Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1972 The oler of propionibacteria in the split defect of Swiss cheese David Howard Hettinga Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Agriculture Commons, and the Food Science Commons Recommended Citation Hettinga, David Howard, "The or le of propionibacteria in the split defect of Swiss cheese " (1972). Retrospective Theses and Dissertations. 5210. https://lib.dr.iastate.edu/rtd/5210 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. 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University Microfilms 300 North Zeeb Road Ann Arbor, Michigan 48106 A Xerox Education Company 72-19,984 HETTINGA, David Howard, 1940- THE ROLE OF PROPIONIBACTERIA IN THE SPLIT DEFECT OF SWISS CHEESE. Iowa State University, Ph.D., 1972 Food Technology University Microfilms, A XEROX Company, Ann Arbor. Michigan THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED The role of propionibacteria in the split defect of Swiss cheese by David Howard Hettinga A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of The Requirements for the Degree of DOCTOR OF PHILOSOPHY Major Subject: Food Technology (Dairy Microbiology) Approved; Signature was redacted for privacy. In Charge of Major Work Signature was redacted for privacy. For the Major Department Signature was redacted for privacy. For the Graduate College Iowa State University Ames, Iowa 1972 PLEASE NOTE: Some pages may have indistinct print. Filmed as received. University Microfilms, A Xerox Education Company ii TABLE OF CONTENTS Page DEDICATION ix INTRODUCTION 1 LITERATURE REVIEW 7 Splits and Their Occurrence in Swiss or Emmental 7 Cheese Carbon dioxide production and the formation 7 of eyes Splits in Swiss cheese 11 Effect of manufacturing procedures 14 Effect of cheese constituents 17 Effect of physical and chemical factors in 20 cheese Effect of packaging films and wrappers 22 Growth and Carbon Dioxide Production of Propioni- 24 bacteria Substrate and product inhibition 25 Associative action of other microorganisms 27 Physical factors affecting growth 29 Effect of copper on growth . 31 Growth in cheese 33 Metabolism and enzyme reactions 35 EXPERIMENTAL MATERIALS AND METHODS 39 Source of Milk 39 Manufacture of Iowa-Type Swiss Cheese 39 Determination of Titratable Acidity of Milk and Whey 42 Sampling Cheese for Analysis 42 iii Proteolysis by Dye Binding in Cheese 42 Determination of Lactic Acid in Cheese 43 Estimation of Carbon Dioxide Production in Cheese . 44 Enumeration of Propionibacteria in Cheese 47 Determination of Sugar in Cheese 47 Strains of.Propionibacteria 54 Preparation of Cultures of Propionibacteria 54 Determination of Gas Production by Propionibacteria 54 Growth of Propionibacteria at Low Temperatures 57 Growth Rate of Propionibacteria 57 Preparation of Cell-Free Extracts of Propionibacteria 58 Determination of Total Protein 59 Preparation of Buffers 59 Determination of Enzyme Activity 62 Heat treatment of cell-free extracts 62 Determination of NADH oxidase activity 62 Isocitrate dehydrogenase activity 62 Glucose 6-phosphate dehydrogenase activity 65 Malate dehydrogenase activity 65 Lactate dehydrogenase activity 65 Pyruvate dehydrogenase activity 69 Spectrophotometrie activity 69 Colorimetric measurement of activity 69 Manometric measurement of activity 69 Measurement of acetyl phosphate formation 69 iv RESULTS AND DISCUSSION 71 Growth of Propionibacteria at Low Temperatures 72 Carbon Dioxide Production by Propionibacteria 72 Growth Rate of Propionibacterium shermanii 59 and 76 Propionibacterium arabinosum 129 Occurrence of Splits in Experimental Swiss Cheese 79 Growth of Propionibacteria in Cheese 79 Disappearance of Lactic Acid in Cheese 86 Proteolysis in Cheese 90 Persistence of Sugars in Cheese 92 Estimation of Carbon Dioxide Production in Cheese 94 Effect of Cheese Wrapping Films 105 Study of Cell-Free Extracts of Propionibacteria 118 SUMMARY AND CONCLUSIONS 137 LITERATURE CITED 145 ACKNOWLEDGMENT 158 APPENDIX 159 V LIST OF TABLES Page 1 Strains of Propionibacterium spp. used in the 55 investigation 2 Concentration aind source of chemicals used in the 63 enzymic studies 3 Estimation of growth of propionibacteria in sodium 73 lactate broth at low temperatures 4 Growth emd carbon dioxide production by strains of 75 Propionibacterium spp. at various temperatures of incubation 5 The occurrence of splits and checks in Swiss cheese 80 made with various strains of Propionibacterium spp. 6 Comparative determination of growth of strains of 84 Propionibacterium spp. in Swiss cheese at various steps in the msuiufacturing and curing process 7 Comparative determination of lactic acid in Swiss 89 cheese at various steps in the manufacturing and curing process 8 Comparison of proteolysis in Swiss cheese manufac- 91 tured with ]P. shermainii 59 aoid P. arabinosum 129 at various steps in the curing process 9 Comparison of four wrapping methods on two lots of 106 6-month old experimental Swiss cheese with each lot manufactured with a different strain of propioni­ bacteria 10 Comparison of two different wrapping films on five 108 lots of 3-month old experimental Swiss cheese with each lot manufactured with one of five strains of propionibacteria 11 Comparison of four different wrapping films on four 112 lots of 3-month old commercial Swiss cheese with each lot memufactured with one of four strains of propionibac ter ia 12 Preparation techniques and protein content of cell- 120 free extracts of strains of Propionibacterium spp. vi 13. Malate dehydrogenase activity and condition of sample 122 of Propionibacterium cell-free extracts at 28 C 14. Lactate dehydrogenase activity of cell-free extracts 127 of Propionibacterium spp. at low temperatures 15. Moisture and milkfat content of experimental Swiss 160 cheeses 16. Carbon dioxide production during curing by 50 kg 161 lots of experimental Swiss cheese mauiufactured with various strains of Propionibacterium spp. 17. Type and properties of packaging films used in the 167 investigation 18. Example of make record for Iowa Swiss-type cheese 168 vii LIST OF FIGURES Page 1. Photographs of typical checks and splits in Swiss 5 cheese 2. Relationship between absorbance and lactic acid 46 concentration 3. Flowsheet of the system employed for the measure- 49 ment of carbon dioxide produced in Swiss cheese 4. Relationship between absorbance and glucose con- 51 centration 5. Relationship between absorbance and galactose 53 concentrat ion 6. Relationship between absorbaunce and protein con- 61 centration 7. Relationship between absorbemce and reduced nico- 68 tinamide adenine dinucleotide concentration 8. Growth curves of Propionibacterium shermanii 59 78 and Propionibac te r ium arabinosum 129 in sodium lactate broth at 21 C 9. Growth of Propionibacterium shermanii 59 and 83 Propionibacterium arabinosum 129 during curing in experimental Swiss cheese 10. Disappearance of lactic acid during curing in ex- 88 perimental Swiss cheese manufactured with Propioni­ bacterium shermanii 59 and cheese manufactured with Propionibacterium arabinosum 129 11. Carbon dioxide production during curing by 50 kg of 97 experimental Swiss cheese manufactured with Pro­ pionibacterium shermanii 59 and by 50 kg of cheese mainufactured with Pr opionibac te r ium arabinosum 129 12. Average carbon dioxide production