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Yeast Diversity in Blue-Mould Ripened Cheeses

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Yeast Diversity in Blue-Mould Ripened Cheeses Copyright Statement I hereby grant to the University of the Free State and its agents the non-exclusive license to archive and make accessible my thesis, in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis. I also retain the right to use, in future works, all or part of this thesis. In addition, I hereby certifiy that, if appropriate, I have obtained written permission from the owner(s) of third party copyrighted matter to be included in my thesis. Use or inclusion of any portion of this thesis in a work intended for commercial use will require clearance by the copyright owner. Copyright by Paul Human de Jager ALL RIGHTS RESERVED Yeast diversity in blue mould ripened cheeses. Paul Human de Jager Yeast diversity in blue mould ripened cheeses. by Paul Human de Jager Submitted in fulfilment of the requirements for the degree of MAGISTER SCIENTIAE in the Faculty of Natural and Agricultural Sciences, Department of Microbiology, Biochemistry and Food Science, University of the Free State Bloemfontein January 2003 Supervisor: Prof. B.C. Viljoen Man wonders over the restless sea, the flowing water, the sight of the sky, and forgets that of all wonders man himself is the most wonderful; St. Augustine, (quoted from Hoebel, 1949, 1.). ACKNOWLEDGEMENTS I would like to thank the following persons and institutions: • Prof. Bennie Viljoen for his guidance and support during this study. • The National Research Foundation for their financial support. • Mr Mike La Grange and Simonsberg cheese for generously donating cheese samples throughout this study. • My friends for their support and encouragement. • My family for their constant support both financially and emotionally. • My father who was a constant inspiration and who set the ultimate example. • My mother for her emotional support. LIST OF PUBLICATIONS Chapter 3: de Jager, P.H., Viljoen, B.C. (2003) Development of yeast populations during the processing and ripening of blue veined cheese. Food Research International. (submitted for review). LIST OF TABLES Chapter 2: Table 1 - Mean populations of yeasts and moulds 70 recovered from blue cheese on ten media. Table 2 - Mean populations of yeasts recovered on 71 three selective media. Chapter 3: Table 1 - Enumeration of environmental dairy associated 93 samples of yeasts and bacteria. Table 2 - Enumeration of lactic acid bacteria and 94 yeasts during processing of blue cheese. Table 3 - Yeasts associated with blue cheese 95 manufacture according to source. Table 4 – The distribution of yeast populations in interior and 96 exterior of Danish-Blue style cheese and Gorgonzola style blue cheese. Chapter 4: Table 1 - Interactions between yeasts isolated and 121 Penicillium roqueforti. Table 2 - Interactions between yeasts and micrococci 122 isolated. Table 3 - Interactions between yeasts and coryneform 123 bacteria. LIST OF FIGURES Chapter 1: Figure 1. - Steps in manufacture of blue veined cheese 53 varieties. Chapter 3: Figure 1. - Survival of yeasts in the interior and exterior 97 of Danish Blue type blue cheese during ripening. Figure 2. - Survival of yeasts on the interior and exterior of 97 Gorgonzola type blue cheese during ripening. Chapter 4: Figure 1. – Proliferation of yeasts during blue veined 124 cheese ripening. Figure 2. - Proliferation of micrococci during blue veined 124 cheese ripening. Figure 3. - Proliferation of coryneform bacteria during 125 blue veined cheese ripening. Figure 4. - Change in pH during blue veined cheese 125 ripening. CONTENTS ACKNOWLEDGEMENTS LIST OF TABLES LIST OF FIGURES CHAPTER 1 - LITERATURE REVIEW 1.1 Introduction 2 1.2 Manufacture of blue cheese varieties 5 1.2.1 Preperation of milk 5 1.2.2 Acid development 5 1.2.3 Addition of coagulant and additives 6 1.2.4 Cutting the curd 7 1.2.5 Stirring of curd 7 1.2.6 Draining of whey 7 1.2.7 Mould inoculation 7 1.2.8 Moulding and pressing 8 1.2.9 Salting 8 1.2.10 Piercing 8 1.2.11 First ripening 9 1.2.12 Second ripening 9 1.3 The occurrence of microorganisms during processing and 10 ripening of blue veined cheese varieties 1.3.1 Primary microflora present in blue veined cheese varieties 10 1.3.1.1 Lactic acid starter bacteria 10 1.3.1.2 Moulds 12 1.3.2 Secondary microflora present in blue veined cheese varieties 14 1.3.2.1 Yeasts associated with blue veined cheese varieties 14 1.3.2.1.1 Diversity of the yeasts present in cheese 15 1.3.2.1.2 Yeasts contribution to cheese ripening 20 1.3.3.2 Non-starter bacteria 21 1.3.2.2.1 Coryneform bacteria 21 1.3.2.2.2 Micrococci 23 1.4 Sources of contamination of yeasts during the ripening of blue 24 veined cheese varieties 1.5 Physical factors influencing microbial growth 26 1.5.1 Temperature 26 1.5.2 Water-activity 26 1.5.3 Oxygen 27 1.6 Chemical factors influencing microbial growth 29 1.6.1 Nutrients 29 1.6.2 pH levels 30 1.7 Processing factors governing the growth of yeasts during ripening 31 1.8 Interactions between yeasts and other microorganisms 34 1.8.1 Interaction between yeasts and Penicillium roquerforti 35 1.8.2 Interactions between yeasts and secondary bacteria 38 1.8.2.1 Interaction between yeasts and coryneform bacteria 38 1.8.2.2 Interaction between yeasts and micrococci 39 1.9 Yeasts as starter cultures in the manufacture of blue veined 40 cheese varieties 1.10 References 45 CHAPTER 2 - STATISTICAL COMPARISON OF TEN MEDIA FOR THE ENUMERATION AND ISOLATION OF YEASTS IN BLUE VEINED CHEESE VARIETIES. Abstract 55 2.1 Introduction 56 2.2 Materials and Methods 59 2.2.1 Samples 59 2.2.2 Media 59 2.2.3 Examination of cheese sample 60 2.2.4 Comparison of media using yeast strains 60 2.2.5 Statistical analysis of results 61 2.3 Results and Discussion 62 2.3.1 Mould counts 62 2.3.2 Yeast counts 63 2.4 Conclusion 66 2.5 References 67 CHAPTER 3 - DEVELOPMENT OF YEAST AND LACTIC ACID BACTERIA POPULATIONS DURING THE PROCESSING AND RIPENING OF BLUE VEINED CHEESE. Abstract 73 3.1 Introduction 75 3.2 Materials and Methods 78 3.2.1 Blue veined cheese manufacture 78 3.2.2 Sampling methods and selection of isolates 78 3.2.3 Sampling during ripening 79 3.2.4 Sample analysis 79 3.2.5 Yeast identification 80 3.3 Results and Discussion. 80 3.3.1 Yeast development during processing 80 3.3.2 Yeast development during the ripening period 83 3.3.2.1 Danish Blue-style 83 3.3.2.2 Gorgonzola-style 86 3.4 References. 89 CHAPTER 4 - GROWTH, SURVIVAL AND INTERACTIONS BETWEEN YEASTS, MOULDS AND BACTERIA DURING THE RIPENING OF BLUE VEINED CHEESE. Abstract 99 4.1 Introduction 100 4.2 Materials and Methods 104 4.2.1 Samples 104 4.2.2 Sampling methods 104 4.2.3 Sample analysis 105 4.2.4 Identification 105 4.2.5 Chemical analysis 106 4.2.6 Microbial interactions 106 4.3 Results and Discussion 108 4.3.1 Growth of yeasts 108 4.3.2 Growth of secondary bacteria 109 4.3.3 Microbial interactions 110 4.3.3.1 Yeast-mould interactions 110 4.3.3.2 Yeast-bacterial interactions 111 4.4 Conclusion 114 4.5 References 115 CHAPTER 5 - GENERAL DISCUSSION AND CONCLUSIONS 126 CHAPTER 6 – SUMMARY 133 HOOFSTUK 7 – OPSOMMING 136 CHAPTER 1 Literature review 1.1 INTRODUCTION The occurrence of yeasts in dairy products, like cheese, is not unexpected as products like these have a high acidity, low moisture content and high salt concentration, all of which contribute to a competitive advantage and proliferation of the species (Fleet, 1990). The role of yeasts in cheese is not well known nor understood and consequently not recognised as a major component of the microflora of cheese nor its contribution to the ripening and flavour development of cheeses (Fleet and Mian, 1987). The presence of yeasts in dairy products is important, because they can either cause spoilage, or contribute to biochemical changes that are desirable, and to the extreme, have an effect on public health (Fleet and Mian, 1987). The desirability of the presence of yeasts in dairy products like cheese, depends mainly on the type of cheese. The presence of yeasts may be extremely desirable in blue veined cheese or Camembert for example, where the growth of the yeast contributes to the ripening and flavour formation of the cheese, while (Jakobsen and Narvhus, 1996) their presence might however not be as desirable in Cheddar and Swiss cheeses. Excessive growth of yeasts during ripening in these types of cheese is a potential source of spoilage (Beresford et al., 2001). Blue veined cheese is manufactured in many countries around the world, each having its own specific procedure and ingredients, which add to the diversity of the cheeses. Further adding to the diversity is the fact that milk from different animals are used to produce the cheese resulting in variation in the end product (Gripon, 1987). A very broad definition for the blue veined cheeses would be, a cheese characterised in appearance and flavour by the growth and development of the fungal-species, Penicillium roqueforti, either naturally or through inoculation, that has a 4-5 % salt content, possesses a spicy, piquant flavour and a moist texture showing slight stickiness with a tendency to crumble (Kosikowski, 1970). According to the International Dairy Federation (IDF) blue veined cheese varieties are classified under two groups of cheeses, namely semi-hard and semi-soft and soft cheese.
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