Environ Microbiol 62:3128–3132. acidic foods. Appl Environ Microbiol 61:3752–3755. 11 A Giovannini, V Prencipe, A Conte, L Marino, CL Little, HA Monsey, GL Nichols, J de Louvois. 12 A Petrini, F Pomilio, V Rizzi, G Migliorati. 2004. 1998. The microbiological quality of ready-to-eat 13 Quantitative risk assessment of Salmonella spp. dried and fermented meat products. Int J Environ 14 infection for the consumer of pork products in an Health Res 8:277–284. 15 Italian region. Food Contr 15:139–144. FK Lücke. 1985. Fermented sausages. In: Microbiol- 16 KA Glass, MP Doyle. 1989. Fate and thermal inactiva- ogy of Fermented Foods. BJB Woods, ed. London: 17 tion of Listeria monocytogenes in beaker sausage and Elsevier Publishers, pp. 41–83. 18 pepperoni. J Food Prot 52:226–231. BM Mackey, CM Derrick. 1979. Contamination of the 19 KA Glass, JM Loeffelholz, JP Ford, MP Doyle. 1992. deep tissues of carcasses by bacteria present on the 20 Fate of Escherichia coli 0157:H7 as affected by pH slaughter instruments or in the gut. J Appl Bacteriol 21 or Sodium chloride and in fermented, dry sausage. 46:355–366. 22 Appl Environ Microbiol 58(8):2513–2516 K Mattila, P Saris S Tyopponen. 2003. Survival of Lis- 23 ME Gonzalez-Fandos, M Sierra, ML Garcia-Lopez, teria monocytogenes on sliced cooked sausage after 24 MC Garcia-Fernandez, A. Otero. 1999. The inﬂuence treatment with pediocin AcH. Int J Food Microbiol 25 of manufacturing and drying conditions on the sur- 89:281–286. 26 vival and toxogenesis of Staphylococcus aureus in C Mena, G Almeida, L Carneiro, P Teixeira, T Hogg, 27 two Spanish dry sausages (chorizo and salchichon). and PA Gibbs. 2004. Incidence of Listeria monocyto- 28 Meat Sci 52:411–419. genes in different food products commercialized in 29 GH Grau. 1986. Microbial ecology of meat and poultry. Portugal. Food Microbiol 21:213–216. 30 In: Advances in Meat Research. Meat and Poultry J Metaxopoulos, C Genigeorgis, MJ Fanelli, C Franti, 31 Microbiology. Westport, Connecticut: AVI Publish- E Cosma. 1981a. Production of Italian dry salami I. 32 ing Company, Inc., pp. 1–48. Initiation of staphylococcal growth in salami under 33 CM Hew, MN Hajmeer, TB Farver, JM Glover DO commercial manufacturing conditions. J Food Prot 34 Cliver. 2005. Survival of Listeria monocytogenes in 44:347–352. 35 experimental chorizos. J Food Prot 68:324–330. ———. 1981b. Production of Italian salami: Effect 36 JC Hinkens, NG Faith, TD Lorang, PH Bailey, D Buege, of starter culture and chemical acidulation on 37 CW Kaspar, JB Luchansky. 1996. Validation of pep- staphylococcal growth in Salami under commercial 38 peroni processes for control of Escherichia coli manufacturing conditions. Appl Envir Microb 39 O157:H7. J Food Prot 59:1260–1266. 42:863–871. 40 M Hugas, M Garriga, M Pascual, MT Aymerichand, TE Minor, EH Marth. 1972a. Staphylococcus aureus 41 JM Monfort 2002. Enhancement of sakacin K activ- and staphylococcal food intoxication. A review II. 42 ity against Listeria monocytogenes in fermented Enterotoxin and epidemiology. J Milk Food Technol 43 sausages with pepper or manganese as ingredients. 35:21–29. 44 Food Microbio 19:519–528. ———. 1972b. Staphylococcus aureus and staphylo- 45 K Incze. 1998. Dry fermented sausages. Meat Sci coccal food intoxication. A review: IV Staphylococci 46 49:S169–S177. in meat bakery products and other foods J Milk Food 47 SC Ingham, DR Buese, BK Dropp, JA Losinski. 2004. Technol 35:228–241. 48 Survival of Listeria monocytogenes during storage of JE Moore. 2004. Gastrointestinal outbreaks associated 49 ready-to-eat meat products processed by drying, fer- with fermented meats. Meat Sci 67:565–568. 50 mentation, and/or smoking. J Food Prot 67: F Naim, D. Leblanc, S. Messier, L. Saucier, G. Piette, 51 2698–2702. A Houde. 2006. Shiga toxin production by sausage- 52 E Lahti, T Johansson, T Honkanen-Buzalski, P Hill, borne Escherichia coli 0157:H7 in response to a 53S E Nurmi. 2001. Survival and detection of Escherichia post-processing in vitro digestion challenge. Food 54N coli 0157:H7 and Listeria monocytogenes during the Microbiol 23:231–240. 40454 42 427-454 r1.qxd 7/20/07 5:11 PM Page 453

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F Naim, S Messier, L Saucier, G Piette. 2003. A model PZ Sakhare, DN Rao. 2003. Microbial profiles during 1 study of Escherichia coli O157:H7 survival in fer- lactic fermentation of meat by combined starter cul- 2 mented dry sausages—Influence of inoculum prepa- tures at high temperatures. Food Control 14:1–5. 3 ration, inoculation procedure, and selected process J Samelis, A Kakouri, IN Savvaidis, K Riganakos, MG 4 parameters. J Food Prot 66:2267–2275. Kontominas. 2005. Use of ionizing radiation doses of 5 ———. 2004. Postprocessing in vitro challenge to 2 and 4kGy to control Listeria spp. and Escherichia 6 evaluate survival of Escherichia coli O157:H7 in fer- coli 0157:H7 on frozen meat trimmings used for dry 7 mented dry sausages. Appl Environ Microbiol fermented sausages. Meat Sci 70:189–195. 8 70:6637–6642. J Samelis, J Metaxopoulos 1999. Incidence and princi- 9 KK Nightingale, H Thippareddi, RK Phebus, JL pal sources of Listeria spp. and Listeria monocyto- 10 Marsden, AL Nutsch. 2006. Validation of a tradi- genes contamination in processed meats and a meat 11 tional Italian-style salami manufacturing process for processing plant. Food Microbiol 16: 465–477. 12 control of Salmonella and Listeria monocytogenes. J Samelis, J Metaxopoulos, M Vlassi, A Pappa. 1998. 13 J Food Protect 69(4):794–800. Stability and safety of traditional Greek salami— 14 H Nissen, A. Holck. 1998. Survival of Escherichia coli A microbiological ecology study. Int J Food Micro- 15 O157:H7, Listeria monocytogenes and Salmonella biol 44:69–82. 16 kentucky in Norwegian fermented, dry sausage. Food T Sameshima, C Magome, K Takeshita, K. Arihara, 17 Microbiol 15:273–279. M Itoh, Y Kondo. 1998. Effect of intestinal Lacto- 18 G-JE Nychas, JS Arkoudelos. 1990. Staphylococci: bacillus starter cultures on the behaviour of Staphylo- 19 Their role in fermented sausages. In: Staphylococci. coccus aureus in fermented sausage. Int J Food 20 D Jones, RG Board, C Collins, eds. Symposium Microbiol 41:1–7. 21 Series No. 19, Society for Applied Bacteriology. C Sauer, J Majkowski, S Green, R Eckel. 1997. 22 London: Blackwell Scientific Publishers. Foodborne illness outbreak associated with a 23 G-JE Nychas, D Marshall, J Sofos. 2007. semi-dry fermented sausage product. J Food Prot 24 Chapter 6: Meat poultry and seafood in food 60:1612–1617. 25 microbiology fundamentals and frontiers. Doyle, C Shadbolt, T Ross, TA McMeekin, 2001. Differentia- 26 Beuchat, and Montville, eds. Herndon, Virginia: tion of the effects of lethal pH and water activity: 27 ASM Press. Food safety implications. Lett Appl Microbiol 28 TJ Pond, DS Wood, IM Mumin, S Barbut, MW 32:99–102. 29 Griffiths. 2001. Modelling the survival of B Siriken, S Pamuk, C Ozakin, S Gedikoglu, M Eyigot. 30 Escherichia coli 0157:H7 in uncooked, semidry, fer- 2006. A note on the incidences of Salmonella 31 mented sausage. J Food Prot 64:759–766. spp., Listeria spp. and Escherichia coli O157:H7 32 MM Pullen, CA Genigeorgis. 1977. A study of coagu- serotypes in Turkish sausage (Soudjouck). Meat Sci 33 lase-positive staphylococci in salami before fermen- 72:177–181. 34 tation. J Food Protect 40:704–708. JL Smith, CN Huhtanen, JC Kissinger, SA Palumbo. 35 K. Rantziou, R Urso, L Iacumin, C Cantoni, 1975. Survival of Salmonellae during pepperoni 36 PCG Comi, L Cocolin. 2005. Culture-dependent and manufacture. Appl Microbiol 30:759–763. 37 -independent methods to investigate the microbial JL Smith, SA Palumbo. 1978. Injury to Staphylococcus 38 ecology of Italian fermented sausages. Appl Environ aureus during sausage fermentation. Appl Environ 39 Microbiol 71:1977–1986. Microbiol 36:857–860. 40 CA Reed. 1995. Challenge study-Escherichia coli D Thévenot ML Delignette-Muller, S Christieans, 41 O157:H7 in fermented sausage. USDA Food Safety C Vernozy-Rozand. 2005a. Prevalence of Listeria 42 and Inspection Service, Washington, DC, 21 August, monocytogenes in 13 dried sausage processing 43 letter to Plant Managers. plants and their products Int J Food Microbiol 102: 44 DCR Riordan, C Duffy, JJ Sheridan, RC Whiting, 85–94. 45 IS Blair, DA McDowell. 2000. Effects of acid adapta- ———. 2005b. Fate of Listeria monocytogenes in 46 tion, product pH, and heating on survival of experimentally contaminated French sausages. Int 47 Escherichia coli O157:H7 in pepperoni. Appl Envi- J Food Microbiol 101:189–200. 48 ron Microbiol 66:1726–1729. J Tilden, Jr., W Young, A-M McNamara, C Custer, 49 M. Rocelle, S Clavero, LR Beuchat 1996. Survival of B Boesel, MA Lambert-Fair, J Majkowski, D Vugia, 50 Escherichia coli O157:H7 in broth and processed SB Werner, J Hollingsworth, JG Morris, Jr. 1996. 51 salami as influenced by pH, water activity, and tem- A new route of transmission for Escherichia coli: 52 perature and suitability of media for its recovery. Infection from dry fermented salami. Am J Pub S53 Appl Environ Microbiol 62:2735–2740. Health 86:1142–I 145. N54 40454 42 427-454 r1.qxd 7/20/07 5:11 PM Page 454

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1 RB Tompkin. 2002. Control of Listeria monocytogenes M Uyttendaele, S Vankeirsbilck, J Debevere. 2001. 2 in the food processing environment. J Food Prot Recovery of heat-stressed E. coli 0157:H7 from 3 65:709–725. ground beef and survival of E. coli 0157:H7 in refrig- 4 RB Tompkin, LN Christiansen, AB Shaparis, H Bolin. erated and frozen ground beef and in fermented 5 1974. Appl Microbiol 28:262–266 sausage kept at 7°C and 22°C. Food Microbiol 18: 6 US Department of Agriculture, Food Safety and 511–519. 7 Inspection Service. 2001. Performance standards for F Villani, L Sannino, G Moschetti, G Mauriello, 8 the production of processed meat and poultry prod- O Pepe, R Amodio-Cocchieri, S Coppola. 1997. 9 ucts; proposed rule. Fed Regist 66:12590–12636. Partial characterization of an antagonistic substance 10 ———. 2003. Control of Listeria monocytogenes in produced by Staphylococcus xylosus1E and determi- 11 ready-to-eat meat and poultry products; ﬁnal rule. nation of the effectiveness of the producer strain to 12 Fed Regist 68:34207–34254. inhibit Listeria monocytogenes in Italian sausages. 13 ———. 2004. Compliance guidelines to control Liste- Food Microbiol 14:555–566. 14 ria monocytogenes in post-lethality exposed ready- T Zhao, MP Doyle, RE Besser. 1993. Fate of enterohe- 15 to-eat meat and poultry products. Available at: http:// morragic Escherichia coli O157:H7 in apple cider 16 www.fsis.usda.gov/OPPDE/rdad/FRPubs/97013F/ with and without preservatives. Appl Environ Micro- 17 CompGuidelines.pdf. Accessed April 19, 2004. biol 59:2526–2530. 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53S 54N 40454 43 455-468 r1.qxd 7/21/07 12:47 PM Page 455

1 2 3 43 4 5 6 Biogenic Amines: Risks and Control 7 8 M. Carmen Vidal-Carou, M. Teresa Veciana-Nogués, 9 M. Luz Latorre-Moratala, and Sara Bover-Cid 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION: BIOGENIC effects caused by vasoactive and/or psychoactive 22 AMINE CLASSIFICATION AND properties. Although these effects are well docu- 23 RELEVANCE mented, information about the mechanisms and toxi- 24 cological doses as well as individual factors that 25 Biogenic amines, also known as biologically active determine the severity of reactions is still incons- 26 amines, are nitrogenous low-molecular weight sub- istent. Therefore, it is difficult to establish the 27 stances of biological origin found in a wide range of maximum tolerable limits of amines at which a 28 concentrations in nearly all types of foods. In food potentially toxic food products should be rejected. 29 products, these compounds can be classified in three However, the severity of the above mentioned reac- 30 groups according to their origin and chemical struc- tion is now reduced, mainly because of three factors: 31 ture: aromatic biogenic amines, aliphatic diamines, (1) most MAOI drugs currently used are selective 32 and natural polyamines (Figure 43.1). Biogenic inhibitors of one MAO isoenzyme, thereby leaving 33 amines are produced mainly by decarboxylation the other available for biogenic amine metabolism; 34 of precursor amino acids by specific microbial (2) patients treated with MAOI drugs generally 35 enzymes. This group includes aromatic amines follow a tyramine-restricted diet; and (3) biogenic 36 (tyramine, phenylethylamine, histamine, and trypta- amine content in food is now lower than in the past 37 mine) and aliphatic amines (putrescine, cadav- thanks to improved manufacturing practices. 38 erine, and agmatine). The physiologically natural Furthermore, biogenic amines are relevant from 39 polyamines, spermine and spermidine, are not asso- the hygienic and technological points of view (Brink 40 ciated with microbial activity and their biosynthesis et al. 1990; Mariné-Font et al. 1995; Izquierdo- 41 follows a more complex process. Low levels of Pulido et al. 1999). Several biogenic amines (mainly 42 putrescine can also be considered of physiological or diamines and histamine) may result from the activity 43 natural origin and are a precursor of spermidine of contaminant bacteria, for which these compounds 44 (Bardócz 1995; Izquierdo-Pulido et al. 1999). are potentially useful chemical indicators of defec- 45 Interest in biogenic amines arose in the 1960s tive hygienic conditions of raw materials and/or 46 after reports of the so-called “cheese reaction,” a manufacturing processes. As microbial metabolites, 47 severe hypertensive crisis after the consumption of biogenic amines (mainly tyramine) have been 48 tyramine-rich cheese. This reaction is caused by the closely associated with milk and meat fermentation, 49 interaction of these compounds with monoamine- and it was even believed that the occurrence of tyra- 50 oxidase inhibitor (MAOI) drugs and, in some cases, mine was inherent to the manufacturing process of 51 may have lethal consequences (Blackwell 1963). fermented products. However, fermented foods can 52 Indeed, studies on biogenic amines in food have tra- also show low levels of this amine in particular 53 ditionally focused on their potentially toxicological and biogenic amines in general. Therefore, it is of S54 N55 455 40454 43 455-468 r1.qxd 7/21/07 12:48 PM Page 456

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Figure 43.1. Chemical structure and formation pathway of biologically active amines. 34 35 36 interest to determine the levels of biogenic amines ated with several acute adverse reactions. The 37 (as a sum of amines or of a particular amine) that mechanisms of action of these compounds involve 38 could be regarded as normal and/or unavoidable, and vasoactive (vasoconstriction for tyramine and 39 thereby to establish a threshold value that would phenylethylamine, and vasodilatation for histamine) 40 indicate improper hygienic practices. and psychoactive reactions. In addition to interac- 41 In this chapter, biogenic amines in fermented tion with MAOI drugs, the ingestion of biogenic 42 sausages are reviewed in relation to safety, hygienic, amines may cause other health complaints, such 43 and technological issues. The toxicological risks for as histaminic intoxication, histamine intolerance due 44 consumers as well as the prospects for manufactur- to enteral histaminosis, and food-induced migraine 45 ers to reduce aminogenesis during fermentation of (Taylor 1986; Bardócz 1995; Mariné-Font et al. 1995; 46 this product are discussed. Shalaby 1996; Amon et al. 1999). The diamines 47 putrescine and cadaverine are not toxic by them- 48 selves, but they may enhance the absorption of 49 HEALTH RISKS OF BIOGENIC vasoactive amines because of saturation of intestinal 50 AMINES IN FERMENTED barriers. Competition for the mucine attachment 51 SAUSAGES sites and detoxiﬁcation enzymes has been proposed 52 to explain the potentiating effect of these diamines 53S Tyramine, histamine, and to a lesser extent phenylethy- (Chu and Bjeldanes 1981; Hui and Taylor 1985; 54N lamine are the main dietary biogenic amines associ- Taylor 1986). 40454 43 455-468 r1.qxd 7/21/07 12:48 PM Page 457

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Despite the recognized wide inter- and intra- occasions, fermented sausages have also been 1 individual variability of susceptibility to biogenic reported to contain small amounts of nitrosamines. 2 amines, the enzymatic intestinal and hepatic barriers For instance, examination of the content of four 3 to these compounds in nonmedicated healthy indi- volatile nitrosamines in dry sausages made in Fin- 4 viduals are efficient, and dietary sources of these land showed very low or even undetectable amounts 5 compounds are not a health risk under normal cir- (Eerola et al. 1997, 1998a). N-nitrosopyrrolidine 6 cumstances. However, certain genetic factors, some was the main and relatively most frequently detected 7 intestinal diseases, the occurrence of dietary poten- nitrosamine in correlation with the amount of 8 tiators (alcohol, other biogenic amines, etc.), and putrescine (its precursor). Some aromatic amines, 9 several drugs that inhibit MAO and diamino oxidase such as tyramine, have been also studied as possible 10 (DAO) enzymes, responsible for biogenic amine precursors of the mutagenic diazotyramine (Ochiai et 11 metabolism, may notably increase susceptibility to al. 1984). Nevertheless, the limiting factor in 12 biogenic amines; consequently the risk of adverse nitrosamine formation is not the availability of 13 reactions may be significant (Mariné-Font et al. precursors but the amount of nitrosating agent 14 1995; Shalaby 1996; Lehane and Olley 2000). (McIntyre and Scanlan 1993). The consequences of 15 Unfortunately, little information on the incidence dietary exposure to nitroso compounds have not been 16 of intoxication by dietary amines is available. More- accurately established. These compounds show car- 17 over, the wide variability of biogenic amine content cinogenic properties in laboratory animals and prob- 18 in food products hinders the estimation of the toxic ably induce cancer in humans, although convincing 19 dose on the basis of epidemiological data. data and epidemiological evidence are still lacking. 20 Although the precise amount of tyramine required Furthermore, nitrosamines may also be formed 21 to produce adverse effects is not known, according endogenously from precursors in the gastrointestinal 22 to the literature, 6 mg of tyramine could cause mild tract (Vermeer et al. 1998). According to several risk 23 crisis and 10–25 mg may lead to a severe headache assessments (i.e., Gangolli et al. 1994; Fernlöf and 24 with intracranial hemorrhage in patients treated with Darnerud 1996), dietary sources of nitrite and N- 25 classical MAOI drugs (McCabe 1986). In contrast, nitroso compounds make a small contribution to the 26 from 50–150 mg of tyramine would be well tolerated body pool of nitroso compounds, and the main 27 by patients receiving a new generation of MAOI source of these compounds would be attributed to the 28 agents (Korn et al. 1988; Dingemanse et al. 1998; intestinal bacterial metabolism of ingested nitrates. 29 Patat et al. 1995). Neither is there consensus regard- 30 ing the dose of histamine that provokes toxic reac- 31 tions. In the risk assessment review of histamine fish AMINOGENESIS IN FERMENTED 32 poisoning, Lehane and Olley (2000) proposed 30 mg SAUSAGES AND MEASURES FOR 33 of histamine as a safe dose, calculated from the ITS CONTROL 34 100 mg/kg histamine level established to protect 35 public health and safety (e.g., Council Directive The fermentation of food in general, and meat 36 91/493/EEC of the EC 1991) and considering 300 g sausages in particular, offers optimal conditions for 37 as a fish serving. The same authors, however, consider biogenic amine accumulation: growth of microor- 38 this calculation of limited value. ganisms (some with recognized aminogenic capac- 39 Biogenic amines in food products are precursors ity), and proteolytic phenomena that increase the 40 of nitroso compounds with potential carcinogenic amounts of precursor amino acids. The production 41 activity, which constitutes an indirect additional risk of weak organic acids is an additional factor favoring 42 associated with dietary amines. Nitrosamines result the aminogenic activity of microorganisms, which 43 from the action of nitrite on secondary amines. The produce biogenic amines as a defense mechanism 44 primary amines, such as the aliphatic diamines and against an unfavorable acidic environment. Although 45 polyamines, may cyclize to secondary amines under a wide number of bacteria have aminogenic potential, 46 certain conditions (Warthesen et al. 1975; Brink amino acid decarboxylases are not homogeneously 47 et al. 1990; Shalaby 1996). Cured and heated/smoked distributed in all microorganisms, but are strain- 48 meat products (mainly cooked and fried bacon) are dependent (Bover-Cid and Holzapfel 1999). Indeed, 49 frequently described as nitrosamine sources. The sausages with comparable microbial profiles may dif- 50 manufacturing process of these products involves fer greatly in their amine content, indicating that the 51 nitrosating agents (such as nitrites and their precursor accumulation of these compounds is governed by a 52 nitrates), mild acidic pH, and high temperatures, all complex interaction of microbial, physicochemical S53 conditions that favor nitrosamine formation. On few and technological factors (Suzzi and Gardini 2003). 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1 BIOGENIC AMINE CONTENT IN FERMENTED fish products. Enterobacteriaceae and Pseudomonas 2 SAUSAGES OF THE RETAIL MARKET are contaminant gram-negative bacteria. They are 3 usually present in low numbers in the final sausage Fermented sausages and cheese are the two food 4 product or even totally inhibited in the first days of products that show the highest biogenic amine con- 5 fermentation because of unfavorable environmental tents. Table 43.1 summarizes the reports describing 6 conditions for growth during production and storage. the occurrence of biogenic amines in fermented meat 7 Consequently, these two strains of bacteria contribute products retailed in several countries. According to 8 to biogenic amine accumulation mainly during the the literature, levels of biogenic amines in fermented 9 early steps of sausage production, particularly when sausages show a large variation among types of prod- 10 raw meat is subjected to lengthy storage or high tem- ucts, manufacturers, and also samples from distinct 11 peratures or when hygienic practices are not followed batches of the same kind of product made by the same 12 (Paulsen and Bauer 1997; Bover-Cid et al. 2000a; producer. However, tyramine is usually the most fre- 13 Bover-Cid et al. 2003). It has been also suggested that quent and most abundant biogenic amine found in 14 a considerable contribution to biogenic amine accu- fermented sausages. The occurrence of diamines, 15 mulation could derive from the activities of viable but putrescine and cadaverine, is also quite common but 16 nongrowing cells, or even from the decarboxylase more variable than tyramine. Most samples show rela- 17 enzymes released at the beginning of fermentation by tively low amounts of diamines and only a few accu- 18 these contaminating flora (Halász et al. 1994; Suzzi mulate higher amounts than tyramine. By contrast, 19 and Gardini 2003). Nevertheless, no specific studies histamine is rare in fermented sausages, but in some 20 confirm these hypotheses. In any case, the occurrence particular samples it may reach quite high levels, usu- 21 of excessive levels of biogenic amines, such as ally accompanied by high amounts of other biogenic 22 diamines and histamine, could be indicative of con- amines. Similarly, the contents of phenylethylamine 23 tamination of raw materials or hygienic failure during and tryptamine are relatively low. These two amines 24 processing, regardless of the presence of aminogenic could be considered minor amines in fermented 25 microorganisms in the final product. sausages and their accumulation appears to depend on 26 Among gram-positive bacteria, lactobacilli strains the occurrence of high contents of tyramine. 27 belonging to L. curvatus, L. buchneri, and L. brevis Fermented sausages are significant sources of 28 are the most potent tyramine producers. Some of physiological polyamines. However, these types of 29 these species may also produce other aromatic amines polyamines have received less attention than other 30 (phenylethylamine or tryptamine) simultaneously to amines (Pavel and Krausová 2005). The natural ori- 31 tyramine, or even diamines (putrescine and/or cadav- gin of these polyamines in raw meat implies that 32 erine) (Straub et al. 1995; Bover-Cid et al. 2001a; their levels are less variable than those of biogenic 33 Aymerich et al. 2006). Histidine-decarboxylase capac- amines derived from microbial metabolism. Also, 34 ity is delimited to some strains of a reduced number of spermine levels are higher than those of spermidine 35 species (for instance L. hilgardii,), which are not com- in food products of animal origin. 36 monly found but arise by specific contaminations 37 (Maijala and Eerola 1993; Paulsen and Bauer 1997). 38 By contrast, L. sakei, L. bavaricus and L. plantarum BIOGENIC AMINE PRODUCTION BY MEAT-RELATED 39 show a lower proportion of aminogenic strains (Bover- MICROORGANISMS 40 Cid et al. 2001a; Aymerich et al. 2006). A very high 41 Microbial flora, either present in raw meat or also number of enterococci strains produce considerable 42 added as starter culture, has a strong effect on bio- amounts of tyramine, and the strongest tyrosine- 43 genic amine formation during the fermentation and decarboxylase-positives also have the capacity to 44 ripening of meat products. The production of these decarboxylate phenylethylamine (Bover-Cid et al. 45 compounds in meat and meat products has often 2001a; Gardini et al. 2001). Much less information is 46 been related to a variety of microorganisms, including available about the capacity to produce biogenic 47 enterobacteria, pseudomonads, lactobacilli, entero- amines of the gram-positive catalase-positive cocci, 48 cocci, carnobacteria, and staphylococci (Table 43.2). namely staphylococci, micrococci, and kocuria. 49 Indeed, many meat enterobacteria and pseudomon- Although some strains produce putrescine, tyramine, 50 ads strains produce putrescine and cadaverine. Par- phenylethylamine, or histamine (Straub et al. 1994; 51 ticular strains, especially among enterobacteria, Montel et al. 1999; Martín et al. 2006), they are usu- 52 have been also described as histamine producers ally described as weak or negative decarboxylase 53S (Roig-Sagués et al. 1996), although less often and to microorganisms (Masson et al. 1996; Martuscelli et al. 54N a lower extent than the enterobacteria isolated from 2000; Bover-Cid et al. 2001a; Martín et al. 2006). 40454 43455-468r1.qxd7/21/0712:48PMPage459

Table 43.1. Biogenic amine contents (mg/kg fresh matter) in fermented sausages of the retail market from several countries. Phenylethyl- Reference Product n Tyramine Histamine amine Tryptamine Cadaverine Putrescine Spermidine Spermine Spanish Vidal- Chorizo 11 131 Mda 46 Md –d ––– –– Sausage Carou 176 Ϯ 149b 76 Ϯ 80 et al. (2–509)c (2–249) (1990) Salchichón 19 135 Md 5 Md – – – – – – 133 Ϯ 62 18 Ϯ 27 (35–270) (1–103) Salami 5 5 Md 73 Md – – – – – – 6 Ϯ 3 66 Ϯ 39 (3–12) (2–102) Sobrasada 3 8 Md 75 Md – – – – – – 8 Ϯ 6 55 Ϯ 36 (3–14) (14–78) Hernandez- Chorizo 20 282 Ϯ 129 18 Ϯ 27 1 Ϯ 3 16 Ϯ 20 20 Ϯ 16 60 Ϯ 141 4 Ϯ 3 26 Ϯ 8 Jover et al. (30–627) (0–314) (0–52) (0–88) (0–658) (3–416) (2–10) (14–44) (1997a) Salchichon 22 281 Ϯ 109 7 Ϯ 14 7 Ϯ 6 9 Ϯ 11 12 Ϯ 23 103 Ϯ 76 5 Ϯ 3 15 Ϯ 8 (53–513) (0–151) (0–35) (0–65) (0–342) (6–400) (1–14) (7–43) Fuet 11 191 Ϯ 73 2 Ϯ 40 2 Ϯ 4 9 Ϯ 8 19 Ϯ 18 72 Ϯ 41 5 Ϯ 3 17 Ϯ 7 (32–743) (0–358) (0–34) (0–68) (5–51) (2–222) (1–11) (9–30) Sobrasada 7 332 Ϯ 131 9 Ϯ 17 2 Ϯ 6 12 Ϯ 23 13 Ϯ 14 65 Ϯ 50 3 Ϯ 2 14 Ϯ 7 (58–501) (3 –143) (0–39) (0–65) (3–42) (2–501) (2–7) (10–18) Bover- Thin fuet 15 68 Md 0.6 Md 1 Md 0 Md 11 Md 15 Md 5 Md 30 Md Cid et al. 92 Ϯ 72 1 Ϯ 2 4 Ϯ 8 5 Ϯ 11 43 Ϯ 48 80 Ϯ 152 7 Ϯ 5 25 Ϯ 14 (1999a) (1–218) (0–5) (0–29) (0–39) (1–115) (1–513) (1–21) (2–44) Fuet 23 103 Md 2 Md 3 Md 4 Md 8 Md 34 Md 6 Md 23 Md 119 Ϯ 64 12 Ϯ 34 8 Ϯ 13 8 Ϯ 11 28 Ϯ 42 49 Ϯ 43 8 Ϯ 9 24 Ϯ 16 (22–272) (0–158) (0–47) (0–36) (2–156) (1–169) (2–45) (2–84)

(Continues) 459 40454 43 455-468 r1.qxd 7/21/07 12:48 PM Page 460

1 9 15 11 2 13 Ϯ Ϯ Ϯ 3 Ϯ 4 5 5 24 1 46 3 25

6 5 22 Ϯ Ϯ Ϯ 7 Ϯ 8 9 10 11 89 6 92 8 96 4 110 6 Ϯ Ϯ 12 Ϯ 13 Ϯ 14 15 38 63 90 91 113 92 16 30 99 Ϯ Ϯ Ϯ 17 Ϯ 18 19 20 25 34 47 33 18 21 12 26 Ϯ Ϯ Ϯ 22 Ϯ 23 24 25 20 32 14 26 28 15 14 7 Ϯ Ϯ 27 Ϯ Ϯ 28 Phenylethyl- 29 30 54 14 37 8 50 10 9 nd nd 103 31 20 12 Ϯ Ϯ Ϯ Ϯ 32 Ϯ 33 34

35 93 14 105 46 120 28 100 6 124 14 Ϯ Ϯ Ϯ Ϯ 36 Ϯ (0–339) (0–39) (0–78) (0–57) (0–28) 37 (3–490) (0–59) (0–126) (0–142) (0–127) (0–325) (1–13) (7–52) 38 141 39 40 41 42 43 (traditional) (1–433)(traditional) (0–205) (84–217) (0–61) (15–16) (0–52) (0–4) (0–156) (0–537) (1–29) (39–110) (2–78) (61–317) (2–6) (82–86) 44 sausage 139

45 Salchichón 19 98 Md 5 Md 5 Md 4 Md 8 Md 86 Md 3 Md 20 Md 46 ) 47 48 (2004)et al. (1990) (60–372) (8–165) (40–310) (nd–53) (1–63) (nd–69) (5–45) (1–150) (1–190) et al. 3(1999) Saucisson (industrial) 164 15 1 et al. nd(2001) (172–268) (16–151) Salsicciaet al. 10 (0–8) 71 223 84 4 Italiani 77 nd (0–9) (0–557) (31–192) 205 nd (0–101) (195–410) (0–20) (4–6) – (59–119) 7 20 3 19 (0–271) (0–416) (0–91) (0–98) et al. (2006) Dry (industrial) (6–675) 67 (0–186) 118 Md (0–186) 1 Md (0–194) 3 Md (2–466) 3 Md (0–410) 9 Md (1–20) (8–44) 33 Md 5 Md 21 Md Capillas (19–214)and (1–16)Jiminéz- Comenero (2004) Arrizado sausage 143 (9–229) (0.8–185) (7–8) (39–59)

49 Continued Brink 14 110 11 14 – 63 52 ––52 – 14 63 11 Brink 14 110 Montel Saucisson 5 SoppressataParente 220 9 71 178 4 Coisson 22 Salamini 4 3 10 103 194 279 – 5 19 Md 91 61 0 Md 99 9 Md 40 36 – – – – Miguélez- Dry 33 128 Md 2 Md 4 Md 5 Md 11 Md 41 Md 4 Md 22 Md Ruiz- Chorizo 3 129 ( Reference Product n Tyramine Histamine amine Tryptamine Cadaverine Putrescine Spermidine Spermine 50 51 52 Sausages 53S Sausages Sausages Dutch French French Italian 54N 43.1. Table

460 40454 43 455-468 r1.qxd 7/21/07 12:48 PM Page 461

1 9 14 2 Ϯ Ϯ 3 4 5 6 7 8 9 10

90 – – 11 239 – 9 123 – 13 Ϯ

Ϯ 12 Ϯ 13 14 15 111 127 2 121 16 –, not reported. Ϯ Ϯ

d 17 18 19 20 25 161 31 1 14 – 75 21 Ϯ Ϯ Ϯ 22 23 24 25

9 25 26 range (minimum-maximum); 20 11 c

Ϯ 27 Ϯ 28 29 30 82 – 49 83 9 17 – – – – – – 151 6 31 Ϯ Ϯ Ϯ Ϯ 32 33 34 35 72 120 92 94 69 Ϯ Ϯ Ϯ 36

(5–320) (0–170)(9–150) (0–5) (0–40)(3–200) (0–7)(5–190) (0–9) (0–54) (0–200) (0–8) (0–16) (0–20) (0–48) (2–580) (0–51) (0–8) (0–42) (3–11) (0–220) (0–71) (0–390) (22–38) (0–210) (0–230) (3–7) (20–40) (1–8) (3–11) (19–45) (21–48) 37 standard deviation when available; available; when standard deviation

Ϯ 38 39

mean 40 b 41 42 43 butcher-made (2–316) (2–478) (0–32) (0–7) (0–7) (0–919) (0–42) sausagesausage (6–240) (0–200) (5–110) (1–33) (1–56) (0–4) (0–43) (3–18) (0–91) (3–310) (0–790)sausage (0–450) (2–8) (23–40) (3–9) (23–47) 44

Danish 8Meatwurst 54 12 9 Lubeck 2 72 21 3 Salami 18 9 6 77 29 6 Pepperoni 13 27 73 6 4 180 130 11 10 33 3 49 5 7 93 3 5 37 30 54 14 20 5 94 21 6 18 82 61 33 6 45 46 47 48 et al. (19–228) (55–291) (19–86) (20–328) (17–275) and factory-Bozkurt made (2004) Sucuk— (1–189) 31 (4–255) 77 (0–87) (0–47) (0–383) (0–50) (2004) (2004) dry (20–87) et al. (1998b) Russian 4 sausage 110 89 11 (4–200) 22 (0–180) 10 93 (2–248) 5 33 (0–43) (0–270)(1996) (0–230) sausages (2–7) (10–53) (19–46) (7–41) (2–81) (3–34) (6–39) (12–100) (5–12) (2–5) 49 Riebroy Som-fugRiebroy 7 87 Erkmen Sucuk— 19 62 Eerola Finnish Eerola Finnish 11 88 54 13 14 50 79 4 Egyptian Shalaby 50 31 Ekici et al. 14 Turkish 5 46 – 10 32 13 19 39 2 2 50 51 52 Sausages Sausages Sausages Sausages S53 Md = median value shown when available; available; when shown Md = median value Thai Egyptian Turkish Finnish a N54

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462 Biological and Chemical Safety of Fermented Meat Products

1 Table 43.2. Microorganisms, with a recognized capacity to produce one or more biogenic amines associated 2 with meat and meat products. 3 4 Species Tyramine Phenylethylamine Tryptamine Histamine Putrescine Cadaverine Reference 5 Carnobacterium ++ 9,13 6 divergens 7 Carnobacterium ++ 9,13 8 piscicola 9 Enterococcus faecalis + + 3,11 10 Enterococcus faecium ++ 11 11 12 Lactobacillus bavaricus + + + 3,13 13 Lactobacillus brevis + + + + + 3,13,14 14 Lactobacillus buchneri + + + 13,14 15 Lactobacillus curvatus + + + + + + 2,3,9,10, 16 11,13,14 17 Lactobacillus hilgardii + 6,14 18 Lactobacillus plantarum ++ 9,11 19 Pediococcus + 9 20 pentosaceus 21 Staphylococcus carnosus ++ 1,5,7 22 Staphylococcus +++7 23 epidermidis 24 Staphylococcus xylosus + 8 25 Staphylococcus warneri + 7 26 Kocuria varians ++ + 1,9 27 Citrobacter freundii ++4 28 Enterobacter aerogenes ++ +4,10 29 Enterobacter cloacae ++ +3,10,12 30 Escherichia coli + 14 31 Hafnia alvei ++ +12 32 Klebsiella oxytoca ++3,6,10,14 33 Morganella morganii + 6,14 34 Serratia liquefaciens ++ +3,4, 11,12,14 35 Pseudomonas spp. + 12 36 37 Sources: (1) Ansorena et al. (2002); (2) Aymerich et al. (2006); (3) Bover-Cid et al. (2001a); (4) Durlu-Özkaya et al. (2001); 38 (5) Hammes et al. (1995); (6) Kranner et al. (1991); (7) Martín et al. (2006); (8) Marstuscelli et al. (2000); (9) Masson et al. 39 (1996); (10) Roig-Sagués et al. (1996); (11) Roig-Sagués et al. (1997); (12) Slerm (1981); (13) Straub et al. (1995); 40 (14) Tschabrun et al. (1990). 41 42 It should be highlighted that the results reported occasionally be involved. Histamine formation would 43 in the literature about aminogenic bacterial activity be shared by specific strains of species belonging 44 obtained in laboratory media do not imply the same mainly to enterobacteria but also to some lactic acid 45 behavior in true fermentation processes, because envi- bacteria, which occur rarely in fermented sausages. In 46 ronmental conditions and microbial interactions are contrast, tyramine accumulation would be related 47 not reproducible. For instance, the production of mainly to lactic acid bacteria, enterococci, and specific 48 putrescine by enterobacteria in meat is conditioned to strains of lactobacilli species (especially L. curvatus) 49 a previous deamination of arginine by lactic acid bac- showing a remarkable contribution. 50 teria, which yields the precursor ornithine, a substance 51 that is not naturally present in food protein structures FACTORS AFFECTING BIOGENIC AMINE 52 (Dainty et al. 1986; Brink et al. 1990). It is feasible to ACCUMULATION IN FERMENTED SAUSAGES 53S associate diamines with the activity of undesired con- 54N taminant gram-negative bacteria, although fermenting Meat constitutes a protein-rich product with high flora, such as some lactobacilli and staphylococci, may water activity and a variety of nutrients that support 40454 43 455-468 r1.qxd 7/21/07 12:48 PM Page 463

Biogenic Amines: Risks and Control 463

the growth of a wide number of microorganisms. contaminant bacteria and the consequent forma- 1 During the fermentation and ripening of sausages, tion of biogenic amines (Maijala et al. 1993, 1995a; 2 the initially dominant gram-negative flora (such as Bover-Cid et al. 2000b, 2001c; González-Fernández 3 Enterobacteriaceae and Pseudomonas) is replaced et al. 2003). In summary, the type and the amount 4 by the more salt- and nitrite-tolerant and competitive of biogenic amines in a product depend on multiple 5 gram-positive species (such as lactic acid bacteria and complex variables, all of which interact, mak- 6 and staphylococci and micrococci). Among the vari- ing difficult to characterize the effects of each tech- 7 ables affecting aminogenesis in fermented sausages, nological factor on aminogenesis during sausage 8 those related to microbial contamination of raw mate- fermentation and ripening. Therefore, it is impor- 9 rials as well as the conditions governing the selection tant to study each particular case in order to eluci- 10 of the microbial communities and the activity of date when and why the aminogenesis occurs 11 amino acid decarboxylase bacteria are of crucial and then specifically apply the best measures of 12 relevance. control. 13 Therefore, in fermented sausage production, par- 14 ticular attention should be paid to the hygienic qual- 15 CONTROL MEASURES TO REDUCE BIOGENIC AMINE ity of meat and ingredients and also to the use and 16 ACCUMULATION IN FERMENTED SAUSAGES type of starter culture (Maijala et al. 1995a; Paulsen 17 and Bauer 1997; Bover-Cid et al. 2000a,b; Ansorena Literature reported biogenic amine levels for fer- 18 et al. 2002). Indeed, high amounts of cadaverine and mented sausages ranging from very small amounts 19 putrescine, as well as histamine, have been related to to nondetectable levels. These observations indicate 20 the poor hygienic quality of raw materials. In partic- that it is possible to produce fermented meat prod- 21 ular, enterobacteria contributes to cadaverine accu- ucts free or nearly free of biogenic amines. With this 22 mulation during the ripening of fermented sausages aim, several alternatives have been put forward. 23 made from meat stored for long periods (Paulsen Several authors have proposed the removal of 24 and Bauer 1997; Bover-Cid et al. 2000a) and also amines after their formation. Some microorganisms 25 from intentionally contaminated raw meat (Bover- of the sausage environment, such as a number of 26 Cid et al. 2003). micrococci and staphylococci strains, break down 27 In addition, the ingredients (sugars, additives such tyramine and/or histamine in vitro by means of 28 as curing agents, species, etc.) and diameter of the amine oxidase enzymes (Leuschner and Hammes 29 sausage, as well as the temperature and humidity 1998; Martuscelli et al. 2000). However, under real 30 during ripening affect biogenic amine accumulation conditions of sausage fermentation, amine-oxidizing 31 (Maijala et al. 1995b; Bover-Cid et al. 1999a, 2001b; microorganisms show a limited effect on tyramine 32 Parente et al. 2001; González-Fernández et al. 2003; and histamine levels (Leuschner and Hammes 1998; 33 Suzzi and Gardini 2003; Bozkurt and Erkmen 2004; Gardini et al. 2002). This ineffectiveness may be 34 Komprda et al. 2004; Garriga et al. 2005; Latorre- explained by low oxygen availability in the sausage 35 Moratalla et al. 2007). However, most of these and also amine-oxidizing bacteria below the 36 variables can show paradoxical effects on the accu- minimum 107 cfu/g required for amine degradation 37 mulation of these compounds because they exert (Leuschner and Hammes 1998). Degradation of bio- 38 influences through several phenomena associated genic amines has also been achieved by means of 39 with aminogenesis, including growth and interaction gamma irradiation. Doses from 2.5 kGy to 25 kGy 40 among microbial communities, acidification, prote- can destroy from 5% to 100% of biogenic amines 41 olysis, decarboxylase enzyme production and activ- dissolved in distilled water (Kim et al. 2004). In 42 ity, etc. For instance, the pH regulates the activity of sliced pepperoni sausages, radiolysis causes a slight 43 amino acid decarboxylases as a physiological system reduction of putrescine, tyramine, spermidine, and 44 of bacteria to neutralize an unfavorable acidic envi- spermine, but no effect on phenylethylamine and 45 ronment. Indeed, the maximum rate of tyramine cadaverine (Kim et al. 2005). Nevertheless, given 46 accumulation during sausage fermentation usually that biogenic amines may indicate poor hygienic 47 occurs when pH falls. A relationship between acidi- conditions, their removal a posteriori should be con- 48 fication and biogenic amine content has been sidered inappropriate or nonethical because it would 49 reported (Santos-Buelga et al. 1986; Buncic et al. imply lack of control over the quality of raw materi- 50 1993; Teodorovic and Buncic 1999; Bover-Cid et al. als and processing conditions. 51 1999b; Miguélez-Arrizado et al. 2006). However, Therefore, to reduce or prevent biogenic amine 52 the effect of pH on overall aminogenesis in fer- accumulation in fermented sausages “prevention is S53 mented sausages is controversial, because it is also better than cure.” It would be more suitable to apply N54 accepted that rapid and sharp acidification inhibits measures focused on the minimization of potentially 40454 43 455-468 r1.qxd 7/21/07 12:48 PM Page 464

464 Biological and Chemical Safety of Fermented Meat Products

1 aminogenic microorganisms, their growth, and their (Maijala et al. 1995a; Hernández-Jover et al. 1997b; 2 amino acid decarboxylase activity during production Bover-Cid et al. 1999b; Ayhan et al. 1999). The 3 and merchandising. Given the importance of the species L. sakei and L. curvatus are well adapted to 4 hygienic quality of raw meat material, the selection the meat fermentation environment, which make 5 of these materials, the control of storage time, and them good candidates for starter cultures because 6 temperature and freezing-thawing process are cru- they are highly competitive to outgrow spontaneous 7 cial to prevent excessive accumulation of biogenic fermenting flora and can inhibit gram-negative con- 8 amines during sausage manufacture (Maijala et al. taminants (Hugas and Monfort 1997). Indeed, the 9 1995a; Bover-Cid et al. 2000a, 2001d). In contrast to literature confirms that starter cultures, including 10 other fermented products, such as cheese, the raw decarboxylase-negative strains of L. sakei are the 11 materials for fermented sausages can not be pasteur- most protective because they reduce the overall amine 12 ized nor sterilized because these processes lead to accumulation by up to 95% in comparison with other 13 detrimental changes from both sensorial and techno- species commercially used as starters, such as L. plan- 14 logical points of view. However, attempts to optimize tarum, Pediococcus spp., and S. carnosus (González- 15 the hygienic quality of raw materials by reducing the Fernández et al. 2003; Bover-Cid et al. 2000b, 2000c; 16 loads of potentially aminogenic microorganisms in Latorre-Moratalla et al. 2007). Nevertheless, the 17 raw materials have been made by means of freezing effectiveness of the starter culture is strongly condi- 18 meat and lard (Bover-Cid et al. 2006) and also by tioned by raw materials of appropriate hygienic stan- 19 applying high hydrostatic pressure to meat batter dards (Maijala et al. 1995a; Eerola et al. 1996; 20 before fermentation (Latorre-Moratalla et al. 2007). Paulsen and Bauer 1997; Bover-Cid et al. 2001d). 21 These two strategies inhibit enterobacteria develop- Moreover, optimization of the technological condi- 22 ment and, as a result, contribute to a notable reductions favors proper implantation and development 23 tion of cadaverine accumulation. of the starter. Therefore, the extent of aminogene- 24 However, the microorganisms responsible for fer- sis reduction also depends on sausage formulation, 25 mentation, such as lactic acid bacteria and gram- the type and amount of sugar added (González- 26 positive catalase-positive cocci (staphylococci and Fernández et al. 2003), the addition of additives 27 micrococci), can also produce biogenic amines. (Bozkurt and Erkemn 2002), and the temperature 28 Enterococci may also be present, and even multiply, and relative humidity of the ripening conditions 29 during sausage fermentation because these strains (Maijala et al. 1995b; Bover-Cid et al. 2001e). 30 are tolerant to acid, sodium chloride, nitrite, and 31 other environmental stress factors, and they have CONCLUDING REMARKS 32 also been associated with the production of biogenic 33 amines, mainly tyramine (Bover-Cid et al. 2001a; A multidisciplinary approach combining the chemi- 34 Ansorena et al. 2002). Therefore, to control these cal, microbiological, and technological aspects of bio- 35 gram-positive aminogenic microorganisms, which genic amine accumulation in foods has been a 36 are inevitably in raw materials, specific technologi- cornerstone of the research in this field. This approach 37 cal measures are required. In this field, the use of has provided reliable answers about the origin and sig- 38 selected amino acid decarboxylase-negative starter nificance of amines as well as suitable technological 39 cultures has been repeatedly recommended as one of measures to minimize their occurrence. Biogenic 40 the most reliable tools by which to control fermenta- amines have no favorable effect on foodstuffs; on the 41 tion and, in turn, biogenic amine accumulation. Sev- contrary, in addition to the potential risks for sensitive 42 eral studies have examined the usefulness of starter consumers, they can reduce the overall hygienic qual- 43 cultures, including commercial strains and single ity of the product. Consequently, the food industry 44 and mixed cultures of experimental strains, to reduce faces the challenge of producing foods with low levels 45 aminogenesis during sausage fermentation. A number of amines, thereby minimizing health risks and assur- 46 of these studies failed to demonstrate a consistent ing the quality of the final product. 47 efficiency of Lactobacillus plantarum, Pediococ- 48 cus acidilatici, P. pentosaceus, or Staphylococcus REFERENCES 49 carnosus to reduce aminogenesis (Rice and Koehler 50 1976; Buncic et al. 1993; Paulsen and Bauer 1997; Amon U, Bangha E, Kuster T, Menne A, Vollrath IB, 51 Bozkurt and Erkemn 2002). Starter cultures consist- Gibbs BF. 1999. 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Int J Food Microbiol 11(1):73–84. 31 storage of dry fermented sausages. J Food Protect Buncic S, Paunovic L, Radisic D, Vojinovic G, 32 63(11):1544–1550. Smiljanic D, Baltic M. 1993. Effects of glucon- 33 Bover-Cid S, Hugas M, Izquierdo-Pulido M, Vidal- odeltalactone and Lactobacillus plantarum on the 34 Carou MC. 2000b. Reduction of biogenic amine for- production of histamine and tyramine in fermented 35 mation using a negative amino acid-decarboxylase sausages. Int J Food Microbiol 17:303–309. 36 starter culture for fermentation of fuet sausages. Chu C, Bjeldanes F. 1981. Effect of diamines, 37 J Food Protect 63(2):237–243. polyamines and tuna fish extracts on the binding of 38 Bover-Cid S, Izquierdo-Pulido M, Vidal-Carou MC. histamine to mucin in vitro. J Food Sci 47:79–80,88. 39 2000c. Mixed starter cultures to control biogenic Coïsson JD, Cerutti C, Travaglia F, Arlorio M. 2004. 40 amine production in dry fermented sausages. 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Changes in biogenic amine and polyamine Durlu-Özkaya F, Ayhan K, Vural N. 2001. Biogenic 52 contents in slightly fermented sausages manufac- amine produced by Enterobacteriaceae isolated from S53 tured with and without sugar. Meat Sci 57:215–221. meat products. Meat Sci 58:163–166. N54 40454 43 455-468 r1.qxd 7/21/07 12:48 PM Page 466

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1 EC (European Commission). 1991. Council Directive trations of biogenic amine contents in chorizo dry 2 91/493/EEC of 22 July 1991 laying down the health sausage. Food Microbiol 20:275–284. 3 conditions for the production and the placing on the Halász A, Baráth A, Simon-Sarkadi L, Holzapfel W. 4 market of fishery products. Official Journal of the 1994. Biogenic amines and their production by 5 European Communities L 268:15–34. microorganisms in food. Trends Food Sci Tech 6 Eerola S, Maijala R, Sagues AXR, Salminen M, 5:42–49. 7 Hirvi T. 1996. Biogenic amines in dry sausages as Hammes W, Bosch I, Wolf G. 1995. Contribution of 8 affected by starter culture and contaminant amine- Staphylococcus carnosus and Staphylococcus pis- 9 positive Lactobacillus. J Food Sci 61(6):1243–1246. cifermentans to the fermentation of protein foods. 10 Eerola S, Roig-Sagués A, Lilleberg L, Aalto H. 1997. 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A review of dietary 32 Gangolli SD, Brandt PA, Feron VJ, Janzowsky C, polyamines: Formation, implications for growth and 33 Koeman JH, Speijers GJA, Spiegelhandler B, Walter health and occurrence in foods. Food Chem 90(1–2): 34 R, Wishnow JS. 1994. Assessment: Nitrate, nitrite 219–230. 35 and N-nitroso compounds. Eur J Pharmac Environ Kim JH, Ahn HJ, Jo C, Park HJ, Chung YJ, Byun MW. 36 Toxic Pharmac Section 292:1–38. 2004. Radiolysis of biogenic amines in model system 37 Gardini F, Martuscelli M, Caruso MC, Galgano F, by gamma irradiation. Food Control 15(5):405–408. 38 Crudele MA, Favati F, Guerzoni ME, Suzzi G. 2001. Kim JH, Ahn HJ, Lee JW, Park HJ, Ryu GH, Kang IJ, 39 Effects of pH, temperature and NaCl concentration Byun MW. 2005. Effects of gamma irradiation on the 40 on the growth kinetic, proteolytic activity and bio- biogenic amines in pepperoni with different packag- 41 genic amine production of Enterococcus faecalis. Int ing conditions. Food Chem 89(2):199–205. 42 J Food Microbiol 64:105–117. Komprda T, Smela D, Pechova P, Kalhotka L, Stencl J, 43 Gardini F, Martuscelli M, Crudele MA, Paparela A, Klejdus B. 2004. Effect of starter culture, spice mix 44 Suzzi G. 2002. Use of Staphylococcus xylosus as a and storage time and temperature on biogenic amine 45 starter culture in dried sausage: Effect on the bio- content of dry fermented sausages. Meat Sci 67(4): 46 genic amine content. Meat Sci 61:275–283. 607–616. 47 Garriga M, Marcos B, Martín B, Veciana-Nogués MT, Korn A, Da Prada M, Raffesberg W, Allen S, Gasic S. 48 Bover-Cid S, Hugas S, Aymerich T. 2005. Starter cul- 1988. Tyramine pressor effect in man: Studies with 49 tures and high pressure processing to improve the moclobemide, a novel, reversible monoamine oxi- 50 hygiene and safety of slightly fermented sausages. dase inhibitor. J Neural Transm Suppl 26:57–71. 51 J Food Protect 68(11):2341–2348. Kranner P, Bauer F, Hellwig E. 1991. Investigations on 52 González-Fernández C, Santos E, Jaime I, Rovira J. the formation of histamine in raw sausages. Proc 53S 2003. Influence of starter cultures and sugar concen- 37th Int Congr Meat Sci Tech (ICoMST) 2:889–891. 54N 40454 43 455-468 r1.qxd 7/21/07 12:48 PM Page 467

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Latorre-Moratalla ML, Bover-Cid S, Aymerich T, Marcos Ochiai M, Wakabayashi K, Nagao M, Sugimura T. 1 B, Vidal-Carou MC, Garriga M. 2006. Aminogenesis 1984. Tyramine is a major mutagen precursor in soy 2 control in fermented sausages manufactured with sauce, being convertible to a mutagen by nitrite. 3 pressurized meat batter and starter culture. Meat Sci Gann 75(1):1–3. 4 75:460–469. Parente E, Martuscelli M, Gardini F, Grieco S, 5 Lehane L, Olley J. 2000. Histamine fish poisoning Crudele M, Suzzi G. 2001. Evolution of microbial 6 revisited. Int J Food Microbiol 58(1–2):1–37. populations and biogenic amine production in dry 7 Leuschner RGK, Hammes WP. 1998. Tyramine degra- sausages produced in Southern Italy. J Appl Micro- 8 dation by micrococci during ripening of fermented biol 90:882–891. 9 sausage. Meat Sci 49(3):189–196. Patat A, Berlin I, Durrieu G, Armand P, Fitoussi S, 10 Maijala R, Eerola S. 1993. Contaminant lactic acid bac- Molinier P, Caille P. 1995. 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Histidine decarboxylase activity of bacteria 30 catalase-positive cocci from slightly fermented isolated from raw and ripened salchichón, a Spanish 31 sausages. Int J Food Microbiol 107:148–158. cured sausage. J Food Protect 59(5):516–520. 32 Martuscelli M, Crudele M, Gardini F, Suzzi G. 2000. Roig-Sagués AX, Hernández-Herrero M, Rodríguez- 33 Biogenic amine formation and oxidation by Staphy- Jerez JJ, López-Sabater EI, Mora-Ventura MT. 1997. 34 lococcus xylosus from artisanal fermented sausages. Occurrence of tyramine producing microorganisms 35 J Appl Microbiol 31:228–232. in “salchichón” and tyramine production in sausages 36 Masson F, Talon R, Montel M. 1996. Histamine and inoculated with a tyramine producing strain of Lac- 37 tyramine production by bacteria from meat products. tobacillus brevis. J Food Safety 17:13–22 38 Int J Food Microbiol 32:199–207. Ruiz-Capillas C, Jiménez-Colmenero F. 2004. Biogenic 39 McCabe BJ. 1986. Dietary tyramine and other pressor amine content in Spanish retail market meta products 40 amines in MAOI regimens: A review. J Am Diet treated with protective atmosphere and high pres- 41 Assoc 86(8):1059–1064. sure. Eur Food Res Technol 218:237–241. 42 McIntyre T, Scanlan R. 1993. Nitrosamines produced in Santos-Buelga C, Peña-Egido MJ, Rivas-Gonzalo JC. 43 selected foods under extreme nitrosation conditions. 1986. Changes in tyramine during Chorizo-sausages 44 J Agric Food Chem 41:101–102. ripening. J Food Sci 51:518–527. 45 Miguélez-Arrizado MJ, Bover-Cid S, Vidal-Carou MC. Shalaby AR. 1996. Significance of biogenic amines to 46 2006. Biogenic amine contents in Spanish fermented food safety and human health. Food Res Int 29: 47 sausages of different acidification degree as a result 675–690. 48 of artisanal or industrial manufacture. J Sci Food Slerm J. 1981. Biogene Amine als potentieller chimis- 49 Agric 86:549–557. cher qualitätsindikator für Fleish. Fleischwirtsch 61: 50 Montel M, Masson F, Talon R. 1999. Comparison 921–926. 51 of biogenic amine content in traditional and industrial Straub BW, Kicherer M, Schilcher SM, Hammes 52 French dry sausages. Sci Aliment 19: 247–254. WP. 1995. The formation of biogenic amines by S53 N54 40454 43 455-468 r1.qxd 7/21/07 12:48 PM Page 468

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1 fermentation organisms. Z Lebensm Unters Forsh A Tschabrun R, Sick K, Bauer F, Kranner P. 1990. Bil- 2 201: 79–82. dung von Histamin in schnittfesten Rohwürsten. 3 Straub BW, Tichaczek PS, Kicherer M, Hammes WP. Fleischwirtsch 70:448–452. 4 1994. Formation of tyramine by Lactobacillus Vermeer IT, Pachen DM, Dallinga JW, Kleinjans 5 curvatus LTH-972. Z Lebensm Unters Forsh A 199: JC, van Maanen JM. 1998. Volatile N-nitrosamine 6 9–12. formation after intake of nitrate at the ADI level 7 Suzzi G, Gardini F. 2003. Biogenic amines in dry fer- in combination with an amine-rich diet. Environ 8 mented sausages: A review. Int J Food Microbiol Health Perspect 106(8):459–463. 9 88:41–54. Vidal-Carou MC, Izquierdo ML, Martín MC, Mariné 10 Taylor SL. 1986. Histamine food poisoning— A. 1990. Histamina y tiramina en derivados cárnicos. 11 Toxicology and clinical aspects. Crit Rev Toxicol Rev Agroquim Tecnol Aliment 30:102–108. 12 17(2):91–128. Warthesen JJ, Scanlan RA, Bills DD, Libbey LM. 1975. 13 Teodorovic V, Buncic S. 1999. The effect of pH on tyra- Formation of heterocyclic N-nitrosamines from the 14 mine production in fermented sausages. Fleis- reaction of nitrite and selected primary diamines and 15 chwirtsch 79(5):85–88. amino acids. J Agric Food Chem 23(5):898–902. 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53S 54N 40454 44 469-476 r1.qxd 7/20/07 5:23 PM Page 469

1 2 3 44 4 5 6 Chemical Origin Toxic Compounds 7 8 Fidel Toldrá and Milagro Reig 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION nitrosylmyoglobin, its contribution to the oxidative 22 stability of lipids, and its contribution to the devel- 23 The development of modern analytical technologies opment of typical and distinctive cured meat ﬂavor. 24 linked to epidemiologic studies and investigations on Nitrite can be converted to nitric oxide, which is a 25 safety aspects of food components have revealed in nitrosating agent. This agent can react with secondary 26 the latest decades that some toxic compounds may be amines and produce potent carcinogenic nitrosamines. 27 present or generated in certain types of meat process- Compounds such as N-nitrosodimethylamine have 28 ing. This is the case of N-nitrosamines when using been shown to be carcinogenic in a wide range of ani- 29 nitrite as a preservative under certain conditions, the mal species. The presence of volatile N-nitrosamines 30 polycyclic aromatic hydrocarbons generated in cer- have been largely studied in cured meat products. 31 tain smoking processes, oxidation of lipids and pro- Some of the most important are N-nitrosodimethy- 32 teins, and other products that may be present in the lamine, N-nitrosopirrolidine, N-nitrosopiperidine, 33 raw materials used for processing (veterinary drug N-nitrosodiethylamine, N-nitrosodi-n-propylamine, 34 residues, environmental contaminants, etc.). Even N-nitrosomorpholine and N-nitrosoethylmethylamine. 35 though meat fermentation is an old technology used In addition, a large number of nonvolatile nitroso 36 for generations in many countries, corrective mea- compounds, higher in molecular weight and 37 sures (i.e., reduction in the addition of nitrite, control more polar, have also been reported. Some of the 38 of raw materials, etc.) have been taken to minimize most important are N-nitrosoaminoacids such 39 this problem. All these hazardous compounds are as N-nitrososarcosine and N-nitrosothiazolidine- 40 brieﬂy described in this chapter. Biogenic amines, 4-carboxylic acid, hydroxylated N-nitrosamines, 41 which are generated by microbial decarboxylation of N-nitroso sugar amino acids, and N-nitrosamides 42 certain amino acids are dealt with in Chapter 43, such as N-nitrosoureas, N-nitrosoguanidines, and 43 “Biogenic Amines and Nitrosamines.” N-nitrosopeptides (Pegg and Shahidi 2000). The 44 amount of N-nitrosamines in meat products depends 45 N-NITROSAMINES on many variables, such as the amount of added and 46 residual nitrite, processing conditions, amount of 47 Nitrite is the main additive used as a preservative in lean meat in the product, heating if any, and presence 48 fermented meats because of its powerful inhibition of catalysts or inhibitors (Hotchkiss and Vecchio 49 of the outgrowth of spores of putrefactive and patho- 1985; Walker 1990). Table 44.1 lists some carcino- 50 genic bacteria like Clostridium botulinum. Nitrite genic nitrosamines. 51 also has some other technological roles, such as its The presence of N-nitrosamines in certain cured 52 contribution to the color through the formation of meat products was the reason for a serious debate 53 S54 N55 469 40454 44 469-476 r1.qxd 7/20/07 5:23 PM Page 470

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1 Table 44.1. List of N-nitrosamines with carcinogenic rubber nettings. These nitrosamines were generated 2 properties. because the reaction of nitrite with amine additives 3 in the rubber nettings (Sen et al. 1987). N-nitrosamines 4 Even though nitrate has no preservative effect, it 5 N-nitrosodiethylamine acts as a reservoir of nitrite in traditional dry meat 6 products, typical of the Mediterranean countries, N-nitrosodiethanolamine 7 like long-ripened dry-fermented sausages and dry- 8 N-nitrosopirrolidine cured ham. Long-ripening processes, with a slow 9 N-nitrosopiperidine and mild pH drop, are typical of traditional dry- 10 N-nitrosodi-n-propylamine fermented sausages. In these cases, the added nitrate 11 is progressively reduced to nitrite and contributes to 12 N-nitrosodi-n-butylamine safety as well as to particular sensory properties. 13 N-nitrosomethylbenzylamine The detection of nitrosamines is complex. 14 Volatile N-nitrosamines can be extracted from the 15 meat matrix by aqueous distillation and then ana- 16 lyzed by gas chromatography coupled to a thermal 17 held in the 1970s on residual nitrite in cured meats. energy analyzer. Other extraction methods can be 18 As a consequence, the levels of nitrites were low- solid phase extraction with specific solvents (Raoul 19 ered, and the use of ascorbate or erythorbate were et al. 1997) or supercritical fluid extraction (Fiddler 20 recommended to inhibit nitrosamines formation and Pensabene 1996). When specific identification 21 (Cassens 1997). Ascorbate reacts with nitrite 240 and confirmation of N-nitrosamines are necessary, 22 times faster than ascorbic acid does, and this is the mass spectrometry detectors are coupled to the gas 23 reason why it is preferred (Pegg and Shahidi 2000). chromatographs or, in other cases, liquid chromatog- 24 Erythorbic and ascorbic acids compete with amines raphy atmospheric pressure chemical ionization 25 for nitrite. mass spectrometry and tandem mass spectrometry 26 It must be mentioned that nitrite and nitric oxide (Eerola et al. 1998). 27 are normal human metabolites. Nitric oxide can be 28 generated in the human organism by enzymatic 29 (nitric oxide synthase) reaction of arginine. This nitric POLYCYCLIC AROMATIC 30 oxide can then be converted to nitrate and nitrite, HYDROCARBONS (PAH) 31 which then can be excreted (Leaf et al. 1989). In addi- 32 tion, the endogenous generation of N-nitrosamines Traditionally, smoking has consisted of the exposi- 33 from precursors has also been reported in the gas- tion of meat products to the smoke generated by con- 34 trointestinal tract (Vermeer et al. 1998). trolled combustion of certain natural hardwoods, 35 A key factor in the N-nitrosamines generation is sometimes accompanied by aromatic herbs and 36 the amount of nitrite added to the meat product. The spices. The pyrolysis of wood generates smoke 37 fate of nitrite is dependent on many variables, such through different oxidation routes. Moist wood chips 38 as the pH of the product, the processing tempera- can also be used for direct generation of smoke. The 39 tures used, and the addition of reducing substances smoke is condensed and adsorbed on the surface of 40 such as ascorbate or isoascorbate. In general, the the meat product. It can penetrate to a certain depth 41 levels of residual nitrite decrease quite rapidly dur- into the product depending on the conditions of the 42 ing processing, remaining at low percentages in the process. The smoke contains flavoring substances 43 final product in relation to the initial added nitrite that exert typical smoke flavor but also contain some 44 (Hill et al. 1973). According to different surveys, health-hazardous compounds like polycyclic aro- 45 made in the late 1990s and early 2000s in European matic hydrocarbons (PAH), phenols and formalde- 46 countries, the residual nitrite content in fermented hyde (Bem 1995). Formaldehyde has been identified 47 sausages was found to be below 20 mg/kg in most of as a cause of cancerous tumours. Some polycyclic 48 the products (EFSA 2003). Nitrosodimethylamine aromatic hydrocarbons, especially benzo-a-pyrene, 49 and nitrosopiperidine were the main nitrosamines are known to possess cancer-inducing and carcino- 50 found at levels above 1 µg/kg. In other surveys made genic properties. The most important polycyclic aro- 51 on European fermented sausages, the levels of matic hydrocarbons are listed in Table 44.2. 52 nitrosamines were rather poor or even negligible Furthermore, some smoke phenols could react to 53S (Demeyer et al. 2000). Some N-nitrosamines have form highly toxic nitrosophenols that could further 54N also been reported in hams packaged in elastic react to form toxic reaction products like nitrophenols, 40454 44 469-476 r1.qxd 7/20/07 5:23 PM Page 471

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Table 44.2. List of polycyclic aromatic compounds These flavorings are produced from primary prod- 1 (PAH) potentially present in primary products used for ucts obtained from different woods after specific 2 the production of smoke flavorings. pyrolisis conditions and extraction protocols. In 3 addition to forming part of the smoke flavorings, 4 Polycyclic Aromatic Compounds (PAH) these primary products can be used as such in foods. 5 Anthanthrene Smoke flavorings have a wide variability of com- 6 pounds, including polycyclic aromatic hydrocarbons 7 Benz[a]anthracene (Jennings 1990; Maga 1987). The toxicological 8 Benzo[b]fluoranthene effects of smoke flavorings can vary significantly 9 Benzo[j]fluoranthene among preparations because these effects depend on 10 Benzo[k]fluoranthene many factors, such as the production process of the 11 primary products, the qualitative and quantitative 12 Benzo[ghi]perylene composition, the concentration used in the flavoring, 13 Benzo[a]pyrene and the final use levels (SCF 1995). Recently, the 14 Chrysene application of smoke flavoring primary products is 15 Cyclopenta[cd]pyrene controlled in the European Union through the Coun- 16 cil Regulation 2065/2003 of 10 November 2003 on 17 Dibenz[a,h]anthracene smoke flavorings used or intended for use in or on 18 Dibenzo[a,e]pyrene foods (EC 2003). Under this regulation, the use of a 19 Dibenzo[a,h]pyrene primary product in and on foods shall be authorized 20 only if it is sufficiently demonstrated that it does not 21 Dibenzo[a,i]pyrene present risks to human health. The European Food 22 Dibenzo[a,l]pyrene safety Authority (EFSA) will issue a list of Primary 23 Indeno[1,2,3-cd]pyrene Products allowed for use as such in/or on food and/or 24 5-Methylchrysene for the production of derived smoke flavorings. 25 Studies on subchronic toxicity and genotoxicity 26 Perylene must be performed to evaluate the potential toxico- 27 Phenanthrene logical effects of the primary products used for the 28 Pyrene smoke flavoring. 29 Detection of PAHs can be performed with gas 30 chromatography coupled to a flame ionization detec- 31 polymeric nitrosic compounds, and other toxic tor or high-performance liquid chromatography cou- 32 compounds or even catalyzed the formation of pled to ultraviolet or fluorescence detectors. In other 33 nitrosamines (Bem 1995). The worse situations are cases, mass spectrometry detectors are coupled to 34 found in heavily smoked meat products with old or both types of chromatographies for the identification 35 inadequate smokehouses. In any case, the content in and confirmation of PAHs. 36 PAH is highly variable because it depends on many 37 variables like the use of direct or indirect smoking, 38 the type of generator used, the type and composition OXIDATION 39 of wood and herbs, the accessibility to oxygen, and 40 LIPID-DERIVED COMPOUNDS the temperature and time of the process. 41 Some alternative processes have been designed to Triacylglycerols, phospholipids, lipoproteins, and 42 reduce the contamination of the smoked meat prod- cholesterol are the main lipid compounds. Phospho- 43 ucts with hazardous compounds. Some of these lipids are very susceptible to oxidation due to its 44 strategies, which can reduce significantly the PAH high content in polyunsaturated fatty acids. Lipid 45 content in smoked meat products, consist of the fil- oxidation follows a free radical mechanism consist- 46 tration of particles, use of cooling traps, use of lower ing in three steps: initiation, propagation, and termi- 47 temperatures, and/or reduction of the duration of the nation. Hydroperoxides are the primary products of 48 process. Alternatively, liquid smoke can be obtained oxidation but they are flavorless, whereas the sec- 49 through distillation and subsequent condensation of ondary products of oxidation can contribute to off- 50 volatile compounds and then applied to the surface flavors, color deterioration, and potential generation 51 of the meat product. Another strategy with extended of toxic compounds (Kanner 1994). Lipid oxidation 52 use consists of the use of smoke flavorings incorpo- may also be induced by hydrogen peroxide gener- S53 rated at concentrations within the range of 0.1–1.0%. ated by peroxide-forming bacteria grown during N54 40454 44 469-476 r1.qxd 7/20/07 5:23 PM Page 472

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1 meat fermentation. Some products of lipid oxidation • Therapeutic agents: medicines used to control 2 may be chronic toxicants and have been reported to infectious diseases 3 contribute to aging, cancer, and cardiovascular dis- • Prophylactic agents: drugs given to prevent 4 eases (Hotchkiss and Parker 1990). In any case, the outbreaks of diseases and control parasitic 5 low levels are far from any acute toxicity. infections 6 Cholesterol oxidation may occur through an • Growth-promoting agents—Anabolic agents: 7 autooxidative process or in conjunction to fatty acid substances added to improve the feed conversion 8 oxidation (Hotchkiss and Parker 1990). Cholesterol efficiency by increasing the lean:fat ratio 9 oxides are considered as prejudicial for health due to • Growth-promoting agents—Antimicrobial 10 its role in arteriosclerotic plaque and also as a muta- agents: substances added to make more 11 genic, carcinogenic, and cytotoxic (Guardiola et al. nutrients available to the animal and not to the 12 1996). No cholesterol oxides have been detected after gut bacteria 13 heating of pork sausages (Baggio and Bragagnolo 14 2006). Studies made on European sausages revealed The abuse of antibiotics has increased the concern 15 the generation of cholesterol oxides up to 1.5 µg/g on the development of bacteria-resistant to antibi- 16 even though the percentage of cholesterol oxidation otics as detected in recent years (Butaye et al. 2001). 17 was below 0.17. The major cholesterol oxide found in The administration of most veterinary drugs was 18 Italian sausage was reported to be 7-ketocholesterol banned in the European Union because of fears 19 whereas 5,6α-5,6-epoxycholesterol was the major about health effects from residues. Some of them 20 end product in the other analyzed sausages may exert genotoxic, immunotoxic, carcinogenic, or 21 (Demeyer et al. 2000). In any case, the values were endocrine effects on consumers. These substances 22 quite below those considered as toxic based on in can be administered to animals only for therapeutic 23 vivo tests with laboratory animals (Bösinger et al. purposes under strict control of a responsible veteri- 24 1993). narian (Van Peteghem and Daeselaire 2004). Main 25 veterinary drugs and substances with anabolic effect 26 are listed in Table 44.3. If these substances are ille- 27 PROTEIN-DERIVED COMPOUNDS gally added to farm animals to improve feed conversion rate and promote animal growth, they could 28 The generation of hydrogen peroxide by certain bac- remain in the treated-animal–derived foods and 29 teria during meat fermentation may induce oxidation could constitute a health concern. The meat process- 30 of proteins. The main modifications of amino acids ing industry also faces some problems when using 31 by oxidation consist of the formation of carbonyl treated meat as raw materials for the manufacture of 32 groups, thiol oxidation, and aromatic hydroxylation fermented meat products. Some of these problems 33 (Morzel et al. 2006). Sulfur amino acids of proteins include lower quality of the resulting products, prob- 34 are those more susceptible to oxidation by peroxide lems in fermentation if there are antibiotic residues, 35 reagents, like hydrogen peroxide. So, cystine is oxi- and less fat with the subsequent loss in juiciness and 36 dized only partly to cysteic acid, whereas methionine poorer flavor development (Brockman and Laarveld 37 is oxidized to methionine sulfoxide and methionine 1986). For all these reasons, but very especially the 38 sulfone in a minor amount (Slump and Schreuder health concern, the presence of these substances in 39 1973). Sulfinic and cysteic acids can also be pro- farm animals and foods of animal origin must be 40 duced by direct oxidation of cysteine (Finley et al. monitored (Croubels et al. 2004). The development 41 1981). The oxidation of homocystine can generate of new methodologies for the analysis of antimicro- 42 homolanthionine sulfoxide as the main product bials and hormone residues was recently supported 43 (Lipton et al. 1977). Peptides like the reduced glu- by the EC Quality of Life Programme (Boenke 44 tathione can also be oxidized by hydrogen peroxide. 2002). 45 The oxidation rates increase with the pH, and most In the European Union, the presence of these sub- 46 of the cysteine in the glutathione is oxidized to the stances in foods and the number of samples to be 47 monoxide or dioxide forms. 48 tested each year are regulated by the EC Directive 49 96/23/EC on measures to monitor certain substances 50 VETERINARY DRUG RESIDUES and residues in live animals and animal products. 51 The analytical methodology for the monitoring of 52 Veterinary pharmaceutical drugs have been used in compliance was given in Decisions 93/256/EEC and 53S animals for different purposes (Dixon 2001) such as 93/257/EEC. The Council Directive 96/23/EC was 54N the following: recently implemented by the Commission Decision 40454 44 469-476 r1.qxd 7/20/07 5:23 PM Page 473

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Table 44.3. List of veterinary drugs and substances with anabolic effect according to classification in Council 1 Directive 96/23/EC. 2 3 Group A: Substances Having Some Main Substances within 4 Anabolic Effect Each Subgroup 5 1. Stilbenes Diethylstilbestrol 6 7 2. Antithyroid agents Thiouracils, mercaptobenzimidazoles 8 3. Steroids 9 Androgens Trenbolone acetate 10 Gestagens Melengestrol acetate 11 Estrogens 17-β-estradiol 12 4. Resorcycilic acid lactones Zeranol 13 5. β-agonists Clenbuterol, mabuterol, salbutamol 14 15 6. Other substances Nitrofurans 16 Group B:Veterinary Drugs 17 1. Antibacterial substances Sulfonamides, tetracyclines, β-lactam, 18 —macrolides (tylosin), quinolones, 19 —aminoglycosides, carbadox, olaquindox 20 21 2. Other veterinary drugs 22 Antihelmintics Benzimidazoles, probenzimidazoles, 23 —piperazines, imidazothiazoles, avermectins, 24 —tetrahydropyrimidines, anilides 25 Anticoccidials Nitroimidazoles, carbanilides, 26 —4-hydroxyquinolones, pyridinols, ionophores 27 Carbamates and pyrethroids Esters of carbamyc acid, type 1 and 2 pyrethroids 28 Sedatives Butyrophenones, promazines, β-blocker 29 —carazolol 30 Nonsteroideal antiinflammatory drugs Salicylates, pyrazolones, nicotinic acids, 31 —phenamates, arylpropionic acids, pyrrolizines 32 Other pharmacologically active substances Dexamethasone 33 Group B: Contaminants 34 3. Environmental contaminants 35 Organochlorine compounds PCBs, compounds derived from aromatic, 36 —ciclodiene, or terpenic hydrocarbons 37 Organophosphorous compounds Malathion, phorate 38 Chemical elements Heavy metals 39 Mycotoxins Aflatoxins, deoxynivalenol, zearalenone 40 Dyes 41 Others 42 43 44 45 46 2002/657/EC, which has been in force since National Residue Program, the exploratory residue 47 September 1, 2002. This decision provided rules for testing programs, and inspector generated in-plant 48 the analytical methods to be used in testing of offi- residue test samples (Croubles et al. 2004). The FDA 49 cial samples and specific common criteria for the Center for Veterinary Medicine issues the analytical 50 interpretation of analytical results of official control criteria. The USDA designed an Additional Testing 51 laboratories for such samples. In the United States, Program to ensure the control of residues of these 52 the Food Safety and Inspection Services (FSIS) substances in the meats exported to the European S53 establishes the surveillance programs, including the Union (Croubles et al. 2004). N54 40454 44 469-476 r1.qxd 7/20/07 5:23 PM Page 474

474 Biological and Chemical Safety of Fermented Meat Products

1 The controls for the detection of these substances Z Bem. 1995. Desirable and undesirable effects of 2 are usually based on screening tests, most of the times smoking meat products. Die Fleischerei 3:3–8. 3 ELISA test kits or antibody-based automatic tech- A Boenke. 2002. Contribution of European research to 4 niques. In the case of antibiotics, microbiological tests anti-microbials and hormones Analytica Chimica 5 such as the European Four Plate Test are used. Screen- Acta 473:83–87. 6 ing tests are relatively rapid but give only qualitative S Bösinger, W Luf, E Brandl. 1993. Oxysterols: Their 7 or semiquantitative data that require further confirma- occurrence and biological effects. Inter Dairy J 8 tion. Thus, those samples suspected to be noncompli- 3:1–33. 9 ant are confirmed through gas or high-performance RP Brockman, R Laarveld. 1986. Hormonal regulation 10 liquid chromatography coupled to mass spectrometry of metabolism in ruminants. Rev. Livestock Prod Sci 11 or other sophisticated methodologies for accurate 14:313–317. 12 characterization and confirmation (Toldrá and Reig P Butaye, LA Devriese, F Haesebrouck. 2001. Differ- 13 2006). Immunochromatography has been used recen- ences in antibiotic resistance patterns of Enterococ- 14 tly for specific veterinary drugs for further cleaning of cus faecalis and Enterococcus faecium strains 15 samples in order to increase sensitivity. isolated from farm and pet animals. Antimicrob 16 Agents Chemother 45:1374–1378. 17 ENVIRONMENTAL RG Cassens. 1997. Composition and safety of cured 18 meats in the USA. Food Chem 59:561–566. 19 CONTAMINANTS S Croubels, E Daeselaire, S De Baere, P De Backer, 20 There are numerous environmental contaminants D Courtheyn. 2004. Feed and drug residues. 21 that may be present in the meats used as raw materi- In: Encyclopedia of Meat Sciences. W Jensen, 22 als for the manufacture of fermented meat products. C Devine, M Dikemann, eds. London, UK: Elsevier 23 These contaminants include dioxins, organophos- Applied Science, pp. 1172–1187. 24 phorous and organochlorine compounds including DI Demeyer, M Raemakers, A Rizzo, A Holck, A De 25 PCBs, mycotoxins, and heavy metals among others. Smedt, B Ten Brink, B Hagen, C Montel, E Zanardi, 26 In the case of PCBs (polychlorinated biphenyls), its E Murbrek, F Leroy, F Vanderdriessche, K Lorentsen, 27 use was phased out but they remain in the environ- K Venema, L Sunesen, L Stahnke, L De Vuyst, 28 ment for many years, especially in stream and lake R Talon, R Chizzolini, S Eerola. 2000. Control of 29 sediments (Moats 1994). bioflavor and safety in fermented sausages: First 30 The contaminants described above can be present results of a European project. Food Res Inter 31 in feeds given to the animals as a direct cause of con- 33:171–180. 32 tamination of the meat. The contamination of feeds SN Dixon. 2001. Veterinary drug residues. In: Food 33 may have many reasons, such as inappropriate for- Chemical Safety. Volume 1: Contaminants. DH 34 mulation, lack of control, use of contaminated ingre- Watson, ed. Cambridge. England: Woodhead 35 dients, inadequate processes, etc. (Croubels et al. Publishing, Ltd., pp. 109–147. 36 2004). For instance, some molds like species of EC. 2003. European Parliament and Council Regula- 37 Fusarium, Aspergillus, and Penicillium can grow in tion 2065/2003 of 10 November 2003 on smoke 38 feed grains and meals and contaminate them with flavourings used or intended for use in or on foods. 39 mycotoxins. Some of these mycotoxins are toxic, for S Eerola, I Otegui, L Saari, A Rizzo. 1998. Application 40 of liquid chromatography atmospheric pressure example, aflatoxin B1, which is a genotoxic and car- 41 cinogenic substance. chemical ionization mass spectrometry and tandem 42 The assessment of environmental contaminants mass spectrometry to the determination of volatile 43 differs from the previous residues because the latest nitrosamines in dry sausages. Food Addit Contam 44 are subject of safety evaluation (analytical controls), 15:270–279. 45 whereas some contaminants are unavoidable EFSA. 2003. The effects of nitrites/nitrates on the 46 even though they can exert great potential toxicity microbiological safety of meat products. The EFSA J 47 (Heggum 2004). 14:16. 48 W Fiddler, JW Pensabene. 1996. Supercritical fluid 49 REFERENCES extraction of volatile N-nitrosamines in fried bacon 50 and its drippings: Method comparison. J AOAC Inter 51 SR Baggio, N Bragagnolo. 2006. The effect of heat 79:895–901. 52 treatment on the cholesterol oxides, cholesterol, total JW Finley, EL Wheeler, SC Witt. 1981. Oxidation of 53S lipid and fatty acid contents of processed meat prod- glutathione by hydrogen peroxide and other oxidiz- 54N ucts. Food Chem 95:611–619. ing agents. J Agric Food Chem 29:404–407. 40454 44 469-476 r1.qxd 7/20/07 5:23 PM Page 475

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F Guardiola, R Codony, PB Addis, M Rafecas, M Morzel, P Gatellier, T Sayd, M Renerre, E Laville. 1 P Boatella. 1996. Biological effects of oxysterols: 2006. Chemical oxidation decreases proteolytic sus- 2 Current status. Food Chem Toxicol 34:193–198. ceptibility of skeletal muscle myofibrillar proteins. 3 C Heggum. 2004. Risk analysis and quantitative risk Meat Sci 73:536–543. 4 management. In: Encyclopedia of Meat Sciences. RB Pegg, F Shahidi. 2000. Nitrite curing of meat. 5 W Jensen, C Devine, M Dikemann, eds. London, Trumbull, Connecticut: Food & Nutrition Press, 6 UK: Elsevier Applied Science, pp. 1192–1201. pp. 175–208. 7 LH Hill, NB Webb, LD Mongol, AT Adams. 1973. S Raoul, E Gremaud, H Biaudet, RJ Turesky. 1997. 8 Changes in residual nitrite in sausages and luncheon Rapid solid-phase extraction method for the detec- 9 meat products during storage. J Milk Food Technol tion of volatile nitrosamines in food. J Agric Food 10 36:515–519. Chem 45:4706–4713. 11 JH Hotchkiss, RS Parker. 1990. Meat and Health. AM SCF. 1995. Smoke flavorings. Report of the Scientific 12 Pearson, TR Dutson, eds. London, UK: Elsevier Committee for Food. Opinion adopted on 23 June 13 Applied Science, pp. 105–134. 1993. 34 series Food Science Techniques. European 14 JH Hotchkiss, AL Vecchio. 1985. Nitrosamines in fired- Commission. 15 out bacon fat and its use as a cooking oil. Food Tech- NP Sen, PA Baddoo, SW Seaman. 1987. Volatile 16 nol 39:67–73. nitrosamines in cured meats packaged in elastic rub- 17 WG Jennings. 1990. Analysis of liquid smoke and ber nettings. J Agric Food Chem 35:346–350. 18 smoked meat volatiles by headspace gas chromatog- P Slump, HAW Schreuder. 1973. Oxidation of methio- 19 raphy. Food Chem 37:135–144. nine and cystine in foods treated with hydrogen per- 20 J Kanner. 1994. Oxidative processes in meat and meat oxide. J Sci Food Agric 24:657–661. 21 products: Quality implications. Meat Sci 36: F Toldrá, M Reig. 2006. Methods for rapid detection of 22 169–189. chemical and veterinary drug residues in animal 23 CD Leaf, JS Wishnok, SR Tannenbaum. 1989. foods. Trends Food Sci Technol 17:482–489. 24 L-arginine is a precursor for nitrate biosynthesis in C Van Peteguem, E Daeselaire. 2004. Residues of 25 humans. Biochimica Biophysica Res Commun Growth Promoters. In: Handbook of Food Analysis, 26 163:1032–1037. 2nd ed. LML Nollet, ed. New York: Marcel Dekker, 27 SH Lipton, CE Bodwell, AH Coleman, Jr. 1977. Amino Inc., pp. 1037–1063. 28 acid analyzer studies of the products of peroxide IT Vermeer, DM Pachen, JW Dallinga, JC Kleinjans, 29 oxidation of cystine, lanthionine and homocystine. JM van Maanen. 1998. Volatile N-nitrosamine for- 30 J Agric Food Chem 25:624–628. mation after intake of nitrate at the ADI level in com- 31 JA Maga. 1987. The flavour chemistry of wood smoke. bination with an amine-rich diet. Environ Health 32 Food Rev Inter 3:139–183. Perspect 106:459–463. 33 WA Moats. 1994. Chemical residues in muscle foods. In: R Walker. 1990. Nitrates, nitrites and nitrosocom- 34 Muscle Foods. Meat, Poultry and Seafood Technology. pounds: A review of the occurrence in food and diet 35 DM Kinsman, AW Kotula, BC Breidenstein, eds. New and the toxicological implications. Food Addit Con- 36 York: Chapman and Hall, pp. 288–295. tam 7:717–768. 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 S53 N54 40454 45 477-480 r1.qxd 7/21/07 12:49 PM Page 477

1 2 3 45 4 5 6 Disease Outbreaks 7 8 Colin Pierre 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION 1990; Chiou et al. 2002). It is hypothesized that use 22 of these containers promotes an anaerobic environ- 23 Foodborne disease is caused by consuming foods ment in which C. botulinum thrives. 24 contaminated by microbes (bacteria, viruses, or 25 parasites) or poisonous chemicals, including toxins 26 produced by some bacteria such as Clostridium STAPHYLOCOCCUS 27 botulinum or Staphylococcus aureus. 28 A foodborne outbreak occurs when a group of Staphylococcal food poisoning is an intoxication caused by the consumption of foods containing 29 people consume the same contaminated food and 30 two or more of them come down with the same enterotoxins produced by certain strains of Staphylo- coccus aureus (Wieneke et al. 1993) and is of major 31 illness. But it could be mentioned that the majority 32 of reported cases of foodborne illness are not part concern for public health. It seems that the type of foods involved differ widely among countries (Le 33 of recognized outbreaks and occur as individual or 34 “sporadic” cases. Loir et al. 2003). Nevertheless, ham, sausages, and other fermented meat could be associated with out- 35 Some fermented products are sometimes associ- 36 ated with foodborne outbreaks. The main example breaks (Anon. 1977, 1979; Bergdoll 1989; Wieneke et al. 1993). 37 should be, in the past, the relationship between 38 “home-made” fermented pork meat and the presence 39 of Clostridium botulinum producing a powerful SALMONELLA, 40 paralytic toxin in the food during the shelf life CAMPYLOBACTER 41 (Hauschild and Gauvreau 1985; Anon. 2003). More 42 recently, in Alaska in 2001, 3 persons out of 14 con- Salmonellosis and campylobacteriosis remain the 43 suming fermented beaver tail and paw had symp- two most common human foodborne outbreaks in 44 toms suggestive of botulism, and type E toxin was industrialized countries. 45 detected in serum specimen and in stools. The type E In 2004, 5,067 outbreaks (out of 6,860) caused by 46 toxin was also detected in three beaver paws impli- Salmonella spp. were reported in the European 47 cated in this meal (Anon. 2001). The “fermented” Union, affecting a total of 30,638 persons, with 48 tail and paws had been wrapped in a paper rice sack 10.8% hospitalized and 12 deaths (Anon. 2005). 49 and stored for up to 3 months. It seems that changing There is a very strong relationship between outbreaks 50 the food preparation, and particularly using plastic due to the presence of Salmonella Enteritidis and the 51 or glass containers for fermentation of food in this consumption of eggs and egg products. The second 52 country, increase the risk of botulism (Shaffer et al. serovar involved is Salmonella Typhimurium, mainly 53 S54 N55 477 40454 45 477-480 r1.qxd 7/21/07 12:49 PM Page 478

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1 associated with the consumption of meat and meat VEROTOXIGENIC STRAINS OF 2 products. Unfortunately, no information on the type ESCHERICHIA COLI 3 of meat (fresh, cured, fermented) was done. Never- 4 theless, during the last decades, some outbreaks Verotoxigenic strains of Escherichia coli (VTEC), 5 involving Salmonella spp. and fermented meat and in particular E. coli O157:H7, have been identi- 6 occurred in Europe: fied in some raw fermented finished products associ- 7 ated with foodborne outbreaks: 8 • In 1986, Van Netten et al. described a small 9 outbreak in August, 1985 (17 patients, without • For Tilden et al. (1996), studying an outbreak in 10 deaths or complications) due to the presence of Washington and California involving E. coli 11 Salmonella Typhimurium (106 per gram) in O157:H7, dry-fermented salami can serve as a 12 Bologna sausage. vehicle of transmission of these bacteria which 13 • In 1987–88, an outbreak of Salmonella can survive “currently accepted processing 14 Typhimurium DT 124 affected 101 patients methods.” 15 in England. The epidemiological and micro- • In 1995, a large outbreak of hemolytic-uremic 16 biological investigations identified a salami syndrome (HUS) occurred in Australia (Paton 17 stick as the vehicle of infection (Cowden et al. et al. 1996). Twenty-one cases with one fatality, 18 1989). from 4 months to 12 years old, were identified. 19 • In Italy, Pontello et al. (1998) described a Different strains of E. coli were isolated, proving 20 community-based outbreak of Salmonella that outbreaks are not necessarily caused by 21 Typhimurium PT193 associated with the contamination with a single “outbreak” strain. 22 consumption of salami. Nevertheless, a large number of these strains, and 23 • More recently, an outbreak due to Salmonella others, were isolated from samples from a 24 Typhimurium associated with a traditional salted, semidry-fermented sausage (mettwurst) 25 smoked, and dried ham, after a family party, suspected and collected from the homes of 26 was described (Mertens et al. 1999). Salmonella patients and from retail outlets. This product was 27 Typhimurium PT20 was found in Coburger ham made from a mixture of raw pork, beef, and lamb. 28 and in feces of patients. Only the consumption of • In Canada, in 1998, 39 cases of E. coli O157:H7 29 Coburger ham and “bone ham,” originated from infection were identified, and 2 children were 30 the same batch of meat and prepared in the same diagnosed with HUS. Consumption of Genoa 31 manner in the same salt bath, was significantly salami was significantly associated with the 32 related to a positive patient’s stools culture. illness. Phage typing and Pulse Field Gel 33 It was concluded that “traditional salting, Electrophoresis (PFGE) patterns of E. coli strains 34 drying, and smoking of raw pork meat was isolated from patients’ stools and from samples of 35 not antimicrobiological-effective against salami produced by the most commonly identified 36 Salmonella Typhimurium. manufacturer were identical (Williams et al. 37 • In 2001, another serovar, Salmonella Goldcoast, 2000). 38 was identified as the causative agent of an • Finally, in British Columbia (Canada), in 1999, 39 outbreak in Germany, linked to the consumption a new outbreak of E. coli O157:H7 was reported, 40 of fermented sausage (Bremer et al. 2001). involving 143 cases, including 6 HUS. Again, 41 Twenty-four cases were identified with a the case-control study found an association with 42 correlation with the consumption of a raw a salami production. The mixture of raw beef, 43 fermented sausage manufactured by a local raw beef suet, raw pork, and ingredients was 44 company; a part of this product was sold after fermented and dried without a cooking step 45 only 4 days of fermentation. during the production (MacDonald et al. 2004). 46 Strains of E. coli O157:H7 isolated from the 47 In the European Union, Campylobacter jejuni and stools of patients and from salami samples has 48 Campylobacter coli are the second agents associated the same PFGE patterns. 49 with outbreaks: 1,243 (out of 6,860), affecting 3,749 50 persons, have been reported in 2004 (Anon. 2005). It YERSINIA, LISTERIA 51 is well known that these bacteria are present in the 52 pork and poultry chain productions but, to date, no Interestingly, a case-control study found an epidemi- 53S outbreaks associated with the consumption of fer- ologic association between human cases of yersin- 54N mented meat have been reported. iosis and consumption of salami (Anon. 2000), 40454 45 477-480 r1.qxd 7/21/07 12:49 PM Page 479

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conﬁrming a Norwegian study concerning different Anonymous. 2004. FAO/WHO Risk assessment of Lis- 1 types of sausages (Ostroff et al. 1994). teria monocytogenes in ready to eat foods—Interpre- 2 The risk of human listeriosis from fermented tative summary. Microbiological Risk Assessment 3 ready-to-eat meat is very low (Anon. 2004). However, series 4. http://www.fao.org. 4 Schwartz et al. (1989) reported an outbreak due to the Anonymous. 2005. Trends and sources of zoonoses, 5 consumption of salami in Philadelphia (U.S.A.). zoonotic agents and antimicrobial resistance in the 6 European Union in 2004. EFSA J—310. 7 MS Bergdoll. 1989. Staphylococcus aureus. In: MP 8 PARASITES Doyle, ed. Foodborne Bacterial Pathogens. New 9 Finally, the consumption of meat and meat products York: Marcel Dekker, Inc., pp. 463–523. 10 may present a risk of parasitic infection (Pozio V Bremer, K Leitmeyer, F Jensen, U Metzel, H Meczulat, 11 2003), mainly by Trichinella spiralis, Taenia solium, E Weise, D Werber, H Tschaepe, L Kreienbrock, S 12 and Toxoplasma gondii (Gamble 1997). Neverthe- Glaser, A Ammon. 2001. Outbreak of Salmonella 13 less, these parasites are inactivated by cooking, Goldcoast infections linked to consumption of fer- 14 freezing, or curing and dry-cured and fermented mented sausage, Germany 2001. Epidemiol Infect 15 meat present little danger (Van Sprang 1984). 132(5):881–887. 16 LA Chiou, TW Hennessy, A Horn, G Carter, JC Butler. 17 2002. Botulism among Alaska natives in the Bristol 18 CONCLUSION Bay area of southwest Alaska: A survey of knowl- 19 edge, attitudes, and practices related to fermented 20 Following this review, it can be concluded that out- foods known to cause botulism. Int J Circumpolar 21 break cases due to the consumption of fermented Health 61(1):50–60. 22 meat represent the minority of all laboratory- JM Cowden, M O’Mahony, CL Bartlett, B Rana, 23 conﬁrmed cases of gastrointestinal infections B Smyth, D Lynch, H Tillet, L Ward, D Roberts, 24 (Moore 2004). In general, on a worldwide basis, Sal- RJ Gilbert. 1989. A national outbreak of Salmonella 25 monella spp., verotoxigenic strains of Escherichia Typhimurium DT 124 caused by contaminated 26 coli (VTEC) and Staphylococcus aureus seem to be salami sticks. Epidemiol Infect 103(2):219–225. 27 the main concerns (Adams and Mitchell 2002). HR Gamble. 1997. Parasites associated with pork and 28 In particular, raw fermented sausage (salami), con- pork products. Rev Sci Tech 16(2):496–506. 29 taminated with verotoxigenic strains of Escherichia AHW Hauschild, L Gauvreau. 1985. Food-borne 30 coli, has recently been implicated in several out- botulism in Canada, 1971–84. Can Med Assoc 31 breaks, including hemolytic-uremic syndrome. J 133(December 1):1141–1146. 32 Y Le Loir, F Baron, M Gautier. 2003. Staphylococcus 33 REFERENCES aureus and food poisoning. Genet Mol Res 34 2(1):63–76. 35 M Adams, R Mitchell. 2002. Fermentation and DM MacDonald, M Fyfe, A Paccagnella, A Trinidad, 36 pathogen control: A risk assessment approach. Int J K Louie, D Patrick. 2004. Escherichia coli O157:H7 37 Food Microbiol 79:75–83. outbreak linked to salami, British Columbia, 1999. 38 Anonymous. 1977. The Staphylococcal Enterotoxin Epidemiol Infect 132:283–289. 39 Problem in Fermented Sausage. U.S. Department of PL Mertens, JF Thissen, AW Houben, F Sturmans. 40 Agriculture. Task Force Report. 1999. An epidemic of Salmonella Typhimurium 41 Anonymous. 1979. Staphylococcal food poisoning associated with traditional salted, smoked, and 42 associated with Genoa and hard salami. U.S. Depart- dried ham. Ned Tijdschr Geneeskd 143(20): 43 ment of Health, Education and Welfare. Morbid Mor- 1046–1049. 44 tal Weekly Report 28:179–180. JE Moore. 2004. Gastrointestinal outbreaks associated 45 Anonymous. 2000. Case-control study assessing the with fermented meats. Meat Sci 67:565–568. 46 association between yersiniosis and exposure to sal- SM Ostroff, G Kapperud, LC Hutwagner. 1994. 47 ami. Canada Communicable Disease Report 26(19). Sources of sporadic Yersinia enterocolitica infec- 48 Anonymous. 2001. Botulism outbreak associated with tions in Norway: A prospective case-control study. 49 eating fermented food—Alaska 2001. CDC, Epidemiol Infect 112:133–141. 50 MMWR Weekly, 50(32):680–682. AW Paton, RM Ratcliff, RM Doyle, J Seymour-Murray, 51 Anonymous. 2003. The effects of nitrites/nitrates on the D Davos, JA Lanser, JC Paton. 1996. Molecular 52 microbiological safety of meat products. EFSA J microbiological investigation of an outbreak of S53 14:1–31. Hemolytic-Uremic Syndrome caused by dry fermented N54 40454 45 477-480 r1.qxd 7/21/07 12:49 PM Page 480

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1 sausage contaminated with Shiga-like toxin- J Tilden, Jr, W Young, AM McNamara, C Custer, 2 producing Escherichia coli. J Clin Microbiol 34(7): B Boesel, MA Lambert-Fair, J Majkowski, D Vugia, 3 1622–1627. SB Werner, J Hollingsworth, JG Morris. 1996. A new 4 M Pontello, L Sodano, A Nastasi, C Mammina, route of transmission for Escherichia coli: Infection 5 M Astuti, M Domenichini, G Belluzi, E Soccini, MG from dry fermented salami. Am J of Public Health 6 Silvestri, M Gatti, E Gerosa, A Montagna. 1998. 86(8):1142–1145. 7 A community-based outbreak of Salmonella enterica P Van Netten, J Leenaerts, GM Heikant, DA Mossel. 8 serotype Typhimurium associated with salami con- 1986. A small outbreak of salmonellosis caused by 9 sumption in Northern Italy. Epidemiol Infect 120(3): Bologna sausage. Tijdschr Diergeneeskd 111 (24): 10 209–214. 1271–1275. 11 E Pozio. 2003. Foodborne and waterborne parasites. AP Van Sprang. 1984. Possibilities of survival of vari- 12 Acta Microbiol Pol 52:83–96. ous parasites in meat and meat products. Tijdschr 13 B Schwartz, D Hexter, CV Broome, AW Hightower, Diergeneeskd 109(1):344–348. 14 RB Hirschhorn, JD Porter, PS Hayes, WF Bibb, AA Wieneke, D Roberts, RJ Gilbert. 1993. Staphylo- 15 B Lorber, DG Faris. 1989. Investigation of an out- coccal food poisoning in the United Kingdom, 16 break of listeriosis: New hypotheses for the etiology 1969–1990. Epidemiol Infect 110:519–531. 17 of epidemic Listeria monocytogenes infections. RC Williams, S Isaacs, ML Decou, EA Richardson, 18 J Infect Dis 159:680–685. MC Buffett, RW Slinger, MH Brodsky, BW Ciebin, 19 N Shaffer, RB Wainwright, JP Middaugh. 1990. Botu- A Ellis, J Hockin, and the E. coli O157:H7 Working 20 lism among Alaska Natives: The role of changing group. 2000. Illness outbreak associated with 21 food preparation and consumption practices. West Escherichia coli O157:H7 in Genoa salami. CMAJ 22 J Med 153:390–393. 162(10):1409–1413. 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53S 54N 40454 46 481-490 r1.qxd 7/21/07 1:28 PM Page 481

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1 2 3 46 4 5 6 Basic Sanitation 7 8 Stefania Quintavalla and Silvana Barbuti 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION that there is the need to improve on general food 22 safety practices. 23 Basic sanitation within food processing facilities, Meat companies have to write and follow basic 24 often referred to as prerequisite programs or standard sanitation programs in order to ensure effective 25 operating procedures (SOPs), are the foundation of a cleaning and sanitizing of the manufacturing equip- 26 successful food safety management system (Wallace ment and environment. 27 and Williams 2001). They consist of hygienic prac- Examples of concerns addressed by the programs 28 tices designed to maintain a clean and healthy envi- mentioned above include the following: sanitary 29 ronment for the food product. The application design and construction of food facilities, cross- 30 of proper sanitation technique is important in main- contamination control, personal hygiene and sanitary 31 taining food safety. The significance of hygienic food handling, waste product disposal, and pest 32 design of plants and equipment has been recently control. 33 recognized in European (EC 2004) and United 34 States legislations (USDA 2000; FDA 2003, 2004). 35 The WHO puts emphasis on the role of prerequisite SANITARY DESIGN AND 36 programs defined as “practices and conditions needed CONSTRUCTION OF FOOD 37 prior to and during the implementation of HACCP FACILITIES 38 and which are essential for food safety” (WHO 39 1993). Sanitary design and layout permit appropriate main- 40 Sanitation is a dynamic and ongoing function and tenance, cleaning, and disinfection and minimize 41 cannot be sporadic; first, it is necessary that the full airborne contamination (Codex Alimentarius Com- 42 meaning of sanitation and its wide economic scope mission 1999). 43 be accepted by everyone concerned in the food sys- Establishment buildings, including their struc- 44 tem, including management. tures, rooms, and compartments, must be kept in 45 Sanitation means preventing product contamina- good repair and be of sufficient size to allow pro- 46 tion by microorganisms, allergens, and foreign cessing, handling, and storage of product in a manner 47 materials. that does not result in product adulteration (Schmidt 48 Poor sanitation practices can contribute to out- 1997; AMI 2003). Rooms or compartments in which 49 breaks of foodborne illnesses. Many of the microbio- edible product is processed, handled, or stored must 50 logical food safety problems result from basic be separate and distinct from rooms or compartments 51 sanitation failures that occur in food production, in which inedible product is processed, handled, or 52 processing, storage, transportation, retailing, and stored to prevent product adulteration by unsanitary 53 handling in the home (CDC 2002). This may indicate conditions. S54 N55 483 40454 46 481-490 r1.qxd 7/21/07 1:28 PM Page 484

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1 Because there is the possibility that the raw mate- important to establish a regular schedule for clean- 2 rial may carry a low number of pathogens, e.g., Sal- ing and sanitizing based on microbiological moni- 3 monella spp. and Listeria monocytogenes, primary toring: daily or more often when required for food 4 building construction should be designed to provide contact surfaces, and daily or weekly for nonfood 5 separate areas for raw materials and a finished prod- contact surfaces. 6 uct with a flow pattern being in one direction and An adequate supply of hot and/or cold potable 7 following a logical sequence from raw material han- water should be available throughout the establish- 8 dling to finished product storage. In the layout of the ment; if nonpotable water is used, for example, for 9 plant, an area to store raw material on reception must fire control, no cross-connections between potable 10 be arranged (Cramer 2003). and nonpotable water supplies must be present. 11 There should be a physical separation between Adequate natural or artificial lighting of sufficient 12 processing areas by the installation of walls and intensity to ensure that sanitary conditions are main- 13 doorways and by an air handling system to provide tained must be provided in areas where food is 14 positive pressure in the finished product rooms. processed, handled, or stored; where equipment and 15 Additional steps—for example, separation of pro- utensils are cleaned; and in hand-washing areas, 16 cessing times—should be taken to minimize the risk dressing and locker rooms, and toilets. Lighting 17 of cross-contamination if complete separation is not apparatus should be protected to prevent food from 18 possible. Color-coding is recommended, with differ- contamination by breakages. 19 ent colors identifying different areas. This can Positive air pressure is required in microbiological 20 be applied to clothing, cleaning supplies, containers, sensitive areas—for example, in slicing and packag- 21 and any other equipment. Separation can also be ing areas. 22 accomplished by the installation of sanitizer systems Dressing rooms and toilet rooms must be sufficient 23 (foot baths, spray systems) inside entrance doors to in number, ample in size, conveniently located, and 24 critical areas. maintained in a sanitary condition and in good repair 25 The establishment buildings should have floor to ensure cleanliness of all persons handling product. 26 and wall surfaces constructed of materials that are They must be separate from, and not directly entered 27 durable, smooth, easily cleaned, and, where neces- from, food processing and handling areas. 28 sary, disinfected. Drains should be operative and Lavatories with running hot and cold water, soap, 29 clean without standing water; pipes and insulation and towels must be placed in or near toilets and in 30 should be dry and in a good repair; overheads and food processing areas. 31 conveyors should be accessible, cleanable, and free 32 of condensate (Lelieveld 2000). 33 Equipment for food processing and handling CROSS-CONTAMINATION 34 operations should be cleanable and maintained in CONTROL 35 such a manner as to prevent contamination. 36 Chapter II of European Regulation 852/2004 (EC Cross-contamination occurs when biological, chem- 37 2004) lists the specific requirements for nonfood ical, or physical contaminants are transferred to food 38 contact and food contact surfaces in rooms where products. 39 foodstuffs are prepared. The type of cross-contamination most frequently 40 The basic principles of sanitary design are also implicated in foodborne illnesses occurs when path- 41 explained in the U.S. document FDA Food Code ogenic bacteria are transferred to ready-to-eat (RTE) 42 (2005): All surfaces in contact with food must be foods. Contaminants, especially bacteria, are trans- 43 inert, smooth, and not porous, visible for the inspec- ferred by people with dirty hands, clothing, or shoes; 44 tion, readily accessible for normal cleaning; the non- by dirty utensils, such as knives; and by dirty pro- 45 food contact surfaces should be arranged to prevent cessing equipment—for example, mincing machine, 46 harboring of soils, bacteria, or pests; equipment mixing machine, stuffing machine, tables, and con- 47 must be designed to protect the contents from exter- veyer belts. 48 nal contamination. Many pathogens, like Escherichia coli, Salmo- 49 Adequate space must be provided within and nella, and L. monocytogenes, enter the food process- 50 around equipment, and equipment must be accessible ing environment via contaminated raw materials. Raw 51 for cleaning, sanitizing, maintenance, and inspection. material contamination can affect any industry, but it 52 All food contact and nonfood contact surfaces, is more common in industries that use animal-derived 53S including utensils, should be cleaned as frequently products or products at risk of cross-contamination 54N as necessary to avoid contamination of food. It is by animal feces (Franco et al. 1995). 40454 46 481-490 r1.qxd 7/21/07 1:28 PM Page 485

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During processing of fermented meat products, A product can become cross-contaminated with 1 fresh meat comes in contact with many solid sur- allergens on the production line. To minimize the 2 faces: examples include conveyors, peelers, colla- risk of cross-contamination, equipment must be 3 tors, belts, slicers, and tables (Mafu et al. 1990). cleaned and sanitized to remove all traces of aller- 4 Both employees and equipment that touch raw meat gens when the next run includes product that should 5 can transmit pathogenic bacteria to the finished not contain allergens. Wash-down techniques may 6 product; raw and RTE products should be physically need adjustment to ensure that they remove allergens 7 separated in coolers or other storage areas. as well as pathogens (Higgins 2000). Rinsing with 8 Bacteria from fresh meat can affix to wet equip- water only or cleaning only at the end of the day 9 ment surfaces, form biofilm and/or niche environ- could be inadequate. Manufacturers may choose 10 ments, and supply a source of cross-contamination. to physically separate lines for allergen- and nonal- 11 Biofilms occur when bacteria form a slime layer lergen-containing products (Morris 2002). 12 upon a surface and provide an environment for Visitors, suppliers, laboratory personnel, truck 13 pathogens to proliferate. The adhesion of pathogenic drivers, inspectors, and management should be made 14 bacteria to a biofilm is a food safety hazard because aware of operating procedures; tours should be done 15 the biofilm can detach and become a significant in a traffic direction counter to production, starting 16 source of food contamination. Cleaning to remove with finished product rooms and ending in the raw 17 biofilms prior to sanitation is often sufficient to pre- material handling areas. 18 vent this problem. However, studies have shown that 19 attached bacteria may survive conventional cleaning 20 methods (Stopforth et al. 2002). Adequate cleaning PERSONAL HYGIENE 21 prior to sanitizing is therefore paramount to control- Good personnel hygiene practices are the foundation 22 ling this problem (Chmielewski and Frank 2003). for successful food safety and quality assurance in 23 Niche environments are sites within the manufac- all food manufacturing facilities. Bacteria causing 24 turing environment where bacteria can get estab- diseases or spoilage may be carried and transmitted 25 lished, multiply, and contaminate the processed to surfaces and food by plant personnel handling the 26 food. These sites may be impossible to reach and to food products. 27 clean with normal cleaning and sanitizing proce- The transfer of contaminants can occur through a 28 dures: examples include hollow rollers on convey- direct route, such as bacteria transferred from the 29 ors, cracked tubular support rods, the space between body skin, mouth, hands, or hair to the product, or 30 close-fitting metal-to-metal or metal-to-plastic parts, indirectly via personal equipment, such as clothing, 31 worn or cracked rubber seals around doors, and on- footwear, utensils, and other tools used in the daily 32 off valves and switches. Manufacturers must iden- tasks. 33 tify and eliminate niches in order to minimize the Two important areas of focus when setting per- 34 risk of postprocessing contamination from niche sonal hygiene policies to prevent contamination are 35 environments (Tompkin 2002; AMI 2003). protective outer clothing (working clothes, footwear, 36 Conveyors, peelers, collators, belts, gloves, slicers, and hair covering) and hand hygiene. 37 and tables can be sources of direct product contamina- 38 tion. Slicers must be cleaned at the end of the shift, and 39 PROTECTIVE OUTER CLOTHING if the slicer is used for different dry-cured meat prod- 40 ucts, it must be cleaned and sanitized before reuse. The clothing of workers must be clean. The purpose 41 Some equipment may need to be disassembled to is not to protect the worker against contamination 42 be cleaned. Where possible, equipment should be but to protect the meat products against contamina- 43 running during cleaning and sanitizing for complete tion. Working clothes must be used exclusively in 44 exposure to chemicals. The cleaning process should the working area and nowhere else. If possible, it is 45 be verified primarily by visual inspection, and the advisable to avoid admittance from the unclean area 46 effectiveness of sanitation procedures should be to the clean area without changing clothes. 47 confirmed by microbiological monitoring, enzyme- Working clothes should be comfortable and easy 48 linked immunosorbent assay (ELISA) tests, and to wash. Their design should encourage good 49 ATP testing (Griffiths 1996; Griffiths et al. 1997; hygiene habits: light-colored working clothes show 50 Shumaker and Feirtag 1997). the need for cleaning sooner than dark-colored ones. 51 Do not limit sampling to flat surfaces. Microbio- Factory clothing should be hygienically designed 52 logical sampling of the environment and equipment to prevent foreign bodies from being lost directly S53 can detect a niche. (buttons) or indirectly (objects falling out of pockets). N54 40454 46 481-490 r1.qxd 7/21/07 1:28 PM Page 486

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1 During work, jewelry, wristwatches, etc., are prohib- the risk associated with different hand-washing 2 ited, because these objects may be sources for techniques (Monville et al. 2002). Soap with an 3 contamination and make hand-washing difficult. antimicrobial agent was observed to be more 4 Laundering has to be controlled by the company effective than regular soap, whereas there was little 5 in order to ensure the sanitary condition of the pro- difference in the efficacy of alcohol and alcohol- 6 tective clothing. free sanitizers. Hot air-drying had the capacity to 7 Footwear can be a vehicle for the transfer of increase the amount of bacterial contamination on 8 pathogens from production areas considered at high hands, whereas paper towel drying caused a slight 9 risk to low-risk areas. Footwear should be made of decrease in contamination. 10 nonporous material that is cleanable. Foamers are The frictional aspects of hand-drying can help 11 the most commonly used footwear decontamination remove microorganisms missed during hand-washing 12 method in the industry. Foot dips/baths and boot- on precisely the parts of the hands that are missed 13 washers are also common; in this case it is essential (Blackmore 1989). 14 that they are monitored to make sure that a “culture Hand-drying by contact with dispenser mecha- 15 broth” for bacteria has not been created. nisms should be avoided because it can cause cross- 16 Human hair and beards are normally heavily con- contamination. 17 taminated with bacteria; to prevent contamination of For some operations, the use of a special bacterio- 18 food, a hair or beard covering in the process area is a static soap or dipping of the hands after washing in a 19 necessary part of work clothes. Many different types germicidal rinse could be required. Use of a nail 20 of hair coverings are seen in the food industry. Dis- brush is recommended because bacteria often hide 21 posable or washable hair and beard coverings are along and under the nails. 22 recommended. Gloves 23 24 The use of gloves does not eliminate the need for HAND HYGIENE 25 sanitary hand-washing and drying. Gloves can pro- 26 Hand-washing policies should require employees to vide a false sense of security. 27 wash after any type of activity that could contami- If the use of gloves is indicated, they must be 28 nate the hands with pathogens, including using kept in the same good hygienic conditions as 29 the restroom; blowing the nose; touching body parts, hands; otherwise, it is better to avoid their use. 30 waste, or nonfood contact surfaces, such as light Gloves should be changed at regular intervals (at 31 switches or pipes; smoking; and eating. Bacteria least once per hour), after touching contaminated 32 may be transmitted to the hands by these acts and surfaces and utensils, or if punctured. 33 thereafter transmitted to meat products that are han- Gloves may be of rubber or plastic and they are 34 dled by hand. used to protect the meat and meat products against 35 Employees also should wash before entering food- contamination. They may also be used to protect the 36 handling areas, changing clothing, and putting on hands against knife cuts and will then be made of 37 gloves. steel. Great care should be taken to keep a certain 38 Careful and frequent hand-washing will do much hygienic standard of these gloves. 39 to reduce contamination. Therefore, hand-washing A study was conducted to determine whether the 40 facilities must be sufficient if the water supply is level of selected microorganisms differed on foods 41 adequate. Basically there should be two sites where handled by gloved and bare hands at fast food restau- 42 the staff can wash their hands, the restroom and the rants (Lynch et al. 2005). There were no significant 43 working area, where sufficient hand-washing facili- differences between samples handled by gloved or 44 ties must be placed close to the working places. If bare hand. The observed tendency of food workers to 45 hand-washing stations are not placed in convenient wear the same pair of gloves for extended periods 46 locations, employee might skip washing. might account for the apparent failure of glove use to 47 reduce or prevent bacterial contamination. Hand-washing 48 49 Use of an effective soap, vigorous rubbing of hands PERSONNEL HEALTH 50 during the washing process, and thorough hand- 51 drying are key to good sanitary hygiene. Good health is important for workers in the meat 52 Both literature and experimental data were used to industry. Ill persons will often be carriers of more 53S develop a quantitative risk assessment model to assess microorganisms (pathogenic microorganisms) than 54N 40454 46 481-490 r1.qxd 7/21/07 1:28 PM Page 487

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usual and these microorganisms may then be trans- measures against pollution through the application 1 mitted to the meat products with the risk of causing of best available techniques (BAT). On January 2 disease to the consumers. Illness must always be 2006, a Reference Document on Best Available 3 reported to the manager and/or the meat inspector of Techniques (BREF) in the Food, Drink and Milk 4 the plant who will decide whether the worker can industries was published (EC 2006). Both general 5 stay or has to leave. BAT for the whole FDM sector and additional BAT 6 Likewise, persons afflicted with infected wounds, for some processes and unit operations in the indi- 7 skin infections, sores, etc., must also be restricted vidual FDM sectors are listed. Their scope is to 8 from food handling areas. Any persons with open reduce the consumption of water, energy, and 9 cuts or wounds should not handle food unless the packaging. 10 injury is completely protected by a secure, water- For example, for meat and poultry processing 11 proof covering. installations, BAT is to thaw meat in the air to reduce 12 water consumption and wastewater production; to 13 dose spices and other solid ingredients from a bulk 14 WASTE PRODUCT DISPOSAL container rather than from plastic bags to minimize 15 Handling of liquid and solid waste influences both the use of plastic packaging material; and to stop the 16 hygiene in processing and of the environment, the water supply automatically when sausage fillers and 17 latter depending on the precautions taken to avoid similar equipment are not used at breaks or at pro- 18 contamination with liquid and solid waste. duction stops to reduce waste of materials, products, 19 Whenever food is processed, wastewater is gener- and water. 20 ated. Sewage must be disposed of in a sanitary man- 21 ner; it must not become a source of contamination to PEST CONTROL 22 the plant environment. Of particular importance is the 23 prevention of microbiological cross-contamination One of the simplest, yet essential, steps in food 24 and pests. Sewage lines must not connect to drainage safety is pest prevention. Pests, including cock- 25 lines within the plant. roaches, flies, stored products insects, rodents, and 26 Garbage disposal and the handling of waste from birds, harbor and spread disease-carrying organisms 27 within the plant during operations and the waste col- that can easily contaminate food products. However, 28 lection facilities outside the plant must be handled in pest control is often ignored until pests and their 29 a manner to reduce the risk of bacterial contamina- damage are discovered. For example, if rodents or 30 tion and the pest infestation and harboring. insects are found in a food storage room, temporary 31 Within the plant there must be a sufficient number measures are taken to eliminate them. The real 32 of garbage containers so that they are accessible trouble, however, is not corrected. The method of 33 to all employees. Covered containers are required. treating a single outbreak is a poor concept of 34 These containers must be clearly marked and identi- sanitation. 35 fied, leakproof, emptied regularly, and cleaned and Control of pests and use of pesticides are particu- 36 sanitized prior to use. larly critical in places where food is processed 37 Waste disposal facilities that are located outside of and/or packaged. Top management is ultimately 38 the plant must not attract pests. They must have cov- responsible for identifying a competent person to 39 ers and be kept closed and in good conditions. develop a pest prevention and control program. Per- 40 sons who apply pesticides in industrial facilities 41 have a responsibility to use the needed pesticide, to 42 ENVIRONMENTAL PROTECTION—IPPC DIRECTIVE apply it correctly (according to label instructions), 43 In 1996, Council Directive 96/61/EC on integrated and to be certain there is no hazard to man or the 44 pollution prevention and control was published (EC environment. 45 1996). The purpose of the Directive is to achieve 46 integrated prevention and control of pollution aris- 47 INTEGRATED PEST MANAGEMENT (IPM) ing from European industrial activities, such as the 48 activities of the FDM sector (Food, Drink and Milk The many drawbacks and problems associated with 49 industries), leading to a high level of protection chemical pesticides have led pest control experts to 50 of the environment as a whole. Central to this reevaluate control procedures. As a result, pest 51 approach is the general principle given in Article 3 control programs based on predicted ecological 52 that operators should take all appropriate preventative and economic consequences have been developed S53 N54 40454 46 481-490 r1.qxd 7/21/07 1:28 PM Page 488

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1 (Thomas 1999). Integrated pest management (IPM) allow access for pests and allow dust to collect 2 is a dynamic combination of varied control practices should be eliminated. 3 designed and implemented to meet the need of main- • Inspections and monitoring: These activities are 4 taining control of pests using a variety of techniques necessary to guide schedules and locations of 5 without total reliance upon pesticides. treatments, and to monitor the effectiveness of the 6 Economic, social, and environmental advantages overall management strategy. 7 may be attained through IPM. The apparent benefits • Pest identification: Pest identification is necessary 8 are related through lower costs, increased pest con- to select the most appropriate control methods. 9 trol, and reduced pesticide usage by up to 60% • Physical and chemical controls: Examples are 10 (Paschall et al. 1992). rodent traps, glue boards, electric fly traps, and 11 A few steps are necessary for implementing an temperature manipulation. 12 ongoing IPM strategy in a food processing facility 13 (Pest Management Regulatory Agency 1998). When These methods are noncontaminating and can fill 14 designing and building a new facility or renovating some of the gaps left by a restricted pesticide use. 15 an existing one, it is essential that the IPM strategy As for HACCP, implementation of the pest 16 be incorporated at the earliest stages. management plan starts with the commitment of 17 The first step of the IPM strategy is an assessment the facility’s management and decision makers. 18 of the actual or potential pest problem. Identification Employees must be trained with an awareness of the 19 of potential pest problems can be based on past IPM program and its elements, particularly sanita- 20 experience, knowledge of similar facilities, and tion. They should learn to recognize pests, pest habi- 21 knowledge of particular pests in the region in which tats, and pest conditions, and how to deal with them. 22 the plant is located. Written procedures should be available for all 23 Inspections are used to identify the type and vari- aspects of an IPM program, including cleaning, 24 ety of pests that are or may be present. Inspections monitoring, identifying, and correcting of problems, 25 should include raw materials, prepared products, and treatments. 26 interior and exterior of the facility, and equipment. The overall effectiveness of the pest management 27 Monitoring, by trapping for example, could be used plan must be reviewed based on monitoring of pests 28 to determine the intensity and the distribution of a and inspection reports. 29 pest problem in and around a facility. Observations Changes to the pest management plan can be 30 can be used to confirm the results of inspection and made to achieve the desired level of control; to 31 monitoring. adjust to new situations, such as new equipment, 32 The next stage is to develop a pest management new products, and new pests; or to incorporate new 33 plan for implementation, based on the assessment pest management techniques. 34 findings. At this stage, information is needed on the 35 biology of the pest organisms and the management PERSONNEL TRAINING 36 of that particular pest. 37 Elements that should be included in the pest man- Regardless of the type of processing or food han- 38 agement plan for effective IPM are: dling operation, people play a key rule in food sani- 39 tation. That is why the most important aspect of a 40 • Building and materials design and retrofitting: sanitation program is ongoing personnel training. It 41 The facility, its equipment and its exterior is essential that the full meaning of sanitation and its 42 surroundings must not promote pest wide economic scope be accepted by everyone con- 43 populations. cerned in the food system including management. 44 • Exclusion practices: Infestations in incoming Personnel training should include appropriate sanita- 45 food and ingredients should be reduced or tion principles and food handling practices, manu- 46 eliminated through strict purchase specifications, facturing controls, and personal hygiene practices. 47 audits of suppliers, and inspection of incoming Personnel training should encourage an under- 48 material. standing of the processing steps and technologies for 49 • Good sanitation practices: They include thorough the product manufactured and where potential prob- 50 and regular cleaning, prevention of dust lems exist. Production personnel should be trained 51 generation and accumulation, and removal of in the critical elements of the operations for which 52 food sources and harbors for pests. they are responsible, in the importance of these oper- 53S • Building maintenance: Holes and cracks in floors, ations, monitoring these operations, and in action to 54N walls, ceilings, roofs, doors, and windows that be taken when these operations are not controlled. 40454 46 481-490 r1.qxd 7/21/07 1:28 PM Page 489

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Control of Listeria monocytogenes 52 MW Griffiths, CA Devidson, AC Peters, LM Fielding. in the food processing environment. J Food Prot S53 1997. Towards a strategic cleaning assessment 65:709–725. N54 40454 46 481-490 r1.qxd 7/21/07 1:28 PM Page 490

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1 USDA, Food Safety and Inspection Service. 2000. WHO. 1993. Training consideration for the application 2 FSIS Directive 11,000.1, Sanitation Performance of the Hazard Analysis Critical Control Points Sys- 3 Standards. Jan 25. tem to food processing and manufacturing. WHO 4 C Wallace, T Williams. 2001. Pre-requisites: A help or a document, WHO/FNU/FOS/93.3, Division of Food 5 hindrance to HACCP. Food Contr 12:235–240. and nutrition. Geneva. 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53S 54N 40454 47 491-502 r2.qxd 7/21/07 12:50 PM Page 491

1 2 3 47 4 5 6 Processing Plant Sanitation 7 8 Jordi Rovira and Dorota Puszczewicz 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION Plant sanitation programs are an important part of 22 the prerequisite programs, which are the base for the 23 Sanitation is a word that comes from the Latin sani- implementation of a HACCP plan and the food qual- 24 tas, which means health. In that sense, in the food ity management systems, all of them acting as differ- 25 industry, plant sanitation refers to all the measures ent barriers to avoid the penetration of contaminants 26 that can be taken to create or maintain hygienic and in the foodstuffs. In that sense, the more barriers that 27 healthful conditions in the production plant, to avoid can be added to the processing line, the more difﬁ- 28 the entrance of contaminants to foodstuffs that can cult it will be for contaminants to enter it and the 29 give rise to potential risks for consumers. There are safer will be the food product manufactured. So, the 30 three main types of food contaminants according to primary aim in processing plant sanitation should be 31 their nature: physical, chemical, and microbiologi- to set up effective barriers to putative contaminants. 32 cal. These are of major importance in processing Table 47.2 shows different barriers that can be con- 33 plant sanitation. These contaminants can enter into sidered for this purpose. 34 the food chain by different routes: with the raw 35 material, ingredients, or other auxiliary material 36 needed for food elaboration; through the environ- FERMENTED MEAT PRODUCTS 37 ment surrounding the processing line, such as con- AND POULTRY 38 tact surfaces, air, or water, by pests; or by personnel 39 food manipulation (Table 47.1). During different As has been stated in other chapters of this book, fer- 40 processing steps, new contaminants can appear as mented meat products are a group of meat products 41 well if they are not done properly or are done with- whose main characteristic is that their sensory char- 42 out taking some preventive measures. Smoking, for acteristics (ﬂavor, texture, and shelf life) have been 43 example, can produce some hazardous substances subjected to the action of microorganisms. They are 44 known as Polycyclic Aromatic Compounds (PAH), ready-to-eat products, although most of them do not 45 α such as benzo( )pyrene, which has been shown to be receive any heat treatment during their processing, at 46 carcinogenic in experimental animals (Anonymous least in European countries. Some other fermented 47 2002). meats, such as pepperoni, can suffer a slight heat 48 As a consequence of the presence of these con- treatment at 43–53°C (110–128°F) in the core of the 49 taminants, the shelf life of the foodstuffs could be sausage during 1 (or several) hour(s), according to 50 reduced and, even worse, consumer health could be the casing diameter, in the last stages of processing 51 compromised. All measures should be taken to avoid (Hinkens et al. 1996). The lack of a heat treatment 52 food contamination and decrease the risk of food- does not mean that these products are not safe 53 borne illnesses. enough for consumption. S54 N55 491 40454 47 491-502 r2.qxd 7/21/07 12:50 PM Page 492

1 Table 47.1. Sources of contaminants in fermented meat products. 2 3 Source Examples 4 Raw Material 5 Meat and fat Enterobacteria, E. coli, Listeria spp., Salmonella 6 Small pieces of bones 7 8 Spices Microorganisms 9 Environment 10 Air Airborne microorganisms 11 “Light” foreign bodies 12 Chemical taints 13 Water supply Microorganisms 14 Chemical pollution 15 Unwanted waters Microorganisms and insects 16 17 Surfaces Biofilms development 18 Cleaning detergent and sanitizer residues 19 Condensations in walls and ceilings 20 Personnel Food Manipulation Microorganisms associated with human activity 21 Small personal belongings, hair, fingernails 22 Processing Machinery lubricants 23 Chemical compounds as N-nitrosamines, smoke 24 —components as benzo-α-pyrenes 25 Biogenic amines 26 Mycotoxins production by surface molds 27 28 29 30 31 32 33 Table 47.2. Several barriers to avoid penetration of contaminants in food processing plants. 34 Barrier Action 35 36 Suppliers control Raw material inspection and specifications 37 Ingredient specifications 38 Air control quality 39 Water supply quality 40 Factory design Plant location 41 Building design 42 Plant distribution 43 44 Sanitation program Cleaning and sanitation 45 Equipment maintenance plan Careful revision of equipment 46 Lubricant control 47 Personnel GMPs Training 48 49 Waste management Updated plan 50 Pest control Updated plan 51 HACCP plan Safety processing control 52 Quality management system Implementation of a quality system 53S 54N

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Dry and semidry-fermented sausages are gener- FERMENTED SAUSAGE 1 ally considered shelf-stable, safe meat products, and PROCESSING PLANT 2 they have rarely been involved in food contamina- SANITATION 3 tion outbreaks (Barbuti and Parolari 2002). These 4 products are a clear example of application of the As stated previously, the aim of the processing plant 5 hurdle technology. Among these hurdles, several are sanitation programs is to put as many barriers as 6 related to the growth of lactic acid bacteria (LAB), possible to avoid the entrance of contaminants into 7 such as pH reduction, lactic acid, and other organic the foodstuffs along the processing line. This section 8 acids production, and nutrient competitiveness; describes general fermented sausage processing and 9 others are due to the drying process, such as mois- the four main sources that can contaminate the prod- 10

ture loss and aw decrease, as well as the use of uct during the process: raw material, processing 11 nitrates and/or nitrites as preservatives. Moreover, environment, personnel manipulation, and some 12 some of these products are smoked, and although processing steps. 13 they are shelf-stable at room temperature, most of 14 them are sold vacuum- or MAP-packaged and in 15 FERMENTED SAUSAGE PROCESSING refrigerated condition. However, although fermented 16 meat products are traditionally considered as very A flow chart of a typical dry-fermented sausage pro- 17 safe ready-to-eat meat products, some recent out- cessing is shown in Figure 47.1. For the manufactur- 18 breaks, mainly due to E. coli O157:H7 have been ing of dry-fermented sausages, different trimmings 19 detected, in the U.S. (1994), Australia (1995), and of pork meat are used, mainly shoulder, belly, and 20 Canada (1998, 1999). In all these cases, products backfat, although a percentage of beef can also be 21 were elaborated totally or partially from beef meat. It used. Today, and for dietetic reasons, some poultry 22 has been proved that some strains of E. coli can sur- and turkey dry-fermented sausages are made. Trim- 23 vive the acidic conditions required for manufactur- mings are kept chilled or frozen prior to use in fabri- 24 ing of raw fermented sausages (Anonymous 2000). cation. Most of the time these trimmings are used in 25 Salmonella has been detected as well as the main blocks that are cut in small pieces by guillotines 26 source of two outbreaks in Italy due to these prod- before mincing. 27 ucts (Pontello et al. 1998). At the same time, studies Depending on the type of sausage, meat is ground 28 on the behavior of L. monocytogenes have shown to different sizes, and with different machines. Nor- 29 that it may be reduced but not necessarily eliminated mally for dry-fermented sausages with a particle 30 from meat during the fermentation process, particu- size higher than 3 mm, a traditional plate grinder is 31 larly when present in high numbers in the raw meat used—for instance, in chorizo making, and for 32 (Glass and Doyle 1989). Nevertheless, according to sausages with lower particle size, such as some types 33 a risk assessment of L. monocytogenes in ready-to- of salami, it is possible to mince the meat with a 34 eat foods carried out by the Food and Agricultural bowl chopper. Minced meat and fat are mixed 35 Organization (FAO), the risk of listeriosis from fer- together with the rest of the ingredients and additives 36 mented meats is very low (2.1 ϫ 10−12 cases/serv- in vacuum mixers, which promote air leaving the 37 ing) and there have been no reported outbreaks of meat mixture, improve the absorption of additives by 38 listeriosis from such products to this point. the meat mixture, and reduce the redox potential to 39 In summary, although there are limited epidemio- create favorable conditions for lactic acid bacteria 40 logical data related to foodborne disease caused development. After mixing, some traditional factories 41 by dry-fermented products, sausage makers must keep the meat mixture resting in refrigeration for a 42 ensure that their products are not contaminated by couple of days before stuffing; for this purpose, vac- 43 pathogens. Although processing techniques normally uum plate stuffers are used and sausages can be 44 used to manufacture fermented sausages appear to stuffed in natural (pork or beef casings) or in synthetic 45 be effective in pathogen control, there is evidence collagen or fibrous casings. Natural casings should be 46 that raw materials are still a major source of bacterial profusely cleaned before use, and synthetic casings 47 contamination, and current cleaning and sanitation should be dipped in a salty solution prior to use. 48 procedures may fail to prevent such pathogens enter- Once dry-fermented sausages are made, some of 49 ing the production line (Barbuti and Parolari 2002). them can be dipped in a solution with mold spores 50 Although some authors consider to a broad extent or yeast that later will give sausages a whitish sur- 51 that dry-cured ham is a whole fermented meat face. If the idea is to produce sausages without 52 product, in this chapter only dry and semidry- molds on its surface, it is usual to dip them into a S53 fermented sausages are considered and discussed. solution of 20% sodium sorbate, or with a solution N54 40454 47 491-502 r2.qxd 7/21/07 12:50 PM Page 494

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Figure 47.1. Dry-fermented meat processing flow chart (adapted from Martín 2005). 41 42 43 44 45 of pymaricine. After bathing, they are hung and the first 48–72 hours at 20–25°C, and after that 46 placed in bars and transported to the ripening room decrease temperature to 13–15°C. In most craft 47 (green room). Ripening rooms provide the sausages industries, fermentation occurs at lower tempera- 48 with the proper conditions for fermenting, drying, tures (below 12–15°C) and for a longer period of 49 and ripening throughout a certain period of time, time, and the result used to be a milder fermented 50 depending on the conditions and mainly on the sausage. Ripening rooms can be natural, when no 51 sausage diameter. Fermentation takes place with forced air is used for the drying process, or arti- 52 more or less intensity depending on the temperature ficial, when forced air is used. For more details 53S conditions in the first steps of the ripening process; about fermented sausage production, see previous 54N normally the fermentation step occurs during chapters. 40454 47 491-502 r2.qxd 7/21/07 12:50 PM Page 495

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CONTAMINATION SOURCES the point of view of factory design, with the aim of 1 lowering the risk of contamination: the siting of the 2 RAW MATERIAL factory, the factory building design, and the internal 3 The quality state of the raw material is of great plant barriers used to separate the manufacturing 4 importance to assure a good quality and safety of the processes into different sections. 5 final product. In that sense, it is necessary to reduce The design, construction, and maintenance of the 6 to a great extent the contamination level of the raw site surrounding the factory provide an opportunity 7 material and other ingredients related with product to establish the first outer barrier to protect produc- 8 formulation. It must be taken into account that the tion operations from contamination (Wierenga and 9 passage of meat trimmings and other ingredients Holah 2003). In that sense, an excellent location will 10 over the processing surfaces leaves residual food be an area with good air quality and no pollution 11 debris, which encourages the growth of microorgan- problems (far from other polluting industries). Tra- 12 isms. With time, these microorganisms can multiply ditionally, fermented sausage plants were located in 13 to relative high numbers and act as a contamination dry, cold places to obtain a good air quality and favor 14 focus that compromises the safety quality of the the drying process. Normally they used a low- 15 meat product manufactured. It is also well known temperature fermenting process with long ripening 16 that contaminated raw materials are prone to give periods, and sometimes, when the temperature was 17 more fabrication defects (abnormal fermentations) too low, they used a slight smoking stage or heater 18 and to shorten the shelf life of the final product. In fans to raise the temperature. Now, these conditions 19 that sense, it is very important to control the raw have changed due to the use of artificial ripening 20 material, proving that meat has been transported at rooms that are able to reproduce practically any nat- 21 the right temperature: less than 7°C in chilled condi- ural condition for the ripening stage. It is also neces- 22 tion or below −18°C in the case of frozen meat. In sary to take care of the surroundings of the factory, 23 both cases, meat trimmings should be properly pack- avoiding the accumulation of waste material and 24 aged with plastic films inside the boxes, to avoid the stagnant water that can attract birds, animals, and 25 contact of the meat with any dirty surface. Control insects. In that sense, the entire factory should be 26 plant inspectors should check the overall appear- provided with a good pest control plan in order to 27 ance, off-odors, slime production, and strange col- avoid the entrance of animals into the factory. 28 orations in meat lots before unloading them from the The building structure is the second major barrier, 29 trucks. Some samples of meat could be taken period- and the factory should be constructed with the aim 30 ically for microbiological analysis. It is also very of providing protection for raw materials, processing 31 important to have a supplier that is certified to follow facilities, and manufactured product from contami- 32 HACCP plans during slaughter and cutting of ani- nation or deterioration. To review with much more 33 mals. The main contaminants of raw meat are E. coli detail all elements regarding the factory building, 34 O157:H7 (in beef ) and Salmonella, Listeria and see Wierenga and Holah (2003). 35 Staphylococcus aureus. The final sets of barriers to contamination are 36 those within the factory itself, where two levels of 37 barriers are required: the first level separates pro- 38 PROCESSING ENVIRONMENT cessing from nonprocessing areas, and the second 39 The term processing environment refers to all ele- one separates high-risk from low-risk processing 40 ments that surround, or that can enter in contact areas. To avoid cross-contamination, it is very 41 with, the meat along the processing line in the plant. important that the processing flow moves in one 42 It includes proper plant design, the quality of the air straight line of production operations from raw 43 and water, and the contact surfaces (mainly produc- materials to finished products, with the aim of mini- 44 tion machinery). mizing the possibility of contamination between 45 processed and raw materials. This concept is also 46 Hygienic Plant Design more efficient in terms of handling, and it is easier to 47 The primary aim of hygienic plant design should be separate clean and dirty process operations and 48 to set up effective barriers to microbial and other restrict movement of personnel from dirty to clean 49 contaminants, providing an appropriate environment areas. High-risk areas are those that process food 50 for processing operations, while ensuring compli- products that have suffered a decontamination or 51 ance with all applicable building safety and environ- preservation process and where there is a risk of 52 ment regulations (Wierenga and Holah 2003). In that product contamination. Low-risk areas are those S53 sense, three main barriers can be established, from where food components have not yet suffered a N54 40454 47 491-502 r2.qxd 7/21/07 12:50 PM Page 496

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1 decontamination or preservation process. In that contaminate foodstuffs. Finally, chemical taints can 2 sense, in dry-fermented sausage production, most of enter the production area through airborne transmis- 3 the process is developed in low-risk areas, with the sion (Lelieveld 2003). 4 exception of the packaging area after the ripening Water 5 process—although in that stage there is also a low 6 risk of cross-contamination by bad manipulation by In the food industry, water has several important 7 personnel. Nevertheless, there should be strict sepa- applications. It is used as an ingredient, as a produc- 8 ration between the production room, from meat tion aid, and as a medium for cleaning. In all cases, 9 grinding to sausage hanging, and the ripening water can serve as a microbial and chemical contam- 10 rooms, where wild microflora can grow on the sur- ination dissemination media, which is why it is very 11 face of the products. important to check the water quality every day. 12 In dry-fermented sausages industries, the concept Unwanted water, such as steam or water vapor, con- 13 in building design and distribution of the different densation, leaking pipes or drains, or rainwater can 14 stages of the processes changed some decades ago, also be a vector for contamination. Particularly haz- 15 mainly in the fermentation and drying steps, with the ardous is stagnant water, because microorganisms 16 introduction of the artificial ripening rooms. Table 47.3 can multiply quickly under favorable conditions and 17 shows the most relevant differences between tradi- serve as a contamination focus (Dawson 2000). 18 tional and modern fermented sausages facilities, Cleaning and Sanitation 19 pointing out some cleaning and sanitation issues. 20 These are two operations that have a great impor- Air 21 tance for avoiding the entrance of contaminants to 22 Air plays a very important role in the processing the process line, and they are discussed broadly in 23 of dry-fermented sausages, because of the drying this chapter. 24 process that takes place during the fermentation There is a good review of this matter from Holah 25 and ripening stages of production. It also serves as (2003). According to this author, cleaning and disin- 26 transfer media of contamination to food products, fection are applied with the intention of removing 27 and unless the air is treated, microorganisms will microorganisms or material that supports microbial 28 be present in it and can be transported from dif- growth; removing materials that could lead to for- 29 ferent reservoirs outside or inside the plant—for eign body contamination or could provide food or 30 instance, from nonproduct contact surfaces such as shelter for pests; removing food debris, which can 31 floors, ceilings or walls, to the processing line. deteriorate product quality, from the production line; 32 Through the air “light” foreign bodies such as extending the life and preventing damage to surfaces 33 dust and insects can move around the plant and and equipment; providing a safe and clean working 34 35 36 37 Table 47.3. Differences between traditional and modern dry-fermented sausage facilities. 38 39 Traditional Modern 40 41 Plant site Dry and cold places Everywhere 42 Building design Several floors in height One or two floors 43 Ripening rooms Big spaces with many windows Small spaces without windows 44 —in opposite walls to facilitate 45 —air flow 46 Sometimes several productions Batches or lots more homogenous 47 —or products mixed 48 Fixed structures for hanging Mobile structures for hanging 49 —sausages —sausages, such as pallets or mobile 50 —frames by ceiling rails 51 Wooden or metal hanging supports Hanging structures of galvanized 52 —and bars for hanging sausages —metal or stainless steel 53S Difficult to clean properly Easy to clean 54N 40454 47 491-502 r2.qxd 7/21/07 12:50 PM Page 497

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place for employees; and showing a good impression divided into four different areas: (1) chilling rooms 1 to visitors. For all these reasons, sanitation programs and stores, (2) the main production room (from meat 2 are of great importance and form an important pillar grinding to hanging of the sausages), (3) ripening 3 to support the HACCP plan. rooms (aging), and (4) the packaging area. 4 Generally, in sanitation programs different factors Chilling rooms and stores are the places where 5 should be taken into account, such as timing of raw materials and other ingredients are stocked 6 cleaning and disinfection, the sequence in which before they are used in production. These facilities 7 equipment and environmental surfaces must be are very difficult to clean and disinfect because they 8 cleaned and disinfected within the processing area are always full of materials or ingredients that will 9 (see Table 47.4), types of cleaners and disinfectants, be used in the next productions. So, emptying these 10 applying method, frequency, type of inspection and facilities for a full cleaning and sanitation is difficult 11 confirmation. for a company. In the case of chilling rooms, the low 12 To make an effective cleaning and sanitation pro- temperatures themselves act as a barrier for micro- 13 cedure, it is necessary to remember that sanitation bial contaminants. Nevertheless, cleaning these 14 programs employ a combination of four major facilities should be done in another way. It helps to 15 factors: mechanical or kinetic energy, chemical have products well organized, and all of them well 16 energy, temperature or thermal energy, and time. packaged or covered to avoid cross-contamination. 17 Mechanical energy is of great importance for remov- Accidental spillages should be cleaned and disinfec- 18 ing biofilms (Gibson et al. 1999). Chemical energy ted as soon as possible by the production cleaning 19 removes soils from surfaces and suspends them in team. In chilling rooms, special cleaners adapted to 20 the cleaning solution to aid rinsing, following a lower temperatures should be used. 21 chemical sanitizer attack against microorganisms The major part of the processing operation for 22 remaining on the surfaces after cleaning to reduce manufacturing dry-fermented sausages is done in 23 their viability. Temperature increases the effective- one room, where the meat is ground/minced, mixed, 24 ness of cleaning and disinfection. Finally, the longer and stuffed, and where sausages can receive a cover 25 the application times of the chemicals, the more treatment to inhibit or facilitate the growth of molds 26 effective the process. and yeast. These operations are still done in a very 27 Because dry-fermented processing is considered traditional way, which means that they are still oper- 28 mostly a low-risk–producing area, cleaning and sani- ating in a batch-discontinuous system. This type of 29 tation programs are of great importance, together production determines the cleaning system; because 30 with raw material and personnel manipulation con- the use of CIP (cleaning in place) systems is impos- 31 trol, to avoid the presence of contaminants through- sible, a COP (cleaning on place) system is used 32 out the production line. This production line could be instead. 33 34 Table 47.4. Sanitation sequence useful to control the proliferation of undesirable microorganisms. 35 36 1. Remove all food and tidy up the production areas. 37 2. Remove gross soil from production equipment and dismantle if required. 38 3. Remove gross soil from environmental surfaces. 39 40 4. Rinse down environmental surfaces (usually to a minimum of 3 m in height for walls). 41 5. Rinse down equipment and flush to drain. 42 6. Clean environmental surfaces, usually in this order: drains, walls, floors. 43 44 7. Rinse environmental surfaces. 45 8. Clean equipment. 46 9. Rinse equipment. 47 10. Disinfect equipment and rinse if required. 48 49 11. Dry equipment surfaces to avoid condensation. 50 12. Fog if required (optional, or do it periodically, for instance, once a week). 51 13. Clean the cleaning equipment. 52 S53 Adapted from Holah (2003). N54 40454 47 491-502 r2.qxd 7/21/07 12:50 PM Page 498

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1 To effectively clean the main production room, it recommended to remove inorganic stains that can 2 is very important to follow a cleaning and sanitation accumulate on the equipment surfaces. 3 program sequence as is described in Table 47.4. The Step 9. Foam is rinsed with low-pressure water at 4 following sequence should be done daily after pro- 40–50°C from top to bottom. After this step, all 5 duction activities: organic soils should have been eliminated from all 6 Step 1. The first step consists of removing and surfaces. The removal of organic material is 7 keeping the remaining raw materials and the other extremely important for avoiding the formation of 8 ingredients and auxiliary materials (casings, clips, biofilms. 9 etc.) not used in production. It is very important to Step 10. Normally in the meat industry, dis- 10 switch off all the equipment and cover properly all infectants based on hypochlorites or quaternary 11 the electrical parts in order to protect them and the ammonium compounds are used. Disinfectant 12 employees. solutions are sprayed over the surfaces, from top 13 Step 2. It is important to remove all gross soil and to bottom, and left for 10–30 minutes. This step 14 debris from the equipment and conveyors and should eliminate, or at least reduce to safety levels, 15 deposit it in wastebaskets. This operation is done the microbial population. Some disinfectants are 16 manually. Most of the equipment used in dry- left to air-dry, but normally they are rinsed off, 17 fermented sausage processing can be dismantled to a again with low-pressure water. This rinsing step is 18 certain extent; this operation facilitates the following better at lower temperatures (40°C), in order to 19 cleaning and disinfection step. All parts of the avoid condensations. 20 equipment removed should be kept in trays for later Step 11. All equipment surfaces must be dried to 21 cleaning. avoid condensation. Floors are cleaned and dried 22 Step 3. Gross soil and debris should be removed as with the aid of mechanical equipment. 23 well from the environmental surfaces such as walls Step 12. Environmental decontamination is rec- 24 and floors. ommended at least once a week, and it is done pro- 25 Steps 4 and 5. Before applying the cleaners, it is ducing a fog (smoke) of disinfectants and/or an 26 important to rinse down all surfaces (equipment and insecticide that covers all the production area. 27 environmental). Normally walls are rinsed up to 3 m Cleaning and disinfection of walls higher than 3 m 28 in height. For rinsing, temperate water at 40–60°C is and ceilings should be done periodically, depending 29 used and low-pressure machines (max 25 bar). This on the splashes and dirt accumulation, normally 30 temperature range is enough for melting most fat every 3–6 months. 31 debris and aids in its removal. Hotter water could be Step 13. Finally, it is very important to dismantle, 32 used, although it is not advised to do so, because it is clean, and disinfect all cleaning tools after every 33 not economical, cooks the protein debris and pro- cleaning to prevent cross-contamination. 34 duces microdrops that expand contamination, and Ripening rooms are a very particular part of the 35 produces some problems of condensation. The rins- dry-fermented sausage process, where a soft meat 36 ing operation should be done following an appropri- mixture converts to a dry-fermented sausage. The 37 ate order, for instance, from up to down and from time sausages spend in the ripening rooms depends 38 one side of the machine to the other. on the type of product, and mainly on the casing 39 Steps 6–8. Normally for cleaning the different diameter. In modern plants, it is much easier to clean 40 surfaces of the production area, which are rich in the ripening room. It is important to empty the ripen- 41 fat and protein soils, an alkaline detergent to which ing room of products and hanging structures, so the 42 active chlorine is added is used to aid the removal of cleaning occurs between two batch productions. 43 proteinaceous deposits. These detergents are applied First the floor and walls are cleaned and disinfected, 44 at low pressure at concentrations ranging from 1–5% following a sanitation program sequence very simi- 45 and as foams, from top to bottom, covering all sur- lar to the one described for the main production room. 46 faces. Foams have the advantage of not producing Environmental decontamination is very important 47 aerosol, and they allow the use of more concentrated when cleaning the ripening rooms. Disinfection and 48 detergents, can remain on vertical surfaces for disinfestations are done by applying the sanitizer 49 longer periods (10–15 min), can cope with high and insecticides, fogging them in closed ripening 50 levels of soils, and make it is easy to identify rooms, for a period of 2–4 hours. After that period of 51 “missed cleaning” areas. Using foams, however, time, ripening rooms are kept closed for at least 24 52 requires more water for rinsing. Cleaning agents hours more, and nobody should enter during that 53S should be left for 10 to 30 minutes on the surfaces time. Fog is produced by means of a self- 54N to clean. A sporadic acid cleaning (once a week) is producing smoke can or by using some mechanical 40454 47 491-502 r2.qxd 7/21/07 12:50 PM Page 499

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devices that produce a thermonebulization of the methods can be used to monitor surfaces or the envi- 1 sanitizer products (Aerobrumer; José Collado, S.A. ronmental state of the production areas. For surface 2 Barcelona; Spain). The hanging structures, which analysis, classical microbiological methods are nor- 3 could be pallets or metal frames that move by ceiling mally used. Total Viable Count (TVC) is typically 4 rails, are cleaned manually using high-pressure used to check the number of microorganisms 5 water jets or with special washing machines. In remaining after cleaning and disinfection proce- 6 some traditional industries, where natural ripening dures. Samples are taken from the surface with a 7 rooms have fixed hanging structures, fogging is the sterile cotton swab and/or sponges. Attached microor- 8 only way to sanitize the facilities. ganisms are resuspended in a recovery medium, to 9 When dry-fermented sausages end their ripening which a disinfectant neutralizer can be added to stop 10 period, most of them are covered in different grades the disinfectant effect. In the lab, these samples are 11 by molds that could be inoculated or wild. In both processed as usual, making serial dilutions, plating 12 cases, it is normal that, before packaging, this mold in PCA (Plate Count Agar) or TSA (Tryptose Soy 13 cover will be reduced or skipped off. This operation Agar) or other selective media, depending on the 14 is made in water brushing machines, which remove microorganism search. Another way to monitor the 15 this cover almost completely. After this operation, remaining microbial population of the surface is by 16 and before the sausages go to packaging, they are using self-prepared commercial plates, such as con- 17 air-dried for a couple of days in a ripening room. The tact plates (Rodac plates) or contact slides. In all 18 waste produced by these washing machines should cases, results are given as cfu/cm2. 19 be canalized and sent to the drains. Traditional hygiene monitoring techniques do not 20 In packaging areas, it is normal to find different provide a satisfactory solution because they are very 21 packaging materials, such as cardboard boxes, plas- time consuming. Increasing attention is being paid 22 tic foils, etc. It is not possible to use wet cleaning to development of rapid methods able to detect 23 here to a great extent, so it is necessary to use a microbial and/or organic contamination of surfaces. 24 dry cleaning system, which is more manual. Peri- This is possible using the technique of ATP biolumi- 25 odically, every week, an environmental disinfec- nescence. ATP is present in all living organisms, 26 tion using fog methods, as described above, is including bacteria, and in large quantities in food- 27 recommended. stuffs. Usually, there is more nonmicrobial ATP 28 in swab samples coming from processing surfaces 29 than microbial ATP (Hawronskj and Holah 1997). 30 EVALUATING THE SANITATION A bioluminescent reaction is produced when ATP 31 STATE OF THE PLANT entrapped in a swab enters in contact with a solution 32 that contains luciferin and luciferase, which reacts to 33 Because microorganisms are not visible directly to produce the fluorescent compound oxyluciferin 34 human eyes, it is very important to assess the effec- (Deluca and McElroy 1978). Light output is meas- 35 tiveness of the sanitation programs to ensure the ured in a luminometer, and results are given as rela- 36 safety and the quality of the final products. The tive light units (RLUs). This method gives, in a few 37 cleaning and disinfection programs should be moni- minutes, a precise indication of the cleaning and/or 38 tored by different methods. It is possible to distin- disinfection state of a processing surface. A new 39 guish between two different types of methods, which development of this technique, using an amplifica- 40 are complementary. tion system of ATP, is capable of detecting very low 41 The first group of methods is related to visual levels of ATP down to 26 pM (Hawronskj and Holah 42 inspection of the cleaning and sanitizing processes, 1997). 43 and it includes auditing the application of the sanita- Other rapid techniques to detect protein soil in 44 tion program sequences described previously. This surfaces have been developed based on the Biuret 45 auditing should be done at least once a month. In reaction. In that case, a swab rubbed on a testing sur- 46 this method, a visual inspection of the cleaning face changes its color, indicating the presence of dirt 47 process should be done every day after sanitizing to on it. All these techniques could be applied every 48 check the effectiveness of the procedure; it involves day and on every surface. 49 inspecting surfaces under good lighting, smelling Finally, an indication of the contamination level of 50 for taints and off-odors, and looking for greasy and the processing environment can be checked by air 51 encrusted surfaces. samplers, which force the passage of air through an 52 The second monitoring group uses microbiologi- agar plate, which is incubated later. Results are given S53 cal analysis to evaluate sanitation programs. These as cfu/m3. N54 40454 47 491-502 r2.qxd 7/21/07 12:50 PM Page 500

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1 Table 47.5. Personnel as source of food contamination. 2 3 Route Source Example of 4 Contaminants 5 Direct (host) Gastrointestinal tract Fecal microorganisms 6 Bad hygienic practices Enterobacteria, 7 Enterococci 8 Intestinal parasites 9 10 Skin Skin host bacteria 11 Touching foods or desquamation Staphylococcus 12 Hair Staphylococcus aureus 13 Hair loss E. coli 14 Streptococci 15 Mouth and nose Staphylococcus aureus 16 Nasal cavity 17 Ears and eyes Staphylococcus aureus 18 Eye infections Streptococci 19 20 Indirect (vector) Clothing and footwear Soil and microorganisms 21 Knives Microorganisms 22 Personal small belongings Pieces of metal 23 24 25 26 PERSONNEL FOOD any transmissible illness before they start working in 27 MANIPULATION a food plant. Personnel with an acute infectious dis- 28 ease or with wounds in their skin should be kept out 29 According to the processing plant sanitation, there is of the production line. 30 a need to take into account people who are working 31 in the factories. Personnel, and in general people that 32 can move around the production line (mechanics, FINAL CONSIDERATIONS 33 visitors, managers), are a major source of contami- 34 nation because they are reservoirs and vectors of The aim of all prerequisite programs, which are the 35 microorganisms and other contaminants (physical). basis for a good HACCP plan, is to reduce at a maxi- 36 Personnel can act as a direct or indirect source of mum the risks of food contamination. This chapter 37 contamination. Direct contamination implies the has discussed brieﬂy some programs, but there are 38 transfer of microorganisms from people to the food more that should be taken into account, such as plant 39 product by direct physical contact; the indirect route pest control or maintenance of production machinery, 40 involves the transportation (vector) of contaminants which also contribute to reducing food contaminants. 41 by the human actions from one area or surface to 42 another (Holah and Taylor 2003). Table 47.5 shows REFERENCES 43 some examples of direct and indirect human con- 44 tamination of foods. Anonymous. 2000. Interim guidelines for the control of 45 To avoid personnel contamination, it is important verotoxinogenic Escherichia coli including E. coli 46 that factories implement a system of Good Manufac- 157:H7 in ready to eat fermented sausages contain- 47 turing Practices (GMP), and all personnel are trained ing beef or a beef product as an ingredient. Health 48 in their right application. Moreover, strict hygienic Canada Report. 49 and cleaning regulations, appropriate clothing, clean- Anonymous. 2002. Opinion of Scientiﬁc Committee of 50 ing and sanitizing methods, and devices should be Food on the risks to human health of Polycyclic Aro- 51 provided to all personnel before they enter a produc- matic Hydrocarbons in food. European Community. 52 tion area. S Barbuti, G Parolari. 2002. Validation of manufac- 53S Production personnel who are in direct contact turing process to control pathogenic bacteria in typi- 54N with food must pass a medical screening to pinpoint cal dry fermented products. Meat Sci 62:323–329. 40454 47 491-502 r2.qxd 7/21/07 12:50 PM Page 501

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D Dawson. 2000. Water quality for the food industry: JT Holah, JH Taylor. 2003. Personal hygiene. In: HLM 1 Management and microbiological issues. Campden & Lelieveld, MA Mostert, J Holah, B White, eds. 2 Chorleywood Food Research Association. Hygiene in Food Processing. Cambridge: Woodhead 3 M Deluca, WD McElroy. 1978. Purification and proper- Publishing Limited. 4 ties of firefly luciferase. Methods Enzymol 57:3–15. HLM Lelieveld. 2003. Sources of contamination. In: 5 FAO/WHO. 2004. Risk assessment of Listeria monocy- HLM Lelieveld, MA Mostert, J Holah, B White, eds. 6 togenes in ready-to-eat foods. Technical Report. Hygiene in Food Processing. Cambridge: Woodhead 7 H Gibson, JH Taylor, KE Hall, JT Holah. 1999. Effec- Publishing Limited. 8 tiveness of cleaning techniques used in the food B Martín. 2005. Estudio de las comunidades micro- 9 industry in terms of the removal of bacterial biofilms. bianas de embutidos fermentados ligeramente 10 J Appl Microbiol 87:41–48. acidificados mediante técnicas moleculares, 11 KA Glass, MP Doyle. 1989. Fate and thermal inactiva- estandarización, seguridad y mejora tecnolúgica. 12 tion of Listeria monocytogenes in beaker sausage and PhD dissertation. Universitat de Girona, Girona, 13 pepperoni. J Food Protec 52:231–235. Cataluña. Spain. 14 JM Hawronskj, J Holah. 1997. ATP: A universal M Pontello, L Sodano, A Nastasi, C Mammina, 15 hygiene monitor. Trends Food Sci Technol 8:79–84. M Astuti, M Domenichini, E Gerosa, A Montagna. 16 JC Hinkens, NC Faith, TD Lorang, P Bailey, D Buege, 1998. A community-based outbreak of Salmonella 17 C Kaspar, JB Luchansky. 1996. Validation of pepper- enterica serotype typhimurium associated with 18 oni processes for control of E. coli O157:H7. J Food salami consumption in Northern Italy. Epidemiol 19 Protec 59:1260–1266. Infect 120:209–214. 20 JT Holah. 2003. Cleaning and disinfection. In: HLM G Wierenga, JT Holah. 2003. Hygienic plant design. In: 21 Lelieveld, MA Mostert, J Holah, B White, eds. HLM Lelieveld, MA Mostert, J Holah, B White, eds. 22 Hygiene in Food Processing. Cambridge: Woodhead Hygiene in Food Processing. Cambridge: Woodhead 23 Publishing Limited. Publishing Limited. 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 S53 N54 40454 48 503-512 r2.qxd 7/21/07 1:55 PM Page 503

1 2 3 48 4 5 6 Quality Control 7 8 Fidel Toldrá, M-Concepción Aristoy, Mónica Flores, and 9 Miguel A. Sentandreu 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION QUALITY CONTROLS AT 22 EACH STAGE 23 Quality is an important and decisive factor for the 24 commercial transactions throughout the world market The quality control of fermented meat products can 25 and within each particular country and region. Meat be done at different stages of the processing. The 26 processing industries are thus required to implement quality control can be applied to 1) the raw materials 27 control systems to guarantee their products and com- and ingredients used for processing, 2) the process, 28 ply with the exigent market demands and legislative and 3) the final product (Curt et al. 2004). Controls 29 requirements (Toldrá et al. 2004). for each one are shown in Table 48.1 and are briefly 30 The final quality of a fermented meat product described below. 31 depends on many factors, such as the quality of the 32 initial raw materials, ingredients, and additives; the 33 processing conditions, such as fermentation condi- CONTROLS OF RAW MATERIALS 34 tions, extent of drying and ripening and packaging; Several types of control measurements, including 35 and, finally the storage conditions at the factory and instrumental and sensory measurements, are per- 36 during the commercial distribution (Toldrá 2006a). formed. Periodic microbiological controls to check 37 It is necessary to control the quality, not only in the the hygienic conditions of the raw materials are 38 final product but also at different stages of the also applied. Main controls (see Table 48.1) are as 39 process. This allows the possibility to trace back to follows: 40 find out and check whether the conditions used for 41 processing are correct and satisfy the demands of the • Lean meat: The controls mainly consist of the 42 consumer in terms of safety, nutritional value, and measurement of pH, temperature, weight, hygiene 43 sensory characteristics (Toldrá 2006b). inspection, and visual examination of lean 44 Quality control is essential for the standardization color and appearance. These controls are useful 45 of fermented meat products, which is a traditional to verify the technological quality of the lean 46 consumer demand for acceptability. It is important meat. 47 to remember that these products are bought in super- •Fat: These controls consist mainly of instrumental 48 market displays or at retail shops, and consumers measurement of temperature and visual 49 basically evaluate their general appearance (size, examination of color and aroma. 50 shape, distribution of lean and fat, color, and general • Casings: These controls involve checking the 51 presentation). This chapter deals with the quality thickness, pore size, and absence of fat as indicated 52 controls that are generally performed on fermented by suppliers. This can affect the ripening stage by 53 and ripened meat products. affecting the drying process. S54 N55 503 40454 48 503-512 r2.qxd 7/21/07 1:55 PM Page 504

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1 Table 48.1. Quality controls to be performed at each stage. 2 3 Stage Products Controls 4 Raw materials Lean meat Hygiene 5 pH, temperature 6 Weight, color, and appearance 7 Fat Hygiene 8 Temperature 9 Aroma, color, and appearance 10 Casings Hygiene 11 Thickness, pore size, and lack of fat 12 Other ingredients and Compliance with speciﬁcations 13 additives 14 15 Process: Fermentation Curing chamber Temperature, air speed, and relative humidity Sausages pH, temperature, and a 16 w 17 Microbial growth 18 Process: Drying Curing chamber Temperature, air speed, and relative humidity 19 Sausages General appearance

20 pH, temperature, and aw 21 Microbial growth, presence of molds, metabolites 22 Weight losses and moisture content 23 Final product Composition analysis Protein, fat, carbohydrates, moisture, and ashes 24 Microbial quality Control of microbial flora 25 Sensory quality General appearance 26 Color: L, a, and b parameters 27 Texture: texture profile analysis 28 Flavor: aroma and taste 29 Sensory analyses: evaluation of sensory properties 30 31 32 33 • Starter cultures and external molds: These conditions to be checked during drying and ripening 34 controls verify growth ability and compliance are temperature, air speed, extent of drying, and 35 with the specifications indicated by suppliers. hygrometry. Generally, the control parameters are 36 • Spices and seasonings, sugars, and additives: temperature and hygrometry conditions in the cham- 37 These controls verify the specifications indicated ber as well as the weight losses, temperature, and 38 by suppliers. evolution of water activity and the pH of the product. 39 Some products from proteolysis and lipolysis may 40 be analyzed. The growth of molds on the external 41 CONTROLS DURING PROCESSING surface must be controlled, too (Toldrá 2006b). 42 43 The fermentation stage is usually considered the 44 critical control point concerning the safety and sen- CONTROLS IN THE FINAL PRODUCT sory quality of the final product. Both can be either 45 Once the product is ready for distribution and sale, improved or worsened during drying/ripening 46 there are several important controls that must be per- (Toldrá 2004). 47 formed to verify its final quality. The most important The processing conditions to be checked during 48 are given in Table 48.1 and briefly described below. 49 fermentation are temperature, time of fermentation and hygrometry of the chamber. Generally, the con- 50 Composition Analysis 51 trol parameters are temperature and hygrometry 52 conditions in the chamber as well as the rate of The analysis of moisture, fat, protein, and collagen 53S microbial growth and evolution of water activity and is usually performed according to AOAC methods. 54N pH in the product (see Table 48.1). The processing The moisture/protein ratios are required by USDA 40454 48 503-512 r2.qxd 7/21/07 1:55 PM Page 505

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for adequate classification of U.S. dry or semidry The moisture content of sausages and hams 1 products (Sebranek 2004). The measurement of constitutes a good control of processing perfor- 2 water activity is used in Europe for product mance. Therefore, dry-fermented sausages may 3 standardization (Toldrá 2006a). Insufficient dryness reach water losses above 20%, whereas dry-cured 4 may be due to incorrect drying conditions, as will ham may reach nearly 30–34% of weight loss 5 be discussed in the section “Control of Drying,” (Toldrá 2006c). 6 below. The measurement of pH is convenient in 7 order to control the extent of acidification during fer- CONTROL OF SENSORY QUALITY 8 mentation and its final value by the end of the 9 process. The sensory quality of fermented meat products is 10 essential for attracting the consumer. Therefore, it 11 Microbial Quality is essential to study the factors that affect the 12 Microbial quality is essential from the safety point appearance, color, aroma, taste, and texture of the 13 of view. A number of successive hurdles protect the product and its relationship to the sensory charac- 14 fermented meat product against undesirable growths teristics perceived by the consumers. Chapters in 15 (Leistner 1992). The generation of off-odors may be Part IV of this book, “Sensory Attributes,” discuss 16 indicating some kind of microbial spoilage. This these sensory characteristics. Some controls are 17 phenomena may be accompanied by green or gray given below. 18 colorations resulting from pigment degradation as 19 well as holes due to gas generation. The growth of 20 COLOR undesirable microorganisms is generally linked to 21 poor hygienic conditions of the raw materials and/or The assessment of color is a useful tool for process- 22 processing conditions, incorrect refrigeration during ing control. The development of the typical cured 23 processing, or insufficient pH drop (Toldrá et al. color is due to the production of nitrosomyoglobin 24 2004; Toldrá 2006a). The generation of amines must by the reaction of nitric oxide, resulting from 25 be checked and the growth of undesirable molds the enzymatic reduction of nitrite, with the muscu- 26 must be prevented. lar protein myoglobin (Sanz et al. 1997). This reac- 27 tion depends on the activity of starter cultures, 28 Sensory Quality myoglobin content, and muscle pH. The content in 29 The final appearance depends on the manufacturing myoglobin, which is the major pigment in meat, 30 conditions. Therefore, defects such as fissures due to varies according to the physiological role of the 31 a rapid drying at the initial stages, dark stains due to muscle but substantially increases with the age of 32 molds, or yellow stains due to the use of rancid fat the animal (Miller 1994; Aristoy and Toldrá 1998). 33 affect the appearance. Sensory quality is important This is one of the reasons why older pigs give bet- 34 for consumer attraction toward the product and is ter red color than meats from younger animals 35 further discussed in the later section “Control of and are thus preferred for the production of cured 36 Sensory Quality.” meats. 37 The use of nonappropriate meats like pale, soft, 38 CONTROL OF DRYING and exudative (PSE) pork meat or meat from very 39 young pigs may give insufficient color development. 40 The conditions of the curing chamber (temperature, Sometimes, and when nitrate is used, the lack of cor- 41 relative humidity, and air rate) are very important rect color development may be due to an insufficient 42 for an adequate distribution of the water (balance nitrosomyoglobin formation due to the inhibition of 43 between external and internal amounts) in the sausage nitrate-reductase by rapid pH drop (Toldrá et al. 44 (Baldini et al. 2000). The equilibrium between diffu- 2001). The development of brown color can be due 45 sion of the water through the sausage and evaporation to an incorrect nitrification. The hollow center defect 46 rate from the outer sausage is difficult to reach in can be due to an overrapid drying, poor binding abil- 47 sausages of larger diameter. An excessive drying rate ity of the batter due to the lack of acidification, and 48 may give color fading in the center of the sausage poor protein solubilization (use of exudative meats, 49 and a dark color on the outside surface, which is insufficient salt content, etc). Some color deteriora- 50 associated with hardening. On the other hand, tion and/or the development of green areas may be 51 poor drying conditions are detected when the product caused by an excessive acid production or by certain 52 contains excessive moisture (Demeyer and Toldrá strains of Lactobacilli or Streptococci (Roca and S53 2004). Incze 1990; Toldrá et al. 2006b). N54 40454 48 503-512 r2.qxd 7/21/07 1:55 PM Page 506

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1 TEXTURE • Insufficient pH drop 2 • An excess of fat in the mix 3 Consistency is related with the pH and water loss • A very fast drying (high temperature, low relative 4 (Roca and Incze 1990). Some typical defects are humidity, and high air velocity), which produces 5 related to defective drying processes. When the hardening of the outer sausage surface but retains 6 sausages are of small diameter, it is usual to find a most of the moisture inside the sausage. 7 hard texture due to an excessive drying as a result of the use of high temperature, low relative humidity, 8 Fermentation (correct sugar addition, temperature, and/or high air rate. These processing parameters must 9 and starter cultures function) and drying, especially be controlled as consumers tend to reject tough 10 relative humidity and air rate within the chamber, sausages. When the sausages have larger diameters, a 11 must be carefully controlled. The microbial activity hard dry area on the outer sausage surface may be 12 produces a decrease in pH value approaching the formed under similar circumstances and act as a shield 13 isoelectrical point of meat proteins. The water hold- retaining most of the moisture and leaving a soft area 14 ing capacity is reduced, producing an increase in inside the sausage. This is known as case hardening. 15 consistency that also will be accelerated during the Texture measurements in the form of texture pro- 16 drying process. This facilitates the sliceability typi- file analysis may be performed, obtaining parameters 17 cal of the product. The salt added in the formula such as hardness, springiness, cohesiveness, adhe- 18 gives a suitable cohesion and texture during drying siveness, and chewiness (Honikel 1997). Another 19 by solubilizing proteins, which act as a bridge type of test may consist of the assessment of resis- 20 between the constituting meat fragments. However, tance to penetration as an index of hardness during 21 several defects can appear in the final product. This mastication (Tabilo et al. 1999). 22 is the case of the sticky defect or softness (due to An excessive softness inside the sausage may be 23 either an excessive amount of fat or high moisture due to different causes (Toldrá 2002): 24 in the final product), dry-edge defect (due to an 25 • The application of nonefficient drying (i.e., high overrapid drying), and inconsistency (stuffing car- 26 relative humidity), which results in poor water ried out at low pressure producing holes and air 27 loss and an excess of moisture inside the sausage inclusions, which also can produce oxidation, ran- 28 • Fat smearing, which retains water cidity, and mold development). 29 30 Table 48.2. Possible origin and odor descriptions of compounds detected in dry-fermented sausages. 31 32 Dry Fermented Sausage 33 Compound Odor 34 35 Amino acid degradation Methional Cooked potato 36 3-Methyl-butanal Sour cheese 37 2-Methyl-butanal Nail polish 38 2-acetyl-1-pyrroline Roasty, popcorn 39 3-Methyl-butanoic acid Sweaty socks 40 Lipid Oxidation Hexanal Green leaves 41 Heptanal Potatoes 42 Decanal Cucumber, dry grass 43 1-Octen-3-ol Mushroom 44 45 Carbohydrate fermentation 2,3-Butandione Butter 46 Staphylococci Esterification Propyl acetate Sourish apple, candy 47 Ethyl isobutanoate Pineapple, fruit 48 Ethyl butanoate Fruit candy 49 Ethyl 2-methylbutanoate Sweet pineapple 50 Ethyl 3-methylbutanoate Chutney, spicy 51 Spices Myrcene Lemon, fruity 52 Limonene Menthol 53S Terpinolene Fruity, eucalyptus 54N From Meynier et al. (1999); Stahnke (1994); Blank et al. (2001). 40454 48 503-512 r2.qxd 7/21/07 1:55 PM Page 507

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FLAVOR odor of meat has been isolated, it seems likely that 1 meaty aromas depend upon a subtle quantitative bal- 2 The characteristic flavor of fermented sausages ance of various components (Mottram and Edwards 3 is developed by the action of microbial and endoge- 1983). The relative contribution of the chemical 4 nous meat enzymes on carbohydrates, lipids, and compounds to the meat aroma depends on its odor 5 proteins. In addition, the salt and spices added to the 6 meat batter should be taken into account because of threshold, which gives an idea of the odor impact. 7 their important contribution to flavor (Demeyer and Therefore, if the concentration of an odorant in a 8 Toldrá 2004).There also are other nonenzymatic sausage is higher than its threshold, probably the 9 pathways that contribute to the generation of flavor odorant will contribute to the overall flavor. In fer- 10 compounds, such as autooxidation. mented sausages, different odors have been detected, 11 The carbohydrate fermentation is responsible for and their compounds and possible origin were iden- 12 the typical sausage tangy or sour taste (Lücke 1985). tified (see Table 48.2). The development of elec- 13 The interactions between carbohydrates and protein tronic noses has found important applications for 14 metabolites during meat fermentation determine the the aroma control of cured meats (Eklov et al. 1998). 15 rate of pH decline and flavor development (Demeyer The main objective on aroma analysis is the determi- 16 1992). There are internal and external parameters nation of those volatile compounds that have a 17 that influence flavor (Verplaetse 1994). The internal high impact on the food flavor. However, these 18 parameters are chemical (added sugars or spices) or compounds can contribute not only to the general 19 microbiological (starter cultures), whereas external dry-fermented aroma but also can contribute to spe- 20 parameters are physical, such as the temperature and cific aroma that in same cases can be detrimental 21 humidity during the process. (Demeyer et al. 2000). This is the case of an exces- 22 In the case of dry-fermented aroma, because no sive lipid oxidation, where the inadequate process- 23 compound or group of compounds with the unique ing conditions produce the generation of a high 24 25 26 27 Table 48.3. Sensory evaluation techniques used in dry-fermented sausages. 28 Sensory Techniques Panel Scale References 29 30 Free choice profiling Trained sensory panel 6-point intensity scale Berry et al. (1979) 31 —(selection) Untrained panel 8-point hedonic scale Berry et al. (1979) 32 Fixed choice profile Trained panel Unstructured scale with end Beilken et al. (1990) 33 —points defined by consensus 34 Semitrained assessors 9-point intensity scales Roncales et al. (1991) 35 Quantitative descriptive Trained panel Unstructured line scale Stahnke (1995) 36 —method —of 15 cm 37 38 Flavor profile Trained panel Nonstructured scales Rousset-Akrim et al. 39 —(1997) 40 Acceptance test Trained panel Nonstructured 10 cm Bruna et al. 41 —hedonic scale —(2000, 2001) 42 Selgas et al. (2003) 43 Herranz et al. 44 —(2005, 2006) 45 Acceptance test Trained panel Nonstructured 10 cm de la Hoz et al. (2004) 46 —hedonic scale 47 Quantitative descriptive Trained panel Benito et al. (2004) 48 —analysis 49 50 Acceptance test Semitrained panel 6-point intensity scale Moretti et al. (2004) 51 Descriptive sensory panel Trained panel 5-point intensity scale Ruiz Perez-Cacho et al. 52 —(2005) S53 Quantitative descriptive Trained panel 6-point intensity scale Valencia et al. (2006) N54 —analysis 40454 48 503-512 r2.qxd 7/21/07 1:55 PM Page 508 Mold Spice aroma SweetBitterSourSalty Meat impression Sweetness Bitterness Acidness Acidity Salty Fatty Rancid Nauseous Burned Solvent perception Juiciness — of aroma Sourish of flavor adhesion Juiciness Juiciness Softness Elasticity Juiciness Sensory sausages. fermented terms descriptive used for Off-aroma Off-aroma SmokeAcceptability External smell quality— Smell quality Salami Smell intensity Sourdough Cheese Cured aroma Rancid Lactic acid Mold Spice odor Hot/spice Off-flavor Smoke quality Flavor Acceptability — — ChewinessGreasiness mouth Overall Fibrousness Cohesiveness Table 48.4. Table Sensory Terms et al. Beilken Aroma Roncales et al. (1990) Acidic/sour External smell intensity (1991) Odor intensity Odor de la Hoz et al. (1995) Stahnke Aroma intensity Benito et al. (2004) Moretti et al. Ruiz Perez-Cacho (2004) Black pepper (2004) et al. (2005) Flavor Acidic/sour intensity Flavor TasteTexture Initial bite/ Toughness Texture Rancidity Taste Black pepper Hardness Saltiness Hardness Hardness Saltiness Acid

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1 2 3 4 5 6 7 Luminance 8 9 10 11 12 acceptability connection Fat/lean 13 — 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 Color cut perception Overall Overall acceptability Overall 44 45 46 47 48 of texture of appearance cut appearance Visual acceptability Ease of skin peeling 49 Fat contentFat Resistance to pressure Acceptability — Particle size Mold cover Overall Total fat Acceptability — external perception Overall Total Overall — Presence of crust 50 51 52 S53

Other Appearance:Terms Appearance: Color external Visual Color AftertasteintensityAppearance: Color Overall Exudate N54

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1 concentration of aldehydes, compounds responsible The taste terms are the basic tastes, and the texture 2 for rancid aromas. Other important flavor defects terms used are related to the hardness, softness, and 3 that can appear in fermented products are excessive cohesiveness of the sausage. In the evaluation of the 4 acidity (due to large amounts of lactic acid), rancid- appearance, the external appearance has been gener- 5 ity (high oxidation due to the use of previous rancid ally evaluated. 6 fat as a consequence of undesirable storage condi- 7 tions such as high temperatures and long times), 8 putrid aromas (developed by undesirable bacteria REFERENCES 9 due to an inappropriate fermentation or by the use of MC Aristoy, F Toldrá. 1998. Concentration of free 10 highly contaminated raw materials), and mold aro- amino acids and dipeptides in porcine skeletal 11 mas (due to contamination by molds). Unpleasant muscles with different oxidative patterns. Meat Sci 12 urinelike off-flavor is due to the use of raw meats 50:327–332. 13 from adult pigs containing androstenone, the male P Baldini, E Cantoni, F Colla, C. Diaferia, L Gabba, 14 hormone 5-α-androsten-16-en-3, at amounts above E Spotti, R Marchelli, A Dossena, R Virgili, S Sforza, 15 the detection threshold level. P Tenca, A Mangia, R Jordano, MC Lopez, L Medina, 16 S Coudurier, S Oddou, G Solignat. 2000. Dry sausages 17 ripening: Influence of thermohygrometric conditions SENSORY ANALYSES 18 on microbiological, chemical and physico-chemical 19 The appropriate evaluation of sensory properties characteristics. Food Res Inter 33:161–170. 20 involves the use of attribute difference tests, such as SL Beilken, LM Eadie, PN Jones, PV Harris, 1990. 21 paired comparison, scoring, or ratio-scaling tests Sensory and other methods for assessing salami 22 (Meilgaard et al. 1991). The descriptive sensory quality. CSIRO Food Res Quarterly 50:54–66. 23 analysis gives details on sensory changes and is use- MJ Benito, M Rodríguez, A Martin, E Aranda, JJ 24 ful in investigating treatment differences, process Córdoba. 2004. Effect of the fungal protease EPg222 25 changes, quality control, and defining sensory prop- on the sensory characteristics of dry fermented 26 erties of a product (Bett 1993). sausage “salchichon” ripened with commercial 27 The idea of descriptive analysis is based on the starter cultures. Meat Sci 67:497–505. 28 division of total flavor into flavor terms, notes, or BW Berry, HR Cross, AL Joseph, SB Wagner, JA 29 descriptors. Therefore, the flavor of a product is Maga. 1979. Sensory and physical measurements of 30 characterized using descriptive terms and it is also dry fermented salami prepared with mechanically 31 measured giving the descriptor intensity. The panel processed beef product and structured soy protein 32 consists on 5 to 20 members, and they are selected fiber. J Food Sci 44:465–468. 33 based on their abilities to smell. Common descrip- KL Bett. 1993. Measuring sensory properties of meat 34 tive flavor methods are the flavor profile (Caul in the laboratory. Food Technol 11:121–126,134. 35 1957), a nonnumerical intensity scale (simple uni- I Blank, S Devaud, LB Fay, C Cerny, M Steiner, 36 versal scale), with which the panel discusses each B Zurbriggen. 2001. Odor-active compounds of 37 sample and forms a consensus score; the quantitative dry-cured meat: Italian type salami and Parma ham. 38 descriptive analysis (QDA), which is a continuous In: Aroma Active Compounds in Foods. GR 39 line scale converted to numbers for statistical analy- Takeoka, M Günter, KH Engel, eds. pp. 9–20, 40 sis (Stone et al. 1974); and the spectrum method Washington D.C.: American Chemical Society. 41 (Meilgaard et al. 1991), which is a universal inten- ———. 2000. Combined used of Pronase E and a fun- 42 sity scale with defined reference points of commer- gal extract (Penicillium aurantiogriseum) to potenti- 43 cially available foods. ate the sensory characteristics of dry fermented 44 In dry-fermented sausages many different tech- sausages. Meat Sci 54:135–145. 45 niques have been used for sensory evaluation (see JM Bruna, M Fernández, EM Hierro, JA Ordóñez, L de 46 Table 48.3). Frequently, quantitative descriptive la Hoz. 2001. The contribution of Penicillium auran- 47 techniques have been applied, although many tiogriseum to the volatile composition and sensory 48 authors prefer the use of acceptance tests with hedo- quality of dry fermented sausages. Meat Sci 49 nic scales. 59:97–107. 50 On the other hand, many different sensory JF Caul. 1957. The profile method of flavor analysis. 51 descriptive terms have been used in fermented Adv Food Res 7:1–40. 52 sausages (see Table 48.4). 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L de la Hoz, M D’Arrigo, I Cambero, JA Ordoñez. Chemical and microbiological parameters and sen- 1 2004. Development of an n-3 fatty acid and alpha- sory attributes of a typical Sicilian salami ripened in 2 tocopherol enriched dry fermented sausage. Meat Sci different conditions. Meat Sci 66:845–854. 3 67:485–495. DS Mottram, RA Edwards. 1983. The role of triglyc- 4 D Demeyer. 1992. Meat fermentation as an integrated erides and phospholipids in the aroma of cooked 5 process. In: New Technologies for Meat and beef. J Sci Food Agric 34:517–522. 6 Meat Products. FJM Smulders, F Toldrá, J Flores, M Roca, K Incze. 1990. Fermented sausages. Food Rev 7 M Prieto, eds. Nijmegen, The Netherlands: Audet, Inter 6:91–118. 8 pp. 21–36. P Roncalés, M Aguilera, JA Beltrán, I Jaime, JM Peiró. 9 DI Demeyer, M Raemakers, A Rizzo, A Holck, A De 1991. The effect of natural or artificial casings on the 10 Smedt, B Ten Brink, B Hagen, C Montel, E Zanardi, ripening and sensory quality of a mould-covered dry 11 E Murbrek, F Leroy, F Vanderdriessche, K Lorentsen, sausage. Inter J Food Sci Technol 26:83–89. 12 K Venema, L Sunesen, L Stahnke, L De Vuyst, R S Rousset-Akrim, JF Martin, MC Bayle, JL Berdagué. 13 Talon, R Chizzolini, S Eerola. 2000. Control of 1997. Comparison between an odour profile and a 14 bioflavor and safety in fermented sausages: First flavour profile of dry fermented sausages. Inter J 15 results of a European project. Food Res Inter 33: Food Sci Technol 32:539–546. 16 171–180. MPR Ruiz Perez-Cacho, H Galan-Soldevilla, F Leon- 17 DI Demeyer, F Toldrá. 2004. Fermentation. In: Ency- Crespo, G Molina-Recio. 2005. Determination of the 18 clopedia of Meat Sciences. W Jensen, C Devine, M sensory attributes of a Spanish dry-cured sausage. 19 Dikemann, eds. London, UK: Elsevier Science Ltd., Meat Sci 71:620–633. 20 pp. 467–474. Y Sanz, R Vila, F Toldrá, P Nieto, J Flores. 1997. Effect 21 T Eklov, G Johansson, F Windquist, I Lundström. 1998. of nitrate and nitrite curing salts on microbial 22 Monitoring sausage fermentation using an electronic changes and sensory quality of rapid ripened 23 nose. J Sci Food Agric 76:525–532. sausages. Inter J Food Microbiol 37:225–229. 24 B Herranz, L de la Hoz, E Hierro, M Fernandez, JA JG Sebranek. 2004. Semidry fermented aausages. In: 25 Ordonez. 2005. Improvement of the sensory proper- YH Hui, L Meunier-Goddik, ÅS Hansen, J Josephsen, 26 ties of dry-fermented sausages by the addition of free W-K Nip, PS Stanfield, F Toldrá, eds. Handbook of 27 amino acids. Food Chem 91:673–682. Food and Beverage Fermentation Technology. New 28 B Herranz, M Fernandez, L de la Hoz, JA Ordonez. York: Marcel Dekker, Inc., pp. 385–396. 29 2006. Use of bacterial extracts to enhance amino acid MD Selgas, J Ros, ML Garcia. 2003. Effect of selected 30 breakdown in dry fermented sausages. Meat Sci yeast strains on the sensory properties of dry fer- 31 72:318–325. mented sausages. Eur Food Res Technol 217:475–480. 32 KO Honikel. 1997. Reference methods supported by LH Stahnke. 1994. Aroma components from dried 33 OECD and their use in Mediterranean meat products. sausages fermented with staphylococcus xylosus. 34 Food Chem 59:573–582. Meat Sci 38:39–53. 35 F Leistner. 1992. The essentials of producing stable and ———. 1995. Dried sausages fermented with 36 safe raw fermented sausages. In: New Technologies Staphylococcus-Xylosus at different temperatures 37 for Meat and Meat Products. FJM Smulders, and with different ingredient levels. 2. Volatile com- 38 F Toldrá, J Flores, M Prieto, eds. Nijmegen, The ponents. Meat Sci 41:193–209. 39 Netherlands: Audet, pp. 1–19. H Stone, J Sidel, S Oliver, A Woolsey, RC Singleton. 40 FK Lücke. 1985. Fermented sausages. In: Microbiol- 1974. Sensory evaluation by quantitative descriptive 41 ogy of Fermented Foods. BJB Wood, ed. London, analysis. Food Technol 28:24–34. 42 UK: Elsevier Applied Science Pub, pp. 41–81. G Tabilo, M Flores, SM Fiszman, F Toldrá. 1999. Post- 43 M Meilgaard, GV Civille, BT Carr. 1991. Sensory mortem meat quality and sex affect textural proper- 44 Evaluation Techniques. Boca Raton, Florida: CRC ties and protein breakdown of dry-cured ham. Meat 45 Press, Inc. Sci 51:255–260. 46 A Meynier, E Novelli, R Chizzolini, E Zanardi, F Toldrá. 2002. Dry cured meat products. Trumbull, 47 G Gandemer. 1999. Volatile compounds of commer- Connecticut: Food & Nutrition Press, pp. 27–220. 48 cial Milano salami. Meat Sci 51:175–183. ———. 2004. Fermented meats. In: Food Processing: 49 RK Miller. 1994. Quality characteristics. In: Principles and Applications. YH Hui, JS Smith, eds. 50 Muscle Foods. DM Kinsman, AW Kotula, BC Ames, Iowa: Blackwell Publishing, pp. 399–415. 51 Breideman, eds. New York: Chapman and Hall, ———. 2006a. Biochemistry of fermented meat. In: 52 pp. 296–332. Food Biochemistry and Food Processing. YH Hui, S53 VM Moretti, G Madonia, C Diaferia, T Mentasti, MA WK Nip, ML Nollet, G Paliyath, BK Simpson, eds. N54 Paleari, S Panseri, G Pirone, G Gandini. 2004. Ames, Iowa: Blackwell Publishing, pp. 641–658. 40454 48 503-512 r2.qxd 7/21/07 1:55 PM Page 512

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1 ———. 2006b. Meat fermentation. In: Handbook of F Toldrá, R Gavara, JM Lagarón. 2004. Packaging and 2 Food Science, Technology and Engineering, Volume 4. quality control. In: Handbook of Food and Beverage 3 YH Hui, E Castell-Perez, LM Cunha, I Guerrero- Fermentation Technology. YH Hui, LM Goddik, 4 Legarreta, HH Liang, YM Lo, DL Marshall, WK Nip, J Josephsen, PS Stanﬁeld, AS Hansen, WK Nip, 5 F Shahidi, F Sherkat, RJ Winger, KL Yam, eds. Boca F Toldrá, eds. New York: Marcel Dekker, Inc., 6 Raton, Florida: CRC Press, Inc., pp. 181-1–181-12. pp. 445–458. 7 ———. 2006c. The role of muscle enzymes in dry- I Valencia, D Ansorena, I Astiasarán. 2006. Nutritional 8 cured meat products with different drying conditions. and sensory properties of dry fermented sausages 9 Trends Food Sci Technol 17:164–168. enriched with n-3 PUFAs. Meat Sci 72:727–733. 10 F Toldrá, M Flores, Y Sanz. 2001. Meat fermentation A Verplaetse. 1994. Inﬂuence of raw meat properties 11 technology. In: Meat Science and Applications. YH and processing technology on aroma quality of raw 12 Hui, WK Nip, RW Rogers, OA Young, eds. New fermented meat products. Proc 40th Int Congr Meat 13 York: Marcel Dekker, Inc., pp. 537–561. Sci and Technol, The Hague, The Netherlands, p. 45. 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53S 54N 40454 49 513-534 r1.qxd 7/20/07 5:36 PM Page 513

1 2 3 49 4 5 6 HACCP 7 8 Maria Joao Fraqueza, Antonio S. Barreto, and Antonio M. Ribeiro 9 10 11 12 13 14 15 16 17 18 19 20 21 THE HAZARD ANALYSIS sophisticated level, in Europe there are a great 22 CRITICAL CONTROL POINT number of regions where products are manufactured 23 CONCEPT:WHY USE IT in a traditional cottage-style or farmhouse manner 24 (Caplice and Fitzgerald 1999; Tradisausage 2006). It 25 The major goal of a food operator is to produce and is also true that consumers prefer traditional fer- 26 put on the market tasty, convenient, and safe prod- mented meat products produced in small and farm- 27 ucts in order to establish and maintain a trust rela- house units due to their characteristics of taste and 28 tionship with consumers and be rewarded for his aroma. However, for large food corporations or 29 effort. The concepts of safety and genuineness imply small farm or cottage manufactures, the safety of the 30 that food will not cause harm to the consumer, from products must be assured and has to be achieved by 31 when it is prepared to when it is eaten in accordance different preventive proactive measures assisted by 32 with its intended use, and that its nature and origin mandatory legal requirements. 33 may be retrieved along the food chain. These are the The proactive methodology such as HACCP 34 primary concerns of consumers, and therefore con- (Hazard Analysis and Critical Control Point) implies 35 stitute the utmost priorities of EU policy Agenda a systematic application of preventive measures to 36 2000 reported on the White Paper on Food Safety be managed in all the fermented meat production 37 (COM 2000) and supported by EU food safety steps according to an established plan. It is a docu- 38 authorities as well as those of the United States ment where identiﬁcation and evaluation of food 39 through Healthy People 2010 (USDHHS 2000) safety hazards are reported along with preventive 40 supported by the Food and Drug Administration measures for their control (Mortimore and Wallace 41 (FDA), Center for Disease Control and Prevention 1997; FDA 2005). 42 (CDC), Department of Health and Human Services Originally, this system was developed and has been 43 (DHHS) and the Food Inspection Systems of the applied to food production since the early 1960s by 44 U.S. Department of Agriculture (USDA) (FDA the U.S. Pillsbury Company with the cooperation and 45 2005). In a global market, food safety is a top prior- participation of the National Aeronautic and Space 46 ity for food trade, and given even greater emphasis Administration (NASA), Natick Laboratories of the 47 by the aging of the population, the increased number U.S. Army, and the U.S. Air Force Space Laboratory 48 of immunocompromised individuals and changes in Project Group. It was considered the only way to 49 consumer eating habits and production practices. ensure 100% food safety for the astronauts of the 50 If the big food plants in the developed world claim Apollo program (Mortimore and Wallace 1997). 51 to have elevated the production of some fermented The terms of reference for food safety include 52 foods to a large industrial and technologically biological, chemical, or physical agents that may be 53 S54 N55 513 40454 49 513-534 r1.qxd 7/20/07 5:36 PM Page 514

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1 present in a food and which are hazards likely to control points (CCPs) to put them under 2 cause illness or injury to consumers if not put under control 3 control. 4 The HACCP system has been recognized world- Construction and implementation of this system is a 5 wide as an effective system to warrant food safety. big expensive task, the success and financial return 6 The system must integrate every step in the food of which implies both the support and full involve- 7 chain from producer to consumer to assure “food ment of upper management. HACCP development 8 safety from farm to fork.” Before being enforced and implementation supported only by middle 9 by law and praised by global free trade, HACCP management or technical personnel is an impossible 10 was recognized by food operators as the most mission. 11 reliable and least expensive system to assure food 12 safety. 13 THE SEVEN HACCP PRINCIPLES 14 Principle 1 is the identification of biological, chemi- 15 FOUNDATIONS OF SYSTEM DEVELOPMENT AND cal, and physical hazards in all materials and process 16 IMPLEMENTATION steps (Goodfellow 1995; Mortimore and Wallace 17 The development of a HACCP program depends on 1997; FAO 1997). 18 the will of the upper administration and free accept- After assessing a consistent risk and preventive 19 ance by the work force. Work starts by organizing a measures designed to control hazards, Principle 2 20 team of workers previously selected and trained in implies the identification of adequate critical control 21 hazard food analysis and HACCP principles. One points (CCPs) in materials and process steps to con- 22 single person cannot carry out a HACCP plan; it has trol the hazards. 23 to be worked by a multidisciplinary team with com- Principle 3 is the definition of critical limits for 24 petences in different areas such as quality, commod- each CCP. 25 ity purchasing, production, engineering, distribution, Principle 4 is monitoring to warrant fulfillment of 26 microbiology, toxicology, and auditing. When all procedures at each stated CCP. 27 such expertise is not available, as in the case of small When a deviation from the outlined limits occurs 28 units producing fermented meat products, expert at a CCP, Principle 5 states that corrective action is 29 advice should be obtained from outer sources such immediately applied to restore control. 30 as trade and industry associations, independent Principle 6 is verification to test compliance of the 31 experts, regulatory authorities, and assisted HACCP plan with the HACCP system. 32 training with the work force being guide by sector- Principle 7 is a record of operations including 33 specific operative manuals. procedures for monitoring, corrective actions, and 34 The team leader should have organizational verification providing an effective demonstration of 35 and communication skills, familiarity with plant the system at work. 36 processes, and absolute authority to pursue this The final element for HACCP development is to 37 program in the company. The development of update the plan when changes in the process or new 38 HACCP program will include: legal or trade requirements are introduced. 39 The HACCP plan is specific to a certain product, 40 • Product description and distribution, including process, and enterprise. Nevertheless, in many cases, 41 formulation and screening of potential abuse several product lines are so similar that they can be 42 through distribution or by consumers grouped together in generic models. The use of a 43 • Identification of projected uses and consumers generic HACCP model to build specific HACCP 44 of the product, raw or cooked, and whether plans always needs creative adaptation and tuning. In 45 segments of the population are at increased risk, this case, system validation is always needed for 46 such as infants, elderly, immunocompromised, etc. each specific plan. 47 • Construction of a flow diagram, providing a brief To accomplish with success the implementation of 48 description of all operative steps involved, from HACCP plans, some prerequirements need to be ful- 49 the reception of raw materials to finished products filled. They are mandatory for food safety. In small 50 distribution food workshops, they can per se be the foundation of 51 • A hazard analysis based on stated terms so called “light” HACCP. It may be assumed in 52 of reference is the foundation of HACCP extremis that a well-trained person with access to guid- 53S construction, coupled with the critical ance is able to implement in-house HACCP methods. 54N 40454 49 513-534 r1.qxd 7/20/07 5:36 PM Page 515

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HACCP MODEL FOR FERMENTED So for small units, the application of a validated Code 1 MEAT SAUSAGES: TOWARD A of Hygiene practices will be effective to reduce the 2 GENERIC MODEL FOR HACCP risk of hazards and is fundamental for subsequent 3 implementation of a HACCP-based approach. 4 IMPLEMENTATION IN 5 TRADITIONAL WORKSHOPS AND PRODUCT DEFINITION AND INTENDED USES 6 SMALL FERMENTED MEAT A great diversity of products which are fermented 7 SAUSAGE PLANTS dry or semidry sausages with singular organoleptic 8 9 PREREQUIREMENTS characteristics are found in different countries, but they have in common a sequence of process steps to 10 Standard requirements of hygiene for the produc- 11 assure their stability such as a decrease in pH and aw, tion, handling, packaging, storing, and distribution promoted by environmental or technological dedi- 12 of fermented meat sausages are mandatory to assure cated flora (starters), and drying coupled sometimes 13 a healthy and wholesome supply of such products with particular ripening agents, preservatives and 14 and the successful construction and implementation smoke (Leistner and Gould 2003). 15 of a HACCP program. Thus, fermented sausages are the end product of 16 Layout and operative conditions of premises and comminuted pork meat stuffed into casings (salami, 17 equipment coupled with Good Hygiene Practices the Italian fermented sausages also contain beef) 18 (GHP) and Good Manufacturing Practices (GMP) mixed with fat, salt, sugar, preservatives (nitrite/ 19 programs are critical to small meat and poultry pro- nitrate), and seasonings (wine, garlic, pepper, paprika, 20 cessing plants, and are required to fulfill a HACCP and other spices), more-or-less acidified by lactic 21 plan for fermented meat sausages. The traceability fermentation (Caplice and Fitzgerald 1999; Leistner 22 of meat and any other substances used in traditional and Gould 2003). The particular seasoning, structure 23 fermented sausages is also required and is to be 24 (meat particles size), type of casing, acidity, and aw assessed at all stages of production and distribution. gives rise to stability and specific organoleptic char- 25 General GHP and GMP codes for different food acteristics of the sausages coming from a traditional 26 process categories have already been performed by knowhow of different countries and regions from 27 regulatory food standard organizations (FAO/WHO central and southern Europe. 28 2005b). However, enterprises must define and elabo- These sausages are marketed without the assur- 29 rate their own GHP and GMP codes as operative ance of a lethal heat treatment. It is important to a 30 guides. HACCP team to assume what kind of product actu- 31 An evaluation of prerequirements in small units ally is concerned (Table 49.2) and how and by whom 32 and farmhouses producing traditional fermented it will be consumed (Mortimore and Wallace 1997). 33 meat products was performed by the Tradisausage 34 project (2006) in southern European countries, PROCESS DESCRIPTION AND FLUX DIAGRAM 35 including France, Greece, Italy, Spain, Portugal, and 36 Slovakia. The data from a checklist designed to eval- A technological flow diagram of operation depicting 37 uate prerequirements, GHP, and GMP (Table 49.1) all pertinent manufacturing steps from the reception 38 led to the conclusion that approximately 80% of the of raw materials to final product, is outlined by the 39 small units in southern Europe have the main pre- HACCP team (Figure 49.1). This flow diagram is 40 requirements essential to implement the HACCP used for hazard analysis after being validated on the 41 system. However, they have to overcome the prob- premises. 42 lem related to documented evidence and validation The technology of process description must comply 43 of GHP. The project revealed that the integration of with all specified good hygiene and manufacturing 44 existing quality control facilities into an operative practices for the elaboration of a specific fermented 45 HACCP plan by a small team is a difficult task. product, as defined in the prerequirements. 46 It is important to note that Codex Alimentarius 47 TERMS OF REFERENCE FOR HAZARDS: Commission (FAO/WHO 2005) and European Com- 48 RISK EVALUATION mission Health and Consumer Protection Direc- 49 torate–General (EC 2005) permit, when applying A hazard is any factor that may be present in the 50 HACCP, flexibility when appropriate, depending on product causing harm to the consumer either through 51 the context of the application and taking into injury or illness. The identification of hazards in the 52 account the nature and the size of the operation. production of fermented sausages must be related to S53 N54 40454 49 513-534 r1.qxd 7/20/07 5:36 PM Page 516

1 Table 49.1. A checklist for prerequirements evaluation in small units and farmhouse facilities producing 2 traditional fermented meat sausages in southern European countries. 3 Answer (% of Processing Unit) n 55 4 5 Ye s a No 6 Questions Badb Sufficient Good 7 8 Premises: Buildings and Equipment 9 Competent authority approved the plant?a 96.4 3.6 10 There is waste treatment?a 70.9 29.1 11 Selection and separation of solid trash?a 60.0 40.0 12 Water supply? With treatment or not?a 96.4 3.6 13 Evaluation of the layout: Are there crossed lines?a 32.7 67.3 14 Facilities: Materials used in the construction 94.5 5.5 15 —(walls, floor, ceilings, doors, windows) 16 —are appropriate for easy cleaning?a 17 Walls and floor material conservation?b 11.1 38.9 50.0 18 b 19 Walls and floor hygiene level? 16.4 32.7 50.9 20 Do open windows have fine mesh to 80.0 20.0 a 21 —keep out insects? 22 Are there electric flytraps?a 70.9 29.1 23 Are all lights in preparation or processing 87.3 12.7 24 —areas equipped with shields or proper covers?a 25 Is there adequate or sufficient ventilation 76.4 23.6 26 —or equipment to minimize odors and vapors 27 —and prevent water condensation?a 28 Are there foot-washing devices at the entrance 25.5 74.5 29 —of your workshop?a 30 Sanitary Facilities 31 Is the establishment producing in compliance with some 70.9 29.1 32 Code of Hygiene Good Practices?a 33 In the process area are the hand-washing facilities 96.4 3.6 34 —adequate and sufficient?a 35 Hand-washing facilities before entrance?a 83.6 16.4 36 a 37 Effective hand-cleaning and sanitizing preparations? 87.3 12.7 a 38 Presence of hand-drying devices? 76.4 23.6 39 Are there toilets, urinals?a 94.5 5.5 40 Are there toilet rest areas?a 58.2 41.8 41 Are the toilets, urinals, and rest areas kept clean?b 10.9 38.2 50.9 42 Are there bird, insect, and rat control plans provided by a 40.0 60.0 43 —specialized pest control enterprise?a 44 Equipment 45 Equipment and utensils are designed and constructed with 98.2 1.8 46 —material and workmanship as to be adequately cleanable?a 47 Equipment and utensils are made of innocuous material 94.5 5.5 48 —that will not cause food contamination?a 49 50 Hygiene and Sanitation a 51 Is an individual health certificate issued? 93.6 36.4 a 52 Do personnel have routine medical examinations? 78.2 21.8 53S Do all workers use suitable protective clothing 81.8 18.2 54N —(hair nets/caps, boots)?a

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Table 49.1. (Continued) 1 2 Answer (% of Processing Unit) n 55 3 4 Ye s a No 5 Questions Badb Sufficient Good 6 Do all workers keep protective clothing in good hygienic 87.3 12.7 7 —condition before beginning to work?a 8 Are jewels, watches, and other adornments removed 83.3 16.7 9 —before entering working premises?a 10 How frequently is the protective clothing changed?b 9.1 58.2 32.7 11 12 Are workers with illnesses, open lesions, or any other 83.6 16.4 13 —abnormal source of microbial contamination not in 14 —direct contact with food?a 15 Are specific and adequate education and training provided 80.0 20.0 16 —to workers (hygiene and process)?a 17 Cleaning-Disinfecting Methods 18 Facilities cleaning schedule—floor. Daily, twice a 92.6 3.7 3.7 19 —week, others?b Daily Twice a week Others 20 Facilities cleaning schedule—walls. Daily, twice a 29.1 21.8 49.1 21 —week, others?b Daily Twice a week Others 22 Ceilings cleaning schedule. Twice a week, every week, 5.5 9.1 85.5 23 —others?b Twice a week Every week Others 24 Equipment cleaning/disinfection frequency. At the 67.3 30.9 1.8 25 —end of each utilization, daily, others?b At the end of Daily Others 26 each utilization 27 Is removal of solid residues done prior to cleaning operations?a 100 28 Is there specific equipment used for sterilization of 45.5 54.5 29 —knives and other utensils?a 30 Are chemicals (pesticides, herbicides, cleaning agents, 89.1 10.9 31 —lubricants, boiler compounds) stored in concealed 32 —designated areas?a 33 34 Production and Process Controls 35 a Are there specific demands for raw materials characteristics? 61.8 38.2 36 Are raw materials and other ingredients checked 72.7 27.3 37 a —on their preemption or “best before” date? 38 Are records of raw materials and other ingredients 45.5 54.5 39 —received kept on file?a 40 Are food additives stored in concealed areas, more reserved?a 76.4 14.5 41 Is there manipulation of other meat species 41.8 58.2 42 —apart from pork or beef?a 43 Is there an operative process control system?a 54.5 45.5 44 Is there a sampling analysis control plan?a 52.7 47.3 45 Is there some sort of regular monitoring of the 60.0 40.0 46 —process’s time and temperature?a 47 Is there a plan for metrology calibration control 25.5 74.5 48 —with a specialized enterprise?a 49 50 Is a record kept on file with this information?a 34.5 65.5 51 Are relevant documents kept in a specific record?a 47.3 52.7 52 a Is there some kind of documentation control? 38.2 61.8 S53 a Answer in two classes. N54 b Answer in three classes. 517 40454 49 513-534 r1.qxd 7/20/07 5:36 PM Page 518

1 Table 49.2. Product definition of some fermented dried sausages. 2 3 Saucisson, Chouriço, Llonganissa, 4 Product Names Loukanika xoriatika, Salami 5 Product composition (depends on the recipes of Pork (70–80% lean meat and 20–30% fat), beef, 6 —different countries and regions) —salt, sugar, condiments (wine, pepper, paprika, 7 —garlic, other spices), additives (ac. ascorbic, 8 —nitrite) casings 9 10 Product characteristics Size and weight: 40 cm, 3 cmø, 270 g–2 kg —pH: 4.8–6.0 a : 0.85–0.90 11 w 12 How it is to be used Raw ready-to-eat by healthy uncompromised people 13 Packaging No packaging or in modified atmosphere or vacuum 14 —packages 15 Shelf life Best before 4 months 16 17 Places/premises of commercialization or marketing Preferentially in open markets and butcher’s shops, 18 —but also in restaurants, retail dealers, and directly 19 —in the workshop 20 Conditions of storage Dry and cool room without incidence of light 21 Labeling instructions According to label legal requirements 22 Commercialization or marketing conditions Keep in dry and cool space or in a refrigerated display 23 24 25 26 Reception of pork carcass, meat cuts, condiments and other ingredients, casings, packaging materials 27 Whole meat cuts Natural dry or salt casings 28 Packaging Condiments (carcass, pieces, and trimmings) 29 material Storage 30 Storage Storage 31 Storage Meat cuts deboning 32 Weighting 33 Mincing 34 Weighting Casing desalting or re- 35 hydration 36 Mixing 37 38 Resting 39 40 41 Stuffing 42 Smoking 43 Fermentation 44 Drying Stabilization 45 Drying 46 Maturation 47 48 MAP or vacuum packaging 49 50 Storage 51 52 Distribution 53S 54N Figure 49.1. General flow diagram of dry/smoked-fermented sausages.

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all ingredients, incoming materials and steps of are the points where control of hazards is performed. 1 process. An action taken at the CCP designed to control the 2 In Table 49.3 potential biological, chemical, and hazard is called a preventive measure. These include 3 physical hazards are shown. They are usually based physical, chemical, or biological factors or other 4 on literature references and experimental and epi- hurdles required to control a hazard likely to occur at 5 demiological historic data. After considering the particular stages of the production of fermented 6 probability of occurrence and severity of a hazard, its sausages (Table 49.4). More than one preventive 7 risk must be assessed, which then determines which measure may be required to control a hazard. On the 8 hazards are to be addressed in the HACCP plan. other hand, more than one hazard might be effec- 9 The microbial hazards in processing units of fer- tively controlled by one single preventive measure. 10 mented sausages depend on the prevalence of Control of some preventive measures designed to 11 pathogens in meats and premises, considering their eliminate or reduce potential hazards may be assured 12 specific microbial ecology (Tompkin 2002). Inade- by validated GHP/GMP. Critical Control points are 13 quate hygiene practices may result in a loss of micro- those requiring strict and expensive monitoring, so 14 bial control and become a hazard (Metaxopoulos et al. they are to be maintained at low numbers—no more 15 2003). According to Tompkin (2002) and Eisel et al. than three or four in each plan. 16 (1997), the complete elimination of pathogens from Effective monitoring operations of CCPs are fun- 17 raw material and the food processing environment is damental to the safety of the product. CCPs can be 18 rather difficult if not impossible; however, monitoring found by using team knowledge about fermented 19 of their control is badly needed within the preventive meat products, only for real and likely occurring 20 practices prerequired by HACCP methodology. hazards and where preventive measures are available 21 The Tradisausage project assessed the probability for their control. 22 of occurrence of potential microbial hazards (E. coli, To assist in finding CCPs, a Decision Tree can be 23 Salmonella spp., S. aureus, Listeria monocytogenes) used (Figure 49.2). Several versions published by 24 associated with the production of traditional fer- FAO/WHO (2005a) provide a tool for structured 25 mented sausages. The occurrence of pathogens in thinking and ensure a consistent approach (or 26 314 environmental samples (machines, cutting embarrassing difficulty) to CCP finding in process 27 tables, knives, and other utensils) from 54 different steps for the real control of each hazard identified. 28 processing units covering central and southern The same reasoning is performed for raw materials 29 European countries was evaluated: Salmonella spp. (Figure 49.3). Table 49.5 summarizes CCPs identifi- 30 was absent in all but 3 located in Greece; L. monocy- cation in fermented dry sausage process steps using 31 togenes were found in 2.2% of the environmental the CCP Decision Tree. 32 samples; and S. aureus were encountered in 6.1%. For the purpose of Decision Tree reasoning, steps 33 There are no epidemiological data on chemical where safety risk is assured by GHP/GMP are called 34 residues of products used in sanitation and mainte- Control Point-GHP/GMP. 35 nance programs in fermented sausages or raw meat Fermentation and/or smoking and maturation/sta- 36 (Alaña et al. 1996). Albeit these chemicals may be in bilization are important sequential steps in the 37 the final product and reach the consumer, they are not process of fermented traditional sausages contribut- 38 considered an actual risk because they are prevented ing all together, as a clustered CCP, to the reduction 39 by the implemented actions of GHP and GMP. of likely occurring biological (growth of pathogens) 40 The frequency of physical hazards occurring on and chemical (biogenic amines) hazards. 41 fermented sausages is unknown; there is no data 42 related to this kind of hazards, but bone and metal 43 ESTABLISHMENT OF CRITICAL LIMITS fragments must be addressed. In spite of its low fre- 44 quency, there is a high social repercussion with eco- After identification of all CCPs, the next step is to 45 nomic losses to the producer. decide how to control them and establish the criteria 46 that indicate the difference between a safe and an 47 unsafe product. The absolute tolerance at a CCP is 48 CRITICAL CONTROL POINT IDENTIFICATION known as a critical limit. 49 A critical control point (CCP) is defined as a point, It is important to note that the critical limit must 50 step, or procedure where control can be applied and be associated with a measurable factor that can be 51 a hazard can be prevented, eliminated, or reduced to routinely monitored according to a fixed schedule. 52 acceptable levels (Mortimore and Wallace 1997). These critical limits are established based on S53 CCPs are essential for product safety because they published data (scientific literature, in-house and N54 40454 49 513-534 r1.qxd 7/20/07 5:36 PM Page 520

1 Table 49.3. List of potential biological, chemical, and physical hazards to be addressed in fermented 2 sausages. 3 Potential Hazards Identification 4 5 Biological (Bacteria, 6 Parasites, Virus) Chemical Physical 7 8 Incoming Materials 9 Raw meat: pork and beef Nonsporulating bacteria: Antibiotic/drug Bone, plastic, wood, 10 —E. coli, Salmonella spp., —residues —metal particles 11 —S. aureus, Hormones, pesticides 12 —L. monocytogenes, Biogenic amines of 13 —Campylobacter spp., —microbial origin 14 —Yersinea enterocolitica. —(tyramine and 15 Sporulating bacteria: —histamine) 16 —C. botulinum, 17 —C. perfringens 18 Parasites: Trichinella; 19 —Cysticercus cellulosae, 20 —C. bovis, Toxoplasma gondii 21 Spices B. cereus, C. botulinum, Nonfood chemical Plastic, sand and 22 —Salmonella spp., Pesticides, mycotoxines —wood particles, 23 —L. monocytogenes —stones 24 Salt E. coli, Salmonella spp., Nonfood chemical Metal, sand and soil 25 —Vibrio spp., S. aureus; —particles, stones 26 —Clostridia 27 28 Casings natural E. coli, Salmonella spp., 29 —L. monocytogenes, Clostridia 30 Water Not potable, E. coli, Nonfood chemical 31 —Salmonella spp., Vibrio spp., 32 —Clostridia, Cryptosporidium, 33 —virus 34 Starter cultures Contamination by pathogens 35 36 Package Nonfood grade 37 —packaging material 38 —or improper printed 39 —labels 40 Process Steps 41 Reception Reception of noncompliant Nonfood grade Reception of 42 —material with legal or —packaging material —noncompliant 43 —predefined requirements —or improper printed —material with legal 44 Growth of pathogens on —labels —or predefined 45 —fresh meat due to time/ —requirements 46 —temperature abuse 47 Storage Growth of pathogens on 48 —meat due to time/temperature 49 —abuse Bacterial contamination 50 —of spices during storage, growth 51 —of fungi producers of myco- 52 —toxines due to humidity abuse. 53S 54N

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Table 49.3. (Continued) 1 2 Potential Hazards Identification 3 Biological (Bacteria, 4 Parasites, Virus) Chemical Physical 5 6 Pork cuts and trimmings Contamination and growth Bone and metal 7 and beef cuts —of pathogens due to time/ —particles falling 8 —temperature abuse —into meat during — 9 cutting/trimming 10 Mincing Contamination and growth Detergent, disinfectant, Bones and metal 11 —of pathogens due to time/ —and lubricants residues particles falling into 12 —temperature abuse meat during mincing 13 14 Casings desalting Growth of pathogens 15 or rehydration —due to time/temperature 16 —abuse 17 Weighting ingredients Excess of nitrites/ 18 and additives —nitrates or other 19 —additives due to 20 —weighing errors 21 Mixing Growth of pathogens due to Detergent, disinfectant, 22 —time/temperature abuse —and lubricants residues 23 24 Stuffing Contamination with Detergent, disinfectant, Casing bursts, bones 25 —pathogens by poor —and lubricants residues —particles, metal 26 —hygiene practices —clips, cotton string 27 —falling into meat 28 —during stuffing 29 Resting Growth of pathogens due to 30 —time/temperature abuse 31 Fermentation/drying Growth of pathogens Biogenic amines of 32 —due to time/temperature/ —microbial origin 33 —humidity abuse, improper —(tyramine and 34 —fermentation, insufficient —histamine) 35

—reduction of pH and aw 36 Smoking Growth of pathogens due Biogenic amines of 37 —to time/temperature/ —microbial origin 38 —humidity abuse, improper —(tyramine and histamine), 39 —fermentation, insufficient —3,4-benzopyrene, 40 —reduction of pH and a —dioxins 41 w 42 Maturation Growth of pathogens due Biogenic amines of 43 —improper time/temperature/ —microbial origin 44 —humidity, insufficient —(tyramine and 45 —reduction of pH and a —histamine) w 46 Packaging vacuum Pathogen bacterial Allergies to an ingredient 47 and MAP and labeling —contamination by environ- —due to wrong labeling of a 48 —ment or handling during —product or unsatisfied lack 49 —packaging —of advice on allergenic 50 Growth of pathogens by —substances 51 —improper package sealing 52 —or gases concentration S53 N54 (Continues)

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1 Table 49.3. (Continued) 2 3 Potential Hazards Identification 4 Biological (Bacteria, 5 Parasites, Virus) Chemical Physical 6 7 Storage end product Growth of molds due to Mycotoxines 8 —improper time/ temperature/ 9 —humidity 10 Distribution Pathogenic bacterial Nonfood chemical Plastic, wood, 11 —contamination through —residues cross contami- —metal particles 12 —damaged packages —nation through damaged —into damaged 13 Growth of pathogens due to —packages —packages 14 —improper time/ temperature/ 15 —humidity 16 17 Sources: Feng (1995); FESP (1997); Foster (1997); Paukatong and Kunawasen (2001); Hoornstra et al. (2001); Oiye and 18 Muroki (2002); Hugas et al. (2003); Roy et al. (2003); FDA/CFSAN (2005); CDC (2006). 19 20 21 22 Table 49.4. Preventive measures to control potential hazards identified in the production of fermented dry sausages. 23 24 Hazard 25 Process Steps Identification Preventive Measure 26 27 Reception: Biological Suppliers selected and homologated and/or certified 28 raw meat Chemical Correct temperature of delivery (7°C) 29 Physical Visual control 30 Other ingredients Suppliers selected homologated and/or certified 31 Visual control of shelf life dates according to specified 32 —requirements 33 Water According to specified and legal requirements of potability 34 35 Package materials Suppliers selected homologated and/or certified 36 According to specified legal requirements 37 Storage Biological GHP and GMP 38 Corrected temperature and relative humidity 39 Pork cuts and trimming Physical GHP and GMP 40 and beef cuts Biological Visual control 41 Corrected temperature and relative humidity 42 Casing desalting or Biological GHP, GMP 43 rehydration Corrected time/temperature 44 45 Mincing Biological GHP, GMP 46 Chemical Preventive equipment maintenance 47 Physical Use of nontoxic food-compatible cleaning compounds 48 Weighting of ingredients Chemical Adequate weighting control, safe operating 49 and additives practices according to additive instructions and legal 50 requirements, calibration of scales 51 Mixing Biological GHP, correct time/temperature, preventive equipment 52 Chemical —maintenance, use of nontoxic food-compatible 53S —cleaning compounds 54N

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HACCP 523

Table 49.4. (Continued) 1 2 Hazard 3 Process Steps Identification Preventive Measure 4 5 Stuffing Biological GHP, GMP, correct time/temperature, use of nontoxic 6 Chemical —food-compatible cleaning compounds, preventive equipment 7 Physical —maintenance, correct stuffing pressure machine 8 Visual check, metal detector 9 Resting Biological GHP 10 Correct time/temperature, batch meat pH 11 12 Fermentation/drying Biological GHP, GMP 13 Chemical Correct time/temperature/relative humidity 14 Smoking Biological GHP, GMP 15 Chemical Corrected time/temperature/relative humidity 16 Maturation/stabilization Biological GHP, GMP 17 Chemical Corrected time/temperature/relative humidity, control of pH 18

—and aw of end product 19 Packaging (vacuum and Biological GHP, GMP, preventive equipment 20 MAP) and package Chemical —maintenance, control of sealing vacuum and 21 22 labeling —CO2/O2 concentration into packages Label monitoring by photoelectric cell or 23 visual check 24 25 Storage end product Biological GHP, GMP 26 Correct time/temperature/humidity 27 Distribution Biological GHP, GMP 28 Chemical Correct time/temperature/humidity 29 Physical 30 31 32 supplier records, regulatory guidelines), experimen- 1997). The monitoring procedures must be able to 33 tal data, expert advice, and mathematical modelling detect immediately loss of control at a CCP. The fre- 34 (Mortimore and Wallace 1997) and designated to be quency of monitoring will depend on the nature of 35 monitored at CCPs. Verification procedures assure the CCP and the type of monitoring procedure. 36 they have been respected. The procedure could be a continuous on-line 37 So-called target limits are often established at measurement where critical data is continuously 38 CCPs to act before a deviation occurs. Recently, in recorded, or a discontinuous off-line monitoring sys- 39 Europe, new legal standards were defined concern- tem. This last system could be disadvantageous 40 ing food microbial criteria (Commission Regulation because the sample taken to analyze may not be fully 41 [EC] 2073/2005) that must be used in verification representative of the whole batch and periodicity of 42 actions at CCPs. These specifications (end-product sampling could create gaps without information 43 criteria) are intended to prevent adverse health about the process. 44 effects. It is assumed, as a starting point, that values Preferentially, methods used for monitoring must 45 below the fixed criteria do not result in significant produce rapid answers, permitting the previously 46 health effects and those above that limit lead to an stated corrective action to be started when necessary. 47 increased probability of an adverse health effect. According to the CCPs established for fermented 48 sausage (Table 49.6), the measurement of tempera- 49 ture, relative humidity, time, pH, and a are objective 50 MONITORING MEASUREMENTS AND OTHER TESTS w methods that provide a rapid answer to control the 51 Monitoring measurements are immediate measure- process. Also scheduled sensorial inspections can be 52 ments or sensorial or analytical quotable determina- used for monitoring some preventive measures at S53 tions at a CCP assuring that the process is operating CCPs; however, there is some restriction because N54 within the critical limits (Mortimore and Wallace the objectivity and accuracy of human sensorial 40454 49 513-534 r1.qxd 7/20/07 5:36 PM Page 524

524 Processing Sanitation and Quality Assurance

1 1.Is there a Hazard at this process step of fermented sausages? 2 - what is it? 3 4 5 YES NO 6 7 Not a CCP 8 2. Do preventive measure(s) exist at this step 9 or subsequent steps for the identified Stop 10 hazard? 11 12 13 YES Modify step, process NO or product 14 15 3. Does this step eliminate or 16 reduce the likely occurrence of YES 17 a hazard to an acceptable level? 18 Is control at this step 19 NO necessary for safety? 20 21 4. Could contaminaton occur at or 22 increase to unacceptable level(s)? NO 23 YES 24 YES 25 26 5. Will a subsequent step or 27 action eliminate or reduce NO 28 the hazard to an acceptable 29 level? 30 Not a CCP 31 NO YES 32 Stop 33 Critical control point 34 35 Figure 49.2. CCP Decision Tree for establishment of critical control points, adapted from FAO/WHO (2005). 36 37 38 39 evaluation are inﬂuenced by several factors, some of The actions to be taken when a deviation occurs 40 them avoidable by training. The frequency of moni- include those related to the adjustment of the process 41 toring will depend on the nature of the CCP, the type to bring it back under control and those related to the 42 of monitoring procedure, and the amount of pro- amount of product that might not be complying with 43 duction. Records of monitoring data are necessary to hygiene and safety requirements. They include the 44 prove that the process is under control and provides a segregation of any suspect product and holding of it 45 pool of data that will contribute, after trend analysis, for the period of time needed to seek advice and 46 to the establishment of new criteria and new safety more information from the HACCP team or outside 47 objectives, improving the implemented system. experts and to perform analysis to assess safety. All 48 this information will lead to different decisions: 49 rejection and destruction of product, product rework, CORRECTIVE ACTIONS FOR DEVIATIONS AT CCPS 50 and product release. All these actions must be kept 51 A corrective action must be taken when monitoring on record. 52 data shows a deviation from critical limits at a CCP; Table 49.6 gives some examples related to correc- 53S it is mandatory to act quickly. tive actions in a fermented sausage process. 54N 40454 49 513-534 r1.qxd 7/20/07 5:36 PM Page 525

HACCP 525

1. Is there a hazard with this raw material - what is it? 1 2 3 4 NO YES 5 6 Proceed to next 7 raw material 8 9 2. Are you or the costumer going to process this 10 hazard out of the product? 11 12 NO 13 YES 14 15 Sensitive raw material 16 3. Is there a cross-contamination High level of control 17 risk to the facility or to other products, required 18 which will not be controlled? CCP 19 20 21 NO 22 23 24 YES Proceed to next raw material 25 26 Sensitive raw material 27 High level of control 28 required 29 CCP 30 31 Figure 49.3. CCP Decision Tree for raw material (Mortimore and Wallace 1997). 32 33 34 35 HACCP CONTROL CHART FOUNDATION OF study has been carried out correctly and that the 36 WRITTEN HACCP PLAN HACCP plan remains effective. 37 Verification can be carried out by different 38 Fulfillment of the seven principles of HACCP leads actions. Table 49.6 summarizes some of the possible 39 to a finished control chart that has been drawn up, verification actions related to a HACCP plan for 40 step by step, during the system development of the fermented sausages. 41 practical application of each principle (Table 49.6). When CCPs are required for suppliers or dis- 42 It is the foundation, in synthesis, of every specific tribution operators, regular audits to evaluate their 43 HACCP plan. quality systems, GHP/GMP, and HACCP should be 44 performed. 45 VERIFICATION PROCEDURES Verification also includes inspective analysis of 46 The HACCP system must testify that it ensures that operations to assure that CCPs are under control, 47 appropriate measures and related monitoring proce- scrutiny of CCPs monitoring records, examination 48 dures at critical control points established to prevent of metrology certificates of all equipment used 49 all known potential hazards are working effectively for measurements, and examination of records relat- 50 and according to the plan. ing to corrective actions taken when deviations 51 Verification includes procedures (others than those have occurred. To underscore product uses, random 52 used in monitoring) that ensure that the HACCP samplings for physicochemical, sensorial, or S53 N54 40454 49 513-534 r1.qxd 7/20/07 5:36 PM Page 526

2 * * 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 Is there a hazard at this processstep of fermented Do preventivesausages? at this step or subse- measure(s) exist occurrence the likely Does this step eliminate or reduce occur at or increase quent steps for the eliminate or reduce contamination of a hazard to an Could step or action to unacceptable the hazard to an a subsequent Will Yes—Ch.Yes—Ph. Yes YesYes—Ch.Yes—Ph. No No Yes YesYes—Ch.Yes—Bi. NoYes—Ch. No Yes No No Yes Yes No Yes Yes No No Not a CCP Not a CCP No Yes Yes No No Not a CCP Not a CCP Not a CCP Not a CCP Not a CCP 47 48

49 Control points and critical dry control points identification in the production of fermented sausages. 50 51 52 trimming of pork Yes—Bi.meat cut or rehydration Yesingredients and additives No Yes Yes Not a CCP 53S drying Yes—Ch. Yes Yes CCP Yes StorageDeboning and — — Yes—Ph.Casing desalting Yes—Bi.— Yes—Bi.Mincing Yes Yes of Weighting Yes— Yes—Bi.— Yes—Ch.Mixing NoStuffing Yes Yes Yes No Yes—Bi.RestingFermentation/ Yes—Ph. Yes—Bi. Yes No Yes Yes—Bi. Yes No Yes Yes Yes Yes Yes No Yes Yes No No Yes Yes Yes Not a CCP Not a CCP Yes Yes Not a CCP Yes Not a CCP Yes No Not a CCP Not a CCP CCP Yes CCP Yes CCP Yes 54N 49.5. Table Process StepsReception What is it? Yes—Bi. identified hazard? level? acceptable Yes level(s)? level? acceptable NoAnswer Final Yes— Yes Not a CCP

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* * 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 physical.

42 43 44 45 46 Yes—Ch. YesYes—Ph. Yes Yes No No CCP Yes Not a CCP Yes—Ch. Yes No No Not a CCP chemical; Ph. 47 48 49 50 51

biological; Ch. biological; 52

stabilizationand MAP Yes—Ch. Yes Yes CCP Yes S53 All these steps could be monitored by a single CCP at stabilization. All these steps could be monitored by Bi. SmokingMaturation/— Yes—Bi. vacuum Packaging Yes—Bi.— Yes—Bi.LabelingStorage end product Yes—Bi. YesDistribution Yes Yes Yes—Ch. Yes—Bi.* Yes Yes Yes No Yes Yes No Yes No No No No No Yes Not a CCP CCP Yes CCP Yes N54 Not a CCP CCP Not a CCP

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1 2 3 scales thermometer monitoring analysis additives analysis against calibrated thermometer analysis and hygrometer 4 microbial and chemical analysis according to planned of thermometers temperature — — — — — — — — — — Data record — 5 — — — — — — Check — 6 7 8 9 10 11 relative humidity relative of scales temperature and ation of its and safety hygiene on end shelf life operators segre- weight, gation of batch 12 meat and evalu- — — — — — Reject product — — — — — 13 Corrective 14 15 16 17 18 19 20 21

22 Monitoring 23 24 and relative and record Recalibration Calibration of room temperature 20% labels to ensure stock rotation check control of each ingredient humidity of Segregation 25 of certiﬁcates (orstamp of homo- and data logation) 20% “best before” check ingredients meat temperature Contact supplier Change supplier Calibration — — — — — — — Visual check of Visual — 26 — — — — — — 27 28 5%;

29 2°C) Measurement of Suppliers audits 30 85 2 days

31 2°C; 2% 2 h

32 RH tion or desalting of time standards to legal Salt concentration Training 2 time of meat storage 5 33 cates or stamp of homologation, limits legal meat (4 — — — — — — — — — — 34 — — — — 35 36 37 38 39 40 41 42 43 2°C) 44 45 46 of room tion or desalting storage (first-in/first-out) Data “best before” practice weight of each ingredient in each batch relative humidity relative refrigeration rotation, time of selected and homologated and/or certified: agreed specifi-cation (maximum levels) acceptable (“best before” dates) meat (4 of Temperature Data “best before” GHP/GMP — — — — GMP — — — — — stock Effective — Preventive Critical 47 — — — — — — check Visual — — of Temperature — 48

49 control chart fermented/dryHACCP sausages. for 50 51 52 or rehydration ingredients and additives materials 53S materials raw (carcasses/meat cuts, casings, ingredients/ additives) Casing desalting of rehydra- Time CP–GHP/GMP of rehydra- Time Measurement 100% Reject batch Data record Weighting ofWeighting Safe and accurate CP–GHP/GMP according Weight Visual Weight 100% Correction of Chemical Storage of raw Storage of raw and Temperature CP–GHP/GMP Room temp. Measurement of On line Correction of Calibration of 54N 49.6. Table Process StepsReception of Action Suppliers CCP/CP CP–GHP/GMP Presence of certiﬁ- check Visual 20% Limits 100% Reject batch; Procedure materials Raw Frequency Actions Veriﬁcation

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1 Continues

( 2 3 metal detector, maintenance equipment plan thermometer and hygrometer thermometer and hygrometer monitoring analysis thermometer and hygrometer 4 — — — — — — — — — — 5 6 7 8 9 10 11 and smoking time suspected product maintenance, operators training suspected product temperature, humidity, relative temperature and humidity relative sausage formula temperature and humidity relative 12 — — — — — — — — — — Correction of Dates record — — 13 14 15 16 periodically scheduled periodically scheduled 17 — — — — 18 19 20 21 22 23 24 and smoking time check check, visual Notify and resting time of Segregation time room temperature, relative humidity relative room temperature, humidity, relative relative humidity, relative room temperature, and fermentation 25 — — Measurement of On line or Correction of Calibration of — — — Measurement of On line or Correction of Calibration of — — — — — — — 26 27

28 29

2% 30 5°C; RH 5%; time 5%; time 2 days 2°C; RH 1 days 31 85 32 according to particular/ traditional of Segregation according to particular/ traditional practices 20 75 3 12–16°C, time relative 25 days, 4 85 3 humidity practices 33 — — — — — — — — — — — — — — — — — — 34 35 36 37 38 39 40 41 42 43 44 45 46 equipment, metal effective detection, visual inspection maintenance of 0% bones relative humidity relative time, relative humidity control time relative time relative humidity refrigeration room, time of resting, 47 — — — — — — — — GHP/GMP — — GHP/GMP — — — 48 49 50 51 52

drying S53 Fermentation/temp. CP–GHP/GMP Temperature, Room Stufﬁng Preventive CCP 0% metal particles; Metal detection, On line Reject product Calibration of Smoking Temperature, CP–GHP/GMP Room temp. CP–GHP/GMP Smoking Temperature, Room Resting period of Temperature CCP Room temp. Measurement of On line Correction of Calibration of N54

529 40454 49 513-534 r1.qxd 7/20/07 5:36 PM Page 530 2 /O 2 1 2 3 thermometer and according to plan residual CO concentration equipment analysis equipment plan, data record monitoring analysis equipment plan, data record monitoring analysis scales, 4 hygrometer and chemical end product analysis — — — — — — — — — — — — — — — — 5 — — — — 6 7 8 9 10 11 relative relative product replace package and notify maintenance or replace label temperature, 12 humidity or period of stabilization suspected Microbiological — — — Corrective Corrective — — — — 13 — — — — 14 15 16 scheduled 17 periodically — 18 — 19 20 21 end w 22 Monitoring 23 2 /O 24 2 relative humidity, humidity, relative 10% of end product batchmeasurement of pH and a Reject product CO sausage sampling residual room temperature, concentration in packages package vacuum photoelectric cell labeling check 25 maturation time, of and weight products till lossdesirable of weight of Segregation — — — — — — — — — — — — Selling test — — — 26 — — — — — 27

28 2 29 30 4.8–6 2°C; RH 31 5%; time 0.85–0.90

32 2%; w according to particular traditional fermented sausage a package, 0% fail 12 residual head space package limits for legal MAP packaging check of Visual label Maintenance 75 of vacuum, O of vacuum, according to loss desirable related of weight to product pH 33 of maturation — — — — — — — — — — — — — — — — — — — — — 34 — 35 36 37 38 39 40 41 42 43 control 44 2 w

45 /O 2 46 ) and a selling and time, relative time, relative CO concentration in packages with cell photoelectric or visual humidity, pH humidity, 47 and vacuum — — Preventive Critical — GHP/GMP — — — — — — — — 48

49 Continued ( 50 51 52 air-conditioned vacuum vacuum stabilization, drying, 53S and MAP Packaging Packaging Control of CP–GHP/GMP 0% fail of seal Measure of On line Stop line, Calibration of Table 49.6. Table Process StepsMaturation, Action Temperature, CCP CCP/CP Limits Room temp. Measurement of Procedure On line or Correction of Frequency Calibration of Actions Veriﬁcation 54N Labeling Control of label CCP 0% absence of or Visual On line Label product Maintenance

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1 2 3 thermometer temperature thermometer temperature car temperature 4 — — Audit records of — — — 5 6 7 8 9 10 11 temperature temperature end product and of its evaluation and safety hygiene on end shelf life 12 — — — — — Reject product — 13 14 15 16 periodically scheduled of Segregation Check 17 — — 18 19 20 21 22 23 24 car temperature room temperature and storage time 25 — — — Measurement of On line or Correction of Calibration of 26 27 28

29 2°C or Measurement of On line Correct Calibration of 30

2°C 31

32 MAP packed MAP packed sausages drivers Training Check other speciﬁed for 18 33 — — — — 34 35 36 37 38 39 40 41 42 43 44 45 46 control, control “Best before” date temperature GHP, GMP GHP, — — GHP/GMP — 47

control point of GHP and GMP. 48

49 50 51 52 S53 Distribution Temperature CP–GHP/GMP 18 Temp. product Storage end Time/ CP–GHP/GMP Room temp. CP–GHP/GMP N54

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532 Processing Sanitation and Quality Assurance

1 microbiological analyses are further examples of qualitative or quantitative evaluation of the nature of 2 verification procedures. the adverse health effects associated with biological, 3 Microbial analysis of fermented sausages, namely chemical, and physical agents that may be present in 4 those related to the quantification of certain microbial the food. Factors such as virulence, microbial vari- 5 groups that are indicators of spoilage and hygiene ability, and consumer sensitivity influence the nature 6 level and also those for detection/quantification of of the adverse health effect. Exposure assessment is 7 pathogens, offer information to evaluate whether the the qualitative or quantitative evaluation of the likely 8 system is reaching the targets. However, an efficient intake of hazardous biological, chemical, and physi- 9 system must establish the real significance of each cal agents via foods. Risk factors in exposure assess- 10 parameter analyzed in order to avoid wasting time ment must be focused on the quality of the raw 11 and money. materials, the process steps, and the process environ- 12 All planned verification actions must be stated on ment as well as on the composition, packaging, and 13 the HACCP plan; however, others can be taken with- storage conditions of the product. 14 out any previous communication. These actions Gonzalez-Miret et al. (2001) applied statistical 15 must take in account any new safety information univariate and multivariate methods to identify the 16 about the product or when the product has been most useful and discriminative microbial parameters 17 implicated as a disease vehicle. (total counts, Pseudomonas, Enterobacteriaceae, 18 Verification reports must be produced containing and Staphylococcus aureus) used in verification 19 all the information from the above-mentioned related to poultry meat production, such as refrigera- 20 actions, modifications introduced into the plan, and tion, cutup, and packaging operations. The authors 21 the training of the persons responsible for CCP mon- concluded that Enterobacteriaceae and total counts 22 itoring and surveillance. were the most discriminating to be used for validat- 23 ing parameters used on HACCP system at CCPs/ 24 RECORD-KEEPING OF HACCP PLAN DATA AND GHPs/GMPs. Similarly, microbiological parameters 25 EXTRAORDINARY EVENTS can be used for verification of goodness of monitoring 26 pH, aw, and time/temperature measurements at 27 As stated above, all data produced by the HACCP CCPs to assure safety control after the Maturation/ 28 operating procedures has to be kept on file for easy Drying/Stabilization step. 29 consultation by the team, auditors, and government Quantitative microbial risk assessment with 30 food inspection authority agents. It is of great value L. monocytogenes counts performed with data 31 to prove existing workable HACCP and provides due obtained in southern European fermented sausages 32 diligence in court trials. (Tradisausage 2006) revealed that if initial counts 33 are higher than 102 CFU/g, no significant reduction 34 VALIDATION OF THE OPERATIVE is to be expected, mainly because of the insufficient 35 HACCP PLAN lowering of pH whose mean value was higher than 36 pH 5.0 in final products. However, L. monocytogenes 37 All the evidence that preventive measures are effec- is not able to grow to more than two logs in any prod- tive in eliminating or reducing hazards to an accept- 38 uct due to the low aw of traditional fermented saus- 39 able level in the HACCP plan at CCPs, GHP/GMP, ages, which is the most crucial hurdle for safety of or other prerequirements are used for validation. If 40 these products. According to these findings, aw and 41 the selected preventive measures are not able to pH coupled with time/temperature are the most 42 achieve the control of identified hazards, they must important parameters to assure safety and stability 43 be modified and reevaluated. and have to be used for monitoring CCP at the matu- 44 According to Hoornstra et al. (2001) quantitative ration/stabilization step. 45 risk assessment is a powerful tool for food compa- 46 nies for the validation of a HACCP system, particu- REVISION OF THE HACCP PLAN 47 larly for validation of critical limits at critical control 48 points. The authors give examples for risk assess- HACCP plan needs must be revised when any modi- 49 ment referring to the fact that for raw fermented fication in production or equipment occurs. The 50 products the risk manager is the government. Quan- HACCP team is responsible for the revision based 51 titative risk assessment needs hazard identification, on internal and external audit reports, records of cor- 52 hazard characterization, exposure assessment, and rective actions, and client complaints. Revision will 53S risk characterization. Hazards characterization is the contribute to a continual improvement of the plan. 54N 40454 49 513-534 r1.qxd 7/20/07 5:36 PM Page 533

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CERTIFICATION OF FOOD FAO. 1997. Hazard analysis and critical control point 1 SAFETY MANAGEMENT principles and application guidelines. Food and Agri- 2 SYSTEMS culture Organization of the United States, U.S. 3 Department of Agriculture, National Advisory Com- 4 Audits of all data recorded and direct measurements mittee on Microbiological Criteria for Foods. 5 produced in a HACCP working system have to be http://www.cfsan.fda.gov/~comm/nacmcfp.html. 6 performed with technical competence and compliance FAO/WHO. 2003. Recommended International Code of 7 with international standards (ISO/FDIS 22000: Practice General Principles of Food Hygiene. Codex 8 2005) or other normative references for food safety Alimentarius Commission. Food and Agriculture 9 management systems. These audits conducted by an Organization of the United States, World Health 10 independent and qualified institution certify food Organization, CAC/RCP1-1969, Rev.4-2003:1–31. 11 safety management systems, giving recognition to ———. 2005a. Codex Alimentarius, Food Hygiene. 12 clients and consumers that the producer is able to Basic texts. Third Edition. Codex Alimentarius Com- 13 control food safety hazards. Unfortunately, these mission. Food and Agriculture Organization of the 14 external audits are too expensive for small compa- United States, World Health Organization. 15 nies to afford. However, the above-mentioned nor- ———. 2005b. Code of hygiene practice for meat. 16 mative reference can be used by small developed Codex Alimentarius Commission. Food and agricul- 17 industries to evaluate their own safety management ture Organization of the United States, World Health 18 system. Organization, CAC/RCP 58-2005, 1–52. 19 FDA. 2005. Food Code 2005. Recommendations of the 20 REFERENCES United States Public Health Services, Food and Drug 21 Administration, National Technical Information 22 GH Alaña, JC Crespo, AH Marteache, M Aguado, JG Service Publication PB-2005-102200. Springfield. 23 Gonzalez. 1996. Implantacion del sistema HACCP http://www.cfsan.fda.gov/~dms/fc05-toc.html. 24 en la industria cárnica. Ed. Servicio Central de Publi- FDA/CFSAN. 2005. Foodborne Pathogenic microor- 25 cationes del Gobierno Vasco. Vitoria-Gasteiz. ganisms and Natural Toxins handbook. Center for 26 España, 222 pp. Food Safety & Applied Nutrition. U.S. Food & Drug 27 E Caplice, GF Fitzgerald. 1999. Food fermentations: Administration. http://www.cfsan.fda.gov/~mow/. 28 Role of microorganisms in food production and P Feng. 1995. Escherichia coli serotype O157:H7: 29 preservation. Inter J Food Microbiol 50:131–149. Novel vehicles of infection and emergence of pheno- 30 CDC. 2006. Preliminary foodNet data on the incidence typic variants. Emerg Infect Dis 1:2,47–52. 31 of infection with pathogens transmitted commonly EM Foster. 1997. Historical overview of key issues 32 through food—10 states, United States, 2005. in food safety. Emerging Infectious diseases, 3: 33 Department of health and human services. Centers 4,481–482. 34 for disease control and prevention. Morbid Mortal FSEP. 1997. HACCP Generic Model: Fermented 35 Weekly Rep 55:14,392–395. smoked sausages. Food Safety Enhancement Program. 36 COM. 2000. White paper on food safety. Commission Canadian Food Inspection Agency. http://www. 37 of the European Communities. COM (1999) 719 inspection.gc.ca/english/fssa/polstrat/haccp/smsaufu 38 final. pp. 1–52. http://ec.europa/comm/dgs/health_ m/smsaufumie.shtml. 39 consumer/library/pub/pub06_en.pdf. ML Gonzalez-Miret, MT Coello, FJ Heredia. 2001. 40 Commission Regulation (EC) 2073/2005 of 15 Novem- Validation of parameters in HACCP verification 41 ber 2005 on microbial criteria for foodstuffs. Official using univariate and multivariate statistics. Applica- 42 Journal of the European Union L 338. 26 pp. tion to the final phases of poultry meat production. 43 WG Eisel, RH Linton, PM Muriana. 1997. A survey of Food Contr 12:261–268. 44 microbial levels for incoming raw beef, environmen- SJ Goodfellow. 1995. Implementation of HACCP pro- 45 tal sources and ground beef in a red meat processing gram by meat and poultry slaughterers. In: HACCP 46 plant. Food Microbiol 14:273–282. in Meat, Poultry and Fish Processing. Advances in 47 European Commission (EC). 2005. Guidance docu- Meat Research—Volume 10. AM Pearson, TR 48 ment on the implementation of procedures based on Dutson, eds. London: Blackie Academic & Profes- 49 the HACCP principles, and on the facilitation of the sional, pp. 58–71. 50 implementation of the HACCP principles in certain E Hoornstra, MD Northolt, S Notermans, AW 51 food business. European Commission, Health & Barendsz. 2001. The use of quantitative risk assess- 52 Consumer Protection Directorate General. pp. 1–29. ment in HACCP. Food Contr 12:229–234. S53 N54 40454 49 513-534 r1.qxd 7/20/07 5:36 PM Page 534

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1 M Hugas, M Garriga, MT Aymerich. 2003. Functional- KV Paukatong, S Kunawasen. 2001. The hazard analy- 2 ity of enterococci in meat products. Inter J Food sis and critical control points (HACCP) generic 3 Microbiol 88:223–233. model for the production of Thai fermented pork 4 ISO/FDIS 22000:2005. Food safety management sys- sausage (Nham). Berliner und Munchener Tier- 5 tems—Requirements for any organization in the food arztliche Wochenschrift 114:9–10, 327–330. 6 chain. International standard organization. Geneva. SL Roy, AS Lopez, PM Schantz. 2003. Trichinellosis 7 31 pp. surveillance—United States 1997–2001. Morbid 8 J Mexatopoulos, D Kritikos, EH Drosinos. 2003. Mortal Weekly Rep 52:SS–6,1–12. 9 Examination of microbial parameters relevant to the RB Tompkin. 2002. Control of Listeria monocytogenes 10 implementation of GMP and HACCP system in in the food processing environment. J Food Protect 11 Greek meat industry in the production of cooked 65:709–725. 12 sausages and cooked cured meat products. Food Tradisausage. 2006. Final report. Assessment and 13 Contr 14:323–332. improvement of safety dry sausages from producers 14 S Mortimore, C Wallace. 1997. HACCP. A practical to consumers. Tradisausage, QLK1 CT-2002-02240. 15 approach. London: Chapman & Hall, 296 pp. 189 pp. 16 National Advisory Committee on Microbiological U.S. Department of Health and Human Services. 2000. 17 Criteria for Foods, 1992. Hazard analysis and critical Healthy people 2010: Understanding and improving 18 control point system. Inter J Food Microbiol 16:1–23. health, 2nd ed. Washington, DC: U.S. Government 19 SO Oiye, NM Muroki. 2002. Use of spices in foods. Printing Ofﬁce, 76 pp. 20 J Food Technol Africa 7:33–44. 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53S 54N 40454 50 535-544 r1.qxd 7/21/07 1:57 PM Page 535

1 2 3 50 4 5 6 Quality Assurance Plan 7 8 Friedrich-Karl Lücke 9 10 11 12 13 14 15 16 17 18 19 20 21 INTRODUCTION also Chapter 49, “HACCP”). Even though HACCP 22 refers to the protection of consumer’s health only, a 23 The objective of food quality assurance is to make similar approach should also be used to assure over- 24 sure that the consumer receives foods that meet all product quality. Hence, food businesses should 25 his/her needs in terms of safety, nutritional value, 26 convenience (including shelf life), and eating qual- establish and maintain an integrated, process- oriented system that covers all criteria of product 27 ity. An ever-increasing number of consumers also 28 demand “authentic” foods, i.e., foods of defined ori- quality. An effective system of quality assurance also reduces the producer’s risk (of economic losses due 29 gin (regional foods) and/or foods manufactured and 30 to process failure) and cuts production costs by min- distributed by use or nonuse of certain production 31 imizing unnecessary work and inputs. and processing methods (e.g., according to regula- 32 This chapter provides the key elements of quality tions on organic and fair-trade foods, to requirements 33 for kosher foods; without application of ionizing irra- assurance plans specific for the manufacture of 34 diation or genetic technology, etc.). These require- fermented sausages and raw hams, respectively. The 35 ments (process quality) are also quality criteria information on processes is based on the publica- 36 according to ISO 9000:2000 standards, even though tions of Stiebing (1996, 1997), Lücke (2000), Toldrá 37 these properties of a food cannot always be readily (2002), and Lautenschläger (2006). An example for 38 detected by sensory testing or in the laboratory. Offi- a quality assurance plan for the design and the pro- 39 cial regulations require that food must be safe, and duction of a Mediterranean-type salami is provided 40 that the consumer must not be misled by inadequate by Thorn (2006). Reference is also made to other 41 labeling about weight, composition (constituents contributions to this book. The reader will note some 42 and additives), shelf life, nutritive value, origin bias on products common in Germany, but the sys- 43 (traceability), and production method. Official regu- tem presented may be easily adapted to fermented 44 lations on food hygiene (such as EU regulation no. 45 852/2004) require that food businesses implement meat products common in other countries. However, it is essential to note that not all standards and speci- 46 and maintain a system to ensure safety, and specify 47 that such a system must be based on HACCP. For fications given are transferable to all types of fermented meats. For example, it is possible to avoid 48 information on the implementation and maintenance 49 the use of curing agents if proper raw material and of HACCP in meat processing, the reader is referred 50 salting/ripening conditions are selected. to official documents (e.g., the Generic HACCP 51 Finally, this chapter gives some information on the model for not heat-treated, shelf-stable meat and 52 poultry products in the USA [USDA-FSIS 1999]) or structure of the documents on the processes (plans 53 the literature (Brown 2000a,b; Sheridan 2000; see and records). S54 N55 535 40454 50 535-544 r1.qxd 7/21/07 1:57 PM Page 536

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1 GENERAL REMARKS ON THE unnecessary paperwork, it is advisable to stamp and 2 PURCHASE AND SELECTION OF fill in a “miniature checklist” on the delivery note. 3 THE RAW MATERIAL The supplier should also be able to provide evidence 4 that the process hygiene criteria specified in the EU 5 Specifications should include the following: Regulation 2073/2005 on microbiological criteria 6 for foodstuffs has been fulfilled, and it is advisable to • Hygienic aspects, in particular temperature 7 verify this by regularly testing the microbiological (including temperature history) of the meat 8 quality of the meat supplied. In the United States, and fat, integrity of containers and transport 9 suppliers must provide certificates indicating that equipment 10 the meat has been sampled and tested negatively • Traceability 11 for salmonellae and E. coli O157:H7 if the manu- • pH of the meat: generally, 5.8 or below, but 12 facturing process of fermented sausages does not definitely not higher than 6.0 13 guarantee a 5 log reduction of these organisms 14 The suppliers of meat and fat should be carefully (USDA-FSIS 1999). 15 selected on the base of their ability to reliably meet 16 the specifications established. This is a complex 17 process, which should involve responsible staff from QUALITY ASSURANCE PLANS 18 the processing, purchase, and quality assurance AND RECORDS FOR FERMENTED 19 department. A Standard Operation Procedure (SOP) SAUSAGES 20 should be in place to organize this process. 21 On reception, the documents indicating the origin Figure 50.1 summarizes the processing steps in the 22 of the raw material should be inspected, and the tem- manufacture of fermented sausages. Control at these 23 perature and cleanliness within the transport equip- steps is important to assure quality of the final prod- 24 ment, the integrity of the containers, and the overall uct even though not all of these steps are “critical 25 appearance of the meat and fat should be examined control points” in terms of the HACCP concept. 26 and recorded. Measuring and recording the internal 27 temperature and pH value of the cuts is also advis- PURCHASE AND SELECTION OF MEAT AND FAT 28 able even though experienced butchers are able to (TABLE 50.1) 29 select cuts of appropriate pH by visual inspection 30 only. The same is true for the firmness and whiteness Table 50.1 lists quality criteria for the raw material, 31 of fatty tissue, which correlates well with the con- indicates why these factors are important for the 32 tent of saturated fatty acids in the tissue. To avoid quality of the final product, and suggests how the 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53S 54N Figure 50.1. General processing scheme for fermented sausages. 40454 50 535-544 r1.qxd 7/21/07 1:57 PM Page 537

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Table 50.1. Purchase and selection of raw material for fermented sausages (process step A). 1 2 Specification Objective Monitoring and Documentation 3 Origin of the material Traceability in case of defects Check of documents and stamps on 4 —caused by process failure in —cuts/containers 5 —primary production, slaughtering, 6 —and/or butchering 7 8 Type of cut pH affects product safety; fat, Visual inspection 9 —collagen, and myoglobin content 10 —affects sensory properties of the 11 —final product 12 pH of lean meat р5.8 Slower growth of undesirable or Measurement of pH or visual 13 —hazardous microorganisms —inspection 14 Reduction of water binding capacity 15 Microbiological quality Reduction of microbiological risk Visual inspection 16 Temperature measurement 17 Check of hygiene-related 18 —documentation provided by 19 —supplier 20 21 Maximum mol% of Less fat oxidation, slower rancidity Visual inspection 22 polyunsaturated Check of documents on feeding 23 fatty acids —regime and age of slaughter 24 —animals 25 Maximum content of fat Sensory and nutritive value Sorting of cuts on the basis of visual 26 and connective tissue —appearance (e.g., using the 27 German GEHA scheme based on 28 the Leitsätze), and/or analysis of 29 preblends (e.g., by NIR) 30 Age of slaughter animals Age affects myoglobin content and Visual inspection 31 —thereby appearance of the Reflectometry 32 —final product 33 34 35 36 quality of the raw material should be assessed and Extended chill storage results in poor microbiologi- 37 recorded. cal quality, and extended frozen storage results in fat 38 deterioration. 39 40 PRETREATMENT OF MEAT AND FAT: 41 CHILLING/FREEZING PREPARATION OF SAUSAGE MIXTURE (TABLE 50.2) 42 Trimmings with the appropriate content of lean The final temperature of the mix should be low, 43 meat, fat, and connective tissue are sorted; at this preferably between −5 and 0°C. This is important to 44 stage, it is important to clearly record the identity, ori- avoid fat deterioration and “smearing” and the 45 gin, and classification of the material (e.g., accord- resulting sensory defects and problems associated 46 ing to the German Code of Practice [Leitsätze]). The with drying the sausages. For some sausage varieties 47 material is subsequently chilled and partly frozen; with a coarse, crumbly structure, a meat grinder is 48 freezing the meat may also somewhat reduce water used instead of a cutter. In this case, higher tempera- 49 binding capacity and improve salt diffusion. The tures of the meat (up to 5°C) are still acceptable. 50 flow of raw material should be carefully controlled The appropriate choice of raw material and non- 51 and documented, in order to ensure traceability, to meat ingredients, such as salt, nitrite, ascorbate, sug- 52 avoid mislabeling, and to minimize storage time ars, starter cultures, and spices, is critical for the S53 according to the “first in, first out” principle. quality and safety of the final product. Hence, N54 40454 50 535-544 r1.qxd 7/21/07 1:57 PM Page 538

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1 Table 50.2. Preweighing of ingredients, preparation of sausage mixture (process step C). 2 3 Subprocess; Examples for 4 Specifications/Critical Limits Objective Monitoring and Documentation 5 Addition of meat and fat Fat, total protein, and collagen Weighing protocol 6 —within specified limits 7 у 8 Addition of 0.3% of a rapidly Sufficient rate and extent of Weighing protocol 9 —fermentable sugar —acid formation 10 Addition of lactic acid bacteria Weighing protocol; specifica- 11 —(105– 106/g) capable of growing —tions issued by manufacturer 12 —and metabolizing in sausage mixture —of starter culture 13 Addition of catalase-positive cocci Nitrate reduction, aroma Weighing protocol; specifications 14 —(105– 106/g) capable of growing —formation, protection from issued by manufacturer of starter 15 —and metabolizing in sausage —deleterious effects of O2 —culture; lot identification 16 —mixture 17 Addition of 2.4– 3.0% NaCl Reduction of microbiological Weighing protocol 18 —risk texture formation 19 20 Addition of nitrite (100–150 mg Formation of NO myoglobin Specification of the nitrite content 21 —NaNO2/kg, as nitrite curing salt, —(curing color) —in curing salt; weighing protocol 22 —in combination with 300– 500 mg Inhibition of oxidative changes 23 —sodium ascorbate/kg) —in unsaturated lipids 24 Inhibition of salmonellae at 25 —early stages of fermentation 26 Addition of nitrate Improved color and aroma of Weighing protocol р 27 —( 300 mg KNO3/kg) —some long-ripened products 28 Addition of defined spice mixture Aroma and taste Weighing protocol; specifications 29 —issued by manufacturer; lot 30 —identification 31 Comminution to specified Appearance of the final Appearance of the mixture; final 32 —particle size —product; prevention of fat —temperature of the mixture; lot 33 —deterioration and drying —identification 34 —problems 35 36 37 appropriate records (weighing protocols) are neces- During filling, inclusion of oxygen should be min- 38 sary. Moreover, the quality of additives (e.g., nitrite imized in order to prevent hole formation with sub- 39 content in curing salt, activity of starter cultures under sequent oxidative deterioration and microbial 40 the fermentation conditions selected) should be spoilage. This can be achieved by using a vacuum 41 defined in specifications issued by the manufacturers. filler or manually. 42 43 SELECTION OF CASINGS AND FILLING SURFACE INOCULATION (TABLE 50.3) 44 45 The choice of casings has a strong effect on product Most French- and Mediterranean-type sausages are 46 quality and safety. Specifications should include covered with surface molds. Some manufacturers 47 mechanical stability and—especially for dry rely on the “house flora”; others dip the sausages 48 sausages—moisture permeability and ability to (after temperature equilibration) into a suspension of 49 shrink during drying. Use of poorly purified and pre- starter culture consisting of molds and sometimes 50 treated natural casings may lead to bacterial contam- yeasts. The starter organisms should be able to rap- 51 ination of the surface and to insufficient drying. For idly colonize the surface after completion of lactic 52 convenience, the casing must be easy to peel off. The fermentation, and this and other relevant properties 53S meat processor should set up specifications and (sensory and safety aspects) should be defined in 54N select a supplier able to comply with them. specifications issued by the manufacturers. 40454 50 535-544 r1.qxd 7/21/07 1:57 PM Page 539

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Table 50.3. Sausage fermentation, smoking, drying, storage of unpackaged product (process steps E, F). 1 2 Subprocess; Examples for 3 Specifications/Critical Limits Objective Monitoring and Documentation 4 Equilibration at 20– 25°C and Prevention of moisture Time-temperature protocol 5 —60% RH for about 6 hours —condensation on sausage surface 6 7 Surface inoculation with Standardization of sensory Specifications issued by 8 —molds/yeasts —properties —manufacturer of starter culture; 9 —preparation of suspension 10 Fermentation: Acid formation to Adjustment of ripening chamber; 11 • 20– 25°C • Inhibit undesired bacteria —monitoring of temperature, 12 • RH ca. 90% • Transform nitrite to nitrous —humidity, and air velocity; 13 • air velocity ca. 1 m/s acid and NO —monitoring of pH decrease 14 • 2– 3 days until pH р5.3 • Reduce water binding capacity 15 and facilitation of drying 16 Smoking Prevention of mold growth Specifications for wood; adjustment 17 Inhibition of oxidative processes —and monitoring of charring and 18 —at the sausage surface —chamber temperature and 19 Aroma formation —smoking time 20 21 Drying/aging: Prevention of Adjustment and monitoring of 22 • 10– 15°C • Undesired mold growth —climate (temperature, humidity, 23 • RH 70– 80% • Uneven drying —and air velocity) in ripening 24 • air velocity ca. —chamber 25 0.2 m/s until a р0.93 w 26 Storage: Prevention of Adjustment and monitoring of 27 • 10– 15°C • Undesired mold growth —climate (temperature, humidity, 28 • RH 70– 80% • Uneven drying —and air velocity) in storage room 29 • Air velocity ca. 0.05 m/s 30 31 32 33 34 35 36 FERMENTATION,SMOKING, AND DRYING QUALITY ASSURANCE PLANS 37 (TABLE 50.3) AND RECORDS FOR RAW 38 The climate in the fermentation and ripening cham- DRY HAMS 39 bers is critical for product safety and uniform drying Figure 50.2 summarizes the processing steps in the 40 of the sausages. Fermentation and drying/aging con- manufacture of raw dry hams. 41 ditions (temperature, relative humidity, air velocity) 42 should be clearly specified and monitored. The 43 proper function of measuring devices should be PURCHASE AND SELECTION OF MEAT AND FAT AND 44 checked regularly and the results documented. For PRETREATMENT OF CUTS (TABLE 50.4) 45 the manufacture of smoked sausages, the smoking 46 process (usually performed after fermentation) As outlined in the section on fermented sausages, 47 should be well controlled by specifying and record- the origin of the meat and its quality must be speci- 48 ing the type of the wood (or liquid smoke prepara- fied and recorded. For raw hams, it is even more 49 tions), the charring temperature, and the time and important to carefully select cuts and inspect them 50 temperature during smoking. Poor control may for integrity, to reduce the risk of internal contami- 51 result in elevated amounts of polycyclic hydrocar- nation and of poor appearance of the final product. 52 bons and other undesired compounds, and in poor To achieve this, it is very important to establish and S53 sensory quality. maintain fair, long-term cooperation with reliable N54 40454 50 535-544 r1.qxd 7/21/07 1:57 PM Page 540

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Figure 50.2. General processing scheme for dry raw hams. 20 21 22 Table 50.4. Purchase and selection of raw material for raw dry hams (process step A). 23 24 Specification Objective Monitoring and Documentation 25 26 Origin of the material Traceability in case of defects Check of documents and stamps on 27 —caused by process failure in —cuts/containers 28 —primary production, slaughtering, 29 —and/or butchering 30 Age and weight of slaughter Age affects myoglobin content and Visual inspection 31 —animals (pigs: Ͼ 7 months) —thereby appearance of the final Reflectometry 32 —product 33 Type and integrity of cuts pH affects product safety Visual check inspection 34 Fat, collagen and myoglobin 35 —content affects sensory properties 36 —of the final product 37 р 38 pH 5.8 (pork: 24 hours, Reduction of water Visual inspection; 39 —beef: 36 hours postmortem) —binding capacity —pH measurement 40 Fat quality: maximum mol% of Less fat oxidation, slower rancidity Visual inspection 41 —polyunsaturated fatty acids Check of documents on feeding 42 —regime and age of slaughter 43 —animals 44 Microbiological quality Reduction of microbiological risk Visual inspection 45 Temperature measurement (core 46 —temperature below 4°C) 47 Check of hygiene-related 48 —documentation provided by supplier 49 50 51 suppliers. A low pH of the meat is essential for prod- smooth, even surface. Freezing and thawing the cuts 52 uct safety; in general, it should not exceed 5.8, and may increase the salt diffusion rate somewhat, but 53S only in certain pork muscles, pH values up to 6.0 this process must be carefully controlled to avoid 54N may be tolerated. Cuts should be trimmed to obtain a spoilage and fat deterioration. 40454 50 535-544 r1.qxd 7/21/07 1:57 PM Page 541

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SALTING AND SALT EQUILIBRATION (TABLE 50.5) POSTSALTING TREATMENT 1 2 Hams are salted by rubbing or tumbling them with Some hams are washed and subsequently dried in 3 salt (dry salting), by immersing them in a brine, order to remove excessive salt from the surface lay- 4 by injection of brine, or a combination of these ers and to obtain a dry surface before smoking. For 5 methods. Salt diffusion through the meat is slow, quality and safety of the product, extensive washing 6 resulting in the risk of growth of pathogens and should be avoided, and the surface should be dried at 7 spoilage bacteria in the core of the cut if it has been low temperatures; otherwise, there is a risk of 8 contaminated during slaughtering and butchering. growth of Staphylococcus aureus and molds or 9 Hence, salting and salt equilibration should be per- yeasts during drying. Temperatures and times of the 10 formed at 5°C or below until the target water activity washing and drying process should be specified and 11 of 0.96 is reached in all parts of the ham. monitored. 12 A work instruction should specify the composi- 13 tion of the salt (in particular, the levels of nitrite SMOKING AND DRYING/AGING 14 and/or nitrate in the salt), the salt content of the brine 15 (if applicable), the amount of salt and/or brine to be Before smoking and/or aging, it has to be ascer- 16 added per kg of meat, the size of the cut (maximum tained that the water activity has been reduced to 17 distance from the surface to the geometric center), below 0.96 in all parts of the cut. Most German-type 18 the temperature (р5°C). and the minimal time for hams are smoked. An appropriate time-temperature 19 salting and salt equilibration. Records should combination is critical for the quality and safety of 20 include weighing and time-temperature protocols. the products. At 18°C and below, products may be 21 Also, the results of visual inspections and/or meas- smoked for longer periods, whereas at higher tem- 22 urements of the final salt content or a value in the peratures, shorter smoking times should be applied w 23 core should be recorded because the salt diffusion to avoid microbiological problems, and a smoking 24 rate may vary between individual cuts. temperature of 25°C should not be exceeded. The 25 26 27 28 Table 50.5. Manufacture of raw ham: salting and salt equilibration (process step B). 29 30 Subprocess; Examples for Monitoring and 31 Specifications/Critical Limits Objective Documentation 32 33 Purchase and preparation of salt Correct levels of curing agents Weighing protocol 34 —in salt and/or brine to obtain 35 —curing color without exceeding 36 —legal limits 37 Preparation of brine: 8–20% salt, Weighing protocol 38 —depending on salting method and 39 —size of the cut 40 Application of salt Dry cure: e.g., ca. Maximum rate of salt Amount of salt or brine 41 —60 g/kg meat —diffusion, minimal —per kg of meat; time 42 Brine cure: e.g., ca. —“oversalting” of —and temperature of 43 —1 liter brine —surface layers; —tumbling 44 —per 2 kg meat —even distribution 45 Injection cure: —of the salt and 46 —depending on salt —curing agents 47 —content in brine 48 Reduction of water Salting (at < 5° C) Inhibition of Time and temperature 49 —activity to < 0.96 Salt equilibration —microorganisms —in salting room 50 —in all parts by —(at < 5° C and —possibly present in Time, temperature, and 51 —< 85% RH) —the interior of the meat —relative humidity in 52 —salt equilibration room S53 N54 40454 50 535-544 r1.qxd 7/21/07 1:57 PM Page 542

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1 precautions against undesirable aroma and excessive is desired (Stiebing 1992). On the other hand, mold- 2 levels of polycyclic hydrocarbons have been outlined ripened sausages should be packaged in material 3 in the section on smoking of fermented sausages. sufﬁciently permeable to oxygen. At any rate, the 4 Mediterranean-type raw hams are usually sub- properties of the packaging material must be clearly 5 jected to extensive aging. By drying, this process speciﬁed. 6 improves shelf life at ambient temperatures but, even The necessary storage temperature depends on the 7 more important, leaves enough time for enzymatic type of products. Most fermented sausages and hams 8 processes, which makes the hams tender and tasty. are stored at 10–15°C; undried sausages may require 9 Aging normally takes place at temperatures above a storage temperature at 7°C or below, whereas prod- 10 15°C. The hams are stable at ambient temperatures if ucts dried to lower water activities (e.g., sausages

11 the water activity is below 0.90. Aging time, temper- with aw below 0.90) may be stored at ambient tem- 12 ature, and relative humidity, as well as the intended peratures (р25°C). Illumination in display cabinets 13 and measured weight loss should be specified and may be detrimental to fat quality, and the light inten- 14 recorded. sity should be adjusted to below 600 lux. 15 16 SLICING, PACKAGING,AND 17 END PRODUCT TESTING 18 STORAGE OF FERMENTED Specifications for the final product may include pH, 19 SAUSAGES AND RAW DRY HAMS water activity (or weight loss), selected sensory 20 (TABLE 50.6) properties (such as firmness), and the compliance 21 The climate in the slicing and packaging room with official or internal microbiological standards. 22 should be adjusted so as to avoid moisture condensa- Testing whether the final products meet their specifi- 23 tion and undesired surface growth of microorgan- cations is useful to verify that the production process 24 isms. As a rule, the relative humidity in the slicing was under control, but it cannot replace control 25 and packaging room should be below 60%. The tem- measures and process monitoring. The parameters 26 perature depends on the type of product and the time used should be fit for the purpose, i.e., provide a 27 the product is held in the packaging room. A clean- maximum of information with minimum input. For 28 ing and disinfection plan for the slicing device example, it is pointless to carry out total microbial 29 should be in place to avoid contamination of the counts or coliform counts of fermented sausages, 30 product. To prevent mold growth, the residual oxy- and it is much more effective to test for pH than to 31 gen level in a package of sliced fermented sausage count lactic acid bacteria. In contrast, Enterobacteri- 32 should be kept below 0.5%. This is achieved by pack- aceae counts could be an indicator of process 33 aging under vacuum or—increasingly common— hygiene, in particular for undried, fresh sausages. 34 under a modified atmosphere containing about 70% Standards referring to the absence of pathogens (sal- 35 N and 30% CO . Moreover, the oxygen permeability monellae, Listeria monocytogenes) from the final 36 2 2 of the packaging material should be below 25 ml m−2 product are defined in the EU Regulation 2073/2005 37 d−1 or even lower if a shelf life of more than 1 month on microbiological criteria for foodstuffs. 38 39 40 Table 50.6. Slicing and packaging (in this case, under modified atmosphere as an example) of fermented 41 sausages and raw hams (process steps G and E, respectively). 42 Subprocess; Examples for 43 Specifications/Critical Limits Objective Monitoring and Documentation 44 45 Removal of Casings 46 Slicing Prevention of moisture Adjustment and monitoring of 47 —condensation on sausage surface —climate in packaging room 48 Packaging Protection against recontamination, Specifications for packaging 49 —oxygen, and weight loss —material (permeability, etc.); 50 weighing protocol; gas 51 composition; lot identification 52 53S Bulk Packaging Lot identification 54N 40454 50 535-544 r1.qxd 7/21/07 1:57 PM Page 543

Quality Assurance Plan 543

GENERAL REMARKS ABOUT THE REFERENCES 1 2 STRUCTURE AND EXTENT OF LITERATURE CITED DOCUMENTATION 3 MH Brown. 2000a. Implementing HACCP in a meat 4 The purpose of process plans is to ascertain that plant. In: MH Brown, ed. HACCP in the Meat Indus- 5 processes work reliably even though the responsible try. Cambridge, UK: Woodhead Publishing, Ltd., 6 persons may change. Moreover, they are necessary pp. 177–2001. 7 to provide evidence that the management has dele- ———. 2000b. Validation and verification of HACCP 8 gated responsibility in an appropriate manner. Basi- plans. In: MH Brown, ed. HACCP in the Meat Indus- 9 cally, the better trained and experienced the workers try. Cambridge (UK): Woodhead Publishing, Ltd., 10 are, the less paperwork is necessary. The purposes of pp. 231–272. 11 records on the processes are to provide evidence of R Lautenschläger. 2006. Rohpökelware. In: W 12 appropriate process control, e.g., compliance with Branscheid, ed. Qualität von Fleisch und Fleis- 13 “due diligence” as defined in product liability legis- chwaren, 2nd ed. Frankfurt/M.: Deutscher Fachverlag. 14 lation. Records such as the Shewhart control charts FK Lücke. 2000. Fermented meats. In: BM Lund, 15 are also a tool to identify problems and start correc- AC Baird-Parker, GW Gould, eds. The Microbiologi- 16 tive actions before critical limits are exceeded and it cal Safety and Quality of Food. Gaithersburg, Mary- 17 becomes necessary to reject the batch. land: Aspen Publishers pp. 420–444. 18 On the other hand, overdocumentation and prob- JJ Sheridan. 2000. Monitoring CCPs in HACCP sys- 19 lems related to information flow and distribution of tems. In: MH Brown, ed. HACCP in the Meat Indus- 20 documents are a major cause of frustration for staff try. Cambridge, UK: Woodhead Publishing, Ltd., 21 and of noncompliance with the system. In particular, pp. 203–230. 22 overdocumentation A Stiebing. 1992. Verpackung—Anforderung bei 23 • Prevents effective use of documents (increased Fleisch und Fleischerzeugnissen. Fleischwirtschaft 24 time for searching) 72:564–575. 25 • Increases the workload necessary for updating ———. 1996. Herstellung von Rohpökelware. 26 • Reduces flexibility In: F Wirth, J Barciaga, UM Krell, ed. Handbuch 27 Fleisch und Fleischwaren. Hamburg: Behr’s Verlag, 28 The key question must be “what could go wrong if Chapter A 6.6. 29 we do not have this document.” Hence, each docu- ———. 1997. Herstellung von Rohwurst. In: F Wirth, 30 ment should be checked using the following ques- J Barciaga, UM Krell, ed. Handbuch Fleisch und 31 tionnaire: Fleischwaren. Hamburg: Behr’s Verlag, Chapter 32 • Is the document necessary to reach its purpose? A 6.5. 33 • Can the purpose of the document be reached with F Toldrá, ed. 2002. Dry-cured meat products. Trumbull, 34 less words/pages? Connecticut: Food & Nutrition Press. 35 • Can the document replace other documents? V Thorn. 2006. Qualitätsmanagement in der Lebensmit- 36 • Is the style and layout of the document suitable telindustrie. In: W Frede, ed. Taschenbuch für Lebens- 37 for use by its target group? mittelchemiker. Berlin: Springer, pp. 183–213. 38 • Can the purpose of the document also be reached 39 by training and education? 40 OFFICIAL DOCUMENTS 41 To avoid confusion, it is also essential that docu- 42 ments are paginated (page x of y) and that every Commission Regulation (EC) no. 2073/2005 of 15 43 page of a document has a header or footer indicating November 2005 on microbiological criteria for food- 44 stuffs. 2005. Official Journal of the EU L338:1–26. • What it covers 45 Regulation (EC) no. 852/2004 of the European Parlia- • Who has written it 46 ment and Council of 29 April 2004 on the hygiene of • Who has approved it 47 foodstuffs. 2004. Official Journal of the EU • The date and number of the version 48 L226:1–19. 49 It is also important to make sure that everyone has USDA-FSIS. 1999. Generic HACCP model for not heat 50 exactly those documents available that he/she needs, treated, shelf stable meat and poultry products. http:// 51 and that any previous versions of the document are www.fsis.usda.gov/OPPDE/nis/outreach/models/ 52 removed. HACCP-15.pdf. S53 N54 40454 Index 545-558 r0.qxd 7/21/07 2:00 PM Page 545

1 2 3 4 5 Index 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Acidification, 140, 303 Factors affecting, 463 22 Additives, 77 Related bacteria/microorganisms, 458, 462 23 Acids, 78, 303 Classification, 455–456 24 Advantages, 77 Contents, 458–461 25 Antioxidants, 66–67, 70–71, 78, 249, 264 Health risk, 456–457 26 Colorants, 79 Histamine intolerance, 456–457 27 Emulsifiers, 80 Histamine intoxication, 456–457 28 Flavor enhancers, 81 MAOI interaction, 457 29 Flavoring agents, 83 Migraine, 456 30 Legal regulations, 77 Nitrosamine formation, 457 31 Multipurpose, 85 Resistance, 142 32 Preservatives, 83 Antioxidants, 141 33 Affective analysis, 197, 199 Appearance, 198–199 34 Aging, 539–542 Aroma, 538, 542 35 Air-Conditioning, 43 Compounds, 227 36 Air relative humidity, 43 Dry-cured Ham, 227–229 37 Dehumidifying, 44 Fermented sausage, 229–232 38 Humidifying, 44 Formation, 232–233 39 Psychrometric chart, 44 Identification, 227 40 Aldehydes, 66 Origin, 232 41 Allergies, 523 Ascorbate (see Ascorbic acid) 42 American, 327–331 Ascorbic acid, 21, 79, 204, 470, 538, 540 43 Manufacturing, 328–331 Ascorbic esters, 79 44 Sausage types, 328 Assessor, 197–200 45 Amines, 129–130, 142 ATP, 32, 56, 499 46 Amino acids, 52–53, 141, 233 Attribute, 197 47 Antioxidative enzymes, 56 Audit, 525, 532–533 48 Catalase, 56 Auditors, 532 49 Superoxide dismutase, 56 Authors, ancient, 4 50 Antimicrobial compounds, 126, 131, 149–150, 152, Cato, 4 51 154, 455–464, 519 Diocletian, 4 52 Aminogenesis, 457–458, 462–464 Homer, 4 53 Control measures, 463–464 Autoxidation, 232, 234 S54 N55 545 40454 Index 545-558 r0.qxd 7/21/07 2:00 PM Page 546

546 Index

1 Bacteriocins (see Antimicrobial compounds) Chilling, 536–537 2 Bacteriophages, 180 Cholesterol, 13, 257 3 Benzoates, 84 Cholesterol oxides, 248 4 Betanin, beet red, 80 Chromosome, 177–179, 181, 183–184 5 BHA, 260 Citric acid, 78, 303 6 BHT, 260 Cochineal, 80 7 Biogenic amines (see Amines) Red, 80 8 Biological hazards, 513, 519–522 Code of Federal Regulations, U.S. (CFR), 267 9 Black pepper, 306 Cofactors, 235 10 Blood protein, 81 Collagen, 59, 68, 70, 244, 304 11 Boar taint (see Off-flavor) Colonization, 140 12 Botulism, 477 Color, 218, 505 13 Bovine, beef, 59, 243–245, 247 Sausage, 218 14 Brine, 206, 541 Competence, 180 15 Buffalo, 10, 370, 372 Composition, 13 16 Buffering capacity, 32 Computed tomography (CT), 408 17 By-products, 70 Conjugation, 180 18 Connective tissue, 59–60, 63, 66, 68, 70 19 Calpains, 54–55 Consumer 20 Camel, 371 Identification, 514 21 Carbohydrates, 12–13, 126, 190 Test, 197–198 22 Content, 190 Consumption, 407, 410–412 23 Dextrose (see Glucose) Contaminants, 469–474, 491–492 24 Glucose, 12, 190–191 Environmental, 473–474, 492, 495 25 Lactose, 190 Nitrosamines, 469–470 26 Maltose, 190 Polycyclic aromatic hydrocarbons, 27 Sucrose/saccharose, 190 470–471, 491 28 Carcass, 245, 275 Veterinary drug residues, 472–474 29 Carmine, 80 Control, 427–428, 436–437, 450 30 Caseinates, 81 Chart, 528–531 31 Casings, 12, 101, 192, 305, 337, 421–423, 538, 542 Measures, 450 32 Cellulose casings, 107 Cooking, 305 33 Collagen casings, 107 Coriander, 306 34 Determining quality, 109 Creatine, 248 35 Handling casings, 109 Criteria, 437 36 Natural casings, 101 Critical control points (see HACCP) 37 Beef, 104 Critical limits, 514, 519, 523–524 38 Glossary of terms, 106 Cross contamination, 484 39 Hog, 104 Barriers, 491–492 40 Horse, 106 Biofilm, 421–422, 485, 492 41 Removal of the viscera, 102 Niche environnements, 485 42 Sheep, 104 Sources, 492 43 Plastic casings, 108 Cured meat, 17, 248 44 Regulatory compliance, 108 Dry-curing process, 17 45 Test procedures, 110 High pressure application, 23 46 Catalase, 141, 212 Polyphosphates, 23 47 Cathepsins (see Proteases) Red cured color, 18 48 Cellar, 394–395, 398 Saltpeter, 17 49 Cereal protein, 81 Curing agents, 126, 153, 188, 536–538, 540, 542 50 Certification, 533 51 Challenge (testing), 431, 435–437, 443–444, 447, Deamidation (see Amino acids) 52 449–450 Decarboxylation (see Amino acids) 53S Checklists, 515–517 Deer meat, 372–373 54N Chemical hazards, 513, 519–522 Defense, 428 40454 Index 545-558 r0.qxd 7/21/07 2:00 PM Page 547

Index 547

Dehydration (see also Drying), 60–61, 63, 65, 70, Sun-drying, 416 1 72, 393–394, 425 Types of 2 Dehydrogenation (see Amino acids) Bayonne, 400, 402–405 3 Denmark, 407, 412 Black Forest (see Schwarzwald) 4 Descriptive test, 198–199 Cinta Senese, 402, 405 5 DFD meat, 63 Country ham, 377 6 Diet, 61, 64–67, 72 Fenalar, 407–408 7 Difference test, 198–199 Iberian, 26, 206, 394–398, 401, 404–405 8 Discoloration, 204, 210, 212 Ibérico (see Iberian) 9 Diseases, 243 Jinhua ham, 415 10 Documentation, 537, 540, 543 Nero Siciliano, 402–403, 405 11 Instructions, 542 Noir the Bigorre, 403, 12 Records, 539, 541, 543 Parma, 26, 206–207, 393–394, 399–405, 415 13 Donkey, 10 San Daniele, 401–402 14 Dried meat products, 5 Sauna ham, 5 15 Carne seca, 5 Schwarzwald, 408, 411 16 Charque, 5 Serrano, 393–396, 400, 402, 404 17 Hangikjöt, 410 Spekenskinke, 409 18 Hjallur, 410 Westphalia, 411 19 Pinnekjøtt, 409 Washing, 416 20 Skerpikjøt, 409–410 Dry-fermented sausages (see also Sausages), 21 Dry-cured ham, 415 9–11, 147 22 Commercial curing, 380 Decoloration, 424 23 Cooking, 385 North European 24 Cure ingredients, 384 Country characteristics, 351–357 25 Grading, 417 General characteristics, 349 26 Green ham preparation, 416 Ingredients, 350 27 Bamboo leaf, 416 Processing technology, 350 28 Hind-leg, 416 Quality criteria, 349 29 Jinhua “Liangtouwu” pig, 416 Ripening program, 351 30 Trimming, 416 Souring, 423 31 History, 415 Drying, 3, 9–10, 41, 113–114, 125–126, 333, 32 South Song Dynasty, 415 393–395, 397–398, 401, 403–404, 407, 33 Tang Dynasty, 415 410–411, 539–541 34 Home curing, 383 Case hardening, 42–43 35 Intramuscular fat, 396–397, 402–404 Drying rate, 43 36 Juiciness, 398, 402, 404 Effective diffusion, 43 37 Postripening, 417 Fat melting, 43 38 Processing, 415 Kinetics, 43 39 Production, 379 Modeling, 43 40 State production, 381 Shrinkage, 43 41 Raw hams, 396, 535, 539, 542 Water diffusion, 43 42 Ripening, 393–395, 397, 403–405, 417 Smearing, 42 43 Centipede rack, 417 Duck, 10 44 Molds, 417 45 Taste substances, 417 Electrophoresis 46 Salting, 393–395, 397–400, 416 Pulse ﬁeld gel electrophoresis, 177 47 Drain salt, 416 Two-dimensional, 181–182 48 Salting duration, 416 England, 412 49 Temperature, 416–417 Enterotoxins, 142 50 Sensory features, 398 Environment, 275 51 Shaping, 416 Epidemiological data, 519 52 Soaking, 416 Erythorbate (see Erythorbic acid) S53 Storage, 417 Erythorbic acid, 79, 204, 206 N54 40454 Index 545-558 r0.qxd 7/21/07 2:00 PM Page 548

548 Index

1 Esterases (see Lipases) Oat, 262 2 EU policy, 513 Wheat, 262 3 European Food Safety Authority (EFSA), 280 Filling, 538 4 Expert, 514, 524 Flavor, 10, 140, 198–200, 227–236, 415, 417, 5 507–510 6 Faeroe Islands, 409 Precursors, 235 7 Hjallur, 410 Wheel, 200 8 Skerpikjøt, 410–411 Flavorings, 470 9 Fat, 53–54, 60, 62, 65, 72, 243–244, 246, 249–250, Food facilities, 483 10 536, 540 Food operators, 513 11 Backfat, 245, 259–260, 263 Food product definition, 515, 518 12 Color, 66 Intended uses, 514–515, 518 13 Consistency, 66 Food safety, 513 14 Content, 59, 61–62, 65 Food Safety and Inspection Service 15 Deterioration, 537 (FSIS), 267 16 Intramuscular, 60, 64–65, 245 Management systems, 533 17 Oxidation, 211, 537, 540 Objectives (FSO), 278 18 Phospholipids, 54 Policy, 513 19 Physical state, 65 Proactive methodology, 513 20 Reduction, 61 Terms of reference, 513, 515 21 Replacers, 71, 259 Formaldehyde, 470 22 Triacylglycerols, 53, 60, 72 Frozen 23 Fatty-acids, 65–66, 72, 243, 339, 536 Lard, 60 24 Free, 53, 56, 62 Meat, 59 25 Monounsaturated, 60, 261 Freezing, 536–537 26 Polyunsaturated, 60, 65–67, 244–245, 251, 259, Functional foods, 154–155 27 261, 537 28 DHA, 260 Game, 59, 372–373 29 EPA, 260 GC-O, 227, 229, 232 30 Linoleic acid, 65–67 Gel, 63, 69–71 31 n-3 fatty acids, 66–67, 260–261 Gelation, 217 32 n-6 fatty acids, 66, 260 Protein, 219, 222 33 Omega-6/omega-3 ratio, 245 Myofibril, 221 34 Saturated, 60, 257, 261, 536 Storage modulus, 219 35 Fermentation, 3, 10, 113–114, 125, Genome, 139 36 126–130, 333 Glucono-delta-lactone, 78, 303 37 Acidification, 188–192 Glutamic acid, 82 38 Backslopping, 5–6, 32 Glycogen, 33 39 Lag phase, 189, 191–192 Glycolysis, 32 40 Style, 351 Goat, 370, 371 41 Fast, 351 Angora goat, 371 42 Traditional, 351 Chevon, 370 43 Very fast American style, 351 Good hygiene practice (GHP), 279, 515, 519 44 U.S. standards, 269 Good manufacturing practices (GMP), 515, 519 45 Fermented sausages, 9–13, 125–131, 324, 493–494, Grinding, 305 46 535–536, 542 Growth, 427–428, 430–432, 436–438, 445, 47 Dry (see Dry-fermented sausages) 448, 450 48 Importance, 324 Guanylates, 82 49 Semi-dry, 9–11 50 Fiber, 248, 250 HACCP, 270, 278–279, 303–304, 331, 450, 491, 51 Albedo, 264 500, 513–514, 525, 535–536 52 Cereal, 262 Concept, 513 53S Fruit, 262 Control points-GHP/GMP, 519, 528–531 54N Inulin, 249, 262 Corrective actions, 514, 524, 528–531 40454 Index 545-558 r0.qxd 7/21/07 2:00 PM Page 549

Index 549

Critical control points (CCP), 514, 519 Embden-Meyerhof mechanism, 32 1 Decision tree, 519, 524–527 Lactate, 32–33 2 Identification, 519 Lamb, 369–370, 372, 407–411, 413 3 Flow diagram, 514–515, 518 Fjellmorr, 370, 372 4 Foundations, 514 Lambaspaeipylsa, 370 5 Plan, 513–514, 518, 525, 528–531, 532 Merguez-type, 370 6 Revision, 514, 532 Lapp traditions, 411 7 Potential Hazards Identification, 513, 515, Latin American typical meat products, 387, 389 8 519, 520–522 Comminuted meat, 389 9 Prerequirements, 515–517 Dry and salted meat, 390 10 Principles, 514, 525, 528–531 Fermented meat, 390 11 Team, 514–515 Processed meat, 389 12 Ham (see Dry-cured ham) Safety risks, 389 13 Hangikjöt, 409–410 Lean, 59, 304 14 Hazard analysis (see HACCP) Leek, 71 15 Health, 243, 247–249, 252, 257 Lipases 16 Herbs, 250, 341 Esterases, 56 17 High-pressure processing, 212 In adipose tissue, 56 18 Histamine, 457–458, 462 In muscle, 13, 55–56 19 Horse, 10, 372 Phospholipase, 55 20 Kazy, 372 Lipids (see Fats) 21 Kotimainen sausage, 372 Lipolysis, 140–141, 173, 251, 408–409 22 Shuzhuk, 372 Listeriosis, 479 23 Sognemorr, 372 Live weight, 59 24 Stabbur, 372 Lupin, 71–72 25 Trøndermorr, 372 26 Humidity (see Moisture), 539, 541 Manganese, 192 27 Relative, 416–417, 422–423 Manufacturing processes, 304–305 28 Hurdle technology, 40, 148 Marbling, 64 29 Biopreservation, 42 Meat 30 Curing, 41 Compositional aspects, 243, 245–246, 249 31 Fermentation, 42 Meat color, 64 32 Packaging, 41 Preserved meats, 248 33 Refrigeration, 41 Red meat, 257 34 Salting, 41 Metabolism, 140–141 35 Spicing, 41 Microbial fermentation, 303, 305 36 Storage, 41 Microbiological safety, 250, 275, 304 37 Hygiene, 516–517, 524–535 Appropriate level of protection (ALOP), 278 38 Regulation, 535 E. coli (see Pathogens) 39 Enterocci, 115–117, 128–129 40 Inactivation, 431, 436–437 Hygiene criteria, 285–286, 288 41 Ingredients, 224 Listeria monocytogenes (see Pathogens) 42 Acid, 219, 222 Microbiological guidelines, 282 43 Enzymes, 221 Microbiological standard, 282 44 Fatty acids, 220, 224 Salmonella (see Pathogens) 45 Glucono-delta-lactone, 219, 222 Staphylococcus aureus (see Pathogens) 46 Inulin, 225 Technological commodity, 277 47 Salt, 225 Total viable counts, 499 48 Inoculum, 192 Toxins/metabolites, 280 49 Inosinates, 82 Zoonotic agents, 275 50 Isoascorbate (see Ascorbate) Microorganisms (see also starter cultures) 51 Amino acid decarboxylase activity, 458, 462 52 Lactate (see Lactic acid) Bacillus, 210 S53 Lactic acid, 32–33, 78, 126, 129, 131, 303 Bifidobacterium, 212 N54 40454 Index 545-558 r0.qxd 7/21/07 2:00 PM Page 550

550 Index

1 Microorganisms (see also starter cultures) (continued) Molds, 11–12, 188, 340 2 Biogenic amine / relation to, 458, 462 Aspergillus, 172 3 Brochothrix, 130 ochraceus, 172–175 4 Candida (see Yeasts) Competition, 173, 175 5 Clostridium botulinum, 469 Growth rate, 173–175 6 Debaryomices (see Yeasts) Hydrophilic, 171–172 7 Enterobacteriaceae, 130, 424–425, 492, Lipolytic activity, 173 8 500, 542 Mucor, 172 9 Escherichia coli (see Pathogens) Proteolytic activity, 172–173 10 Gram-positive catalase-positive cocci (GCC+), Relative humidity (RH), 171–175 11 113–116, 120, 122 Temperature, 171, 173–174 12 Gram-positive coagulase-negative cocci, Toxic metabolites, 171–173 13 126, 129 Xerophilic, 172 14 Kocuria, 139, 188, 190–191 Xerotolerant, 172 15 Staphylococcus carnosus, 120, 122 Penicillium, 172–175, 188–190, 220 16 LAB (see Lactic acid bacteria) camemberti, 172–174 17 Lactic acid bacteria, 9, 11, 33–35, 113–118, 122, chrysogenum, 172–175 18 138, 140, 148, 153, 188–190, 209, 219, gladioli, 172–175 19 321, 423 nalgiovense, 172–175, 422–423, 425 20 Biogenic amine/relation to, 458, 462, 464 verrucosum, 172–175 21 Lactobacillus curvatus, 118–119, 122, Molecular markers, 386 22 127–128, 131, 138–139, 189 Monitoring, 514, 519, 523 23 Lactobacillus plantarum, 11–12, 118–119, Monosodium glutamate (see Glutamic acid) 24 122, 137–139 Mustard, 306 25 Lactobacillus sakei, 12, 33–34, 118–119, Mutant construction, 180 26 122, 127–128, 131, 137–139, 177–182, Mutton (see Lamb) 27 188–189 Myoglobin, 17, 24, 64, 66, 537–538, 540 28 Lactobacillus, 33–34, 188, 209 Deoxymyoglobin, 205 29 Lactococcus, 149 Metmyoglobin, 204, 205 30 Leuconostoc, 423–424 Nitrosylmyoglobin, 18, 24, 204–207 31 Listeria monocytogenes, 270, 279, 285, 304 Oxymyoglobin, 18, 25, 205 32 Micrococcaceae, 11, 190, 204, 208 Perferrylmyoglobin, 207 33 Nocardia, 210 34 Pediococcus, 11, 32, 137, 139, 188 Natamicin, 84 35 acidicaltici, 11–12 National Country Ham Association, 379 36 pentosaceus, 12 Nisin, 84, 152–154 37 Penicillium (see Molds) Nitrate reductase, 140, 208–209, 424 38 Pseudomona, 130, 208 Nitrate, 9–10, 19–20, 31–32, 34, 84, 189–190, 39 Salmonella (see Pathogens) 204, 206, 249, 268, 416, 470, 493, 40 Staphylococcus, 31, 33, 188, 190–191, 208, 232, 538, 542 41 235 Nitric acid, 20 42 Formerly Micrococcus, 34–35 Nitric oxide synthase (NOS), 210 43 Staphylococcus aureus, 35, 189, 270 Nitric oxide, 20, 204, 210, 470 44 Staphylococcus carnosus, 11, 139, 177–182 Nitrite reductase, 208–209 45 Staphylococcus equorum, 120–122, 129, 139 Nitrite, 9–10, 17–20, 31–32, 34–35, 84, 189–190, 46 Staphylococcus saprophyticus, 120–122, 129 204, 206, 248–250, 268, 304, 321, 416, 47 Staphylococcus xylosus, 12, 120–122, 469–470, 493, 538–539, 542 48 129, 131, 139, 177–182 Nitrogendioxide, 20 49 Milk protein, 81 Nitrosamines, 28, 410, 469–470 50 Minerals Nitrosylating agent, 205 51 Iron, 246, 257 Nitrosylmyochrome, 204 52 Manganese, 246, 250 Nitrous acid, 20 53S Mixing, 305 NO-dimethylamines, 28 54N Moisture, 9, 10, 13 Non-meat ingredients, 304 40454 Index 545-558 r0.qxd 7/21/07 2:00 PM Page 551

Index 551

Nutrition, 243, 248 Listeria monocytogenes, 115–117, 130, 148, 189, 1 Energy, 243–244, 247 270, 279, 285, 304, 427, 429–430, 436, 2 Nutritional guidelines, 247 438, 450, 492–493, 495 3 Salmonella, 12, 130, 189, 270, 279, 285, 304, 4 Obesity, 243, 247 428–430, 444–445, 450, 477–479, 492, 5 Off-flavor, 200 495, 536, 538 6 Oils Staphylococcus aureus, 115–117, 130, 270, 7 Fish oil, 245, 249–261 427–430, 448, 450, 477, 479, 495, 500, 542 8 Linseed, 259 Pediocin, 150, 152 9 Olive, 13, 259, 264 Peptides, 52 10 Soy, 261 Performance (lethality) standards, 431, 437, 444 11 Oleoresins, 83 Personal hygiene, 485 12 Ostrich, 13, 371–372 Clothing, 485 13 Ovine, 59 Contamination, 500 14 Oxidation, 60, 64, 66–67, 70, 72, 220, 246, 251, Hand hygiene, 486 15 471–472 Hand washing, 486 16 Of amino acids, 472 Footwear, 485–486 17 Of cholesterol, 472 Gloves, 486 18 Of peptides, 472 Working clothes, 485 19 Of phospholipids, 471 Personnel, 486, 488 20 TBARS, 252, 260 Health, 486 21 Oxidative enzymes, 56 Training, 488 22 Lipoxygenase, 56 Pest control, 487–488 23 Integrated pest management (IPM), 487–488 24 Packaging, 289–290, 298, 305–306, 521, pH, 63, 69, 192, 268, 336, 536–537, 540 25 536, 540, 542 Phages (see Bacteriophages) 26 Functional products, 260 Phosphates, 23, 85 27 Light, 210, 212 Phospholipase (see Lipases) 28 Oxygen, 210, 212 Phospholipids (see also Lipids), 66, 72 29 Packaging functions, 289, 291–292 Physical hazards, 513, 519–522 30 Communication, 289–290 Pigment (see Myoglobin) 31 Containment, 289 Pinnekjøtt, 409 32 Convenience, 289–291 Piramicin, 422 33 Permeability, 542 Plasmid 34 Protection, 289–290, 293, 299 Profile, 128–129, 131 35 Packaging materials, 290–291, 293–296, 298–299 Vectors, 180–181 36 Barrier, 290–296, 298–299 Poland, 412 37 Sealing, 290, 292–295 Polycyclic aromatic hydrocarbons, 470–471, 491, 542 38 Strength, 290, 292–295 Polyphenols, 79 39 Packaging systems, 289, 294, 297–298 Polyphosphates (see Phosphates) 40 Active packaging, 294, 296, 298–299 Pork, 243–245, 247–248, 304 41 Aseptic packaging, 294, 299 Postgenomics, 181 42 Modified atmosphere packaging, 26, 293–295 Poultry meat, 304, 361 43 Vacuum packaging, 293–296, 298 Characteristics, 362 44 Paprika extract, 80 Compositional aspects, 246–248 45 Parasites, 269–270, 304, 479, 520–522 Delicatessen, 361 46 Pathogens (see also Microorganisms), 130, Dry magret, 366 47 519–522, 542 Fat, 362–364, 366 48 Campylobacter sp., 477–478 Safety aspects, 274 49 Clostridium botulinum, 35, 209, 477 Sausages, 361 50 Emerging, 147–148 Dry, 361 51 Escherichia coli O157:H7, 35, 130, 189, 219, Fermented, 361 52 270, 279, 285, 304, 427–432, 436–437, Quality, 365 S53 444, 450, 492–493, 495, 500, 536 Sausage-making process, 363 N54 40454 Index 545-558 r0.qxd 7/21/07 2:00 PM Page 552

552 Index

1 Prebiotics, 263 Sarcoplasmic, 52 2 Preference test, 198–199 Stromal, 52 3 Preservation methods, 3 Proteolysis, 54–55, 141, 172–173, 408–410 4 Drying, 3 Proteomics, 181–182 5 Fermentation, 3 Proton motive force, 32 6 Smoking, 3 PSE meat, 63–64 7 Prevalence, 428, 432–436, 438, 443, 445, Pyruvate, 32 8 447–450 9 Preventive measure, 519, 522–523 Quality, 56, 273, 535–537, 540 10 Probiotics, 263 Assurance, 535, 539 11 Processing, 223 Controls, 503–512 12 Chopping, 224 Drying, 505 13 Control, 331 Final product, 504 14 Acidulation, 327, 331 Microbial, 505 15 Fermentation, 328, 331 Raw materials, 503–504 16 pH control, 331 Sensory, 505–510 17 Description, 12, 515, 518 Criteria, 535 18 Encapsulation, 219, 222 End product, 273 19 Factors (effects), 218, 223–224 Food chain, 273 20 Fermentation, 224 Food commodity, 273 21 Grinding, 24 Health factor, 273 22 Mixing, 224 Meat production line, 274 23 Ripening, 220 Microbiological, 536–537, 540 24 Product development, 197 25 Profile test, 198–199 Rabbit, 10, 372–373 26 Propanoic acid, 33 RAPD-PCR, 128–129, 131 27 Propionic acid, 33 Records, 514, 523–524, 532 28 Prosciutto (see Dry-cured ham) Reindeer, 10, 372–373, 411–412 29 Proteases, 13 Poro sausage, 373 30 Aminopeptidases, 55 Reinsdyr pølse, 373 31 Calpains, 55 Revision, 532 32 Carboxypeptidases, 55 Ribonucleotides, 82 33 Cathepsins, 54, 55 Ripening, 336 34 Dipeptidases, 55 Acceleration, 341 35 Dipeptidylpeptidases, 55 Autooxidative phenomena, 337 36 Lysosomal (see Cathepsins) Casing, 337 37 Proteasome, 55 Exogenous enzymes, 339 38 Tripeptidylpeptidases, 55 Lipid breakdown, 337 39 Proteinases (see Proteases) Microbial activities, 337 40 Proteins, 243–244 Muscular proteases, 339 41 Amino acids Protein fragmentation, 338 42 Essential amino acids, 244 Risk, 427–430, 444, 450 43 Free amino acids, 251, 339 Analysis, 277 44 Leucine, 251 Assessment, 427, 429–431, 450 45 Methionone availability, 244, 252 Characterization, 427–428 46 Tryptophane, 244 Dose, 427, 431 47 Hydrolyzates, 83 Evaluation, 515 48 Moisture/protein ratio, 268, 303 Exposure, 427, 430–431, 436 49 Muscle, 51–52 Hazard, 427–428, 430, 450 50 Myofibrillar proteins, 51–52, 251 Identification, 427–428 51 Myoglobin, 52 Quantitative, 427, 450 52 Protein fragmentation, 338 53S Protein oxidation, 67, 251 Safety (see also Microbiological safety), 141, 409, 411 54N Protein quality, 244, 249, 274 Standards, 377 40454 Index 545-558 r0.qxd 7/21/07 2:00 PM Page 553

Index 553

Salt, 9–10, 12, 31, 33–34, 65, 68–70, 188–189, 204, Filipino sausage, 324 1 243, 248–249, 304, 408–409, 412, 536, 540 Longamisa, 324 2 Diffusion, 537, 542 Gam Ngan Cheong (Gold silver sausage), 323 3 Equilibration, 541–542 Gold silver sausage, 323 4 Reduction, 408 German sausage, 22, 324 5 Substitutes, 258 Goon Chiang, 324 6 Sanitary design, 483–484 Hollow sausages, 425 7 Air pressure, 484 Hungarian salami, 4, 7, 355 8 Cleaning and sanitation, 496–499 Kalofer, 356 9 Establishment buildings, 483, 484 Kantwurst, 354 10 Hygienic plant design, 494–499 Katenrauchwurst, 352 11 Lighting, 484 Kolbász, 355 12 Processing plant sanitation, 493 Korean sausage, 324 13 Surface in contact, 484 Sooday, 324 14 Surface non in contact, 484 Sunday, 324 15 Trafﬁc, 485 Kotimainen, 357 16 Sausages, types of La chang, 323 17 Aap Gon Cheong (Duck liver sausage), 323 Landjäger, 352–353 18 African, 322 Lap Cheong (La chang), 4, 323 19 Beirta, 322 Lebanon Bologna, 306 20 Biltong, 322 Longanisa, 324 21 Kaidu-digla, 322 Loukanka, 355 22 Miriss, 322 Lovecky, 356 23 Mussran, 322 Mediterranean, 321–322, 333–336 24 Ambaritsa, 356 Chorizo, 322 25 American, 328 Saucisson, 322 26 Australian sausage, 324 Mettwurst, 5, 10 27 Vento salami, 324 Middle Eastern, 322 28 Babek, 356 Soudjouk, 322 29 Boerenmetworst, 354 Moldy sausages, 422 30 Budapest salami, 355 Morr sausage, 357 31 Cervelat, 5, 10, 13, 352–354 Moscow-type salami, 356 32 Chicken liver sausage w/wo pig liver, 323 Nepalese sausage, 324 33 Chinese sausage, 323 Nhum (Thai fermented sausage), 323 34 Aap Gon Cheong (Duck liver sausage), 323 North American, 321 35 Chicken liver sausage w/wo pig liver, 323 Lebanon bologna, 321 36 Gam Ngan Cheong (Gold silver sausage), 323 Pepperoni, 321 37 La chang, 323 Northeastern sour Thai-style sausage, 323 38 Lap Cheong (La chang), 4, 323 Northern European, 322 39 Chorizo, 10, 258, 269 Greuβner salami, 322 40 Crusty sausages, 425 Jalowcowa, 322 41 Danish salami, 357 Katwurst, 322 42 Dried Northern Thai sausage (Sai Ua), 323 Krakowscha sucha, 322 43 Dry-cured loin, 65 Metwurst, 322 44 Dry fermented, 62, 64, 66, 71–72, Paprikas, 256 45 268–269, 334 Pepperoni, 10, 13, 324, 369 46 Duck liver sausage, 323 Pig liver sausage, 323 47 Eastern European, 322 Polican, 356 48 Hungarian salami, 322 Polska, 356 49 Winter salami, 322 Raw fermented sausages, 333 50 Eichsfelder, 352 Rotten sausages, 424 51 European-style sausage, 324 Russian salami, 356 52 Feldkieker, 352–353 Sai Ua (Dried Northern Thai sausage), 323 S53 Fermented, 59–60, 63 Salami, 4, 10–11, 13, 258, 269 N54 40454 Index 545-558 r0.qxd 7/21/07 2:00 PM Page 554

554 Index

1 Sausages, types of (continued) Color, 18, 198–199, 259 2 Salchichón, 11, 259 CIE Lab, 259 3 Sausage for pizza, 306 Features, 61–62, 64, 66 4 Sausages of Germany, 22 Flavor, 59, 64, 66, 70–71, 198–200, 339 5 Schlackwurst, 352–353 Formation, 64, 69 6 Semidry sausages, 268–269, 306 Objectionable, 67 7 Singapore sausage, 323 Rancid, 66, 199–200 8 Chicken sausage, 323 Juiciness, 60, 62, 64 9 Pig liver sausage, 323 Oxidation, 220 10 Special grade pork sausage, 323 Panel, 197 11 Slimy sausage, 421 Taste, 199, 200, 258–259 12 Snack Sticks, 306 Sour, 200 13 Snijworst, 354 Texture, 199–200, 258, 260, 262, 508–509, 538 14 Sooday, 324 Traits, 62, 65, 70 15 Soudjouk (Sucuk), 370–372 Sensory science, 197 16 South American, 321 Shelf stable sausages, 268 17 Cacciaturi, 321 Skerpikjøt, 410–411 18 Special grade pork sausage, 323 Slicing, 536, 540, 542 19 Spegepølse, 357 Smoke, 3, 13, 41, 252, 305, 423, 470–471, 491, 20 Sri Lankan sausage, 324 536, 540–541 21 Stabbur, 357 Smoke extracts, 83 22 Stapelpack salami, 352 Smoking (see Smoke) 23 Sticky sausage, 421 SOD, 212 24 Sucuk, 260 Sodium chloride (see Salt) 25 Summer sausage, 5, 10–11, 13, 269, 306, Sodium lactate (see Lactic acid) 26 321, 369, 373 Soja protein, 81 27 Sunday, 324 Sorbate, 84 28 Swedish Medwurst, 357 Spices, 12, 87, 126, 192, 250, 252, 304, 341, 29 Sweet Bologna, 306 536, 538 30 Szegediner salami, 355 Applications, 97 31 Teewurst, 10, 352–353 Botanical properties, 88 32 Thai fermented sausage (Nhum), 323 Chemical properties, 89 33 Thai sausage, 323–324 Ethnic preference, 87 34 Goon Chiang, 324 Product forms and appearances, 88 35 Nhum (Thai fermented sausage), 323 Quality standards, 94 36 Northeastern sour Thai-style sausage, 323 Sanitation criteria, 95 37 Sai Ua (Dried Northern Thai sausage), 323 Sensory properties, 97 38 Thuringer Bratwurst, 10–11, 352 Spoilage, 117, 130, 541–542 39 Traditional sausages, 113–114, 117, 122 Standards, 535 40 Turkish type salami, 263 ISO, 535 41 U.S. products, 269 Microbiological, 542 42 Venäläinen, 357 Specification, 535–536 43 Vento salami, 324 Starch, 243, 248 44 Westphalian salami, 352 Starter cultures, 12–13, 32–34, 126, 147, 154–155, 45 Scientific Committee on Foods (SCF), 280 166, 177–181, 183–185, 204, 258, 46 Scientific Panel on Biological Hazards 328–331, 336, 536, 538 47 (BIOHAZ), 280 Amino acid decarboxylase negative, 464 48 Scopulariopsis, 172 Catalase-positive cocci, 538 49 Sensory, 11, 218 Lactic acid bacteria, 126–127, 131, 336, 538 50 Acceptability, 62–64 Micrococcaceae, 129, 131, 336 51 Appearance, 260 Molds, 171–175, 538 52 Aroma, 60, 66, 199–200 Yeasts, 538 53S Volatile compounds, 66, 71 Storage, 289–291, 293, 295–296, 299, 539, 542 54N Characteristics, 62, 64, 67–68 Stress, 431, 436 40454 Index 545-558 r0.qxd 7/21/07 2:00 PM Page 555

Index 555

Structure, 222 Verification, 514, 523–525, 528–531 1 Acid addition (effect), 222 Veterinary drug residues, 472–474 2 Encapsulation (effect), 219 Antibiotics, 472–473 3 Micro-structure, 222 Substances with anabolic effect, 472–473 4 Stuffing, 305 Virus, 520–522 5 Substrate level phosphorylation, 32 Vitamins, 13, 246 6 Sugars, 243–244, 247, 304, 536 C (see Ascorbic acid) 7 Survival, 428, 431–432, 435–438, 443–445, Vitamin-E, 64, 246, 250 8 447–449 Volatile compounds (see also Flavor), 415, 417 9 Sweden, 407, 411–412 VTEC, 478–479 10 11 Taste compounds, 233–234 Warmed-Over-Flavor, 210 12 ATP derivatives, 233 Waste disposal, 487 13 Free amino acids, 234 IPPC Directive, 487 14 Organic acids, 233 BAT, 487 15 Peptides, 234 BREF, 487 16 Salt, 233 Water 17 Sugars, 233 Activity, 31, 33, 61, 65, 69, 171, 173, 175, 336, 18 Temperature, 187–189 417, 421–422, 425, 539, 541–542 19 Tenderness, 201 As an ingredient, 69 20 Texture, 199–200, 217, 506, 508–509, 538 Holding capacity, 63, 67, 69–70 21 Elasticity, 218 In muscle, 63 22 Fermentation, 217 Losses, 65 23 pH effect, 218–219 Migration, 63, 65 24 Storage modules, 219 Water binding capacity, 537, 539–540 25 Tocopherols, 64, 67, 79, 259 26 Toxins, 142 Yeast, 115–117, 159–167, 188–190 27 Traceability, 515, 537, 540 Dry cured hams, 161, 165–166 28 Traditional workshops, 515, 518 Contribution to the flavour, 165–166 29 Training, 514, 524 Mycobiota composition, 161–163 30 Transamination, 233 Fermented sausages, 159–161, 164–165 31 Transformation protocols, 180 Contribution to the flavor, 164–165 32 Transposition, 180 Mycobiota composition, 160–163 33 Triacylglycerol (see Triglycerides) Growth conditions, 160 34 Trichinae (see Parasites) Hydrolyzates, 83 35 Trichinella spiralis, 269–270, 304 Lipolytic and proteolytic activities, 164–166 36 Typing, 129, 131 Presence in meat products, 159–160 37 Tyramine, 457–458, 464 Starter cultures, 166 38 Types of 39 United States, 327–331 Candida, 160–164, 166 40 USDA—United States Department of Debaryomyces, 129, 160–166, 188–189 41 Agriculture, 327–328, 330–331 Hansenula, 160–161, 163 42 USDA—Food Safety Inspection Service Pichia, 160–161, 163–164 43 (FSIS), 328, 331, 377 Rhodotorula, 160–161, 163 44 Yarrowia, 160, 164–165 45 Validation, 514, 519, 532 Yersiniosis, 478 46 Vegetables, 71 47 Venison (deer meat), 373 Zn porphyrin, 206–207, 213 48 49 50 51 52 S53 N54