APPLICATION OF MUSTARD AND ITS DERIVATIVBS FOR CONTROL OF ESCHERICHIA COLI Ot57zH7IN DRY-CIIRED FERMBNTED HAM AND SAUSAGE By Gary Helmut Graumann A Thesis Submitted to the Faculty of Graduate Studies In Partial Fulfillment of the Requirements For the Degree of MASTER OF SCIENCE Department of Food Science Faculty of Agricultural and Food Sciences The University of Manitoba Winnipeg, Manitoba OCopyright Gary Helmut Graumann 2008 THE TINIVERSITY OF MANITOBA FACULTY OF G.R A.DUATE STUDIES COPYRIGHT PERMISSION Application of Mustard and its Derivatives for Control of Escherichia Coli OtSl:i,lll in Dry-Cured Fermented Ham and Sausage BY Gury Helmut Graumann A Thesis/Practicum submitted to the Facutty of Graduate Studies of The University of Manitoba in partial fulfillment of the requirement of the degree MASTER OF SCIENCE Gary Helmut Graumann @ 2008 Permission has been granted to the University of Manitoba Libraries to lend a copy of this thesis/practicum, to Library and Archives Canada (LAC) to lend a copy of this thesis/practicum, and to LAC's agent (UMI/ProQuest) to microfilm, sell copies and to publish an abstract of this thesis/practicum. This reproduction or copy of this thesis has been made available by authority of the copyright owner solely for the purpose of private study and research, and may only be reproduced and copied as permitted by copyright laws or with express written authorization from the copyright owner. I hereby declare that I am the sole author of this thesis' I authorize the University of Manitoba to lend this thesis to other institutions or individuals for the purpose of scholarly research- Gary Helmut Graumann I further authorize the University of Manitoba to reproduce this thesis by photocopying or by other means, in total or in part, at the request of other institutions or individuals for the purpose of scholarly research. Gary Helmut Graumann 111 There is no coming to consciousness without any pain' - CarI Jung - 1V Acknowledgments I would like to begin by thanking my mother for her numerous sacrifices, rock-steady support and unconditional love she provided for me over all of my years' i thank Raik Meissner (Piller's Sausages and Delicatessens) for his guidance and timely replies to my numerous questions regarding both the dry-curing process and the artisanal aspects of Westphalian ham production. I would also like to thank the Department of Food Science students and staff both past and present who helped and taught me in different ways' I especially appreciate my lab members Boon-Fei, Peter and Janice for more than your assistance but for your friendships which made school a place that I always looked forward to. I am also grateful for the funding of this project that was provided by the Natural Sciences and Engineering Research Council of Canada (NSERC) through a Strategic Grant and by Piller's Sausages and Delicatessens Ltd., Waterloo, ON, Canada. But most of all I would like to thank Dr. Holley. I truly respect how hard that you work for your students and this department. I do not think I could have found a better advisor for my graduate studies. You provided me with an opportunity to develop and hone numerous skills with an excellent balance of academic structure and freedom. I am truly a better person because of my time here. And to Susan...you were with me from the beginning and through it all. You stood, listened, encouraged, cared and loved me more than you can imagine' '. Organization of the thesis The thesis is organized to include three manuscripts in various stages of publication, in press or under peer review (Chapters 4-6) which are indicated below. These chapters were stand atdized for presentation in the thesis format' Chapter 1 gives an overall introduction to the thesis; Chapter 2 is a comprehensive Iiterature review; Chapter 3 includes an extensive survey of the Winnipeg market place for Listeria contamination on various dry-cured ham products; Chapter 7 is a general discussion of all thesis research and Chapter 8 represents the general conclusions of this thesis work followed by bibliographic references. Chapter 4 was published with authorship by Graumann, G'H. and Holley, R.A. in 2007 as "survival of Escherichia coli Ol57:H7 in needle-tenderized dry cured Westphalian ham" in the International Journal of Food Microbiology 118:L13-I79. Chapter 5 is submitted for peer review for the purposes of publication to the Joumal of the Science of Food and Agriculture. Authorship by Graumann, G.H' and Holley, R'A' entitled "survival of E. coli OL57:Hl during manufacture of dry-cured Westphalian ham surface-treated with allyl isothiocyanate or hot mustard powder." Chapter 6 is currently ín press with authorship by Graumann, G'H. and Holley, R'A' in 2008 as "Inhibition of Escherichiq coli OI51:Hi in ripening dry fermented sausage by ground yellow mustard" in the Journal of Food Protection 7I(4). VI Table of Contents Page Acknowledgements. iv Organization of the thesis. V List of Tables. X List of Figures. xii Abstract x11t Chapter L L.0 Introduction. Chapter 2 2.0 Literature Review 5 2,1 Dry-cured fermented sausage and ham: Product irientity. '. 5 2.2Ftole of microorganisms in dry-cured fermented meat products' 5 2.2.1 Starter cultures used in dry sausage manufacture" '. .' 5 2.2.2 Indigenous microflora of dry-cured ham. 7 2.3 Pathogen survival in dry-cured fermented sausage and ham. 2.3.1 Staphylococcus aureus...... 8 2.3.2 Salmonella 9 2.3.3 Listeria monocytogenes. 10 2.3.4 Toxigenic moulds. L2 2.3.5 Clostridium botulinum. Í2 2.3.6 Trichinella 13 2.4 Foodborne disease significanc e of Escherichia coli Ol51:H7 . L3 2.4.1 E. coli Ol51:H7 reservoirs. '- 14 2.4.2 E. coli OI57:Hi survival in fermented dry-cured sausage" 15 2.4.3 E. coti 0157:H7 survival in fermented dry-cured ham'. .. 16 2.5Dry sausage antimicrobial treatments..... 17 2.6 Antimtcrobial activity of mustard and its derivatives I7 2.6.1 Mustard condiments '. 17 2.6.2 Mustard essential oils... 18 2.6.3 Yellow mustard powder. 19 vll Chapter 3 3.0Isolation of Listerín spp. from ready-to-eat, dry-cured ham products: A Winnipeg market survey. 24 3.1 Abstract...... 24 3.2 Introduction. 25 3.3 Materials and Methods' 26 3.3.1 Enrichment and analysis of Listeria spp. on dry-cured ham samples. 26 3.3.2 Westphalian ham physico-chemical analysis' 27 3.4 Results and Discussion.. 28 3.5 Conclusions. 30 Chapter 4 4.0 Survival of Escherichin colí Ol57:H7 in needle-tenderized dry cured Westphalian ham. JJ 4.1 Abstract.. " '. JJ 4.2 Introduction. 34 36 4.3 Materials and methods. ' ' 4.3.I Westphalian ham manufacture. 36 4.3.2 Inocuiation of hams. 38 38 4.3.3 SamPling Procedures 4.3.4 Enumeration of bacteria.."'. 39 4.3.5 pH and water activitY' 4I 4.4 Resuits and discussion'.. 4l 4.4.1 westphalian ham physico-chemical changes during ripening. 4l 42 4.4.2 Effects of ham Processing' 4.4.3 Development of naturally occurring internal microflora during drying. 44 4.4.4 Changes in staphylococci numbers. '.. 45 4.5 Conclusions. 46 Chapter 5 5.0 Survival of E. coli Ol57zHl7 during manufacture of dry-cured Westphalian ham surface-treated with allyl isothiocyanate or hot mustard powder. ....'.: 55 5.1 Abstract...... 55 56 5.2 Introduction . 58 5.3 Materials and methods. ' - 5.3.1 E. coli Ol57:H7 preparation '. '. '. 58 5.3.2 Westphalian ham manufacture. '.. ' ' 59 viii 5.3.3 AIT microencapsulation and mustard powder' 5.3.4 Wesrphalian ham physico-chemical and microbial anaiysis. 60 5.3.5 Statistical analYsis. 62 62 5.4 Results and discussion.. ' 5.4.1 Westphalian ham physico-chemical changes' 62 5.4.2 Westphalian ham microbiological changes' " ' ' 63 5.5 Conclusions' 67 Chapter 6 6.0lnhibition of Escherichin coli O157:H7 in ripening dry fermented 16 sausage by ground Yellow mustard. 76 6.I Abstract. .... 6.2 Introduction. 77 6.3 Materials and methods. 80 6.3.I Bacterial strain preparation 80 6.3.2 Dry fermented sausage manufacture" " " 81 6.3.3 Ground mustard seed'. 82 6.3'4MicrobialandphysiochemicalanalysisofdrySausage.'..'. 82 6.3.5 Texture evaiuation of sausage' 83 6.3.6 Statistical analysis'.:."..' 84 6.4 Results and discussion.... 84 6.4.I Dry salami physicochemical changes' 84 6.4.2 Reductions of E. coli ol57:H7 in dry fermented sausage..' 85 6.4.3 Effect of mustard powder on starter cultures in sausage formulations...... 6.4.4 Effect of mustard powder on dry fermented sausage texture. 90 6.5 Conclusions. 91 Chapter 7 99 7.0 General discussion..... ' 7.1 Physico-chemical properties of dry-cured sausage and ham. 99 T.ZDevelopment of the indigenous microflora Westphalian ham, 100 i .3 The antimicrobial activity of mustard and its derivatives on fermented meat microorganisms' r02 1.3.1 Effects of mustard compounds on the dry-cured ham microflora. r02 7.3.2 Effects of mustard compounds on dry fermented sausage starter cultures. 103 7.4 Antimicrobial effects of mustard compounds towards E. coli Ol57:H7 in fermented meat products' 104 IX Chapter 8 8.0 General conclusions. 108 r10 References List of Tables Page Table geographical locations ' ' ' 22 Table 2. I Isolation of E. coli Ol51 :H] from swine animals in different ' of dry- Table2.2 Incidence of Escherichia coLi Ol51:H1 illness associated with consumption 23 cu¡ed fermented sausage.. 31 Table 3.1 Characterization of dry-cured ham samples subjected To Listería enrichment" needle-tenderized Table 4.1 changes in pH plus surface and interior water activities of 48 westfhalian úams during dry curing (n = 4) '.