Microbiology and Biochemistry of Cheese and Fermented Milk

Microbiology and Biochemistry of Cheese and Fermented Milk JOIN US ON THE INTERNET VIA WWW, GOPHER, FTP OR EMAil: WWW: http://www.thomson.com GOPHER: gopher.thomson.com fri'\® A service of I(!)P FTP: ftp.thomson.com EMAIL: [email protected] Microbiology and Biochemistry of Cheese and Fermented Milk Second edition

Edited by

B.A. LAW Research Consultant Burghfield Common Berkshire, UK

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ISBN-13: 978-1-4612-8427-7 e-IS81\-13: 978-1-4613-1121-8 DOl: 10.1007/978-1-4613-1121-8 Apart from any fair dealing for the purposes of research or private study, or criticism or review, as permitted under the UK Copyright Designs and Patents Act, 1988, this publication may not be reproduced, stored, or transmitted, in any form or by any means, without the prior permission in writing of the publishers, or in the case of repro graphic reproduction only in accordance with the terms of the licences issued by the Copyright Licensing Agency in the UK, or in accordance with the terms of licences issued by the appropriate Reproduction Rights Organization outside the UK. Enquiries concerning reproduction outside the terms stated here should be sent to the publishers at the London address printed on this page. The publisher makes no representation, express or implied, with regard to the accuracy of the information contained in this book and cannot accept any legal responsibility or liability for any errors or omissions that may be made. A catalogue record for this book is available from the British Library Library of Congress Catalog Card Number: 96-86400

@) Printed on acid-free text paper, manufactured in accordance with ANSI/NISO Z39.48-1992 (Permanence of Paper). Contents

List of contributors xi Preface xiii Preface to the first edition xvii

1 Rennets: their role in milk coagulation and cheese ripening 1 P.F. FOX and P.L.H. McSWEENEY

1.1 Introduction 1 1.2 The milk protein system 2 1.3 Primary phase of rennet action 4 1.4 Rennet and rennet substitutes 7 1.5 Immobilized rennets 10 1.6 Factors affecting the hydrolysis of K-casein 11 1.6.1 pH 11 1.6.2 Ionic strength 12 1.6.3 Temperature 12 1.6.4 Degree of glycosylation 12 1.6.5 Heat treatment of milk 12 1.7 Secondary (non-enzymatic) phase of coagulation and gel assembly 13 1.8 Curd tension and gel syneresis 14 1.9 Industrial manipulation of rennet coagulation time 14 1.10 Proteolysis during ripening 15 1.11 Proteolytic agents in cheese 16 1.12 Significance of secondary coagulant proteolysis 16 1.13 Specificity of chymosin and rennet substitutes in cheese 18 1.14 Specificity of indigenous milk proteinases 21 1.14.1 Plasmin 21 1.14.2 Cathepsin D 23 1.15 Proteolytic enzymes from microorganisms in cheese 23 1.16 Characterization of proteolysis in cheese 25 1.17 Influence of chymosin on rate of cheese ripening 39 1.18 Possible future developments 39 1.19 References 40

2 Classification and identification of bacteria important in the manufacture of cheese 50 B.A. LAW and E.B. HANSEN 2.1 Introduction 50 2.2 Molecular phylogeny as a basis for bacterial taxonomy 50 2.3 Current classification of cheese bacteria 51 2.4 Taxonomy in relation to safety and technology of dairy bacteria 54 2.5 Conclusions 55 2.6 References 56 VI CONTENTS

3 Microbiology and technology of fermented milks 57 A.Y. TAMIME and V.M.E. MARSHALL

3.1 Introduction 57 3.1.1 The diversity of fermented milks 58 3.1.2 Annual production figures 59 3.2 Microbiology of fermented milks 61 3.2.1 Starter organisms and types 61 3.2.2 Technology of manufacture 63 3.3 Fermented milks with lactic acid 76 3.3.1 Use of mesophilic strains 77 3.3.2 Use of thermophilic strains 85 3.3.3 Use of pro biotic strains 95 3.4 Fermented milks with alcohol and lactic acid 109 3.4.1 Kefir 109 3.4.2 Koumiss, kumys or coomys 118 3.4.3 Acidophilus-yeast milk 122 3.4.4 Acidophiline or acidophilin 122 3.5 Fermented milks with mould and lactic acid 123 3.5.1 Background 123 3.5.2 Viili 123 3.6 Concentrated fermented milks 125 3.6.1 Ymer 125 3.6.2 Skyr 126 3.6.3 Chakka and shrikhand 128 3.6.4 Strained yoghurt or labneh 128 3.7 Conclusions 132 3.8 References 133

4 Physiology and biochemistry of fermented milks 153 V.M.E. MARSHALL and A.Y. TAMIME

4.1 Introduction 153 4.1.1 Milk as a medium for microbial growth 155 4.1.2 Associative growth 155 4.1.3 Non-traditional organisms 157 4.2 Carbohydrate metabolism 158 4.2.1 Homolactic fermentation 158 4.2.2 Heterolactic fermentation 160 4.3 Nitrogen metabolism 163 4.4 Pathways leading to flavour compounds 164 4.4.1 Metabolic routes to flavour production 165 4.4.2 Yeast and mould fermentation 168 4.5 Polysaccharide secretion 169 4.5.1 Exopolymers from mesophiles and thermophiles 169 4.5.2 Microbial physiology and polysaccharide production 170 4.6 Therapeutic properties of fermented milks 171 4.6.1 Antagonistic compounds 171 4.6.2 Bacteriocins 172 4.6.3 Lactose malabsorption 181 4.6.4 Bile salt resistance and cholesterol 'assimilation' 183 4.6.5 Stimulation of the immune system 184 4.7 Conclusions 185 4.8 References 186 CONTENTS VB

5 Flavour and texture in soft cheese 193 J.-c. GRIPON

5.1 Introduction 193 5.2 The flora of surface mould-ripened cheeses 193 5.3 Biochemical reactions involved in soft cheese ripening 194 5.3.1 Glycolysis 194 5.3.2 Proteolysis 195 5.3.3 Lipolysis 198 5.4 Aroma development 199 5.5 Textural development 201 5.6 Controlling the defects of surface-moulded cheeses 202 5.7 References 203

6 Flavour and texture in low-fat cheese 207 Y. ARDO

6.1 Introduction 207 6.2 Role of fat in cheese 208 6.3 Consequences of measures taken to slow down syneresis 210 6.4 Casein breakdown in reduced-fat cheese 211 6.5 Use of enzymes and bacteria to improve quality of reduced-fat cheese 212 6.6 References 216

7 Control and enhancement of flavour in cheese 219 M. EL SODA

7.1 Introduction 219 7.2 Strategies used for the enhancement of cheese flavour 221 7.2.1 Elevated ripening temperature 221 7.2.2 Slurry systems 222 7.2.3 Addition of enzymes 223 7.2.4 Entrapped enzymes 226 7.2.5 Modified bacterial cells 230 7.2.6 Genetically modified starter cells 236 7.2.7 Other cheese flavour enhancement methods 237 7.3 Enzymes release from cheese microorganisms 238 7.4 Flavour enhancement in non-conventional cheeses 240 7.4.1 Cheese made from recombined milk 240 7.4.2 Cheese made by ultrafiltration 240 7.4.3 Cheese made from ovine, caprine or buffalo's milk 240 7.4.4 Low-fat cheese 242 7.5 Economic aspects 244 7.6 Future perspectives 244 7.7 References 246

8 The chemical and biochemical basis of cheese and milk aroma 253 G.URBACH

8.1 Introduction 253 8.2 Isolation and identification of volatiles from dairy products 253 8.2.1 Tower extraction 254 viii CONTENTS

8.2.2 Steam stripping 254 8.2.3 Supercritical fluid extraction 254 8.2.4 Molecular distillation and concentration 255 8.2.5 Headspace analysis 258 8.3 Quantitative estimations 262 8.3.1 Estimation of acids 262 8.3.2 Estimation of IX-dicarbonyls 262 8.3.3 Estimation of glutathione (GSH) 262 8.3.4 Determination of redox potential 263 8.4 Aroma compounds in cheese and fermented milks 264 8.4.1 General considerations 264 8.4.2 Ketones and alcohols 266 8.4.3 Acids 267 8.4.4 Phenolic compounds 271 8.4.5 Sulphur compounds 272 8.4.6 Terpenes 279 8.4.7 IX-Dicarbonyls and related compounds 280 8.4.8 Esters 280 8.4.9 Amines, ami des and other nitrogen-containing compounds 281 8.4.10 Lactones 282 8.5 Some less-common cheese varieties 282 8.6 Cheese with high linoleic acid content 282 8.7 Enzyme-modified cheese 283 8.8 Off-flavours 283 8.9 Conclusions 285 8.10 References and bibliography 287

9 Proteolytic systems of dairy lactic acid bacteria 299 F. MULHOLLAND

9.1 Introduction 299 9.2 Milk as a growth medium 299 9.3 Proteolysis 300 9.3.1 Localization of the proteolytic system 300 9.3.2 Extracellular proteolytic enzymes 301 9.3.3 Amino acid and peptide transport systems 303 9.3.4 Intracellular proteolytic enzyme: peptidases 305 9.4 Conclusions and topics for further research 313 9.5 References 314

10 Molecular genetics of dairy lactic acid bacteria 319 M.J. GASSON

10.1 Introduction 319 10.2 Genetics of industrially relevant traits 319 10.2.1 Lactose fermention 319 10.2.2 Casein degradation 321 10.2.3 Bacteriophage resistance 325 10.2.4 Starter cell lysis 327 10.2.5 Antimicrobial peptides 328 10.3 Gene cloning techniques 330 10.3.1 Food-compatible cloning systems 332 10.3.2 Natural gene transfer systems 333 10.4 Whole genome analysis 334 CONTENTS IX

10.5 Future prospects 335 10.6 References 336

11 Sensory evaluation of dairy flavours 341 1. BAKKER

11.1 Introduction 341 11.2 Sensory mechanisms 342 11.3 Panels for sensory analysis 342 11.3.1 Quantitative descriptive analysis 343 11.3.2 Time-intensity measurements 344 11.3.3 Experimental design and statistical analysis 346 11.4 Flavour release and perception 348 11.4.1 Mastication 350 11.4.2 Sensors 351 11.4.3 Fat and flavour release 352 11.4.4 Protein and flavour release 353 11.4.5 Flavour release in the mouth 354 11.5 Cheese 355 11.5.1 Low-fat cheese 357 11.6 Yoghurt 358 11. 7 Ice-cream 358 11.8 Conclusions 359 11.9 References 359

Index 363 Contributors

Y. Ardo The Royal Veterinary and Agricultural University, Department of Dairy and Food Science, Dairy Science, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark J. Bakker Institute of Food Research, Reading Laboratory, Earley Gate, Reading, RG6 6BZ, UK P.F. Fox Department of Food Chemistry, University College, Cork, Ireland M.J. Gasson IFR, Norwich Laboratory, Norwich Research Park, Colney, Norwich, NR4 7UA, UK J.-c. Gripon Unite de Recherche de Biochimie et Structure des Proteines, INRA, 78350 Jouy-en-Josas, France E.B. Hansen Chr Hansen A/S, DD Division, R&D Management, Hoersholm, Denmark B.A. Law (formerly of) Institute of Food Research, Reading Laboratory, Earley Gate, Whiteknights Road, Read• ing, RG6 6BZ, UK P.L.H. McSweeney Department of Food Chemistry, University College, Cork, Ireland V.M.E. Marshall The University of Huddersfield, School of Applied Sciences, Queensgate, Huddersfield, HD1 3DH, UK F. Mulholland Institute of Food Research, Food Macromolecular Sciences Department, Reading Laboratory, Earley Gate, Whiteknights Road, Reading, RG6 2EF, UK M. El Soda Laboratory of Microbial Biochemistry, Department of Dairy Technology, Faculty of Agriculture, Alexan• dria University, Alexandria, Egypt A.Y. Tamine SAC-Auchincruive, Food Science and Technology Department, Ayr, KA6 5HW, UK G. Urbach CSIRO Division of Food Science and Technology, Melbourne Laboratory, Highett, Australia 3190 Preface

The first edition of Advances in the Microbiology and Biochemistry of Cheese and Fermented Milk was aimed at the gap in the literature between the many excellent technical texts on the one hand, and the widely scattered scientific literature on the other. We tried to present the state of the art in pre• competitive research in a predigested, yet scientifically coherent form, and relate it to the marketable properties of fermented dairy products. In this way, researchers could use the book to mentally step back from their specializations and see how far they had progressed as a community; at the same time we hoped that R&D-based companies could use it to assess the utility (or lack of it) of the research output in setting out their research acquisition strategy for product improvement and innovation. In a sense, the first edition could claim to have initiated Technology Foresight in its limited field before Government caught the idea, and it certainly gave the science base an opportunity to display its talents and resources as a potential source of wealth creation, well before this became an 'official' function of publicly funded science and technology. Thus, the first edition was intended as a progressive move within the growing science and technology literature, and judged by its market success, it seems to have served precisely that purpose. This second edition strives to continue the progression, both by collecting and discussing new research findings under the first edition headings, and by introducing completely new approaches to the subject matter. For example, Chapter 1 on the subject of coagulants refects the realization over the past 12 years that these enzymes play a pivotal, rather than peripheral role in cheese maturation; the remarkable new developments in coagulant production technology, arising from advances in molecular biology, have to be viewed not only as changes in the vat stage of cheese technology, but also in the maturation stage. The initial breakdown of caseins to peptide substrates for enzymes of the starter culture and adventitious cheese flora to act on is a function of the coagulant after it has done its work in the initial formation of the curd gel. Thus, the coagulant initiates the entire cascade of protein breakdown to taste and aroma compounds, described in chemical detail in Chapter 8. Molecular biology has also been harnessed increasingly by starter culture researchers in academe and industry to investigate and manipulate the industrially important functions of these bacteria. These developments are dealt with in several chapters, including revised coverage of the molecular xiv PREFACE genetics of lactic acid bacteria (Chapter 10) and a new Chapter (9) on culture enzymology, dealing with the enzymes of cheese bacteria which are most clearly implicated in texture and flavour development. The increasing range and market penetration of fermented milks is reflected in increased basic research on their microbiology, especially of those milks to which health-promoting properties are assigned. Thus the original chapter on the research base underpinning these products has been extensively revised and extended to two chapters (3 and 4) covering science-driven technological developments as well as new findings in micro• biology, physiology and biochemistry. The taxonomy of bacteria has undergone such upheavals in the last 12 years, particularly arising from the development and application of nucleic acid sequences in the design of molecular probes, that there is no single authority on cheese and fermented milk bacteria, as there was when the first edition was published. For this edition, I have chosen to defer to existing reviews and formal texts for phylogenetically based classifications of the dairy lactic acid bacteria. For non-Iactics, I have been fortunate to have enlisted the help of an industry-based molecular biologist involved directly in dairy culture development to select the important genera and to guide the reader through their inter-relatedness. Thus Chapter 2, dealing with taxon• omy, is very brief and highly focussed; it should be used as a supplement to the original chapter (Garvie, 1984), rather than as a stand-alone work. The classification of fermented milk bacteria has been covered by the specialist authors in this area, in the context of their technology, physiology and product biochemistry (Chapter 4). Although diagnostic and determinative microbiology has taken enor• mous strides since the first edition was published, this area was not specifically covered then, and will not be in this new edition. This is because the research base is generic, dealing with pathogenic and spoilage bacteria which occur in a wide range of foods, including cheese, and the reader is referred to the reviews by Grant and Kroll (1993) and Lee and Morgan (1993) to gain an appreciation of molecular probe technology applied to the detection and isolation of food pathogens. For those who need to keep abreast of the occurrence and behaviour of pathogens in cheese, the 1994 International Dairy Federation survey (Spahr and Url, 1994) is an ideal source to supplement national surveys of food poisoning outbreaks, pub• lished annually by Public Health Laboratories. Also, the Food Micromodel Database, held and managed by the Institute of Food Research, Reading, UK, is a PC-based system which predicts the growth and toxin-producing potential of a range of commonly occurring pathogens in foods, in response to selected conditions of temperature, pH, water activity, and preservative concentration (McClure et ai., 1994); this has obvious application in dairy product research to determine the spoilage and poisoning potential of specific microorganisms. Specific research data on the anti-pathogen activity PREFACE xv of cheese and fermented milk cultures appears in a number of chapters dealing with the dairy lactic acid bacteria. Finally, I should comment on the book's coverage of flavour and texture in cheese; since the first edition was published, the range of cheese varieties under detailed and intensive investigation has increased to an extent that a single chapter is no longer appropriate. I have therefore dealt with this in the second edition by including a separate chapter (5) exclusively on soft cheeses, with their unique chemistry and microbiology, and a new chapter (6) on challenges to research from the special problems of making low-fat cheeses. All other cheese varieties feature more or less in the other chapters within the discussions of, for example, coagulants, starter culture enzy• mology, flavour chemistry and accelerated ripening. The only omission, if it is such, is a detailed coverage of pasta filata cheeses used as pizza toppings; most of the literature in this area is technically based, rather than research-based, and deals with process technology. I have relied on authors of the research area chapters to note developments in pasta filata, as appropriate.

Barry A. Law March,1996

References

Garvie, E.1. (1984) Taxonomy and identification of bacteria important in cheese and fermented dairy products, in Advances in the Microbiology and Biochemistry of Cheese and Fermented Milk (eds F.I. Davies and B.A. Law), Elsever Applied Science Publishers, London, New York, pp. 35-66. Grant, K.A. and Krol1, R.G. (1993) Molecular biology techniques for the rapid detection and characterisation of food borne bacteria. Food Sci. Technol. Today, 7, 80-3. Lee, H.A. and Morgan, M.R.A. (1993) Food immunoassays: application of polyclonal, monoclonal and recombinant assays. Trends Food Sci. Techno!., 4, 129-33. McLure, PJ., Blackburn, C.W., Cole, M.B., Curtis, P.S., Jones, J.E. and Legan, J.D. (1994) Model1ing the growth, survival and death of microorganisms in foods; the UK Micromodel approach. Int. J. Food Microbiol., 23, 265-75. Spahr, U. and Uri, B. (1994) The behaviour of pathogens in cheese; summary of experimental data. Bull. Int. Dairy Fed., 298, 2-16. Preface to the first edition

The manufacture of cultured dairy products constitutes a major fermenta• tion industry, particularly in the developed countries. For example, in 1980, 3.5 million tonnes of cheese and over 1500 million litres of yoghurt and fermented milks were produced in the EEC. Milk and milk products account for about 15% of domestic food expenditure in the UK. Although liquid milk consumption is declining in many countries, the market for cheese is increasing slowly, with a tendency towards more complex varieties whose maturation is most difficult to control. Fermented milks enjoy not only increased interest in a wider variety of products but also an overall increase in popularity. It is clear, therefore, that the fermentation of milk to nutritious, stable and interesting products is an expanding activity which requires a constant input of both new technology and fundamental research. Although the industry can call upon a comprehensive technical literature as an aid to solving some of its problems, an equivalent coverage of basic research from which to draw new ideas is not at present available. Such information is widely scattered in specialized publications. We have there• fore attempted to provide one volume in which research relating to the major stages of fermented dairy product manufacture is reviewed. In this way we hope to provide some insight into possible future directions for development in the industry, based on current efforts in research labora• tories. Studies into the physico-chemical nature of cheese have been important in investigations into modified processes such as the use of concentrated milk and the increased incorporation of whey proteins. The starter cultures used in these fermentations can now be more accurately identified and their relationships determined by the use of techniques such as DNA-DNA hybridization. The physiological mechanisms by which they utilize lactose and milk proteins to produce lactic acid and other essential end products are only now being well characterized, and the genetic control of these mechanisms is a subject of increasing study. Thus modification and im• provement of starter cultures for traditional fermentations and the extension of their use to new food areas are important new possibilities. The applica• tion of genetic knowledge will also be prominent among approaches being taken to eliminate or control the attack of starter cultures by bacterio• phages. Elucidation of the biochemical pathways of flavour development in fermented milks has led to new possibilities for product development and in xviii PREFACE TO THE FIRST EDITION the case of cheese this knowledge has been supplemented by important new work which could substantially reduce the traditionally long ripening periods required to achieve a mature flavour. Early detection of key flavour volatiles could be important in prediction of final mature cheese quality and the use of non-sensory instrumental methods will permit more objective assessments to be made. It is the advances in these areas with which this book is concerned. All the contributors are actively engaged in the research they describe and the individual chapters are seen as current 'state of the art' reviews directed at those working at this level in industry, government and education.

Barry A. Law F. Lyndon Davies