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Cheese Chemistry, Physics and Microbiology Volume 1

General Aspects

This Page Intentionally Left Blank

Cheese Chemistry, Physics and Microbiology Volume 1

General Aspects

Third edition

Edited by Patrick F. Fox, Paul L.H. McSweeney, Timothy M. Cogan and Timothy P. Guinee

AMSTERDAM • BOSTON • HEIDELBERG • LONDON • NEW YORK • OXFORD PARIS • SAN DIEGO • SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO

This book is printed on acid-free paper First published 1987 by Elsevier Applied Science Second edition 1993 by Chapman & Hall Third edition 2004 Copyright © 2004, Elsevier Ltd. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the publisher. Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone: (44) 1865 843830, fax: (44) 1865 853333, e-mail: [email protected]. You may also complete your request on-line via the Elsevier homepage (http:/ /www.elsevier.com), by selecting ‘Customer Support’ and then ‘Obtaining Permissions’. Elsevier Academic Press 84 Theobald’s Road, London WC1X 8RR, UK http:/ /www.elsevier.com Elsevier Academic Press 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http:/ /www.elsevier.com British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Library of Congress Control Number: 2004105782 Volume 1 ISBN Volume 2 ISBN Set ISBN

0-1226-3652-X 0-1226-3653-8 0-1226-3651-1

Cover images: Bacteria (detail): reprinted with permission from Donald J. McMahon, Food Structure, 1993, Vol. 12, pp. 251–258, Fig. 3A. Sensory Circle: adapted with permission from Pierre Lavanchy et al., A Guide to the Sensory Evaluation of Texture of Hard and Semi-Hard Cheeses, 1994, Institut National de la Recherché Agronomique, Paris. Electropheresis gel: published with kind permission of Vivek Upadhyay, University College Cork, Republic of Ireland.

Typeset by Integra Software Services Pvt. Ltd, Pondicherry, India Printed and bound in Italy 04 05 06 07 08 9 8 7 6 5 4 3 2 1

Contents

Foreword List of Contributors Preface to the First Edition Preface to the Second Edition Preface to the Third Edition Cheese: An Overview P.F. Fox and P.L.H. McSweeney

vii ix xiii xv xvii 1

Rennets: General and Molecular Aspects M.J.C. Crabbe

19

Rennet-induced Coagulation of Milk D.S. Horne and J.M. Banks

47

The Syneresis of Rennet-coagulated Curd P. Dejmek and P. Walstra

71

Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels J.A. Lucey

105

Starter Cultures: General Aspects E. Parente and T.M. Cogan

123

Starter Cultures: Genetics M.J. Callanan and R.P. Ross

149

Starter Cultures: Bacteriophage S. McGrath, G.F. Fitzgerald and D. van Sinderen

163

Secondary and Adjunct Cultures J.-F. Chamba and F. Irlinger

191

Salt in Cheese: Physical, Chemical and Biological Aspects T.P. Guinee and P.F. Fox

207

Application of Membrane Separation Technology to Cheese Production V.V. Mistry and J.-L. Maubois

261

The Microbiology of Cheese Ripening T. Beresford and A. Williams

287

Raw Milk Cheeses E. Beuvier and S. Buchin

319

Biochemistry of Cheese Ripening: Introduction and Overview P.L.H. McSweeney

347

Metabolism of Residual Lactose and of Lactate and Citrate P.L.H. McSweeney and P.F. Fox

361

Lipolysis and Catabolism of Fatty Acids in Cheese Y.F. Collins, P.L.H. McSweeney and M.G. Wilkinson

373

vi Contents

Proteolysis in Cheese during Ripening V.K. Upadhyay, P.L.H. McSweeney, A.A.A. Magboul and P.F. Fox

391

Catabolism of Amino Acids in Cheese during Ripening Á.C. Curtin and P.L.H. McSweeney

435

Sensory Character of Cheese and its Evaluation C.M. Delahunty and M.A. Drake

455

Cheese Flavour: Instrumental Techniques J.-L. Le Quéré

489

Rheology and Texture of Cheese D.J. O’Callaghan and T.P. Guinee

511

Growth and Survival of Microbial Pathogens in Cheese C.W. Donnelly

541

Toxins in Cheese N.M. O’Brien, T.P. O’Connor, J. O’Callaghan and A.D.W. Dobson

561

Nutritional Aspects of Cheese N.M. O’Brien and T.P. O’Connor

573

Factors that Affect the Quality of Cheese P.F. Fox and T.M. Cogan

583

Index

609

Foreword

The art of cheesemaking has been augmented steadily by greater knowledge on the science of cheesemaking. This evolution has resulted from basic and applied research and from the increased need to understand and control the characteristics of milk, the microorganisms used in the manufacture and maturation of cheese, the manufacturing technologies, and the physical properties and flavour of cheese. Traditional methods of cheese manufacture have been modified by the need for greater efficiencies in the manufacture and maturation of cheese and by changes in the marketing channels for cheese. Accommodating these changes while maintaining the characteristics of a given cheese variety has been accomplished by the application of scientific principles. The need for greater understanding of the characteristics of cheese has also been driven by the increased use of cheese as an ingredient in other foods. This has required specific control of selected properties of cheese to impart the desired properties to the food, and to retain characteristics of the cheese during various food processing technologies. The successive editions of Cheese: Chemistry, Physics and Microbiology have documented the application of science to the art of cheesemaking. Certain characteristics are common in all editions: a thorough description and evaluation of scientific and technological advances, prodigious referencing to direct readers to more in-depth discussion of topics, and careful editing to impart consistency of discussion and a smooth transition between chapters. However, each edition has been revised to incorporate new information and to reflect recent trends in describing the science of cheesemaking and maturation and in the use of cheese as a food ingredient. Scientific principles emphasised in Volume 1 cover microbiological, chemical and physical attributes of cheese as in previous editions. Greater emphasis is given to the genetics and metabolic activity of lactic starters and on the secondary microflora in the third edition. Conversion of components (lactose, lactate, citrate, lipids, proteins) by microbial metabolism and enzymatic action is discussed in several chapters. Inclusion of modern sensory evaluation techniques and instrumental identification of flavour compounds recognises the relationship between these areas. A new chapter on acid gels provides the basic background for discussion in Volume 2 on cheese varieties made by acid or heat plus acid coagulation that are becoming more important as food ingredients. Volume 2, as in previous editions, focuses on various types of cheese, but the cheeses have been grouped into more logical categories based upon characteristics rather than geographical regions of production. The first chapter of Volume 2 provides an overview of the diversity of cheese varieties and systems of categorising varieties. A similar approach in the second chapter familiarises the reader with the general aspects of cheese technology to emphasise that there are common elements in cheesemaking and maturation and that cheese varieties result from specific deviations from or additions to these common elements. The last chapter is appropriately a discussion of cheese as an ingredient, which recognises recent trends in the science of cheese. A substantial bank of knowledge has been accumulated on cheese and this has been rigorously incorporated into the two volumes. It is inevitable that this bank of knowledge will be revised and expanded. The third edition of Cheese: Chemistry, Physics and Microbiology provides the base upon which these revisions and expansions can be undertaken objectively. N.F. Olson Department of Food Science, University of Wisconsin, Madison


List of Contributors

Dr J.M. Banks CHARIS Food Research Hannah Research Institute Ayr KA6 5HL Scotland Dr T. Beresford Dairy Products Research Centre Teagasc, Moorepark Fermoy Cork Ireland Dr E. Beuvier Station de Recherches en Technologie et Analyses Laitières Institut National de La Recherche Agronomique F-39801 Poligny Cedex France Dr S. Buchin Station de Recherches en Technologie et Analyses Laitières Institut National de La Recherche Agronomique F-39801 Poligny Cedex France Dr M.J. Callanan Dairy Products Research Centre Teagasc, Moorepark Fermoy Cork Ireland Dr J.-F. Chamba Institut Technique Francais de Fromages 74801 La Roche sur Foron Cedex France Professor T.M. Cogan Dairy Products Research Centre Teagasc, Moorepark Fermoy Cork Ireland

Ms Y.F. Collins Dairy Products Research Centre Teagasc, Moorepark Fermoy Cork Ireland Professor M.J.C. Crabbe Division of Cell and Molecular Biology School of Animal and Microbial Sciences The University of Reading Whiteknights Reading RG6 6AJ UK Dr Á.C. Curtin Department of Food and Nutritional Sciences University College Cork Ireland Professor P. Dejmek Department of Food Engineering Lund University Box 124, 221 00 Lund Sweden Dr C.M. Delahunty Department of Food and Nutritional Sciences University College Cork Ireland Dr A.D.W. Dobson Department of Microbiology University College Cork Ireland Dr C.W. Donnelly Department of Nutrition and Food Sciences University of Vermont 200 Carrigan Building Burlington VT 05405-0044 USA

x

List of Contributors

Dr M.A. Drake Department of Food Science North Carolina State University Campus Box 7624 Raleigh NC 27695-7624 USA Dr G.F. Fitzgerald National Food Biotechnology Centre Departments of Microbiology and Food & Nutritional Sciences University College Cork Ireland Professor P.F. Fox Department of Food and Nutritional Sciences University College Cork Ireland Dr T.P. Guinee Dairy Products Research Centre Teagasc, Moorepark Fermoy Cork Ireland Dr D.S. Horne CHARIS Food Research Hannah Research Institute Ayr KA6 5HL Scotland Dr F. Irlinger Institut National de La Recherche Agronomique 78850 Thiverval-Grignon Cedex France Dr J.-L. Le Quéré Institut National de la Recherche Agronomique Unité Mixte de Recherche sur les Aromes 17 rue Sully F-21065, Dijon France Dr J.A. Lucey Department of Food Science University of Wisconsin-Madison 1605 Linden Drive Madison, WI 53706-1565 USA

Dr A.A.A. Magboul DAL Food Industries Industrial Area No. 1/15 Block 4F Khartoum North, PO Box 708 Sudan Professor J.-L. Maubois Laboratoire de Recherches Laitières Institut National de la Recherche Agronomique 35012 Rennes Cedex France Dr S. McGrath National Food Biotechnology Centre Department of Microbiology University College Cork Ireland Dr P.L.H. McSweeney Department of Food and Nutritional Sciences University College Cork Ireland Professor V.V. Mistry Dairy Science Department South Dakota State University Brookings SD 57007 USA Dr N.M. O’Brien Department of Food and Nutritional Sciences University College Cork Ireland Dr D.J. O’Callaghan Dairy Products Research Centre Teagasc, Moorepark Fermoy Cork Ireland Dr J. O’Callaghan Department of Microbiology University College Cork Ireland

List of Contributors xi

Dr T.P. O’Connor Department of Food and Nutritional Sciences University College Cork Ireland

Mr V.K. Upadhyay Department of Food and Nutritional Sciences University College Cork Ireland

Dr E. Parente Dipartimenta Biologia Università della Basilicata Campus di Macchia Romana 85100 Potenza Italy

Professor P. Walstra Department of Food Science The Argricultural University 6703 HD Wageningen The Netherlands

Dr R.P. Ross Dairy Products Research Centre Teagasc, Moorepark Fermoy Cork Ireland

Dr M.G. Wilkinson Department of Life Sciences University of Limerick Castletroy Limerick Ireland

Dr D. van Sinderen Department of Microbiology University College Cork Ireland

Professor A. Williams CHARIS Food Research Hannah Research Institute Ayr KA6 5HL Scotland


Preface to the First Edition

Cheese manufacture is one of the classical examples of food preservation, dating from 6000–7000 BC. Preservation of the most important constituents of milk (i.e. fat and protein) as cheese exploits two of the classical principles of food preservation, i.e.: lactic acid fermentation, and reduction of water activity through removal of water and addition of NaCl. Establishment of a low redox potential and secretion of antibiotics by starter microorganisms contribute to the storage stability of cheese. About 500 varieties of cheese are now produced throughout the world; present production is ⬃107 tonnes per annum and is increasing at a rate of ⬃4% per annum. Cheese manufacture essentially involves gelation of the casein via iso-electric (acid) or enzymatic (rennet) coagulation; a few cheeses are produced by a combination of heat and acid and still fewer by thermal evaporation. Developments in ultrafiltration facilitate the production of a new family of cheeses. Cheeses produced by acid or heat/acid coagulation are usually consumed fresh, and hence their production is relatively simple and they are not particularly interesting from the biochemical viewpoint although they may have interesting physico-chemical features. Rennet cheeses are almost always ripened (matured) before consumption through the action of a complex battery of enzymes. Consequently they are in a dynamic state and provide fascinating subjects for enzymologists and microbiologists, as well as physical chemists. Researchers on cheese have created a very substantial literature, including several texts dealing mainly with the technological aspects of cheese production. Although certain chemical, physical and microbiological aspects of cheese have been reviewed extensively, this is probably the first attempt to review comprehensively the scientific aspects of cheese manufacture and ripening. The topics applicable to most cheese varieties, i.e. rennets, starters, primary and secondary phases of rennet coagulation, gel formation, gel syneresis, salting, proteolysis, rheology and nutrition, are reviewed in Volume 1. Volume 2 is devoted to the more specific aspects of the nine major cheese families: Cheddar, Dutch, Swiss, Iberian, Italian, Balkan, Middle Eastern, Mould-ripened and Smear-ripened. A chapter is devoted to non-European cheeses, many of which are ill-defined; it is hoped that the review will stimulate scientific interest in these minor, but locally important, varieties. The final chapter is devoted to processed cheeses. It is hoped that the book will provide an up-to-date reference on the scientific aspects of this fascinating group of ancient, yet ultramodern, foods; each chapter is extensively referenced. It will be clear that a considerably body of scientific knowledge on the manufacture and ripening of cheese is currently available but it will be apparent also that many major gaps exist in our knowledge; it is hoped that this book will serve to stimulate scientists to fill these gaps. I wish to thank sincerely the other 26 authors who contributed to the text and whose co-operation made my task as editor a pleasure. P.F. Fox


Preface to the Second Edition

The first edition of this book was very well received by the various groups (lecturers, students, researchers and industrialists) interested in the scientific and technological aspects of cheese. The initial printing was sold out faster than anticipated and created an opportunity to revise and extend the book. The second edition retains all 21 subjects from the first edition, generally revised by the same authors and in some cases expanded considerably. In addition, 10 new chapters have been added: Cheese: Methods of chemical analysis; Biochemistry of cheese ripening; Water activity and the composition of cheese; Growth and survival of pathogenic and other undesirable microorganisms in cheese; Membrane processes in cheese technology, in Volume 1 and North-European varieties; Cheeses of the former USSR; Mozzarella and Pizza cheese; Acid-coagulated cheeses and Cheeses from sheep’s and goats’ milk in Volume 2. These new chapters were included mainly to fill perceived deficiencies in the first edition. The book provides an in-depth coverage of the principal scientific and technological aspects of cheese. While it is intended primarily for lecturers, senior students and researchers, production management and quality control personnel should find it to be a very valuable reference book. Although cheese production has become increasingly scientific in recent years, the quality of the final product is still not totally predictable. It is not claimed that this book will provide all the answers for the cheese scientist/technologist but it does provide the most comprehensive compendium of scientific knowledge on cheese available. Each of the 31 chapters is extensively referenced to facilitate further exploration of the extensive literature on cheese. It will be apparent that while cheese manufacture is now firmly based on sound scientific principles, many questions remain unanswered. It is hoped that this book will serve to stimulate further scientific study on the chemical, physical and biological aspects of cheese. I wish to thank sincerely all the authors who contributed to the two volumes of this book and whose cooperation made my task as editor a pleasure.

P.F. Fox


Preface to the Third Edition

Very considerable progress has been made on the scientific aspects of cheese since the second edition of this book was published in 1993. This is especially true for the Microbiology of Cheese and the Biochemistry of Cheese Ripening; consequently those sections have been expanded very considerably. The general structure of the book is similar to that of the earlier editions, with the more general aspects being treated in Volume 1 and the more applied, variety-related aspects in Volume 2. The book contains 36 chapters. Reflecting the very extensive research on cheese starters in recent years, four chapters have been devoted to this topic in the third edition. Another new feature is the inclusion of two chapters on cheese flavour; one on sensory aspects, the other on instrumental methods. In Volume 2 of the second edition, cheese varieties were treated mainly on a geographical basis. While some elements of the geographical distribution remain, cheese varieties are now treated mainly based on the characteristic features of their ripening. Obviously, it is not possible to treat all 1000 or so cheese varieties, but the 10 variety-related chapters in Volume 2 cover at least 90% of world cheese production and it is very likely that your favourite cheese is included in one of those 10 chapters. Cheese is the quintessential convenience food and is widely used as an ingredient in other foods and in the USA approximately 70% of all cheese is used as a food ingredient. The use of cheese as a food ingredient is a major growth area; consequently, a chapter has been devoted to the important features of cheese as an ingredient, including a section on Enzyme-modified Cheese. Each chapter is extensively referenced to facilitate further exploration of the extensive literature on cheese. While the book is intended for primarily lecturers, senior students and researchers, production management and quality control personnel should find it to be a very useful reference book. We wish to thank sincerely all authors who contributed to the two volumes of this book and whose cooperation made our task as editors a pleasure. Special thanks are due to Ms Anne Cahalane for very valuable assistance. P.F. Fox P.L.H. McSweeney T.M. Cogan T.P. Guinee


Cheese: An Overview P.F. Fox and P.L.H. McSweeney, Department of Food and Nutritional Sciences, University College, Cork, Ireland

Historical Cheese is the generic name for a group of fermented milk-based food products, produced in a wide range of flavours and forms throughout the world. Although the primary objective of cheesemaking is to conserve the principal constituents of milk, cheese has evolved to become a food of haute cuisine with epicurean qualities, as well as being highly nutritious. Sandine and Elliker (1970) suggested that there are more than 1000 varieties of cheese. Walter and Hargrove (1972) described more than 400 varieties and listed the names of a further 400, while Burkhalter (1981) classified 510 varieties (although some are listed more than once). Jim Path (University of Wisconsin) has compiled a list of 1400 varieties (visit www.cdr.wisc.edu). As discussed in detail in ‘Diversity of cheese varieties: An Overview’, Volume 2, a number of attempts have been made to classify cheese varieties into meaningful groups. The most common criterion for the classification is texture (very hard, hard, semi-hard, semi-soft, soft) which is related mainly to the moisture content of the cheese. Various attempts have been made to improve on this basis of classification, for example, by including the milk-producing species, moisture to protein ratio, method of coagulation, cooking temperature, microflora. These classification schemes are discussed in ‘Diversity of cheese varieties: An Overview’, Volume 2. However, no classification scheme developed to date is completely satisfactory; the inclusion of chemical indices of ripening would be useful. It is commonly believed that cheese evolved in a region known as the ‘Fertile Crescent’, i.e., from the Tigris and Euphratres rivers, through what is now southern Turkey to the Mediterranean coast, some 8000 years ago. The so-called ‘Agricultural Revolution’ occurred in this region with the domestication of plants and animals. Presumably, humans soon recognized the nutritive value of milk produced by domesticated animals and contrived to share the mother’s milk with her offspring. Apparently, goats and sheep, which are gregarious and docile, were the first dairy animals domesticated, but cattle have become the dominant dairy species in most parts of the world (c. 85% of the total world supply of milk is obtained from cows).

Milk is also a rich source of nutrients for bacteria which contaminate the milk, some species of which utilize milk sugar, lactose, as a source of energy, producing lactic acid. Bacterial growth and acid production would have occurred during storage or during attempts to dry milk in the prevailing warm, dry climate to produce a more stable product – air-drying of meat, fruits and vegetables appears to have been practised as a primitive form of food preservation at this period in the development of civilization. When sufficient acid has been produced, the principal proteins of milk, the caseins, coagulate, i.e., at their isoelectric points – ⬃pH 4.6, to form a gel in which the fat is entrapped. The rate of acidification by the adventitious microflora would usually be slow, allowing the (unhomogenized) fat globules to form a cream layer. This layer of sour cream could be blended into the lower protein gel or scooped off for the production of butter. Thus originated three of our classical fermented dairy products: fermented milks, sour cream and lactic butter, all of which are still produced widely, sometimes depending on the adventitious microflora for acidification, but now usually through the growth of cultures of lactic acid bacteria. The first fermented dairy foods were produced by a fortuitous combination of events – the ability of a group of bacteria, the lactic acid bacteria (LAB), to grow in milk and to produce enough acid to reduce the pH of milk to the isoelectric point of the caseins, at which these proteins coagulate. Neither the LAB nor the caseins were designed for this outcome. The caseins were ‘designed’ to coagulate following limited proteolysis in the stomach of neonatal mammals, the gastric pH of which is around 6, i.e., very much higher than the isoelectric point of the caseins. The ability of Lactococcus lactis to ferment lactose, a sugar specific to milk, is plasmid-encoded, suggesting that this characteristic was acquired relatively recently in the evolution of these bacteria. Their natural habitats are vegetation and/or the intestine, from which they presumably colonized the teats of dairy animals, contaminated with lactosecontaining milk; it is likely that through evolutionary pressure, these bacteria acquired the ability to ferment lactose. When an acid-coagulated milk gel is broken, e.g., accidentally by movement of the storage vessel or intentionally

Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 1: General Aspects ISBN: 0-1226-3652-X Set ISBN: 0-1226-3651-1

Copyright © 2004 Elsevier Ltd All rights reserved

2 Cheese: An Overview

by breaking or cutting, it separates into curds and whey. It would have been realized quickly that the acid whey is a pleasant, refreshing drink for immediate consumption while the curds could be consumed fresh or stored for future use. In fact, whey was long considered to have medicinal benefits (see Hoffmann, 1761). It was probably soon realized that the shelf-life of the curds could be extended by dehydration and/or by adding salt; heavily salted cheese varieties are still widespread throughout the Middle East and small quantities of a number of dehydrated cheeses are produced in North Africa and the Middle East, e.g., Tikammart and Aoules (Algeria), Djamid (Jordan), Ekt (Saudi Arabia) and Madraffarah (Syria) (see Phelan et al., 1993). It is presumed that one of the principal families of cheese, the acid cheeses, modern members of which include Cottage cheese, Cream cheese and Quarg, originated in this way. While lactic acid, produced in situ, is believed to have been the original milk coagulant, an alternative mechanism was also recognized from an early date. Many proteolytic enzymes can modify the casein system in milk, causing it to coagulate under certain circumstances. Enzymes capable of causing this transformation are widespread in nature, e.g., bacteria, moulds, plant and animal tissues, but an obvious source would have been animal stomachs. It would have been observed that the stomach of young mammals after slaughter contained curds, especially if the animals had suckled shortly before slaughter; curds would also have been observed in the vomit of human infants. Before the development of pottery (⬃5000 BC), storage of milk in bags made from animal skins was probably common (as it still is in many countries). Stomachs of slaughtered animals provided ready-made, easily sealed containers; under such circumstances, milk would extract enzymes (chymosin and some pepsin) from the stomach tissue, leading to its coagulation during storage. The properties of rennet-coagulated curds are very different from those produced by isoelectric (acid) precipitation, e.g., they have better syneresis properties which makes it possible to produce low-moisture cheese curd without hardening. Rennet-coagulated curds can, therefore, be converted to a more stable product than acid curds and rennet coagulation has become predominant in cheese manufacture, being exploited for c. 75% of total world production. Although animal rennets were used from early times, rennets produced from a range of plant species, e.g., fig and thistle, also appear to have been common in ancient times. However, plant rennets are not suitable for the manufacture of long-ripened cheese varieties and gastric proteinases from young animals became the standard rennets until a recent shortage of supply made it necessary to introduce ‘rennet substitutes’.

While the coagulation of milk by the in situ production of lactic acid was, presumably, accidental, the use of rennets to coagulate milk was intentional. It was, in fact, quite an ingenous invention – if the conversion of milk to cheese by the use of rennets was discovered today, it would be hailed as a major biotechnological discovery! The advantages accruing from the ability to convert the principal constituents of milk to cheese would have been apparent from the viewpoints of storage stability, ease of transport and, presumably, as a means of diversifying the human diet and cheese manufacture became well established in the ancient civilizations of the Middle East, Egypt, Greece and Rome. There are numerous references to cheese and other foods in the Bible (see MacAlister, 1904). Milk and dairy products formed an important part of the diet of peoples of the Near East during Biblical times; indeed Palestine was praised as ‘a land flowing with milk and honey’ (Exodus 3.8). Animals herded during Biblical times for milk production included goats (e.g., Proverbs 27.27), sheep (e.g., Deuteronomy 14.4) and possibly camels (Genesis 32.15). Cows’ milk is rarely specified in the Old Testament, presumably because of the unsuitability of the terrain of the Holy Land for cow pasture. In addition to milk, other foods of dairy origin mentioned in the Bible include curds (perhaps fermented milk: Genesis 18.8; Isaiah 7.22) and butter (Psalms 55.21). There are several clear references in the Old Testament to cheese, e.g., Job (1520 BC, where Job remarks to God ‘did Thou not pour me out like milk and curdle me like cheese’; Job 10.10) and Samuel (1170–1017 BC; as a delicacy sent by Jesse to his sons (I Samuel 17.18) and as a gift presented to David (II Samuel 17.29)). Cheese is represented in the tomb art of Ancient Egypt and in Greek literature. Vegetable rennets are mentioned in the first work of European literature; Homer (c. eighth century BC) implies the use of fig rennet in the Iliad (‘. . . as when fig juice is added to white milk and rapidly coagulates, and the milk quickly curdles as it is stirred, so speedy was his healing of raging Ares.’ Iliad 5) and describes the Cyclops, Polyphemus, making ewes’ milk cheese in the Odyssey (Book 9) using ‘well made dairy vessels’ and ‘pails swimming with whey’. Other Greek authors who mention cheese include the Father of History, Herodotus (484–408 BC), who referred to ‘Scythian cheese’ and the philosopher, Aristotle (384–322 BC), who noted that ‘Phrygian’ cheese was made from the milk of mares and asses. Apparently, cheese was prescribed in the diet for Spartan wrestlers in training. Cheese manufacture was well established in the Roman Empire and was a standard item in the rations

Cheese: An Overview 3

issued to Roman soldiers. Cheese must have been popular with Roman civilians also and demand exceeded supply, forcing an emperor, Diocletian (AD 284–305), to fix a maximum price for cheese. Many Roman writers, e.g., Cato the Elder (234–149 BC), Varro, Columella and Pliny the Elder, described cheese manufacture and quality and the culinary uses of cheese. Pliny the Elder (AD 23–79) mentioned cheese in his encyclopedia, Historia Naturalis (Book 28) and described its uses in the diet and in medicinal applications. Varro (c. 116–27 BC; De Agricultura 2.3–2.6) distinguished between ‘soft and new cheese’ and that which is ‘old and dry’ and described the Roman cheesemaking season in the spring and summer. Varro briefly described cheese manufacture: to about 2 congii (c. 5.7 L) of milk was added a piece of rennet from the hare or kid (in preference to that from the lamb). Varro described the quantity of rennet to be added as ‘the size of an olive’, implying that the rennet was solid, perhaps a piece of stomach tissue. If so, then this rennet may be analogous to rennet paste, which is used today for the manufacture of certain Italian cheese varieties (see ‘Biochemistry of Cheese Ripening: Introduction and Overview’, ‘Lipolysis and Catabolism of Fatty Acids in Cheese’, Volume 1). Fig latex and vinegar were mentioned by Varro as an alternative rennet and vinegar is also mentioned as a means for coagulating milk (as practised today in the manufacture of some forms of Queso Blanco and Ricotta). However, the most complete ancient description of cheesemaking is given by Lucius Junius Moderatus Columella, a Roman soldier and author from Gades (modern Cadiz), in his treatise on agriculture, De Re Rustica (c. AD 50). A manufacturing procedure for Roman cheese, based on the description of Columella, is given in Fig. 1, which includes many observations and practices recognizable by modern cheesemakers. He recommends that the (raw) milk be held at ‘some degree of heat’ but warns against over-heating by placing the pail on the flames of a fire. Columella distinguished between cheese with a ‘thin consistency’ (soft?) which must be sold quickly ‘while it is still fresh and retains its moisture’ and that with a ‘rich and thick consistency’ (hard?) which may be held for a long period. Since the concept of pH and the existence of bacteria were unknown in antiquity, no mention is made of starter; the cheese curd was acidified using the adventitious microflora of the raw milk. However, Columella did discuss different types of rennets in some detail. He recommended coagulation using rennet from lamb or kid but states that milk can also be coagulated using flowers of certain thistles (perhaps Cynara cardunculus), seeds of the safflower (Carthamus tinctorius), or sap from the fig tree. Interestingly, Col-

PAIL OF MILK (Sheep or goat) Rennet, > weight of denarius (c. 3.4 g) (Lamb, kid or other)

‘Some degree of heat’ (Stand not far from the fire) Coagulum

WHEY

Drain whey quickly when milk coagulated using wicker baskets or moulds. Aid whey drainage using weights Curds Place cheese in a cool shady place

Surface application of dry salt

Rind formation Pressing using weights Further application of dry salt Repeat for 9 days Wash cheeses using water

Place cheeses in rows on wickerwork trays Allow them to become ‘moderately dry’ Pack closely on shelves in an enclosed place not exposed to the wind Cheese becomes ‘more tender’ Cheese can be ‘exported beyond the sea’

CHEESE

Figure 1 Flow diagram for the manufacture of a type of Roman cheese based on the description of Columella (De Re Rustica, 7.8.1–7.8.7).

umella recommended that the smallest amount of rennet possible be used to ensure high quality cheese. This may be related to the excessive proteolytic activity of plant proteinases used as rennets which often produce bitter cheese. Whey drainage was through wicker baskets, perhaps analogous to the drainage of whey through moulds in the manufacture of certain soft cheeses (e.g., Camembert). No mention was made by Columella of cooking the curds/whey mixture prior to whey drainage; moisture control seems to have been by pressing the curds during whey drainage or pressing the cheese after salting. Salting was by means of the repeated application of dry salt to the cheese surface (which is still practiced, e.g., in the manufacture of Blue cheese), which encouraged further loss of moisture (‘acid liquid’). However, Columella also mentioned brine salting as a method of ‘hardening’ cheese. The cheeses were washed with water, allowed to form a rind and placed on shelves in an enclosed place ‘so that the cheese may remain more


tender’. Interestingly, the comparative form of the adjective used in the Latin text (tenerior) can also be translated as ‘more soft’; if this is the intended meaning, it is the first recorded mention of the changes which occur in cheese during ripening. Columella also discussed defects which may occur in cheese, including being ‘full of holes’ (perhaps mechanical openings as the remedy recommended is increased pressing), too salty or too dry. According to Columella, cheeses were flavoured with herbs and coloured with smoke, practices which persist to a certain extent today. He also described briefly the manufacture of ‘hand-pressed’ (manu pressum) cheese in which hot water is poured over the curds which are then shaped by hand, a practice perhaps related to the kneading and stretching steps for pasta-filata varieties. Thus, cheesemaking practice appears to have changed little from the time of Columella until the nineteenth century! The great migrations of peoples throughout Europe immediately before and after the fall of the Western Roman Empire must have promoted the further spread of cheese manufacture, as did the Crusaders and other pilgrims of the Middle Ages. Probably, the most important agents contributing to the development of cheese ‘technology’ and to the evolution of cheese varieties were monasteries and feudal estates. In addition to their roles in the spread of Christianity and in the preservation and expansion of knowledge during the Dark Ages, the monasteries made considerable contributions to the advancement of agriculture in Europe and to the development and improvement of food commodities, notably wine, beer and cheese. Many of our current well-known cheese varieties were developed in monasteries, e.g., Wenslydale (Rievaulx Abbey, Yorkshire), Port du Salut or Saint Paulin (Monastery de Notre Dame du Port du Salut, Laval, France), Fromage de Tamie (Abbey of Tamie, Lac d’Annecy, Geneva), Maroilles (Abbey Moroilles, Avesnes, France) and Trappist (Maria Stern Monastery, Banja Luka, Bosnia). The inter-monastery movement of monks would have contributed to the spread of cheese varieties and probably to the development of new hybrid varieties. The great feudal estates of the Middle Ages were self-contained communities. The conservation of surplus food produced in summer for use during winter was a major activity on such estates and undoubtedly cheese represented one of the more important of these conserved products, along with cereals, dried and salted meats, dried fruits, dried and fermented vegetables, beer and wine. Cheese probably represented an item of trade when amounts surplus to local requirements were available. Within these estates, individuals acquired special skills which were passed on to succeeding generations. The feudal estates evolved into

villages and some into larger communities. Because monasteries and feudal estates were essentially selfcontained communities, it is readily apparent how several hundred distinct varieties of cheese evolved from essentially the same raw material, milk or rennet-coagulated curds, especially under conditions of limited communication. Traditionally, many cheese varieties were produced in quite limited geographical regions, especially in mountainous areas, where communities are isolated. The localized production of certain varieties is still apparent and indeed is preserved for those varieties with controlled designations of origin (Appelation d’Origine Contrôlée). Regionalization of certain cheese varieties is particularly marked in Spain, Portugal and Italy, where the production of many varieties is restricted to very limited region. Almost certainly, most cheese varieties evolved by accident because of a particular set of local circumstances, e.g., a peculiarity of the local milk supply, either with respect to chemical composition or microflora, an ‘accident’ during storage of the cheese, e.g., growth of mould or other microorganisms. Presumably, those accidents that led to desirable changes in the quality of the cheese were incorporated into the manufacturing protocol; each variety thus underwent a series of evolutionary changes and refinements. The final chapter in the spread of cheese throughout the world resulted from the colonization of north and south America, Oceania and Africa by European settlers who carried their cheesemaking skills with them. Cheese has become an item of major economic importance in some of these ‘new’ countries, notably the US, Canada, Australia and New Zealand, but the varieties produced are mainly of European origin, modified in some cases to meet local requirements. Cheese was not manufactured in these regions before colonization by Europeans; in fact, there were no cattle, sheep or goats in Australia, North or South America and there were no land mammals in New Zealand before the arrival of Europeans. For further information on the history of cheese, the reader is referred to Squire (1937), Cheke (1959), Davis (1965), Kosikowski (1977), Scott (1986), Kosikowski and Mistry (1997) and Robinson and Wilbey (1998). For references on Roman agriculture, see White (1970). Cheesemaking remained an art rather than a science until relatively recently. With the gradual acquisition of knowledge on the chemistry and microbiology of milk and cheese, it became possible to direct the changes involved in cheesemaking in a more controlled fashion. Although few new varieties have evolved as a result of this improved knowledge, the existing varieties have become better defined and their quality more consistent.


Considering the long history of cheesemaking, one might be inclined to the idea that what have come to be regarded as standard varieties have been so for a long time. However, although the names of many current varieties were introduced several hundred years ago (Table 1), these cheeses were not standardized; for example, the first attempt to standardize the wellknown English varieties, Cheddar and Cheshire, was made by John Harding in the mid-nineteenth century. Prior to that, ‘Cheddar cheese’ was that produced in a particular area in England around the village of Cheddar, Somerset, and probably varied considerably depending on the manufacturer and other factors. Cheese manufacture was a farmstead enterprise until the mid-nineteenth century – the first cheese factory in the US was established near Rome, NY, in 1851 and the first in Britain at Longford, Derbyshire, in 1870. Thus, there were thousands of cheese manufacturers and there must have been great variation within any one general type. This situation persists in a modified form today in Switzerland and Italy where there are a large number of small cheese factories, often grouped together into consortia for the purposes of marketing and quality control. When one considers the very considerable inter-factory, and indeed intra-factory, variations in quality and characteristics which occur today in well-defined varieties, e.g., Cheddar, in spite of the very considerable scientific and technological advances, one can readily appreciate the variations that must have existed in earlier times. Some major new varieties, notably Jarlsberg and Maasdamer, have been developed recently as a consequence of scientific research. Many other varieties have evolved very considerably, even to the extent of becoming new varieties, as a consequence of scientific research and the development of new technology – notable examples are (US) Queso Blanco, various cheeses produced by ultrafiltration and various forms of Quarg. There has been a marked resurgance of farmhouse cheesemaking in recent years; many of the cheeses being produced on farms are not standard varieties and some of these may evolve to become new varieties.

Table 1 First recorded date for some major cheese varieties (Scott, 1986) Goronzola Schabzieger Roquefort Maroilles Schwangenkäse Grana Taleggio

897 1000 1070 1174 1178 1200 1282

Cheddar Parmesan Gouda Gloucester Stilton Camembert St Paulin

1500 1579 1697 1783 1785 1791 1816

A major cause of differences in the characteristics of cheese is the inter-species differences in the composition and physico-chemical characteristics of the milk used. Although milks from several species are used in cheese manufacture, the cow is by far the most important; sheep, goat and buffalo are commercially important in certain areas. Approximately 85, 11, 2 and 2% of total milk is produced from cows, buffalo, sheep and goats, respectively. However, most sheep’s and goats’ milk is used for cheese manufacture and therefore are disproportionately important; many famous cheese varieties are made from sheep’s milk, e.g., Roquefort, Manchego, Feta and all the various Pecorino and Canestrato varieties. There are very significant inter-species differences in the composition of milk which are reflected in the characteristics of the cheeses produced from them. Major inter-species differences of importance in cheesemaking are the concentration and types of caseins, concentration of fat and especially the fatty acid profile, concentration of salts, especially of calcium. There are also significant differences in milk composition between breeds of cattle and these also influence cheese quality, as do variations due to seasonal, lactational and nutritional factors and of course the methods of milk production, storage and collection.

Cheese Science and Technology Cheese is the most diverse group of dairy products and is, arguably, the most academically interesting and challenging. While many dairy products, if properly manufactured and stored, are biologically, biochemically, chemically and physically very stable, cheeses are, in contrast, biologically and biochemically dynamic, and, consequently, are inherently unstable. Throughout manufacture and ripening, cheese production represents a finely orchestrated series of consecutive and concomitant biochemical events which, if synchronized and balanced, lead to products with highly desirable aromas and flavours but when unbalanced, result in off-flavours and odours. Considering that, in general terms, a basically similar raw material (milk from a very limited number of species) is subjected to a manufacturing protocol, the general principles of which are common to most cheese varieties, it is fascinating that such a diverse range of products can be produced. No two batches of the same variety, indeed probably no two cheeses, are identical. A further important aspect of cheese is the range of scientific disciplines involved: study of cheese manufacture and ripening involves the chemistry and biochemistry of milk constituents, fractionation and chemical characterization of cheese constituents, microbiology, enzymology, molecular genetics, flavour


chemistry, nutrition, toxicology, rheology and chemical engineering. It is not surprising, therefore, that many scientists have become involved in the study of cheese manufacture and ripening. A voluminous scientific and technological literature has accumulated, including a range of books (e.g., Sammis, 1948; Van Slyke and Price, 1949; Kosikowski and Mocquot, 1958; Davis, 1965, 1967; Kosikowski, 1977; Davies and Law, 1984; Eck, 1984; Scott, 1986; Fox, 1987, 1993; Buch Kristensen, 1995; Kosikowski and Mistry, 1997; Law, 1997, 1999; Robinson and Wilbey, 1998; Eck and Gilles, 2000; Fox et al., 2000) and chapters in many others. In addition, there are numerous encyclopedias or pictorial books, with brief descriptions of cheese, e.g., Simon (1956), Layton (1973), Mair-Waldburg (1974), Cantin (1976), Eekhof-Stork (1976), Christian (1984), Robinson (1995), Jenkins (1996) and Harbutt (1999, 2002). There are also a number of country-specific or variety-specific books, e.g., Squire (1937), Cheke (1959), Fraser (1960), Meyer (1973), Montandon (1981), Rance (1982), Gonzalez and del Cerro (1988), Berger et al. (1989), Anifantakis (1991), Robinson and Tamime (1991), Zehren and Nusbaum (1992), Resmini et al. (1992), Masui and Yamada (1996), Vizzardi and Maffeis (1999), Ottogalli (2001) and Kammerlehner (2003). Most of the above books deal mainly with cheese technology; the present book concentrates on the more scientific aspects of cheese. The book is in two volumes. The more general aspects of cheese manufacture, i.e., molecular properties of rennets, coagulation mechanism, curd syneresis, starters, salting, rheology, the biochemistry of ripening, pre-concentration by ultrafiltration and nutritional aspects, which apply, more or less, to most cheese varieties, are considered

Milk

Manufacture (5–24 h) Preparation of milk Selection Standardization Pasteurization *Others Acidification Coagulation Syneresis (dehydration) Cut Cook Agitation Other operations, e.g., Cheddaring Kneading/stretching Pressing Salting

in the first volume. The second volume deals with specific aspects of the principal families of cheese. The principal objective of this introductory chapter is to provide an integrated overview of cheese manufacture and to provide some general background for the more detailed later chapters that follow.

Outline of Cheese Manufacture Almost all acid-coagulated and a little rennet-coagulated cheese is consumed fresh, i.e., the flavour, texture and appearance of the cheese are in their final form at the end of curd production and the curds are not subjected to a period of maturation/ripening. The production of acid-coagulated cheeses can be summarized as:

Milk

Acidification

Cut/break Curds and whey

Coagulum

(biological or chemical)

Cook Separate Wash Curds

Cold-pack cheese

Optional flavours/dressings heat homogenize Package Hot-pack cheese

However, the production of the majority of rennetcoagulated cheese varieties can be sub-divided into two well-defined phases, manufacture and ripening, both of which involve a number of processes:

Cheese curd

Ripening (2 week–2 year) Development of characteristic microflora Metabolism of residual lactose Citrate metabolism Proteolysis Lipolysis Secondary reactions Fatty acid catabolism Amino acid catabolism Lactate metabolism

* e.g., bactofugation, microfiltration, addition of colour (annato)

Mature cheese


The manufacturing phase might be defined as those operations performed during the first 24 h, although some of these operations, e.g., salting and dehydration, may continue over a longer period. Although the manufacturing protocol for individual varieties differs in detail, the basic steps are common to most varieties; these are: acidification, coagulation, dehydration (cutting the coagulum, cooking, stirring, pressing, salting and other operations that promote gel syneresis), shaping (moulding and pressing) and salting. Cheese manufacture is essentially a dehydration process in which the fat and casein in milk are concentrated between 6- and 12-fold, depending on the variety. The degree of dehydration is regulated by the extent and combination of the above five operations, in addition to the chemical composition of the milk. In turn, the levels of moisture and salt, the pH and the cheese microflora regulate and control the biochemical changes that occur during ripening and hence determine the flavour, aroma and texture of the finished product. Thus, the nature and quality of the finished cheese are determined largely by the manufacturing steps. However, it is during the ripening phase that the characteristic flavour and texture of the individual cheese varieties develop. Selection and pre-treatment of cheese milk

Cheese manufacture commences with the selection of milk of high microbiological and chemical quality. The adventitious microflora of milk is normally heterogeneous. Some of these microorganisms, especially the LAB, may be beneficial. Previously, and still for some minor artisanal cheeses, the indigenous LAB were responsible for acid production but selected starter LAB cultures are used for acidification in most cases. Non-starter LAB (NSLAB) probably contribute positively to the ripening of raw milk cheese (see ‘Microbiological changes during ripening’, ‘Biochemistry of Cheese Ripening: Introduction and Overview’, ‘Metabolism of Residual Lactose and of Lactate and Citrate’, ‘Lipolysis and Catabolism of Fatty Acids in Cheese’, ‘Proteolysis in Cheese during Ripening’ and ‘Catabolism of Amino Acids in Cheese During Ripening’, Volume 1) but they are variable and uncontrolled and may be responsible for some of the variability in raw milk cheese. For large-scale cheesemaking operations, it is preferable to kill the NSLAB by pasteurization (although this is not the primary objective of pasteurization). There is increasing interest in inoculating pasteurized milk with selected lactobacilli as an adjunct culture (see ‘Secondary and Adjunct Cultures’, Volume 1).

Some members of the adventitious microflora are undesirable. The most important are number of pathogens, the killing of which is the primary objective of pasteurization (see ‘Growth and Survival of Microbial Pathogens in Cheese’, Volume 1). Raw milk may also contain several spoilage microorganisms, e.g., coliforms (which are unlikely to be a problem today), psychrotrophs (especially if the milk is cold-stored for a long period) and Clostridium tyrobutyricium. Growth of this sporeforming organism during ripening of most cheese varieties results in a defect known as late gas blowing caused by an anaerobic metabolism of lactate to butyrate and H2. Contamination with Cl. tyrobutyricum is minimized by good on-farm hygiene, contaminants may be removed by bactofugation or microfiltration, or their growth may be prevented by NaNO3 or lysozyme. Cheesemilk must be free from antibiotics, which totally, or partially, inhibit the starter bacteria; delayed acidification results in an abnormal composition and microflora and consequently in flavour and textural defects and perhaps very significantly in the growth of harmful, pathogenic or food-poisoning microorganisms. All aspects of cheese curd production (rennet coagulation, gel firmness, syneresis) are affected by the chemical composition of the cheesemilk, especially the concentrations of casein, calcium and pH. The specific effects of compositional parameters on various aspects of curd formation will be described in detail in several subsequent chapters. For a comprehensive description of the chemistry of milk and milk constituents, the reader is refered to Fox (1982, 1983, 1985, 1987, 1992, 1995, 1997) and Fox and McSweeney (1998, 2003). In modern commercial practice, milk for cheese is normally cooled to 4 °C immediately after milking and may be held at about this temperature for several days on the farm and at the factory. Apart from being selective for the development of an undesirable psychrotrophic microflora, cold-storage causes physico-chemical changes (e.g., shifts in calcium phosphate equilibrium and dissociation of some micellar caseins) which have undesirable effects on the cheesemaking properties of milk; these changes are reversed on heating, e.g., at 50 °C for 10–20 min or under HTST pasteurization conditions and hence are of no practical significance. However, cold storage after heat treatment aggravates the adverse effects of heating on the rennet coagulation of milk; this effect is known as rennet hysteresis. The composition of most cheeses falls within certain compositional ranges, sometimes with legal status. The most important compositional factors are fat-in-dry matter (FDM), moisture in non-fat substances (MNFS; which is, in effect, the ratio of moisture to protein), moisture, salt (best expressed as salt-in-moisture, S/M) and pH. The values for FDM and MNFS are determined


mainly by the composition of the cheesemilk and extent of syneresis, respectively. The composition of milk should be adjusted to give the desired values of fat and protein. Previously, the ratio of fat:protein was altered by natural creaming (which is still used for Parmigiano-Reggiano, Grana Padano and some other Italian cheeses) or by the addition of cream or skim milk. It is now possible, and commercially practiced to an increasing extent, to adjust the concentrations, as well as the ratio, of fat and protein in the cheesemilk by manipulating the fat content of the milk together with protein standardization using low concentration factor ultrafiltration. These operations improve the cheesemaking characteristics of the milk and increase and standardize the yield of curd. Owing to the importance of Ca2 in various aspects of cheese manufacture and quality (see ‘Rennets: General and Molecular Aspects’, ‘Rennet-induced Coagulation of Milk’ and ‘The Syneresis of Rennet-coagulated Curd’, Volume 1), it is common practice to supplement cheesemilk with CaCl2. pH is also a critical factor in cheesemaking and since the pH of milk varies (e.g., increases with advancing lactation and during mastitic infection), it is recommended that the pH should be standardized, preferably using the acidogen, gluconic acid--lactone. Although raw milk is still used in both industrial and farmhouse cheesemaking, most cheesemilk is pasteurized, usually immediately before use. Pasteurization alters the indigenous microflora and facilitates the manufacture of cheese of more uniform quality, but unless due care is exercised, it may damage the rennet coagulability and curd-forming properties of the milk, as will be discussed in ‘Rennet-induced Coagulation of Milk’, Volume 1. Even when milk is properly pasteurized, the resulting cheese develops a less intense flavour and ripens more slowly than raw milk cheese. Several heatinduced changes, e.g., killing of indigenous microorganisms, inactivation of indigenous milk enzymes and partial denaturation of whey proteins and their interaction with micellar -casein, could be responsible for these changes. The relative contribution of these factors to the differences between cheeses made from raw or pasteurized milk has been an active area of research in recent years (see Fox et al., 2000; ‘The Microbiology of Cheese Ripening’, ‘Raw Milk Cheeses’ and ‘Biochemistry of Cheese Ripening: Introduction and Overview’, Volume 1). A number of approaches have been used to render cheesemilk free from its indigenous microflora or to inhibit the growth of NSLAB in order to study their contribution to ripening. Non-starter lactic acid bacteria have been removed physically from raw

skim milk by microfiltration (e.g., McSweeney et al., 1993; Beuvier et al., 1997), environmental contamination has been minimized by manufacturing cheese under strictly controlled microbiological conditions (McSweeney et al., 1994), ripening cheese at a low (c. 1 °C) temperature to reduce the growth rate of NSLAB (Shakeel-Ur-Rehman et al., 2000b,c) and the use of antibiotics to inhibit the growth of NSLAB (ShakeelUr-Rehman et al., 1999). Attempts have been made to mimic the NSLAB microflora of raw milk cheese by adding selected strains of NSLAB (see Lynch et al., 1999) to pasteurized cheesemilk or by inoculating pasteurized cheesemilk with raw milk (by blending a low level, e.g., 1%, of raw milk with pasteurized milk; Shakeel-Ur-Rehman et al., 2000a). The results of these studies suggest that heat-induced changes to the microflora of raw milk are the major cause of the differences between raw and pasteurized milk cheeses. However, denaturation of certain indigenous enzymes, particularly lipoprotein lipase, may contribute to the observed differences. Pasteurization of cheesemilk minimizes the risk of cheese serving as a vector for food-poisoning or pathogenic microorganisms, so that even high-quality raw milk may be unacceptable for cheese manufacture. In addition to rendering milk safe from a public health viewpoint, pasteurization renders good quality raw milk almost free of bacteria and improves the consistency of cheese. Pasteurization of milk is essential for the production of cheese of consistent quality in the large, highly mechanized factories that are common today. Although more consistent than cheese made from raw milk, it is also less highly flavoured. To increase the intensity of the flavour of cheese made from pasteurized milk, it is becoming increasingly common to inoculate pasteurized milk with selected organisms, usually lactobacilli, isolated from good quality raw milk cheese (see Lynch et al., 1999; ‘Secondary and Adjunct Cultures’ and ‘The Microbiology of Cheese Ripening’, Volume 1). Thermization (⬃65 °C 15 s) of cheesemilk on arrival at the factory is common or standard practice in some countries. The objective of thermization is to control psychrotrophs and the milk is normally pasteurized before cheesemaking. Microfiltration and bactofugation may be used to remove spores from milk to avoid the defect known as late gas blowing (see ‘The Microbiology of Cheese Ripening’, Volume 1). Not more than 75% of the total protein in milk is recovered in rennet-coagulated cheeses. Obviously, a considerable economic advantage would accrue if some or all of the whey proteins could be incorporated into the cheese. Ultrafiltration (UF) offers a means for accomplishing this, with considerable


success in the case of semi-soft or soft cheeses, especially Feta and Quarg, but with less success for hard and semi-hard varieties. The application, and associated problems, of UF in cheese manufacture is comprehensively reviewed in ‘Application of Membrane Separation Technology to Cheese Production’, Volume 1. An alternative approach is to heat denature the whey proteins (e.g., 90 °C 1 min) to induce their interaction with the casein micelles. Normally, such severe heat treatments are detrimental to the renneting properties of milk but the effects can be off-set by acidification or supplementation with calcium (see ‘Rennet-induced Coagulation of Milk’ and ‘The Syneresis of Rennet-coagulated Curd’, Volume 1). In the authors’ experience, yield increases of up to 8% can be achieved by this approach, while retaining acceptable quality. However, to the authors’ knowledge, the technique is not used commercially except for Quarg, e.g., the thermo-Quarg process (see ‘Acid- and Acid-Rennet-Curd Cheeses: Part A Quark, Cream Cheese and Related Varieties’, Part B Cottage Cheese’, and Part C ‘Acid-heat Coagulated Cheeses’, Volume 2). Acidification

One of the basic operations in the manufacture of most, if not all, cheese varieties is a progressive acidification throughout the manufacturing stage, i.e., up to 24 h, and, for some varieties, during the early stages of ripening also, i.e., acidification commences before and transcends the other manufacturing operations. Acidification is normally via in situ production of lactic acid, although pre-formed acid or acidogen (usually gluconic acid--lactone) is now used to directly acidify curd for some varieties, e.g., Mozzarella, UF Feta-type and Cottage cheese. Until relatively recently, and still in some cases, especially in artisanal varieties, the indigenous microflora of milk was relied upon for acid production. Since this was probably a mixed microflora, the rate of acid production was unpredictable and the growth of undesirable bacteria led to the production of gas and off-flavours. It is now almost universal practice in industrial cheesemaking to add a culture (starter) of selected lactic acid-producing bacteria to raw or pasteurized cheesemilk to achieve a uniform and predictable rate of acid production. For cheese varieties that are cooked to not more than 40 °C, a starter consisting of Lactococcus lactis subsp. lactis and/or Lc. lactis subsp. cremoris is normally used while cultures of Streptococcus thermophilus and a Lactobacillus spp. (Lb. delbrueckii subsp. bulgaricus, Lb. delbrueckii subsp. casei, Lb. delbrueckii subsp. lactis or Lb. helveticus) or a Lactobacillus culture alone is

used for varieties that are ‘cooked’ to higher temperatures, e.g., Swiss and hard Italian varieties. Probably the earliest form of starters were ‘slopback’ cultures – a sample of whey from one day’s cheesemaking was incubated overnight and used as a starter culture on the following day. Such starters are still used for some high-cook cheese varieties (e.g., Parmigiano-Reggiano and Grana Padano). Incubation of hot whey is selective for thermophilic microorganisms and although slop-back cultures are very heterogeneous, they work well if managed carefully. Originally, and still for many varieties, mixed-strain mesophilic starters were used for low-cook cheese. Because the bacterial strains in these starters may be phage-related (i.e., subject to infection by a single strain of bacteriophage) and also because the strains in the mixture may be incompatible, thereby leading to the dominance of one or a few strains, the rate of acid production by mixed-strain starters is variable and unpredictable, even when the utmost care in their selection and handling is exercised. To overcome these problems, single-strain mesophilic starters were introduced in New Zealand about 1935. Unfortunately, many of the fast acid-producing, single-strain starters produced bitter cheese, the cause(s) of which will be discussed in ‘Starter Cultures: General Aspects’, Volume 1. This problem was resolved by using selected pairs of fast and slow acid producers. Defined-strain mesophilic starters are widely used in many countries, frequently consisting of a combination of 2–6 selected, phage-unrelated strains which give very reproducible rates of acid production if properly selected and maintained. The use of defined-strain thermophilic starters is becoming more common. The science and technology of starters have become highly developed and specialized; ‘Starter Cultures: General Aspects’, Volume 1, is devoted to these developments. Acid production at the appropriate rate and time is the key step in the manufacture of good quality cheese (excluding the enzymatic coagulation of the milk, which is a sine qua non for rennet-coagulated cheese varieties). Acid production affects several aspects of cheese manufacture, many of which will be discussed in more detail in later chapters, i.e.: • Coagulant activity during coagulation. • Denaturation and retention of the coagulant in the curd during manufacture and hence the level of residual coagulant in the curd; this influences the rate of proteolysis during ripening, and may affect cheese quality. • Strength of the coagulum, which influences cheese yield.


• Gel syneresis, which controls cheese moisture and hence regulates the growth of bacteria and the activity of enzymes in the cheese; consequently, it strongly influences the rate and pattern of ripening and the quality of the finished cheese. • The rate of pH decrease determines the extent of dissolution of colloidal calcium phosphate which modifies the susceptibility of the caseins to proteolysis during manufacture, influences the rheological properties of the cheese, e.g., compare the texture of Emmental, Gouda, Cheddar and Cheshire cheese, and determines the meltability and stretchability of cheese curd (e.g., Mozzarella and Pizza cheese). • Acidification controls the growth of many species of bacteria in cheese, especially pathogenic, food poisoning and gas-producing microorganisms; in fact, properly made cheese is a very safe product from the public health viewpoint. In addition to producing acid, many starter bacteria produce bacteriocins that also restrict or inhibit the growth of non-starter microorganisms. Mesophilic Lactococcus spp. are capable of reducing the pH of cheese to ⬃4.6 and Lactobacillus spp. to somewhat lower values, perhaps 3.8. The natural ultimate pH of cheese curd falls within the range 4.6–5.1. However, the period required to attain the ultimate pH varies from ⬃5 h for Cheddar to 6–12 h for Blue, Dutch and Swiss varieties. The differences arise from the amount of starter added to the cheesemilk (0.2–5%), the cooking temperature and schedule which may retard the growth of the starter microorganisms and the rate of subsequent cooling of the curd. The level and method of salting have a major influence on pH changes in cheese. The concentration of NaCl in cheese (commonly 0.7–4%, i.e., 2–10% salt in the moisture phase) is sufficient to halt the growth of starter bacteria. Some varieties, mostly of British origin, are salted by mixing dry salt with the curd towards the end of manufacture and hence the pH of curd for these varieties must be close to the ultimate value (⬃pH 5.1) at salting. However, most varieties are salted after moulding by immersion in brine or by application of dry salt on the surface; as discussed in ‘Salt in Cheese: Physical, Chemical and Biological Aspects’, Volume 1, salt diffusion in cheese moisture is a slow process and thus there is ample time for the pH to decrease to ⬃5.0 before the salt concentration becomes inhibitory. The pH of the curd for most cheese varieties, e.g., Swiss, Dutch, Tilsit, Blue, etc., is 6.2–6.5 at moulding and pressing but decreases to ⬃5 during or shortly after pressing and before salting. The significance of various aspects of the concentration and distribution of NaCl in cheese will be discussed in

‘Salt in Cheese: Physical, Chemical and Biological Aspects’, Volume 1. In a few special cases, e.g., Domiati, a high level of NaCl (10–12%) is added to the cheesemilk, traditionally to control the growth of the indigenous microflora. This concentration of NaCl has a major influence, not only on acid development, but also on rennet coagulation, gel strength and syneresis (cf., ‘Rennet-induced Coagulation of Milk’ and ‘The Syneresis of Rennet-coagulated Curd’, Volume 1). Coagulation

The essential characteristic step in the manufacture of all cheese varieties is coagulation of the casein component of the milk protein system to form a gel which entraps the fat, if present. Coagulation may be achieved by: • limited proteolysis by selected proteinases; • acidification to pH ⬃4.6; • acidification to about pH 5.2 in combination with heating to ⬃90 °C. The majority of cheeses are produced by enzymatic (rennet) coagulation. With a few exceptions (e.g., Serra da Estrêla (Portugal) in which acid proteinases from the flowers of the cardoon thistle, Cynara cardunculus, are used), acid (aspartyl) proteinases of animal or fungal origin are used. Rennet from the stomachs of young animals (calves, kids, lambs, buffalo) was used traditionally. The principal enzyme in rennet prepared from young animal stomachs is chymosin (⬃95% of total milk-clotting activity), with a little pepsin. However, limited supplies of such rennets (due to the birth of fewer calves and an increasing trend in many countries to slaughter calves at an older age than previously), concomitant with a worldwide increase in cheese production, have led to a shortage of calf rennet and consequently rennet substitutes (usually bovine or porcine pepsins and less frequently, chicken pepsin, and the acid proteinases from Rhizomucor miehei and less frequently R. pusillus or Cryphonectria parasitica) are now used widely for cheese manufacture in many countries with more or less satisfactory results. The calf chymosin gene has been cloned in Kbryveromyces lactis, E. coli and Aspergillus niger and chymosin from these organisms is now widely used. Reviews on rennet substitutes include Sardinas (1972), Ernstrom and Wong (1974), Nelson (1975), Sternberg (1976), Green (1977), De Koning (1979), Phelan (1985), Fox and McSweeney (1997). The molecular and enzymatic properties of calf chymosin and other acid proteinases used as rennets are reviewed in detail in ‘Rennets: General and Molecular Aspects’, Volume 1.


Although it appears to have been recognized since 1917 (see Berridge, 1942) that milk is not coagulated by rennet at low temperatures, Berridge (1942) is usually credited with clearly demonstrating that the rennetcatalysed coagulation of milk occurs in two phases: a primary enzymatic phase and a secondary nonenzymatic phase. The primary phase has a temperature coefficient (Q10) of ⬃2 and occurs in the range 0–50 °C, while the secondary phase has a Q10 of ⬃16 and occurs very slowly or not all at temperatures ⬃18 °C. The two phases can thus be readily separated by performing the primary phase at a low temperature, e.g., ⬃10 °C; when cold-renneted milk is warmed, coagulation occurs very quickly. Cold renneting, followed by rapid warming, forms the basis of attempts to develop methods for the continuous coagulation of milk but such approaches have not been successful commercially. Normally, the two phases of rennet coagulation overlap to some extent, the magnitude of overlap being quite extensive at low pH, high temperature and in milk concentrated by ultrafiltration. The primary phase of rennet action appears to have been recognized, in general terms, by Hammersten, during the period 1880–1890, who reported the formation of small peptides during renneting. Richmond (1899) described the action of rennet as follows: ‘the action of rennet is to split the casein into a dyscaseose, the calcium salt of which is insoluble and which forms a curd, and a soluble caseose; the insoluble curd carries down with it a large portion of the fat.’ The coagulation of milk attracted quite a lot of interest during the early part of the twentieth century. For example, Alexander (1910, 1912) proposed that the casein in milk exists as an unstable colloid which is protected and stabilized by the whey protein, lactalbumin (He was probably the first to use the idea of a ‘protective colloid’ in casein chemistry); he proposed that rennet coagulated milk by hydrolysing (destroying) the protective colloid. The protective colloid (Schutzcolloid) hypothesis of the colloidal stability of casein and the rennet coagulation of milk was supported by Marui (1926) and Linderstrøm-Lang (1929) but not by Palmer and Richardson (1925) and Palmer (1935) who claimed that increased sensitivity of rennetaltered casein was responsible for the rennet-induced coagulation of milk rather than the destruction of a protective colloid. The early literature on the rennet coagulation of milk was reviewed by Palmer and Richardson (1925) and Palmer (1935) and in a long series of articles in Le Lait by Porcher (1929, 1930, 1931). However, a full explanation of the process had to await the isolation of the casein micelle-protective protein, -casein, by Waugh and von Hippel (1956). These workers showed that the protective capacity of

-casein was destroyed on renneting and Wake (1959) demonstrated that -casein is the only milk protein hydrolysed during the primary phase of rennet action. Only one peptide bond, Phe1059Met106, is hydrolysed (Delfour et al., 1965), resulting in the release of the hydrophilic C-terminal segment of -casein (the (caseino)macropeptides, some of which are glycosylated). The unique sensitivity of the Phe9Met bond of -casein, hydrolysis of which occurs optimally at pH 5.1–5.5 has been the subject of extensive study since 1965 and this work is reviewed in ‘Rennetinduced Coagulation of Milk’, Volume 1. The visual coagulation of milk is really only the start of the gelation process which continues for a considerable period thereafter. Although these post-coagulation changes determine many of the critical cheesemaking properties of the gel, e.g., curd tension (which influences cheese yield) and syneresis properties (which determine the moisture content and hence the ripening profile of the cheese), it is perhaps the least well-understood phase of the cheesemaking process. The recent literature on aspects of the post-visual coagulation phase is reviewed in ‘Rennet-induced Coagulation of Milk’ and ‘The Syneresis of Rennet-coagulated Curd’, Volume 1. Post-coagulation operations

A rennet-coagulated milk gel is quite stable if maintained under quiescent conditions but if it is cut or broken, it syneresis rapidly, expelling whey. The rate and extent of syneresis are influenced, inter alia, by how finely the coagulum is cut (small pieces promote syneresis; the coagulum for high-moisture cheeses is not cut but is ladeled into moulds), milk composition, especially [Ca2] and [casein], pH, cooking temperature, rate of stirring of the curd–whey mixture and of course, time (see ‘The Syneresis of Rennet-coagulated Curd’, Volume 1). The composition of the finished cheese is to a very large degree determined by the extent of syneresis and since this is under the control of the cheesemaker, the differentiation of the individual cheese varieties really begins at this stage, although the composition of cheesemilk, the amount and type of starter and the amount and type of rennet are also significant in this regard. The temperature to which the curds are cooked varies from ⬃30 °C (i.e., no cooking) for high-moisture cheeses (e.g., Camembert) to ⬃55 °C for low-moisture cheese (e.g., Parmigiano-Reggiano). After cooking, the curds and whey are separated by various, variety-specific techniques. The curds for most varieties are transferred to moulds where further drainage and acidification occur. Curds that have undergone extensive syneresis in the vat (i.e., have a


low moisture content) are pressed in the moulds, sometimes according to a programmed increase in pressure, with the objective of fusing the curds and rendering the cheeses free from mechanical openings and reducing the moisture content further. The curds for two families of cheese, Cheddar and pasta-filata, are subjected to special treatments prior to moulding. Cheddar-type cheese undergoes a process called ‘cheddaring’. In the traditional process, the drained curds are piled into two beds in the vat, separated by a channel for whey drainage. The beds of curd are cut into blocks, ⬃10 cm side, which are inverted every 15 min and later piled two or three blocks high. This process continues for ⬃2 h until the pH decreases to ⬃5.4. During cheddaring, the blocks of curd flow slightly and the cheese acquires a fibrous texture similar to that of cooked chicken breast meat. In the modern mechanized process, the drained curds are transferred pneumatically to a tower (⬃10 m tall) or to a moving belt; in the tower or on the belt, the mass of curds flows slightly but much less than in the traditional process. Previously, it was believed that the flow during cheddaring was essential for the texture of Cheddar, but it is likely that the most important change during cheddaring is acidification which dissolves the CCP – when the Ca:protein ratio decreases to a certain value, the texture assumes the characteristics of Cheddar cheese. The manufacture of Mozzarella curd is similar to that for Cheddar up to the point at which the pH decreases to ⬃5.4. The acidified curds are then heated in hot water to 60–65 °C, kneaded and stretched. It is claimed that the kneading and stretching are essential for the characteristic fibrous texture and stretchability of Mozzarella. However, it may be that the function of heating and kneading is simply to inactivate enzymes and kill bacteria and, in effect, to stabilize the characteristics of the cheese. Heating and kneading were probably introduced originally to control the microflora of cheese curd produced from milk of poor microbiological quality. The unique manufacturing schedule for the specific varieties is not considered in this book and the interested reader is referred to appropriate texts (e.g., Van Slyke and Price, 1949; Davis, 1965, 1967; Kosikowski, 1977; Eck, 1984; Scott, 1986; Robinson, 1995; Kosikowski and Mistry, 1997; Robinson and Wilbey, 1998; Eck and Gilles, 2000). Some chemical and physico-chemical aspects of the manufacture of the major cheese families are discussed in Volume 2. Flow diagrams for some important cheese varieties are shown in Fig. 2. The last manufacturing operation is salting. While salting contributes to syneresis (⬃2 kg H2O are lost per kg NaCl taken up), it should not be used as a means of

controlling the moisture content of cheese. Salt has several functions in cheese which are described in ‘Salt in Cheese: Physical, Chemical and Biological Aspects’, Volume 1. Although salting should be a very simple operation, quite frequently it is not performed properly, with consequent adverse effects on cheese quality. As indicated previously, cheese manufacture is essentially a dehydration process. With the development of ultrafiltration as a concentration process, it was obvious that this process would have applications in cheese manufacture, e.g., for standardizing cheesemilk with respect to fat and casein, or for the preparation of a concentrate with the composition of the finished cheese, commonly referred to as ‘pre-cheese’. Standardization of cheesemilk by adding UF concentrate (retentate) is now common in some countries and the manufacture of precheese has been successful commercially for a range of soft and semi-soft cheese varieties (see ‘Application of Membrane Separation Technology to Cheese Production’, Volume 1). Ripening

Some cheeses, mainly acid-coagulated varieties, are consumed fresh and such cheeses constitute a major proportion of the cheese consumed in some countries; the principal acid-curd cheeses are described in ‘Acid- and Acid-Rennet-Curd Cheeses: Part A Quark, Cream Cheese and Related Varieties’, Part B Cottage Cheese’ and Part C ‘Acid-heat Coagulated Cheeses’, Volume 2. However, most cheese varieties undergo a period of ripening (curing, maturation) which varies from ⬃2 weeks (e.g., for Mozzarella) to 2 years (e.g., Parmigiano-Reggiano or extramature Cheddar), the duration of ripening being generally inversely related to the moisture content of the cheese. Many varieties may be consumed at any of the several stages of maturity, depending on the flavour preferences of consumers and economic factors. Although curds for different cheese varieties are recognizably different at the end of manufacture (mainly as a result of compositional and textural differences arising from differences in milk compositional and processing factors), the unique characteristics of the individual cheeses develop during ripening, although in most cases the biochemical changes that occur during ripening, and hence the flavour, aroma and texture of the mature cheese, are largely pre-determined by the manufacturing process, i.e., by composition, especially moisture, NaCl and pH, by the type of starter and in many cases by secondary inocula added to, or gaining access to, the cheesemilk or curd.

(a)

Cows’ milk Standardize casein:fat to 0.7:1.0 Pasteurize (HTST) Cool to 31°C

Starter (1–2%, v/v): Lactococcus lactis subsp. cremoris and/or Lc. lactis subsp. lactis

(b)

(c)

Cows’ milk Rennet, thermophilic starter, ±propionibacteria

30–32 °C

Cows’ milk Standardize, pasteurize, cool to 30 °C Rennet (mesophilic) DL starter nitrate

Coagulum

CaCl2, 0.02%, w/v Rennet (1:15,000)

(d)

Raw cows’ milk Evening milk Gravity creaming overnight

Cream

Semi-skimmed milk

Raw cows’ milk Morning milk

Cut coagulum, Cook to c. 55 °C

Coagulum

Coagulum Copper-plated conical vat Hot water

Whey Cut [~1 cm cubes]

Calf rennet

Stir for c. 30 min Curds and whey

Acidification by natural whey cultures

c. 36 °C

Pitch curds-whey into moulds

Coagulum

Whey Press

Break coagulum Press under whey

Cook

[Raise temperature: 30 °C to 37–39 °C over ~30 min; hold for ~1 h]

Drain

[~pH 6.1]

Dry cheese surface Apply salt to the surface

Cook to 53–55 °C Place curds in moulds Whey

1–2 days 5– 6 h

Whey

Place curds in mould

Brine salt Curds

2 days, 8–10 °C

Brine salt

Further whey drainage, light pressure

3– 5 days

Mill

Dry salt

Brine salt

Cool storage Dry rind forms

Cheddar [pH ~5.4]

[NaCl, ~2%, w/w]

Wax

10–14 days 10–15 °C, 90% ERH Hot room 20–24 °C, 80–83% erh

Fresh cheese Fresh cheese

3 weeks–2 months Eye development

Mould, press and package

Ripen Ripen ~2 years

Cool storage 1–2 months 7 °C

Fresh cheese

2–3 months at ~5 °C Parmigiano-Reggiano cheese Gouda cheese

Ripen

[3–24 months; 4–12 °C]

Emmental cheese

Mature Cheddar cheese

Figure 2 Flow diagrams for the manufacture of (a) Cheddar, (b) Emmental, (c) Gouda and (d) Parmigiano Reggiano cheeses.

13


During ripening, an extremely complex set of biochemical changes occur through the catalytic action of the following agents: • coagulant; • indigenous milk enzymes, especially plasmin and lipoprotein lipase, which are particularly important in cheese made from raw milk; • starter bacteria and their enzymes; • secondary microflora and their enzymes. This secondary microflora may arise from the adventitious microorganisms in milk that survive pasteurization or gain entry to the milk after pasteurization, e.g., Lactobacillus, Pediococcus, Micrococcus, or they may be inoculated as secondary starter, e.g., Propionibacterium in Swiss cheese, Penicillium roqueforti in Blue varieties, P. camemberti in Camembert or Brie, or the cheese may acquire a surface microflora from the environment during ripening, e.g., the complex Gram-positive microflora of smear-ripened cheeses such as Tilsit, Limburger, etc. In many cases, the characteristics of the finished cheese are dominated by the metabolic activity of these microorganisms. The primary biochemical changes which occur during ripening involve the metabolism of residual lactose and of lactate and citrate, lipolysis and proteolysis which are described in ‘Biochemistry of Cheese Ripening: Introduction and Overview’, ‘Metabolism of Residual Lactose and of Lactate and Citrate’, ‘Lipolysis and Catabolism of Fatty Acids in Cheese’, ‘Proteolysis in Cheese during Ripening’, Volume 1. These primary changes are followed and overlapped by a host of secondary catabolic changes, including the various reactions involving amino acid catabolism (transamination, deamination, decarboxylation and various lyase activities ‘Catabolism of Amino Acids in Cheese During Ripening’, Volume 1), fatty acid catabolism and related reactions (-oxidation of fatty acids, esterification, formation of thioesters) and the catabolism of lactic acid to CO2 and H2O or the propionic, acetic or butyric acids and CO2 or H2. While it is not possible to review the biochemistry involved in the ripening of all individual cheese varieties, an overview of the principal ripening reactions is presented in ‘Biochemistry of Cheese Ripening: Introduction and Overview’, ‘Metabolism of Residual Lactose and of Lactate and Citrate’, ‘Lipolysis and Catabolism of Fatty Acids in Cheese’, ‘Proteolysis in Cheese during Ripening’, ‘Catabolism of Amino Acids in Cheese During Ripening’, Volume 1. The rheological properties of cheese are reviewed in ‘Rheology and Texture of Cheese’, Volume 1. Microbiological changes which occur during ripening are discussed

in ‘The Microbiology of Cheese Ripening’, Volume 1 and the volatile flavour compounds in cheese and sensory properties of cheese are discussed in ‘Sensory Character of Cheese and its Evaluation’ and ‘Instrumental Techniques’, respectively, of Volume 1. More detailed discussions of specific aspects of the ripening of the principal families of cheese, extra-hard varieties, Cheddar, Gouda, Swiss-type cheese, mould ripened varieties, pasta-filata cheeses and sheep’s and goats’ milk are given in Volume 2 together with discussion of processed cheese products and the uses of cheese as a food ingredient. While most people consume cheese principally for its organoleptic qualities, it must be remembered that cheese is a very valuable source of nutrients, especially protein, calcium and phosphorus; see ‘Nutritional Aspects of Cheese’, Volume 1. Cheese is the quintessential convenience food which can be consumed in many forms without preparation. In addition, a large proportion of cheese (50–70%) is used as an ingredient (see ‘Cheese as an Ingredient’, Volume 2) or converted to more stable, more convenient products by heat treatment to yield processed cheeses which are discussed in ‘Pasteurized Processed Cheese and Substitute/Imitation Cheese Products’, Volume 2.

Cheese Production and Consumption World cheese production was ⬃16.5 106 tonnes in 2002 and has increased at an average annual rate of ⬃3% over the past 20 years. Europe, with a production of ⬃8.7 106 tonnes per annum (⬃53% of world production) is the largest producing region; North and Central America produces ⬃28% of world cheese. The production of cheese by country and region are shown in Table 2. Cheese consumption varies widely between countries, even within Europe; it is noteworthy that with the exceptions of Israel and the Dutch Antilles, no Asian, African or South American country is listed among the top 23 cheese-consuming countries (Table 3). Cheese consumption in most countries for which data are available has increased considerably since 1970. Thus, while cheese manufacture is practised worldwide, it is apparent from Tables 2 and 3 that cheese is primarily a product of European countries and those populated by European emigrants. However, cheese in some form is produced in most countries throughout the world and some interesting minor varieties are produced in ‘non-dairying’ countries (see Phelan et al., 1993).


Table 2 Production of all types of cheese (tonnes) in 2001 (source: www.FAO.org) World Africa Asia Europe (total) European Union (15) North and Central America South America Oceania Developed countries Developing countries Albania Algeria Angola Argentina Armenia Australia Austria Azerbaijan, Republic of Bangladesh Belarus Belgium-Luxembourg Bhutan Bolivia Bosnia and Herzegovina Botswana Brazil Bulgaria Canada Chile China Colombia Costa Rica Croatia Cuba Cyprus Czech Republic Denmark Dominican Republic Ecuador Egypt El Salvador Eritrea Estonia Ethiopia Finland France Georgia Germany Greece Guatemala Honduras Hungary Iceland Iran, Islamic Rep of Iraq

16507068 704227 1039789 8674772 6834006 4653978 709686 724615 14145817 2361251 12050 1540 1230 420000 2616 444000 145320 10750 1000 54497 65000 44 6834 8700 2214 38500 46150 359720 57184 217250 52500 6861 21879 14500 5030 139074 300000 2500 7265 465000 2400 312 15500 3975 106000 1666850 75 1773000 236200 11100 8975 89240 4860 199168 30586

Ireland Israel Italy Japan Jordan Kazakhstan Kenya Kyrgyzstan Latvia Lebanon Lithuania Macedonia,The Fmr Yug Rp Malta Mauritania Mexico Moldova, Republic of Mongolia Morocco Myanmar Namibia Netherlands, The New Zealand Nicaragua Niger Nigeria Norway Oman Panama Peru Poland Portugal Romania Russian Federation Slovakia Slovenia South Africa Spain Sudan Sweden Switzerland Syrian Arab Republic Tajikistan Tanzania, United Rep of Tunisia Turkey Turkmenistan Ukraine United Kingdom United States of America Uruguay Uzbekistan Venezuela, Boliv Rep of Yemen Yugoslavia, Fed Rep of Zambia Zimbabwe

93750 102029 1020712 123000 3662 6750 210 3500 12400 21564 57900 1540 282 2058 153861 5250 1260 7716 31976 70 660000 280615 13195 14655 6955 81700 824 7866 8934 460100 72800 37500 433000 54660 21684 36000 180374 151000 132000 162300 93475 6715 2000 6420 126156 1600 109000 382000 4073000 29320 20675 89150 11185 11500 773 2100


Table 3 Supply of cheese (kg per caput per annum) in 2000 (source: www.FAO.org) World Greece France Netherlands, The Italy Austria Germany Sweden Israel Netherlands Antilles Iceland Denmark Norway United States of America Switzerland Finland Belgium-Luxembourg Czech Republic Estonia Malta Argentina Canada Poland New Zealand Lebanon Slovakia Australia United Kingdom Hungary Portugal Ireland Slovenia Egypt Kuwait Spain Bermuda Barbados Cyprus Saint Lucia Syrian Arab Republic Grenada Croatia Lithuania French Polynesia New Caledonia Sudan Bulgaria Trinidad and Tobago Venezuela, Boliv Rep of Albania Panama Latvia United Arab Emirates Bahamas Belarus Belize Bosnia and Herzegovina Chile

2.6 25.4 23.6 22.5 20.5 19.2 18.9 17.4 16.7 16.2 15.5 15.1 15.1 14.9 14.8 14.1 13.7 13.5 13.1 13.0 12.2 11.8 11.1 10.2 9.7 9.4 9.2 9.2 8.9 8.8 8.5 8.1 7.0 6.7 6.3 6.0 5.8 5.7 5.5 5.4 5.3 5.2 5.0 4.8 4.8 4.8 4.5 4.3 4.2 4.1 4.1 3.9 3.9 3.8 3.8 3.8 3.7 3.7

Iran, Islamic Rep of Russian Federation Saudi Arabia Uruguay Botswana Japan Antigua and Barbuda Swaziland Jordan Mauritius Mexico Turkey Macedonia,The Fmr Yug Rp Dominica Romania Saint Vincent/Grenadines El Salvador Cuba Honduras Libyan Arab Jamahiriya Jamaica Costa Rica Guyana Iraq Ukraine Yugoslavia, Fed Rep of Azerbaijan, Republic of Niger Colombia Guatemala Moldova, Republic of Saint Kitts and Nevis Suriname Tajikistan Uzbekistan Seychelles South Africa Tunisia Yemen Bolivia Mauritania Algeria Armenia Dominican Republic Kyrgyzstan Myanmar Ecuador Korea, Republic of Nicaragua Cape Verde Kazakhstan Mongolia Brunei Darussalam Fiji Islands Maldives Morocco Paraguay Peru

3.0 2.9 2.8 2.8 2.7 2.6 2.3 2.3 2.2 2.2 2.0 2.0 1.9 1.8 1.8 1.8 1.7 1.6 1.6 1.6 1.5 1.4 1.4 1.4 1.4 1.4 1.3 1.3 1.2 1.2 1.2 1.2 1.1 1.1 1.1 1.0 0.9 0.9 0.9 0.8 0.8 0.7 0.7 0.7 0.7 0.7 0.6 0.6 0.6 0.5 0.5 0.5 0.4 0.4 0.4 0.4 0.4 0.4

Brazil Gabon Turkmenistan Vanuatu China Malaysia Philippines Zimbabwe Angola Djibouti Eritrea Ethiopia Gambia Georgia Haiti Kiribati Lesotho Liberia Nigeria Senegal Tanzania, United Rep of Zambia Bangladesh Benin Burkina Faso Burundi Cambodia Cameroon Central African Republic Chad Comoros Congo, Dem Republic of Congo, Republic of Côte d’Ivoire Ghana Guinea Guinea-Bissau India Indonesia Kenya Madagascar Malawi Mali Mozambique Namibia Nepal Pakistan Papua New Guinea Sao Tome and Principe Sierra Leone Solomon Islands Sri Lanka Thailand Togo Uganda Viet Nam

0.3 0.3 0.3 0.3 0.2 0.2 0.2 0.2 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0


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MacAlister, A. (1904). Food, in, A Dictionary of the Bible Dealing with Its Language, Literature and Contents, Including Biblical Theology, Vol. 2, Hastings, H., ed., T and T Clark, Edinburgh. pp. 27–43. Mair-Waldburg, H. (1974). Handbook of Cheese: Cheeses of the World A to Z. Volkwertschaftlecher Verlag GmBH, Kempten Allgan, Germany. Marui, S. (1926). Unterschungen über des halferment. III. Mitteilung die ersetzerkeit der phosphate druch andre substance. Biochem. Z. 173, 381–388. Masui, K. and Yamada, T. (1996). French Cheeses. Dorling Kindersley, London. McSweeney, P.L.H., Fox, P.F., Lucey, J.A., Jordan, K.N. and Cogan, T.M. (1993). Contribution of the indigenous microflora to the maturation of Cheddar cheese. Int. Dairy J. 3, 613–634. McSweeney, P.L.H., Walsh, E.M., Fox, P.F., Cogan, T.M., Drinan, F.D. and Castelo-Gonzalez, M. (1994). A procedure for the manufacture of Cheddar cheese under controlled bacteriological conditions and the effect of adjunct lactobacilli on cheese quality. Irish J. Agric. Food Res. 33, 183–192. Meyer, A. (1973). Processed Cheese Manufacture. Food Trade Press, London. Montandon, J. (1981). Käse aus der Schweiz. Edita SA, Lausanne, Switzerland. Nelson, J.H. (1975). Application of enzyme technology to dairy manufacturing. J. Dairy Sci. 58, 1739–1750. Ottogalli, G. (2001). Atlante dei Formaggi. Ulrico Hoepli, Milan. Palmer, L.S. (1935). Coagulation of milk, in, Fundamentals of Dairy Science. Associates of Lore, A. Rogers, Reinhold Publishing Corp., New York. pp. 205–249. Palmer, L.S. and Richardson, G.A. (1925). The colloidal chemistry of rennet coagulation. Third Colloid Symposium Monograph. Chemical Catalogue Co. Inc., New York. pp. 112–134. Phelan, J.A. (1985). Milk Coagulants – An Evaluation of Alternatives to Standard Calf Rennet. PhD Thesis. National University of Ireland, Cork. Phelan, J.A., Renaud, J. and Fox, P.F. (1993). Some nonEuropean cheese varieties, in, Cheese: Chemistry, Physics and Microbiology, Vol. 1, 2nd edn, Fox, P.F., ed., Chapman & Hall, London. pp. 421–465. Porcher, C. (1929). La methode synthetique dans l’etude du lait, le lait au point de vue colloidal, recherches sue le mechanisme de l’action de la pressure. Le Lait 9 (several articles). Porcher, C. (1930). La methode synthetique dans l’etude du lait, le lait au point de vue colloidal, recherches sue le mechanisme de l’action de la pressure. Le Lait 10 (several articles). Porcher, C. (1931). La methode synthetique dans l’etude du lait, le lait au point de vue colloidal, recherches sue le mechanisme de l’action de la pressure. Le Lait 11 (several articles). Rance, P. (1982). The Great British Cheese Book. Macmillan, London. Resmini, P., Pompei, C., Volonterio, G., Lembo, P., Lodi, R., Riva, M. and Spedicato, E. (1992). I Prodotti Caseari del Mezzogiorno. Consiglio Nationale delle Ricerche, Roma.

Richmond, H.D. (1899). Dairy Chemistry: A Practical Handbook. Charles Griffin and Co. Ltd, London. Robinson, R.K., ed. (1995). A Colour Guide to Cheese and Fermented Milk. Chapman & Hall, London. Robinson, R.K. and Tamime, A.Y. (1991). Feta and Related Cheeses. Ellis Horwood Ltd, Chichester. Robinson, R.K. and Wilbey, R.A. (1998). Cheesemaking Practice, 3rd edn, Scott, R., ed., Aspen Publishers, Gaithersburg, MD. Sammis, J.L. (1948). Cheesemaking. Cheesemaker Book Co., Madison, WI. Sandine, W.E. and Elliker, P.R. (1970). Microbiologically induced flavors and fermented foods: flavor in fermented dairy products. J. Agric. Food Chem. 18, 557–562. Sardinas, J.L. (1972). Microbial rennets. Adv. Appl. Microbiol. 15, 39–73. Scott, R. (1986). Cheesemaking Practice. Applied Science Publishers, London. Shakeel-Ur-Rehman, McSweeney, P.L.H. and Fox, P.F. (1999). A study on the role of the indigenous microflora on the ripening of Cheddar cheese. Milchwissenschaft 54, 388–392. Shakeel-Ur-Rehman, McSweeney, P.L.H., Banks, J.M., Brechany, E.Y., Muir, D.D. and Fox, P.F. (2000a). Ripening of Cheddar cheese made from blends of raw and pasteurized milk. Int. Dairy J. 10, 33–44. Shakeel-Ur-Rehman, Banks, J.M., McSweeney, P.L.H. and Fox, P.F. (2000b). Effect of ripening temperature on the growth and significance of non-starter lactic acid bacteria in Cheddar cheese made from raw of pasteurized milk. Int. Dairy J. 10, 45–55. Shakeel-Ur-Rehman, Banks, J.M., Brechany, E.Y., Muir, D.D., McSweeney, P.L.H. and Fox, P.F. (2000c). Influence of ripening temperature on the volatiles profile and flavour of Cheddar cheese made from raw or pasteurized milk. Int. Dairy J. 10, 55–65. Simon, A.L. (1956). Cheeses of the World. Faber & Faber, London. Squire, E.H., ed. (1937). Cheddar Gorge: A Book of English Cheeses. Collins, London. Sternberg, M. (1976). Microbial rennets. Adv. Appl. Microbiol. 20, 135–157. Van Slyke, L.L. and Price, W.V. (1949). Cheese. Orange Judd, New York. Vizzardi, M. and Maffeis, P. (1999). Formaggi Italliane: StoriaTecnologia e Microbiologia lattiero-casearia. Edizioni Agricole della Calderini, Bologna. Wake, R.G. (1959). Studies on casein. V. The action of rennin on casein. Aust. J. Biol. Sci. 12, 479–489. Walter, H.E. and Hargrove, R.C. (1972). Cheeses of the World. Dover Publications, Inc., New York. Waugh, D.F. and von Hippel, P.H. (1956). -Casein and the stabilization of casein micelles. J. Am. Chem. Soc. 78, 4576–4582. White, K.D. (1970). Roman Farming. Thames and Hudson, London. Zehren, V.L. and Nusbaum, D.D. (1992). Process Cheese. Cheese Reporter Publishing Co., Madison, WI.

Rennets: General and Molecular Aspects M.J.C. Crabbe, Division of Cell and Molecular Biology, School of Animal and Microbial Sciences, The University of Reading, UK

Introduction Natural chymosin may consist of up to six molecular species, corresponding to genetic variants A and B, each of which is a mixture of three forms differing at the N-terminus, with one being three residues longer, and the other two residues shorter, than the mature chymosin (Lilla et al., 2001). The function of chymosin is to coagulate milk in the stomach. Rennet may be considered as a functional enzyme preparation that is effectively and naturally adapted to the purposes of cheesemaking (Ye et al., 2000). Proteolytic enzymes can be classified on the basis of their catalytic activity into one of the four groups – serine, cysteine, metallo and aspartic proteinases (Kay, 1985). Chymosin (rennin; EC 3.4.23.4) is a neonatal gastric aspartic proteinase and is of commercial importance in cheesemaking. It belongs to the aspartic proteinase family which is widely distributed in many organisms and tissues with different physiological and functional properties (Chitpinityol and Crabbe, 1998). The nucleotide and amino acid sequences and the three-dimensional structures of several aspartic proteinases are available and provide information for the protein engineering design of this protein family. Enzymes can now be produced recombinantly in various expression systems in sufficient amounts for structural and functional studies.

Chymosin and Other Aspartic Proteinases Aspartic proteinases contain two aspartyl residues (Asp32 and Asp215, pepsin numbering) at the active site (Tang et al., 1973). They are susceptible to inhibition by pepstatin, a pentapeptide naturally produced by Streptomyces strains (Umezawa et al., 1970), and to affinity labelling at the catalytic aspartates using either diazoacetylnorleucinemethyl ester (DAN) in the presence of cupric ions (Rajagopalan et al., 1966) or 1,2epoxy-3-(p-nitrophenoxy) propane (EPNP). Natural sources

Aspartic proteinases can be found throughout nature from viruses to higher plants and mammals. They are

generally divided into two major groups – pepsin-like and retroviral enzymes. These enzymes have been isolated from five major sources: a. The stomach. Several types of gastric enzyme, pepsin (EC 3.4.23.1), pepsin B (EC 3.4.23.2), gastricsin (EC 3.4.23.3) and chymosin (EC 3.4.23.4), are produced in the abomasal mucosa as inactive precursors, zymogens. Pepsin is the predominant proteinase in adult mammals (Tang et al., 1973). Gastricsins are found in all parts of the mammalian stomach, -cells of pancreatic islet, prostate gland and seminal vesicles. Chymosin is produced early on during gestation (in utero) in the abomasal mucosa of newborn mammals, including calf (Foltmann, 1970), piglet (Foltmann et al., 1978), kitten (Jensen et al., 1982), seal (Shamsuzzaman and Haard, 1984) and lamb (Baudys et al., 1988; Pungercar et al., 1991). The production of these enzymes varies, depending on the age of the animal and the feeding regime (Andrén and Björck, 1986). b. Lysosomes of many cell types contain cathepsin D (Hurley et al., 2000) and cathepsin E. Cathepsin E is found in gastric mucosa, thymus, spleen and blood cells (Kageyama, 1995). Human cathepsin D is possibly involved in the degradation of intracellular and endocytosed proteins, and is a prognostic indicator of breast tumour invasiveness. There appears to be a role for this enzyme during proteolysis in cheese ripening, most clearly in cheese where rennet activity is low, such as Swiss cheese, Quarg and Feta. c. Tissues such as kidney and sub-maxillary gland produce renin (Kay, 1985). d. Plants, including squash, cucumber, tomato, barley, rice, wheat, sorghum and lotus (Doi et al., 1980; Morris et al., 1985; Polanowski et al., 1985; Belozersky et al., 1989). e. Micro-organisms. Several aspartyl proteinases are secreted by fungi, including Cryphonectria parasitica (Sardinas, 1968), Penicillium janthinellum (Hofmann and Shaw, 1964), Rhizomucor pusillus (Arima et al., 1970), Rhizomucor miehei (Sternberg, 1971), Rhizopus chinensis (Fumamoto et al., 1967), Aspergillus awamori



20 Rennets: General and Molecular Aspects

(Ostoslavskaya et al., 1986), Aspergillus niger (Koaze, et al., 1964) and Trichoderma reesei (Pitts, 1992). Proteinases have been found in the yeasts Saccharomyces cerevisiae (MacKay et al., 1988), Candida tropicalis (Togni et al., 1991) and Yarrowia lipolytica (Yamada and Ogrydziak, 1983). Thermopsin is secreted by Sulfolobus acidocaldarius, a thermophilic archaebacterium (Lin and Tang, 1990). Retroviral aspartic proteinases are dimeric, and each monomer is about half the size of a eukaryotic aspartic proteinase and carries only one catalytic aspartic residue. Retropepsins have been found in several viruses, including human immunodeficiency virus (HIV), Rous sarcoma virus, avian myeloblastosis virus and simian immunodeficiency virus (SIV) (Toh et al., 1985; Kotler et al., 1989). These proteinases are required for processing for RNA dimerization within the virion, and hence for infectivity. Physical properties and stability of aspartyl proteinases Molecular weight and isoelectric point

Chymosin and aspartic proteinases have a molecular weight in the range 32–39 kDa, with a multiplicity of isoelectric points corresponding to a number of isozymes, auto-degradation and post-translationally modified products. N-linked glycosylation has been found in several proteinases such as cathepsin D (N67 and N183), S. cerevisiae proteinase A (N67 and N263), rhizomucor protease (N173) and human renin (N67). Specific receptors for phosphorylation have been found in porcine pepsin at S68 (Tang et al., 1973), bovine pepsin and human cathepsin D (Martin and Corre, 1984; Metcalf and Fusek, 1993). Transgenic sheep chymosin appears to be identical to calf chymosin (Mezina et al., 2001). Enzyme stability

Chymosin is most stable at pH values between 5.3 and 6.3. However, even at pH 2, chymosin is relatively stable (Foltmann, 1959a). Under acidic conditions (pH 3–4), the enzyme loses its activity rapidly, probably caused by auto-degradation, while at alkaline pH values (above 9.8), loss is due to an irreversible conformational change (Cheeseman, 1965). Loss of activity of chymosin A is higher than for chymosin B (Foltmann, 1966). Chymosin is more stable at 2 °C than at room temperature (Foltmann, 1959b). Kawaguchi et al. (1987) reported the rapid loss of the activity of chymosin when the temperature is increased from 45 to 55 °C. Photo-oxidation of histidine, as well as modification of the -amino group of lysine, slightly affects the activity of chymosin (Hill and Laing, 1965; Smith et al., 1991b,c). Chymosin

loses approximately half of its activity after incubation in 4.6 mol/l urea at 37 °C for 30 min (Sugrue et al., 1990). It has been shown that both pro-part and cysteine residues are essential for refolding of chymosin after denaturation (Sugrue et al., 1990; Huang et al., 1992). Chymosin in a crystalline form appears to be very stable (Foltmann, 1992). Prochymosin is more stable than chymosin at neutral pH (Foltmann, 1966). At pH values below 5.0, prochymosin is converted to chymosin whereas at pH above 11.0 the stability of prochymosin is lost due to a conformational change. Pseudochymosin is stable at acidic pH for days but is quickly converted to chymosin if the pH is increased above 4.5 (Barkholt et al., 1979). Rhizomucor protease, cryphonectria protease and S. cerevisiae proteinase A are stable at pH 3.5–7.0 (Sardinas, 1968; Dreyer et al., 1986; Bailey and Siika-aho, 1988). Pepsin shows greater general stability than chymosin; for example, after incubation in 6 mol/l urea at 37 °C for 30 min, only 10% of the original activity is lost (Cheeseman, 1965). The thermostability of pepsin is reduced in solution at high pH, in the presence of urea or salt solution, but is increased in the presence of pepstatin (Privalov et al., 1981). At pH 6.0, pepsin is more stable than pepsinogen. At pH values between 8.5 and 10.5, pepsinogen is less stable than prochymosin and cannot be converted to the active form in an acidic environment (McPhile, 1975). Inactivation of pepsin can be initiated by dissociation of the N-fragment, and the sequence of this portion is a major determinant of enzyme stability (Tanaka and Yada, 2001). Prochymosin can be efficiently refolded in high yields by controlled air oxidation (Menzella et al., 2002). Aspartic proteinases containing carbohydrate are more stable towards high temperature, denaturants and degradation than proteins without carbohydrate (Aikawa et al., 1990; Berka et al., 1991; Brown and Yada, 1991). Glycosylation of rhizomucor protease by either chemical or genetic modification resulted in a loss of stability and an increase in the ratio of milk-clotting activity to proteolytic activity (C/P ratio) (Brown and Yada, 1991; Aikawa et al., 1992). The stability of rhizomucor proteinase was reduced by pre-treatment with acid, oxidation of methionine or modification of the -amino group of lysine (Hubble and Mann, 1984; Smith et al., 1991b). Enzyme solubility

The solubility of chymosin is affected by pH, temperature and ionic strength of the solution (Foltmann, 1959b). Non-crystallized chymosin is soluble in solution containing 1 mol/l NaCl and at pH 5.5. In a solution of 2 mol/l NaCl, chymosin appears to be insoluble. Crystallized chymosin shows higher solubility at 25 °C

Rennets: General and Molecular Aspects 21

than at 2 °C (Foltmann, 1970); however, amorphous precipitates of chymosin are more stable at 2 °C than at 25 °C. At pH values close to the isoelectric point, chymosin is very insoluble at an ionic strength of 0.005; its solubility is increased by increasing its ionic strength. Structure of chymosin and other aspartic proteinases Gene sequence and primary structure

The genomic DNA of avian and mammalian aspartic proteinases, chicken embryonic pepsinogen (Hayashi et al., 1988), human renin (Miyazaki et al., 1984), bovine chymosin (Hidaka et al., 1986) and human pepsinogen (Sogawa et al., 1983), is comprised of nine exons separated by eight introns, and all exon–intron junction points are highly conserved. These results support the belief that the genes for these enzymes have evolved from a common ancestral gene. Conversely, in several microbial aspartic proteinases, including those of S. cerevisiae (Ammerer et al., 1986), C. tropicalis (Togni et al., 1991), R. pusillus (Tonouchi et al., 1986) and R. miehei (Gray et al., 1986), no intron was found in the genes for these enzymes. However, in the genes for the aspartic proteinases of R. niveus (Horiuchi et al., 1988) and A. awamori (Berka et al., 1990), one and three short introns, respectively, were found, but their exon–intron junctions were at different positions from those in the genes for mammalian and avian aspartic proteinases. Calf chymosin is found in two major forms, A and B, chymosin B being more abundant. Chymosins A and B differ at only one amino acid position: chymosin A has an aspartate residue at position 243 (pepsin numbering), whereas this is a glycine residue in chymosin B. A third form, chymosin C, appears to be a degradation product of chymosin A that lacks three residues, D244–F246 (Danley and Geoghegan, 1988). It is likely that chymosins A and B are synthesized from different alleles of the same polymorphic gene, rather than a multiple gene family, as only one locus of the chymosin gene was found from the hybridization of the calf genome with the chymosin gene (Donnelly et al., 1986). Fig. 1 illustrates the nucleotide (cDNA) and amino acid sequences of calf chymosin B. The secretion signals of aspartic proteinases are approximately 15–24 residues long with low sequence homology (Fig. 2). These secretion sequences tend to be rich in hydrophobic amino acids. The known pro-regions of aspartic proteinases are shown in Fig. 3. The pro-peptides are 38–54 amino acid residues in length and are rich in basic residues. Although sequence identity is high among the closely related enzymes, there are variables in the cleavage site between the pro-segment and the mature enzyme.

A lysine residue (K36P; pepsinogen numbering) is conserved in all proteinases, except lamb prochymosin and barley aspartic proteinase, and this residue has been postulated to interact with the catalytic aspartate residues in the zymogen molecule (James and Sielecki, 1986; Foltmann, 1988). The pro-segments are probably important for correct folding, targeting and control of the activation of zymogens (Koelsch et al., 1994). Chymosin is a single polypeptide chain enzyme of 323 amino acid residues with a low content of basic residues, and rich in dicarboxylic and -hydroxy amino acid residues (Foltmann et al., 1977, 1979; Harris et al., 1982; Moir et al., 1982; Hidaka et al., 1986). The sequence alignment of calf chymosin with those of lamb chymosin, porcine pepsin, penicillopepsin, rhizopus protease and S. cerevisiae proteinase A is illustrated in Fig. 4. There are variable numbers of cysteine residues in their sequences but their positions, when present, are conserved. Therefore, there is the potential for two disulfide bridges in the Rhizomucor and Rhizopus enzymes, a single disulfide bridge in the Cryphonectria, Penicillium and Aspergillus enzymes, and no disulfide bridges in the Irpex aspartic proteinase. Secondary structure

The secondary structure of chymosin consists mainly of -sheets with a few small -helical segments. The secondary structure of chymosin is illustrated in Fig. 5. The sheets and helices are named by analogy to the scheme adapted for cryphonectria protease (Blundell et al., 1985, 1990). The strands are named aN, bN, cN, dN, aN, bN, cN, dN, qN and rN in the N-terminal domain and aC, bC, cC, dC, aC, bC, cC, dC, qC and rC in the C-terminal domain. The helices are named hN and hC in the N- and the C-terminal domains, respectively. The antiparallel -strands form three welldefined sheets (Newman et al., 1991). The sheets, 1N and 1C, are formed by seven or eight strands in a similar pattern in both lobes and are related by a topological two-fold axis. The b, c, b and c strands form sheets 2N and 2C which occur beneath 1N and 1C, respectively. Sheet 3 is formed by six -strands, aN, rN, qN, qC, rC and aC, all of which are antiparallel. This sheet resides beneath the strands forming the base of the active site cleft. In each lobe, strands labelled a, b, c, d are related to a, b, c, d by the intra-lobe diad and these strands are related to their equivalents in the opposite lobe by the inter-lobe diad. The helices hN, hN, hC and hC occur in topological intra- and interdomain two-fold symmetry in that they all occur after the d strands. The fifth helix occurs between the cN and dN strands and the sixth occurs at a large insertion in the C-terminal domain.


CCC AGA TCC AAG ATG AGG TGT CTC GTG GTG CTA CTT GCT GTC TTC GCT CTC TCC CAA GGC GCT M R C L V V L L A V F A L S Q G A PP1 P1 GAG ATC ACC AGG ATC CCT CTG TAC AAA GGC AAG TCT CTG AGG AAG GCG CTG AAG GAG CAT GGG G I T R I P L Y K G K S L R K A L K E H G P10 P20 CTT CTG GAG GAC TTC CTG CAG AAA CAG CAG TAT GGC ATC AGC AGC AAG TAC TCC GGC TTC * GGG L L E N F L E K E E Y G I S S K Y S G F G P30 P40 1 GAG GTG GCC AGC GTG CCC CTG ACC AAC TAC CTG GAT AGT CAG TAC TTT GGG AAG ATC TAC CTC E V A S V P L T N Y L D S Q Y F G K I Y L 10 20 GGG ACC CCG CCC CAG GAG TTC ACC GTG CTG TTT GAC ACT GGC TCC TCT GAC TTC TGG GTA CCC G T P P N E F T V L F D T G S S D F W V P 30 40 TCT ATC TAC TGC AAG AGC AAT GCC TGC AAA AAC CAC CAG CGC TTC GAC CCG AGA AAG TCG TCC S I Y C K S N A C K N H Q R F D P R K S S 50 60 ACC TTC CAG AAC CTG GGC AAG CCC CTG TCT ATC CAC TAC GGG ACA GGC AGC ATG CAG GGC ATC T F Q N L G K P L S I H Y G T G S M Q G I 70 80 CTA GGC TAT GAC ACC GTC ACT GTC TCC AAC ATT GTG GAC ATC CAG CAG ACA GTA GGC CTG AGC L G Y D T V T V S N I V D I Q Q T V G L S 90 100 ACC CAG GAG CCC GGG GCA GTC TTC ACC TAT GCC GAA TTC GAC GGG ATC CTG GGG ATG GCC TAC T Q E P G D V F T Y A E F D G I L G M A Y 110 120 CCC TCG CTC GCC TCA GAG TAC TCG ATA CCC GTG TTT GAC AAC ATG ATG AAC AGG CAC CTG GTG P S L A S E Y S I P V F D N M M N R H L V 130 140 GCC CAA GAC CTG TTC TCG GTT TAC ATG GAC AGG AAT GGC CAG GAG AGC ATG CTC ACG CTG GGG A Q D L F S V Y M D R N G Q E S M L T L G 150 160 GCC ATC AAC CCG TCC TAC TAC ACA GGG TCC CTG CAC TGG GTG CCC GTG ACA GTG CAG CAG TAC A I N P S Y Y T G S L H W V P V T V Q Q Y 170 180 190 TGG CAG TTC ACT GTG GAC AGT GTC ACC ATC AGC GGT GTG GTT GTG GCC TGT GAG GGT GGC TGT W Q F T V D S V T I S G V V V A C E G G C 200 210 CAG GCC ATC TTG GAC ACG GGC ACC TCC AAG CTG GTC GGG CCC AGC AGC GAC ATC CTC AAC ATC Q A I L D T G T S K L V G P S S D I L N I 220 230 CAG CAG GCC ATT GGA GCC ACA CAG AAC CAG TAC GGT GAG TTT GAC ATC GAC TGC GAC AAC CTG Q Q A I G A T Q N Q Y G E F D I D C D N L 240 250 AGC TAC ATG CCC ACT GTG GTC TTT GAG ATC AAT GGC AAA ATG TAC CCA CTG ACC CCC TCC GCC S Y M P T V V F E I N G K M Y P L T P S A 260 270 TAT ACC AGC CAA GAC CAG GGC TTC TGT ACC AGT GGC TTC CAG AGT GAA AAT CAT TCC CAG AAA Y T S Q D Q G F C T S G F Q S E N H S Q K 280 290 TGG ATC CTG GGG GAT GTT TTC ATC CGA GAG TAT TAC AGC GTC TTT GAC AGG GCC AAC AAC CTC W I L G D V F I R E Y Y S V F D R A N N L 300 310 GTG GGG CTG GCC AAA GCC ATC TGA TCACATCGCTGACCA........... V G L A K A I 320 323

Figure 1 Nucleotide and amino acid sequence of calf chymosin B cDNA (adapted from Moir et al., 1982).


Secretion signal peptides Fungi A. awamori 1 A. oryzae 2 R. miehei 3 R. pusillus 4 R. niveus 5 R. chinensis 6

MVVFSKTAALVLGLSSAVSA*A MVILSKVAAVAVGLSTVASA*L MLFSQITSAILLTAASLSLTTA*R MLFSKISSAILLTAASFALTSA*R MKFTLISSCVALAAMTLAVEAA*P MTFTLNSSCIAIAALAVAVNAA*P

Mammals Bovine chymosin7 Lamb chymosin8 Porcine pepsin9 Human pepsin10 Rat pepsin11 Rat cathepsin D12

MRCTVVLLAVFALSQG*A MRCLVVLLAVFALSQG*A MKWLLLLSLVVLSEC*L MKWLLLLGLVALSE*C MKWMVVALLCLPLLEA*S MQTPGVLLLILGLLDASS*S

Yeast MFSLKALLPLALLLVSANQVAA*K S. cerevisiae (YPA)13 S. cerevisiae (BAR1)14 MSAINHLCLYLILASFAIINTITA*L MATIFLFTKNVFIA.LA.FA.L C. tropicalis15 Plant Barley APR16

MGTRGLALALLAAVLLQTVPAASEA*E

Figure 2 Alignment of the secretion signal peptides of aspartic proteinases. The junctions between putative signal sequence and proenzyme are indicated by (*) and possible sites are indicated by (•). References: (1) Berka et al. (1990); (2) Ward and Kodama (1991); (3) Gray et al. (1986) and Boel et al. (1986); (4) Tonouchi et al. (1986); (5) Chen et al. (1991); (6) Horiuchi et al. (1988); (7) Harris et al. (1982) and Moir et al. (1982); (8) Pungercar et al. (1991); (9) Lin et al. (1989); (10) Hayano et al. (1988); (11) Ishihara et al. (1989); (12) Birch and Loh (1990); (13) Ammerer et al. (1986); (14) MacKay et al. (1988); (15) Togni et al. (1991); (16) Runeberg-Roos et al. (1991) (adapted from Orprayoon, 1994).

Tertiary structure

The three-dimensional structure of several aspartic proteinases has been solved by X-ray crystallography (Fig. 6). These include porcine pepsin (Andreeva et al., 1984; Abad-Zapatero et al., 1990; Cooper et al., 1990; Sielecki et al., 1990), pepsinogen ( James and Sielecki, 1986; Hartsuck and Remington, 1988), human renin (Sielecki et al., 1989), cryphonectria protease (Blundell et al., 1990), penicillopepsin ( James and Sielecki, 1983), rhizopus protease (Suguna et al., 1987) and retroviral proteinases (Lapatto et al., 1989; Miller et al., 1989; Wlodawer et al., 1989). Crystals of chymosin obtained by Bunn et al. (1971) showed that the space group was either I222 or I212121, with one molecule in the asymmetric unit. The structure of recombinant bovine chymosin has been independently solved and refined at 2.3 Å resolution (Gilliland et al., 1990) and at 2.2 Å resolution (Newman et al., 1991). Crystallographic studies at 2.0 Å resolution have also been performed on a site-specific mutant of chymosin, in which V111 was replaced by phenylalanine, and the structure has been refined to an R-factor of 19.5% (Strop et al., 1990). All the molecules display very similar secondary and tertiary structures.

The structure of chymosin B described below was solved by Gilliland et al. (1990) and by Newman et al. (1991). The crystals of chymosin have the space group of 1222 with approximate overall dimensions of 40 60 65 Å (Gilliland et al., 1990). The protein has a bilobal folding pattern formed by the N- and the C-terminal domains divided by a deep active-site cleft. A 2.5-Å extended cleft contains the catalytic aspartates and the substrate-binding pockets. These two lobes are related by an approximately 2-fold axis which passes between the two catalytic aspartate residues, 32 and 215, and forms the approximate intra-molecular symmetry (Fig. 7). High symmetry between the N- and the C-lobes is found inside the active site and the core of the enzyme (Newman et al., 1991). Intra-domain pseudo-diad axes in the N- and the C-domains of chymosin have rotations of 180° and 177°, respectively, with negligible translations (Newman et al., 1991). There are three disulphide bridges at positions 45 . . . 50, 206 . . . 210 and 249 . . . 282. In addition, ion-pairs are found between R59 . . . D57, R157 . . . E308, R157 . . . I326 (COO ), R307 . . . D11 and R315 . . . D138 (Gilliland et al., 1990; Newman et al., 1991). Chymosin also contains a single cis-proline, P23, on the -turn connecting strand bN to cN (Gilliland et al., 1990; Newman et al., 1991). In rhizomucor protease, cryphonectria protease and porcine pepsin, a cis-proline is found at an identical position to that in chymosin (Blundell et al., 1990; Cooper et al., 1990; Newman et al., 1993) while two cis-proline residues are found at positions 23 and 324 in rhizopus protease (Suguna et al., 1987) and three cisproline residues are found at positions 111, 194 and 297 in human renin (Dhanaraj et al., 1992). The active site of aspartic proteinases is highly conserved and consists of residues, Asp9Thr9Gly, from each domain of the enzyme. Nine per cent sequence identity is observed between the N- and the C-terminal lobes of chymosin (Newman et al., 1991). A comparison of chymosin structure with that of other aspartic proteinases reveals a high degree of structural homology (Gilliand et al., 1990). Chymosin has the closest structural agreement with porcine pepsin. Of the fungal proteinases, the rhizopus protease molecule has higher structural homology with chymosin than with penicillopepsin or cryphonectria protease. The structural superposition of aspartic proteinases reveals that the N-terminal domain has greater structural similarity than the C-terminal domain (Gilliland et al., 1990). The C-terminal domain is more separated from the rest of the molecule than the N-terminal domain, and the rigid body movement appears in the C-terminal domain (residues 190–302) (Sali et al., 1992). The greatest differences between these proteinases are in the surface loop regions. One remarkable difference is the


Propeptide Fungi A. awamori APR1 A. oryzae APR2 A. miehei APR3 M. pusillus APR4 R. niveus APR5 R. chinensis APR6

APAPRTRKGFTINQIARPANKTRTINLPGMYARS-------LA-KFGGTVPQSVKEA-A*SK LPTGPSHSPHARRGFTINQITRQTARVGPKTASFPAIYSRALA-KYGGTVPAKLKSAVA*GH RPVSKQSESKDKLLALPLTSVSRKFSQTKFGQQQ-------LAEKLAG-----LKPFSE*AA RPVSKQSDADDKLLALPLTSVNRKYSQTKHGQQ--------AAEKLGG-----IK-A-F*AE PNGKKINIPLAKNN----SY-KPSA--KNALNKA------LA-KYNRRKVGSGGITTE*AS PGEKKISIPLAKNP----NY-KPSA--KNAIQKA------IA-KYNKHKINTSTGGIV*AG

Mammals Bovine prochymosin7 Lamb prochymosin8 Chicken pepsinogen9 Porcine pepsinogen10 Human pepsinogen11 Human progastricsin12 Mouse prorenin13 Human prorenin14 Human procathepsin D15 Rat procathepsin D16 Yeast S. cerevisiae APR17 C. tropicalis APR18 Plant Barley APR19

AEITRIPLYKGKSLRKAL-KEHGLLE-DFLQKQQYG-ISSKYS-------GF*GE AEITRIPLYKGKPLRKAL-KERGLLE-DFLQKQQYG-ISSEYS-------GF*GE SIHRVPLKK GKSLRKQL-KDHGLLE-DFLKKHPYN-PASKYHPV------L*TA LVKVPLVRKKSLRQNLIKD-GKLK-DFLKTHKHN-PASKYFPE---AAAL*IG IMYKVPLIRKKSLRRTL-SERGLLK-DFLKKHNLN-PARKYFPQWE-APTL*VD AVVKVPLKKFKSIRETM-KEKGLLG-EFLRTHKYD-PASKYRFGD-----L*SV TFSLPTRTATFERIPLKKMPSVREIL-EERG--V-DMIRLSAEWGVFTK----------R*PS TFGLPTDTTTFKRIFLKRMPSIRESL-KERG--V-DMARLGPEWSQPMK----------R*LT SALVRIPLHKFTSIRRTM-SEVGGSVEDLIAK----GPVSKYSQAV-PAVTE*GP SALIRIPLRKFTSIRRTM-TEVGGSVGDLI----LKGPITKYSMQSSPRTKE*PV KVHKAKIYKHELSDEMKEVTFEQHLAHLGQKYLTQFEKANPEVVFSREHPFFTE*GG LAFALFAQGLTIPD-----GIEKRTDKVVSLDFTVIRKPFNATAHR---LIQKR*SD EGLVRIALKKRP-IDRNSRVATGLSGGEEQP---LLSG------AN---PLR*SE

Figure 3 Alignment of the propeptides of aspartic proteinases. The junctions between proenzyme and mature enzyme are indicated by (*). References: (1) Berka et al. (1990); (2) Ward and Kodama (1991); (3) Gray et al. (1986) and Boel et al. (1986); (4) Tonouchi et al. (1986); (5) Horiuchi et al. (1988); (6) Chen et al. (1991); (7) Harris et al. (1982) and Moir et al. (1982); (8) Pungercar et al. (1991); (9) Baudys and Kostka (1983); (10) Lin et al. (1989); (11) Sogawa et al. (1983); (12) Wong and Tang (1986); (13) Holm et al. (1984); (14) Imai et al. (1983); (15) Faust et al. (1985); (16) Birch and Loh (1990); (17) Ammerer et al. (1986); (18) Togni et al. (1991); (19) Runeberg-Roos et al. (1991) (adapted from Orprayoon, 1994).

position of the flap (residues 73–85 in chymosin). This region participates in the substrate-binding specificity. In chymosin, the position of Y77 is stabilized by interaction with hydrophobic residues F119 and L32 (Gilliland et al., 1990). In other aspartic proteinases, Y77 hydrogenbonds to W39. In pepsin, the location of the hydroxyl group of Y77 is occupied by a water molecule, w424, in the chymosin crystal. This water molecule forms two hydrogen bonds with the hydroxyl group of Y75 and with the conserved water molecule, w403. In the V111F mutant chymosin, the flap appears to occupy two different conformations corresponding to that found in native chymosin and pepsin (Strop et al., 1990). This suggests that chymosin can exist in two alternative structural forms: the active form in which S1 and S3 binding pockets are free for substrate binding and the self-inhibited form in which these pockets are occluded by its own Y77 residue (Andreeva et al., 1992; Gustchina et al., 1996). The structure of S. cerevisiae proteinase A agrees overall with other uninhibited aspartic proteinases, although the conformation of Y75 occupying the S1 substratebinding pocket is similar to that in chymosin, suggesting a functional significance for this conformation (Gustchina et al., 2002). The conversion of chymosin

from the self-inhibited to the active form can be promoted by an allosteric activator, the histidine–proline cluster (9His9Pro9His9Pro9His9) of -casein, thereby explaining the catalytic specificity of chymosin towards -casein. Three-dimensional structures of homodimer retroviral proteinases are to a large extent similar and bear close resemblance to the structure of bilobal fungal and mammalian aspartic proteinases (Lapatto et al., 1989; Miller et al., 1989; Navia et al., 1989; Wlodawer et al., 1989). The structural superpositions between the retroviral enzymes and the eukaryotic aspartic proteinases appear to be similar. It is not clear whether the eukaryotic proteinases are derived from a homodimer enzyme by gene duplication and fusion (Tang et al., 1978) or evolved from a cellular gene by one or more deletion events (Rao et al., 1991). Nevertheless, an engineered homodimer of the pepsin N-terminal lobe, which exhibits a general proteolytic activity, reveals the close relationship between these two aspartic proteinase families (Lin et al., 1992). The active site

The active-site aspartates, D32 and D215, are situated on the corners of the two extended loops (-structures


10

4CMS 4PEP 2APP 2APR 4APE YPE

Strand a´ 60 70

4CMS 4PEP 2APP 2APR 4APE YPE

50

90

Strand c´ 100

110

Strand q 150

140

Strand r 160

Strand b 190

Strand c 200

210

Strand d 220

AIDPSYYTGSLHWVPVTV-QQYWQFTVDSVTISGVVVACEGGCQAILDTGTSKLVGPSSD GIDSSYYTGSLNWVPVSV-EGYWQITLDSITMDGETIACSGGCQAIVDTGTSLLTGPTSA FIDSSKYTGSLTYTGVDNSQGFWSFNVDSYTAGSQ-SG-DG-FSGIADTGTTLLLDDSVV GYDSTKFKGSLTTVPIDNSRGWWGITVDRATVGTSTVA-SS-FDGILDTGTTLLILPNNI FIDTTAYTGGITYTAVSTLQHFWEWTSTGYAVGSGTFKSTS-IDGIADTGTTLLYLPATV GIDESKFKGDITWLPVRRK-AYWEVKFEGIGLGDEYAELES-HGAAIDTGTSLITLPSGL

240

Strand b´ 250

260

Strand c´ 270

280

ILNIQQAI-GATQNQ-YGEFDIDCDNLSYMPTVVFEINGKMYPLTPSAYTSQD---QGFC IANIQADI-GASENS-DGEMVISCSSIDSLPDIVFTIDGVQYPLSPSAYILQD---DDSC VSQYYSQVSGAQQDSNAGGYVFDCST--NLPDFSVSISGYTATVPGSLINYGPSGDGSTC AASVARAY-GASDNS-DGTYTISCDT-SAFKPLVFSINGASFQVSPDSLVFEEF--QGQC VSAYWAQVSGAKSSSS-VGYVFPCSAT--LPSFTFGVGSARIVIPGDYIDFGPISTGSSC AEMINAEI-GAKKGW-TGQYTLDCNTRDNLPDLIFNFNGYNFTIGPYDYTLEV---SGSC Strand d´ 290

4CMS 4PEP 2APP 2APR 4APE YPA

Strand b´ 80

130

Strand a 180

Strand a´ 230


40

AEFDGILGMAYPSLASE---YSIPVFDNMMNRHLVAQDLFSVYMDRNG----QESMLTLG APFDGILGLAYPSISAS---GATPVFDNLWDQGLVSQDLFSVYLSSNG---DSGSVVLLG TNNDGLLGLAFSSINTVQPQSQTTFFDTVKSS-L-AQPLFAVALKHQ-----QPGVYDFG -PNDGLLGLGFDTITTVR--GVKTPMDNLISQGLISRPIFGVYLGKAKN--GGGGEYIFG STIDGLLGLAFSTLNTVSPTSQQTFFDNAKA-S-LDSPVFTADLGY-----HAPGTYNFG GKFDGILGLGYDTISVD---KVVPPFYNAIQQDLLDEKRFAFYLGDTSKDTENGGEATFG

170


30

FDPRKSSTFQNL-GKPLSIHYGT-GSMQGILGYDTVTVSNIVDIQQTVGLSTQEPGDVFTY FNPQDSSTYQST-SGELSITYGT-GSMTGILGYDTVQVGGISDTNQIFGLSETEPGSFLYY YNPSA--TGKELSGYTWSISYGDGSSASGNVFTDSVTVGGVTAHGQAVQAAQQISAQFQQD YDPNQSSTYQAD-GRTWSISYGDGSSASGILAKDNVNLGGLLIKGQTIELAKREAASFASG YTPSKSTSTKLLSGATWSISYGDGSSSSSDVYTDTVSVGGLTVTGQAVESAKKVSSSFTED YDHEASSSYKAN-GTEFAIQYGTG-SLEGYISQDTLSIGDLTIPKQDFAEATSEPGLTFAF Strand d´ 120


20

GEVASVPLTNY-LDSQYFGKIYLGTPPNEFTVLFDTGSSDFWVPSIYCKSNAC-KNHQR IGDEPLENY-LDTEYFGTIGIGTPAQDFTVIFDTGSSNLWVPSVYCSSLAC-TNHNL AASGVATNTPTANDIEEYIPVTIG--GTTLNLNFDTGSSDLWVFSTELP-ASQQSGHSV AGVGTVPMTDYGNDIEYYGQVTIGTPGKKFNLDFDTGSSDLWIASTLCT--NCGSGQTK STYSATTTPIDSLDDAYITPVQIGTPAQTLNLDFDTGSSDLWVFSSETTASE-VDGQTI GGH-DVPLTNYLNA-QYYTDITLGTPPQNFKVILDTGSSNLWVPSNECGSLAC-FLHSK

Strand q 300

Strand r 310 320

TSGFQSENHS----QKWILGDVFIREYYSVFDRANNLVGLAKAI TSGFEGMDVPTSSGELWILGDVFIRQYYTVFDRANNKVGLAPVA LGGIQSNSGI----GFSIFGDIFLKSQYVV FDSDGPQLGFAPQA IAGFGYG-NW----GFAIIGDTFLKNNYVVFNQGVPEVQIAPVAE FGGIQSSAGIG----INIFGDVALKAAFVVFNGATTPTLGFASK ISAITPMDFPEPVGPLAIVGAFLRKYYSIYDLGNNAVGLAKAI

Figure 4 The sequence alignment of calf chymosin (4CMS, Newman et al., 1991) with other aspartic proteinases based on threedimensional structures. References: 4PEP: porcine pepsin (Sielecki et al., 1990); 2APP: penicillopepsin (James and Sielecki, 1983); 2APR: rhizopuspepsin (Suguna et al., 1987); 4APE: endothiapepsin (Pearl and Blundell, 1984); YPA: S. cerevisiae proteinase A (Dreyer et al., 1986) (adapted from Orprayoon, 1994).

within sheets cNdN and cCdC) in the N- and the C-terminal domains. The side chains of these two aspartates are oriented towards each other around the pseudo-interlobe diad axis in a complicated hydrogenbonding network, known as the ‘fireman’s grip’ (Pearl and Blundell, 1984) shown in Fig. 8. This network is

formed by the interaction of two loops (residues 31–35 and residues 214–218) and a central water molecule. The side chain of T33 and its symmetry-related T126 form hydrogen bonds across the diad axis to the carbonyl oxygens of L214 and F31, respectively, and to the peptide N atoms of T216 and T33, respectively. The carboxyl


Figure 5 A schematic diagram of the secondary structure of chymosin. The directions of the strands are indicated by the large arrows. The inter- and intra-lobe two-fold axes are shown as large and small diad markers. The main hydrogen bonds are indicated by arrows in the direction of donor to acceptor (adapted from Newman et al., 1991).

oxygens of D32 and D215 are hydrogen-bonded with nitrogen atoms of the conserved G34 and G217, respectively. In addition, the side chains of S35 and T218 also form hydrogen bonds with the outer oxygen atoms of D32 and D215, respectively. There are some conserved glycine residues in eukaryotic aspartic proteinases which are believed to be important; among them, G34

and G217 are conserved in all aspartic proteinases. Side chains at these positions would interfere sterically with the catalytic aspartates. Residue D303 is conserved among all proteinases with an acidic pH optimum. However, in renins, which have a more neutral pH optimum, this residue is replaced by an alanine. The effect of the side chain at this position on the pKa has


Figure 6 Three-dimensional structures of aspartic proteinases showing the high degree of structure homology among these proteinases (adapted from Pitts et al., 1992).

been revealed by site-directed mutagenesis of renin (Yamauchi et al., 1988) and chymosin (Mantafounis and Pitts, 1990). The hydrogen bond between D303 and T216 may affect the pKa of D215 via the peptide dipole of T216–G217 (Pearl and Blundell, 1984).

Catalytic mechanisms

The catalytic mechanism of aspartic proteinases has been modelled based on the structural analysis of several aspartic proteinase–inhibitor complexes. Early mechanisms (James et al., 1977, 1982; James and


Figure 7 A plot of the C position of chymosin. The approximate molecular symmetry axes are shown as follows: (i) the inter-lobe non-crystallographic 2-fold screw axis relating the N- and C-terminal lobes; (ii) the intra-domain axis for the N-terminal domain; (iii) the intra-domain axis for the C-terminal domain (adapted from Newman et al., 1991).

Sielecki, 1985) proposed that catalysis was initiated by protonation of the carbonyl oxygen of the substrate by a proton from D215, followed by nucleophilic attack on the carbonyl carbon of the substrate aspartate residue by a hydroxide ion generated from water after donation of its proton to D32. These protonation events lead to the formation of the tetrahedral intermediate. The breakdown of the intermediate is generated by

protonation of the nitrogen atom either from bulk solvent or from the catalytic carboxyl group of D215. Protonation of the substrate carbonyl and nucleophilic attack may appear simultaneously during the formation of the tetrahedral intermediate. Similarly, proton transfer from the intermediate to the diad may occur at the same time as the protonation of the nitrogen atom of the substrate during the cleavage of the resultant intermediate (Polgár, 1987). Pearl (1987) suggested that the distortion of the scissile bond towards the enzyme–substrate binding may facilitate the collapse of the intermediate by generating lone pair orbitals antiperiplanar to the C9N bond but not to the hydroxyl C9O bond. Therefore, the leaving product is a free amine rather than the original nucleophile. In addition, the charged oxygen of a solvent molecule forms hydrogen bonds with residues D32 and S35 (Suguna et al., 1987) or residues G76, D77 or Y75 on the flap (Blundell et al., 1987; Pearl, 1987). Veerapandian et al. (1990) have proposed the catalytic mechanistic model outlined in Fig. 9. The proR(statine-like) hydroxyl of the tetrahedral carbonyl hydrate is hydrogen-bonded to the outer oxygen of D32 and D215. The second hydroxyl oxygen of the hydrate is hydrogen-bonded only to the carboxyl oxygen of D32. The scissile bond carbonyl is protoned by D32 and is simultaneously attacked by a water molecule polarized into a nucleophilic state by D215. The rigid movement in the enzyme–substrate complex may impel distortion of the amide bond and facilitate the attack of nucleophilic water on the polarized carbonyl. Thus, in the tetrahedral intermediate I, the negatively charged D31 is stabilized by extensive hydrogen bonding. The amide nitrogen will have been pyramidalized with the new arrangement,

Figure 8 The ‘fireman’s grip’ at the active site of chymosin. Hydrogen bonds (broken lines) involved are: T216N . . . T33O1 (2.8 Å), T33O1 . . . K214O (2.7 Å), T33N . . . T216O1 (2.9 Å) and T216O1 . . . F31O (2.8 Å). Other hydrogen bonds contributing to the stability of D32 and D215 are also shown (adapted from Newman et al., 1991).


Figure 9 A proposed catalytic mechanism for aspartic proteinases (Veerapandian et al., 1990).

favouring protonation. A proton can be transferred from bulk solvent or from D215. A similar mechanistic proposal has been described by James et al. (1992). Pepsin and chymosin have been shown to catalyse peptide synthesis (Fruton, 1982; Jakubke, 1987; Abdel Malak, 1992). Formation of a peptide is catalysed by chymosin optimally at pH 4–5 which is similar for peptide hydrolysis (Abdel Malak, 1992). The pH optimum for peptide synthesis catalysed by pepsin is further from that for peptide hydrolysis. The catalytic ability of the enzyme is sensitive to the amino acid residues flanking the bond to be formed or hydrolysed as well as the nature of adjacent amino acid residues. Zymogen activation

The structure of porcine pepsinogen has been refined at high resolution (James and Sielecki, 1986; Sielecki et al., 1991; Hartsuck et al., 1992). Structural comparisons between pepsin and pepsinogen suggest that the enzyme and proenzyme structures are very similar. Most of the differences occur in the proximity of the cleft which, in pepsinogen, is covered and filled by the pro-part (1P–44P) and the first 13 residues of pepsin.

The extension of 13 residues adopts completely different conformations in the active and the zymogen forms (James and Sielecki, 1986). The secondary structure of the zymogen consists mainly of -sheet, with an approximate 2-fold axis of symmetry ( James and Sielecki, 1986). The activation peptide packs into the active site cleft, and the N-terminus (2P–9P) occupies the position of the mature N-terminus (2–9) since the first ten amino acids of the pro-part form -strand aN of pepsinogen. Therefore, changes upon activation include excision of the activation peptide and proper relocation of the mature N-terminus. At neutral or alkaline pH, the pro-segment of pepsin binds and is stabilized across the active site between the two lobes by electrostatic, hydrogenbonding and hydrophobic interactions which contribute to the binding between the pro-segment and the rest of the protein (Sielecki et al., 1991). Lowering of pH protonates acidic residues on the mature enzyme portion of the molecule, thereby disrupting favourable electrostatic interactions with positively charged amino acid residues on the pro-segment. Subsequent conformational change of the zymogen leads to intramolecular proteolytic cleavage that liberates


the pro-segment from the zymogen (McPhile, 1972; Nielsen and Foltmann, 1993). The mechanisms of activation of zymogens of the aspartic proteinase are different and depend on the pH. At pH below 2.5, conversion of pepsinogen is primarily by an intramolecular mechanism. The propeptide is cleaved monomolecularly at position M16P–E17P, resulting in an active pseudo-enzyme which is enzymatically active and may form a complex with the released pro-segment. At pH values below 4.0, the L44P9I1 bond is not susceptible to proteolytic cleavage but becomes susceptible at higher pH. At low pH, the cleavage sites differ among the aspartic proteinases – F27P9L28P for calf prochymosin, human progastricsin and chicken pepsinogen, M16P9E17P for porcine pepsinogen B and L26P9I27P for procathepsin D (Barkholt and Foltmann, 1975; Barkholt et al., 1979; Truk et al., 1985; Foltmann, 1993; Larsen et al., 1993). Removal of the entire propeptide predominately occurs at pH 3–4 through an intermolecular mechanism. It has been suggested that cleavage of the F42P9G1 bond of prochymosin is faster at pH 2 than at pH 4.5 (Barkholt et al., 1979). The recombinant pepsinogen originally from Rhizopus and produced in E. coli can convert to the active enzyme in an acidic medium by a similar mechanism as for pepsinogen (Chen et al., 1991). The pseudorhizopus protease and rhizopus protease are generated by the cleavage at N38P9T39P and V45P9A1, respectively (p prochymosin). Moore et al. (1995) have studied the crystal and the molecular structures of human progastricsin at 1.62 Å resolution and suggest that human progastricsin has a conformational structure and mechanism of activation analogous to those for pepsinogen. Site-directed mutagenesis at the two sites for autoproteolysis of prochymosin suggests that these processing sites can function independent of one another (McCaman and Cummings, 1986, 1988). Changing the prochymosin sequence from F27P9L28P9Q29P9K30P9Q31P to F27P9P28P9R29P9Q30P9Q31P resulted in the partially activated zymogen at pH 2, while at pH 4.5, normal activation processing and proteolytic processing occurred (McCaman and Cummings, 1986). Conversely, when the seven residues including the processing site at pH 4.5 were removed, a new cleavage site (S37P9V38P) was generated at pH 4.5 while the processing site at pH 2 was not affected (McCaman and Cummings, 1988). The activation reactions are dependent on pH, salt concentration and temperature. At pH 5 and ⬃20 °C, activation is completed in two or three days (Rand and Ernstrom, 1964), while at pH 2, ⬃20 °C and an ionic strength of 0.1, activation is completed in 5–10 min (Foltmann, 1962). However, autoproteolysis alone may

not be able to generate the mature form of the enzymes as shown in procathepsin D, which cannot autoactivate to the mature enzyme at acidic pH (Larsen et al., 1993). Prochymosin is also activated by proteolytic enzymes, including plasmin, Legionella pneumophila metalloproteinase and Aspergillus oryzae thermolysin (Stepanov et al., 1990). Position 36p in the propeptides of gastric aspartic proteinases is generally occupied by lysine or arginine. This has led to the conclusion that a basic residue at this position, which interacts with the active-site aspartates, is essential for folding and activation of the zymogen. Lamb prochymosin has been shown by cDNA cloning to possess glutamic acid at position 36p. To investigate the effect of this natural mutation which appears to contradict the proposed role of this residue, calf and lamb prochymosins and their two reciprocal mutants, K36pE and E36pK, respectively, were expressed in E. coli, refolded in vitro and autoactivated at pH 2 and 4.7 (Francky et al., 2001). All four zymogens could be activated to active chymosin and, at both pH values, the two proteins with E36p showed higher activation rates than the two K36p forms. E36p was also demonstrated in natural prochymosin isolated from the fourth stomach of lamb, as well as being encoded in the genomes of sheep, goat and mouflon, which belong to the subfamily Caprinae. A conserved basic residue at position 36p of prochymosin is thus not obligatory for its folding or autocatalytic activation. The apparently contradictory results for porcine pepsinogen A (Richter et al., 1999) can be reconciled with those for prochymosin. K/R36p is involved in stabilizing the propeptide–enzyme interaction, along with residues nearer the N-terminus of the propeptide, the sequence of which varies between species. The relative contribution of residue 36p to stability differs between pepsinogen and prochymosin, being larger in the former (Francky et al., 2001). B-Crystallin, the small heat shock protein (Plater et al., 1996; Crabbe and Hepburne-Scott, 2001; Derham et al., 2001) can form a complex with prochymosin. After activation, once chymosin is recovered without bound B-crystallin, the yield of activity is increased (Chitpinityol et al., 1998b). Substrate-binding pockets and specificity

A high concentration of NaCl or (NH4)2SO4 increases the hydrolytic activity of pepsin and retroviral proteinases, in addition to broadening their specificities (Kotler et al., 1989; Tropea et al., 1992). Aspartic proteinases have an extended substratebinding pocket that can accommodate at least seven amino acid residues. Detailed structural studies of


aspartic proteinase–inhibitor complexes have been used to identify the amino acid residues in each subsite (Bott et al., 1982; Andreeva et al., 1984; James et al., 1985; Blundell et al., 1987; Cooper et al., 1987; Foundling et al., 1987; James and Sielecki, 1987; Suguna et al., 1987). In chymosin, the subsites S1 and S1 are shallow pockets within the active site cleft. The S1 subsite (for binding of F105) has greater specificity than S1, and is blocked by Y75 (Gilliland et al., 1990). Therefore, a significant movement of the flap is essential to allow binding of the substrate. The S1 is quite hydrophobic compared to S1 in which an additional charged residue, E290, is near to the -casein M106 side chain. The S2 pocket has low specificity and allows the peptide side-chains to adopt a range of conformations whereas at subsites S1 and S3, the conformation of the side chains is strongly restricted (Dhanaraj et al., 1992). Chymosin residues involved in the interactions with the corresponding residues of the substrate are shown together with a sequence identical to that of the -casein cleavage site in Table 1. There are two differences in the S1 subsites of chymosin and the fungal proteinases that promote more hydrophobic S1 subsites (Gilliland et al., 1990). The first is the position of the flap region which is due to the reorientation of Y75 and a deletion of one amino acid residue in this loop. Another difference is the substitution of L30 in chymosin for the D30 or the N30 of the rhizopus protease and penicillopepsin, respectively. In human cathepsin E, the important specificitydetermining interactions are found in the S3 (E13) and S2 (T222, E287, L289, I300) subsites (Raonaik et al., 1995). Figure 10 summarizes the results of cleavage of the B chain of oxidized insulin by chymosin and some related acid proteinases. In chymosin, the S1 subsite has favourable interactions with aromatic amino acids at P1 whereas the S1 subsite is less specific (Bang-Jensen et al., 1964; Foltmann, 1964; Guillou et al., 1991; Nedjar et al., 1991).

The fungal and yeast proteinases have an S1 subsite with a deeper pocket and broader specificity. Therefore, the S1 pocket can accomodate lysine as well as hydrophobic residues at P1 (Oka et al., 1973; Hofmann et al., 1984; Newman et al., 1993). However, in rhizomucor protease, specificity for lysine at P1 was not observed due to the absence of polar residues at positions 30 and 111. In retroviral aspartic proteinases, the primary specificities for HIV-1 and HIV-2 aspartic proteinases at P1 are L, M, Y and F, and at P1 are P, M, F and A (Poorman et al., 1991). Among the isozymes of chymosin, chymosin A has a significantly higher specific activity than chymosin B (Foltmann, 1960) which may be the result of the enhanced binding affinity of -casein through, possibly, the stronger electrostatic interactions between the substrate and chymosin A. In addition, these two isozymes have different pH optima, 4.2 and 3.7 for chymosin A and B, respectively. These different values may be the result of an extensive hydrogen-bonding network near the two catalytic aspartates. The optimum pH for proteolysis by aspartic proteinases depends upon the species from which the enzyme is produced, and the substrate used (Table 2). HIV-1 proteinase and renin have a high pH optimum among aspartic proteinases. The residues S35, T218 and D303 have been postulated to play a role in the pH profile of aspartic proteinases. In vitro mutagenesis of A35S of HIV-1 proteinase (A28S in HIV-1 numbering) showed a lowering of pKa2 (compared to wild type) by 1.2 units but no effect was found in the pKa1 value (Ido et al., 1991). In contrast, mutation of S35A in porcine pepsin lowered pKa1 and pKa2 but raised it for rhizopusprotease. Site-directed mutagenesis of T218A in porcine pepsin, chymosin and rhizopus protease shifted the pH optimum by 0.2–0.5 units (Mantafounis and Pitts, 1990; Tang et al., 1992). Mutation of A303D in renin lowered the pH optimum by 0.5 units (Yamauchi et al., 1988). Similarly, mutation of D303A in chymosin raised the optimum pH by 0.6 units (Mantafounis and Pitts, 1990). The double mutations, T218A/D303A

Table 1 The substrate-binding pockets of chymosin. Chymosin residues involved in the interactions with the corresponding residues of the substrate are shown together with residues at the -casein cleavage site (adapted from Gilliland et al., 1990; Newman et al., 1991) Subsite

-Casein residues

Chymosin residues

S4 S3 S2 S1 S1 S2 S3

His102 Leu103 Ser104 Phe105 Met106 Ala107 Ile108

Ser219, Lys220, Gln288 Ser12, Gln13, Tyr75, Phe117, Gly217, Thr218, Ser219 Gly76, Thr77, Gly217, Thr218, Lys220 Leu30, Asp32, Gly34, Tyr75, Gly76, Phe117, Ile120, Asp215, Gly217, Thr219 Gly34, Tyr189, Asp215, Thr218, Glu289, Ile301 Gly34, Ser35, Tyr189 Tyr189


Chymosin1 Pepsin2 Rhizopuspepsin3 Penicillopepsin3 Endothiapepsin4 Proteinase A5 Cathepsin E6 R. miehei APR7 R. pusillus APR8

1 10 20 30 F V N Q H L C G S H L V E A L Y L V C G E R G F F Y T P K A ↑ ↑ ⇑ ↑ ↑ ↑ ⇑ ↑ ↑ ↑ ⇑ ⇑ ↑ ↑ ↑ ⇑ ↑ ⇑ ⇑ ↑ ↑ ⇑ ↑ ↑ ⇑ ⇑ ↑ ↑ ⇑ ⇑ ↑ ↑ ⇑ ↑ ↑ ↑ ↑ ⇑ ⇑ ⇑ ↑ ⇑ ↑ ⇑ ⇑ ↑ ↑ ⇑ ↑ ⇑ ⇑ ⇑ ⇑ ⇑ ⇑ ⇑ ⇑ ↑ ⇑ ⇑ ↑ ⇑ ⇑ ↑ ⇑ ⇑ ⇑ ⇑ ⇑ ↑ ⇑ ⇑ ⇑ ⇑ ⇑

Figure 10 Comparison of the cleavage specificity of chymosin towards the B-chain of oxidized insulin with those of some other aspartic proteinases. References: (1) Foltman (1964); (2) Sanger and Tuppy (1981); (3) Oka et al. (1973); (4) William et al. (1972); (5) Takahashi (1995); (6) Athauda et al. (1991) (pH 3.0); (7) Rickert (1971); (8) McCullough and Whitaker (1971). Legends: (y) Main cleavage site and (q) other sites of action.

in chymosin affected the pH optimum similarly to that of D303A mutatagenesis (Pitts et al., 1993). The substrate specificity of aspartic proteinases is affected by the operating pH and the presence of salts (Kotler et al., 1989; Athauda et al., 1991; Tropea et al., 1992). The pH dependence of hydrolysis of synthetic substrates demonstrates that secondary specificity occurs at subsite S3 of mammalian aspartic proteinases whereas lower specificity is found in microbial proteinases (Dunn et al., 1986). In chymosin, isoleucine or valine is favoured at P3, and tyrosine, valine or serine at P2 (Guillou et al., 1991). The favourable interaction between K220 (NH3) of chymosin and glutamate (COO ) in P2 of the substrate is suggested to cause the

effects of pH on hydrolysis (Dunn et al., 1987). The specificity at P2 towards both K220 and Q288 has been determined by in vitro mutagenesis (Suzuki et al., 1990; Quinn et al., 1991). We have studied the effect of replacing threonine 77 of chymosin by aspartate (mutant T77D), as well as the addition of two residues (9H9G) (mutant PC 2) to the C-terminus of the protein, on the activity of the enzyme on a synthetic hexapeptide, L9S9 F(NO2)9NI9A9L9OMe, as substrate (Chitpinityol et al., 1996, 1998a). For the recombinant wild type, the optimum pH was 3.7, similar to that reported for the authentic chymosin B using the same substrate (Martin et al., 1980). The PC 2 mutant had an optimum pH

Table 2 pH optimum for general proteolysis by chymosin and other aspartic proteinases

Enzymes

Substrates

Optimum pH

Chymosin

Acid-denatured haemoglobin

3.7

Bovine serum albumin Oxidized B-chain of insulin -,-Caseins

3.4 3.5 4.5

Milk-clotting activity Synthetic peptides -Casein H9P9H9P9H9L9S9F9M9A9I9P9P9K9K Oxidized B-chain of insulin Trypsinogen Haemoglobin Haemoglobin -Casein Milk clotting Hammerten casein z9Phe9Leu9Ala9Ala Acid-denatured haemoglobin

6–6.3 4.7 5.5 5.4 2.0 3–4 2–2.5 4.0 4.5 5.5 3.5 3–4 3.2

Berridge (1945); Fish (1957) Foltmann (1959a) Fish (1957) Lindqvist and Storgads (1960) Okigbo et al. (1985a) Raymond et al. (1972) van Hooydonk et al. (1984) Visser et al. (1976, 1987) Fish (1957) Hofmann and Shaw (1964) William et al. (1972) Arima et al. (1970) Arima et al. (1970) Arima et al. (1970) Arima et al. (1970) Oka et al. (1973) Dreyer et al. (1986)

Haemoglobin

1.1

Takahashi (1995)

Pepsin Penicillopepsin Endothiapepsin Rhizomucorpepsin

S. cerevisiae proteinase A A. niger proteinase A

References


similar to the native enzyme. The optimum pH of T77D mutant chymosin was shifted towards neutrality by 1 pH unit, to pH 4.7 from 3.7. The optimum temperature for the activity of the T77D mutant was increased relative to the wild-type enzyme, from approximately 45 °C for the wild type and PC 2 mutant, to 55 °C for the T77D mutant. These changes may be due to the increased negative charge at the ‘flap’ region that may have altered the network hydrogen bonding and influenced the substrate recognition of the enzyme. Inhibitors

All aspartic proteinases are inhibited by pepstatin, by the binding of the hydroxyl group of statine to the two catalytic aspartates (Marciniszyn et al., 1976a,b). The inhibition constant (Ki) of pepstatin for chymosin determined at pH 6.0 and 3.2 is 2.2 10 7 mol/l and 3.2 10 8 mol/l, respectively (Powell et al., 1985). Pepsin and cathepsin also show pH-dependency of the inhibitory effect (Knight and Barrett, 1976; Baxter et al., 1990), and psuedochymosin is more sensitive to pepstatin than chymosin (McCaman et al., 1985). As pepstatin is relatively ineffective towards calf chymosin, analogue inhibitors have been developed. A series of inhibitors have been designed by Powell et al. (1985), including R(CO)NH9L9S9Sta9A9I9P9P9K9K (R acyl group) which has a Ki value for chymosin almost 20-fold better than pepstatin at pH 6.0 and approximately 10-fold better at pH 3.1 than pepstatin. Chymosin is inhibited by the pro-part of chicken pepsinogen (Ki value of 8 10 8 mol/l at pH 5.6) but not by its own pro-segment (Strop et al., 1990).

Mechanism of Milk Clotting In milk, the primary soluble proteins are the whey proteins, -lactalbumin and -lactoglobulin. The insoluble proteins are found in large colloidal particles, called casein micelles. -Casein is a calcium-insensitive protein which forms a protective layer around the calciumsensitive caseins ( S1-, S2-, - and -), resulting in stable casein micelles. In the presence of chymosin, milk clotting occurs in two separate steps. The first phase starts with the cleavage of -casein at the F1059M106 bond which results in the release of a hydrophilic glycopeptide (residues 106–169) that passes into the whey, and para--casein that remains in the micelles. para--Casein is positively charged at neutral pH and causes a decrease of electric repulsive forces between casein micelles (Green, 1973). Hydrolysis of other proteins in milk, including S1-, S2- and -caseins and -lactalbumin monomer, by chymosin has been reported with a much slower rate

of proteolysis (Carles and Dumas, 1985; Miranda et al., 1989). The proteolytic action of microbial proteases on -casein has been reported (de Koning, 1967; Yu et al., 1968; Larson and Whitaker, 1970). Porcine pepsin A and C, and R. miehei proteinase cleave the same bond as chymosin (F1059M106), but C. parasitica proteinase cleaves the S1049F105 bond (Drønse and Foltmann, 1989). Chymosin causes limited hydrolysis of -casein, with the formation of only macropeptide and para--casein, while fungal proteinases cause extensive non-specific hydrolysis of both -casein and para--casein (Shammet et al., 1992). Visser et al. (1980) suggested that other residues near the cleaved bond are also involved in the hydrolytic reaction. From studies with synthetic peptides, two additional residues at both sides of the hydrolysable bond are required for appreciable reaction (Raymond et al., 1972). The peptide corresponding to residues 98–111 of -casein (H9P9H9P9H9L9S9F9M9A9I9 P9P9K) was found to provide a complete requirement for hydrolysis (Visser et al., 1987, 1988). Initially, the stability of the micelle is destroyed by the action of chymosin. This is followed by a nonenzymatic secondary phase in which the aggregation of para--casein and other caseins occurs under the influence of Ca2 and eventually results in gel formation (Bringe and Kinsella, 1986a; Merin et al., 1989). The formation of a clot is Ca2 dependent. The primary and the secondary phases of milk clotting overlap as the aggregation of micelles begins before the enzymatic process is complete (Brown and Collinge, 1986; Bringe and Kinsella, 1986b). Several factors influence the milk-clotting process, including pH, temperature, ionic strength, enzyme concentration and salts (Foltmann, 1970; Okigbo et al., 1985a,b; Bringe and Kinsella, 1986a,b). The reaction is pH dependent; at high pH (6.6–6.7), the clotting time and the curd firmness are reduced (Okigbo et al., 1985a), while at low pH (3–4), the hydrolytic activity is high and a decrease in curd yield occurs. Generally, milk clotting is performed at pH 6.3–6.6; only when direct acidification is used does rennet coagulation occur at a pH value down to 5.6. The rate of milk clotting increases with temperature as long as the enzyme is stable (Berridge, 1942). Increasing the temperature above 30–32 °C or reducing the pH from 6.6 permits flocculation at a lower percentage of -casein hydrolysis (Dalgleish, 1982). However, the induction of gel formation at 35 °C requires approximately 65% hydrolysis of -casein (Carlson et al., 1986). The differences in milk constituents (both proteins and other chemicals) as well as the pre-treatment process can affect the rate of the primary enzymatic stage. The time


taken to coagulate milk decreases with increasing enzyme concentration, but the formation and firmness of the gel is not altered (Bringe and Kinsella, 1986a). Milk-clotting activity is also dependent on the source of chymosin; for example, porcine chymosin is eight times more active on porcine milk than on bovine milk; calf chymosin is only half as active on porcine milk as on bovine milk and the activity of lamb chymosin is about 20% higher on ovine milk than on bovine milk (Foltmann, 1992). Calcium ion concentration affects milk clotting by forming bridges between micelles to form the coagulum and minimizes variability arising from inconsistency in milk composition (Berridge, 1952; Bringe and Kinsella, 1986b). However, Pyne (1955) reported that other ions, such as strontium, magnesium and barium, could affect the Ca2 requirement for coagulation. Milk clotting is inhibited by anions (Bringe and Kinsella, 1986b). While synthetic substrates have been used to reveal the hydrolytic mechanism of chymosin and other aspartic proteinases (Raymond et al., 1972; Martin et al., 1980; Visser et al., 1987, 1988), milk clotting is much more complicated than the hydrolysis of a synthetic substrate. For cheesemaking, the appropriate enzyme should have a high ratio of milk-clotting activity to proteolytic activity (C/P) (Dalgleish, 1982). The C/P ratio of chymosin is higher than for other enzymes; over 2 times higher than rhizomucor protease, 4 times higher than cryphonectria protease and over 25 times higher than pepsin, trypsin and papain (Martin et al., 1980; Yada and Nakai, 1986).

Recombinant Calf Chymosin Chymosin has been used as the milk-clotting enzyme for the industrial production of cheese. Several rennet substitutes have been used, including bovine pepsin (from adult cows), fungal proteinases and other proteolytic enzymes. However, they have a much greater level of non-specific proteolytic activity, and in some cases higher thermostability that causes more degradation of milk proteins to peptides, leading to a reduction in yield and poor flavour development in some types of cheese. Consequently, there have been numerous attempts to produce chymosin in micro-organisms. Prokaryotic expression

The first report of an attempt to produce chymosin in E. coli was that of Uchiyama et al. (1980). Efforts to express prochymosin cDNA in E. coli led to intracellular accumulation of inactive chymosin in the form of inclusion bodies (Emtage et al., 1983; Nishimori et al., 1984; McCaman et al., 1985; Kawaguchi et al., 1987;

Chitpinityol et al., 1998a). Generally, chymosin was synthesized in the form of M-prochymosin or N-terminal fusion proteins under the control of E. coli lac promoter (Nishimori et al., 1984; McCaman et al., 1985), trp promoter (Beppu, 1983; Emtage et al., 1983; Kawaguchi et al., 1984; Marston et al., 1984; Nishimori et al., 1984), tac promoter (McCaman et al., 1985; Strop et al., 1990), PR promoter (Caulcott et al., 1985), pho A promoter (Little et al., 1989) or T7 promoter (Chitpinityol et al., 1998a). In E. coli expression systems, the recombinant prochymosin was expressed at a high level which resulted in the accumulation of highly refractive inclusion bodies (Emtage et al., 1983; Kawaguchi et al., 1984; Shoemaker et al., 1985). The inclusion bodies produced were up to 40% of the total cell mass and were organized in an irregular mass without any obvious membranelike boundary, with an average diameter of 0.5–1 m (Marston et al., 1984; McCaman et al., 1985; Strop et al., 1990; Kaprálek et al., 1991). The synthesis of prochymosin as intracellular inclusion bodies causes a fragilility of cell membranes, and the loss of cell respiratory activity and their ability to multiply (Marston et al., 1985; Kaprálek et al., 1991). The production of inclusion bodies can be improved by the plasmid construction, plasmid stability, host strain, composition of the cultivation medium and growth temperature (Caulcott et al., 1985; Kawaguchi et al., 1986, 1987; Kaprálek et al., 1991). The N-terminal methionine of Met-prochymosin can be removed together with pro-part during acid activation. The insoluble form of prochymosin requires denaturing condition (8 mol/l urea or 6 mol/l guanidine HCl) to solubilize prochymosin, followed by renaturation to generate correctly folded protein that can be activated (Shoemaker et al., 1985). The deletion of disulfide bonds from prochymosin showed that the presence of disulfide bonds was not reponsible for inclusion body formation (McCaman, 1989). Improvements in the production of recombinant chymosin in E. coli have been successively developed by selection of host strain, the modification of plasmids and the optimization of cultivation conditions (Kawaguchi et al., 1986; Kaprálek et al., 1991). It has also been found that a high yield of active recombinant calf chymosin can be achieved by optimizing solubilization and renaturation conditions (Tichy et al., 1993; Yonezawa et al., 1993; Chitpinityol et al., 1996; Chitpinityol et al., 1998a,b). In our experiments (Chitpinityol et al., 1998a), the recombinant enzyme was refolded by a modified procedure based on that of Marston et al. (1984). Table 3 shows that the yield of chymosin was maximal when the urea mixture was diluted 25-fold (0.32 mol/l final urea concentration). If the solubilization mixture was diluted over 25-fold, the yield of chymosin was


Table 3 Effect of dilution ratios on the yield of chymosin recovered by refolding. Washed inclusion pellets (protein concentration, 7.9 mg/ml) were solubilized in 8 mol/l urea buffer, pH 8. The urea mixture was incubated at 25 °C for 1 h before the insoluble molecules were removed by centrifugation. The urea solution was then diluted in a high pH buffer (pH 10.7) for renaturation of prochymosin. The protein concentration was determined by using a BCA Protein Assay Reagent Dilution ratio of urea mixture in buffer pH 10.7

Final urea concentration (M)

Initial concentration of protein in buffer (mg/ml)

Final amount of activated chymosin (mg)

1:10 1:20 1:25 1:30 1:40

0.80 0.40 0.32 0.27 0.20

0.79 0.39 0.32 0.26 0.20

0.46 0.49 0.50 0.40 0.26

dependent on the protein concentration in the alkaline buffer. Table 4 shows that 0.25 mg/ml protein was optimal under the refolding conditions used. This optimized procedure improves the yield of recombinant enzyme nearly three-fold. Since insoluble proteins require a further refolding process prior to regaining enzymatic activity, there have been attempts to produce prochymosin extracellularly. The N-terminal sequence of prochymosin was fused to a signal peptide of the outer membrane protein A; this resulted in cell lysis after induction (Elliott et al., 1989). Holland et al. (1990) reported that the fusion of hemolysin Hly A8 signal sequence to the C-terminal of prochymosin resulted in the production of hybrid protein up to 25% of the total cell proteins, of which 0.8% was a soluble hybrid product. An expression system for the production of soluble porcine pepsinogen A has been developed by fusing the pepsinogen and the thioredoxin genes and then expressing the fused product in E. coli (Tanaka and Yada, 1996). Other bacterial expression systems used to produce prochymosin include Lc. lactis, Bacillus subtilis and L forms of Proteus mirabilis (Kaprálek et al., 1991; Parente et al., 1991; Simons et al., 1991). In Lc. lactis, the cDNA for prochymosin was expressed under the control of proteinase prtP promoter by fusion with various lengths of Lc. lactis cell envelope-located protease (Simons et al., 1991). Under the control of the T5 phage promoter and

the induction of a two-cistron sequence at the 5 region of the gene, prochymosin was synthesized as insoluble aggregates in B. subtilis cells but the yield was still low (Parente et al., 1991). The extracellular production of prochymosin in B. subtilis can be achieved by fusing the prochymosin gene to the B. subtilis subtilisin signal sequence and production reached up to 100 mg/l (Parente et al., 1991). Using L forms of a P. mirabilis expression system, the fusion of prochymosin cDNA minus codons 1–4 to streptococcal pyrogenic exotoxin type A gene (speA) sequences resulted in the secretion of fusion prochymosin up to 40 g/ml of cell-free culture fluid (Kaprálek et al., 1991). Eukaryotic expression

Several eukaryotes, including yeast, fungi, insect and mammalian cells, have been used for the production of prochymosin and chymosin. In S. cerevisiae, the cDNA coding for preprochymosin, prochymosin or chymosin has been expressed under the control of phosphoglycerate kinase (pgk), galactosidase (gal 1 and gal 10) and triosephosphate isomerase (tpi). The proteins are synthesized mainly as insoluble forms which accumulate in the cells and are difficult to activate (Mellor et al., 1983; Goff et al., 1984; Moir and Davidow, 1991). Expression of preprochymosin cDNA did not allow the secretion of chymosin, while substituting yeast invertase signal

Table 4 Effect of protein concentration on the refolding of recombinant prochymosin. Inclusion bodies solubilized in 8 M urea were diluted to various protein concentrations in phosphate buffer, pH 10.7. The urea final concentrations were kept at 0.32 mol/l. The protein concentrations were determined by using a BCA protein assay reagent Initial protein concentration (mg/ml)

Initial amount of protein (mg)

Final amount of activated chymosin (mg)

% of refolding

0.32 0.28 0.25 0.22 0.19

1.58 1.42 1.26 1.11 0.95

0.33 0.31 0.37 0.28 0.18

20.86 21.85 28.99 25.54 18.66


peptide for the chymosin secretion signal peptide led to the secretion of approximately 10% of the total prochymosin made (Moir et al., 1985). The secretion of prochymosin is critical for obtaining soluble activatable proteins. The failure to form or the incorrect formation of disulfide bonds is characterized by insoluble prochymosin produced in the cytoplasm of both yeast (Smith et al., 1985) and E. coli (Shoemaker et al., 1985). Using yeast secretion signals, integration of the transcriptal units into yeast genome and mutations of the host genome, the secretion of prochymosin increased at least 80-fold which allowed the production of activatable prochymosin to a level of 20 mg/l of culture medium (Smith et al., 1985; Moir and Davidow, 1991). Kluyveromyces lactis has been developed as an alternative host to S. cerevisiae in the expression of recombinant proteins. It has been used successfully to secrete prochymosin under various signal sequences. Efficient synthesis and secretion of prochymosin to more than 95% of the theoretical levels was achieved by using the K. lactis lactase gene (Lac4) (van den Berg et al., 1990). Commercially viable yields have been obtained from this species by DSM Food Specialities, Delft, the Netherlands. The yeast, Yarrowia lipolytica, has also expressed prochymosin using either the Leu2 or the alkaline protease XPR2 promoters (Franke et al., 1988). All of the prochymosin produced by these systems was readily activated to mature chymosin. Filamentous fungi have also been used as hosts for the production of chymosin. In Aspergillus nidulans, chymosin was synthesized as an active extracellular enzyme using the glucoamylase (glaA) promoter from A. niger (Cullen et al., 1987). A commercial strain of A. niger var. awamori has been used to express prochymosin cDNA under different expression cassettes (Ward, 1989; Ward et al., 1990). The level of active extracellular chymosin was 250 mg/l when prochymosin cDNA was fused with the entire coding sequence for glucoamylase and expressed in the host which has been deleted of the aspergillopepsin A gene (pepA) (Ward et al., 1990). The introduction of an N-linked glycosylation site on the flap region resulted in a 10-fold increase in the production of secreted glycosylated chymosin over the wild-type chymosin, possibly as a result of improved secretion efficiency. The milk-clotting activity of glycosylated chymosin was reduced to about 20% of the native enzyme. However, almost all of the activity was recovered after endoglycosidase H treatment (Berka et al., 1991). The production of chymosin by A. niger var. awamori has been obtained up to 1.3 g/l by combining a mutagenesis and an efficient screening programme (Dunn-Coleman et al., 1991). About 10 mg/l chymosin has been produced by A. oryzae using a host -amylase promoter (Boel et al.,

1987) whereas in the same expression system, a more than 3 g/l of rhizomucor protease was obtained (Christensen et al., 1988). The production of chymosin by Trichoderma reesei has also been reported, using chymosin signal peptide, cellobiohydrolase I (cbh I) sequence or the fusion of cbh I-chymosin signal sequence (Harkki et al., 1989). Chymosin A was produced at a level of 40 mg/l (Harkki et al., 1989). A number of chymosin mutants cloned in T. reesei have been reported to exhibit novel properties, including mutants with a shift in the pH optimum, substrate-specificity pocket and an altered surface loop (Pitts et al., 1991, 1993). These mutants might be of interest in commercial investigations. The prochymosin secreted by T. reesei was readily activated to chymosin. In HeLa cells, calf preprochymosin cDNA has been expressed under the CMV-SV promoter (Kolmer et al., 1991). The product was processed to prochymosin prior to secretion into the cultivation medium at a level of 10–20 mg/l and readily activated to chymosin by acid treatment. Recombinant chymosin is now produced in largescale commercial operations using E. coli (California Biotechnology and DSM Food Specialities, the Netherlands), Kluyveromyces lactis (DSM Food Specialities, the Netherlands) or mammalian cells (Upjohn, USA) as the hosts (Hodgson, 1993). Many firms, including Genencor/Genentech, Celltech, Hansen and Novo, produce recombinant enzymes for laboratory use. Varieties of cheese have been made using recombinant chymosin and evaluated in comparison to cheese produced using the natural enzyme. No significant differences could be detected between them, regarding recovery of milk solids, rate of proteolysis during ripening, as well as in the characteristics of the final cheese products (Green et al., 1985; Kawaguchi et al., 1987; Hicks et al., 1988; Bines et al., 1989; Flamm, 1991; Ward and Kodama, 1991). Recombinant lamb chymosin has been used as an alternative coagulant in cheese production, and the overall quality was at least comparable to cheese made with recombinant calf chymosin, and was scored better than cheese made using bovine rennet (Rogelj et al., 2001).

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Rennet-induced Coagulation of Milk D.S. Horne and J.M. Banks, Charis Food Research, Hannah Research Institute, Ayr KA6 5HL, Scotland

Introduction The first stage of cheese manufacture is the conversion of liquid milk to cheese curd. Traditionally, this was achieved by the addition of rennet to coagulate the milk and by the subsequent expulsion of the whey by syneresis. In this chapter, we will be concerned with the first of these steps, the enzyme-induced coagulation. Later chapters will review syneresis and curd-handling procedures. We will consider the basic chemistry and physics underlying aggregation and gel formation, and the technological factors (milk composition, processing) that influence the coagulation process. In doing so, we will be covering ground treated in two separate consecutive chapters on enzymatic coagulation of milk (Dalgleish, 1993) and on post-coagulation phenomena (Green and Grandison, 1993) in the earlier editions, but updating and broadening those reviews. Attention is also drawn to more recent work by Lomholt and Qvist (1999). Milk can also be clotted by acidification or a combination of significant acidification and minor enzymic action. These aspects form the subject of ‘Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels’, Volume 1. After the addition of chymosin or other milk-clotting enzyme to the milk, nothing apparently happens for some time until the milk coagulates rapidly. During this lag phase, the enzyme hydrolyses the -casein which stabilizes the casein micelles. When sufficient destabilization has been brought about, an aggregation reaction is set in train and this eventually leads to a three-dimensional, space-filling gel, the cheese curd. Previously, this overall coagulation reaction has been envisaged as occurring in three stages. The enzymatic proteolysis forms the first or primary phase, and leads to the activation of the aggregating species. In the secondary phase, which overlaps the first, since the milk may begin to clot before the enzymatic cleavage of -casein is complete (Green et al., 1978; Dalgleish, 1979; Chaplin and Green, 1980), the destabilized micelles begin to aggregate and most of the previous discussions stop with this stage (Dalgleish, 1992, 1993; Hyslop, 2003). Those authors recognize the possibility of a third stage but consider this to involve changes largely in the curd structure once it has formed. Just as the first and the second stage overlap, it will be our con-

tention that so too does the aggregation phase overlap the development of curd structure and properties. Indeed conceptually, together they comprise the gelation process. We therefore view this partitioning of second and third stages as wholly (or largely?) artificial and aim to treat the formation of the coagulum, at least to the cutting stage, in a single mechanistic framework. The drastic changes, post-cutting, take the curd into a completely different environment, and their treatment is beyond the remit of this chapter.

Milk Properties Cheesemaking capitalizes on the curdling of milk. To understand the coagulation reaction, we must look more closely at the individual components of milk to discern their role, particularly the fat, the protein and the minerals. Fat

Fat exists in natural milk as small globules surrounded by membrane proteins and in a size range dependent on breed, lactational status and diet of the cow. The fat in milk helps to produce flavour, aroma and body in mature cheese. Unless the milk is homogenized, the fat globules are physically trapped in the protein network created in gel formation. Hence, their size and the network mesh size interact in determining the overall yield of cheese. Otherwise, the fat globules play the part of an inert filler in influencing curd rheological properties but no active role in gel formation. Homogenization of the milk, which creates many more smaller fat droplets, stabilizes these droplets by utilization of adsorbed whey and casein proteins. Where these are caseins, they can be involved in gel formation and influence its development. These aspects are considered in later sections. Protein

Two types of protein are found in milk: the globular whey proteins, which are soluble in the serum phase, and the caseins which exist in a stable colloidal suspension of aggregates known as casein micelles. Cheesemaking exploits the destabilizing mechanisms nature



48 Rennet-induced Coagulation of Milk

has built into this colloidal system by using the natural enzyme, chymosin, originally extracted from the stomach of the calf but nowadays available in cloned form, to hydrolyse the -casein and induce the destabilization of the casein micelle system to form a gel. The properties of the caseins and their micellar form play a major role in defining that reaction and its final outcome. Casein chemistry

The caseins are a family of phosphoproteins. In bovine milk, the family consists of four distinct gene products, designated s1-, s2-, - and -caseins. Together, they constitute around 80% of bovine milk protein and are found in the approximate proportions 4:1:4:1, respectively (Davies and Law, 1980; Walstra and Van Vliet, 1986). Two post-translational modifications of the proteins, newly synthesized in the mammary gland, have a major impact on the physico-chemical, functional and assembly properties of the proteins. These reactions are glycosylation and phosphorylation. In bovine casein, only -casein is found glycosylated with several threonine, and occasionally serine, residues in the hydrophilic C-terminal end of the -casein molecule carrying relatively short sugar chains (Zevaco and Ribadeau-Dumas, 1984; Vreeman et al., 1986). Glycosylation is not complete, however, and the nonglycosylated form is still the major component (Vreeman et al., 1986). These oligosaccharide chains increase the negative charge (through inclusion of sialic acid), the hydrodynamic bulk and the hydrophilic character of the C-terminal end of the -casein molecule. The second post-translational reaction is phosphorylation. All the caseins are phosphorylated at serine, or rarely threonine, residues to varying extents. The phosphorylation reaction requires a particular sequence template, 9Ser9X9A, where X is any amino acid and A is Glu, SerP or, rarely, Asp (Mercier, 1981). The pattern of serine residues along the amino acid sequences of s1-, s2- and -caseins ensures that most of the phosphorylated residues are found in clusters in these molecules, one in -casein, two in s1-casein and three in s2-casein (Swaisgood, 1992). -Casein is unique amongst the caseins in the absence of phosphoseryl clusters along its sequence. Most -casein molecules contain only one phosphoseryl residue but some evidence indicates a minor amount of doubly phosphorylated proteins but still only as singlets (Vreeman et al., 1977). Bovine caseins are almost always fully phosphorylated to the level of their potential. At most only one of the template serines is found without its expected phosphate residue in s1- and -caseins. Most gaps are found with s2-casein where the variability ranges from 10 to 13 moles P per mole protein (Whitney, 1988).

The influence of the varying degrees of phosphorylation of the caseins is mirrored in the sensitivity of these molecules to calcium-induced precipitation. Thus, s2-casein is the most calcium sensitive, precipitating at Ca2 concentrations less than 2 mM (Aoki et al., 1985), while s1-casein precipitates in the range 3–8 mM (Parker and Dalgleish, 1981; Aoki et al., 1985; Farrell et al., 1988) and -casein precipitates in the range 8–15 mM Ca2 at 37 °C but remains in solution at 1 °C (Parker and Dalgleish, 1981; Farrell et al., 1988). -Casein remains soluble at all these calcium concentrations and prevents the precipitation reaction when present with the other casein types, producing instead a colloidal suspension. Casein structures

Controversy still exists over the level of secondary structure present in the caseins. Previously, much of this was designated to the random coil in line with the open, highly hydrated state presented by the molecules in solution. Because of this, the caseins have been described as rheomorphic proteins, indicating that their conformational structure is dictated by, and is responsive to, the molecular environment (Holt and Sawyer, 1993). Views have shifted, however, and current opinion suggests that parts of - and -caseins might adopt the polyproline II-helix structural motif (Farrell et al., 2001; Syme et al., 2002). From the point of view of their self-association and micellar assembly, the amphiphilicity of the caseins may play a more crucial role than recognizable secondary structural elements (Horne, 2002). The clustering of the phosphoseryl residues has already been mentioned, and these groupings are further flanked by polar and charged residues making these regions very hydrophilic. Other regions of the casein molecules have a high concentration of hydrophobic residues, conferring on the molecules an almost block copolymerlike structure. Thus, the N-terminal peptide of -casein with the phosphoseryl cluster is very hydrophilic and the C-terminal is very hydrophobic. The behaviour of this protein on adsorption at a hydrophobic interface reflects this segregation, with the hydrophobic C-terminus adsorbing strongly and the hydrophilic N-terminal sticking out into solution (Horne and Leaver, 1995). Ample experimental evidence from dynamic light scattering, neutron reflectivity, enzyme proteolysis and surface force measurements confirm this view (Horne and Leaver, 1995). Further support comes from selfconsistent field calculations to determine the segment density function of a polymer model of -casein, normal to the adsorbing surface (Dickinson et al., 1997a,b). Similar calculations carried out on s1-casein suggest that it can be sub-divided into three blocks, a hydrophobic

Rennet-induced Coagulation of Milk 49

N-terminal region, a hydrophilic central loop, containing the phosphoseryl clusters which extends out into the aqueous phase on adsorption of the molecule to a hydrophobic surface, and a hydrophobic C-terminal peptide (Dickinson et al., 1997a). Calculations suggest that these hydrophobic regions are entrained close to the adsorbing surface. Such block structures reflect the general distribution of hydrophilic and hydrophobic residues along these casein protein sequences. By analogy, a block polymer structure can be drawn for s2casein, depicting it as having four blocks. Moving from the N- to the C-terminus, these are a hydrophilic N-terminal tail with a phosphoseryl cluster, a hydrophobic train, a hydrophilic loop, containing further clusters of phosphoseryl residues, and finally a second hydrophobic train at its C-terminus. -Casein is seen to be a mirror image of -casein, with a hydrophilic C-terminus, the caseinomacropeptide cleaved off by chymosin, and a hydrophobic N-terminal block preceding the Phe1059Met106 bond. Importantly, however, the macropeptide has no phosphoseryl cluster. Casein self-assembly

Individual caseins in solution exhibit strong tendencies to self-associate, and the shape and topography of the structure adopted reflects the distributions of hydrophobic/hydrophilic residues just described. Thus, -casein, which resembles a detergent molecule with a hydrophilic head and a hydrophobic tail, forms detergent-like micelles with the tails forming a central core and the hydrophilic heads sticking out into the aqueous solution like the bristles on a hedgehog (Payens et al., 1969). In like fashion, s1-casein selfassociates in solution to form a worm-like chain polymer with the hydrophobic ends of one molecule interacting with those of different molecules (Schmidt, 1970). -Casein also self-associates in solution, interacting via its hydrophobic C-terminal, exhibiting a monomer 4 micelle equilibrium but, here, polymer growth is also influenced by the intermolecular disulphide linkages produced by reaction of its cysteine residues (Vreeman et al., 1977; Vreeman, 1979). For s1- and -caseins, it has been demonstrated that the size of the s1-casein polymer or the -casein micelle produced by self-association is dependent on pH and ionic strength, and also sensitively on temperature in the case of -casein. Temperature is important for the strength of hydrophobic attraction, but pH and ionic strength govern electrostatic repulsion and its range. The balance of these attractive and repulsive components in the overall interaction free-energy equation thus controls the aggregate size and, more importantly in a local situation, the strength of individual intermolecular bonds.

Casein micelle assembly

From these concepts, Horne (1998) devised a polymerization scheme for the assembly of casein micelles. Cross-linking of the molecules is envisaged as proceeding via two routes, hydrophobic interactions between groups on different molecules forming one pathway, with more than two molecules possibly joining at such junctions, and a second pathway where chain extension is through calcium phosphate nanoclusters, small calcium phosphate crystallites, the precipitation of which is regulated by the presence of the caseins. A calcium phosphate nanocluster acts as a neutralizing bridge between two phosphoseryl clusters on different molecules of s1-, s2- or -casein. Again, more than two casein molecules could be involved with any one calcium phosphate nanocluster. If the casein molecule is -casein, further extension of this chain is through a hydrophobic linkage. Both routes permit branching and hence lead to a three-dimensional network structure. -Casein can link only to a hydrophobic region on another molecule. Because it has no phosphoseryl cluster on the opposite end of the molecule to permit further extension, the polymer chain ends there. No further growth occurs beyond this point. This occurs for each growing chain and hence the proportion of -casein limits the micelle size. In consequence, the micelle acquires an external coat of -casein which acts as a steric stabilizer for the micelle. In devising this mechanism for micellar assembly, no new features are ascribed to the casein molecules. The ability to bond and the strength of those bonds is the resultant of a localized favourable balance of attractive hydrophobic interaction and electrostatic repulsion. Reducing that hydrophobic interaction by lowering the temperature, or increasing electrostatic repulsion by dissolving out calcium phosphate but maintaining pH, weakens those bonds and causes (partial) disintegration of the micelle. Casein micelle properties

Almost all the casein proteins present in bovine milk are incorporated into the casein micelles, together with a high proportion of the available calcium and inorganic phosphate. These micelles have an average molecular weight of ⬃108 Da and a mean diameter of ⬃100 nm (range 50–600 nm). The micelles are very open, highly hydrated structures with typical hydration values of 2–4 g H2O g 1 protein, depending on the method of measurement. The structure is not rigidly fixed but dynamic. Cooling the milk from the 37 °C of the udder to storage at refrigeration temperatures brings about solubilization of a significant fraction of -casein, some -casein and much lower levels of s1- and s2-caseins from the micelles, and all


of this is pH-dependent also (Dalgleish and Law, 1988). Raising the temperature back to 37 °C reverses this process. Almost complete disintegration of the micelles can be achieved either through the addition of a strong calcium sequestrant such as EDTA (Griffin et al., 1988) or through the addition of high concentrations of urea (McGann and Fox, 1974). Dissociation to molecular level is not achieved, and the dissociated species have average diameters of the order of 10–15 nm and are also of variable composition (Aoki et al., 1985). All of these experimentally observed properties, temperature- or reagent-dependent dissociation, variable composition with size, location of -casein, inverse relationship of micellar size and -casein content are predicted or are manifest as a consequence of the dualbinding assembly model described above. As far as the mechanism of chymosin-induced aggregation of casein micelles is concerned, the proposed theories largely neglect internal micellar structure or at least regard it as of no consequence to the outcome of the reaction. Whilst we do not deviate from this view, as described below, we feel that internal micellar structure and the modifications to it as a consequence of pH, salt and temperature changes occurring during curd manufacture should also be considered and that these must impact on curd properties. Micelle stability

The casein micelle system is an excellent example of colloidal dispersion. Repulsive forces hold the micelles in suspension until removed by some external influence. Because the casein micelles were negatively charged, resulting in a zeta potential of about 20 mV, and this charge is reduced by ⬃50% on rennet treatment (Green and Crutchfield, 1971; Pearse, 1976; Darling and Dickson, 1979; Dalgleish, 1984), attempts were made to explain the stability of the casein micelle using the DLVO (Derjaguin-Lamdau-Verwey-Overbeek) theory of the stability of lyophobic colloids (Verwey and Overbeek, 1948). Such concepts envisage stability as arising from the presence of a repulsive energy barrier, the resultant of ubiquitous attractive Van der Waals forces and repulsive electrostatic forces. Unfortunately, as Payens (1979) calculated, this energy barrier is located at such a short inter-surface distance (⬃0.1 nm) as to be physically meaningless, lying well within the orbit of surface roughness, the loops and the tails of the protein molecules in the outer micellar regions. Though the complete DLVO theory is rendered inapplicable by the above and other failures related to ionic strength changes, the general concept of micellar stability being due to the presence of a repulsive energy barrier is still valid. It is now accepted that micellar stability arises from the presence of a sterically stabilizing

outer layer of -casein molecules, the C-terminal portion of which extends out into the solution (Holt, 1975; Walstra, 1979; Holt and Horne, 1996). Repulsion arises due to the increase in free energy brought about when the protein layer of one micelle is brought into contact with (or overlaps) the layer of another micelle. The role of chymosin is to proteolyze -casein, splitting it at the Phe1059Met106 bond and thus shave off the hairy layer, so that the subsequently exposed micelle cores begin to aggregate, once sufficient of their -casein has been hydrolysed. The overall milk clotting process is shown diagrammatically in Fig. 1. Of these stages, only the proteolytic cleavage can be monitored totally independently of the others by following the release of the glycomacropeptide, -CNf 106–169, or the formation of para--casein, residues 1–105. The aggregation reaction of the destabilized micelles is a consequence of this proteolysis. Its rate cannot be separated easily from that of the proteolysis reaction. The aggregation overlaps the proteolysis reaction; the latter is certainly not complete before the aggregation begins. Aggregation leads to bigger and bigger clusters until eventually the system acquires the solid-like nature of the gel. Again, there is a smooth continuum through this point and beyond as the gel matures. As we discuss in greater detail later, the separation into aggregation and gelation stages is largely artificial, driven in most instances by the requirements of the experimental technique or the mechanistic model.

Primary Enzymatic Phase The -casein molecules provide a steric stabilizing layer with their hydrophilic C-terminal peptides protruding into the aqueous phase. Gel formation is initiated by the proteolysis of the -casein molecules which is accompanied by the release of a hydrophilic peptide, termed the caseinomacropeptide, into the serum (whey) phase. The remaining N-terminal region of the -casein, termed the para--casein remains bound in the casein network. Gradual loss of the caseinomacropeptide is accompanied by a decrease in the micellar zeta potential which results in destabilization of the micelles and aggregation into a gel. Proteases capable of initiating the required proteolysis of -casein are aspartic proteinases (EC 3.4.23). Milk clotting enzymes were obtained originally by extraction from the stomachs of ruminants, and calf and adult bovine rennets are widely used in cheese manufacture today. Concerns in the 1960s that world cheese production had increased to such an extent that the production of rennet products derived from


(A) Micelles (O) + enzyme ( )

(B) Partially renneted micelles

(C) Aggregating micelles in small clusters

(D) Percolating clusters

Figure 1 A schematic description of the various stages envisaged in the enzymatic coagulation of milk, starting from the initial mixture of casein micelles and enzyme (A) and proceeding through proteolysis (B), initial aggregation into small clusters (C) and reaching a gel point at percolation (D).

animal tissue would be insufficient to meet future demand led to the development of alternative products. Rhizomucor miehei, R. pusillus and Cryphonectria parasitica were used to produce aspartic proteinases by fermentation, and these new coagulants were successfully introduced to the market. In the late 1980s, recombinant DNA technology was used to clone the gene for chymosin, the main clotting component of calf rennet. E.coli, Aspergillus niger and Kluveromyces

lactis were used as host organisms (Teuber, 1990; Harboe, 1992). The chymosin products generated are now referred to as fermentation-produced chymosin (FPC). A wide range of clotting agents are now available for cheese manufacture and the use of these coagulants in cheese manufacture has been reviewed extensively (Guinee and Wilkinson, 1992; Wigley, 1996; Fox and McSweeney, 1997; Harboe and Budtz, 1999). Calf rennet and adult bovine rennet still dominate in cheese


production, but market share for FPC continues to increase, and microbial coagulants derived from R. miehi are the third most commonly used coagulants (Harboe and Budtz, 1999). Chymosin (EC 3.4.23.4) is a gastric proteinase which is secreted in the abomasal mucosa of new-born ruminants and other mammals during the first days of life (Foltmann, 1992). It is the main clotting enzyme in calf rennet. The activity of chymosin differs markedly from that of other gastric aspartic proteinases in that it exhibits low general proteolytic activity but is particularly active in hydrolysis of the Phe1059Met106 of -casein. Milk clotting enzymes differ in the rate at which they continue to degrade casein following the hydrolysis to initiate gel formation. Only those enzymes with a high ratio of milk-clotting activity to general proteolytic activity are considered suitable for cheese manufacture. A high level of non-specific proteolysis can lead to a weak gel structure, high losses of protein and fat in the whey and reduced cheese yield. The higher the level of proteolysis, the greater is the reduction in cheese yield. Chymosin activity on -casein is limited with only formation of the caseinomacrpeptide and para--casein, while in the case of fungal proteinases, extensive non-specific hydrolysis of both -casein and para--casein occurs (Shammet et al., 1992). The use of microbial rennets is generally considered to result in reduced cheese yield, compared with calf rennet (Olson, 1977; Emmons et al., 1990a; Lucey and Kelly, 1994). The fermentation-produced chymosins have a high ratio of milk clotting to general proteolytic activity and no significant differences in cheese yield have been reported between recombinant chymosin and calf rennet (Green et al., 1985; Hicks et al., 1988; Ustinol and Hicks, 1990; Emmons et al., 1990b; Banks, 1992; van den Berg, 1992). The properties of chymosin and other aspartic proteinases have been reviewed comprehensively by Chitpinityol and Crabbe (1998) (see ‘Rennets: General and Molecular Aspects’, Volume 1). Rennet preparations are generally prepared from multiple calf stomachs and are heterogeneous in their chymosin content. Calf chymosin occurs in three forms, A, B and C, chymosin B being the most abundant in natural rennet. Chymosins A and B are allelic variants which differ at only one amino acid position; Asp243 in chymosin A is replaced by Gly243 in chymosin B. Chymosin C appears to be a degradation product of chymosin A which lacks three residues, Asp244–Phe246 (Danley and Geoghegan, 1988). The three variants show differences in clotting activity, and of the three forms, chymosin A has the highest specific clotting activity and chymosin C the lowest. The A and B forms are equally efficient in cheese manufacture (Harboe and Budtz,

1999). The cloned chymosins derived from Aspergillus niger and Kluveromyces lactis are B variants (Harboe and Budtz, 1999). The specificity of chymosin for the Phe9Met bond has been studied extensively (see Fox and McSweeney, 1997). The length of the peptide and the sequence around the sectile bond are important determinants of enzyme–substrate interactions. Observations that synthetic di-, tri- or tetra-peptides containing a Phe9Met bond were not susceptible to hydrolysis by chymosin suggested that other residues close to the cleaved bond are also required for the hydrolytic reaction (Visser et al., 1980). Kinetic studies on synthetic peptides indicated that two additional residues at either side of the hydrolysable bond are required for appreciable reaction (Hill, 1968, 1969; Raymond et al., 1972). The sequence of His989Lys111 includes all the necessary determinants (Visser et al., 1980), and this tetradecapeptide is hydrolysed with a kcat/Km of ca 2M 1 s 1, which is similar to that of intact -casein (see Fox and McSweeney, 1997). The Phe and Met residues in the chymosin-sensitive bond of -caseins are not essential for chymosin action on caseins. The residues in the chymosin-sensitive bond differ across species which suggests that it is the amino acid sequence surrounding this bond rather than the residues in the bond itself, which contain the important determinants of hydrolysis (see Fox and McSweeney, 1997). Porcine pepsin (A and C) and R. miehei proteinase cleave the Phe9Met bond in a similar fashion to chymosin but the C. parasitica proteinase cleaves the Ser1049Phe105 bond (Dronse and Foltmann, 1989). Calf rennet is the standard product against which all other coagulants are assessed. Adult bovine rennet contains a higher proportion of pepsin and therefore a more general proteolytic activity. Rennets extracted from ovine, caprine and porcine stomachs are the most efficient at clotting milk of their own species (Foltmann, 1992). Coagulants extracted from the flowers of the thistle Cynara cardunculus are used in the production of artisanal cheeses in the Iberian peninsula (Sousa et al., 2001). The coagulants are aspartic proteinases and comprise of two enzymes, cardosins A and B. Both enzymes hydrolyse the Phe1059Met106 bond of -casein (Esteves et al., 1995). Kinetic parameters of cardosin A are similar to those of chymosin while those for cardosin B are similar to pepsin (Verissimo et al., 1995). The ratio of clotting to proteolytic activity is low compared to chymosin, and non-specific casein hydrolysis results in lower gel firmness compared to that obtained with chymosin (Esteves et al., 2002).


Measurement of Clotting Time and Curd-Cutting Time The most easily detected outcome of chymosin proteolysis and rennet clotting is the visible observation of the presence of flocs in a milk sample in a rotating tube. The time taken for their appearance is defined as the rennet coagulation time, and for the cheesemaker interested in the activity of an enzyme preparation, this may be the only quantity of interest. The importance of its determination is reflected in the number of techniques tested over the years (see Lucey, 2002; O’Callaghan et al., 2002, for reviews of these methods). Since the coagulum is cut sometime after the coagulation point when it has attained sufficient firmness, the more technically successful techniques are those which continuously monitor the development of the coagulum with time by measuring changes in some particular physical attribute, such as viscosity (Scott Blair and Oosthuizen, 1961), reflectivity (Hardy and Fanni, 1981; Ustinol et al., 1991), thermal conductivity (Hori, 1985) or ultrasound transmission (Benguigui et al., 1994) to name only a few. Few of these techniques have entered commercial practice for in-vat applications not only because the instruments are often difficult to clean and maintain properly but also because the changing processing conditions and schedules in response to such instrumental readings are not always an attractive option to a large modern cheese factory. In such cases, the standardization of milk protein is the preferred approach, since this minimizes differences in coagulation and it has been observed that cheese yield does not seem to be very sensitive to small changes in gel firmness, at cutting, in such standardized circumstances. Many of the techniques described by Lucey (2002) and O’Callaghan et al. (2002) have also been developed from research tools used to study the influence of reaction variables such as temperature, pH and milk composition and pre-treatment on gel development. The most useful of these techniques are those where the behaviour of the variable of interest can be predicted by a mathematical model based on a mechanistic description of the reaction. Few, if any, techniques provide direct relationships applicable over the entire course of the reaction from micelle to gel. Indeed, this is perhaps one reason why gel formation has been split into primary and secondary phases, since the early aggregation phase can be followed readily by turbidity or light scattering (Payens et al., 1977; Dalgleish et al., 1981a,b; Dalgleish, 1983; Bauer et al., 1995; Lomholt et al., 1998) whereas the gel formation and development is most easily monitored in the laboratory by rheometry (Tokita et al., 1982; Bohlin et al., 1984; Van Hooydonk

and Walstra, 1987; Zoon et al., 1988a,b,c, 1989a,b; Van Vliet et al., 1991; Horne, 1995, 1996; Lopez et al., 1998; Mellema et al., 2002). Each technique suffers from limitations. Light scattering requires a dilute dispersion of particles so that only singly scattered photons are collected at the detector. Direct conversion to molecular weight and/or size is also limited by the ratio of particle size to light wavelength. Studies using light scattering are thus limited to early stages of aggregation, where growth of molecular weight or degree of polymerization is obtained as a function of reaction time. Rheological measurements suffer from the opposite failing. There, the limitation is instrument-sensitivity and a detectable signal is realized only after the reaction has progressed to a significant extent. The relationship between measured viscoelasticity and gel structure and bonding is also highly model-dependent, as we shall see, and interpretations are often controversial.

Kinetic Models of Rennet Coagulation The earliest attempt to describe the kinetics of the clotting process was made in the 1870s by Storch and Segeleke (see Foltmann, 1959, 1971). This simply stated that the clotting time was inversely related to the concentration of rennet used to clot the milk. A further refinement was postulated by Holter (1932) and rearranged by Foltmann (1959) to give the familiar equation: RCT

k A [E]

(1)

where k and A are constants and [E] is the enzyme concentration, RCT being the rennet coagulation time. This relationship is purely empirical, but it is an important relationship which has to be satisfied by any more descriptive mechanistic model, even if only over a restricted range of enzyme concentration and RCT values. The Holter modification separated the coagulation time into two components, an enzyme proteolysis stage and a secondary coagulation phase. The equation assumes that there is no overlap between the proteolysis and the coagulation phases and that the extent of proteolysis is always the same at RCT (Foltmann, 1971). Experimental evidence suggests that the proteolyzed fraction of -casein is very high. Dalgleish (1979) suggested 60–80% of the -casein must be hydrolysed, though his plot shows no significant aggregation below 90% proteolysis. Green et al. (1978) found that aggregation did not start until after about 60–80% of the RCT had passed, by which time the


enzyme action was almost complete. In a separate experiment, Green et al. (1978) found that the viscosity of milk rose sharply when the enzyme reaction was ⬃86% complete. Other groups have found that viscosity increases well before the visible coagulation time indicated by the presence of flocs (Guthy and Novak, 1977), confirming an inescapable fact that the detection of an aggregation point is determined by the sensitivity of the measuring technique to the presence of aggregates and also that the two processes, proteolysis and subsequent aggregation, overlap in time. The extent of overlap, or the percentage of -casein proteolyzed before aggregation becomes detectable, is also dependent on milk pH and ionic calcium content, decreasing as pH is decreased (Van Hooydonk et al., 1986; Carlson et al., 1987a,b) or as Ca2 content increased (De Kruif, 1999; Horne and McCreight, unpublished observations). The functional description of the overall kinetics of coagulation must therefore take both proteolysis and aggregation reactions into account. The kinetics of the proteolysis reaction has been discussed by Dalgleish (1993) and Hyslop (2003). In milk, the reaction appears to be of first order, but whether the reaction is truly first order throughout or follows standard Michaelis–Menten kinetics for a single-step enzymecatalysed reaction with a relatively high value for the dissociation constant of the chymosin–-casein complex, where the Michaelis–Menten equation approximates to a first-order picture, is still undecided (Hyslop, 2003). Many attempts have been made to model the aggregation reaction, the models differing in how the aggregation rate constant is modelled and how it depends on the enzymatic proteolysis of -casein. Beginning with the model calculations of Payens (1976, 1977, 1989), Payens et al. (1977) Payens and Brinkhuis (1986), and Hyslop (2003) has outlined the nuances of the various schemes, highlighting their differences and critically pointing out their shortcomings. Depending on the assumptions made and the experimental circumstances involved, he concluded that three models, step-function (Dalgleish, 1980a,b), energy barrier (Darling and Van Hooydonk, 1981) and functionality theory (Hyslop, 1993; Hyslop and Qvist, 1996) may be used to describe the aggregation reaction. The step-function model (Dalgleish, 1980a,b) is based on the idea of a critical level of proteolysis before aggregation is possible, but does not explain why this should be necessary. A plausible explanation arises if an energy barrier is gradually reduced by rennet proteolysis, leading to a gradually increasing probability of reaction on collision in the aggregation reaction. Introduced by Darling and Van Hooydonk (1981), the

energy barrier model has been tested extensively (Van Hooydonk and Walstra, 1987; Dalgleish, 1988; Hyslop, 1989; Payens, 1989; Hyslop and Qvist, 1996). Energy barrier models are mean-field models. They imply the existence of a uniform repulsive force that decreases gradually with time, and therefore uniform micellar surfaces. Since the -casein molecules are hydrolysed individually, a more realistic approach might be the creation of attractive patches on the micelle surface by the removal of sufficient macropeptide hairs, as envisaged in the geometric model of Dalgleish and Holt (1988). Continuing removal of -casein hairs would lead to multiple patches and the creation of conditions necessary for the operation of a polyfunctional model of the Flory–Stockmayer type (Stockmayer, 1943), which gives the rate constant as: kij K{4 2(f 2) (i j) (f 2)2 ij}

(2)

where K proportionality factor; f number of functional sites (functionality); i, j number of particles of type i, j. If f 1, only dimers are possible, if f 2, linear polymers are predicted and if f 2, chain-branching occurs and gelation is possible. In the beginning at t 0, f 0, the micelles have no reactivity or inclination to aggregate, and a realistic model has to account for the growth of f during the course of the reaction. This is generally done by proposing that f is some function of the degree of proteolysis of -casein, most frequently linear. A further refinement in this model is to allow the proportionality factor to depend on the energy barrier height (Bauer et al., 1995). All the various models describe essentially the growth in average molar mass of the micellar aggregate with time of reaction. Average molecular weight is most readily measurable by static light-scattering techniques. Due to problems encountered with multiple light scattering, where the detected photon has encountered more than one scatterer in its passage through the suspension, these techniques are applicable only in highly diluted suspensions (Dalgleish et al., 1981a,b; Brinkhuis and Payens, 1984; Bauer et al., 1995), or over very short path lengths, as in the turbidity measurements of Lomholt et al. (1998). Increasing complexity in the models increases the number of parameters the value of which can be varied to fit experimental data. Possibly their most stringent test so far has been carried out by Lomholt et al. (1998) who considered most variations involving energy barriers. They found that they could obtain good representations of the initial stages of renneting, up to aggregates of ⬃5–10 micelles, with plausible values for the variable parameters, energy barrier height for native casein


micelles and after completion of rennet proteolysis. They reproduced the growth of aggregate size with time, adequately accounting for the effect of enzyme concentration and to some extent micellar casein concentration. They were, however, unable to differentiate between the different model expressions, all giving equally acceptable fits, and thus were unable to reliably relate experimental data to any particular mechanistic picture. A major disadvantage of these experiments is that they are concerned only with the initial stages of the coagulation reaction, largely because of constraints relating to the experimental techniques. Early lightscattering studies (Dalgleish et al., 1981a,b; Brinkhuis and Payens, 1984) operated in dilute solution because of this but even in the case of the later work of Lomholt et al. (1998) the effects of multiple scattering by large aggregates produced an upper limit to the size which can be extracted from such data (Worning et al., 1998). Consequently, although they operated in a concentration regime where coagulation eventually occurred, Lomholt et al. (1998) were unable to derive any information on the progress of the reaction in this region.

Adhesive Sphere Models and Viscosity Viscometry was one of the techniques used in early attempts to monitor the course of the rennet coagulation reaction (Scott Blair and Oosthuizen, 1961). The relationship between viscosity and molecular weight is rather complex and the results therefore do not lend themselves to ready testing of the theoretical models outlined above. On addition of chymosin to milk, there is an initial decrease in the viscosity which then passes through a minimum before increasing sharply as the coagulation progresses. Rather than interpreting this rise as due to the formation of aggregates and from them a gelling network, De Kruif and coworkers (De Kruif et al., 1992; De Kruif, 1999) have proposed a different mechanism based on treating casein micelles as sterically stabilized hard spheres which become sticky, or adhesive, as the -casein is proteolyzed. They write the relative viscosity of skim milk as:

冢

r 1 2.5 5.9

冣

1.9 2

B2 4

1

(4)

冢

冢冣冣

B2

4 12 1 exp VHS kT

冢 [P][P] [P] 冣

hkT ln

(5)

(6)

where [P] is the concentration of macropeptide released at time t, and the other parameters are defined in Fig. 2, VHS being the hard sphere volume 4/3 a3, where a is the effective micelle radius. The only fitting parameter is h, which is of order 2 (De Kruif, 1999). The initial decrease in the viscosity arises because of the decrease in hydrodynamic volume fraction, , as the -casein hairs are sheared. This decrease in hydrodynamic size has been measured experimentally using dynamic light scattering techniques in both dilute (Walstra et al., 1981) and concentrated (Horne and Davidson, 1993) micellar suspensions. In the adhesive sphere model, however, the loss of the -casein-stabilizing hairs also causes the attractive well depth to increase in proportion to the logarithm of the normalized hair loss. This stickiness then produces the observed increase in viscosity in this model. At later times, however, the viscosity also increases due to aggregation and network formation. Passage to this status is seamless, and with no indication of its occurrence, making realistic assessment of the validity of the model at least problematical. Nevertheless, on its basis, De Kruif (1999) has demonstrated that the micellar system can be flocculated at higher hair density (lower levels of proteolysis) when ionic calcium level is increased and that renneting time can be shortened by the inclusion of increasing amounts of ethanol. A major disadvantage of the adhesive sphere model is that its time dependence relates only to the proteolysis reaction. Indeed, it is confined purely to

(3)

where is the micellar volume fraction and a stickiness parameter related through the second virial coefficient (B2) to the depth ( ) of an attractive square well potential created as the -casein hairs are proteolyzed. The relevant equations are:

Figure 2 A schematic defining the terminology of the adhesive sphere model. The micelle of diameter () has a hairy layer of thickness () equivalent to the width of the square well potential. The depth of this attractive potential ( ) deepens as the hairs are proteolyzed by chymosin.


pre-aggregation events and can say nothing of the kinetics of aggregation and gel formation, highly significant events in the definition of gel properties.

Development of Rheological Properties during Rennet Coagulation Possibly the most direct way to measure gel formation is to monitor the evolution of rheological properties (Bohlin et al., 1984; Dejmek, 1987; Zoon et al., 1988a; Horne, 1995, 1996). Dynamic rheology applies an oscillatory shear stress (0 ) or strain (0) and measures the response from the developing gel. The measurement yields the elastic or storage modulus (G), which is a measure of the energy stored per oscillation cycle and reflects how the sample behaves as an elastic solid, and the viscous or loss modulus (G) which is a measure of the energy dissipated per cycle and indicates how much the sample behaves as a viscous liquid. Their ratio is tan , the tangent of the phase angle of the response to the applied stress or strain. The shear moduli are defined as follows:

G

冢冣cos

(7)

冢冣sin

(8)

0

0

G

0

0

G G

(9)

90

90

80

80

70

70

60

δ

60

G′

50

50

40

40

30

30

20

20 G″

10

Phase angle (deg)

Shear moduli, G ′, G ″ (Pa)

tan

Thus, when 45°, the viscous component dominates, whereas when 45°, the sample appears more like an elastic solid. Ideally, measurements should be made in the linear viscoelastic region, i.e., deformation should be proportional to the applied stress. When the gel is very weak in the earliest stages of reaction, it is debatable whether this situation is achievable. Minimum forces must be applied for the instrument to produce a discernible movement in its geometry, or in the case of a constant strain rheometer, the movement has to be detectable but a minimum force will be required to overcome inertia, friction in bearings, etc. This minimum movement or stress may be sufficient to damage the gel at this point in its development. Fortunately, the rennetinduced gel develops viscoelasticity rapidly and so moves into linear response quickly but, nevertheless, the gel point may vary between instruments or instrument settings, and too large applied stresses or strains should be avoided. Figure 3 shows an example of the development of viscoelasticity in a skim milk sample during renneting. The sharp decrease in the phase angle, from close to the 90° of a Newtonian liquid to around 20° coincides approximately with the visible coagulation time. In the same time frame, gel firmness as indicated by the growth of shear moduli, becomes apparent. Although not visible on the scale plotted, G initially lags on growth of viscosity or viscous modulus, G, but quickly crosses and dominates as gel elasticity rapidly develops, the transition point occurring at 45°. Plots of shear moduli versus reaction time evolve as sigmoidal curves which tend to approach a constant

10

0

0 0

0.5

1.0

1.5

2.0

2.5

3

Time (ks) Figure 3 A typical example of the changes in viscoelastic parameters observed during gel formation, showing increases in shear moduli (G, G) and decreases in phase angle () with time after enzyme addition.


value appropriate to each milk sample at very long times. In practice, of course, the gel is generally cut at a fixed time after rennet addition or after visible coagulation is noted, perhaps after a period of no more than 30 min. In either instance, the gel firmness is likely to be of the order of 30 Pa and possibly has not passed its maximum rate of firming. A rennet-induced milk gel is described as a viscoelastic solid. In rheological terms, the critical gelation point is often taken as the time at which the elastic modulus exceeds the viscous modulus (Ross Murphy, 1995). Others demand compliance with more stringent conditions, such as the Winter and Chambon (1986) criterion which gives the gelation as occurring at the time when both G and G show power-law dependence on oscillation frequency, both dependencies with the same positive exponent. Derived for chemically cross-linked polymers, this time does not necessarily coincide with that for G and G cross-over in a single frequency experiment. Depending on the instrument sensitivity and the reaction, it may not even be possible to detect this cross-over. In some concentrated systems, the value of G may have already exceeded G, whilst in others it may occur early in the reaction below the instrument detection threshold. In the latter circumstance, an acceptable working definition of the gel point would be when instrument response rises above the background noise level. What physically defines the gel point and, beyond that critical point, what defines the dynamic evolution of the viscoelasticity of the gel in terms of a mechanism, describing network, creation and growth are the subjects of the next section.

Theoretical Basis of Viscoelasticity Continuity of structure and permanency of that structure are general features of a gel. We continue to visualize the rennet-induced milk gel as a particle gel. The spatial distribution of the casein micelles (the particles) in the gel network and the strength of the bonding between those elements define the existence and the rheology of the gel. By considering the deformation of the network following application of a shearing force, Van Vliet and Walstra (1985) derived the following equation for the modulus of the network:

G CN

d2F dx2

(10)

where N is the number of stress-bearing strands per unit area in a cross-section perpendicular to x, the direction of the external force or stress, C is a charac-

teristic length determining the geometry of the network, dF is the change in Gibbs free energy when the elements are moved apart over a distance dx, and is therefore related to the bond strength in systems where enthalphic effects dominate over entropic, as argued by Van Vliet and Walstra (1985). In the case of particles which are homogeneously distributed over the available space, or at least homogeneously on the length scales of the experimental measuring device, all particles involved in the network will contribute to the network modulus to the same extent. N will then be directly proportional to the volume fraction of particles in the network. In describing the formation of the rennet-induced milk gel, G will be a function of reaction time, and thus the growth of the shear modulus with time will depend on the rate of incorporation of particles into the network and the rate of change of bond strength (or increase in well depth) with time. Two possible scenarios present themselves. In the first, only the well depth is considered as a function of time. This is akin to the adhesive sphere concept of De Kruif and coworkers (1992); De Kruif (1999) and De Kruif and Holt (2003) would assume that all of the casein micelles would be considered as a network from time zero, the point at which -casein proteolysis is initiated and the attractive well begins to deepen. Dickinson (2003) has emphasized that the hard sphere model operates within the framework of equilibrium statistical mechanics. It therefore describes the gelation for relatively weak, short-range attractions under reversible conditions. That is, the particles are free to dissociate, as well as to associate, the dynamic equilibrium constant or bond lifetime being a function of well depth. With well depth increasing as proteolysis progresses, the bonds become increasingly stronger and could become effectively irreversible within the time-span of the experiment. This bond lifetime or relaxation time has an important bearing on the response of the network in the rheometer geometry, where the lifetime of a bond must be correlated with the oscillation time of the applied deformation. Strong bonds with a high energy content will generally have a long relaxation time. They establish the permanent or elastic character of the gel. Weak bonds generally break and reform spontaneously over much shorter timescales. They contribute to the temporary character of the gel network, appearing as the viscous component. Thus, non-relaxing bonds only contribute to G whereas rapidly relaxing bonds only contribute to G. Bonds with relaxation times in the timescale of the measurement contribute to both G and G. Because bond lifetime changes smoothly during proteolysis and gel formation, in this model from very short to


very long, the gelling system passes monotonically from a viscous liquid to a firm elastic solid and the phase angle, , passes through the critical 45° value. Changes in phase angle may therefore more sensitively reflect changes in the nature of the bonds (Roefs, 1985). As Fig. 3 shows, however, during the formation of a rennet-induced milk gel, the phase angle decreases rapidly in the early stages through the gel point but remains substantially constant throughout the main growth period in the shear moduli, as was also observed by Dejmek (1987) and Lopez et al. (1998). This indicates that the nature and strength of the bonds does not change significantly during this growth. It is therefore likely that the increase in gel firmness is caused by an increase in the number of bonds with time, the other time-dependent factor in our theoretical expression for the modulus, G, in equation 10. In the second scenario, the micelles aggregate into clusters and these clusters eventually form the gel network. The kinetics of this aggregation can be viewed as an increase in the number of bonds with time. What point in the time course of the progress of this reaction can be identified as the gel point, and can this be detected experimentally? Again, two extreme scenar-

ios can be envisaged (Fig. 4). The first is a strong gelation where bond energy is kT and bond formation is virtually irreversible. As depicted in Fig. 4, gelation corresponds to the achievement of a percolation cluster spanning the container (Stauffer, 1976; DeGennes, 1979). This critical percolation cluster is present only for a very short moment. Gel curing continues as the remaining free particles and clusters are incorporated into the network, creating more stress-carrying strands. This is what is observed as the growth of shear modulus with time. In the second scenario in aggregate growth (Fig. 4b), bonds are weak, aggregation is reversible, and clusters break up as readily as they form. The critical gelation condition is reached when the clusters achieve a pseudoclose-packed arrangement as depicted in Fig. 4b. The majority of potential bonds are satisfied at this juncture, and increases in shear moduli occur as the system rearranges itself in its quest for an equilibrium structure. Can the measured kinetics of gel formation assist in differentiating these two extremes? As is shown in Fig. 5a, gel cure curves, plotted as complex modulus, G*, versus t, are typical for each milk. In the set shown, the same reaction conditions, rheometer settings and

(a)

(b)

Figure 4 Two different scenarios defining the occurrence of the gel point. (a) The percolation cluster model where gelation is sensed by the measuring device whenever clusters extend across the container. Note the number of clusters and monomers which have still to be incorporated into the gel, creating more links and strands to carry applied stress. (b) The formation of a weak gel where most particles are weakly linked into the network but reversibility allows rearrangements. The larger circles depict the fractal ‘blob’ concept where the system is fractal within but closed packed outside.


1.5

150 (a) 4

5

1.25 1 1.00

100

3

75

G ′/G ′ at 3*tg

Shear modulus, G ′ (Pa)

125

(b)

7

6

50

8

0.75 0.50

0.25

25

2 0

0 0

2.0 4.0 6.0 8.0 10.0 12.0 14 Time from rennet addition (ks)

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Reduced time, t/tg

Figure 5 Depiction of the derivation of the master curve plot from a series of gel cure curves obtained by renneting individual milks (1–7) under the same reaction conditions (left-hand plot). The data in the right-hand plot were obtained by dividing the reaction time (time after enzyme addition) by the clotting time (tg) and then normalizing each individual curve against its particular value at 3tg.

chymosin concentration were used in each case but the individual milks exhibit their own gelation times, tg, rates of curd firming and ultimate gel firmness. When these curves are re-plotted against a reduced time, t/tg, and each normalized against its particular value at a low multiple of tg, usually two or three, all curves collapse onto a master curve (Fig. 5b) (Horne, 1995). Such behaviour is known as ‘scaling’. The fact that the curves can be scaled has several implications. It implies that the mathematical expression describing the time-dependent growth of the shear modulus of the curve can be factorized into the product of two terms and written as: G(t) G f

冢 tt 冣

(11)

g

where G is a simple constant, the asymptotic value of the shear modulus at infinite reaction time for a particular milk system. As a constant, it embodies all the static attributes of the gel. The second term, f(t/tg), is more complicated, but it is nevertheless a function of a single variable, the reduced time, t/tg, and all data sets follow this master function which describes the dynamics of the gel-formation process. It is important to realize the significance of the dependence on the critical gel time, tg. It is defined as the time required to form the critical gel network and is the rate parameter controlling the aggregation reaction leading to that network. Its presence as the parameter also controlling the rate of gel cure indi-

cates that essentially the same kinetics is dominating that reaction. In essence, this is a pointer to the first, the percolation, scenario as being the more true picture, with the further incorporation of micelles and clusters being simply another part of the ongoing aggregation reaction throughout all phases of gel development. The gel-development kinetics clearly depends on the rate of the proteolysis reaction, the rate controlling the achievement of the gel time. Lomholt and Qvist (1997) have raised doubts about this scaling approach, showing that multiplying reaction time by the rate constant for proteolysis, ke, does not produce a master cure curve in a series of experiments conducted with varying enzyme and protein concentrations. Our results use tg 1 as the reducing factor, and other groups have found that the product of tg and ke is not always unity, as attempting to consider them equivalent would imply (Tokita et al., 1982). Perhaps the answer lies in the mode of action of the enzyme and how the activation of the micelle towards aggregation comes about. Proteolysis estimates give a global figure for the suspension as a whole which may not relate to the status of individual micelles, whereas gel times are a direct measure of the aggregation behaviour. Further work in this area is undoubtedly required. A further advantage of the scaling approach is the observation that f(t/tg) is a single-valued function, in the sense that any one value of the ratio t/tg leads to a single value for f(t/tg). That being the case, the value of G at a defined value of the reduced time, say t/tg 3,


will always be the same fraction of G. Thus, whilst G is essentially unattainable, all information on its behaviour is accessible through the value of the shear modulus as the multiple of tg selected for comparison purposes. Based on this accessibility to G, Horne (1996) has developed a mechanical model for the gel. If it is assumed that each micelle has the potential for forming an average number of bonds, k, with its neighbours, then these will all be fully linked into the network attained at infinite time and will therefore define the value of G. With all of the micelles incorporated into the network, the number of bonds will be proportional to the number of micelles present. Hence, according to equation 10, G will be proportional to the number of micelles, assuming that all bonds are of equal strength. If the average micelle is of diameter, d, then at a given concentration of c g/ml, the number of micelles is proportional to c/d3. Hence, this simple model predicts: G

c d3

(12)

and since G at 3tg is a fixed fraction of G by the scaling requirements, then Gat 3tg

c d3

(13)

Horne (1996) fractionated casein micelles according to size from individual cow milks using a sequence of eight consecutive centrifugation steps, applying the centrifugation to the supernatant of the previous step, as described in detail by Dalgleish et al. (1989). These pellets were re-suspended in milk ultrafiltrate to the same casein concentration and their average size was measured independently by dynamic light scattering. The micellar suspensions were then renneted under the same incubation conditions. Growth of the elastic modulus was monitored as a function of time. G* at 3tg was found to be inversely proportional to the cube of micelle size, as predicted by equation 13. In a separate experiment, milk from a single source was concentrated by ultrafiltration and then diluted back to give a series of milks of the same micellar size distribution but differing in protein concentration. When these milks were renneted, the value of G* at 2tg was found to be a linear function of the concentration factor, again as predicted by Horne (1996). Niki et al. (1994) also prepared sized fractions separated from skim milk by differential centrifugation. They confirmed that smaller micelles gave firmer gels but not as strongly as the inverse cubic dependence predicted. Their micellar fractions were, however, used as prepared

and not adjusted to a constant protein concentration. Obviously, those fractions from the wings of the size distribution have a lower protein content than those obtained around the mean size. Niki et al. (1994) do not give details of the protein content or composition of their fractions, making it impossible to recalculate their data on the basis of the behaviour predicted by equation 13.

Modelling the Gel-Firming Kinetics Many attempts have been made to produce mathematical equations to predict the growth of gel firmness with time. These range from the purely empirical (Scott Blair and Burnett, 1958) to those rooted in some kinetic mechanism (Tuszynski, 1971; Douillard, 1973; Carlson et al., 1987a; Clark and Amici, 2003). Tested simply on their ability to fit the observed growth curves, some are more successful than others which fail to reproduce salient features. Others have no theoretical basis which makes them less useful as predictive tools. Scott Blair and Burnett (1958) proposed an empirical model of the form:

冢 (t t ) 冣

G(t) Gexp

(14)

g

to describe the increase in shear modulus (G) with time beyond the gel point occurring at tg, being a constant characteristic of the sample and determined by fitting. Dejmek (1987) demonstrated that this model provides a good fit to experimentally obtained cure curves but has non-random residuals, indicating that the function may not be appropriate for its intended purpose. Dejmek (1987) also proposed equations relating the parameters of the Scott Blair equation to the value of the experimental cure curve at its inflexion point, obviating the need for measurements at extended times, but relying perhaps too heavily on a few points in that particular region of the cure curve. In favour of this model is the observation that this function shows an acceleratory growth phase immediately following the gel point, that it evinces a maximum rate of firmness and tends to a plateau as t tends to infinity. Against, it is its purely empirical nature which makes prediction of the dependence of its parameters on reaction variables such as temperature, pH or enzyme concentration all but impossible. Arguing that the shape of the growth curve was similar to that for simple autocatalytic reactions, Tuszynski (1971) proposed that the growth kinetics be fitted by the model: dG kG(G G) dt*

(15)


where t* t tg, G is the value of the shear modulus at t and k is the rate constant for the process but essentially a fitting parameter. Again, the model provides no indication how changes in reaction variables will influence the gel-firming process. Like the Scott Blair model, it too gives sigmoidal behaviour but it is a symmetric function, predicting that the inflexion occurs when G 0.5G, and this is not observed in practice. Douillard (1973) proposed a model in which the rate of change of shear modulus with time followed first-order kinetics: dG k(G G) dt*

t* 0

or

t tg

(16)

This equation can be integrated to give: G G[1 exp ( kt*)]

(17)

with the initial condition that G 0 at t tg. This equation has recurred several times in the history of studies on the rennet coagulation of milk. Tokita et al. (1982) fitted their cure curves to an nth-order reaction equation and determined that the first-order form, the Douillard equation above, gave the best fit. They further demonstrated that the gel time, tg, obtained in these studies varied inversely with enzyme concentration, and that the rate parameter, k, had a power-law dependence on enzyme concentration with an exponent of 0.8. In their discussion, Tokita et al. (1982) extended the consideration of the Smoluchowski equation and its use in the kinetics of polymer gelation by Ziff (1980) and Ziff and Stell (1982) to rennet gel formation. Assuming that ‘gel’ reacts with ‘polymer’ in the sol phase and that ‘gel’ does not cross-link by itself, they show that the concentration of gel is proportional to 1 exp( t). With the further assumption that is proportional to the rate constant of the Smoluchowski equation and that the elastic modulus is proportional to the concentration of gel, the Douillard equation emerges. Tokita et al. (1982) thus reached the important conclusion that beyond the gel point, most of the growth in gel firmness arises as a result of aggregation between the infinite cluster and the smaller clusters and micelles in the sol phase. In a later paper, Tokita (1989) considered gel formation as a percolation process. In percolation, the bond formation probability is defined by N/Ntotal where N is the number of bonds formed up to that point and Ntotal is the total number of bonds possible in the system. Ntotal is the product of the number of lattice sites multiplied by their functionality, Z, the number of bonds

allowed for each site. The percolation probability, P(p), is defined as the probability that a site chosen at random will belong to the infinite cluster. With the assumption that P(p) 1 exp( Zp) for p pc, the critical percolation probability, he equated G to P(p) and went on to derive the Douillard equation. Fitting his latest data to this equation, Tokita (1989) found that the reaction rate constant was best described as a linear function of the enzyme concentration used, slightly different from their earlier result. The Douillard equation also emerges as a limiting case of the Carlson model (Carlson et al., 1987a) discussed below. Whilst it is thus widely applied, the Douillard equation does not reproduce one of the most significant features experimentally observed in the gel cure curves obtained with modern more sensitive rheometers, namely the acceleratory phase immediately after the gel point, and hence further refinements to it are required. Some of these are to be found in the Carlson model. Carlson et al. (1987a) derived the rather complex model given by:

冤冢 k k k 冣exp( k t*) l

G G 1

l

f

f

冢 k ⴚk k 冣exp( k t*)冥 f

l

f

l

(18)

where t* t tg, kl is the rate constant for the creation of ‘active’ sites and kf is the rate constant for the destruction of these sites as they are incorporated into the gel network. Essentially, the model envisages ‘active’ sites being created on micelles, which then go on to react with one another in forming bonds in the gel network. Both reactions, activation and destruction, are considered as first-order processes. Activation requires the enzymatic hydrolysis of -casein, and therefore kl emerges as proportional to the enzyme concentration. That the site destruction reaction should also be a firstorder process is an empirical observation which fits in well with the idea of the gel network mopping-up smaller clusters and micelles still requiring to be activated beyond the gel point. When the enzyme concentration is large, -casein hydrolysis is rapid compared to the removal of activated micelles and the Douillard equation is recovered with a rate constant now equal to that for the activation reaction and therefore governed by the enzymatic hydrolysis process. In the hands of the present authors, application of the Carlson model to gel firming curves gave excellent fits with very low standard errors (Horne, unpublished observations). This confirms the views of Esteves et al. (2001) who compared its performance to those of the


Scott Blair and Douillard models, although they finally considered the Scott Blair model superior because it gave a smaller standard error and lower fluctuations in the systematic oscillations of the residuals. Unfortunately, as we commented above, the Scott Blair model has no basis in theory which would permit predictive use of its parameters. Further efforts should therefore be directed to more extensive tests of the Carlson model with perhaps extensions to incorporate the refinements of aggregation models now extant. More quantitative testing of the model would also discover whether the many parameters involved possess realistic values or whether they are merely ‘best-fits’. Along yet another avenue, Clark and Amici (2003) have compared the predictions of cascade theory, a random cross-linking polymerization theory, with experimental biopolymer gelation curves. The comparison was made of log (G/G) versus tg/t, a linear transform of the Scott Blair equation, for calculated and experimental data. For rennet-induced milk gels they obtained reasonable fits when the critical gelling concentration (C0) was set much less than the micellar concentration (C). The calculations require that the ratio C/C0 be set, so the experimental data were compared to a series of theoretical curves calculated for a range of these values. Accepting that the fits are not outstanding, Clark and Amici (2003) point out that the cascade model used does not contain any pre-gelation kinetic terms and, in other studies of polymer gelation carried out with this theoretical approach, their inclusion markedly influenced later events beyond the gel point. These theoretical approaches are very interesting and tantalizing but more work is needed to fully explore the implications of their results before a definitive model of the cure curve can be achieved.

Fractal Models of Rennet-Induced Milk Gels and Rearrangements Fractal aggregation theories have been applied to the flocculation of casein particles by Bremer and coworkers (Bremer et al., 1989; Bremer, 1992). Aggregates can be considered fractal if their geometry is scale invariant which implies that their structure is similar when viewed over a reasonably large range of length scales or magnifications. The emphasis of the fractal concept is therefore on structure. It is a mathematical description of the distribution of a particle cluster or network in space. Various models are then used to predict gel or cluster properties based on that structural organization. The number of particles in an aggregate or cluster (Np) is given by: Np

冢 Ra 冣

Df

(19)

where R is the radius of the floc, a is the primary particle size and Df is the fractal dimension. The latter is usually a non-integer and is always less than the geometric or Euclidean dimension of three. This equation implies that the cluster becomes ever more tenuous as it grows, as verified in computer simulations of aggregation reactions (Kolb et al., 1983; Meakin, 1983) and experimental measurements on dilute colloidal systems (Lin et al., 1990). These results demonstrate that extremes of reaction probability give rise to different fractal dimensions, ranging from 1.7 for a diffusionlimited cluster–cluster aggregation to 2.5 for a reaction-limited particle–cluster process. Since the number of particles that could be present in a close-packed cluster is given by: Nc

冢 Ra 冣 , 3

(20)

the volume fraction of the cluster is given by:

cluster

Np Nc

冢 aR 冣

Df

(21)

The average volume fraction therefore decreases as the cluster grows. When it reduces to the volume fraction of particles in the system, 0, the clusters fill the total space available and the gel is formed. Bremer and coworkers (Bremer et al., 1989; Bremer, 1992) define the gel point by this event which implies that all particles present in the system are incorporated in the clusters. The real question is whether this can be equated to the rheological gel point recognized experimentally but this appears to be the assumption made. The decrease in density can be accomplished only if the growing cluster develops holes or voids of everincreasing size as the cluster grows. This is the meaning of scale invariance. When such a cluster grows to macroscopic size, it should have macroscopic holes on that length scale. No such holes are seen in particle gels, of which rennet-induced milk gels are our examples. Instead, they appear as a homogeneous, solid-like mass. Brown (1987) circumvented this difficulty by introducing the concept of the fractal blob, suggesting that clusters grow to a size, Rblob, and these then closepack homogeneously to give a uniform volume fraction defined by that of the blob at that point. The picture of the network is then similar to that depicted in Fig. 4b, and at the gel point the volume fraction achieved is again that originally in the suspension, 0. With all of the particles (micelles) already bonded into the network at the gel point, the only way in which gel firmness can grow with time thereafter is


through rearrangements of the bonds already in the structure, a phenomenon referred to as ageing by Mellema et al. (2002). Various models have been elaborated relating the elastic modulus of the gel to the volume fraction through a power-law equation, with the exponent of this equation written as a simple function of the fractal dimension and other possible parameters (Bremer, 1992). Such models allow the stress-carrying strands to be straight or curved. Further, in the models of Shih et al. (1990), the elasticity of the gels may be determined by the elasticity of the flocs or blobs (strong links between blobs) or dominated by the elastic content of the inter-floc links (weak-link regime). It perhaps should be mentioned here that Shih et al. (1990) categorically state that their models apply well above the gelation threshold. This creates a total of four possible expressions for the exponent, yet the experimentally observed power-law dependence of G on volume fraction gives a single value. In some instances, model candidates can be eliminated because they yield unphysical values for the fractal dimension. In others, no choice can be selected without other independent information. In a summarizing expression, Mellema (2000) has written the exponent as:

3 Df

where

2 1

(22)

is the number of junctions or links per strand (0, 1 or 2), and the value of is set by the dominant type of macroscopic deformation: bending ( 1) or stretching ( 0).

With this background, Mellema (2000) went on to consider four levels of rearrangement in rennetinduced gels, operating on different length scales. These were intra-micellar rearrangement, individual micelle shifts, strand rearrangements and in the whole gel (syneresis), the latter as a result (mainly) of the previous three categories listed. These possibilities are depicted in Fig. 6. Applying this picture to the cure curves, Mellema (2000) accommodated increases in elastic modulus with time by postulating changes in the parameter , as the gel aged. This necessitated assuming that the fractal dimension, Df, was constant throughout, or that its variation with time was measured independently in separate experiments (Mellema et al., 2000). Gel cure was therefore interpreted as arising from changes in strand thickness, strand conformation and number of linkages depending on how was modified but no kinetic mechanism was derived to predict the dynamics of these changes and directly test the speculations, no matter how reasonable.

Milk Processing and Gel Formation Rennet clotting activity as influenced by milk processing

Milk coagulation by rennet can be influenced by a number of processing treatments applied to the milk (Harboe and Budtz, 1999). The gel formation characteristics of high-pressure and heat-treated milks for cheesemaking have been studied extensively in recent years. Interest in these areas will be sustained as both treatments can be used to maximize cheese yield.

Figure 6 Diagrams of the various pathways open in rearrangements of the network. These can occur on several length-scales, at internal micellar links where the individual proteins rearrange themselves and allow more and more links to be formed between the original particles, along the chains where particles can detach and reattach forming new links (centre drawing) and the detachment of chains either at one or both ends leaving them to find a new home elsewhere on the network. All three processes contribute to macroscopic syneresis of the maturing curd (adapted from Mellema et al., 2002).

64 Rennet-induced Coagulation of Milk High pressure

High pressure treatment influences the coagulation and cheesemaking properties of milk indirectly through a number of effects on milk proteins, including a reduction in the size of casein micelles, denaturation of -lactoglobulin and possible interaction of lactoglobulin with micellar -casein (Trujillo et al., 2000, 2002; O’Reilly et al., 2001; Huppertz et al., 2002). Gel firmness and cheese yield can be improved by highpressure treatment of milk through an increased recovery of whey proteins and increased moisture content. Treatment of milk at pressures of up to 200 MPa for 30 min reduces the RCT while higher pressures, up to 600 MPa, result in RCT values similar to those of untreated milk (Lopez-Fandino et al., 1996, 1997; Needs et al., 2000). Changes in RCT observed are associated with changes in both the enzymatic primary phase of coagulation and the secondary phase of aggregation. These changes would be expected to be associated with changes in micelle size resulting from high-pressure treatment. The average casein micelle size is unchanged in reconstituted skim milk treated at pressures of 150–250 MPa (Desobry-Banon et al., 1994; Gaucheron et al., 1997), although one report (Needs et al., 2000) suggests a small increase of 9% in micelle size in raw skim milk using a pressure of 200 MPa. At pressures between 250 and 600 MPa, micelle size is reduced by 40–50% in reconstituted skim milk (Desobry-Banon et al., 1994) or raw skim milk (Needs et al., 2000). The effect of pressure treatment on micelle size in reconstituted skim milk is temperature-dependent (Gaucheron et al., 1997). Pressure treatment of milk at 4 °C reduced micelle size, at 20 °C resulted in no change and at 40 °C causes an increase in micelle size which may be associated with interactions between fragments of casein micelles and denatured whey proteins (Buchheim et al., 1996). Transfer of individual caseins from the colloidal to the soluble phase has been observed at pressures of 100–400 Mpa (Law et al., 1998; Lopez-Fandino et al., 1998). Treatment of raw milk at a pressure of up to 200 MPa for 30 min reduced the RCT, while further increases in pressures up to 400 MPa resulted in RCT values close to those for untreated milk (LopezFandino et al., 1996, 1997; Needs et al., 2000). The RCT of pressure-treated milk is affected by both the temperature of treatment and the pH of the milk. Treatment at 200 MPa at 60 °C or 300 MPa at 50 °C inhibits the rennet coagulation of milk (Lopez-Fandino and Olano, 1998). Acidification of milk to pH 5.5 prior to high pressure treatment reduced its RCT whereas increasing pH to 7.0 had the opposite effect (Arias et al., 2000).

The reduced release of CMP during the primary phase for samples treated at 400 MPa or at 300 MPa at 40 °C has been associated with the interaction of high-pressure-denatured -lactoglobulin with glycosylated -casein, which would hinder the action of chymosin on -casein (Lopez-Fandino et al., 1997; Lopez-Fandino and Olano, 1998). Blocking agents have been used to show that high pressure effects observed on RCT are associated with sulphydryl interactions which cause the -lactoglobulin to bind to the surface of micelles via interaction with -casein (Needs et al., 2000). However, Needs et al. (2000) reported that the release of glycosylated CMP was unaffected by high-pressure treatment, and only the second phase of rennet coagulation (rate of micelle aggregation) was affected. Rates of aggregation and gel formation of milk treated at 200 MPa were considerably higher than for untreated milk, but these rates decreased at higher pressures. Samples treated at 400 or 600 MPa produced higher gel strengths than samples treated at 200 MPa or untreated samples. The authors concluded that two opposing mechanisms operate to control the rate of aggregation – there was a direct effect of pressure on the properties of the micelles, which resulted in their rapid aggregation (following the hydrolysis of -casein), while increasing -lactoglobulin denaturation reduced the rate of aggregation. Coagulation time was not related to the degree of -casein hydrolysis, which suggested that pressure favoured the aggregation stage. Heat treatment

Heat treatment of milk results in a number of changes in physico-chemical properties. These include the denaturation of whey proteins, the interactions between the denatured whey proteins and the casein micelles and the conversion of soluble calcium to the colloidal state. High heat treatment of milk for cheesemaking provides a potential route for maximizing cheese yield by the inclusion of whey proteins in curd (Singh and Waungana, 2001). However, milk which has been heated at a temperature in excess of pasteurization has poor renneting and gel formation characteristics (Morrissey, 1969; Dalgleish, 1992), and a number of studies have explored the extent to which the primary enzymatic and the secondary phases of aggregation are influenced by heat treatment (Van Hooydonk et al., 1987; Dalgleish, 1990; Leaver et al., 1995; Waungana et al., 1996). Thermal denaturation of -lactoglobulin is known to affect the cheesemaking properties of milk. It has been claimed that heating milk affects the clotting process by slowing or inhibiting the primary phase of rennet action as -casein--lactoglobulin cross-linking


reduces the susceptibility of -casein to hydrolysis by chymosin (Van Hooydonk et al., 1987; Leaver et al., 1995). The decrease in the rate of gel formation and final gel firmness in heated milks can also be attributed to the association of whey protein aggregates with casein micelle surfaces through the formation of a -Lg–-casein complex which may protrude from the micelle surface (Singh and Waungana, 2001). This association would affect the close approach of the reactive sites formed on the micelles by the action of rennet. Following the hydrolysis of -casein to para-casein, aggregation would occur mostly between micelles not fully covered with -Lg, resulting in the formation of fewer bridges with fewer and weaker bonds. The severity of heat treatment will determine the extent of inhibition of either the primary enzymatic phase or the secondary aggregation phase (Singh and Waungana, 2001). The rennet coagulation properties of heated milk can be partially restored either by (i) acidification of heated milks to pH values below 6.2, (ii) acidification of heated milk to low pH values (⬃5.5) followed by reneutralization to 6.7, which is termed pH cycling or (iii) heating at elevated pH combined with pH cycling and CaCl2 addition (see Singh and Waungana, 2001, for a review). Acidification or pH cycling has been used in the manufacture of Cheddar cheese from severely heated milks (Banks et al., 1987, 1993; Banks, 1988; Imafidon and Farkye, 1993). Improvements in cheese yield of up to 4.0% on a dry solids basis were achieved (Banks et al., 1993).

Conclusions Despite intensive research effort, now spanning many decades, there is still no definitive overall description of the kinetics of gel formation which would allow prediction of the cutting time from a knowledge of milk composition and treatment. Even now, we are perhaps only realizing that we can treat the reaction as a continuum and that we have largely forgotten that it was mainly the constraints of earlier theories and methodologies that artificially divided the process and confined studies to particular stages of aggregation and curd development. In rheometry, we now have the instrumentation to directly measure cure curves and as these instruments become ever more sensitive, the effects of possible gel damage in the early stages of the reaction lessen, allowing aggregation and gelation to come seamlessly together instrumentally. Exciting new developments in the fields of microscopy and image analysis allow the potential to

follow the mobility of particles through the gel point as they are confined and incorporated into a gel network. This will perhaps settle the question as to whether gel cure is the result of the firming up of a percolated initial structure for the gel as sol material is included into the network or whether the rearrangement process dominates beyond the gel point, as the fractal models demand. The fractal picture is important, however, because it forces us to confront the role of rearrangement in determining gel firmness, particularly the shifts in bonding within the micelle, which we would argue should be considered within the context of the dualbinding model of the micelle described earlier. Whilst the majority of model studies of rennetinduced gelation have been carried out (fortuitously) at high pH where micellar integrity seems assured, many cheesemaking procedures involve a lowering of the pH by straightforward adjustment or by fermentative growth of starter cultures. Lowering the pH leads to a solubilization of calcium phosphate, a consequent decrease in the number of bonds preserving micellar integrity and an increased propensity for rearrangements of protein molecules within and between aggregated micelles. Horne (2001, 2003) has demonstrated the influence of such breakdown in micellar integrity in the context of gel development in studies of model yoghurt systems. The rate at which such processes occur will impact on the rate of increase of the elastic modulus of the gel with time but this aspect is yet to be considered in the context of the dynamics of rennet-induced gel development. Further work in this area is required to assess that impact, perhaps by looking for deviations in the scaling behaviour as the gel cures, perhaps by including terms in the Carlson (1987a,b) model to accommodate reversibility in the removal of activated micelles, or perhaps by considering altogether new models for the network structure and the manner in which the elastic modulus is related to that structure.

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The Syneresis of Rennet-coagulated Curd P. Dejmek, Department of Food Engineering, Lund University, Lund, Sweden P. Walstra, Department of Agrotechnology and Food Sciences, Wageningen University, Wageningen, The Netherlands

Introduction Gels formed from milk by renneting or acidification under quiescent conditions may subsequently show syneresis, i.e., expel liquid (whey), because the gel (curd) contracts. Under quiescent conditions, a rennetinduced milk gel may lose two-thirds of its volume, and up to 90%, or even more, if external pressure is applied. Often, syneresis is undesired, e.g., during storage of products like yoghurt, sour cream, cream cheese or quark; hence, it is useful to know under what conditions syneresis can be (largely) prevented. In making cheese from renneted or acidified milk, syneresis is an essential step. Consequently, it is useful to understand and quantitatively describe syneresis as a function of milk properties and process conditions, particularly when new methods or process steps are introduced in cheesemaking. This concerns several aspects: • regulation of the water content of the cheese implies controlling syneresis; • the rate of syneresis affects the method of processing, and thereby the equipment and time needed, and the losses of fat and protein in the whey; • rate of syneresis in relation to other changes (e.g., acidification, proteolysis, inactivation of rennet enzymes) affects cheese composition and properties; • the way in which syneresis of curd grains proceeds may affect the propensity of the grains to fuse into a continuous mass during shaping and/or pressing; • differences in syneresis throughout a mass of curd cause differences in the composition of the cheese between loaves of one batch and between sites in one loaf; • after a cheese loaf has been formed, it may still show syneresis and hence loss of moisture.

(based on Chapter 5 in the 2nd edition of ‘Cheese: Chemistry, Physics and Microbiology’, P.F. Fox, ed., Chapman & Hall, London 1993, by P. Walstra, revised and updated by P. Dejmek).

(Note: throughout this chapter we will use the word ‘moisture’ for any liquid that may move through curd or cheese; it is thus generally an aqueous solution and not just water). Thus, the importance of syneresis is obvious. Accordingly, numerous research reports have been published, providing many important data on the influence of various factors on the rate, and sometimes on the end-point, of syneresis. However, the results vary considerably according to the conditions during the test method employed and are difficult to interpret. Grounds to a deeper understanding of syneresis were laid in the late 1980s and early 1990s (van Dijk, 1982; van den Bijgaart, 1988; Akkerman, 1992; Walstra et al., 1985). Recent overviews were given by van Vliet and Walstra (1994) and Lucey (2001).

Gel Formation and Properties The casein micelle

As shown in ‘Rennet-induced Coagulation of Milk’, the caseins of milk occur under physiological conditions as large polydisperse aggregates, i.e., casein micelles, up to 0.5 m. Details of the internal structure of the micelles are still being discussed, but there is little doubt that the existence of this aggregated state is dependent on hydrophobic interactions and on calcium phosphate nanoclusters connected to the phosphoserines of the individual casein molecules. The solution stability of the micelles is dependent on the presence of charged groups and steric stabilization (Walstra, 1990). Both of these can be manipulated in dairy processing with the aim of destabilizing the micelles and promoting further aggregation of the caseins. The aggregation may lead to a gel and then to gel shrinkage, syneresis. Most of the -casein of the micelles is at the surface and the strongly hydrophilic C-terminal part of these molecules apparently sticks out from the micelle surface as a flexible chain that perpetually changes its conformation by Brownian motion (Walstra and Jenness, 1984), thereby causing steric repulsion, though only a third of



72 The Syneresis of Rennet-coagulated Curd

the surface appears to be covered by -casein (Dalgleish, 1998). The micelles are thus said to be ‘hairy’. They also have a negative charge, causing electrostatic repulsion between them. Steric and electrostatic repulsion provide complete stability of the micelles against aggregation under physiological conditions. There are multiple dynamic equilibria between caseins, and different forms of calcium and phosphate in the micelles and in the solution. The micelles may change considerably due to changes in their environment. At low temperatures, a part of the casein, especially -casein, goes into solution and additional ‘hairs’ of partly protruding -casein molecules are presumably formed. A small part of the micellar calcium phosphate also goes into solution. The micelles attain a higher voluminosity (i.e., they swell). These changes are reversible, although it is not quite certain that the micelles regain exactly their native structure after cooling and rewarming. At high temperatures, the amount of micellar calcium phosphate increases somewhat. At temperatures high enough for serum proteins to denature, association of denatured serum proteins with the micelles occurs, to an extent greatly dependent on pH – the lower the pH, the stronger the association. Lowering the pH causes considerable change. Some trends are illustrated in Fig. 1; in as far as it concerns properties of a rennet gel, these are discussed later. The main change is that micellar calcium and phosphate go into solution, thereby loosening the bonds keeping the micelles together. This leads to dissolution of casein, especially at low temperature. At still lower pH, electrostatic bonds between positive and negative groups on the caseins keep the micelles together, and at the isoelectric pH, these bonds are quite strong, again. In fact, the casein particles at this pH are very different from the micelles at physiological conditions, although their size distribution has not changed greatly (Roefs et al., 1985). It should also be noted that a lower pH in milk leads to a higher calcium ion activity, which also lowers the negative charge on the micelles. Starting at about pH 5 (Vasbinder et al., 2001) and at not too low a temperature, the casein particles begin to aggregate; electrostatic repulsion is now absent and the -casein hairs, which provide steric repulsion, are also lost (they are presumably ‘curled up’). Addition of calcium at a constant pH to milk reduces the negative charge on the micelles and increases the amount of micellar phosphate. This reduces the stability of the micelles and high levels of added calcium cause their aggregation. Renneting

During the renneting of milk, the proteolytic enzymes in the rennet (mainly chymosin) hydrolyse the -casein molecules to para--casein and soluble caseino-

40 Ca

%

Pin

20 30 °C 0 –ζ (mV) 10 20 °C 0 500

20 °C

G ′ (Pa) 250 0 0.4 tan δ 0.2

20 °C

0 4.5

5.5 pH

6.5

Figure 1 The proportion of calcium (Ca) and inorganic phosphate (Pin) and the electro-kinetic potential () of casein micelles, as well as the dynamic shear modulus (G, frequency 1 s 1) and the loss tangent (tan , frequency 0.01 s 1) of rennet-induced skim milk gels, as a function of pH (from Walstra, 1990).

macropeptides (the C-terminal region), thereby largely removing the hairs and greatly reducing steric and electrostatic repulsion. The micelles can now approach one another closely and it is observed that they flocculate, i.e., remain close together. The kinetics of renneting is intricate since two reactions are involved. The enzymic reaction is essentially first order and the flocculation can be described, in principle, by Smoluchowski kinetics (van Hooydonk and Walstra, 1987). The caseinomacropeptide segments are removed from the micelles one by one (a micelle contains in the order of 1000 -casein molecules, and the number of micelles is roughly 100 times the number of chymosin molecules normally added to cheese milk). Consequently, the reactivity of the micelles, i.e., the probability that micelles which encounter each other will become flocculated, at first remains low but strongly increases as a greater proportion of the -casein has been hydrolysed (see also Fig. 2). The reactivity is roughly an inverse exponential function of the concentration of unhydrolysed -casein molecules on the

The Syneresis of Rennet-coagulated Curd 73

100

50 S

G

V

0 0

20

40

Time (min) Figure 2 Approximate example of the changes that occur in milk after adding rennet. Degree of hydrolysis of -casein (S ), aggregation of para-casein micelles as measured by viscosity (V ) and shear modulus (G) of the gel formed as a percentage of the values after 40 min, as a function of time.

micelles. As long as less than about 70% is hydrolysed, the flocculation rate is virtual1y zero, at least at physiological pH and 30 °C. If the pH is lowered, the enzymic reaction becomes much faster and, moreover, flocculation starts at a lower proportion of hydrolysed -casein molecules (van Hooydonk et al., 1986). It appears that at low pH, the chymosin becomes adsorbed onto the micel1es and this causes the hydrolysis of the -casein to be not quite random any more. Presumably, a chymosin molecule now often makes a ‘bare’ patch on the micelle before becoming desorbed and diffusing away, to find another (or possibly the same) micel1e on which to act. At such a bare spot, the micel1e is reactive. This implies that at a lower pH, flocculation starts at a stage where less -casein has been hydrolysed. The reactivity of ful1y renneted micel1es, i.e., those that are ful1y converted into paracasein micel1es, depends little on pH, increases with Ca2 concentration, decreases with increasing ionic strength (NaCl) and increases markedly with temperature, especial1y from 15 to 30 °C (Dalgleish, 1983). Above 50 °C, the flocculation rate becomes almost independent of temperature, being roughly equal to that predicted by Smoluchowski’s equation for diffusion-controlled coagulation (Dalgleish, 1983). The temperature dependence is often taken as indicative of hydrophobic interactions being responsible for the reaction between the para-casein micelles (Dalgleish, 1983). An alternative explanation is that with decreasing temperature only the activation free energy for flocculation increases, presumably because of protrusion of -casein chains. Gel formation

After a while, flocculation leads to the formation of a gel (see Fig. 2). Microscopically, one can observe that aggregates are formed, at first irregular, but often

somewhat thread-like; these grow to form large tenuous flocs, until they start to touch and form a continuous network (Mulder et al., 1966; Henstra and Schmidt, 1970; Walstra et al., 1985). Electron microscopy reveals (e.g., Kalab and Harwalkar, 1973; Knoop and Peters, 1975a; Green et al., 1978) that the network can be described as consisting of strands of micelles, 1–4 micelles thick and some 10 micelles long, alternated by thicker nodes of micelles and leaving openings up to 10 m in diameter. The essential requirement for gel formation is of course a thermodynamic instability of the system, i.e., an attraction between the particles high enough (relative to thermal energy) to bring about the formation of a condensed phase at the existing particle volume fraction. Gels are typically formed in systems where the range of the interparticle attraction is short compared to particle size. In addition to short range, the attraction needs to be sufficiently strong. A weak attraction allows the particles to asociate and dissociate until they find a position binding with many neighbours, and thus form a compact aggregate. When the attraction is sufficiently large, particles will stick at first contact, and a ramified structure may span the entire system, provided that the kinetics of aggregation is faster than the sedimentation of the aggregates formed. The topology of the resulting network can readily be described by the theory of ‘fractal’ aggregation (Family and Landau, 1984; Meakin, 1988). For particle gel formation, the fractal mechanism was pointed out in a qualitative sense by Walstra et al. (1985) and quantitatively applied to the flocculation of casein particles by Bremer and coworkers (Bremer et al., 1989, 1990; Walstra et al., 1990; Bremer, 1992). The current view of fractal particle gels in foods was summarized by Walstra (2000). Assuming random aggregation of particles and of aggregates already formed (called cluster–cluster aggregation), computer simulations show the aggregates formed to be stochastic fractals, i.e., structures that are on average scale-invariant at scales larger than that of the primary particles (radius a). The number of particles in an aggregate or floc is given by: Na

冢 Ra 冣

D

(1)

where R is the radius of the floc and D the fractal dimensionality, which is always smaller than three. This implies that the floc becomes ever more tenuous as it becomes larger; computer simulations show rarefied structures, consisting mainly of long irregular strands of particles, which are in most places only one particle thick. Equation (1) has been shown to hold


remarkably well over a wide range of R and under many conditions, both in simulations and experiments; colloidal interaction forces and geometrical constraints determine the value of D. The number of particles that could be present in a floc if the particles were closely packed, obviously is: Na

冢 Ra 冣

3

(2)

This implies that the average volume fraction of particles in a floc is given by:

floc

NP Na

冢 Ra 冣

D 3

(3)

The average volume fraction of the flocs thus decreases during flocculation, and when it has reached the volume fraction of particles in the system, (for para-casein micelles at 30 °C, about 0.09), the flocs fill the total space available and a gel has formed. It also follows that the average radius of the flocs at the moment of gelation is given by: Rgel a1/(D 3)

(4)

In the above derivation, it has been implicitly assumed that flocculation proceeds undisturbed. But if the liquid is stirred during flocculation, gel formation may be hindered. Another disturbance may be appreciable sedimentation of the flocs occurring before a gel can be formed. The casein micelles in milk are small enough, and differ little enough in density from the milk serum, for sedimentation to be negligible. It may thus be assumed that under normal renneting conditions, gel formation occurs unhindered. If equal-sized spherical particles flocculate in Brownian motion and if each encounter leads to lasting contact (so-called diffusion-limited cluster–cluster aggregation), the fractal dimensionality turns out to be about 1.8. Several deviations from this simplest model, for instance a situation (as during renneting) in which only a certain small proportion of the encounters of particles leads to their lasting contact (so-called chemically limited aggregation), or rearrangements occurring in the floc structure, lead to higher D values. Moreover, during gel formation, the flocs interpenetrate to some extent and this also causes a higher dimensionality. One type of change that certainly does occur in the flocs is a rearrangement of just-flocculated particles in such a way that each particle will touch more than two other particles; this leads, in principle, to strands of thickness of

about three particles rather than one (Meakin, 1988). This is in agreement with microscopical observations on casein gels (Bremer, 1992). Such a rearrangement does not detract from the initial fractal nature of the flocs or the gel formed from the flocs. Brownian dynamics is a tool which has been used to probe theoretically how different choices of interaction potentials can affect the properties of the gel network (Bijsterbosch et al., 1995; Bos and van Opheusden, 1996; Mellema et al., 1999; Dickinson, 2000; Rzepiela et al., 2001). The findings modulate the simple irreversible fractal description; the low cut-off of the fractal regime, i.e., the size of the building block typically increases in time, and the fractal dimension may depend on both the interactions and the volume fraction. For low-capture efficiency, the fractal dimensionality tends to 2.35 (Walstra, 2000; Mellema et al., 2002b). For real casein gels, volume fractal dimensions have been determined experimentally by a variety of methods, including wavelength dependence of turbidity, angle dependence of light scattering and analysis of electron or confocal microscopy images. The values most commonly found are D 2.2 2.4 (Bremer et al., 1989, 1990; de Kruif et al., 1995; Mellema et al., 2000). The experimental values of D obtained are, however, much dependent on the evaluation procedure (Mellema et al., 2000) and the assumptions involved in interpretation of the experimental data (Bushell et al., 2002). Worning et al. (1998) questioned the validity of the turbidity approach, and the same group found by light scattering, D 2 (Lehner et al., 1999). Assuming the radius of para-casein micelles to be 55 nm and their volume fraction in milk to be 0.09, it is calculated that the average radius of the flocs at the onset of gelation is about 2.5 m and that these flocs contain several thousand para-casein micelles. There is, however, considerable spread in these values within one gel, and the gel is thus fairly inhomogeneous (see Fig. 3). The average pore size in the gel is of the order of Rgel but some pores are larger. Average pore size is related to the permeability, B, in the equation of Darcy: v

冢 B 冣p

(5)

which relates the superficial velocity, v, of a liquid of viscosity, , flowing through the gel due to a pressure gradient p. The permeability of a ‘fractal’ gel is, under some assumptions, given by: B const. a22/(D 3)

(6)

The constant is not easily calculated; it is much smaller than unity. For D 2.3, the power of is about 2.9


Figure 3 Optical sections, made by confocal scanning laser microscopy in fluorescent mode, of rennet-induced skim milk gels, aged for 1 h (top) or 18 h at 30 °C. The bars indicate 10 m (from Bremer, 1992).

(in agreement with experiments), which implies that the permeability of the gel depends strongly on the initial and thus on casein concentration. A similar strong dependence on holds for some other properties and for the size of the flocs at the onset of flocculation. Above, it has been tacitly assumed that skim milk is renneted. In the presence of fat globules, flocculation and gel formation proceed somewhat differently, but not greatly. The pores in the gel of para-casein micelles are roughly large enough (about 4 m) and sufficient in number (about 2.1016 m 3) to accommodate the fat

globules (average diameter – about 3.4 m; number of globules larger than 1 m – 3.1015 m 3; Walstra and Jenness, 1984). Nevertheless, the pore size distribution in the gel is, of course, somewhat influenced by the presence of the fat globules, and most fat globules are entrapped in the gel. Rheological characteristics

The discussion will be based mainly on the extensive results of Zoon et al. (1988a,c, 1989a,b). A convenient and easily observed characteristic of a gel is its small


deformation modulus, i.e., the ratio of the applied stress over the resulting strain (relative deformation). Mostly, the dynamic shear modulus, G, is determined (which implies that the deformation type is simple shear) as a function of the frequency of deformation, . Most gels are visco-elastic materials and these are characterized by two parameters. The storage modulus, G, is a measure of the true elastic property of the gel, the loss modulus, G, of the viscous property; G/ can be seen as a viscosity. We further have G2 G2 G2. In these dynamic measurements, the material is brought under an oscillating small strain, and G and G can be determined separately, each as a function of ; the timescale of the deformation is about 1. Values of G are shown in Fig. 1. The moduli were observed to depend generally on and to increase steeply with , in agreement with the theory of fractal gels (Bremer et al., 1990; Bremer and van Vliet, 1991). The model predictions linking fractal dimensionality to rheological properties need to take into account the topology and the connectivity of the network, information which is not contained in the fractal dimensionality (Roberts and Knackstedt, 1996; Mellema et al., 2002a). An important parameter is the loss tangent (tan G/G), as it is a measure of the preponderance of viscous (or liquid-like) or over-elastic (or solid-like) properties of the gel. It is related to the relaxation of bonds in the gel during its deformation, and therefore it mostly increases with increasing timescale (decreasing oscillation frequency); this is because, in general, a greater proportion of the bonds that are under stress can relax when the timescale is longer. For rennet milk gels at physiological pH and 30 °C, tan 0.45 0.6 at 10 3 s 1, i.e., under conditions relevant for syneresis. This implies that a rennet milk gel has a significant viscous component in its rheological behaviour. In accordance with this, it is observed that its relaxation time, i.e., the time needed for the stress to decrease to 1/e of its initial value if a certain small deformation is applied to the material, is of the order of only 1 min. The loss tangent does not depend on casein concentration and is virtually independent of the age of the gel once formed. The modulus of the gel strongly increases after it is formed (see Fig. 2). Potentially, the increase could be

a

b

due to two phenomena. One is that additional junctions are formed between casein particles, partly because there are strands of particles that are attached to the gel at only one end, partly because additional casein particles and small clusters thereof become incorporated into the gel. The latter situation will always occur to some extent during the formation of a particulate gel, but more strongly during normal renneting, since at the moment of gel formation not all casein micelles have been fully transformed into paracasein micelles. However, only a negligible amount of free casein was found in the whey from a rennet milk gel quite early in the renneting process, at G less than 10% of its ultimate value (Mellema et al., 2002b), and similar values were found in simulations (Mellema et al., 1999). The other phenomenon is illustrated in Fig. 4, which is derived from electron microscopical studies (Knoop and Peters, 1975b). Any ‘junction’, by which is meant a contact region between two original micelles, must contain several bonds, and the number of bonds per junction increases on ageing. One may say that the micelles more or less fuse, and after some hours the original particles making up the gel can no longer be distinguished. If no starter is added and the proteolytic enzymes of milk have been inactivated, the increase in modulus continues for about 24 h (Zoon et al., 1988a). The lower the temperature, the slower and the longer-lasting is the increase in modulus. As mentioned, the increase in the number of bonds does not lead to a significant change in the loss tangent. For deformations (in shear) larger than about 3%, the rheological behaviour of developed rennet milk gels becomes non-linear; during the early stages of gel development, the linear range is wider. In curd-making practice, the stresses applied are often too large for linear behaviour. Figure 5 shows what happens when a relatively large stress is applied (Zoon et al., 1989b). After the instantaneous (elastic) response, the deformation soon becomes virtually viscous, i.e., d/dt is constant. After some, often fairly long, time, the deformation rate increases and eventually becomes infinite – the gel fractures. Fracture does not mean falling into pieces, but rupture of the gel matrix only – the cleft formed fills

c

d

Figure 4 Schematic picture of the change in conformation of flocculated para-casein micelles during ageing of the gel (from Walstra and van Vliet, 1986).


2

Deformation (γ)

238 Pa

68 Pa 1 35 Pa

0 0

100

200

300

Time (s) Figure 5 Deformation in shear () of a gel of renneted skim milk as a function of time, applying a constant stress. Temperature: 30 °C, pH: 6.65, gel aged for 3.5 h. The stress applied is indicated near the curves. At 35 Pa, fracture occurred after 1350 s (from results of Zoon et al., 1989b).

with whey immediately. Presumably, local fracture occurs already at an early stage, as soon as the linear deformation range is exceeded; the small cracks formed increase slowly in size and number, and coalesce until a fracture plane throughout the whole test piece has formed. This implies that long before macroscopic fracture, the gel structure has been altered markedly, which has been confirmed in loading–unloading experiments (Zoon et al., 1989b). Note that the shear at fracture is very large; values between 1 and 5 have been obtained (van Dijk, 1982; Zoon et al., 1989b), according to conditions. It is seen (Fig. 5) that a higher stress leads to smaller deformation at fracture and to a much shorter time than needed for fracture to occur. In other words, at a shorter timescale, the fracture stress is higher. Like the modulus, the fracture stress increases with ageing of the gel. The results of experiments at large deformations depend on the type of test applied (e.g., creep or dynamic), but the same trends are observed. Attempts to gain insights into the causal relationships between the range and the strength of forces between particles and network flow behaviour are being made by simulations (Whittle and Dickinson, 1998; Dickinson, 2000; Rzepiela et al., 2002). Temperature has a big effect on gel properties (Zoon et al., 1988b, 1989b). One should, however, distinguish between temperature of renneting and of measuring rheological properties. If renneting is at a lower temperature, gel formation is much slower and the modulus of the gel may consequently be smaller when measured at the same time, but this is not a true representation of the effect of temperature on gel properties. Lowering the

temperature of a formed rennet milk gel generally causes a very brief decrease in modulus, but the latter subsequently starts to increase to reach a constant higher level after, say, 1 h. At 10 3 s 1, the storage modulus at 20 °C is about 2.4 times that at 30 °C. From the effect of temperature on the loss tangent it is seen that a rennet milk gel is much more solid-like at lower temperatures; similar behaviour could be seen even in a non-renneted, centrifuged pellet of casein micelles which gels at a low temperature (Horne, 1998). The permeability tends to be higher at higher temperatures; this will be considered later. At large deformations, a higher temperature causes a larger deformation and a lower stress at fracture. Some effects of acidity (Zoon et al., 1989a; Roefs et al., 1990) are shown in Fig. 1. Again, one should distinguish the pH of renneting from that at measurement, since renneting at a lower pH causes faster gelation. Figure 1 gives results obtained several hours after renneting at the pH values indicated, and it is seen that the storage modulus at first increases with decreasing pH, to decrease again at still lower pH values; the loss tangent continues to increase, until the pH range where a ‘rennet reinforced acidic gel’ (Tranchant et al., 2001) begins to develop. At large deformations, the effects of moderate acidity are not great (Zoon et al., 1989b); the fracture stress is somewhat higher for a lower pH, if determined at the same timescale. Acid gels

The casein particles at pH 4.6 are rather different from those at physiological pH, as is illustrated in Fig. 1. They


are very prone to aggregation (except at low temperature), they contain no undissolved inorganic phosphate and they have a (presumed) voluminosity at 30 °C of about 3.4 ml g 1 (leading to 0.08 in skim milk). Casein particles from strongly heat-treated milk differ significantly in their properties, and due to their associated whey proteins may behave as -lactoglobulin during acidification (Vasbinder et al., 2001) and recently it was recognized that thiol cross-linking occurs in acid gels from heat-treated milk (Vasbinder et al., 2003). Unmodified caseins form a gel at temperatures above about 10 °C. Gel properties have been studied extensively (Roefs and van Vliet, 1990; Roefs et al., 1990a; Lucey et al., 1997a,b) including those of gels made by the combined action of acid and rennet (Roefs et al., 1990b; Lucey et al., 1998, 2000, 2001; Tranchant et al., 2001) and gels from transglutaminase-cross-linked micelles (Schorsch et al., 2000). The gels are in many respects quite comparable to renneted milk gels (Table 1). They are also of a fractal nature and have roughly the same fractal dimensionality and thereby about the same dependence of modulus and of permeability on casein concentration (Bremer et al., 1989, 1990). The absolute value of the permeability is also roughly the same, as is the pore size distribution. The rheological properties are, however, rather different. They are treated separately in

Table 1 Properties of skim milk gels obtained by renneting (aged for about 1 h) or by acidification (aged for 6–16 h). Acid gels are of type 1 (obtained by cold acidification and subsequent warming) or of type 2 (obtained by slow acidification with glucono-lactone at 30 °C). Approximate results at 30 °C (from various sources) Acid gel Property

Rennet gel

Type 1

Type 2

pH G at 0.01 rad s 1 (Pa) Tan at 0.01 rad s 1 Fracture stressa (Pa) Fracture straina ( ) Permeability B (m2) Fractal dimensionalityb dB/dt (nm2 s 1) Initial syneresis ratec

6.65

4.6

4.6

32

180

20

0.55

0.27

0.27

10

100

100

3.0

0.5

1.1

0.25

0.15

0.15

2.23 20

2.39 1

2.36 –

15

1

1

a Loading time 1000 s. b From the relation between concentration and B. c Arbitrary units.

‘Formation, Structural Properties and Rheology of Acidcoagulated Milk Gels’, Volume 1.

Syneresis Mechanism of syneresis

Various mechanisms have been held responsible for syneresis (for an early review, see Walstra et al., 1985). Summarizing, the following types of mechanism were distinguished: • A decrease in solvation or water binding of the material making up the gel. For a particulate gel, this way of explaining syneresis does not appear suitable and there is no indication that an ongoing change in solvation is involved. • Shrinkage of the building blocks of the gel, i.e., the para-casein micelles in our case. This may happen when the pH is lowered or the temperature increased, but syneresis also occurs under constant conditions. • Rearrangement of the network of para-casein micelles. This is the main cause of syneresis. The para-casein particles in the gel form junctions with a limited number (mostly 2–4) of others. (Strictly speaking, this is not true. As mentioned earlier, there is a rapid rearrangement into thicker strands, leading to a higher coordination number. However, one may use the same arguments by considering the ‘particles’ to be aggregates of, on average, three micelles.) However, the particles are expected to be reactive over their entire surface (or to contain numerous reactive sites smeared out over their surface), and in the initial fractal network, by far the greater part of the surface of each particle does not touch (form bonds with) another one. Rearrangement of the particles into a more compact network would thus increase the number of bonds and hence decrease the total free energy (the counteracting loss in mixing entropy is very small). But the particles cannot easily attain a more compact configuration because they are almost immobilized in the network. In other words, the network has to be deformed locally to form new junctions. Thermal motion of the strands may occasionally bring two particles in different strands close to each other so that a new junction is formed, especially shortly after renneting. This would lead to the build-up of a tensile stress in at least some of the strands. The fusion process illustrated in Fig. 4 may also cause such a stress to develop. As a result, strands may occasionally break, providing a possibility for the formation of more new junctions, thereby tending to make the network contract. These events are illustrated in Fig. 6. Even if


Figure 6 Schematic representation of strands of para-casein micelles forming new cross-links, leading to breaking of one of the strands (from van Dijk, 1982).

no syneresis would follow, the changes mentioned would cause the strands of particles to become straightened. This is indeed in agreement with the relation found between the modulus and the volume fraction of particles making up the gel (Bremer et al., 1990). The propensity of the strands to break has been carefully studied by van Vliet et al. (1991). They concluded that spontaneous breakage is possible if (1) the bonds in a junction can relax, and (2) the number of bonds in a junction is not too high. If the first condition is met, this is reflected in the loss tangent being fairly high on the timescale considered; the second is met if the strands are (locally) only one-particle thick and the junction zones fairly small (small particles, little fusion). For normal para-casein micelle gels, the critical loss tangent appears to be about 0.4, syneresis being stronger at a higher tan . To say it in other words, the activation free energy for the breaking of bonds should be fairly low for syneresis to be possible. But also the activation free energy for bond formation should be fairly low, since otherwise no new junctions will be formed. Attempts to unify the approach to gel formation and syneresis

It has been earlier recognized that casein gels are transient networks (Bremer, 1992; Bijsterbosch et al., 1995). In the last decade, a unified theoretical framework for the formation of more or less transient particle gels has been proposed (Poon and Haw, 1997; Tanaka, 1999; Prasad et al., 2003), not least due to the convenient experimental system of colloid particles in a noninteracting polymer solution. In such a system, because the centre of gravity of the dissolved polymer is sterically excluded from the region close to the particle surface, i.e., the surface region is depleted of the polymer, an effective attractive short range potential between the particles is created. Both the strength of the attraction (via osmotic pressure of the solution) and its range (via the polymer size) can be manipulated easily.

The framework attempts to unify the description of all phase separation phenomena, including gel formation. The gel is considered as a possible transient phenomenon on the way to full phase separation or to a final arrest of the phase separation process through glass transition. A general theory of viscoelastic phase separation has been developed by Tanaka and collaborators (recently reviewed by Tanaka, 2000). The theory proposes the formation of a transient gel as a possibility in any system in which one of the emerging separating phases has dynamics much slower than the other, covering both polymers and colloidal systems. In this approach, after the initial diffusioncontrolled spinodal decomposition, diffusion is hindered by the viscoelasticity, or more specifically the bulk and the shear moduli of the emerging ‘slow’ phase. The bulk modulus of the gel phase, which is assumed to include contributions from the excluded volume, the network topology and the particle binding energy, and which need not necessarily scale the same way as the shear modulus, is also the origin of gel contraction, i.e., microscopic syneresis. In very late stages of the process, it becomes once again dominated by the, by then slow, diffusional processes. In simulations, the model correctly predicts the morphology of the resulting gels both in polymers (Araki and Tanaka, 2001) and in colloids (Tanaka et al., 2003). The transient gel model clearly emphasizes that the apparent ‘equilibrium’ properties of the gel are the consequence of a drastic slowing down of an ongoing process and can thus not be expected to correlate neatly with the thermodynamic state variables such as composition, temperature and pH. There are several attractive features in the viscoelastic phase separation model. One is that it offers a predictive dynamic model which relates the thermodynamic driving force for separation on a molecular level to the relaxation bulk and the shear moduli of the gel. Another is that it allows in the same general framework modelling of both the early stages of the aggregation, where the casein micelles can be treated


as individual colloid particles, and the late stages where a fused casein phase may be better modelled as a viscoelastic fluid. The thermodynamical driving force, the gradient of free energy of mixing, can be related to independently measurable properties of the system. For renneted casein, the attractive energy has been derived from measurements of viscosity and light scattering, evaluated with the adhesive hard sphere model pioneered in the dairy context by de Kruif (de Kruif et al., 1992, 1995; Mellema et al., 1999) and it should even be accessible to direct measurement in AFM (atomic force microscope). AFM could also give a direct answer to the much-discussed issue of whether a renneted or acidified casein micelle can be regarded as uniformly attractive or as possessing ‘hot spots’. As mentioned earlier, attempts have been made to relate the rheological properties of the network to observables such as fractal dimensionality. It has become clear that the fractal dimensionality and volume fraction of the gel phase do not completely determine the rheological properties of the network; further topological assumptions or facts are needed (Roberts and Knackstedt, 1996; Mellema et al., 2002a,b). Confocal scaning microscopy (CSM) can provide topographical information which can then be transformed directly into more model-independent rheological properties (Mellema et al., 2000), or, with time resolved CSM it is possible to evaluate directly the rheological properties of the network based on the observed movement of the network components under thermal (Brownian) excitation (Dinsmore and Weitz, 2002). The above-mentioned hopes for more general predictive theories may be overoptimistic (Walstra, 2003); gels are not homogeneous and relevant phenomena occur on many length scales, involving different types of bonds, and are therefore not easily interpretable by localized studies such as microscopy, and pose formidable problems to Brownian simulations.

After the gel has become firmer, it can withstand a greater disturbance without exhibiting spontaneous syneresis. Usually, it does not show syneresis at the milk surface, either. The composition of the milk surface is not known with certainty. It may be lipid-rich or may be covered by protein, presumably -casein (Holt and White, 1999), oriented with its hydrophobic chains towards the air. If this surface layer is bonded to the para-casein matrix, the hydrophobic air interface must be wetted to allow the serum to leave the matrix, and thus the matrix to shrink, thus it is essentially the capillary forces that prevent spontaneous syneresis. As soon as the gel is cut or the surface (locally) wetted, syneresis occurs. This effect permits experimentally starting syneresis at any desired moment after a gel has formed. These observations imply that in a constrained milk gel, no syneresis occurs. However, the processes depicted in Fig. 6 will nevertheless occur; there is no reason to suppose they would not. This implies that on a local scale, the gel network becomes more dense; this has been called microsyneresis (van Dijk, 1982; Walstra et al., 1985). At the same time, the network will become less dense at other sites; these changes are shown in Fig. 3. The surface-weighted average pore size will thus increase and it is indeed observed that the permeability of a constrained gel keeps increasing (see Fig. 7). It may be argued that the rate of change of the permeability, dB/dt, is a measure of the tendency of the gel to exhibit syneresis. Until now, only the inherent or endogenous tendency of a gel to show syneresis has been considered. Exerting a stress on the gel may be expected to speed up greatly the expulsion of whey, because of the increased pressure (see equation 7). Moreover, it may enhance syneresis by

1.5

5.35 5.75

Syneresis of renneted milk 1.0 B (μm2)

Under typical cheesemaking conditions, if the gel is formed undisturbed and sticks completely to the wall of the vessel in which it is formed (e.g., clean glass), it usually shows no apparent syneresis, at least if the vessel is not too large and has vertical walls, and if the temperature is not too high (e.g., 30 °C) (van Dijk, 1982). Apparently, the gel is now constrained and cannot shrink. Spontaneous syneresis is observed if the milk is renneted in a conical flask; presumably, the gel tears loose from the glass wall by gravity before it is fully set. Similarly, spontaneous syneresis may occur in a cylindrical glass if it is tilted slightly for a moment during setting.

5.97 0.5

6.33 6.68

0 0

1

2

3

Time (h) Figure 7 Permeability, B, of rennet-induced skim milk gels of various pH (indicated near the curves) as a function of time after renneting. Temperature 30 °C (from results by van den Bijgaart, 1988).


bringing strands of the network closer to each other and, perhaps more importantly, it will enhance breaking of strands, thereby providing a greater possibility for the number of junctions to increase. As was discussed above in relation to Fig. 5, deformation of the gel also causes local rupture of the network, thereby increasing its permeability. Indeed, local densification of the structure and the formation of empty holes was predicted in simulations which specifically included imposed deformation (Rzepiela et al., 2002). One-dimensional syneresis at constant conditons

In this section, the detailed investigations by van Dijk, van den Bijgaart and coworkers (van Dijk, 1982; van Dijk et al., 1984; van Dijk and Walstra, 1986; van den Bijgaart, 1988), as complemented by the recent work by Lodaite (2002), Lodaite et al. (2000, 2002), Unger Grundelius (2004) and Unger Grundelius et al. (2000), will be discussed. They studied horizontal slabs of renneted milk, the top of which was moistened at a predetermined time after renneting, after which syneresis was followed by measuring the change in height, h, of the slab; whey could flow out only at the top. The milk was brought to the desired pH and the apparatus was kept at a constant temperature. Examples of results are shown in Fig. 8. The diameter of the cylindrical slabs was much larger than their thickness (mostly 5 mm). In this way, one-dimensional syneresis under constant conditions could be determined. This is, of course, an over-simplification of the situation during actual curdmaking, but it allowed precise and unequivocal determination of syneresis under various conditions, providing insight into the processes occurring, and permitting the development and the testing of a simple mathematical model. 10

h (mm)

8 6 4 2 0 0

2

4

6 Time (h)

8

10

∞

Figure 8 The height of slabs of renneted skim milk of various initial height, h, as a function of time after initiation of syneresis. Temperature 30 °C, pH 6.7. The values at infinite time are from extrapolation of log–log plots (from results by van Dijk, 1982).

In parallel experiments, the permeability, B, and its change with time, dB/dt, were determined. Unless stated otherwise, the results pertain to renneted skim milk. Modelling the process

The gel can be considered as two inter-penetrating continuous spaces, one consisting of the para-casein matrix and the other of whey. If there is to be macroscopic syneresis, the para-casein matrix must contract and the whey move in the opposite direction. This relative movement is accompanied by a friction force, proportional to the relative velocity between the matrix and the whey. This frictional resistance against flow can be described by the equation of Darcy (equation 5), which is conveniently written as: v

冢 B 冣 " 冢 pl 冣

(7)

where v is the relative superficial velocity of the liquid in the direction of l, the distance over which the liquid has to flow. The pressure causing the syneresis can, in general, be written as: p ps pg pc

(7a)

which terms are, respectively, the endogenous syneresis pressure, the pressure exerted by the network itself due to gravity and any external pressure applied to the network. Results for pc 0 will be discussed first; note that pg varies from zero at the top of the slab to a maximum of g h ! at the bottom, e.g., 1 Pa for a 1 cm slab (! density difference between the para-casein network itself and the interstitial whey). Attempts to directly measure ps failed; its value was too small. Only the order of magnitude could be estimated, and it was 1 Pa (van Dijk et al., 1979; van Dijk, 1982). This is a very small pressure – it corresponds to the pressure exerted by a water ‘column’ of 0.1 mm and, as seen in Fig. 8, unaided syneresis is indeed very slow – it takes 7 h at 30 °C for a 6-mm slab to be reduced to 3 mm. Since equation (7) must hold, and since the rate of syneresis, v, B, l and can be measured, it is possible to determine p indirectly and thereby ps, because pg can also be calculated. The calculation is, however, very intricate, because: • permeability increases with time (Fig. 7); • permeability becomes smaller because of syneresis; • most likely, endogenous syneresis pressure also varies with ongoing syneresis; • pressure due to gravity changes as well; • the coordinates change with syneresis.


Consequently, most variables vary with time and location. Syneresis will start in the uppermost layer, thereby altering its permeability, etc., and progressively reach deeper layers. A finite difference model was developed (van Dijk et al., 1984), in which the slab was divided into parallel thin slices, to each of which equation (7) and the equation of continuity were applied to calculate the outflow of liquid in small time intervals. By inserting various values for ps and comparing the computed results with the observed h as a function of time, the endogenous syneresis pressure could be derived. By assuming p and B to be constant, which may be assumed to be the case at the very beginning of syneresis, an analytical solution can be found (Biot, 1941), which is mathematically equivalent to the solution of the diffusion equation (Tanaka and Fillmore, 1979). This would imply that h changes proportionally to the square root of time and directly yields the initial endogenous syneresis pressure, ps0. This proved not to be the case. In careful experiments, an initial proportionality with time t, the power of about 3/4 was obtained by van den Bijgaart (1988) and about 1 by Lodaite et al. (2000). The explanation probably is as follows (van den Bijgaart, 1988) – an implicit assumption in the application of equation (7) is that the network can without significant resistance comply with the outflow of whey. But the initial shrinkage rate of the outermost layer would then be very high (the proportionality with square root of time even implies an infinite rate at t 0) and that is clearly not possible. A significant effort went into the refinement of the model and the numerical fitting to calculate the intrinsic syneresis pressure as a function of time and process conditions, such as in Fig. 9. The observed trends in the intrinsic pressure could be qualitatively explained

6.33

ps (Pa)

2

6.48 1 6.68 0 0

1 Time (h)

2

Figure 9 The endogenous syneresis pressure (ps) of rennetinduced skim milk gels as a function of the time elapsed after renneting when syneresis was initiated, at various pH (indicated near the curves). Temperature, 30 °C. The broken lines are assumed (from results by van den Bijgaart, 1988).

a posteriori, however, the prediction power of the model remained poor. A possible explanation could lie in the fact that equation (7) is formulated for the liquid space of the network. This means that the forces needed for the deformation of the matrix are not considered explicitly. The intrinic syneresis pressure is thus the observable difference between the balance of the thermodynamic forces attempting to reach a new equilibrium, the eventual external forces and the forces caused by the viscoelastic properties of the matrix. The magnitude of the latter depends on the deformation of the matrix, the deformation rate and the deformation history at each point in time and space. An order of magnitude estimate can be based on Fig. 10. Some 8 Pa external pressure doubled the initial rate of syneresis; thus, the viscoelastic resistance of the matrix would seem to be almost an order of magnitude larger than the observed intrinsic syneresis pressure. While the derived intrinsic pressure is not easily interpreted, one can argue that the same balance of thermodynamic and viscoelastic forces that causes the macrosyneresis is also reflected in microsyneresis, and thus the rate of change of permeability should be a direct index to the intrinsic rate of syneresis. Even in the measurement of permeability there are some caveats. While the observed constrained permeability and its rate of change, measured by the standard tube method was found to be independent of curd column length and driving pressure difference by van Dijk (1982), it was found to depend on both the size of the tube and the driving pressure difference by Unger Grundelius (2004). Some important results are given in Fig. 11. The variables, temperature and pH, affect dB/dt in a similar way, in accordance with the ideas outlined above. All these trends would cause faster syneresis at a higher temperature and lower pH; this is indeed observed. An additional complication, which will lead to syneresis being given an additional dependency on the physical dimensions of the synerising sample, is that rapid syneresis also leads to the rapid formation of a highly shrunken outer layer, which implies that the permeability of that layer becomes very low and its modulus high, thereby slowing down further syneresis. The close correlation between B and dB/dt is presumably due to the latter factor already causing an increase in B before the gel is firm enough to allow the estimation of B. Temperature has a very large effect – below 20 °C, endogenous syneresis is virtually zero. The relations for the effect of concentrating the milk by ultrafiltration are different. Naturally, B decreases with increasing concentration, and the network becomes denser. The bonds remain of the same type, however, which is reflected in


0.4 B (μm2) 0.2 0 dB/dt 0 syn

0 40

20

5.5

T (°C)

0

6.5 pH

1

0.1

CaCl2 (%)

2

3

UF

Figure 10 Properties of rennet-induced skim milk gels. Permeability (B), rate of change of permeability (dB/dt), and approximate initial syneresis rate (syn, arbitrary scale) as a function of temperature (T ), pH, added quantity of CaCl2 and preconcentration by ultrafiltration (UF, degree of concentration) (mostly from results by van Dijk, 1982; van den Bijgaart, 1988).

tan remaining constant (Zoon et al., 1988a). Nevertheless, dB/dt decreases with increasing concentration. The overall result is that the rate of syneresis decreases with increasing concentration. The relative shrinkage rate, i.e., relative to one minus the volume fraction of para-casein particles in the gel, increases somewhat with increasing concentration (van den Bijgaart, 1988). It also appears that the rate of syneresis of a gel from pre-concentrated milk (by UF) is higher than that of a gel of the same concentration but caused by syneresis; at least part of the explanation is presumably that in the latter case, considerable relaxation of the internal stresses in the network has occurred, implying a lower syneresis pressure.

The effect of adding CaCl2 is somewhat puzzling – B and dB/dt are not affected and neither is tan (Zoon et al., 1988b), whereas syneresis rate increases. It should be noticed, however, that the effect of adding CaCl2 is rather variable and that the time elapsed between addition and doing the experiments also affects the results (van den Bijgaart, 1988). It would require painstaking investigations to settle these fine points. Some other variables also (slightly) affect endogenous syneresis. The quantity of rennet added has very little effect, provided that the time elapsed after rennet addition has been sufficient to ensure almost complete hydrolysis of the -casein. Adding NaCl has very little effect, unless a large quantity is added. Comparison of renneted milk with

Initial rate of syneresis

62

6.33

62

6.68

27 8 0

34 °C 0 20

30 Temperature (°C)

0

2

4

6

8

pe/Pa

Figure 11 Effects of temperature and external pressure (pe in Pa, indicated near the curves in the left-hand graph) on syneresis of renneted skim milk. pH was 6.68 (filled circles) or 6.33 (open circles) (from results by van den Bijgaart, 1988).


0.4 pe T (°C) (Pa)

pH

30

0

6.67

34

0

6.67

30 30

8 8

6.67 6.33

30

62

6.33

equilibrium voluminosity of para-casein micelles as a function of pH, temperature and salt content; however, no such work has been reported. Syneresis during curdmaking

After renneting has led to a gel of sufficient firmness, it is usually cut into pieces to promote whey release. For most types of cheese, the mixture of curds and whey is then stirred, often, part of the whey is removed, and it is fairly common to increase the temperature of the mixture after some time (scalding or cooking), all of which are measures aimed at enhancing syneresis. Moreover, during this process of curdmaking, the pH decreases, again enhancing syneresis. An example of the water content of the curd during the course of the process is given in Fig. 13. Note how time, temperature, acidity and pressure affect the water content; the effect of pressure is seen when the curd is taken out of the whey for moulding, by which action the pressure due to gravity increases by a factor of about 30. All these effects are in qualitative agreement with the above results. In this section, the effect of several variables under conditions during actual curdmaking, or conditions more or less mimicking these, will be considered. This is because most published experiments were done in such a way, and methods for estimating syneresis will be reviewed briefly. Some effects of milk composition and pre-treatment will also be discussed. 90

% Water

skim milk shows that the presence of fat globules causes a somewhat lower permeability; dB/dt is not affected, and syneresis is a little slower. As an example, after 5 h a slab had shrunk to 53% of its height, compared to 48% in the case of skim milk (van den Bijgaart, 1988). The effect of ethanol, a less-polar solvent than water, was investigated by Renault et al. (1997). Syneresis decreased in the presence of ethanol, which may suggest that para-casein was more strongly solvated; micellar casein was found to dissociate on heating in the presence of ethanol (O’Connell et al., 2001). Experiments on the influence of an external pressure, pe, were performed by placing a porous disc on top of the syneresing slab. Some results are shown in Fig. 11. It is seen that the effect is considerable and is about proportional to the square root of pressure. The effect of external pressure cannot be seen as an amplification of syneresis – it is about additive to the endogenous syneresis. These results are in accordance with those of model calculations. Figure 11 also suggests that in the presence of an external pressure, the lowest temperature at which syneresis can occur is lower, the more so for a higher pressure. Although this has not been verified by experiments, the effect must exist at least to some extent. Presumably, syneresis has an endpoint. Eventually, the system will be close-packed. Such an end-point has, however, not been observed. Figure 12 gives some results up to 50-h syneresis. It is seen also that after a long time, the shrinkage is greater for a higher temperature, a lower pH and a higher external pressure. A higher fat content also causes somewhat less shrinkage after long time. It would be of great importance for theory formation to establish the apparent

70

i

0.3

0.2

0.1

0 0

20

30

40 t (h)

50

Figure 12 Effects of temperature (T), external pressure (pe) and pH on the shrinkage of renneted skim milk. Relative remaining volume (i ) as a function of time (t) after renneting. Initial slab height 5 mm (from results by van den Bijgaart, 1988).

50

0

2

4 Time (h)

6

8

Figure 13 Examples of changes in the water content of curd (determined by oven drying) as a function of time after renneting. The gel was cut after 0.5 h and the curd and whey mixture was stirred continually. At two moments (indicated by arrows), curd was removed from the whey and put into a cheese mould. Experiments with (filled line) and without (dashed line) adding starter. Temperature of the whey was 32 °C throughout, temperature in the mould gradually fell to 20 °C (recalculated from van de Grootevheen and Geurts, 1977; Kwant et al., unpublished).


Methods for estimating syneresis

The ultimate result of syneresis is reflected in the water content of the cheese after pressing. Determining only this quantity yields, however, little understanding. It is much more interesting to follow syneresis during the curdmaking process, but it is not easy to do this unequivocally. The various methods and their pros and cons have been reviewed extensively (Walstra et al., 1985); only the salient points will be described here. The methods may be classified as follows: 1. Measuring the shrinkage of the curd, either the height of a slab (as discussed earlier) or the volume or mass of a slab or pieces of curd (in air or in whey). These methods are typically applied in laboratory experiments. 2. Determining the amount of whey expelled. This can be done in two ways: a. determination of the volume of whey drained off. The results strongly depend on conditions, especially the often imprecisely known external pressure. It also may be fairly uncertain how much interstitial whey is left between the curd grains. b. determination of the degree of dilution of an added tracer. This method has an inherent uncertainty, in as much as the tracer may adhere onto or diffuse into the curd. 3. Determination of the dry matter content of curd pieces taken out of the whey. The main uncertainty is the unknown quantity of whey adhering to the curd particles; trying to remove the adhering whey may introduce the opposite error. 4. Determining the density of the curd grains by putting them in solutions of various density. This method is fairly crude, but is hardly biased if carefully executed. In addition to the classical methods, the use of low-resolution NMR was introduced by Tellier et al. (1993). The method showed great promise, as it was able to monitor both the degree of syneresis and give a quantitative measure of pore size distribution in

the curd, but was not further pursued for syneresis studies. All these methods can, of course, be executed under various conditions that affect syneresis, e.g., temperature, pH and effective pressure, and at various times after renneting or cutting. Most authors have used method 2a, but methods 2b and 3 have also been fairly popular, especially in experiments involving stirring of the curds–whey mixture. Hardly ever have different methods been compared on the same curd. Figure 14 gives an example, and it is seen that the difference is considerable. It may be concluded that absolute values are hard to obtain and that most methods provide only trends. Even then, one has to be careful, since the method may not be linear. In relation to this, it should be realized that at the beginning of syneresis a large amount of whey has to be removed for the moisture content of the curd to become appreciably lower, whereas at the end of the process the opposite is true (see Fig. 15). Rate equations

As was discussed above, even for a very simple model for the case of one-dimensional syneresis under constant conditions, solution of equation (7) in conjunction with the equation of continuity leads to complicated relations. This will be even more so for the situations considered here, where the geometric boundary conditions are more complicated and changing, and where the physico-chemical conditions affecting syneresis are not constant either. Nevertheless, some authors have tried to give simple analytical expressions for the process.

90

Water content (%)

It goes without saying that curdmaking is aimed at other things besides regulating the rate and the extent of syneresis. The main aspect is that a higher moisture content goes along with a higher sugar content of the curd, which, in turn, leads to a lower pH. This can be modified by ‘washing’ the curd. A lower pH at the moment of separating curds and whey causes the cheese to contain less calcium phosphate. Other aspects are the limitation of the loss of curd fines, the inclusion and the activity of rennet in the cheese and, in some types, the killing of undesired micro-organisms (caused by scalding).

80

0

20

60 40 Time (min)

80

Figure 14 Water content of curd from skim milk renneted and kept at 31 °C as a function of time after cutting, determined from the concentration in the whey of added polyvinyl alcohol (filled circles) and by oven drying of pieces of curd strained off (open circles) (from Kwant et al., unpublished).


40

0

20

60 % Whey removed

100

Figure 15 Calculated relation between the water content of curd (from whole milk of 12.3% dry matter) and the quantity of whey (6.8% dry matter) expelled as % (w/w) of the original milk.

Kirchmeier (1972) reported that the change in volume, V, of a piece of curd is, under ‘constant conditions’, given by: V Vo exp( Kt)

(8)

where Vo original volume, t time after starting syneresis and K would be a first-order rate constant, linearly dependent on temperature. A similar relation, albeit with some ‘extra’ syneresis immediately after cutting, was observed by Marshall (1982). Apart from the lack of theoretical justification for equation (8), it predicts that V approaches zero for very high t, which is clearly impossible. Weber (1984), therefore, modified equation (8) to: V Vo [0.15 0.85 exp( Kt)]

(9)

where it was assumed that the curd eventually shrinks to 0.15 times its original volume (actually, Weber used mass rather than volume). A further modification was made by Peri et al. (1985) who introduced the final (relative) volume, Vinf, as a variable and obtained: V Vinf [(Vo Vinf) exp( Kt)]

(10)

This would be a correct equation for a simple relaxation process where 1/K is the relaxation time. As we have seen, syneresis can certainly not be considered such a process; nevertheless, Peri et al. (1985) found a good agreement with their results, obtained under a fairly wide range of conditions. The good fit may have been due to equation (10) containing two adjustable parameters. Caron et al. (2001) chose yet another asymptotic twoparameter fit, of the same form as the Michaelis–Menten equation. Daviau et al. (2000c) used two exponential relaxation times, and thus five adjustable parameters. Several workers (Koestler and Petermann, 1936; Stoll,

1966; Lawrence and Hill, 1974) found that the amount of whey expel1ed (Vo V) from pieces of curd was proportional to t1/2, and concluded that ‘rate of syneresis is substantially diffusion-controlled’. Such a conclusion had also been reached for syneresis in cross-linked polymer gels (Beltman, 1975). The results quoted above are incompatible; for Kt 1, equations (8–10) predict that (Vo V) is proportional to t, not t l/2; more generally, different authors find different relations. The only conclusion can be that, under constant conditions, the rate of syneresis ( dV/dt) decreases as syneresis proceeds. This need not always be true for the relative rate of syneresis ( d ln V/dt), although this quantity will also eventually approach zero. Effects of curd grain size

Cutting the renneted milk gel into pieces creates a free surface through which syneresis can occur. Before cutting, the gel mostly sticks to the wall and its top surface does not show syneresis, unless it is wetted. Moreover, the distance over which the whey has to flow through the curd is greatly reduced. Empirically, cutting strongly enhances syneresis. Tests using the standard cheesemaking procedures, where a body of curd is cut into pieces, compress the curd and can thus induce structural changes or even cracks in the gel. They thus do not allow to differentiate between the effect of size of the curd grains on one hand and the cutting-induced effects on the other. Unger Grundelius et al. (2000) used curd cylinders of different sizes, produced by rennetting in plastic syringes, but even with the best care, initial whey expulsion could not be avoided. An indication of the effect of grain size is given in Fig. 16; the trends agree with the corresponding onedimensional syneresis data. The time for a given (low) Time [s] to 10% (slab) or 20% (grain) shrinkage

% Water in curd

80

10000 pH 6.4

pH 6.0

1000

100 1

10

100

Slab thickness or 1/2 grain size (mm) Figure 16 Initial one-dimensional syneresis in curd slabs (lines) and three-dimensional syneresis in curd grains (symbols) as a function of size (from Unger Grundelius et al., 2000 and Lodaite et al., 2000).


level of initial syneresis scaled more or less proportionally to curd grain size at pH 6.4, but at a power less than one at a lower pH. Small pieces of curd shrink more than large ones. The latter implies that uneven cutting will cause local variations in moisture content and acidity in the fresh cheese. Stirring

Stirring enhances syneresis (see, for example, Fig. 17). The main factor may be the prevention of sedimentation of the curd particles. Although in a sedimented layer the pressure on the curd may be higher, the possibility of the whey flowing out of the curd layer soon becomes small, thereby strongly impeding syneresis (see further below). Another factor is that stirring causes some pressure to be exerted on the curd grains, and external pressure has a large effect (see below). van den Bijgaart (1988) has made some rough calculations. Stirring causes velocity gradients and consequently, according to Bernoulli’s law, pressure differences. In laminar flow, these remain fairly small; they may amount to several Pa during curd-making. Mostly, flow will be turbulent and pressures up to 160 Pa were calculated, although these exist only for short times. Collision of curd particles with each other or with the stirrer gives rise to brief pressure bursts of the order of 100 Pa, although the average external pressure will probably be about 10 Pa. It has indeed been observed that more vigorous stirring (Patel et al., 1972) or removing more whey (Lawrence, 1959; Birkkjaer et al., 1961), which causes more frequent collisions between curd grains, hence a higher average pressure, leads to somewhat more whey expulsion. The intermittent deformation of the curd grains occurring during stirring may have another effect. As discussed in relation to Fig. 5, large deformation of a

80

% Whey

Stirring

40

0

No stirring

1

2

3

Time (h) after cutting Figure 17 The volume of whey expelled (as % of the original milk volume) from curd kept in the whey at 38 °C as a function of time after cutting, with or without stirring (from Lawrence, 1959).

renneted milk gel causes cracks to be formed in it. Experiments in which an amount of renneted milk gel between two concentric cylinders was brought temporarily under shear and the permeability determined before and afterwards (van Dijk and Walstra, 1986), yielded the following results. Up to a shear of 0.35, B had altered little, a shear of about 0.7 caused an increase by, on average, 20%, and a larger shear could cause a much higher permeability. Unger Grundelius (2004) observed enhanced permeability with time at constant pressure 7.5 kPa/m curd height. It has also been observed (Akkerman, 1992) that an external pressure of the order of 100 Pa can, under certain, not very well-known, conditions cause several small cracks to appear at the outside of shrunken curd grains. (Perhaps the cracks are always formed if the local pressure is high enough, but are often sealed again.) To what extent these phenomena mitigate the strong inhibition of further syneresis due to the formation of a dense outer layer on the curd grains is unknown. Practical conditions of modern curd-making usually allow few opportunities to markedly affect syneresis by varying cutting, stirring, etc. For instance, the size to which the renneted milk gel is cut certainly has an effect on the water content of the cheese, but the effect is small (Sammis et al., 1910; Wurster, 1934; Thomé et al., 1958; Kammerlehner, 1974), at the most some 1% water in the cheese (Straatsma and Heijnekamp, 1988). The main reason presumably is that curd size cannot be varied greatly. If the initial particles are very large, they will inevitably be broken into smaller ones during stirring as long as they are still soft. If one tries to make very small particles, a considerable loss of curd fines occurs. Stirring for a longer time causes a lower moisture content (see, e.g., Fig. 13), but a certain minimum duration of stirring is needed to give the particles sufficient firmness. After that, any longer stirring leads to a slope of, for instance, 0.04% water in the cheese per minute stirring for semi-hard cheese (Straatsma and Heijnekamp, 1988). Consequently, other measures should be taken to influence the water content, especially varying the temperature. After stirring, the curd particles are usually allowed to sediment. If they are sufficiently rigid (which implies mainly after they have lost sufficient whey), they will deform and fuse only to a limited extent in the sedimented layer, implying that any additional external pressure leads to a considerable loss of whey. This is illustrated in Fig. 18; the lower pressures in this graph were due to stirring curds and whey, the higher ones due to pressure exerted on the sedimented curd layer, either by the curd itself or by the perforated plates lying on top.


effect of heating, presumably because -lactoglobulin now reacted primarily with -casein in the serum during heating, thus affecting the casein micelles less. Ovine milk was less sensitive to heat treatment, and caprine even less so (Calvo and Balcones, 2000).

% Whey

100

50

Homogenization of the milk

50

0

100 104

External pressure (Pa) Figure 18 The amount of whey expelled from curd (as % of the original milk volume) after 2 h at 30 °C as a function of the external pressure applied to the curd (approximate results, recalculated from van Dijk et al. (1979) (100 Pa 10 3 bar)).

Effects of other process variables

Numerous authors have studied the effects of product and process variables on syneresis rate, beginning with Sammis et al. (1910). Other extensive studies were by, successively, Wurster (1934), Koestler and Petermann (1936), van der Waarden (1947), Thomé et al. (1958), Stoll (1966) and Daviau et al. (2000a,b,c,d). Several others have studied one or a few variables. As will be seen below, the results often vary somewhat. Results that are obviously in error in view of our present understanding have generally been omitted. But even then, differences in the individual milk samples, in the methods used and in the conditions employed, cause variation. Particularly, the stage at which syneresis is measured affects the results. Moreover, the effect of one variable may be influenced greatly by the level of another, and altering one factor often causes other conditions to change also. Heat treatment of the milk

Heat treatment of milk to such an extent that serum proteins are denatured, increasingly diminishes the syneresis rate of renneted milk, according to many authors (Wurster, 1934; van der Waarden, 1947; Dimov and Mineva, 1962; Stoll, 1966; Kammerlehner, 1974; Nilsen, 1982; Pearse et al., 1985; Daviau et al., 2000c). Some found even a slight decrease caused by mild heat treatments (Siegenthaler and Flückiger, 1964; Stoll, 1966; Nilsen, 1982), but the others did not. Pearse et al. (1985) found the decrease in syneresis to be almost linearly correlated with denaturation of -lactoglobulin. Heat treatment of synthetic milk free of serum proteins hardly affected syneresis. Addition of -casein to milk diminished the detrimental

Homogenization or recombination of milk significantly decreases syneresis rate (Vaikus et al., 1970; Kammerlehner, 1974; Emmons et al., 1980; Humbert et al., 1980; Green et al., 1983; Storry et al., 1983; Ghosh et al., 1994). This is related to the incorporation of micellar casein in the surface coat of the fat globules, which causes the fat globules to become part of the para-casein network, which, in turn, may hinder shrinking of the network. A comparable effect on syneresis was observed if milk had been concentrated by evaporation and diluted again before clotting (Cheeseman and Mabbitt, 1968) which has a similar consequence for the fat globules (Mulder and Walstra, 1974). If fat is homogenized into whey, so that the fat globules do not contain much casein on their surface layers, the detrimental effect of homogenization on syneresis is clearly less (Emmons et al., 1980). Various additions to the milk

Additions meant to modify specific residues of the milk proteins, in order to study the clotting reaction, will not be considered here. Adding sugars, which are fairly unreactive, has been reported to cause no effect (Stoll, 1966; Grandison et al., 1984a), a slight decrease (van der Waarden, 1947) or a slight increase (Cheeseman, 1962) in syneresis rate. About the same holds for addition of up to 10% urea (van der Waarden, 1947; Cheeseman, 1962). In cheesemaking, some CaCl2 is frequently added to enhance coagulation. Most authors report that small additions (e.g., up to 10 mM) of CaCl2 enhanced syneresis somewhat (Wurster, 1934; van der Waarden, 1947; Stoll, 1966; Kammerlehner, 1974; Lelievre and Creamer, 1978) while others found little or no effect (Lawrence, 1959; Cheeseman, 1962; Emmons et al., 1980); larger additions were generally found to reduce syneresis (Wurster, 1934; Gyr, 1944; Tarodo de la Fuente and Alais, 1975). van der Waarden (1947) clearly showed that the main enhancing effect of CaCl2 is due to its lowering the pH; if the pH was kept constant, addition of CaCl2 caused syneresis to decrease, while MgCl2 caused a marked increase (van der Waarden, 1947; Stoll, 1966; Kovalenko and Bocharova, 1973). Presumably, one has to consider two points – increasing of the calcium ion activity (enhancing syneresis) and of the colloidal calcium phosphate (diminishing syneresis). Presumably, Mg2


act much the same as Ca2, whereas Mg-phosphates are much more soluble than Ca-phosphates (addition of MgC12 may thus cause some dissolution of colloidal phosphate). Lowering the pH causes, of course, a dissolution of colloidal phosphate and an increase in Ca2 activity. Addition of phosphate, citrate, oxalate or EDTA (van der Waarden, 1947; Stoll, 1966) at constant pH, all reduce syneresis; these additions considerably reduce Ca2 activity and adding phosphate also increases colloidal phosphate content. The salt equilibria in milk are intricate, depend on several conditions and often exhibit slow changes, as discussed by, for instance, by Walstra and Jenness (1984). Increasing the ionic strength of milk with univalent ions (e.g., NaCl) has been reported to cause at first no change (Cheeseman, 1962) or a slight increase in syneresis (Stoll, 1966); it tends to reduce the amount of colloidal phosphate and possibly the Ca2 activity. A large increase in ionic strength causes a decrease in syneresis (van der Waarden, 1947; Cheeseman, 1962; Stoll, 1966), but then, milk with added salt coagulates very poorly on renneting. Decreased ionic strength increased syneresis (Daviau et al., 2000c). Addition of AlCl3 reduces syneresis (Stoll, 1966).

the free surface was kept constant, such disturbance caused syneresis rate to increase by 20–30% in some experiments (Cheeseman and Chapman, 1966). This may have been due to the disturbance affecting the structure of the network. Curd formed solely by acidification shows very little syneresis if left undisturbed. In milk clotted below about pH 5, the presence of rennet was found to enhance syneresis considerably, the more so when the amount of added rennet was increased (Emmons et al., 1959). Presumably, this signifies a gradual change from an acid to a rennetinduced gel and is of importance in the production of fresh cheese types (Weber, 1984). Temperature

Temperature greatly affects syneresis rate of rennet curd; some results are summarized in Fig. 19. All authors agree as to the trend and all results show that the rate of change of syneresis with temperature (Q10 or d ln V/dT) decreases with increasing temperature, but otherwise the results are fairly different. At 25 °C, reported values of Q10 vary from about 2.5 to 15, at 45 °C from about 1.1 to 1.5. 100

Coagulation

80

60 % Whey

Most authors agree that rennet concentration has no effect on syneresis (Sammis et al., 1910; Wurster, 1934; Stoll, 1966; Lelievre, 1977). Others found that more rennet gave a slight increase (Gyr, 1944; Kammerlehner, 1974; Lelievre and Creamer, 1978; Kaytanli et al., 1994) or decrease (Kovalenko and Bocharova, 1973) in syneresis, or observed an optimum concentration (Weber, 1984). These effects should be considered in relation to the time of cutting (Weber, 1984; Stoll, 1966; Lelievre, 1977). As made clear by, for instance, Weber (1984), it is the stage of the coagulation process or the firmness of the curd at the moment of cutting that is the variable; if cutting is very late, syneresis may be somewhat less. It has also been observed that a higher coagulation temperature leads to slightly less syneresis (Straatsma and Heijnekamp, 1988); this may be due to the cutting starting at an effectively later stage. Whether milk is renneted by chymosin or pepsin makes no significant difference (Andersson and Andrén, 1990). Renneting with proteolytic enzymes from Rhizomucor miehei or Gyphonectria parasitica caused somewhat slower syneresis, but curd firming was slower also, and when cutting 45 rather than 30 min after adding the rennet, the syneresis rate was observed to be normal (Gouda and El-Shabrawy, 1987). Disturbance of the gel during setting may considerably enhance syneresis rate (Wurster, 1934). This was ascribed to the increase of free surface, but also when

40

20

0 20

40

60

Temp. (°C) Figure 19 The volume of whey expelled (as % of the original milk volume) from curd set, kept and treated at different (constant) temperatures. Most results were obtained 1 h after cutting (recalculated from Sammis et al. (1910), Wurster (1934), Koestler and Petermann (1936), Gyr (1944), Lawrence (1959), Stoll (1966), Kirchmeier (1972), Kammerlehner (1974), Marshall (1982)).


The initial rate is increased, but the final amount of syneresis may even decrease, as temperature is raised above 45 °C (Huber et al., 2001). It appears that the rate at which temperature is changed (dT/dt) does not, as such, affect syneresis (Wurster, 1934; Patel et al., 1972). Keeping the milk for some time at a low temperature before renneting has been reported to have no effect (Johnston et al., 1983), a small detrimental effect on syneresis (Nilsen, 1982) or a considerable effect – holding for 20 h at 5 °C reduced syneresis by about 30% (Kammerlehner, 1974). Raynal and Remeuf (2000) observed a similar decrease for bovine milk, but no effect on caprine and ovine milk. Any detrimental effect of precooling is probably reversed by prewarming the milk to a fairly high temperature before renneting, as is commonly done in cheesemaking to ensure normal setting. Acidity

If milk has been acidified to a lower pH before renneting, syneresis rate is faster. Some observations are summarized in Fig. 20; other authors have reported similar results (Sammis et al., 1910; Koestler and Petermann,

100

Washing of the curd

80

% Whey

60

40

20

0

1936; van der Waarden, 1947; Tarodo de la Fuente and Alais, 1975; Lelievre and Creamer, 1978). Although the observed trends were mostly the same, there were, again, considerable quantitative differences. The deviating relation found by Berridge (1970) is not due to inaccuracy but may be related to the different experimental set-up (syneresis of a cylinder of curd attached to a grid, in air). The inflection points in the curves near pH 6 are also realistic. Stoll (1966) observed that the effect of pH was relatively greater at lower temperature and in the absence of stirring, i.e., if syneresis was slower. There were no appreciable differences according to the acid used (van der Waarden, 1947). If the pH falls during syneresis this may enhance syneresis rate to a greater extent than is found when the pH is previously brought to the same value (Stoll, 1966; van de Grootevheen and Geurts, 1977) because the building blocks of the protein network tend to shrink due to the change in pH. This is also exemplified by some results by Emmons et al. (1959) shown in Fig. 20. Here, the milk contained variable numbers of starter bacteria, and the pH values indicated in the figure are those at the moment of cutting. A higher pH at that stage implies a greater drop in pH after cutting, hence more syneresis. Milk that has already been soured to a very low pH (e.g., 4.5) exhibits only weak syneresis, even after renneting (Sammis et al., 1910).

5

6

7

pH Figure 20 The volume of whey expelled (as % of the original milk volume) from curd set, kept and treated at different pH; in some cases, pH decreased slightly during the experiment. Most results were obtained 1 h after cutting (recalculated from Wurster (1934), heavy line average of several experiments; Gyr (1944); Cheeseman (1962); Stoll (1966); Berridge (1970); Patel et al. (1972); Marshall (1982); Pearse et al. (1984); Weber (1984). See text for the results of Emmons et al. (1959)).

Washing, i.e., adding water after part of the whey has been removed, has been reported to enhance syneresis (Kammerlehner, 1974), and to give a slightly, possibly insignificantly, lower water content (Casiraghi et al., 1987). However, washing may coincide with a change in temperature and a difference in the effectiveness of stirring, both of which affect syneresis. In studies by van de Grootevheen and Geurts (1977), either water or an equal quantity of whey at the same temperature was added at a certain stage during cheesemaking and the water content of the curd determined at various times. The water content of the curd to which water had been added was up to two percentage units higher, but the difference could be fully explained by taking into account the difference in dry matter content of the moisture (liquid) in the curd. Hence, the osmotic effects of washing are negligible. Ultrafiltration

Ultrafiltration of cheese milk and renneting the retentate allows the manufacture of curd in such a way that less, or even no, syneresis occurs. The latter, i.e., concentrating the milk to a composition roughly equal to that of the (unsalted) cheese to be made, is feasible only for soft-type cheese; it usually involves


diafiltration also. For harder cheeses, partial ultrafiltration can be applied and an important point then is to what extent syneresis is affected. Some results were already given in Fig. 11. The comparison of the conclusions of different studies may be sometimes misleading, as the authors may relate their syneresis rates to the original amount of milk, or the amount of whey to be expelled. Extensive studies were made by Peri et al. (1985), applying equation (10). They concentrated the milk up to 5.2-fold. The rate constant of the first-order equation, which is thus a measure of the rate relative to the amount of whey yet to be removed, varied little with the degree of concentration; clear correlations with pH or extent of diafiltration were not observed either. The extrapolated proportion of whey eventually expelled (Vo Vinf) varied roughly linearly with the reciprocal of the degree of concentration. A lower pH resulted in a lower (extrapolated) final moisture content. The effects of diafiltration, pre-acidification and sequestering of Ca were also studied (Casiraghi et al., 1987). When adjusting casein cocentration with UF or MF retentate powders, Caron et al. (2001) found faster syneresis for milks adjusted with MF retentate of preacidified milk. Other workers obtained slightly different results (Green et al., 1983; Storry et al., 1983). This may have been due to variation in the time elapsed after renneting before cutting. When renneting normal milk, about 2% of the -casein is still unhydrolysed at the moment of cutting, whereas this may be about 12% for a milk concentrated two-fold by ultrafiltration (van Hooydonk and van den Berg, 1988); this proportion is higher for a more concentrated mi1k. Consequently, the early stages of gel formation and syneresis probably proceed somewhat differently, depending on the moment of cutting. High-pressure treatment

The effects of high-pressure treatment are twofold, the breakdown of the casein micelles at pressures of about 400 MPa, and denaturation of -lactoglobulin at higher pressures, similar to the effects of heating. Casein micelle disruption causes faster aggregation and a finer gel structure with stiffer gels, but syneresis was only affected at pressures above 400 MPa (Needs et al., 2000). Effect of milk composition

Milk composition may clearly affect syneresis, but the effect is usually not large. A higher fat content in the milk on average is accompanied by somewhat slower syneresis (Beeby, 1959; Feagan et al., 1965; Stoll, 1966; Kammerlehner, 1974; Emmons et al., 1980; Storry et al., 1983; Weber, 1984; Grandison et al., 1984a). In practice,

milk is usually standardized as to fat content. A higher casein content goes along with a slower absolute rate of syneresis, but a hardly different relative rate (see under Ultrafiltration). Minor components may have a larger influence and it must be presumed that the calcium ion activity is an especially important variable. For instance, separate milkings of individual cows may vary by a factor of three in syneresis rate (Koestler and Petermann, 1936; Thomé et al., 1958; Kammerlehner, 1974; Grandison et al., 1984a,b), but addition of some CaCl2 greatly reduces the variation. Minor variation has been observed with the stage of lactation (Kammerlehner, 1974; Grandison et al., 1984b) and this may possibly be related to the calcium ion activity also. Milk from cows suffering severe mastitis exhibits poor clotting by rennet and somewhat diminished syneresis (Thomé and Liljegren, 1959; Kiermeier and Keis, 1964; Kiermeier et al., 1967). Extensive growth of pseudomonads in milk was shown to reduce syneresis markedly (Lelievre et al., 1978). On the other hand, considerable proteolysis caused by plasmin activity hardly affected whey expulsion (Pearse et al., 1986b). The effect of casein composition has been studied. Pearse et al. (1986a) made milk with synthetic micelles of variable casein composition. The proportions of - and -caseins clearly affected clotting time, but syneresis far less. Dephosphorylation of -casein caused the clotting time to increase and the syneresis rate to decrease. Interpretation of these results is very difficult without knowing such variables as micelle size and voluminosity, or loss tangent and permeability of the renneted milk. There also appears to be some correlation between syneresis and genetic variants of milk proteins, especially with the -lactoglobulin variant (McLean and Schaar, 1989). This may, again, be due to differences in the calcium ion activity, which correlates with the genetic variants. Other conditions being equal, renneted goats’ milk exhibited greater syneresis than cows’ milk, and ewes’ milk syneresed less (Storry et al., 1983). It may be noted that the latter usually has a clearly higher casein content (Walstra and Jenness, 1984). The effect of several variables on syneresis of renneted cows’ milk, as discussed above, was often different for either ewes’ (Raynal and Remeuf, 2000) or buffaloes’ milk (Dimov and Mineva, 1962).

Concluding Remarks The results on syneresis during practical cheesemaking presented here (see also a review by Pearse and Mackinlay, 1989) generally agree with experiments on


undisturbed curd, although only in a qualitative sense. In a practical situation, quantitative predictions on syneresis rate can hardly be made. Nevertheless, it may be concluded that the main variables affecting syneresis rate are: • the geometrical constraints (dimension of the curd grain); • pressure applied to the curd (grains), where the relative effect is greatest in the low pressure range; • pH; • temperature, where the relative effect is the greatest in the low temperature range. The effect of the other variables is generally small (with the exception of intense heat treatment) and tends to be relatively smaller when overall syneresis rate is higher. Stoll (1966) observed, for instance, that stirring the curd–whey mixture almost eliminated differences caused by some variables observed when studying syneresis under quiescent conditions. This was explained by van den Bijgaart (1988) from the over-riding effect of the permeability of the outer layer of the curd grains. Any condition leading to very rapid syneresis also causes rapid development of a poorly permeable layer, which then markedly slows down any further syneresis. In a qualitative sense, this has been observed before, e.g., by Koestler and Petermann (1936). Cheesemakers speak of a ‘skin’ around the curd grains, and it is even assumed that very rapid initial syneresis may lead to an ultimately higher water content in the curd, as compared to a situation where syneresis proceeds more slowly; cf. the high-temperature syneresis results of Huber et al. (2001).

Behaviour of Curd during Processing When the curd grains are sufficiently dry, they are usually allowed to sediment into a ‘bed’ in the cheese vat or in a drainage pipe. The layer of curd grains compacts, more whey is expelled from the grains and the

grains partly fuse to form a coherent mass. Compaction may be due to pressure exerted by the layer itself or by perforated plates laid on top. Effective pressure ranges from about 100–500 Pa. The compaction is either allowed to proceed for a considerable time, after which the curd mass is cut into small pieces (‘milling’, as for Cheddar types), or, after a short while, blocks of curd are cut from it, which then are subjected to moulding and pressing. The phenomena that occur during compaction and drainage are complicated. At first, compaction occurs due to sedimentation and reorientation of the curd grains. Further events are summarized in Table 2. The different processes are mutually dependent, and especially the deformation of the grains, and the expulsion of whey from them are coupled. Moreover, fusion of curd grains is greatly enhanced when they are deformed. By and large, processes 1 and 2 lead to a lower moisture content, whereas 3 impedes the loss of moisture. Fusion may proceed until the pores between the grains are no longer interconnected, when further drainage virtually stops. On the other hand, some initial fusion will promote drainage, as it can prevent further reorientation of the grains into a denser packing. These conclusions may be true enough, but they are only qualitative and thus not very helpful. To arrive at quantitative relations, the processes were studied in some detail by Akkerman (1992) and Akkerman et al. (1994) for Dutch-type cheese. Lodaite (2002) and Lodaite et al. (2002) studied curd deformation and fusion for the conditions corresponding to soft cheeses. The Akkerman group used whole milk, mostly without added starter, and made curd grains that had been left to synerese to roughly a quarter of their initial volume while Lodaite and collaborators used reconstituted skim milk, and allowed little or no syneresis to occur prior to deformation. The temperature was mostly 35 °C. Expression of single grains, fusion of a collection of grains, compaction of a column and change in pore size (distribution) in a column of grains were studied separately. Because of the many variables of importance, the

Table 2 Processes occurring during compaction and drainage of a curd–whey column Process

Results in

Depends on

Expulsion of whey from grains

Lower whey content of grains Grains less deformable Closer packing of grains Narrower pores

Degree of concentration, temperature, pH, effective pressure, free surface area of grains External pressure, pore size distribution (hence, grain size distribution and shape), geometrical constraints Deformability of grains (hence degree of concentration, temperature, pH), external pressure, duration

Drainage of whey from column

Deformation and fusion of grains

Narrower pores Smaller free surface area


intricacy of the processes and the experimental difficulties, the results are to some extent uncertain, but they clearly show quantitative trends. Syneresis under pressure

Some results of Akkerman (1992) on uniaxial expression of single grains are shown in Fig. 21. The situation was quite different from that leading to the results in Figs 11 and 12. A synerized curd grain was now involved, which implied that the outer layer was much more dense than the centre. Moreover, when the pressures were generally higher, the curd grain could deform sideways and most of the outflow of whey was in directions perpendicular to the applied force. It is seen that the grain showed an immediate, i.e., elastic, deformation, followed by a viscous one that became ever slower. If the pressure was released within a few seconds, the grain more or less regained its original shape, but after some minutes the deformation was permanent; this agrees with the average relaxation time of renneted milk of about 1 min found earlier (Zoon et al., 1989b, 1990). Analysis of the results led to the conclusion that the deformability of the grain, more precisely its effective biaxial elongational viscosity, was rate-determining, the elongational viscosity markedly depending on (decrease with) the stress applied. Akkerman (1992) defined a pseudo Poisson number, , as:

冢

0.5 1 d ln

冣

V

H d

(11)

where V is grain volume and H is the relative deformation expressed as the Hencky strain. He found, as an average over the first 15 min, 0.27, almost independent of conditions. This implies that a (nearly) constant part of the decrease in height of the grain is due to shrinkage. The constancy of agrees with the deformability (i.e., the rheological properties) of the

6

0.30 t = 900 s

3

i

h (mm)

pe = 1200 Pa

0

0.15

0 0

500 Time (s)

1000

0

2 4 pe (kPa)

Figure 21 Uniaxial compression of curd grains at constant stress. h is the height of the curd grain, t duration of pressing, i the relative remaining volume and pe the pressure applied (from Akkerman, 1992).

curd now being rate-determining. As shown in Fig. 21, the deformation, and thereby the expression of whey, depends on the remaining volume, i, pressure and time. It is little dependent on the grain size. The expression reasonably followed the relation: (it iinf) exp( Kpe sqrt(t)) (i0 iinf)

(12)

with iinf 0.1 and where the rate constant K 4 10 5 Pa l s 0.5. Whereas Akkerman (1992) followed compression at various constant pressures, Lodaite (2002) followed the (non-lubricated) compression of non-synerized curd at different constant (Cauchy) deformation rates. Some results are given in Fig. 22. The pressure was seen to increase more or less proportionally with deformation, but not in proportion with the deformation rate. The pressures observed were much lower than what would correspond to the permeability of the curd; this presumably means that during deformation, minute cracks appeared in the curd, vastly increasing its permeability. An empirical formula of the form of Hooke’s law, with a compression rate-dependent modulus, described the data: pe E (1 i)

(13)

with E K (d(1 i)/dt)3/4 and K 40 kPa s 3/4. The effect of pH was not studied, but in view of the effect of pH on the rheological properties of renneted milk (see e.g., Fig. 1) and cheese, it must be significant. Curd fusion

Akkerman (1992) and Akkerman et al. (1993) evaluated the fusion of curd grains in a curd–whey column by determining the fracture stress when pulling two parts of the column, separated by a perforated plate, away from each other. The force divided by the contact area of the grains was taken as the fracture stress. The pressure and time during which it was applied had a strong effect. At 34 °C, the fracture stress obtained was about 60% higher than at 32 °C, whereas at 36 °C it was, maybe, somewhat lower again. The less the grains had shrunk prior to the experiment, the higher the fracture stress. Lodaite (2002) and Lodaite et al. (2002) devised a method for measuring fusion on a single grain pressed against a thin layer of curd, allowing the simultaneous measurement of deformation. Fusion pressure, fusion time and syneresis time, temperature, pH (6.0 and 6.4)


Compaction of a curd column

25

The compaction of a curd column was studied (Akkerman, 1992; Akkerman et al., 1994) for radial drainage. The results depended strongly on the geometrical constraints, especially the radius of the column. The curd particles tended to stick to the wall, and leakage along the wall was also of importance. The total pressure exerted was, following Schwartzberg et al. (1985), presumed composed of three terms:

20

15

10

5 u = 0.1 mm min–1

Compressive stress (Pa)

0 500 400 300 200 100 u = 1 mm min–1 0 1500 1200 900 600 300

pe pc pl pw

(14)

A part of the pressure is lost by friction to the wall (subscript w), and the remainder may not only be on the network of curd particles (subscript c), but also on the liquid (subscript l). As soon as the outflow of whey is hindered by a lack of interconnected pores, the pressure on the liquid increases rapidly. If the pores become completely disconnected, all the pressure is exerted on the liquid (except for pw) or, in other words, the pressure is isotropic, and expression from the curd grains stops. Some results are shown in Fig. 24. The total pressure exerted appears to be the dominant variable. For a low pe, the expression increases strongly with pressure, roughly following equation (12). But above a certain pe, called the threshold pressure, any higher pressure leads to a progressively increasing pl. Also, the pressure loss at the wall, pw, markedly increases with total pressure, being, for instance, proportional to pe2.5. Altogether, at a high pe the effective pressure on the grains, pc, soon becomes very small and expression (almost) stops. The other results in Fig. 24 speak for themselves; note the very strong decrease of the

u = 10 mm min–1

0 0.0

0.1

0.2

0.3

0.4

0.5

2.5

0.6

Relative deformation

and casein and rennet concentrations were varied. It was concluded that the total deformation (which is of course dependent on fusion pressure and time) was the best predictor of fusion, and for a given total deformation, the fusion was independent of pH and rennet concentration. The fusion–deformation relationship appears to hold even for the results of Akkerman (1992) and Akkerman et al. (1993) (see Fig. 23). Lodaite et al. (2002) confirmed the strong effect of temperature in the studied range 28–33 °C.

Fracture stress [kPa]

2 Figure 22 Uniaxial compression of a curd slab at different constant Cauchy strain rates. The initial slab height 5 mm, u linear compression rate (from Lodaite, 2002).

0–2 kPa, 500 s 1 kPa, 0 –1500 s

1.5 1 0.5

0 0

0.2

0.4

0.6

0.8

1

Relative deformation Figure 23 Strength of fusion of curd grains in a column expressed as the fracture stress f after pressing for various times at various pressures as a function of deformation (recalculated from Equation 12 from results by Akkerman, 1992).


10 h (cm)

30 450 5 2100

Φv

0

0.1 530

d (mm)

0

2

530

1

B* (m2)

0

10–10 820 10–12 0

0.5

1

Time (ks) Figure 24 Compression of a curd–whey column (radius 6 cm) under uniaxial compression and with radial drainage. Column height (h), volume fraction of pores (v), volume- average apparent pore diameter (d ) and average radial permeability (B*) calculated as a function of time after applying pressure (indicated near the curves, Pa) (from results by Akkerman, 1992).

permeability of the curd column with time, despite pe being fairly small. Other variables affecting drainage are the degree of concentration of the curd grains at the beginning, given as i0, and the temperature. For a higher i0 and a higher temperature, the initial rate of compaction is higher, but the threshold pressure mentioned above is lower; in other words, the highest pressure that can be applied for the drainage to proceed satisfactorily is smaller (Akkerman et al., 1996). Threshold pressures are mostly somewhere between 800 and 2000 Pa; they depend considerably on the geometry of the system. The presence of curd fines may strongly lower the

drainage rate, as the fine particles tend to block the pores between the grains; the threshold pressure now is much lower. It must be assumed that the curd particle size (average and spread) has some influence, but not a lot within the range studied. The effect of pH was not estimated. Akkerman et al. (1995) further compared the results obtained for single grains with the compaction of a curd column by developing a computer model of the process. Up to pressures of a few hundred Pa, the calculated results agreed reasonably well with the experimental data. He also concluded that at a high pe the curd grains can, in principle, initially be expressed quite fast, without the pressure on the liquid, pl becoming substantial. This opens up possibilities for improving the drainage process. In the Wageningen laboratory, some preliminary studies on axial drainage in a curd column were performed under a wider range of conditions (Heerink and Geurts, 1981); actually there may have been considerable radial drainage as well. Curd was made from skim milk, without a starter. After cutting and stirring (and removing some whey), a column of curd and whey, 30 cm high, was taken; the curd sedimented almost immediately to a height of about 20 cm, after which pressure was applied via a perforated disc, and the curd column gradually compressed to a height of, for instance, 5 cm; the compression was allowed to proceed for 90 min. The final moisture content of the curd column was determined and the earlier values were calculated from the change in height and expressed as the mass fraction of moisture (moisture means liquid containing dissolved substances, i.e., whey in this case). The proportion of moisture between the grains was originally about 40%. Also here, very high pressures gave little improvement. At 20 °C, very little whey was expelled from the curd grains, in accordance with the strong dependence of the apparent curd viscosity (Zoon et al., 1988b) and of syneresis on temperature. Some of the conclusions drawn in this section may in a sense be derived from earlier observations (Vas, 1931; Tarodo de la Fuente and Alais, 1975; Lelievre, 1977; Lelievre and Creamer, 1978; Johnston and Murphy, 1984; Grandison et al., 1984a). Especially interesting is the work of Scott Blair and Coppen (1940), who reasoned that firmer curd grains would permit faster drainage of whey from a mass of grains, and made use of this in devising a test method to determine the ‘pitching point’ of the curd, i.e., the moment at which the grains have lost sufficient moisture, and stirring can cease. A volume of curds and whey is put into a perforated cylinder and allowed to drain for a fixed time; now the ‘superficial density’ is determined,


i.e., the weight of curd divided by the height of the curd column. They found a fairly good positive correlation between the water content and the superficial density, indicating that high moisture, and, thus, soft grains deformed rapidly to close the channels between them, thereby greatly hindering further drainage. Firm (i.e., ‘dry’) grains permitted ongoing drainage, leading to a low superficial density, because the voids between particles now become filled with air. As is to be expected, other factors affect the draining rate. Scott Blair and Coppen (1940) found for the same value of superficial density, a range in water content of about 14 percentage units. There was a tendency for rapid initial syneresis, hence presumably the presence of a more or less rigid ‘skin’ around the curd grains, to lead to a lower superficial density. Likewise, curd at a lower pH (Cheshire as compared to Cheddar) tended to have a lower superficial density. It would be useful to study these and other variables in greater detail. The water content of cheese

There obviously is a lowest possible water content of (freshly made) cheese – the para-casein particles have a given voluminosity. This aspect was reviewed by Walstra et al. (1985) and it was concluded that few hard conclusions can be drawn. The equilibrium voluminosity of para-casein micelles at room temperature and physiological pH was roughly estimated to correspond to 1.4 g water per g protein; this would come down to a water content of an unsalted full-cream cheese of about 40%. The voluminosity would be lower for a lower pH and a higher temperature. The latter effect is considerable. It is also known that a cheese or curd may take up moisture when the temperature is lowered (e.g., Delbeke and Naudts (1970), on Herve cheese; results obtained in the Wageningen laboratory on renneted milk ultrafiltration retentate at pH 5.2; observations in the Lund laboratory on renneted microfiltration retentates at pH 6, observations in practice on Feta cheese kept in brine). The dependence is corrobated by the results of Teo et al. (1996) on reconstituted renneted casein. Moderate NaCl concentrations increase the voluminosity of both native (Famelart et al., 1999) and renneted (Creamer, 1985) casein micelles. Electron microscopy observations indicated that the volume of the casein matrix increased, and the volume of the interstitial dilute phase decreased in salt-injected Muenster cheese (Pastorino et al., 2003) and salted non-fat Mozzarella (Paulson et al., 1998). This would appear to be in contradicton to a large number of studies showing that a higher salt content correlates with a lower level of moisture in cheese, but during salting in

brine or in dry salt, cheeses lose water by pseudoosmosis to the highly concentrated brine (Geurts et al., 1974), and thus other things being equal, the more heavily salted the cheese is the more drier it will be. In a cheese, the lowest possible water to protein ratio may be slightly lower for a lower fat content; in practice, a lower fat in dry matter content always goes along with a distinctly lower water to protein ratio, but this presumably is due to faster syneresis. Altogether, the final moisture content of most cheeses is determined primarily by the rate and the duration of the processes causing whey expulsion, rather than by the equilibrium-swelling state of the para-casein. After curd-making and drainage, one of the following procedures is usually applied. • Moulding the curd, followed by further drainage under its own weight; this is applied only for fairly soft cheese. • Moulding and pressing the curd; this is the common method for semi-hard and several hard cheeses. • Letting the curd rest for a considerable time to develop sufficient acidity (often while allowing the curd to flow, e.g., cheddaring) after which the coherent curd mass is cut into fairly small pieces (milling), salted, moulded and pressed. • Intensively working the already acidified curd (pH 5.3) at a quite high temperature, as is done in making pasta-filata cheeses. During several of these process steps, the curd may lose considerable moisture. Merely taking the curd out of the whey, allowing further whey to leak out, has already a marked effect; see e.g., Figs 13 and 25. Sometimes, the curd grains are worked after removal from the whey, which leads to a much drier cheese with an open texture (numerous small, irregularly shaped holes). The pastafilata treatment also causes appreciable loss of moisture (high temperature and pressure), although the fairly large size of the lumps of curd formed has a mitigating effect (long distance and relatively small surface area). Pressing of the curd mass is aimed at obtaining a coherent mass with a closed rind. The formation of a rind, i.e., an outer layer in which all the curd grains are fully fused with their neighbours, is greatly favoured by the possibility of rapid removal of moisture from the outer layer, for instance by application of a cloth around the curd mass (Mulder et al., 1966). The closed rind greatly reduces further expression of moisture. Figure 24 shows that the effective permeability of a drained mass of curd is about 10 12 m2, which still allows considerable flow of moisture under a pressure of 10–100 kPa, which are common in practice. The permeability in the outer layer may be as low as 10 16 m2 or less, and even a layer of a few mm then makes a substantial barrier.


The effects of moulding, pressing and resting on the moisture content have been carefully studied by Geurts (1978) and some results are shown in Fig. 25. It is seen that the lower the moisture content before pressing, the less the further loss of moisture. The initial water content has other important consequences. First, consider the situation where it is high, say about 55% at the beginning of pressing. Now, pressing at an earlier stage or at a higher pressure leads to a higher water content (more precisely, a less reduced water content); the difference is of the order of 1% water.

% Water

60

50

S

40 0 0

5 Time (h)

10

Figure 25 Water content of a loaf of curd as a function of time after moulding. The dotted lines give the assumed course during pressing. Spherical loaves of about 22 cm diameter, except for one of 12 cm (designated S) (from Geurts, 1978).

The explanation is that a closed rind is formed at an earlier stage or of a greater thickness. Pressing at a higher initial temperature or having a larger loaf of curd led to a lower water content. Although a higher temperature implies a softer curd, and thereby presumably easier rind formation, the overriding effect seems to be the effect of temperature itself on syneresis (see e.g., Fig. 10). A smaller loaf will cool faster and thereby lose less moisture (see Fig. 25). These relations are rather different if the curd mass has a low water content at the beginning of pressing, say 40%. (Such a low water content can be obtained only by prolonged stirring at a high temperature and letting the pH decrease appreciably.) A higher pressure, a smaller loaf and a lower temperature all lead to a lower water content. Presumably, it takes a longer time to obtain a closed rind, the more so for a lower temperature, whereby more moisture can be pressed out of the loaf before the rind is formed. Geurts (1978) also studied the distribution of moisture in unsalted cheese. Some results are shown in Fig. 26. Apart from a thin outer layer, i.e., the rind, which has a slightly reduced water content, the lowest water content is at the centre. This is the region where the temperature has remained highest, especially in a large loaf. It was even observed that the temperature increased in the greater part of a large loaf, undoubtedly due to the heat generated by the growing starter bacteria. If an unsalted cheese is left to rest, the water content tends to become somewhat lower at the bottom side. It may thus be concluded that the moisture moves away from regions where the temperature is higher and/or the pressure higher than elsewhere. Soon, however, the process of fusion of curd grains

Moisture content of cheese (%)

49

1-kg CHEESE 45

6-kg CHEESE

41 0

40

80

120

160

200

mm Figure 26 Water distributions in unsalted spherical cheeses (1 and 6 kg), moulded from the same curd, lightly pressed and kept for a few days. The broken lines indicate the average water content (from Geurts, 1978).


becomes complete, say after two days (Luyten, 1988), and the permeability of the cheese mass becomes too low to permit appreciable transport of moisture. In several types of cheese, the drained mass of curd is allowed to spread laterally for a considerable time (‘cheddaring’). Olson and Price (1970) found that this led to a higher moisture content (1–2% more water), compared to curd kept for the same time but which was prevented from spreading. Although cheddaring may have caused a slightly lower average temperature, the main cause for the differences was presumably that the flow of curd promoted deformation of the curd grains; hence, closing of pores between grains and hindering drainage of any moisture, still leaving the grains due to syneresis. The water content of the cheese must, to a considerable extent, depend on the amount of syneresis during curd preparation, and the results for syneresis as given earlier, indeed qualitatively, agree with results on the water content of cheese (e.g., Sammis et al., 1910; Whitehead, 1948; Whitehead and Harkness, 1954; Birkkjaer et al., 1961; Feagan et al., 1965; Straatsma and Heijnekamp, 1988). Whether there is exact agreement is uncertain. The water content of cheese always shows considerable random variation (e.g., Straatsma et al., 1984) and this makes exact comparisons difficult. During cheesemaking, conditions usually change, for instance pH, temperature and effective pressure acting on the curd, so that one has to take some kind of average. Moreover, the factors are inter-related; for instance, temperature affects the rate of acidification. The latter is also affected by oxygen content (Gillies,

1959) and this may explain why some authors found a negative correlation between stirring rate of the curds–whey mixture and the final water content of the cheese (Kiermeier and von Wüllerstorf, 1963); presumably, faster stirring caused a higher oxygen content, hence inhibition of starter bacteria, slower acidification, and consequently less syneresis. The main causes of discrepancy may be, however, the considerable effects of the conditions of curd drainage and further treatment, such as pressing. If these processes are kept constant, as is nearly always more or less the case during modern cheesemaking, the correlation between syneresis and final water content may be fairly good; one should then take into account the remark made earlier about the inhibiting effect of the formation of a dense outer layer around the curd grains. Some interesting practical results for the case of semi-hard brine-salted cheese were obtained by Straatsma and Heijnekamp (1988), and some of these are summarized in Fig. 27. It is seen that most variables have, within the variation that can reasonably be applied in practice, a fairly small effect. Only the scalding (cooking) temperature and the acidity of the curd had a significant influence. The acidity depends primarily on type and quantity of starter added, any preacidification applied and temperature and duration of acid development. The rate at which the acidity was reached appeared to make little difference. It may finally be mentioned that the water content of cheese also depends, of course, on salting (by pseudoosmosis, Geurts et al., 1974), drying and proteolysis (which causes water to be converted into dry matter).

Change in water content (% units)

+2

0

–2 60

70 80 Past. temp. (°C)

5.5

6.0 6.5 33 35 37 pH after 4 h Scalding temp. (°C)

+1 0 –1 40

60 Time (min)

80

5

6

Grain size (mm)

0

2

4

CaCl2 (mmolar)

Figure 27 The effect of some variables in treatment of milk and in curd-making on the water content of unsalted Gouda type cheese, 5.5 h after renneting, other conditions being equal; time means time after cutting. The water content under standard conditions was about 46% (from Straatsma and Heijnekamp, 1988).


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Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels J.A. Lucey, Department of Food Science, University of Wisconsin-Madison, Madison, USA

Introduction Fresh acid-coagulated cheese varieties include Cream cheese, Cottage cheese and Quarg and other cheeses where the coagulation of milk occurs by acid rather than by rennet, as in most other cheese varieties (e.g., Cheddar). Fresh acid cheeses differ from fermented milk products in having a significant amount of the moisture (whey) removed after coagulation. Whey removal methods, such as centrifugal separation and ultrafiltration (UF), are used for Quarg and Cream cheese whereas cutting of the coagulum into granules and a high cook temperature are used for Cottage cheese. Cultures of mesophilic lactic acid bacteria (i.e., usually Lactococcus spp. and Leuconostoc spp.) and sometimes probiotic species are used as cultures for most fresh acid curd cheeses. A common factor in all of these acid cheese products is that the initial step involves the formation of an acid-induced gel, which is then further processed. The formation and physical properties of acidified milk gels have been reviewed recently (Lucey and Singh, 1997, 2003; Horne, 1999; Lucey, 2002a). There has been considerable research on acid milk gels made with thermophilic cultures for the production of yogurt (e.g., Tamime and Robinson, 1999). The manufacture and technologies involved in the production of fresh acid cheeses have also been reviewed (Guinee et al., 1993; Puhan et al., 1994; Kosikowski and Mistry, 1997; Fox et al., 2000; Lucey, 2002b). This chapter focuses primarily on the formation of these acid-milk gels and their physical, rheological and microstructural properties. Casein micelles

Caseins constitute approximately 80% of the protein in bovine milk, with four main types ( s1-, s2-, - and -caseins (CN)) in combination with appreciable quantities of micellar or colloidal calcium phosphate (CCP) in the form of aggregates called casein

micelles. The stability of casein micelles of milk is attributed to their net negative charge and steric repulsion by the flexible macropeptide region of -casein (the so-called ‘hairs’). Different types of interaction are responsible for micelle integrity, including Ca-induced interactions between protein molecules, electrostatic, hydrophobic and hydrogen bonding. These interactions are probably also involved in the formation and structural properties of acid casein gels. Various models for the structure of casein micelles have been proposed, and it has been the source of controversy over the years. The latest model by Horne (1998) envisages a polymerization scheme (dual-binding model) for the assembly of casein micelles. Cross-linking of the molecules proceeds via two routes, hydrophobic interactions between groups on different molecules forming one pathway, with more than two molecules possibly joining at such junctions, and a second pathway where chain extension is through a CCP nanocluster acting as a neutralizing bridge between two phosphoseryl clusters on separate molecules of s1-, s2- or -casein. Both routes permit branching and hence lead to a three-dimensional network. However, -CN can link only to hydrophobic residues on another CN molecule. Because it has no phosphoseryl cluster to permit further extension, the polymer chain ends there. As a consequence, the -CN acquires an external surface position where it acts as a steric stabilizer. As the pH of milk is reduced, CCP dissolves and the caseins are liberated into the serum phase (Dalgleish and Law, 1988). The extent of liberation of caseins depends on the temperature at acidification (Dalgleish and Law, 1988), which has little effect on the solubilization of CCP. Apparently, little change in the average hydrodynamic diameter of casein micelles occurs during acidification of (unheated) milk to pH ⬃5.0 (Roefs et al., 1985; de Kruif, 1997), although the internal structure of the casein micelles is altered due to the loss of CCP (Walstra, 1993). Aggregation of casein occurs as the isoelectric point (pH ⬃4.6) is



106 Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels

Casein in solution (%)

0.25 0.20 0.15 0.10 0.05 0.00 1

2

3

4

5

6

7

8

pH Figure 1 Solubility of whole casein in water as a function of pH (figure replotted with permission from Strange et al., 1994).

approached (Fig. 1); under conditions of rapid acidification and/or agitation, the casein aggregates precipitate from solution and this is the basis of acid casein manufacture.

Coagulation Mechanisms Theoretical models

Acid milk gels are examples of particle gels and at least three theoretical models, namely fractal, adhesive hard spheres and percolation models, have been used to model the formation of acidified milk gels (Horne, 1999; Lucey and Singh, 2003). Only a brief overview is given here and interested readers can refer to these review articles. Fractal aggregation theory has been applied to the formation of various casein gels (Bremer et al., 1989, 1990, 1993; Vetier et al., 2000). From the fractal approach, a number of scaling laws have been used to derive relations between the physical properties of gels and the fractal dimensionality (Bremer, 1992). The fractal approach has successfully described semiquantitative features of casein gels (e.g., rheological properties), but appears to have some deficiencies, including the lack of any allowance for aggregate rearrangement or interpenetration, and the assumption that all aggregates have the same size at the gel point (Dickinson, 1997). If there are only limited rearrangements, the fractal dimensionality probably increases, but after severe rearrangements a fractal description of the clusters will no longer hold (van Vliet, 1999). Horne (1999) has also questioned the fractal definition of the gelation point (i.e., the suggestion that all casein particles become part of the gel matrix at the point of gelation).

Casein aggregation during the acidification of milk has also been modelled using the adhesive hard sphere theory (de Kruif et al., 1995; de Kruif and Roefs, 1996; de Kruif, 1997, 1999). In this model, it is proposed that the caseinomacropeptide (CMP) part of -casein sterically stabilizes casein micelles and is considered as a polyelectrolyte brush, which collapses on the surface of the micelle as the pH of the system approaches the pKa of the charged (carboxylic acid) groups on the brush. Horne (1999, 2003) pointed out that this model assumes that only the surface features of casein particles have any bearing on the structural properties of acid milk gels. However, it has been shown recently that the loss of CCP from casein micelles dramatically influences the properties of casein gels (Lucey et al., 1998c; Horne, 2001, 2003). Horne (1999) reviewed the suitability of percolation models for acid milk gels and suggested that such models may be suitable only at the gel point and that it is difficult to use this theory to model the mechanical properties of acid milk gels. Physico-chemical mechanisms involved in the formation of gels from unheated milk

Native casein micelles (in milk of normal pH) are stabilized by a negative charge and steric repulsion (Walstra, 1990; Mulvihill and Grufferty, 1995). Some of the techniques that have been used to study the acid coagulation process are listed in Table 1. The surface charge of casein micelles can be approximated from the zeta potential and a plot of the changes in zeta potential as a function of pH is shown in Fig. 2. Casein micelles exhibit some unusual zeta potential behaviour. There is a minimum at pH 5.4 (negative) and a maximum at pH 5.1 (Schmidt and Poll, 1986; Anema and Klostermeyer, 1996). It has been suggested that the shape of the zeta potential–pH profile is due to subtle dissociation and association phenomena of the caseins in this pH region (Heertje et al., 1985).

Table 1 Some of the various techniques used to study the acid coagulation process Viscometry Rheometry Thrombelastography Texture analysis Dynamic light scattering Diffusing wave spectroscopy Turbidity Colorimetry Confocal laser scanning microscopy Electron microscopy Permeability

Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels 107

–12

Zeta potential (mV)

–10

–8

–6

–4

–2

0 4.4

5.0

6.0 pH

7.0

8.0

Figure 2 Dependence on pH of the zeta-potential of washed unheated casein micelles on pH (from Schmidt and Poll, 1986 reproduced with permission from Elsevier).

They proposed that at pH ⬃5.5, there is preferential dissociation of -casein and that at pH ⬃5.2 it reassociates with the micelles and this coincides with a ‘stage of contraction and rearrangement’. However, recent studies (Law, 1996; Singh et al., 1996) have shown that at temperatures 20 °C, which are commonly used for the formation of acid milk gels, no preferential dissociation of -casein from the micelles occurs during the acidification of milk. It is more likely that this unusual behaviour of the zeta potential is caused by the solubilization of CCP, which modifies the ionic environment around casein micelles. Three pH regions in the acidification of milk from pH 6.7 to 4.6 (which is the pH range of interest for the various types of acid-type cheeses) can be distinguished: 1. pH 6.7 to ⬃6.0. The decrease in pH causes a decrease in the net negative charge on the casein micelles, thereby reducing electrostatic repulsion. Only a relatively small amount of CCP is dissolved above pH 6.0, so the structural features of the micelles are relatively unchanged (e.g., size). As a consequence of this reduced repulsion (as the pH is

lowered) there is a decrease in the gelation time and an increase in gel firmness if rennet is used to coagulate milk (Zoon et al., 1989). 2. pH ⬃6.0 to ⬃5.0. The decrease in pH causes a decrease in the net negative charge on the casein micelles, thereby reducing electrostatic repulsion. The -casein ‘hairs’ on the micelle surface are charged, so their charged ‘hairs’ may shrink as the pH decreases. The net result is a decrease in both electrostatic repulsion and steric stabilization, the two factors that are primarily responsible for micelle stability. The CCP within casein micelles is dissolved completely by pH ⬃5.0 in the case of milk, but a considerable proportion of CCP remains intact in the manufacture of natural, rennet-coagulated cheese (Lucey and Fox, 1993), presumably due to a protective effect of the higher solids. The dissociation of casein from the micelle is very dependent on temperature and pH. The pH of maximum dissociation (at temperatures #20 °C) is 5.2–5.4 (Dalgleish and Law, 1988), presumably due to loosening of the molecular interactions between caseins due to the loss of CCP, which causes increased electrostatic repulsion between the newly exposed phosphoserine groups. At


low temperatures, for e.g., 5 °C, considerable dissociation occurs, especially at pH 5.4–5.2; some dissociation occurs at 20 °C, but dissociation decreases rapidly 20 °C, and at 30 °C there is virtually no liberation of caseins (Dalgleish and Law, 1988). -Casein dissociates to a greater extent than the other caseins during acidification at low temperatures (Dalgleish and Law, 1988); since the -casein ‘hairs’ provide a stabilizing layer (both sterically and electrostatically), any reduction in this stabilization should render the micelles more sensitive to aggregation. 3. pH #5.0. The net negative charge on the casein micelles declines with the approach of the isoelectric point and there are increased electrostatic interactions and reduced electrostatic repulsion, which allow increased hydrophobic interactions (Horne, 1998). In unheated milk gels, in which acidification is the only coagulation method, gelation occurs around pH 4.9 unless acidification is performed at a very high temperature when a higher gelation pH is observed.

On acidification, casein particles aggregate as a result of (mainly) charge neutralization, the main titratable groups in milk are shown in Table 2. Acidification eventually leads to the formation of chains and clusters that are linked together to form a three-dimensional network (Mulvihill and Grufferty, 1995). Acid casein gels can be formed from sodium caseinate and gelation also occurs at pH ⬃5.0 (Lucey et al., 1997b,c). Direct acidification of milk at a low temperature may allow solubilization of CCP prior to gelation and therefore these gels may undergo less change in their mechanical properties (e.g., syneresis) than traditional cultured products. Glucono-lactone (GDL) is also used to acidify milk but these acidinduced gels probably have different rheological and structural properties from gels produced by in situ acid production by bacterial cultures (Lucey et al., 1998d). Hydrophobic interactions are unlikely to play a direct role in the strength of acid gels as the G of these gels increases with decreasing assay temperature. Cooling such gels results in an increase in G, probably due to swelling of casein particles (caused by the weaker hydrophobic interactions) and an increase in the contact

Table 2 Main titratable groups in milk (reprinted with permission from Singh et al., 1997)

Group

Approximate concentration (mM)a,b

Expected pKac,d

pKa (in milk)a

Salts Inorganic phosphate Citrate Organic phosphate esters Carbonate Lactic acid Formic acid Acetic acid Various amines

21.0e 9.0–9.2 2.5–3.5 2.0 0.4 0.2–1.8 0.05–0.8 1.5

2.1, 7.2, 12.3 3.1, 4.7, 5.4 1.4, 6.6? 6.4, 10.1 3.9 3.6 4.7 ⬃7.6

3, 5.8, 6.6f 3, 4.1, 4.8f 1.7, 5.9f 6.4, 10.1 3.9 3.6 4.8 7.6

Concentration (mM)a 19 50 6 12 20 7 0.5

Expected pKaa,c,d,g 4.6 4.6 7.0 9.6 10.2 1.5, 6.5 2.6 3.7 7.9

pKa (in milk)a 4.1 4.6 6.5

Ionizable groups of proteins Aspartic acid (-COOH) Glutamic acid (-COOH) Histidine (imidazole) Tyrosine (phenol) Lysine ( -NH3) Phosphoserine (phosphate) N-acetylneuraminic acid (COOH) Terminal carboxyl ( -COOH) Terminal amino ( -NH3) a b c d e f g

}

1.5

Data from Walstra and Jenness (1984). Data from Jenness (1988). Data from Tanford (1962). Data from Edsall and Wyman (1958). About 10 mM colloidal phosphate, 11 mM in solution (at pH 6.6). pKa values from titration with Ca(OH)2. Data from Damodaran (1996).

10.5 2.6 5.0 3.7 7.9


area between particles. With increasing ionic strength, charged groups on casein would be screened, thereby weakening interactions between casein particles.

Physical Properties of Acid-Induced Gels Rheological properties of acid milk gels

Acid milk gels are viscoelastic and exhibit shear thinning when sheared and slow recovery after shearing is stopped. The textural and rheological properties of acid milk gels can be assessed by a range of fundamental and empirical methods such as small amplitude oscillatory rheology (SAOR), large amplitude oscillatory shear, penetration, texture profile analysis, rotational viscometry and flow through an orifice such as a Posthumus funnel (Benezech and Maingonnat, 1994; Velez-Ruiz and Barbosa Canovas, 1997). A combination of techniques should ideally be used to monitor the gel formation phase, as well as the impact of further processing steps on the gel properties (e.g., stirring). The rheological parameters characterizing acid casein gels depend on the number and strength of bonds between the casein particles, on the structure of the latter and the spatial distribution of the strands making up these particles (Roefs et al., 1990a). Some techniques (e.g., texture profile analysis) may give only a single-point measurement and damage the sample. The initial step in the manufacture of most acidtype cheeses is gelation, so dynamic non-destructive techniques, such as SAOR, are needed to study this process. The SAOR technique involves the application of an oscillating strain or stress that is within the linear viscoelastic region for that material (usually less than 5% strain for most milk gels). Some of the main parameters determined from these tests include the elastic or storage modulus (G), which is a measure of the energy stored per oscillation cycle, the viscous or loss modulus (G), which is a measure of the energy dissipated as heat per cycle, and the loss tangent (tan ), which is the ratio of the viscous to the elastic properties (Lopes da Silva and Rao, 1999). These parameters are defined as follows: G'

冢冣cos

(1)

G"

冢冣sin

(2)

tan

G G

(3)

where 0 is the amplitude of the shear stress, 0 is the amplitude of the strain and is the phase angle. The rheological properties of acid milk gels have been studied extensively over the past 15 years or so (see reviews by Benezech and Maingonnat, 1994; Lucey and Singh, 1997). In general, unheated skim milk forms a weak gel (G 50 Pa), and the pH at gelation is generally ⬃4.8–5.0. An example of some rheological properties of a high-fat acid milk gel (similar to cream cheese) is shown in Fig. 3. After gelation, G increases rapidly and only starts to plateau during ageing of the gel (in the region of pH ⬃4.6), tan decreases to 0.4 soon after gelation and decreases to ⬃0.2–0.3 during the ageing of acid milk gels. Roefs (1986) demonstrated that for acid gels, G could continue to increase for up to several days, due, presumably, to slow ongoing fusion/rearrangements of casein particles. An unusual rheological phenomenon is observed soon after the formation of an acid-induced gel from heated milk; tan decreases initially but then increases to a maximum value before decreasing again (e.g., Biliaderis et al., 1992). A high tan indicates an increased susceptibility of bonds and strands in the gel to break or relax, thus facilitating more rearrangements of the gel (van Vliet et al., 1991). The maximum in tan may be a consequence of a partial loosening of the weak initial gel network due to the solubilization of CCP, while at lower pH values there are increased protein–protein attractions between casein particles as the net charge decreases with the approach of the isoelectric point (Lucey et al., 1998c). The maximum in the value of tan occurs in acid gels that have a high gelation pH, e.g., gels made from heated or unheated milk to which some rennet is added (Lucey et al., 1998c, 2000). An example of some rheological properties of an acid skim milk gel made from severely heated milk (similar to quarg cheese) is shown in Fig. 4. The very low incubation temperature (23 °C) and the slow acidification and gelation processes result in a smaller maximum for tan (more of a flattening of the curve) compared to gels made at a higher temperature (e.g., 30 °C). Presumably, under those gelation conditions there is a slower and less dramatic impact of solubilization of CCP on the mechanical properties of casein gels (more CCP is solubilized pre-gelation). An example of some rheological properties of an acid skim milk gel made from unheated milk with a small amount of rennet added (similar to Cottage cheese) is shown in Fig. 5. Gelation occurs at a high pH due to the action of rennet and a clear maximum in tan is observed due to the high incubation temperature (32 °C)


250

7.0

0.45

200

6.5

Loss tangent

150 0.35 100 0.30 50

0.25

0

0.20 0.15

6.0

5.5

pH

0.40

Storage and loss modulus, Pa

0.50

5.0 4.5 4.0

0

100

200

300

400

500

600

700

800

900 1000

Time (min) Figure 3 Rheological changes during the formation of the initial cream cheese gel. Storage modulus (), loss modulus (), loss tangent () and pH changes (solid line) as a function of time during the incubation of 12% fat milk with 2% of a mesophilic starter culture at 23 °C. Milk was homogenized at 17.5 and 5 MPa double stage at ⬃60 °C. The applied frequency was 0.1 Hz and the strain was 1% (unpublished data of Lucey et al., 2003).

and the fast acidification rate (5% starter culture added). The G increases rapidly initially but the profile flattens in the vicinity of the maximum in tan . These trends are similar to that observed for model gels made with a combination of rennet and acid (GDL) (Lucey et al., 2000).

Horne (1999) reported that the rheological properties of acidified milk gels exhibit a form of scaling behaviour. For acid gels made from unheated or heated milk, there are two distinct ‘master curves’, which implies that there are fundamental differences in the kinetics and dynamics of the gel formation process in these two types of gels.

160

0.50

7.0

Loss tangent

0.40

100 80

0.35 60 0.30

40 20

0.25

6.5

6.0 pH

120


140 0.45

5.5

5.0

0 0.20

4.5 0

100 200 300 400 500 600 700 800 900 1000 1100 Time (min)

Figure 4 Rheological changes during the formation of the initial quarg cheese gel. Storage modulus (), loss modulus (), loss tangent () and pH changes (solid line) as a function of time during the incubation of skim milk with 1% of a mesophilic starter culture at 22 °C. Milk was pre-heated at 90 °C for 5 min. The applied frequency was 0.1 Hz and the strain was 1% (unpublished data of Lucey et al., 2003).


80

1.0

6.6 6.4

Loss tangent

0.8 40

0.7 0.6

20

0.5 0

0.4

6.2 6.0 5.8

pH

60


0.9

5.6 5.4 5.2 5.0

0.3

4.8 0

50

100

150

200

250

Time (min) Figure 5 Rheological changes during the formation of the initial cottage cheese gel. Storage modulus (), loss modulus (), loss tangent () and pH changes (solid line) as a function of time during the incubation of skim milk with 5% of a mesophilic starter culture at 32 °C. Approximately 1 ml of standard strength rennet was added per 450 kg milk just after culture addition. The applied frequency was 0.1 Hz and the strain was 1% (unpublished data of Lucey et al., 2003).

In experiments where the time-scale of the applied deformation was varied (frequency sweeps), log G versus log angular frequency gave linear curves with a slope of ⬃0.15 for various types of acid casein gels (Roefs and van Vliet, 1990; Lucey and Singh, 1997). This suggests that similar (fundamental) structural components (bonds) are present in all types of (aged) acid casein gels. Acid casein gels are very brittle and fragile compared with rennet-coagulated milk gels. It is difficult to form a gel suitable for cutting and this approach is used for only a few cheeses (e.g., Cottage). Most acid gels when stirred or mixed have a smooth, non-curdy texture. Improper equipment design and excessive pumping can damage or shatter this fragile gel and result in yield losses. There is little published information on the fundamental large deformation properties of acid milk gels although this would provide useful information on properties that may be related to the consistency of the gel during consumption, cutting or shearing. Mixing and stirring of acid milk gels prior to rheological testing means that many reported ‘fracture’ (yield) properties are not those of the original ‘set’ gel (Lucey and Singh, 1997). Another problem that can affect viscometric measurements of acid milk gels is slip when using flow curves (Suwonsichon and Peleg, 1999). Unrealistically low values (0.5) that have been reported for the flow index (n) of stirred acid milk gels could be due to these problems (Suwonsichon and Peleg, 1999). Acid milk gels exhibit time-dependent

flow behaviour (Benezech and Maingonnat, 1994; VelezRuiz and Barbosa Canovas, 1997). Fundamental large deformation rheological properties of acid casein gels have been reported (Bremer et al., 1990; van Vliet et al., 1991; van Vliet and Keetels, 1995; Lucey et al., 1997a,b, 2000). Gross fracture of acid casein gels made with GDL was observed at a strain of 0.5–0.6. The shear stress at fracture increases with decreasing gelation temperature and with ageing of the gel. The strain at fracture decreases with ageing of the gel. Heat treatment of milk prior to acidification (with GDL) results in a large reduction in the strain at fracture, from ⬃1.5 for gels made from unheated milk to 0.5–0.8 for gels made from milk samples heated at a temperature 80 °C. Partial rebodying (structural recovery) of acid milk gels occurs after the structure has been disrupted by shearing (Arshad et al., 1993), which presumably reflects reforming of some of the weak (electrostatic, hydrophobic) interactions between casein particles. Texture and sensory properties

The microstructure of acid milk gels has a marked effect on their texture and sensory attributes (Langton et al., 1996). An excessively firm texture can be caused by factors such as a very high total solids content of the mix (both fat and casein) or an excessive amount of added stabilizers. A weak body can be caused by factors such as a low solids (fat) content of the mix, insufficient heat treatment of the milk, low acidity (high pH) and a too low gelation temperature.


For Cream cheese it is considered that if the pH of the cheese is too high (i.e., 4.7) the texture will be soft and the cheese will lack flavour. At a very low pH (4.6), Cream cheese may become too grainy and the flavour too acid. Defects in Cream cheese include whey separation from the product during storage, lack of spreadability and a grainy chalky texture, especially in the lower fat types. Textural defects described as ‘chalkiness’ or ‘grainy’ are objectionable, as consumers usually expect a smooth, fine-bodied product (Bodyfelt et al., 1988). Excessive aggregation of protein has been associated with this kind of chalky or gritty defect. Hot-pack cheese has a more brittle texture than cold-pack product due to the additional heating and shearing treatments. Cream cheese should have a spreadable consistency as it is commonly used on bagels and in cheesecakes. Quarg from skim milk is smooth and white with a mild clean, acid flavour. Addition of fat improves smoothness. In contrast to most other fresh acid cheeses, Cottage cheese has a granular, curdy texture instead of being a viscous, smooth or pasty product. The sensory or flavour attributes of acid milk products are very important. For many markets, fruits, sweeteners, spices and condiments are added, which can, to a large extent, determine the sensory properties of these products.

Confocal laser scanning microscopy (CSLM) is a relatively new (but expensive) technique which enables samples to be observed with minimal preparation procedures due to its unique optical sectioning capabilities and high spatial resolution (Brooker, 1995) and is very suitable for observing the overall microstructure of milk gels (Hassan et al., 1995; Lucey et al., 1997c, 1998b,e, 2001). A confocal micrograph of an acidinduced gel made from heated milk is shown in Fig. 6; its structure appears more interconnected than unheated milk gels, especially if a small concentration of rennet is added (Fig. 7) (Lucey et al., 2001). Confocal images are very amenable to image analysis since the images are already in a digital form. Permeability

Permeability measurements provide information about inhomogenities at the level of the gel network (i.e., largest pores). A simple tube method was developed by the Wageningen group (van Dijk and Walstra, 1986) to determine the permeability coefficient of milk gels. Gels are made in open-ended glass tubes. After gelation the gel tubes are placed in a measuring container full of whey, where the level of whey is above the height of the gel in the tubes. The pressure gradient resulting from the difference in height of the (top of the) whey container (actually an empty reference tube) and the gel tube is enough to cause the flow of serum through the gel. The permeability coefficient can be calculated as follows:

Microstructure

Electron microscopy (EM) and confocal scanning laser microscopy (CSLM) studies on acid milk gels have shown that these gels consist of a coarse particulate network of casein particles linked together in clusters, chains and strands (Kalab et al., 1983; Lucey and Singh, 1997). The network has pores or void spaces in which the aqueous phase is confined; in fat-containing products, the presence of (large) fat globules obscures the finer details of pores and strands. The diameter of these pores varies considerably, with larger pores in gels made at a high gelation temperature (usually 30 m) or from milk with a low protein content. There have been several EM studies on the microstructure of gels formed by acidification of heated milk (Davies et al., 1978; Parnell-Clunies et al., 1987; Mottar et al., 1989). Harwalkar and Kalab (1980) proposed, based on the examination of electron micrographs, that acid milk gels made from unheated milk had larger protein clusters (coarse structure) than gels made from heated milk, which they described as highly branched (fine structure). Similar trends have been reported for GDL-induced gels (Lucey et al., 1998e).

ht2) H ht1) [!g(t2 t1)]

冤ln (h(h

B

冥

(4)

where B is the permeability coefficient (m2), h is the height of the whey in the reference tube (m), ht1 is the height of the whey in the gel tube at t1 (m), ht2 is the height of the whey in the gel tube at t2, is the viscosity of the whey, H is the height of the gel (m), ! is the density of the whey and g is acceleration due to gravity. For most acid milk gels formed at 30 °C, the value of B is in the range ⬃1 2 10 13 m2 (Roefs et al., 1990a; van Marle and Zoon, 1995; Lucey et al., 1998e). In general, a very high incubation temperature, use of rennet and conditions of rapid acidification, e.g., GDL-induced gels, can all result in gels with high permeability. The permeability of rennet-induced gels is in the same range as acid-induced gels but for rennetinduced milk gels, B increases with time, which has been taken as evidence of ‘microsyneresis’ or breakage of strands in the network, resulting in the formation of larger pores (Walstra, 1993). Studies on the permeability of acid-induced gels have shown that the value of B


Figure 6 Confocal laser scanning micrographs of Quarg cheese gel made from skim milk fermented with 1% (w/w) of a mesophilic starter culture at 22 °C. Milk was pre-heated at 90 °C for 5 min. The pH of the gels was ⬃4.7. Scale bar 20 m (unpublished data of Lucey et al., 2003).

does not change with time (Roefs et al., 1990a; Lucey et al., 1997c); however in these studies, B was determined in aged gels. It is possible that the value of B for acid milk gels could change with time, at least for a short period during after gel formation. Gels with larger pores (higher permeability) are generally less stable and are more susceptible to whey separation (syneresis) (Lucey et al., 1997c). Appearance

Most acid milk gels should have a smooth, semisolid consistency, with no surface whey even if they are subjected to further processing such as the addition of stabilizers. The appearance of a set gel should be smooth with no cracks or ‘blemishes’. Defects that

are apparent at the initial gelation stage would probably require the addition of more stabilizers to prevent whey separation during storage. Acid gels made from severely heated milks with GDL had a ‘rough’ surface, with visible cracks and some whey separation (Lucey et al., 1998a,e). It was suggested that rearrangement of the network during after gel formation might be responsible for these defects. Gels made from severely heated milk have a lower strain at fracture than gels made from unheated milk and this may make heated gels more susceptible to localized fracturing of strands in the network (Lucey et al., 1997a). The ‘transition’ in the rheological properties (as indicated by the maximum in loss tangent) may increase the susceptibility of protein–protein bonds to relax and if these bonds have a relatively short lifetime,


Figure 7 Confocal laser scanning micrographs of Cottage cheese gel made from skim milk fermented with 5% of a mesophilic starter culture at 32 °C. Approximately 1 ml of standard strength rennet was added per 450 kg milk just after culture addition. The pH of the gel was ⬃4.7. Scale bar 20 m (unpublished data of Lucey et al., 2003).

this may lead to yielding or breaking of strands (van Vliet et al., 1991). Whey separation and syneresis

Whey separation (wheying-off) refers to the appearance of liquid (whey) on the surface of a milk gel and is a common defect in yogurt. However, in fresh acid cheeses, such as Cream cheese, several techniques are used to remove some of the whey from the original gel. Whey removal increases the total solids content of the product, which results in increased firmness and viscosity. Syneresis is defined as shrinkage of a gel and this occurs concomitantly with the expulsion of whey. It is useful to define spontaneous syneresis as the contraction of a gel without the application of an external force (e.g., centrifugation), and this is related

to instability of the gel network (i.e., due to largescale rearrangements) (see review by Walstra, 1993). In practice, fresh acid cheese manufacturers often try to prevent whey separation in the retail product by adding stabilizers (e.g., xanthan gum, locust bean gum, carrageenan) or whey protein concentrate (WPC) prior to packaging. The causes of wheying-off during retail storage include post-acidification, temperature fluctuations, proteolysis by the starter culture, and physical abuse (e.g., vibration, shaking, improper stacking). The amount of spontaneous whey separation in acid milk gels can be quantified using simple approaches such as determining the amount of surface whey that is expelled during gelation (Lucey et al., 1998a). The quantity of whey expelled from acid milk gels as a result of high speed centrifugation may be a useful indicator


of the amount of whey that can be removed during the mechanical separation process used for Quarg or Cream cheese products. Quantifying the amount of whey drainage from a broken gel distributed over a screen gives a measure of water-holding capacity, and is more relevant to products such as Cottage cheese where screens are used to separate the curds/whey mixture (Lucey et al., 1998a). Some acid milk gels may be produced by concentrating the milk (e.g., by UF) to the desired total solids of the final product, thus eliminating the need for a whey removal step. It has been shown (van Dijk and Walstra, 1986) that the one-dimensional syneresis of milk gels is related to the flow of liquid (whey) through the network and is governed by the equation of Darcy: v

B p x

(5)

where v is the superficial flow velocity of the syneresing liquid through the gel (i.e., volume flow rate over the cross-sectional area through which the liquid flows), B is the permeability coefficient, is the viscosity of the liquid, p is the pressure acting on the liquid and x the distance over which the liquid must flow. In milk gels, a key factor in controlling syneresis is the degree of rearrangement that occurs in the casein network (van Vliet and Walstra, 1994). The relations between rheological parameters (e.g., G, tan , yield strain and stress, and the frequency dependency of tan ) and syneresis of acid milk gels have been discussed by Lucey (2001). Parameters that affect the time-scale for rearrangements of bonds in a gel include the dynamic moduli, which indicates the strength and number of bonds in the network, the yield stress and shear deformation at yielding, which determine the susceptibility of the strands to breakage, and tan , with higher values favouring the relaxation of bonds (van Vliet et al., 1991; Lucey et al., 1997a,c). In freshly made gels, the number of bonds between each junction is not yet very high, as indicated by the low dynamic moduli, and tan is higher than in aged gels; these factors might explain why wheying-off occurs sometimes in young but less often in aged gels. Syneresis of acid milk gels made with GDL increases at high gelation temperatures, high pH values and in the presence of even low rennet levels (van Vliet et al., 1997; Lucey, 2002a). It has been shown recently that endogenous syneresis pressure is generally small in acid gels made from sodium caseinate and this results in a lesser tendency of these gels to shrink compared to rennet-induced gels (Lucey et al., 1997c). Acid-induced milk gels formed

by slow acidification of milk at a low temperature and under quiescent heating exhibit little wheying-off or spontaneous syneresis (Roefs, 1986). Surface whey expelled during gelation is sometimes reabsorbed by the product on cooling and storage at a low temperature (Lucey et al., 1997c). Post-acidification, product mishandling and temperature abuse are common causes of wheying-off in acidified milk products. It should be noted that acid milk gels undergo much less syneresis than rennet-induced gels even when they are subjected to centrifugation. For this reason, acid-coagulated cheeses have a very high moisture content (e.g., 50%).

Effects of Compositional and Processing Parameters on the Textural Properties of Acid Milk Gels The effects of each processing step on the textural properties of acid milk gels are considered in the following section. A summary of the effects of some of the main processing factors is given in Table 3. Inoculation and gelation temperature

Acidification of fresh acid products by cultures is generally performed by either of two methods: slow, 12–16 h at 20–23 °C (long set) or 4–6 h at 30–32 °C (short set). Cultures of mesophilic lactic acid bacteria (i.e., mainly Lactococcus spp. and Leuconostoc spp.) and sometimes probiotic species are used as cultures for most acid-coagulated cheeses. Sometimes, fresh cheeses are made by the addition of acid, e.g., phosphoric or lactic acid (direct-acid-set or direct acidification) and/or GDL. Compared with gels made at 20 °C, acid casein gels made at 40 °C are coarser, show more rearrangements, are weaker and less stable (Lucey et al., 1997b,c). In practice, other process variables (e.g., fat content, stabilizers, heat treatment) can help to stabilize this type of gel. In general, an excessive rate of acid development (e.g., use of GDL) at a high incubation temperature (e.g., 45 °C) contributes to the ‘wheying-off’ defect and poor gel formation. In various types of acid milk gels formed with GDL, a lower gelation temperature (e.g., 30 °C) results in a longer gelation time but these gels can have higher G values than gels made at a much higher gelation temperature (e.g., 40 °C) (Cobos et al., 1995; Lucey et al., 1998d). This is due to a coarser gel structure (greater rearrangements) in GDL gels formed at high temperatures (Lucey et al., 1997c). In cultured products, these gelation temperature-related trends may be less obvious due to the large differences in the rate of pH decline between cultured and GDL-induced


Table 3 Summary of the effects of some processing conditions on the acid coagulation of milk and properties of the resulting gel Condition

Impact on acid coagulation and gel properties

Incubation temperature

Faster acid production at higher temperatures leads to shorter gelation times. At a very high temperature (e.g., 35 °C) there are more rearrangements of casein particles in the network leading to lower plateau values for gel stiffness and an increased likelihood of whey separation than gels made at a lower temperature (e.g., 26 °C). At very high temperatures, the gelation pH may increase. At very low temperatures (e.g., 4 °C), no coagulation of casein occurs even at pH 4.6. Heat treatment of milk at a temperature 78 °C for 15 min causes enough whey protein denaturation to greatly increase gelation pH, decrease gelation time and increase viscosity/firmness. The high isoelectric point (5.3) of the main whey protein, -lactoglobulin, is responsible for this effect. Disulfide cross-linking of casein strands increases gel stiffness but solubilization of CCP occurs in casein particles that are already participating in the gel matrix, which triggers greater rearrangements and is responsible for the increase in loss tangent observed in rheological tests. Aggregation occurs as the isoelectric point of casein (#4.9) is approached. Maximum gel firmness occurs around pH 4.6. In general, a slower rate of acidification results in slightly higher gel firmness (may also provide more time at a low pH which should favour additional bond formation). At very high ionic strength (e.g., 0.1 M NaCl), no aggregation of casein particles occurs at pH 4.6 due to screening of electrostatic charges. A minimum concentration of Ca2 is required for acid coagulation. Gel stiffness is proportional to casein concentration The use of a very small amount of rennet in some fresh-type cheeses results in gelation occurring earlier (i.e., at a higher pH), and greater syneresis during processing (e.g., cooking).

Heat treatment

pH

Ionic strength Casein content Use of rennet

gels. In cultured products, gels that are made at very low temperatures (e.g., 21 °C) are weaker than gels made at slightly higher temperatures (eg., 26 °C). The dynamic moduli of acid gels increase with decreasing measuring temperature (Lucey et al., 1997a,b). Whey separation also decreases in GDL gels made at a lower gelation temperature (Lucey et al., 1997c, 1998a). Acid-induced milk gels can be formed by slow acidification of milk with acid (e.g., HCl) at a low temperature (e.g., 5 °C) followed by quiescent heating (Roefs, 1986). The casein particles at pH values close to 4.6 are very different from those at the normal, physiological pH (Walstra, 1993). In this type of gel, it has been proposed that the decrease in the voluminosity of the casein particles after a gel has formed during the heating step results in a ‘straightening’ of the normally tortuous strands in the network (Walstra, 1993). Hammelehle et al. (1997, 1998) used citric acid to form milk gels by this cold acidification procedure. They found that close to the isoelectric point it was harder to get homogeneous gels when the samples were subsequently warmed. Gels were formed at a lower heating temperature when the acidification pH was lower. The use of a higher setting temperature (e.g., 40 °C compared with 30 °C) resulted in firmer gels, which is the opposite trend compared with GDLacidified gels. It is likely that the structure of GDL- and directly-acidified acid milk gels is different. The method of acidification and gel formation (e.g., GDL, cold acidification or bacterial fermentation) has a major impact on the structure and physical properties of acid milk gels (Roefs, 1986; Lucey et al., 1998d).

Rapid heating of cold-acidified gels to a high temperature (e.g., 50 °C) resulted in firm gels but considerable syneresis. Heat treatment

Heat treatment of milk is one of the most important process parameters affecting the texture of acid milk gels (Mulvihill and Grufferty, 1995). Milk used for some fresh acid cheeses, such as Quarg, is subjected to an extensive heat treatment. Incorporation of whey protein into fresh cheese is an important aspect of fresh cheese manufacture because of an increased yield. Acid whey is also considered less valuable than rennet whey in terms of its use for the manufacture of high value-added whey products. High heat treatment of milk is not usually practised for Cottage cheese since heating reduces whey syneresis, which causes textural defects, including excessive softness and brittleness. Forming a gel that is suitable for cutting is a step unique to Cottage cheese and is not used in the manufacture of most other fresh cheeses. Cream cheese is manufactured from pasteurized milk (72–75 °C for 30–90 s) (Guinee et al., 1993) as a higher heat treatment causes difficulties due to not being able to remove sufficient whey during the centrifugal separation process. When milk is pre-heat treated, denatured whey proteins associate with casein micelles and they cross-link the gel network when aggregation occurs during subsequent acidification of milk. The firmness and viscosity of acid gels has been related to the extent of


denaturation of whey proteins during heat treatment (e.g., Dannenberg and Kessler, 1988). Heat treatment also results in a reduction in the gelation time. In gels that are formed from pre-heated milk, gelation occurs at a higher pH (e.g., 5.2–5.4) than from unheated milk (pH ⬃5.0); these pH values depend on the gelation temperature. The higher gelation pH can be attributed to the higher isoelectric pH (⬃5.2) of the main whey protein, -lactoglobulin, which initiates isoelectric precipitation/aggregation at a higher pH than for caseins which have an isoelectric point of ⬃4.6 (Lucey et al., 1998c). In gels produced from heated milk, the solubilization of CCP in casein particles that are already part of the gel network can loosen the gel network, which assists in curd syneresis. At lower pH values, electrostatic repulsion is weaker which facilitates greater hydrophobic interactions and as a result, the gel becomes firmer again and exhibits less syneresis. In acid milk gels, syneresis is virtually absent at pH 4.6, although this pH is the point of maximum gel firmness (Roefs, 1986). Moderate heat treatment prior to acidification had little effect on the extent of solubilization of CCP from the micelles (Law, 1996; Singh et al., 1996). High heat treatments also increase the dynamic moduli of acid milk gels (van Vliet and Keetels, 1995; Lucey et al., 1997a, 1998b,c) although the fracture strain decreases with increasing heat treatment, making these gels more brittle (Lucey et al., 1997a). Heat treatment may increase the susceptibility of GDL gels to wheying-off as the gel may undergo greater rearrangement (Lucey et al., 1998a). There have been a number of reports on the effects of heat treatment on the rheological properties of acid milk gels determined by dynamic low amplitude (strain) oscillation (van Vliet and Keetels, 1995; Lucey et al., 1997a, 1998b,c). van Vliet and Keetels (1995) reported that acid skim milk gels made from reconstituted low-heat skim milk powder (SMP) had much lower dynamic moduli than gels made from high-heat SMP. Lucey et al. (1997a) reported that heating milk at a temperature 78 °C greatly increased G compared to unheated milk (⬃15 Pa) and produced gels with G in the range 350–450 Pa. Increased cross-linking or bridging, by denatured whey proteins, within gels made from heated milk may be responsible for the increased rigidity of the network (Lucey et al., 1997a, 1998c). Rennet addition

Rennet is sometimes added in the manufacture of some acid cheeses (e.g., Cottage and Quarg). The concentration of rennet added is very low (e.g., 0.2–10 ml of standard strength rennet per 1000 l of milk) and it

is often an optional additive in fresh cheeses. Rennet may be added shortly after the point when the starter culture is added or more commonly during the fermentation process as long as the pH is not too low (typical range: pH 6.0–6.3). Some rennet (pre-diluted rennet and other ingredients are added in a product called ‘coagulator’) is often added when making largecurd Cottage cheese but is rarely used when making small curd–style, as the curd may be more easily shattered during cutting. When rennet is added, the Cottage cheese gel is ready to be cut at a higher pH (e.g., 4.8) than in its absence (e.g., 4.6) or there would be excessive loss of fines. Increasing the amount of rennet added increases the pH of the curd at the point of coagulation (Emmons et al., 1959); if sufficient rennet is added, milk will coagulate close to the starting pH value, as for rennet-coagulated cheeses. Rennet hydrolyses some -CN and the resulting CMP diffuses away from the micelles, leading to a decrease in the zeta potential, by ⬃5–7 mV (⬃50%), which reduces electrostatic repulsion between rennet-altered micelles. Removal of the ‘hairs’ also results in a decrease in the hydrodynamic diameter by ⬃5 nm, and a loss of steric stabilization. In acid cheeses, the rennet coagulation process proceeds slowly due to the very low rennet level and low temperature that are commonly used. There have been a number of recent reports on model acid milk gels made with combined rennet addition and concomitant acid production (Roefs et al., 1990b; Lucey et al., 2000, 2001; Tranchant et al., 2001). The rheological profiles are often complex due to the effects of solubilization of CCP from micelles that are already part of a gel network, and changes in casein–casein interactions as the pH decreases. Solids non-fat (SNF) content

It is well known that increasing the solids non-fat (SNF) content of milk increases the firmness and viscosity of acid milk gels. The protein or SNF content of milk can be increased by concentrating milk, e.g., by reverse osmosis, UF or thermal evaporation or by dry-matter enrichment. The sources of dry-matter are usually SMP and WPC. At similar protein levels, acid milk gels enriched with Na caseinate have a higher viscosity or firmness than gels enriched with SMP or WPC. The addition of 1% WPC to milk followed by heat treatment, resulted in an increase in G and a reduction in the gelation time for acid milk gels (Lucey et al., 1999). It was suggested that during heat treatment, the added whey proteins, as well as the original whey proteins in milk, were denatured and associate with the casein micelles to provide additional


cross-linkages within acid-induced gels (Lucey et al., 1999). Substitution of up to 10–15% of the casein by WPC has little effect on the final viscosity or sensory attributes of acid milk gels but at higher levels of substitution, flocculation and off-flavors can occur in the product. The firmness of acid gels made from milk with various casein to whey protein ratios was similar ( Jelen et al., 1987). Milk with a very high (11%) solids content is not commonly used for Cottage cheese manufacture due to problems with cutting the coagulum if it becomes too firm, texture defects, reduced syneresis, increased buffering capacity causing a reduced rate of pH decline, and ‘stratification’ or layering of the solids can occur in the vat during the slow coagulation process. Both the total solids and the fat content of milk used to make Cream cheese influence the ease of whey separation, e.g., at a low fat content there may be a high concentration of cheese in the separator whey as the density of the cheese (which is greatly influenced by the fat content) becomes similar to the whey. Fat content and homogenization

Fat provides a perception of creaminess and improves the mouth-feel of acid dairy products. Homogenization of milk for fresh cheese manufacture helps to prevent fat separation during storage, improves consistency, increases whiteness and reduces whey separation. Milk is usually homogenized at a pressure in the range 10–20 MPa, at a temperature in the range 55–65 °C prior to heat treatment of the mix. It is considered that homogenized full-fat milk produces a firmer gel than those made from skim milk (Becker and Puhan, 1989). An increase in homogenization pressure has been reported to increase the viscosity of full-fat acid milk gels (Puhan, 1988). In the manufacture of high-fat acid milk gels, the use of a higher homogenization pressure or multiple passes to produce smaller milk fat globules in the milk results in an increased G value and a higher yield stress value calculated from flow curves (Sanchez et al., 1995). The nature of the fat globule membrane determines the types of interaction that can occur between fat globules and the protein matrix. Fat globules act as an inert filler if the native fat globule membrane is intact since this membrane does not interact with casein particles (van Vliet and Dentener-Kikkert, 1982; van Vliet, 1988). The G value of acid milk gels decreases with an increasing volume fraction of fat, which has an intact native fat globule membrane (van Vliet and DentenerKikkert, 1982; van Vliet, 1988). In homogenized or recombined milk, the native membrane is replaced largely by casein and some whey proteins so that the

surface of fat particles can interact with the protein matrix (largely casein but some denatured whey proteins when the gel is made from heated milks) of acid milk gels (van Vliet and Dentener-Kikkert, 1982; van Vliet, 1988). In acid milk gels made from recombined milk, G increases with an increasing volume fraction of fat (van Vliet and Dentener-Kikkert, 1982; van Vliet, 1988; Lucey et al., 1998b). Cho et al. (1999) showed that the G of acid milk gels made from either heated or unheated milk is influenced by the nature of the fat globule membrane; gels containing fat globules stabilized by sodium caseinate or denatured whey proteins had very high G values compared with those stabilized with SMP or native whey proteins. Homogenization or shearing of high-fat acid gels, such as is practised in the manufacture of Cream cheese, results in increased firmness, and brittleness becomes more evident if shearing of the hot cheese is excessive (Guinee et al., 1993). There is a general trend for lower fat products to be more susceptible to a ‘chalky’ or ‘grainy’ defect (Muir, 2000). This may be due partly to the creamy mouth-feel imparted by fat to dairy products. pH and calcium content

It is usually considered that the optimum pH for the firmness/viscosity of acid milk gels is ⬃4.6. If the pH falls below about 4.2, the gel may become weaker and more susceptible to syneresis. A pH of 4.6–4.75 at cutting is often recommended for Cottage cheese; a higher pH gives a firmer coagulum while a lower pH gives a softer curd (Emmons and Tuckey, 1967; Emmons and Beckett, 1984). A higher pH at cutting probably results in a greater retention of Ca as CCP within casein particles. Electrostatic repulsion between casein molecules is increased by dissolving the CCP (Horne, 1998). Acid casein gels with a very high pH value (e.g., 4.8) have a much greater tendency to synerese than gels with a low pH value (#4.6) (van Vliet et al., 1997). The total calcium content of most fresh cheeses is low due to the low pH achieved during fermentation which solubilizes a high proportion of the CCP, which is lost in the whey during drainage. Calcium chloride is often added to reconstituted milk used for Cottage cheese manufacture (e.g., White and Ryan, 1983), although it probably has only minor effects of gel firmness. Calcium fortification of fresh cheese is attractive from a nutritional perspective. The addition of CaCl2 (even up to 0.1% which is well above the legal permitted level) did not increase the Ca content of Cottage cheese curd (Wong et al., 1976). Presumably, most of the added CaCl2 was soluble at the low pH of acid milk gels. Acidification of milk to pH ⬃4.9 solubilizes all the CCP (Pyne and McGann, 1960). In acid casein


manufacture, Jablonka and Munro (1986) considered that residual Ca2 on casein particles forms bridges between negatively charged groups of the caseins (e.g., phosphoserine), resulting in tighter, more compact curds and larger curd particles. Increasing the CaCl2 concentration from 0 to 50 mM reduced the rate of casein aggregation (Bringe and Kinsella, 1993), presumably via charge neutralization/screening effects as well as ‘salting-in’ of the proteins. The addition of Ca-chelating agents, e.g., citrate, oxalate or ethylenediaminetetraacetic acid, to milk resulted in increased firmness of GDL-induced gels (Johnston and Murphy, 1992). The structure of casein micelles is disrupted when CCP is chelated and this open structure presumably provides additional possibilities for casein–casein interactions in acidified milk products. Excessively high calcium levels have been associated with a ‘bitter’ taste in fresh products, such as Quarg, although no clear mechanism has been proposed for how this ‘bitterness’ develops.

Acknowledgements The author would like to thank Tao Wang, Wonjae Lee and Chanokphat Phadungath for preparing various milk gel samples. The author is grateful for the financial support for this research by the Wisconsin Center for Dairy Research, Wisconsin Milk Marketing Board, and the USDA Cooperative State Research, Education and Extension Service (CSREES) project WISO4363.

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Roefs, S.P.F.M., van Vliet, T., van den Bijgaart, H.J.C.M., de Groot-Mostert, A.E.A. and Walstra, P. (1990b). Structure of casein gels made by combined acidification and rennet action. Neth. Milk Dairy J. 44, 159–188. Sanchez, C., Maurer, K. and Hardy, J. (1995). Influence of fat globule size on the rheological properties of a model acid fresh cheese, in, Food Macromolecules and Colloids, E. Dickinson and D. Lorient, eds, Royal Society of Chemistry, Cambridge. pp. 512–516. Schmidt, D.G. and Poll, J.K. (1986). Electrokinetic measurements on unheated and heated casein micelle systems. Neth. Milk Dairy J. 40, 269–280. Singh, H., Roberts, M.S., Munro, P.A. and Teo, C.T. (1996). Acid-induced dissociation of casein micelles in milk: effects of heat treatment. J. Dairy Sci. 79, 1340–1346. Singh, H., McCarthy, O.J. and Lucey, J.A. (1997). Physicochemical properties of milk, in, Advanced Dairy Chemistry, Vol. 3, Lactose, Water, Salts and Vitamins, 2nd edn, P.F. Fox, ed., Chapman & Hall, London. pp. 469–518. Strange, E.D., van Hekken, D.L. and Holsinger, V.H. (1994). Effect of sodium chloride on the solubility of casein. J. Dairy Sci. 77, 1216–1222. Suwonsichon, T. and Peleg, M. (1999). Rheological characterisation of almost intact and stirred yogurt by imperfect squeezing flow viscometry. J. Sci. Food Agric. 79, 911–921. Tamime, A.Y. and Robinson, R.K. (1999). Yoghurt: Science and Technology, 2nd edn, CRC Press, Boca Raton, FL. Tanford, C. (1962). The interpretation of hydrogen ion titration curves of proteins. Adv. Protein Chem. 17, 69–165. Tranchant, C.C., Dalgleish, D.G. and Hill, A.R. (2001). Different coagulation behaviour of bacteriologically acidified and renneted milk: the importance of fine-tuning acid production and rennet action. Int. Dairy J. 11, 483–494. van Dijk, H.J.M. and Walstra, P. (1986). Syneresis of curd. 2. One-dimensional syneresis of rennet curd in constant conditions. Neth. Milk Dairy J. 40, 3–30. van Marle, M.E. and Zoon, P. (1995). Permeability and rheological properties of microbially and chemically acidified skimmilk gels. Neth. Milk Dairy J. 49, 47–65. van Vliet, T. (1988). Rheological properties of filled gels. Influence of filler matrix interaction. Colloid Polym. Sci. 266, 518–524. van Vliet, T. (1999). Factors determining small-deformation behaviour of gels, in, Food Emulsions and Foams: Interfaces, Interactions and Stability, E. Dickinson and J.M. Rodriguez Patino, eds, Royal Society of Chemistry, Cambridge. pp. 307–317. van Vliet, T. and Dentener-Kikkert, A. (1982). Influence of the composition of the milk fat globule membrane on the rheological properties of acid milk gels. Neth. Milk Dairy J. 36, 261–265. van Vliet, T. and Keetels, C.J.A.M. (1995). Effect of preheating of milk on the structure of acidified milk gels. Neth. Milk Dairy J. 49, 27–35. van Vliet, T. and Walstra, P. (1994). Water in casein gels; how to get it out or keep it in. J. Food Eng. 22, 75–88. van Vliet, T., van Dijk, H.J.M., Zoon, P. and Walstra, P. (1991). Relation between syneresis and rheological


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Starter Cultures: General Aspects E. Parente, Dipartimento di Biologia DBAF, Università della Basilicata, Potenza, Italy T.M. Cogan, Dairy Products Research Centre, Teagasc, Fermoy, Co. Cork, Ireland

Cheese cannot be made without the use of certain species of lactic acid bacteria (LAB), the major functions of which are to produce lactic acid from lactose during manufacture and cause biochemical changes during ripening, which help to develop the characteristic flavour of the cheese being made. The LAB involved are called Primary Cultures. These organisms are also called starter bacteria because they ‘start’ (initiate) the production of lactic acid. Generally, the starter bacteria are carefully selected and deliberately added to the milk before cheesemaking but, for some cheeses, particularly Spanish and Italian varieties, no starter is added. Instead, the cheesemaker relies on adventitious contaminants present in the milk used to make the cheese. The main species involved include Lactococcus lactis, Leuconostoc sp., Streptococcus thermophilus, Lactobacillus delbrueckii subsp. lactis, Lb. delbrueckii subsp. bulgaricus and Lb. helveticus but not all of them are used in every cheese variety. The first two organisms are used in most cheese varieties while the latter are used in cheeses like Emmental and Parmigiano Reggiano and Pizza/Mozzarella cheese, which are heated to a high temperature during manufacture. In many artisanal cheeses, especially those produced in Mediterranean countries, other LAB, including Lb. casei, Lb. plantarum, Ec. faecalis, Ec faecium, Lb. salivarius, and Staphylococcus species are also found. Other microorganisms are also used in cheesemaking, e.g., Propionibacterium freudenreichii, Brevibacterium linens, Debaryomyces hansenii, Geotrichum candidum, Penicillium roqueforti and P. camemberti. These organisms have no function in acid production and are called Secondary Cultures. Their major role is to produce organoleptic and biochemical changes in or on the cheese. These include the production of CO2 by P. freudenreichii in Emmental cheese (‘Cheese With Propionic Acid Fermentation’, Volume 2), the blue veins in Blue cheese, caused by growth of P. roqueforti (‘Blue Cheese’, Volume 2) or the velvet-like coat of P. camemberti (mainly) which develops on Camembert cheese during ripening (‘Surface Mould-Ripened Cheese’, Volume 2). Since the last edition of this book (Fox, 1993) there has been an explosion in scientific information on starter LAB. This has necessitated the division of the

review of starter cultures into four chapters. This chapter is devoted to general aspects of starter cultures but it cannot be considered to be exhaustive because of the time required to digest and assimilate the considerable literature currently available on these important bacteria. The approach taken is to highlight recent studies on important aspects of starter cultures. Further information on many aspects of starter cultures can be obtained in the symposia on LAB held every three years in The Netherlands (Venema et al. 1996; Konings et al., 1999; Siezen et al., 2002), the colloquia held every few years in France (Anonymous 1996, 1998, 2000, 2001), Cogan and Accolas (1996) and Salminen and von Wright (1998).

Taxonomy and Strain Identification Except for Sc. thermophilus, there have been no changes in the taxonomy of the starter bacteria since the previous review by Cogan and Hill (1993). In 1984, Sc. thermophilus was classified as a sub-species of Sc. salivarius (Farrow and Collins, 1984) but extensive DNA:DNA hybridization studies under stringent conditions and physiological data have provided evidence to re-confer species rank on the organism (Schleifer et al., 1991). A new species, Streptococcus madedonicus, has been recently isolated from Kasseri cheese (Tsakalidou et al., 1998). It does not hybridize with Sc. thermophilus and differs from it in not producing -galactosidase and in producing acid from cellobiose, maltose and N-acetyl glucosamine; Sc. macedonicus shows 96% similarity in 16S and 23S rDNA sequences with those of Sc. thermophilus. Another new species, “Sc. waius”, isolated from biofilms formed on exposure of stainless steel surfaces to pasteurized milk, is identical to Sc. macedonicus (Manachini et al., 2002). Until recently, it was difficult to distinguish between strains of the same species but the advent of modern molecular techniques, particularly sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), randomly amplified polymorphic DNA (RAPD) and pulse field gel electrophoresis (PFGE) has changed this significantly. Many isolates from natural cheese cultures show considerable heterogeneity and these



124 Starter Cultures: General Aspects

techniques have proved to be very useful in determining how many strains are present (Giraffa et al., 1998). Separation of whole-cell proteins by SDS-PAGE or DNA fragments by agarose gel electrophoresis results in characteristic patterns, which can be scanned, normalized and compared. RAPD is a polymerase chain reaction (PCR) technique in which a random primer of 10 nucleotides is used as a template to generate DNA fragments which are then separated by gel electrophoresis. This is a rapid procedure. PFGE is a procedure where the total DNA is extracted from the cells and hydrolysed with rare-cutting restriction enzymes into large fragments, which are then separated by gel electrophoresis. This is a slow and labour-intensive technique but the band patterns are very reproducible and allow one to discriminate objectively between different strains and decide unequivocally if strains are the same or not. PFGE was used by Boutrou et al. (1995) to classify 18 strains of Sc. thermophilus into two groups, and by O’Sullivan and Fitzgerald (1998) to separate 16 strains of the same species into three groups, which corresponded broadly with their proteolytic and acidifying properties. Moschetti et al. (1998) showed that in 51 strains of Sc. thermophilus, the 16S–23S rDNA intergenic spacer region gave a single amplification product of 350 bp; cleavage of the product with HaeIII gave two different restriction patterns. Considerable heterogeneity was found among 40 strains of Sc. thermophilus using RAPD-PCR and the M13 primer; three broad clusters were found, which were partly correlated with the source of the isolates (Giraffa et al., 2001). Various molecular techniques, including RAPD, PFGE and SDS-PAGE, have also been used to characterize different Lb. helveticus isolates. Lombardi et al. (2002) showed that 67 strains of Lb. helveticus isolated from whey starters and cheese could be grouped using a combination of genotypic (RAPD) and phenotypic methods. The grouping corresponded with the cheese from which the strains were isolated in the case of Monte Veronese and Provolone cheeses but not Grana cheese. In contrast, RAPD provided clear differentiation between 23 strains of Lb. helveticus isolated from Grana and Provolone cheeses (Giraffa et al., 1998). The number of isolates (23) in the latter study was small and a greater number may have allowed less clear conclusions. SDS-PAGE of cell-wall proteins clearly separated isolates of Lb. heleveticus from Grana and Provolone cheese (Gatti et al., 1999). PFGE was used to show that at least 15 different strains of Lb. helveticus are in use in the US as starter cultures, including mixed-cultures containing one to four strains ( Jenkins et al., 2002). The RAPD technique has also been shown to be useful for discriminating between a large group of strains of

lactococci (Tailliez et al., 1998). The analysis resulted in three major groups, two of which, G1 and G3, contained Lc. lactis subsp. lactis and the other Lc. lactis subsp. cremoris. PFGE has also been used to characterize lactococci (Tanskanen et al., 1990), and unrelated strains showed quite different patterns. Phage-resistant derivatives yielded identical or almost identical patterns to that of the parent strain, indicating the usefulness of PFGE to discriminate between closely related strains. PFGE has also been used to follow the diversity of Lc. lactis in Pecorino Sardo cheese (Mannu et al., 2000). The divergence in the DNA sequences of Lc. lactis subsp. lactis and Lc. lactis subsp. cremoris is estimated to be between 25 and 30% (Godon et al., 1992). Lc. lactis subsp. lactis differs from Lc. lactis subsp. cremoris in 9–10 bp in the sequence of the V1 region of the 16S rRNA gene and this has allowed specific DNA probes for the different species of lactococci and leuconostocs to be designed (Klijn et al., 1991). A novel method for distinguishing between Lc. lactis subsp. lactis and Lc. lactis subsp. cremoris was proposed by Nomura et al. (1999), who showed that Lc. lactis subsp. lactis produced -aminobutyric acid by decarboxylation of glutamate while Lc. lactis subsp. cremoris did not. A recent study (Kelly and Ward, 2002) has shown that strains of Lc. lactis subsp. cremoris having a lactis phenotype can be isolated in low numbers from dairy and plant environments; the opposite, i.e., Lc lactis subsp. lactis with a cremoris phenotype can also be found but is rare.

Types of Cultures Starter cultures may be classified on the basis of their function, their temperature of growth or their composition. Some examples are presented in Table 1. Primary starters are involved mainly in the production of lactic acid from lactose, which occurs early in cheese production. Therefore, high numbers of active cells are added to the cheese milk. However, many of them also produce volatile compounds, e.g., diacetyl from citrate, which is an important flavour component of fresh cheese, and CO2 from lactose (heterofermentative species) and citrate (homofermentative and heterofermentative species) which contribute to the open texture of some cheeses. Their proteolytic systems are also involved in flavour and aroma development in ripening cheeses. Moreover, by lowering the pH and Eh, by competing with spoilage and pathogenic microorganisms and by producing antimicrobial compounds, they also contribute to the microbial safety of cheese. The secondary microflora is more varied, both from a taxonomical and a functional point of view: non-starter

Table 1 Examples of cheese types, starter cultures and their function. Cheeses are ordered from soft to hard Starter microflora Primary c Cheese

Starter function a

Starter type b

Cottage, Quarg, Cream

LA, D

Camembert, Brie

LA, P, L

Mozzarella (Italian)

LA, AR

Pizza cheese Roquefort, Stilton

LA, P LA, C, P, L LA, P, L LA, P, SM

DSS, MSS MSS, DSS NS, MSS, DSS DSS DSS, MSS DSS NS DSS

LA, P LA, C, D, P LA, C, PG, P LA, P

DSS MSS MSS DSS NS

Gorgonzola Tilsit

Cheddar Gouda, Edam Emmentaler, Sbrinz, Gruyere Parmigiano-Reggiano

Ln

Lc

Lc Cit

Ec

Lb

Ll

Lh

Geotrichum candidum, Penicillium camemberti

Other

13

Sc

See Volume 2, Chapter

11

Penicillium roqueforti staphylococci, yeasts Penicillium roqueforti Brevibacterium linens, other coryneforms, staphylococci, yeasts

7

Propionibacterium shermanii

11 8 8 9

4 5 6 3

a LA, lactic acid; D, diacetyl; AR, aroma, other than diacetyl; C, CO2; P, proteolysis; L, lipolysis; PG, propionic acid and gas; SM, surface smear. b NS natural starter, MSS mixed strain starter, DSS defined strain starter. c Ln, Leuconostoc mesenteroides subsp. cremoris; Lc, Lactococcus lactis subsp. lactis (Cit ) and/or cremoris; Lc, Cit citrate-utilising Lc. lactis subsp. lactis; Ec, Enterococcus faecium and/or faecalis; Sc, Streptococcus thermophilus; Lb, Lactobacillus delbrueckii subsp. bulgaricus; Ll, Lb. delbrueckii subsp. lactis; Lh, Lb. helveticus, , dominating species; species occurring in lower numbers or occasionally.

125


lactic acid bacteria (NSLAB), propionibacteria, coryneforms, staphylococci, yeasts and moulds may all contribute to the organoleptic properties of cheeses. Because these microorganisms play their role during ripening, high initial numbers are not needed and natural contamination, from milk and the cheese environment, is still relied upon in many cheese varieties. However, improvement in the hygiene of milk and the need for standardization and acceleration of ripening have resulted in blander-flavoured cheese. This, in turn, has prompted the use of many secondary starters or adjuncts to improve the sensory properties of cheese, or its health benefits (probiotics) (see ‘Secondary and Adjunct Cultures’, Volume 1). Primary starters are usually classified as mesophilic or thermophilic. The latter are characteristics of Italian (Grana, Pecorino, Mozzarella) and Swiss (Emmentaler, Sbrinz, Gruyère) cheese varieties, where a high temperature (37 °C but generally 48–52 °C) prevails during the early phases of cheesemaking. Mesophilic starters are used in all cheese varieties in which the temperature of the curd during the early stage of acid production does not exceed ⬃40 °C (Cheddar, Gouda, Edam, Camembert, etc.). However, this distinction is losing some of its meaning, since mesophilic and thermophilic species are often found (or used) together in both mixed and defined starters for the manufacture of cheeses like Mozzarella (Limsowtin et al., 1996; Parente et al., 1997) and Cheddar (Beresford et al., 2001). Probably, the most common classification of starter cultures is based on the complexity of the culture and the way it is reproduced (Limsowtin et al., 1996). All starter cultures available today are derived in one way or another from natural (or artisanal) starters of undefined composition (i.e., containing an undefined mixture of different strains and/or species), reproduced daily in cheese factories by some form of backslopping. Reports on adding sour cream or buttermilk to cream to improve the quality of butter in Denmark date back to the 1860s and the use of natural whey cultures (i.e., the addition of whey from a previous cheesemaking batch to cheesemilk) for Grana production dates from 1890 (Bottazzi, 1993). Natural starters are still used widely in Europe (Limsowtin et al., 1996; Beresford et al., 2001) and in Argentina (Reinheimer et al., 1996). However, for many cheeses they have been replaced by commercial mixed-strain starters (MSS), derived from the ‘best’ natural starters and reproduced under controlled conditions by specialized institutions (Dairy Research Centres or commercial starter companies) and distributed to cheese plants which use them to build up bulk starter or for directvat inoculation (see below). While the composition of MSS is undefined, their reproduction under more con-

trolled conditions reduces the intrinsic variability associated to the use of artisanal starters. Natural starter cultures and commercial MSS, because of their long history, are called traditional starters (Limsowtin et al., 1996) as opposed to definedstrain starters (DSS). These are composed of one or more strains (cultures with up to 13 strains are used in Switzerland) which were first used in New Zealand for Cheddar cheesemaking in the 1930s. Like MSS, DSS are selected, maintained, produced and distributed by specialized institutions. Because of their optimized, highly reproducible, performance, and their high phage resistance, DSS have replaced traditional starters in the production of many cheese varieties, including some PDO European varieties. While the development of DSS is still based largely on the isolation and selection of strains from raw milk, cheese or traditional starters (Limsowtin et al., 1996; Wouters et al., 2002), the need to improve the control of phage under the high selective pressure imposed by production schedules in large-scale cheesemaking and the availability of food-grade cloning and gene transfer systems have led to the development and use of genetically enhanced strains in DSS by the introduction of natural phage resistance mechanisms into industrial strains (Coffey and Ross, 2002; ‘Starter Cultures: Bacteriophage’, Volume 1). These strains are not classified as GMOs according to current European and US definitions (Kondo and Johansen, 2002) and their use is not restricted. Issues related to consumer acceptability have limited the commercial use of engineered strains of starters, developed to show improved autolysis, improved aroma-producing properties, over-expression of peptidases, novel phage resistance mechanisms, etc. (Kondo and Johansen, 2002). Natural starter cultures

Natural starter cultures are reproduced daily at the cheese plant by some form of backslopping (i.e., the use of an old batch of a fermented product to inoculate a new one) and/or by application of selective pressure (heat treatment, incubation temperature, low pH). No special precautions are used to prevent contamination from raw milk or from the cheesemaking environment and control of media and culture conditions during starter reproduction is very limited. As a result, even in any given cheese plant, natural starters are continuously evolving, undefined mixtures composed of several strains and/or species of LAB. The composition and techniques for the production of artisanal starters have been reviewed by Limsowtin et al. (1996). Two subtypes are recognized, whey- and milk-starters, depending on the medium and techniques used for their reproduction.

Starter Cultures: General Aspects 127

Natural whey cultures are prepared by incubating some of the whey drained from the cheese vat overnight under more or less selective conditions. The composition and the biological diversity of the culture are strictly dependent on the selectivity of the incubation conditions. In the manufacture of Parmigiano Reggiano and Grana Padano cheese (see ‘Extra Hard Varieties’, Volume 2), whey is removed for the cheese vat at the end of cheesemaking at 48–52 °C and is incubated overnight at a controlled temperature (45 °C), or in large containers in which the temperature decreases to 37–40 °C, to a final pH as low as 3.3 (Limsowtin et al., 1996). The resulting whey culture (siero-fermento, siero-innesto) is dominated by aciduric and/or thermophilic strains; Lb. helveticus usually dominates (85%), but other species (Lb. delbrueckii subsp. lactis, Lb. fermentum, Sc. thermophilus) may be present. Seasonal and geographical variations in the composition and performance of the culture have been observed. In a recent molecular ecology study (Cattivelli et al., 2002) it was shown that a limited number of strains (maximum 6) dominates the cultures, that a ‘house’specific flora can be identified in different plants, and that Sc. thermophilus is found only in wheys incubated at a low temperature. Similar whey cultures are used in the production of pasta-filata cheese varieties in Italy (Limsowtin et al., 1996; Parente et al., 1997), hard cheese varieties in Argentina (Reinheimer et al., 1996), and Comté cheese in France (Bouton et al., 2002). Other types of whey cultures include deproteinized whey starters (scotta-innesto) used for the manufacture of Pecorino cheese (Limsowtin et al., 1996; Mannu et al., 2002; see ‘Cheeses Made from Ewes’ and Goats’ Milk’, Volume 2), and deproteinized whey starters with rennet (Fettsirtenmagenlab, Présure à la ‘recruite’) which are used for the manufacture of Swiss-type cheeses (Emmental, Sbrinz, Gruyère; see ‘Cheese With Propionic Acid Fermentation’, Volume 2) in small cheese factories in the Alps. Invariably, thermophilic lactobacilli (Lb. helveticus, Lb. delbrueckii subsp. lactis) dominate cultures produced under selective conditions (high temperature) while streptococci (Sc. thermophilus, but also lactococci and enterococci) often dominate cultures incubated at a relatively low temperature (42 °C), which usually show higher microbial diversity (Parente et al., 1997). Natural milk cultures (colture naturali in latte, lattoinnesti, lattofermento) are still used in small cheesemaking plants in both Southern and Northern Italy for the production of traditional cheeses. The selective pressure used for the development of the desired microflora includes thermization/pasteurization of raw milk (62–65 °C for 10–15 min) followed by incubation at a high temperature (37–45 °C) until the desired titrat-

able acidity is reached. These cultures are usually dominated by Sc. thermophilus but other species may be present (Sc. macedonicus, enterococci, mesophilic lactobacilli; Limsowtin et al., 1996; Andrighetto et al., 2002). The use of natural starter cultures has both advantages and disadvantages. They are an extremely valuable source of strains with desirable technological properties (phage resistance, production of antimicrobials, aroma production), although many strains show limited acid production ability when cultivated as pure cultures (Cogan et al., 1997). Fluctuations in composition result in variable performance and this may not be acceptable in modern cheesemaking practice. Natural starters are considered to be highly tolerant to phage infection. Like the ‘practice’ MSS used in Dutch cheese manufacture (Stadhouders and Leenders, 1984), natural cultures are reproduced in the presence of phage, which exert selective pressure, which ultimately leads to the dominance of resistant or tolerant strains. Occasionally, the development of highly virulent phage attacking the dominant strains may severely reduce culture activity, and time will be needed for the establishment of a new equilibrium. The presence of bacteria, like coliforms and enterococci (Coppola et al., 1988; Parente et al., 1997), in some natural starter cultures may also raise some concern. In Europe, the standards of identity of many PDO cheeses require the use of natural starter cultures, because a strict relationship is believed to exist between the use of given natural starter cultures and cheese properties. Molecular and technological characterization of Lb. helveticus strains isolated from natural starters used for Provolone and Grana cheese in Italy (Gatti et al., 1999; Giraffa et al., 2000) has indeed shown that strains from the cultures used for the two cheeses are different. On the other hand, Sc. thermophilus strains isolated from natural milk cultures used as starters for PDO cheeses produced under very similar conditions (Asiago d’Allevo, Montasio, Monte Veronese) cannot be distinguished by RAPD-PCR (Andrighetto et al., 2002). Mixed-strain starters

When undefined cultures are propagated under controlled conditions with a minimum of subcultures, the stability of their composition and performance is greatly improved, without losing the advantage of tolerance to phage infection (Stadhouders and Leenders, 1984). Mixed-strain starters, obtained by careful selection of natural starters, are maintained, propagated and distributed by starter companies and research institutions, and are widely used for the production of


cheese in Europe (Table 1). The traditional method for the reproduction of MSS, which required several transfers in the cheesemaking plant to build up the bulk starter, starting from small amounts of stock cultures, has been replaced by the use of concentrated cultures for the inoculation of the bulk starter tank or for direct inoculation of the cheese milk, thus minimizing the need for transfers within the factory and the risk of fluctuations in starter composition and activity. Mixed-strain starters are usually classified as mesophilic or thermophilic, with an optimum growth temperature of 28–30 °C and 42 °C, respectively (Limsowtin et al., 1996). Mesophilic MSS can be further classified on the basis of citrate fermentation and composition, as citrate-negative ‘O’ starters (which contain acidproducing Cit Lc. lactis subsp. lactis and cremoris) or citrate-positive L, D and DL starters (containing Leuc. mesenteroides subsp. cremoris, Cit Lc. lactis subsp. lactis, or both, respectively, in addition to acid-producing strains). Thermophilic MSS are used for the production of Italian and Swiss cheese varieties, and usually contain Sc. thermophilus alone or in mixtures with thermophilic lactobacilli (Lb. delbrueckii subsp. lactis, Lb. helveticus) (Glättli, 1990). Like artisanal starters, MSS contain undefined mixture of strains, which differ in their physiological and technological properties (including phage resistance). Plasmid profiles and phage sensitivity have been used to estimate the diversity of strains in MSS, although other molecular methods (PFGE, RAPD-PCR, etc.) may provide a better estimate of strain diversity. In a recent study, Bissonnette et al. (2000) evaluated the diversity of Lc. lactis subsp. cremoris in seven MSS used for the manufacture of Cheddar cheese in Canada by isolating and typing a relatively large number of strains (30) from each culture. Two MSS were dominated by 2–3 strains, three by 7–9 strains but two had a high diversity, with 18–24 distinct strains; 32 different strains have been claimed to be present in an MSS used for Cheddar cheesemaking in Denmark (Josephsen et al., 1999). Because they are derived from cultures which were reproduced in cheese plants without protection from disturbing phages, MSS contain many phage-resistant strains but also harbour their own phages (Stadhouders and Leenders, 1984; Limsowtin et al., 1996; Josephsen et al., 1999; Bissonnette et al., 2000). The development of MSS for the production of Dutch cheeses at NIZO (Stadhouders and Leenders, 1984) and thermophilic MSS (Rohmischkulturen) for the manufacture of Swiss cheese varieties by the Swiss Federal Dairy Research Station (Glättli, 1990) are two examples of the successful development and long-term use of MSS (Limsowtin et al., 1996; ‘Gouda and Related

Cheeses’ and ‘Cheese With Propionic Acid Fermentation’, Volume 2). Even if MSS have a long history of successful use without severe inhibition by phage, one should not be overly confident that phage infection will never be experienced. Published studies on long-term monitoring of phage/starter interaction in cheese plants using MSS are rare. Josephsen et al. (1999) have documented the development of virulent phages in a factory which had been using the same MSS almost continuously before occasional slow acidification problems were experienced. The isolates from the MSS for which homologous phages were detected in cheese whey increased from 16 to 97% over 11 years, and their virulence increased greatly. In fact, while phages isolated when no acidification problem was experienced had restricted host range, long latent times (38–52 min) and relatively low burst sizes (35–84), phages isolated in recent year had broader host ranges (and were able to multiply on strains which were highly phage resistant), reduced latent times (35 min) and greatly increased burst sizes (120–200). Defined-strain starters

Mesophilic DSS originated in New Zealand in the 1930s, as a response to the occurrence of open texture defects in Cheddar cheese produced with MSS containing Cit strains. The history of mesophilic DSS systems in New Zealand, Australia, USA and Ireland has been reviewed by Limsowtin et al. (1996). Since the strain and/or species ratio in DSS is defined, their technological performance is extremely reproducible. This is obviously a highly desirable property in modern cheese plants with large throughputs of milk and tight production schedules. Since only a limited number of strains are used (commonly 2–6), phage infection may have destructive consequences on starter activity. In fact, the history of DSS is a continuous fight to devise measures to control phage infections. Singlestrain starters were used initially in New Zealand, but rapid onset of destructive phage infections, with complete loss of activity, occurred. These were then replaced by pairs of phage-unrelated strains, which were rotated daily, and measures to ensure aseptic reproduction of the starters were implemented (Whitehead and Cox, 1936). Rotations were cumbersome to maintain and they were replaced by an approach based on the selection of bacteriophage insensitive mutants (BIM; Heap and Lawrence, 1976). This approach allowed development of 3-day rotations with highly phage-resistant strains, which were subsequently used together in a single multiple-strain starter containing six strains


(Limsowtin et al., 1977). The difficulty of replacing strains led to the reduction of the number of components from six to five and finally to three. Such DSS are in use in Australia, New Zealand, USA and Ireland. In general, highly phage-resistant DSS are available either through research institutions (e.g., the Australian Starter Cultures Research Centre, or Fonterra Research in New Zealand) or from commercial suppliers. The strategy used for the management of DSS in Australia has been documented thoroughly (Limsowtin et al., 1997). Today, the selection of BIM has been largely replaced by strategies based on the introduction of natural phage resistance mechanisms into industrial strains (Coffey and Ross, 2002; see also ‘Starter Cultures: Bacteriophage’, Volume 1). Thermophilic DSS are also commercially available for the production of a variety of Italian- and Swisstype cheeses. Starters composed of single or multiple strains of Sc. thermophilus are still preferred in Italy for the production of high-moisture Mozzarella cheese, but associations of Sc. thermophilus and Lb. delbrueckii subsp. bulgaricus (rod:coccus starter cultures) are used for the manufacture of low-moisture Mozzarella cheese (Kinstedt, 1993; Oberg and Broadbent, 1993). The use of Lb. helveticus in place of Lb. delbrueckii subsp. bulgaricus has been claimed to present several advantages (Oberg et al., 1991) such as reduced make time and improved functional properties. Phage-resistance mechanisms are apparently less widespread among thermophilic starter cultures than in lactococci (Coffey and Ross, 2002). Due to the relatively narrow host range of Sc. thermophilus phage, use of rotations and BIMs is still relied upon to control phage infection in thermophilic starter cultures (Moineau, 1999). New sources of starters

Most, if not all, of the LAB found in starter cultures can be isolated from cheese made without the deliberate addition of a starter culture. Such strains are natural contaminants of milk which grow and produce acid during cheesemaking. The ultimate source of these bacteria remains to be determined. However, it is generally thought that plants and plant material are the natural habitat of Lc. lactis subsp. lactis. The habitat of Lc. lactis subsp. cremoris has not been determined but it can be isolated from dairy products. Many of the pure cultures of starter bacteria used in defined cultures are phage-related, implying that the number of different strains of starter bacteria is generally limited. Therefore, efforts have been made to isolate ‘new’ strains from raw milk, plants and other natural sources (Salama et al., 1995; Cogan et al., 1997; Wouters et al., 2002). Any potential new starter

strain must produce acid rapidly, lack off-flavour development in milk and be resistant to a mixture of common phage. Lc. lactis subsp. lactis but not Lc. lactis subsp. cremoris has been isolated from red nettles, common sow thistle, Himalayan blackberries, potato, cucumber, corn, sweet pea, beans, cantaloupe, corn and broccoli and many of them were good acid producers, coagulating milk in 18 h at 21 °C (Salama et al., 1995). In contrast, very few strains of Lc. lactis (the sub-species was not determined) isolated from artisanal dairy products were good acid producers (Cogan et al., 1997). Some of them produce unusual flavours in milk. For example, the combination of a ‘wild’ starter, which had low protolytic activity and high amino acid decarboxylase activity, with a commercial strain, which had high proteolytic activity and low decarboxylase activity, resulted in the production of chocolate flavour in milk, due to several branched chain aldehydes and acids (Wouters et al., 2002).

Genome Sequence Arguably, the most significant advance in starter cultures in the past 30 years has been the determination of the complete genome sequence of the chromosome of Lc. lactis IL 1403 (Bolotin et al., 1999). Almost 1500 genes were located and their functions classified on the basis of homology to human proteins. Five potential or rudimentary prophages were identified in the genome, implying that the ultimate source of phage is probably the starter cell itself. The analysis also showed that Lc. lactis has the potential to synthesize 20 amino acids and 4 co-factors. However, the presence of these genes does not mean that Lc. lactis will not require these compounds for growth. Since then, the genomes of three other LAB, Lb. plantarum, Lb. johnsonii and Lb. acidophilus, have also been sequenced, and 24 other LAB including other strains of Lb. lactis subsp. cremoris, Lb. debreuckii subsp. bulgaricus, Lb. casei, Lb. helveticus, Sc. thermophilus are on-going. Genome sequence projects for other nonLAB which are important in cheese ripening have either been completed (P. freudenreichii) or are ongoing (B. linens). The information that these data will generate will be of considerable benefit in understanding the fundamental metabolism of these bacteria, including the production of lactic acid, proteolytic systems, tolerances to heat, acid and salt stresses, production of bacteriocins and other anti-microbials. As many of them also have widely different ecological niches, the data should also be very useful in determining why particular species occupy a particular niche. Such data will also help in the development of


new strains or modification of common strains used as starter cultures (Klaenhammer et al., 2002).

Metabolism of Starter Cultures Sugar metabolism

Lactose is the major sugar in milk and its transport, metabolism and regulation in several different starter cultures have been reviewed (Poolman, 1993, 2002; Cocaign-Bousquet et al., 1996) and will not be reviewed further here. The salient features of the pathways used by different starter cultures are summarized in Table 2. Application of NMR has been very useful in understanding the flux through different pathways during growth and in understanding regulation of different aspects of metabolism in LAB and the literature has been reviewed by Ramos et al. (2002). NMR has also been useful in understanding exopolysaccharide (EPS) production. In the case of glucose metabolism, the results have shown that the rate of fructose1,6-bisphosphate consumption and the magnitude of the PEP potential (PGA PEP) are considerably higher when Lc. lactis is grown under aerobic than under anaerobic conditions, implying that NADH oxidase activity is important. Citrate metabolism

Citrate is present at a low concentration in milk and is metabolized by Leuconostoc subsp. and some strains of Lc. lactis subsp. lactis to CO2, which is responsible for eye formation in some cheeses, and diacetyl and acetate, which are important flavour components in fermented milks. The latter organism was called Sc. diacetylactis in the old literature and more recently Lc. lactis subsp. lactis biovar diacetylactis. This name has no taxonomic status and the correct way to refer to it is citrate-utilizing (Cit) Lc. lactis subsp. lactis. Cit

strains of Lc. lactis differ from the more normal noncitrate-utilizing (Cit ) strains in containing a plasmid which encodes the transport of citrate. Citrate metabolism in LAB has been reviewed by Hugenholtz (1993). In recent years, considerable effort has been devoted to understanding the energetics of citrate transport in Leuc. mesenteroides and Lc. lactis (Garcia-Quintans et al., 1989; Marty-Teyssett et al., 1996; Magni et al., 1999). In the absence of any other carbon source, Leuc. mesenteroides and Lc. lactis transport citrate in symport with a proton, which leads to the generation of a pH or proton motive force. In the presence of D-lactate and glucose, citrate is transported by an antiport system with lactate being extruded; in this case, citrate metabolism is also more rapid. This is due to the fact that the exchange between citrate and lactate is much faster than the citrate/H symport system. Since D-lactate is a product of sugar metabolism, the transporter operating under physiological conditions is likely to be that for citrate/lactate. An electrogenic citrate/D-lactate exchange occurs, generating a proton electro-chemical gradient across the membrane. This may contribute significantly to the enhanced growth of Leuc. mesenteroides during co-metabolism of glucose and citrate. Co-metabolism of glucose and citrate by Leuconostoc subsp. results in a faster growth rate. This has been attributed to a metabolic shift in the glucose pathway, leading to increased ATP production (Cogan, 1987). The results of Marty-Teysset et al. (1966) suggest that the citrate/D-lactate exchange is also involved in producing energy. In Lc. lactis, co-metabolism of citrate and sugar does not result in a major effect on growth rate at neutral pH. However, at acid pH values (4.5), the citrate transport system is induced. Metabolism of citrate results in an increase in pH to a value at which the consumption of glucose begins (Garcia-Quintans et al., 1989). More recently, it has been suggested

Table 2 Salient features of lactose metabolism by starter organisms

Organism

Transport a

Pathway b

Cleavage enzyme c

Lactococcus lactis Leuconostoc spp.

PEP-PTS Permease

GLY PK

pgal gal

Sc. thermophilus Lb. delbrueckii Lb. helveticus

Permease Permease Permease

GLY GLY GLY

gal gal gal

a b c d

PEP PTS, phosphotransferase system. GLY, glycolysis; PK, phosphoketolase. gal, phospho--galactosidase; gal, -galactosidase. These species metabolize only the glucose moiety of lactose.

Products (mol/mol lactose) 4 Lactate 2 Lactate ethanol 2 CO2 2 Lactated 2 Lactated 4 Lactate

Isomer of lactate L D L D DL


Therefore, further growth requires the hydrolysis of milk proteins. In fact, the growth of many LAB is diauxic in milk; an initial fast growth rate, during which free amino acids and peptides are used up, is followed by a slightly slower rate during which further peptides and amino acids are obtained by hydrolysis of casein. Proteolysis is a major event in cheese ripening; the proteolytic system of the primary starter and of the secondary microflora contributes the production of hundreds of flavour compounds through the production of low-molecular weight peptides and amino acids and their subsequent catabolism. The role of proteolysis and amino acid catabolism in cheese has been addressed by several recent reviews (Sousa et al., 2001; Yvon and Rijnen, 2001) and is described in detail in ‘Proteolysis in Cheese during Ripening’ and ‘Catabolism of Amino Acids in Cheese During Ripening’, Volume 1. The proteolytic system of LAB is composed of a cell-wall bound proteinase, transport systems for amino acids, di- and tripeptides and oligopeptides, a number of intracellular peptidases and some intracellular proteinases (Fig. 1). Several excellent reviews have been published on this topic (Kunji et al., 1996;

(Magni et al., 1999) that the induction of the citrate metabolic pathway under acidic conditions makes the cells more resistant to the inhibitory effects of lactate. Nitrogen metabolism

Nitrogen metabolism by starters has an enormous impact on their activity and on cheese quality. To perform their main function of acid production in milk and curd, LAB must grow to high numbers, from ⬃1 106 cfu/ml in the inoculated milk to ⬃1 109 cfu/g in the cheese curd; syneresis of the curd due to expulsion of whey also contributes to the increase in cell numbers. Lactic acid bacteria are fastidious microorganisms and are unable to synthesize many amino acids, vitamins and nucleic acid bases. Depending on the species and the strain, LAB require from 6 to 14 different amino acids (Chopin 1993; Kunji et al., 1996). Although milk is rich in nitrogen, it is present mainly as protein. It has been calculated that the amount of free amino acids and low-molecular weight peptides present in milk can support only limited growth (10–20% of the final biomass of a fully grown culture of lactococci; Thomas and Pritchard, 1987).

OUT cell wall membrane

IN

PrtP

peptides (2–18 aa)

Opp DtpT DtpP

casein

peptide transport systems

peptidase

amino acids transport systems

Pro-specific peptidases s • XDAP (PepX ) • proline iminopeptidase PepI) m • prolidase (PepQ ) m • prolinase (PepP ) general peptidases • aminopeptidases m c (PepN , PepC ) m • dipeptidases (PepV ) m • tripeptidases (PepT ) • endopeptidases m m (PepO , PepF )

Glu-specific peptidases m • aminopeptidases (PepA )

aa amino acid catabolism aa

arginine deiminase pathway aldolases aminotransferases decarboxylases dehydrogenases

aa

biosynthetic pathways protein synthesis aroma compounds

lysis

Figure 1 Schematic representation of nitrogen metabolism in lactic acid bacteria. The abbreviations refer to enzymes of Lactococcus lactis. The superscripts refer to classes of peptidases (c, cysteine peptidases; m, metallo peptidases; s, serine peptidases) (adapted from Kunji et al. (1996) and Sousa et al. (2001), Christensen et al. (1999) and Yvon and Rijnen (2001)).


Christensen et al., 1999; Siezen, 1999) and only a general overview will be presented here. Proteinase

Lactic starters degrade casein and large casein-derived peptides produced by milk and coagulant enzymes by a cell-envelope proteinase (CEP, lactocepin, EC 3.4.21.96, also called the cell wall-bound proteinase; Kunji et al., 1996; Siezen, 1999). All CEPs from LAB described to date are serine-proteinases related to subtilisins. The CEP of Lc. lactis (PrtP) is the most extensively characterized. The proteinase gene (prtP), which may be located on plasmids or on the chromosome, encodes a protein of 1902 (Lc. lactis WG2 and NCDO763) or 1962 (Lc. lactis SK11) amino acids; the larger size is due to a duplication near the C-terminus. To date, several domains have been identified in PrtP (Fig. 2). A pre–pro domain (PP) is needed for secretion and processing. A signal sequence of 31–39 residues at the N-terminus is responsible for Sec-dependent (general) translocation of the pro-proteinase across the cell membrane, and a proteinase maturase (PrtM), encoded by a gene immediately upstream of prtP, cleaves the pro-region between Thr187 and Asp188, producing a catalytically active CEP. The catalytically active domain (PR, residues 188–699 of PrtP) is responsible for activity and substrate specificity of CEPs and is highly conserved. The three-dimensional structure of the PR domain of all subtilases has been predicted (Fig. 3; Siezen and Leunissen, 1997) and this has allowed scientists to engineer the stability, catalytic activity and specificity of the lactococcal proteinase (Siezen, 1999). Amino acid substitutions in positions 96–107 and

125–130 of PrtP, corresponding to the substrate binding cleft, result in a variety of specificities towards degradation of s1-, - and -casein. All CEPs have broad substrate specificity, and no consensus sequence for cleavage sites has been identified. Three further domains (I, A and B) may be important for the stability, specificity and regulation of the activity of the PR-domain, while a helix (H) domain positions PR, A and B domains away from the cell surface. The C-terminus of the proteinase is involved in binding to the cell wall; deletion analysis show that truncated forms lacking 130 or more residues are released into the medium. Incubation of the cells in a Ca-free buffer results in auto-proteolysis and release of a fragment of 135–145 kDa, which is still catalytically active. Two further domains, the W-domain, which is a cell-wall spacer and spans the peptidoglycan layer and a cell-wall anchor, the AN-domain, part of which is cleaved during translocation, anchor PrtP to the cell wall. Further CEPs have been characterized in thermophilic (Lb. helveticus, PrtH; Lb. delbrueckii subsp. bulgaricus, PrtB) and mesophilic lactobacilli (Lb. paracasei, Lb. rhamnosus). They all belong to the subtilase family and share many properties with the lactococcal PrtP, although specificities and domain structure may be different (Fig. 2). The catalytic domains of PrtP, PrtB and PrtH show higher degrees of homology than the other domains. The release of the CEP of thermophilic lactobacilli into the medium requires drastic treatments (lysozyme, osmotic shock, membrane solubilization); although they lack the AN domain, the W-domain is very basic and may bind to the cell wall by electrostatic interaction. Transport systems and peptidases

PrtP

PP

PrtH

I PR

PP

PP

B

A I PR

H

B

A

W

H

W

B

W

AN

cell wall membrane

PrtB

I PR

A

Figure 2 Representation of the predicted domain structure of the cell-envelope proteinase (CEP) of Lc. lactis (PrtP), Lb. helveticus (PrtH) and Lb. delbrueckii subsp. bulgaricus (PrtB). PP, prepro domain; PR, protease domain; I, insert domain; A, A-domain; B, B-domain; H, helical domain; W, cell-wall domain; AN, anchor domain (redrawn from Siezen, 1999).

The activity of CEPs on caseins releases a large variety of oligopeptides; although most are in the range of 4–10 residues, peptides of up to 30 residues can be produced from -casein. No appreciable amounts of free amino acids, di- or tripeptides are produced by the action of CEP. It is now well recognized that all peptidases of LAB are located intracellularly and therefore peptides can be hydrolysed only if they are transported into the cell. Although lactococci (and other LAB) have many amino acid, and di- and tripeptide transport systems, the oligopeptide transport system (Opp) is essential for growth in milk. Opp can transport oligopeptides containing from 4 to 18 amino acids without any significant specificity for their composition (Detmers et al., 1998). Once peptides reach the cytoplasm, they are sequentially degraded by a large variety of peptidases (Kunji et al., 1996; Christensen et al., 1999). Due to the presence of high numbers of Pro and Glu residues


Figure 3 Three-dimensional model of the -carbon backbone structure of the protease (PR) domain of the CEP of Lactococcus lactis (PrtP). The conserved core of subtilases (in grey), the position where residues are inserted or deleted (in white), the substrate binding cleft, the N- and C-termini of the PR-domain and the predicted position of bound calcium ions are shown (from Siezen, 1999).

in the caseins, general, Pro-specific and Glu-specific peptidases are needed to liberate essential amino acids for growth. Figure 1 shows the most important peptidases in Lactococcus lactis, some of which (PepN, PepC, PepX, PepV) have also been found in other dairy LAB. Many other peptidases have been characterized in other LAB (Christensen et al., 1999). Studies with single or multiple peptidase-deficient mutants have shown that, although lack of any single peptidase does not result in complete inhibition, the growth rates of peptidase-deficient mutants are usually lower than the wild type (7–120% increase in generation time; Christensen et al., 1999), with severe inhibition for multiple mutants. The contribution of the peptidases of lactic starters to the release of free amino acids in cheese is now well recognized. Although these enzymes are intracellular, they are liberated in cheese following autolysis of the

cells. Acceleration of autolysis and over-expression of peptidases have been used to accelerate cheese ripening (see ‘Proteolysis in Cheese during Ripening’ and ‘Catabolism of Amino Acids in Cheese During Ripening’, Volume 1). Amino acid degradation

Degradation of amino acids has important implications for the metabolism of starter cultures (e.g., by providing energy in the sugar-depleted environment of cheese), for the safety of cheese (e.g., by production of biogenic amines by decarboxylation of Tyr, His, Trp), and for the production of flavour and aroma compounds. The breakdown of para-casein to amino acids and peptides by a combination of chymosin and proteinases and/or peptidases of the starter bacteria is generally considered to be the most important aspect of cheese ripening. However, amino


acids and peptides, of themselves, are not responsible for flavour development in cheese. The products of the catabolism of amino acids include alcohols, aldehydes, amines and organic acids and are considered to be of major significance in flavour foundation (see ‘Proteolysis in Cheese during Ripening’ and ‘Catabolism of Amino Acids in Cheese During Ripening’, Volume 1). Amino acid catabolism in LAB and in other dairy organisms and its relationship to cheese flavour have been reviewed recently (Christensen et al., 1999; Weimar et al., 1999; Yvon and Rijnen, 2001; ‘Catabolism of Amino Acids in Cheese During Ripening’, Volume 1). The arginine deiminase pathway yields energy directly through substrate-level phosphorylation. In addition, decarboxylation of Asp, Glu, His, Tyr and Trp to the corresponding amines yields energy through amine extrusion and the consequent generation of electrochemical gradients. Histamine, tyramine and tryptamine are biogenic amines, which have been involved in monoamine intoxication (Christensen et al., 1999). While some non-lactic microorganisms (Brevibacterium linens, yeasts, micrococci) initiate amino acid catabolism by elimination reactions, and threonine is catabolized by a threonine aldolase (which produces Gly and acetaldehyde), the first step in amino acid catabolism in LAB is usually a transamination reaction. Aminotransferases (AT) of LAB have broad and often overlapping specificities. Aromatic amino acid ATs (AraT) and branched chain amino acids AT (BcaT) catalyse the first step in the catabolism of aromatic and branched chain amino acids, respectively, resulting in the production of -ketoacids which are potent aroma compounds, e.g., -keto isovaleric acid, which is produced from Thr, or are converted further to aroma compounds (alcohols, aldehydes, esters, etc.) by a variety of enzymes. Transamination reactions generally require -ketoglutaric acid as a substrate, and the concentration of this ketoacid is limiting in cheese. In fact, addition of -ketoglutaric acid ( KG) to cheese has been shown to greatly enhance flavour formation. Alternatively, -ketoglutaric acid can be produced from glutamate by strains with glutamate dehydrogenase activity (Yvon and Rijnen, 2001; Tanous et al., 2002). Of necessity, these reactions must be capable of progressing at relatively low pH (⬃5.0) and the relatively high salt concentrations (⬃6%), which occur in most cheeses during ripening. Such conditions limit enzyme activity significantly but small activities acting over the protracted period of cheese ripening are of considerable importance in generating flavour.

Lipases and esterases

Except for Parmigiano Reggiano, Pecorino and related Italian cheeses, and Blue cheeses, limited lipolysis occurs in cheese during ripening. Nevertheless, the limited level, which does occur, is considered to be important for flavour and taste perception. Esterases have been purified from several starter and LAB, including Lc. lactis (Holland and Coolbear, 1996; Chich et al., 1997), Sc. thermophilus (Liu et al., 2001) and Lb. plantarum (Gobbetti et al., 1997). All of them are serine enzymes that preferentially hydrolyse butyrate esters and are optimally active at pH 7. Some of them have no activity at pH 5.0; nevertheless, a very small amount of activity over a long time could result in significant hydrolysis of fat during cheese ripening. The major tributyrin esterase of Lc. lactis has been cloned, over-expressed and characterized (Fernandez et al., 2000). The purified enzyme showed a preference for short-chain acyl esters and also phospholipids, suggesting that it may be involved in phospholipid metabolism in vivo. Growth

Chemically defined media (CDM) for the growth of Lc. lactis, Leuc. mesenteroides and Sc. thermophilus have been developed ( Jensen and Hammer, 1993; CocaignBousquet et al., 1995; Foucaud et al., 1997; Letort and Juillard, 2001). Maximum specific growth rate ranges from 0.4 to 1.0 h 1. The medium for Sc. thermophilus contains only 20 components, including six amino acids (glutamine, cysteine, methionine, leucine, isoleucine and valine). Addition of pyridoxamine eliminated the need for nucleic acid bases in the case of Sc. thermophilus and Lc. lactis. Growth of Lc. lactis NCDO 2118, which was isolated from a vegetable source, and Lc. lactis IL 1403, which was isolated from a dairy source, in a CDM were compared by Cocaign-Bousquet et al. (1995). NCDO 2118 required no amino acid (prototrophic), while IL 1403 required several amino acids, including glutamate, arginine, methionine, valine, leucine or isoleucine (auxotrophic), when the single omission technique was used to identify a requirement. However, in a simplified CDM, NCDO 2118 required glutamate, methionine, isoleucine, leucine, valine and serine, indicating that proto/auxotrophy is partly dependent on the composition of the medium. The dairy strain showed an additional requirement for arginine, histidine and threonine. Lactic acid bacteria do not have a functional TCA cycle and hence most of them require the glutamate but, surprisingly, not the aspartate, family of amino acids. A gene cluster coding for citrate synthase, aconitase and isocitrate dehydrogenase has been identified


in Lc. lactis C2 (Wang et al., 2000). Lc. lactis NCDO 2118 was able to grow in a CDM containing KG but no glutamate, but the lag phase depended on the concentration of KG added (Lapujade et al., 1998). No glutamate dehydrogenase activity, the enzyme which is used in many bacteria to produce glutamate directly from KG, was detected but transaminase activities, with several amino acids as amino group donors and KG as acceptor, were detected. Indeed, addition of KG to milk before cheese manufacture also increases flavour development during ripening (Yvon and Rijnen, 2001), indicating the importance of amino acid transferase activity in the maturation of cheese (see later). Niven et al. (1998) found biphasic growth of Lc. lactis MG 4685 in milk, with a faster initial rate (0–4 h) followed by a slower (4–8 h) one. There was little change in the concentration of amino acids during the first phase while the second phase correlated with increased production of amino acids; however, significant decreases in glycine and alanine occurred during both growth phases. The growth rates of Lc. lactis ML3 and Wg2 decreased rapidly above pH 7 when grown on a synthetic medium containing glutamate but not glutamine (Poolman and Konings, 1988). If glutamate was replaced by glutamine, the pH of growth was extended to 8.0, indicating that the unionized form of glutamic acid, rather than the ionized form, is transported by the glutamic acid/glutamine transporter in these organisms. At alkaline pH, the rate of growth in the absence of glutamine is limited because less unionized glutamic acid is available at the higher pH values. The effect of different environmental conditions on the rate of growth of starter bacteria, particularly Lc. lactis, has been investigated in several recent studies. Generally, lactococci produce other products of sugar metabolism besides lactate when grown on galactose or a low level of glucose (Thomas et al., 1979). In contrast, Even et al. (2001) showed that Lc. lactis subsp. lactis IL1403 retained its homolactic behaviour on glucose and galactose in two different minimal media of different nutritional complexities, despite significant variations in both growth rate and sugar consumption. Lactic acid bacteria are essentially fermentative organisms but they are also capable of consuming oxygen with the formation of H2O2. Under these conditions, various NADH oxidases and peroxidases are produced to reduce the toxic H2O2 (Duwat et al., 2001; van Niel et al., 2002). LAB are therefore considered to be aerotolerant organisms. An NADH oxidase has been purified from Lc. lactis MG1363 (Lopez de Felipe and Hugenholtz, 2001). Oxygen can be beneficial to Lc. lac-

tis during aerobic growth if heme is also present. Lc. lactis MG1363 grown in M17 (glucose) in the presence of 10 g haemin/ml produced increased biomass and retained almost 100% viability over 70 days at 4 °C (Duwat et al., 2001). Growth also resulted in changes to a heterolactic fermentation. The results were correlated with the production of cytochrome oxidase, which is required for respiration, late in growth. Another recent study (van Niel et al., 2002) has shown that the intracellular concentration of pyruvate in Lc. lactis subsp. lactis ATCC 19435 can reach 93 mM, which rapidly destroys H2O2 non-enzymatically. A plasmid-free strain of Cit Lc. lactis DRC1 grew at a significantly (5%) faster rate in complex broth than the parent strain. The slower growth rate of the parent was due to the presence of a small (7.4 kb) plasmid (Kobayashi et al., 2002). Metabolic engineering

Lactic acid bacteria have a relatively simple sugar metabolism and homofermentative strains convert 90% of metabolized sugar to lactic acid. Other products, some of which are commercially important, e.g., diacetyl, are also produced but in much smaller amounts. Because LAB are GRAS organisms, with a relatively simple metabolism, efforts have been made to get them to over-produce these minor products. The metabolic engineering of LAB to produce these products has been reviewed (Hoefnagel et al., 2002; Hugenholtz et al., 2002). Diacetyl is produced chemically from -acetolactate (AL) which is produced from pyruvate which, in turn, is produced from citrate. -Acetolactate is highly unstable and breaks down to diacetyl when O2 is present and to acetoin when O2 is absent. Acetoin production from AL is also catalysed by AL decarboxylase but there is no enzyme which produces diacetyl from AL. Platteeuw et al. (1995) cloned the AL synthase gene from Lc. lactis MG 1363 into Lc. lactis MG 5267 and obtained a 100-fold increase in AL production. Only lactic acid was produced by the strain under anaerobic conditions but 26 and 42% of the pyruvate was converted to acetoin under aerobic conditions at pH 6.8 and 6.0, respectively. An LDH-deficient strain of Lc. lactis MG 5267, grown anaerobically, produced significant amounts of fumarate, ethanol, acetoin and butanediol. Under aerobic conditions, approximately half of the pyruvate was converted to acetoin and onethird to butanediol. To produce diacetyl in such systems, the acetolactate decarboxylase gene, as well as the ldh gene must be inactivated. This was partially accomplished by random mutagenesis of three strains of a Cit strain of Lc. lactis by Monnet et al. (2000).


The strains were deficient in ALD and had much lower LDH activity than the parent strains. The ‘double’ mutants produced up to four times more AL and acetoin and two times more diacetyl than the parent under partially anaerobic (not defined) conditions and grew very poorly in milk under aerobic conditions. Addition of yeast extract (0.2 g/L) or catalase (70 U/ml) increased the level of AL and resulted in the production of 5 and 6 mM diacetyl, respectively; however, acetoin was still produced. Recently, the ALD in Cit Lc. lactis subsp. lactis has been shown to be a key regulator of valine and leucine biosynthesis as well as in the production of acetoin by controlling the flux of acetolactate (Goupil-Feuillerat et al., 1997). Overproduction of NADH oxidase and inactivation of ALD have also been shown to increase diacetyl production in aerated cultures of Lc. lactis (Hugenholtz et al., 2000). Lb. helveticus has two different LDHs which produce both L and D lactate. Inactivation of the D LDH in Lb. helveticus CNRZ 32 resulted in a strain which produced the same amount of lactic acid as the parent strain but all of it was in the L form (Bhowmik and Steele, 1994). Lb. plantarum also produces both D and L lactate. Inactivation of both enzymes resulted in a strain which produced acetoin (mainly) and small amounts of ethanol and mannitol from glucose (Ferain et al., 1996). Mannitol has a sweetness value about half that of sucrose and, since it cannot be metabolized by humans, it is considered a low-calorie sweetener. Therefore, mannitol over-producing strains may have applications in the production of functional foods. Lc. lactis can metabolize mannitol (Neves et al., 2002) but leuconostocs will produce mannitol during growth on fructose (Grobben et al., 2001). Autolysis

Cell lysis, and the consequent release of intracellular enzymes, particularly peptidases and amino aciddegrading enzymes, is receiving considerable attention as an important aspect of flavour development in cheese since Feirtag and McKay (1987) discovered that temperatures close to the cooking temperature of Cheddar cheese cause the lysis of many starter strains, including Lc. lactis subsp. cremoris SK11, AM1, AM2, US3 but not E8 or KH. This is due to induction of temperate phage. Interestingly, the thermo-inducible strains do not produce bitterness in cheese while the non-inducible strains do. Since then, several groups have identified other thermo-inducible strains (Langsrud et al., 1987; Chapot-Chartier et al., 1994) and the ability to lyse has become an important factor in selecting starters for cheesemaking because of the

increased release of intracellular enzymes. Crow et al. (1995) and Pillidge et al. (2002) reviewed autolysis in LAB, mostly lactococci, and the consequences for cheese ripening, particularly proteolysis. Prophageinduced lysis should be distinguished from true autolysins, which are also found in lactococci (Buist et al., 1998). These findings have stimulated the study of the effect of strains with different lytic and proteolytic properties to make different cheeses, including Cheddar (Wilkinson et al., 1994), St Paulin (Boutrou et al., 1998; Lepeuple et al., 1998) and Gouda (Meijer et al., 1998). Lysis is normally measured by the release of intracellular marker enzymes and concomitant increases in soluble N and free amino acids. In Cheddar cheese ripened at 4 or 10 °C, flavour formation was best in the cheese made with Lc. lactis AM2, the strain showing greatest lysis (Wilkinson et al., 1994). NSLAB numbers reached 106 cfu/g within 1–2 months and flavour was evaluated at 4 months but the contribution of NSLAB to the overall flavour of the cheese does not appear to have been considered. Strain AM2 and a prophage-cured derivative have also been evaluated in St Paulin cheese (Boutrou et al., 1998). The parent strain underwent greater lysis and produced greater amount of amino N; NSLAB counts were 105 cfu/g and the prophage-cured derivative produced a bitter cheese compared with the parent strain. In another study on St Paulin (Lepeuple et al., 1998), five starters with different lytic and proteolytic properties were evaluated for their effect on the flavour of the cheese. Lysis positively influenced the ripening and flavour of the cheese but strains with low peptidase activities and low lytic properties produced bitter cheese. These studies confirm that cell lysis is important in the development of flavour in cheese during ripening. Autolysis appears to be a general property of LAB as Sc. thermophilus, Lb. helveticus and several strains of leuconostocs have also been shown to be lytic (Sandholm and Sarimo, 1981; Lortal et al., 1997; Cibik and Chapot-Chartier, 2000; Husson-Kao et al., 2000). It is not clear if lysis also occurs in NSLAB. Temperate phage is involved in Sc. thermophilus (Husson-Kao et al., 2000) but whether phage are involved with the other organisms was not reported. In the case of Sc. thermophilus, autolysis occurred in response to the depletion of lactose in the medium. Bacteriocins

Bacteriocins are proteins produced by various bacteria, which inhibit the growth of other bacteria. The inhibitory host-range and the molecular mass can be either large or small. Bacteriocins produced by LAB are


divided into three classes: lantibiotics, small heatstable non-lantibiotics and large heat-stable bacteriocins (Nes et al., 1996). Nisin, the best known bacteriocin, is a lantibiotic which is produced by some strains of Lc. lactis, and is used commercially in more than 50 countries as a food preservative to control the growth of spoilage and pathogenic bacteria. Lantibiotics are distinguished by the presence of unusual amino acids, e.g., didehyroalanine and didehydrobutyrine, which are produced by post-translational modificiation of serine and threonine, respectively. Generally, bacteriocins are single compounds but some bacteriocins comprise two components. For more information, see the recent reviews of McAuliffe et al. (2001) and Twomey et al. (2002). Bacteriocins are receiving considerable attention because many of them inhibit a wide range of Grampositive spoilage and pathogenic bacteria, especially Listeria monocytogenes. The latter is a particular problem in cheese because it can grow at high salt concentrations, low temperatures and low pH, all of which typically occur in cheese. The surface of smear-ripened cheeses is a particular problem because of the increase in the pH of the surface during ripening (see ‘Surface Mould-ripened Cheeses’ and ‘Bacterial Surface-ripened Cheeses’, Volume 2). Bacteriocins have also been used to increase lysis of starter cells during cheese ripening resulting in better flavour. The number of bacteriocinproducing cells must be carefully controlled in the latter application so that only limited reduction in the ability of the starter culture to produce lactic acid occurs during cheese manufacture (Ross et al., 1999). They also have potential in cheese ripening to control the growth of NSLAB. Stress responses

Lactic acid bacteria are characterized by their ability to produce large amounts of lactic and sometimes also acetic acid during growth, which cause a significant reduction in pH. Some starter bacteria are also subjected to a considerable range of temperature during cheese manufacture. For example, thermophilic starters are usually grown at 37–42 °C but must withstand temperatures of up to 54 °C in the manufacture of some cheeses, e.g., Emmental and ParmigianoReggiano. Thus, tolerance to acid and temperature and indeed other stresses is being actively studied. Acid tolerances can be of two types, a general stress response, which occurs during the stationary phase of growth and an adaptive response, which develops during the logarithmic phase of growth, called the logarithmic acid tolerance response (LATR) (van de Guchte et al., 2002). Induction of the LATR can also protect LAB against temperature, salt and H2O2

stresses. Acid-adapted cells maintain a slightly higher intracellular pH than non-adapted cells (O’Sullivan and Condon, 1997) so that the maintenance of a transmembrane pH gradient (pH) via the F0F1 ATPase is an important aspect of the ATR (van de Guchte et al., 2002). The ability to produce NH3 from arginine, via the deiminase pathway in many LAB, or from urea as occurs in Sc. thermophilus, may also be important in maintaining the pH gradient. These responses require protein synthesis. Numerous proteins are induced by heat in lactococci and enterococci and similar proteins are also produced during osmotic shock but very few of them have been characterized. Betaine and proline are often called ‘compatible solutes’ because they can be accumulated to high concentrations inside cells without affecting their physiology or metabolism. Lc. lactis grown under high osmotic strength contains high pools of proline or betaine without any apparent effect on the physiology of the cell (Molenaar et al., 1993). Betaine is taken up by a high affinity transport system while proline has a low affinity system which is repressed in complex media. Storage of cells is also receiving attention. Cells of Lc. lactis MG 1363 stored at 10 °C for 4 h showed a 100-fold increase in survival to freezing at 20 °C (Wouters et al., 1999). This was correlated with the synthesis of several cold-induced small (7 kDa) proteins. In another study, the flux of guanine and phosphate was implicated in the stress response of lactococci (Duwat et al., 1999). Cells of Lc. lactis transferred into a medium containing no glucose or limiting glucose remained viable (106 cfu/ml) for at least 1 year at 30 °C. However, considerable rearrangement of plasmids occurred during storage (Rallu et al., 2000). These findings may have potential for the storage of cultures for cheesemaking. Exopolysaccharide production

Smooth and creamy products, which are also low in fat and sugar, have considerable appeal for consumers who are conscious of their health. One way of improving the smooth texture of a product is to add suitable polysaccharides to the product during processing. Most of the polysaccharides used in food as thickeners and stabilizers are obtained from plants (starch and pectin) or different seaweeds (carrageenan, alginates). Except for xanthan, very few are produced by microorganisms. Exopolysaccharide production is an important characteristic of many LAB involved in the production of fermented milks. Producing cultures are generally considered to be ‘ropy’ and result in the thickening of the fermented milk. Such cultures are particularly important in Scandinavian countries, e.g., Langfi in


Sweden and Viili in Finland. The industrial application of EPS produced by LAB is hampered by low yields, typically 50–500 mg per litre, and efforts to improve yields by genetic engineering and by manipulation of culture conditions have been reviewed (Kleerebezem et al., 1999; Jolly et al., 2002). Lactic acid bacteria produce either homopolysaccharides, comprised only of fructose or glucose residues or heteropolysaccharides, which are comprised of repeating units of several different sugars including two or more of the following glucose, galactose, fructose and rhamnose (De Vuyst et al., 2001). They may be involved in a wide variety of biological functions, including prevention of desiccation, protection from environmental stresses, adherence to different surfaces, pathogenesis and symbioses ( Jolly et al., 2002). EPS-producing cultures have also been used to increase the moisture and improve the yield of low-fat Mozzarella cheese (Low et al., 1998; Perry et al., 1998). It is possible to increase EPS production by Sc. thermophilus by altering the levels of enzymes in carbohydrate metabolism, particularly phosphoglucomutase and UDP-glucose pyrophosphorylase (Levander et al., 2002). The biosynthesis of EPS has been studied in an EPS strain of Lc. lactis and a derivative carrying a plasmid encoding the EPS gene cluster (EPS strain) (Ramos et al., 2001). The concentration of UDP-glucose and UDP-galactose, the precursors of EPS, were significantly lower in the EPS strain than in the EPS strain, while the concentration of the UDP-N-acetylmuramoyl pentapeptide, which is part of the cell wall, was greater in the EPS strain, indicating that there is competition between EPS synthesis and cell growth. These data suggest that the production of EPS could be enhanced by increasing both the flux of the EPS precursors and the lipid carrier precursors. Response surface analysis of different fermentation conditions showed that the optimum temperature, pH and casitone concentration for EPS production by Lb. delbrueckii subsp. bulgaricus RR were 38 °C, 5 and 30 g/L, respectively. The actual yield, 354 mg EPS/L, was within the 95% confidence limit of the predicted yield (Kimmel et al., 1998). Oxygen, orotic acid and carbon source were also important for EPS production which was greatest during the stationary phase of growth in a chemically defined medium (Petry et al., 2000). Maximum production of EPS by Lb. helveticus occurred at pH 6.2 (Torino et al., 2001). A model describing the growth and EPS production by Sc. thermophilus LY03 has been developed and some evidence for EPS degradation has been found (Degeest and De Vuyst, 1999). This strain produces two heteropolysaccharides of the same monosaccharide compos-

ition (a 4:1 ratio of galactose and glucose) but different molecular masses. The levels of phosphoglucomutase, UDP-galactose 4-epimerase and UDP-glucose pyrophosphorylase correlated highly with EPS production (Degeest and De Vuyst, 2000). The production of EPS confers no intrinsic resistance to phage (Deveau et al., 2002).

Preparation of Starters Lactic starters must perform one of their technological functions (acid production) early in cheesemaking and a sufficient amount of a metabolically active culture must be used to inoculate cheese milk. Usually, the initial population of starters in cheese milk is around 1–5 106 cfu/ml at inoculation and reaches 1–10 108 cfu/ml when the curd is transferred to the moulds, typically 5–6 h later in the case of Cheddar cheese. In most cheeses, during this time, the pH must decrease from ⬃6.6 to 5.5; cells which are not fully active or are sub-lethally stressed at inoculation will show slower growth and consequently slower acid production, thus increasing the cheesemaking time. Except for natural starters, most cheese plants use cultures provided in one of several forms (liquid, frozen, freeze-dried) by specialized industries or institutions (see above). An outline of current systems for the preparation of starters is presented in Fig. 4. The traditional approach for the build-up of a starter culture for inoculation of cheese milk, requiring a number of steps from a small volume (1 ml or g) of stock culture to a large volume (100–1000 l) of bulk starter, is still in use. However, it is being replaced by the use of frozen or freeze-dried cultures for direct inoculation of bulk starter milk or of cheese milk directly, especially in small cheesemaking plants. While the traditional system based on multiple transfers is cheaper than the direct-to-vat system, it requires skilled personnel and facilities and increases the risk of contamination with phage. Aspects of the commercial production of starter cultures have been thoroughly reviewed by Whitehead et al. (1993) and Sandine (1996). Here, an overview of the issues related to the production, preservation and distribution of starter cultures by companies and of the preparation of starters at the cheese plant is presented. Propagation of starter cultures

The production of starter cultures requires careful selection of media and operating conditions to obtain optimum results in terms of final cell numbers, activity (prompt growth, reduced lag phase, suitable acid production, aroma production, proteolytic ability), stability upon storage and, in mixed cultures, composition of the


Frozen or freeze dried concentrated cultures

Cheese vat 0.1–2% v/v

Frozen or freeze-dried stock culures (1 g, 1 ml)

A Mother culture (RSM)

1% v/v

C

1% v/v

B

1% v/v D Intermediate or feeder culture (RSM)

E Bulk starter (RSM, starter media)

Frozen stocks Figure 4 Examples of the production of lactic starter cultures in a cheese plant. A number of steps are necessary to build up the bulk starter from frozen or freeze-dried stock culture. Duration and inoculum size for each step are variable, depending on the type of culture (mesophilic, thermophilic, mixed, defined) and the temperature of incubation. To reduce time for build-up of bulk starter and the risk of contamination, frozen or freeze-dried starter concentrates can be used to inoculate the bulk starter tank or the cheese vat. The elements of a typical bulk starter tank are shown: A, agitator; B, sterile air inlet/outlet with HEPA filters to prevent the access of phages during cooling and operation; C, ports for inoculation and addition of alkali for pH control; D, pH and temperature probes; E, jacket for circulation of water or steam. Digital or analogic controllers and printer/recorders for temperature and pH, and external alkali tanks are not shown.

starter. These, in turn, are influenced by several factors, including the presence of disturbing phage, medium composition and fermentation conditions (heat treatment, temperature and pH control during fermentation, duration of incubation, storage temperature, etc.). Although cheese milk was the traditional medium for the growth of starters in cheese plants, it has been replaced by pre-tested, antibiotic-free reconstituted skim milk (RSM) and by specially designed starter media, available from starter culture companies. The availability of pre-tested RSM allows better control of growth prior to inoculation of milk in the cheese vat. It can be reconstituted to a higher solid level than that of fresh milk, thus improving the buffering capacity and therefore the growth and activity of the culture. Doubling the concentration of solids in RSM from 8 to 16% usually results in a doubling of the number of viable cells (from 5–7 108 to 10–14 108 cfu/ml) with a higher final pH (from 4.5 to 4.7). A similar result can be obtained by increasing milk solids by ultrafiltration. The need to increase cell numbers and improve the activity and stability of the cultures has prompted the development of specially designed starter media. Although most starter media are milk- or whey-based

(whey permeate can also be used), they may contain a range of ingredients to improve starter growth, to control pH and to inhibit phage adsorption (Table 3) (Whitehead et al., 1993). Two of the most important issues in the design of starter media are phage and pH control. Measures for phage control are described in ‘Starter Cultures: Bacteriophage’, Volume 1. pH control is important for building up starter biomass, preventing acid stress and loss of activity, and controlling the ratio of species and strains in mixed cultures (Oberg and Broadbent, 1993; Whitehead et al., 1993; Sandine, 1996). While lactobacilli and leuconostocs are relatively acid tolerant, mesophilic and thermophilic cocci are rapidly inhibited when the pH falls below 5.5. Therefore, the rod:coccus ratio of thermophilic starters may be affected significantly by the pH and pH-course during incubation. pH control also allows complete consumption of the carbohydrate source and retention of viability during prolonged refrigerated storage of fully grown cultures (Sandine, 1996). Both internal and external pH control are used. Internal pH control is achieved by the use of soluble or insoluble buffering agents. Soluble buffers (phosphates) perform the dual role of pH control and phage


Table 3 Ingredients in starter media and their functions; the most commonly used ingredients are in boldface (adapted from Whitehead et al., 1993) Category

Ingredients

Function and typical concentration range

Carbohydrates

Lactose, glucose, maltose, sucrose

Nitrogen sources Growth factors

Milk proteins, whey proteins, casein hydrolysates, peptones Yeast extract, Corn Steep Liquor

Chelating agents

Phosphates, citrates

Antioxidants

Ascorbic acid, ferrous sulphate

Buffers and neutralizers

Insoluble, for internal buffering: Trimagnesium phosphate, calcium carbonate, encapsulated sodium carbonate. Soluble, for internal buffering: phosphates. Soluble, for external pH control: ammonia, potassium or sodium hydroxide

Energy and carbon source, 10–40 g/L. Glucose 1 g/L may be used to facilitate recovery of stressed cells Sources of amino acids, from 1 g/L for hydrolysates to 20–30 g/L for proteins and peptones Sources of amino acids, vitamins, nucleotides and minerals, 2.5–5 g/L Inhibition of phage adsorption by chelation of available Ca2, 7.5–20 g/L (under external pH control) Prevention of oxidative stress due to H2O2 production, 1 g/L Control of pH during fermentation to 5.5–6.5. Amount added is highly variable depending on carbohydrate concentration and target pH at the end of fermentation

inhibition but the concentration needed to control pH effectively can be inhibitory to same species and may even reduce cheese yield by chelating Ca2 in the cheese milk. Insoluble (calcium carbonate, trimagnesium phosphate) or encapsulated (sodium carbonate encapsulated in ethyl and methyl cellulose; Whitehead et al., 1993) buffers result in a better performance, with high cell numbers (up to 1010 cfu/ml) and prolonged stability (up to 10 days) on refrigerated storage. However, internal pH control is unsuitable if the pH of the culture must be maintained at a fixed value. pHcontrolled bulk starter tanks, fitted with sterilizable electrodes for pH measurement and computer control with automatic addition of alkali to control the pH at the desired set point are now readily available. The most common neutralizers used in external pH control are KOH, NH4OH and gaseous ammonia. NaOH and Na2CO3 are cheaper but some starters may be inhibited by high concentrations Na. Other process factors that affect starter growth and performance are the heat treatment of the growth medium, and the temperature and the duration of incubation. Time/temperature combinations using during heat treatment are much higher (80–90 °C for 10–30 min is typical; higher temperatures can be used in commercial production of thermophilic starters) than commercial pasteurization (72 °C, 16 s). Such conditions drastically reduce the microflora in the medium, ensure the destruction of phage, which are resistant to pasteurization, and reduce the redox potential, driving off oxygen and denaturing proteins, thus improving starter growth. The temperature and duration of incubation are highly dependent on the composition of the starter cul-

ture and on other practical considerations. The temperature of incubation may greatly affect starter composition in mixed cultures. Incubation at 18–21 °C is usually preferred for cultures of lactococci and leuconostocs, because both organisms have approximately the same growth rate in this temperature range, while lactococci grow faster at 30 °C. For thermophilic rod:coccus cultures, a compromise (42 °C) must be found between the optimum temperature of the moderately thermophilic Sc. thermophilus (37–39 °C) and that of the thermophilic Lb. delbrueckii subsp. bulgaricus and Lb. helveticus (45 °C), although symbiosis between culture components may overcome the problems caused by growth at sub-optimal temperatures (Oberg and Broadbent, 1993). In cultures without pH control, the temperature and duration of incubation may be highly related. In general, cultures should be refrigerated shortly after the beginning of the stationary phase of growth; this requirement is less critical when the carbohydrate source is exhausted and the pH is controlled. An active mesophilic culture reaches the stationary phase in milk media in 6–8 h at 30 °C and in 16–18 h at 18–21 °C; the latter combination is obviously more suitable for overnight incubation. Thermophilic cultures may reach the stationary phase in 6–8 h at 37 °C. Preservation and distribution of starter cultures

While stock cultures are usually stored at the cheese plant for only a limited time, the production and distribution of starter cultures on a commercial basis requires suitable means for the preservation and distribution of cultures in a highly active state. The cultures may be preserved by a variety of means (chilling of


liquid cultures, drying, freezing, freeze-drying) which expose the culture to a variety of sub-lethal and lethal stresses (van de Guchte et al., 2002) which negatively affect the vitality and activity (by sub-lethally damaging the cells, by selectively killing some components of the culture thus changing culture composition). Sub-lethally stressed cells need a longer lag phase to recover, which translates into the need for longer resuscitation. Historically, cultures have been produced and distributed in liquid form, in air-dried form (spray dried), as frozen cultures and freeze-dried cultures. The two latter means of preservation are used most widely in the starter industry today.

cryoprotective medium is usually practised. RSM (12–14% with added lactose) may be suitable as a cryoprotectant, but other agents can also be used, e.g., 5–15% glycerol, 5% sodium glutamate, 7% sucrose. Cultures should be cooled as rapidly as possible to below 60 °C. Storage temperature should be between 20 °C and 40 °C. Frozen cultures should be thawed as rapidly as possible to maximize survival. The need to maintain the culture frozen at all times makes frozen cultures less practical than freeze-dried cultures for dispatch to cheese plants. However, because of their high activity, frozen concentrated cultures (see below) are still preferred as the means for distributing starter cultures in some countries.

Liquid and air-dried cultures

Chilling of liquid cultures is the oldest method of preservation and distribution of cultures. CaCO3 (6 g/L) is usually added to milk to maintain a high pH and the cultures are stored at a low temperature (2–5 °C). Stability does not exceed 1 or 2 weeks and several transfers are needed to obtain an active culture. Although chilling is still used for daily distribution of some mixed cultures for the production of PDO cheese in Italy, it has been superseded by freezing and freeze-drying. Air- or vacuum-drying of cultures was used in the past to produce cultures in powdered forms (Sandine, 1996) but because of poor vitality and activity this means of preservation is not used any more. Spray-drying is a fast and economic method for removing water but cultures are exposed to a variety of stresses (heat, dessiccation, oxidation), and survival and activity are usually low (Teixeira et al., 1995; To and Etzel, 1997). Frozen cultures

Freezing at a very low temperature ( 80 °C, 196 °C) in the presence of cryoprotective agents is the best way for preserving the vitality and activity of bacteria, and freezing is a preliminary step in the production of freezedried cultures. Several factors affect the survival of LAB during freezing and their activity after thawing, e.g., species, strain, growth medium composition, culture conditions, growth phase, composition of the medium used for suspending the cells during freezing, type and concentration of the cryoprotective agent, temperature and rate of freezing, storage temperature, temperature and rate of thawing (Sandine, 1996). To obtain high cell densities prior to freezing, cells are grown under pHcontrol. Stationary cells are more resistant to freezing than exponentially growing cells. Mesophilic and thermophilic cocci are more resistant than thermophilic lactobacilli and leuconostocs; therefore, care should be exercised in the freezing of mixed cultures to maintain the correct strain balance. Removal of the cells from the growth medium and their suspension in a suitable

Freeze-dried cultures

While removing water at ambient temperature is detrimental to the survival and activity of starter cultures, freeze-drying, i.e., removing water from a frozen culture by sublimation under high vacuum, results in high levels of survival. Freeze-drying has been used for the preparation of dairy starters for about a century (Sandine, 1996). Freeze-dried stocks containing 108–109 cfu/g are dispatched to cheese factories in vials, serum bottles or pouches containing a few grams of powder and need several transfers for full reactivation. The procedure for the preparation of freeze-dried cultures is similar to that used for the preparation of frozen cultures up to the freezing step, although addition of antioxidants like ascorbic acid, together with cryoprotective agents, is common. Cultures are frozen rapidly in vials connected to manifolds or in trays and dessiccated under high vacuum (10 Pa) for 12–24 h, to a final aW of 0.1. Vials can be closed under vacuum, while powders lyophilized in trays are aseptically packaged in a variety of containers under an inert atmosphere, since dried cells are highly sensitive to oxidative stress. Freeze-dried cultures can be dispatched and stored at ambient temperature, although survival and activity are improved by storage at 4 °C or 20 °C. Concentrated starter cultures

Conventional frozen and freeze-dried cultures do not contain enough cells for inoculation of the bulk starter tank or the cheese milk and therefore several transfers are needed to build up sufficient inoculum for the bulk starter. Frozen and freeze-dried concentrated starters, typically containing 1010–1011 cfu/g and 1011–1012 cfu/g, respectively, for inoculation of the bulk starter (also known as bulk sets) or the cheese milk (direct-to-vat cultures, direct vat set cultures) are now readily available from starter companies, and are widely used in both small and large plants. Although concentrated starters are more expensive than starter build-up from


a stock culture, the use of concentrated starters improves plant flexibility (because of reduction or elimination of the time needed for building up the bulk starter), reduces or eliminates the need for skilled personnel and equipment for the production of starters and reduces the risk of phage contamination in the factory. Starters are grown in pH-controlled milk- or wheybased media and concentrated by bactofugation or by microfiltration. When milk media are used, citrate (1%) is used to solubilize the milk proteins, even when pH control is used. Cells are then resuspended in a suitable medium, containing cryoprotectants and antioxidants, rapidly frozen or frozen and freeze-dried. Frozen concentrated cultures may be packaged directly or frozen in pellet form and then packaged. Freeze-dried concentrated cultures are lyophylized in large tray freeze-dryers and then packaged under vacuum or a nitrogen atmopherein amounts suitable for inoculation of 500–1000 l of bulk starter medium or 1000–5000 l of cheese milk. Frozen concentrates are partially thawed by putting the container in chlorinated (25–50 mg/kg) water at room temperature for 20 min before adding it to milk, where thawing is completed in 15–30 min. Freezedried starters can be added directly to the bulk starter tank or cheese vat, although rehydratation in a small volume of milk is advisable to improve distribution. Because of their high activity, frozen concentrated cultures for direct-vat inoculation do not significantly increase cheesemaking time, and are widely used in the USA and Australia. On the other hand, sub-lethal damage caused by freeze-drying may increase cheesemaking time by 0.5–1 h (Sandine, 1996); however, this disadvantage may be offset by the fact that dispatching and handling of freeze-dried starters is greatly simplified, and freeze-dried concentrates are more widely used in Europe. The choice between concentrated cultures for bulk starter inoculation and those for direct vat inoculation (DVI) may depend ultimately on considerations related to costs and ease and flexibility of use. A comparison of costs for a cheese plant processing about 10 000 tonne/year of cheese is presented in the web site of Australian Starter Cultures Research Centre (http://www.ascrc.com.au/strategy.html); the cost of concentrates for bulk starter inoculation is estimated at US$20/tonne of cheese, compared to US$47/tonne of cheese for cultures for DVI. However, recently aggresive marketing by the culture suppliers has reduced the price of DVI culture to US$24 per tonne of cheese though the cost is higher for smaller cheese plants. Concentrated starter cultures are now the preferred way for the distribution and use of starter cultures and a wide selection of species, strains and combinations is available. A list of websites of some companies and

institutions providing on-line catalogs of starter cultures includes: Chr.Hansen http://www.chr-hansen.com/ Danlac http://www.danlac.com/starter-cultures.shtml Rhodia Dairy http://www.rhodiadairy.com/products/ Swiss Federal Dairy Research Station http://www.sar. admin.ch/ Development of concentrated starters implies higher R&D and production costs, which are reflected in the price of the culture; moreover, not all species and strains are suitable for the production of concentrated starters for DVI, and the wide diffusion of cultures for direct vat has also reduced the diversity of cultures available for cheesemaking.

Acknowledgement We are grateful to Ian Powell for the information he provided and for useful discussion.

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Starter Cultures: Genetics M.J. Callanan and R.P. Ross, Teagasc, Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Ireland

Introduction Within the last three decades there has been intensive study of the genetics of starter bacteria, ranging from plasmid biology to genetic tool development, and leading ultimately to elucidation of the complete genome. This has facilitated such advances as the metabolic engineering of these commercially important bacteria and to the improvement of particularly significant industrial traits, such as increased bacteriophage resistance. The purpose of this review is to discuss some of the major advances which have occurred during this time, culminating in the elucidation of the genomes of several strains. It is worth emphasising that while the chromosomes of a number of strains have been characterised in detail, many commercially significant traits are encoded on mobilisable elements such as plasmids and transposons. In fact, many industrially important strains have rich plasmid complements, and it could be argued that much strain ‘individuality’, in terms of industrial performance, could be attributed to their plasmid genomes. The development of genetic tools for strain improvement, in combination with genomic and metabolomic technologies have opened new possibilities for the routing or re-routing of metabolism towards desirable metabolites such as flavour compounds, e.g., diacetyl and vitamins, e.g., folate. This, together with the huge array of as yet un-mined sequence information should lead to the development of new and improved starter strains for food production.

Genetics of Mesophilic Starters Starter cultures used by the dairy industry can be broadly divided into two types, mesophilic and thermophilic, based on their optimum growth temperature (see ‘Starter Cultures: General Aspects’, Volume 1). Mesophilic starters have a growth optimum of ⬃30 °C and are used in the production of Cheddar, Gouda, Edam, Blue and Camembert cheeses. Only three species of the lactic acid bacteria (LAB) group employed as commercial starters are considered mesophilic, Lactococcus lactis subsp. lactis, Lc. lactis subsp. cremoris and Leuconostoc mesenteroides subsp.

cremoris. Of all starter bacteria, Lc. lactis subsp. lactis IL1403 and Lc. lactis subsp. cremoris MG1363 are the most thoroughly investigated at the genetic level and have been the workhorses for studying starter cultures. The construction of these two plasmid-free strains from cheese starter parents in the early 1980s was instrumental in their selection for further study. Chromosome

The sequencing of the Lc. lactis subsp. lactis IL1403 genome has provided the first comprehensive insight into the genetics of lactococcal starters. Strain IL1403 is a derivative of the Streptococcus (now Lactococcus) lactis strain IL594, isolated from a cheese starter culture (Chopin et al., 1984). The 2365-kb circular chromosome has a G C content of 35.4% (Fig. 1) and encodes 2310 open reading frames (ORFs) (Bolotin et al., 2001). This genome is relatively small when compared to other bacterial genomes, such as Bacillus subtilis (4214 kb, 4099 ORFs), and most likely reflects the specialised adaptation of Lactococcus to growth in the nutrient-rich milk environment. As expected, many genes required for de novo synthesis of essential nutrients and the degradation of complex molecules are absent from the IL1403 genome. Similarly, the machinery that controls gene expression differs from the environmentally responsive Bacillus. The proportion of transcriptional regulators is similar in the two bacteria but IL1403 encodes much fewer genes that respond to changing environmental conditions. B. subtilis encodes 18 sigma factors and 34 twocomponent systems compared with 3 sigma factors and 8 two-component systems in the lactococcal chromosome. There were also a few unexpected revelations from the annotated genome sequence. For example, strain IL1403 requires a number of amino acids (isoleucine, valine, leucine, histidine, methionine and glutamic acid) and vitamins (folic acid, menaquinone, riboflavin and thioredoxin) for growth in defined media despite having the genetic potential to synthesise all these necessary growth factors (Bolotin et al., 2001). The genes in the amino acid biosynthetic operons appear to have mutations that may be specific to laboratory strains. In addition, the ability of



150 Starter Cultures: Genetics

Figure 1 Schematic representation of the L. lactis IL1403 genome. The figure highlights the nucleotide composition of the chromosome and demonstrates the gross organisation of the entire genome into two divergent blocks arranged around the replication initiation (0 kb) and termination (c. 1250 kb) sites. The figure (available at www.cbs.dtu.dk) was generated using Genome Atlas (Jensen et al., 1999; Pedersen et al., 2000).

Lactococcus to respire aerobically has remained controversial but the presence in the genome of the genes for the biogenesis of menaquinone and cytochrome d agrees with data indicating the respiratory capacity of an organism considered to be exclusively fermentative (Gaudu et al., 2002). A recent examination of the genetic organisation of the two reference lactococci, the partially sequenced MG1363 and the completely sequenced IL1403, revealed an average of only 85% identity at the DNA level (Campo et al., 2002). There is, however, a high degree of conserved gene organisation, or synteny. In particular, the order is conserved in the oriC region of the two genomes but not in the vicinity of the mobile element-rich replication terminus. Overall, about 9.2% of the Lc. lactis genome is formed by insertion elements and prophages, and this percentage reflects the contribution of horizontal gene transfer to the composition of the genome. For example, there is a large chromosomal inversion between MG1363 and IL1403 that covers nearly 50% of the genome and contains the six ribosomal RNA operons (Le Bourgeois

et al., 1995). This plasticity of the Lactococcus genome may be an important factor in optimising the genome in response to the nutrient-rich milk environment. Moreover, as noted by Campo et al. (2002), the comparative genomics of the two strains ‘demonstrates that the IL1403 genetic content is probably not representative for the content of all lactococcal subspecies’. This information is important in extrapolating from the IL1403 sequence data to other starter lactococci. Bolotin et al. (2001) commented on the high level of apparent non-functioning genes for amino acid biosynthesis which may be due to the treatments used to cure the parent of the laboratory strain of its plasmids. The accumulation of further genomic data on other strains will be necessary to establish the degree of conservation between laboratory strains and current industrial mesophilic starter cultures. Plasmids

The genetic investigation of starter cultures was effectively initiated by McKay and co-workers at the

Starter Cultures: Genetics 151

University of Minnesota with the demonstration that the lactococci contain plasmids that encode traits essential in the cheesemaking process (McKay and Baldwin, 1975). In addition to the genetic information encoded by the chromosome, Lc. lactis species harbour numerous large cryptic plasmids. The presence of these plasmids helped explain the well-known and problematic instability of key industrial traits. A rapid and reliable method was subsequently developed that allowed the plasmids to be isolated and visualised using agarose gels (Anderson and McKay, 1983). Plasmid profiling remains an important method of differentiating lactococcal starter strains, although their instability precludes the plasmids from providing a permanent fingerprint of strain identity. Indeed, the plasmid content of strains can vary dramatically during long-term storage. It was also quickly recognised that it is possible to mobilise a number of the plasmids via conjugation. Therefore, the enzymes necessary to hydrolyse casein, transport and metabolise lactose and citrate, produce bacteriocins and combat phage could be transferred among industrially important strains. The commercial importance of the plasmidencoded traits has resulted in numerous studies, and some of the plasmids have been partially or even completely sequenced (Dougherty et al., 1998; van Kranenburg et al., 2000; Boucher et al., 2001). However, the physiological role of the majority of these extra-chromosomal elements is yet to be defined fully. In addition to encoding key industrial traits, the native lactococcal plasmids have provided the necessary material for the development of genetic tools required to manipulate Lc. lactis. The original lactococcal cloning vectors, such as pSA3 (Dao and Ferretti, 1985), were ‘shuttle’ vectors and encoded two replication origins in order to maintain the plasmid in both lactococcal and E. coli hosts. The ability to replicate in E. coli was necessary to expose the plasmid to the highly advanced genetic techniques available in that background. Subsequent vectors have been based primarily on more promiscuous replication origins, such as those from the cryptic lactococcal plasmids, pWV01 and pSH71 (de Vos and Simons, 1994). Combined with an antibiotic selection marker, these replicons are stable in Gram-positive and Gram-negative hosts and continue to form the backbone of the most useful lactococcal vectors. Genetics of industrially important traits Lactose and citrate metabolism

The primary function of Lactococcus in an industrial dairy fermentation is to produce lactic acid. The bacteria synthesise this flavoursome and spoilage-preventing acid as a product of the fermentative conversion of lac-

tose. In milk, the disaccharide lactose acts as the carbohydrate source. The genetics of lactose utilisation by lactococcal starters has been well-documented (de Vos and Vaughan, 1994). The plasmid-encoded lac operon consists of nine genes in the order lacABCDFEGX. They are responsible for the transport and incorporation of the galactose and glucose constituents of lactose into the Embden-Meyerhof-Parnas pathway (Fig. 2). Transport occurs via the lactose-specific phosphotransferase system encoded by lacEF. The operon is directly regulated at the transcriptional level by the product of the divergently transcribed lacR gene that represses the pathway in the presence of glucose. A second, more global, mechanism of regulation also appears to act in the absence of lacR (de Vos and Simons, 1994). Citrate metabolism contributes the distinctive diacetyl/acetate flavour and aroma to products manufactured with citrate utilising (Cit) lactococci. It is also required for CO2 production resulting in eye formation in Dutch-type cheese. The ability to metabolise the relatively low concentration of citrate in milk (8 mM) is primarily dependent on the presence of citrate permease (Fig. 2). The gene for the permease, citP, is part of the plasmid-encoded citBRP operon. The transport genes are not induced by citrate but are induced under conditions of lactic acid stress (Garcia-Quintans et al., 1998) reflecting the role of citrate in relieving growth inhibition by lactate (Magni et al., 1999). Following transport, citrate is cleaved to produce oxalacetate and release acetate. Oxalacetate is converted to diacetyl, acetoin and CO2 through pyruvate as an intermediary. The distinctive nature of citrate metabolism has prompted a number of studies aimed at manipulating the pathway in order to increase the concentration of desirable end products, e.g., diacetyl. One approach has been to manipulate the citrate metabolism pathway of Cit Lc. lactis through genetic techniques such as gene inactivation and over-expression (Fig. 2). Inactivation of lactate dehydrogenase, the enzyme responsible for the production of lactic acid from pyruvate, resulted in increased acetoin production (Snoep et al., 1992). However, to efficiently convert sugars to diacetyl, inactivation of -acetolactate decarboxylase (ALDB) and over-expression of NADH-oxidase are required (Hugenholtz et al., 2000). The NADH-oxidase overproduction causes the rerouting of pyruvate through NADHindependent pathways leading to diacetyl and acetoin since the cell no longer needs to regenerate NAD through lactate dehydrogenase. Inactivation of aldB ensures that the rerouted pyruvate is converted to diacetyl and not acetoin, the product of the decarboxylase. These manipulations resulted in a strain with an increased capacity for diacetyl production. These types of metabolic engineering studies will be further enabled


Lactose Cell wall Cell membrane

Transporter

lac genes Glucose

Galactose

galKTE

2,3-butanediol Glucose-6-P Acetoin aldB Diacetyl

α–acetolactate Pyruvate Acetate ethanol

Oxaloacetate

ldh Citrate lysase Lactate

Permease (citP)

Citrate

Figure 2 Schematic of lactose (closed arrows) and citrate (open arrows) metabolism in LAB. Genes inactivated as part of metabolic engineering strategies for overproducing diacetyl are indicated (see text for detail).

by our increasing knowledge of lactococcal metabolic pathways from genome sequence analysis. Proteolysis and amino acid catabolism

The lactococci are fastidious organisms specially adapted to grow in the nutrient-rich growth medium that is milk. Lactose provides a readily utilisable sugar source for energy whereas degradation of the principal milk protein, casein, provides nitrogen sources and supplies the amino acids for protein synthesis. Casein breakdown is initiated by a cell envelope-associated proteinase (CEP). The proteinase gene is another of the key industrial traits located on the indigenous lactococcal plasmids. The multi-domain CEPs are large proteins (more than 1800 amino acids are encoded by the gene) and are classified based on their caseinolytic specificity (Kunji et al., 1996). The plasmid-encoded genes have been sequenced for a number of lactococcal strains and the sequence data exploited to investigate the catalytic properties of these important enzymes. Site-directed and cassette mutagenesis techniques have

identified the substrate-binding domain and active site residues (Siezen et al., 1993). The peptides produced by the proteinase are transported into the cell by three systems. The Opp system is responsible for oligopeptides of 4–18 residues, while the DtpT and DtpP transport hydrophilic and hydrophobic di- and tripeptides, respectively (Kunji et al., 1996). Both Opp and DtpP are multi-gene systems organised in operons. In contrast, the proton motive force-driven DtpT transporter is encoded by a single gene (Fang et al., 2000). Once inside the cell, the peptides are hydrolysed to their constitutive amino acids by a set of peptidases with various specificities. The genes for 13 peptidases have been described for the IL1403 sequence, a number of which had been studied previously (Christensen et al., 1999). Of particular interest are the aminopeptidases, PepN and PepC, and the proline-specific peptidases. PepN and PepC display low substrate specificity and free amino acids by cleaving the N-terminal end of oligopeptides. Casein is a proline-rich substrate and lactococci have multiple peptidases with


activity against various proline-containing peptides. The genes for X-prolyl-dipeptidyl aminopeptidase (pepX), prolidase (pepQ), proline iminopeptidase (pepI) and aminopeptidase P (pepP) are present on the Lc. lactis genome. In addition, a number of peptidases shown to cleave internal peptide bonds (endopeptidases) and enzymes that hydrolyse di- and tripeptides have been described. While inactivation of the individual peptidase genes described above did not eliminate the ability of Lactococcus to grow in milk, mutants exhibit reduced growth rates. Moreover, strains in which multiple peptidase genes were disrupted had significantly reduced growth rates (Mierau et al., 1996). Recently, a comprehensive study on the regulation of the proteolytic system was performed in MG1363 and some interesting observations were made (Guedon et al., 2001a). The transcription of 16 genes encoding 12 peptidases, 2 CEPs and the 3 transport systems were analysed in response to various environmental factors. It was found that neither the sugar source nor temperature effected transcription with the sole exception of pepP, which is modified by sugar. Transcription of the 2 CEP genes, 3 aminopeptidase genes (pepX, pepN and pepC) and the Opp transport system operon were all regulated by the peptide content of the medium. The remaining genes, that appeared to be unregulated, were expressed at lower levels and it was suggested that they probably encode enzymes involved in cellular functions other than peptide utilisation (Guedon et al., 2001a). In a companion study, a transcriptional repressor with homology to the CodY regulator of Bacillus subtilis was shown to regulate expression of the genes that were sensitive to the peptide content of the medium (Guedon et al., 2001b). The catabolism of amino acids in Lactococcus has received significantly less attention than the proteolytic system (see ‘Catabolism of Amino Acids in Cheese during Ripening’, Volume 1). The ability to overcome amino acid auxotrophy through degradation of casein is well documented but the ability to synthesise various amino acids has not been investigated thoroughly. In particular, very little work has focused on the genetic components involved. The availability of the genome sequence will redress this situation but at present the majority of the studies have focused on detecting specific enzyme activities associated with the catabolic pathways in various strains (reviewed by Christensen et al., 1999). The catabolism of amino acids by lactococci is likely to have an essential role in the development of cheese flavour and aroma. Inactivation of an aromatic amino acid aminotransferase gene, araT, of Lc. lactis NCDO 763 has already confirmed the involvement of this enzyme in the conversion of

amino acids to aroma compounds (Yvon et al., 2000). Two further aminotransferases have been cloned and sequenced from Lc. lactis LM0230 (Atiles et al., 2000; Dudley and Steele, 2001) but much more work is needed. Bacteriocins

It is well established that starter bacteria produce a range of substances, including lactic acid and metabolites which aid in the preservation, and contribute to the safety of many fermented food products (Klaenhammer, 1988; Holzapfel et al., 1995). In addition to the production of antimicrobial metabolites such as lactate, almost all the different representative species have been reported to produce antimicrobial peptides and/or proteins which are collectively referred to as bacteriocins. Of the many bacteriocins isolated and studied to date, nisin is the only one whose commercial potential has been realised. Nisin was assessed to be safe for food use by the Joint Food and Agriculture Organisation/World Health Organisation (FAO/WHO) Expert Committee on Food Additives in 1969, and is in use in more than 48 countries. In the dairy industry, nisin is exploited mainly for the prevention of clostridial growth in processed cheese, dairy desserts and cheese spreads. Nisin has been the subject of a wide variety of fundamental studies as to its structure and genetics (Dutton et al., 2002). It is classified as an antibiotic peptide, a term used to describe a heterogeneous group of lanthioninecontaining bacteriocins that undergo extensive posttranslational modification, and has recently been shown to exhibit inhibitory activity at nanomolar concentrations (Wiedemann et al., 2001). A number of genes are involved in the production and export of, and immunity to, nisin (Rodriguez and Dodd, 1996). These genes are tightly linked in the nisin cluster, composing a total of 11 genes of which nisA encodes the nisin precursor itself. Interestingly, the genes responsible for nisin A production and immunity are carried on a 70 kb conjugative transposon called Tn5301 from Lc. lactis NCFB894 (Dodd et al., 1990) or Tn 5276 from Lc. lactis NIZO R5 (Rauch and de Vos, 1992) while the genetic determinants for nisin Z (a natural variant of nisin, in which the histidine at position 27 is replaced by asparagine) are on transposon Tn5278 (Immonen et al., 1995). Nisin synthesis is regulated by a two-component regulatory system made up of the membrane-bound histidine kinase sensor protein, NisK, and the regulator, NisR (Fig. 3). This regulatory system responds to extracellular nisin, which leads to the expression of genes involved in immunity and synthesis/post-translational modification (Kuipers et al., 1995).


nisK

nisR Regulated gene expression

Nisin induction NisK

Pi

Protein X

NisR

Signal transduction

NisR

Nisin gene X

Figure 3 Protein expression using the nisin-inducible expression system (NICE).

Another lactococcal bacteriocin which has received considerable attention in recent years is lacticin 3147, produced by Lc. lactis strain DPC3147, which was originally isolated from an Irish kefir grain (Ryan et al., 1996). The bacteriocin is composed of two post-translationally modified peptides, both of which are required for optimal killing activity (Ryan et al., 1999). Lacticin 3147 has a very broad spectrum of action which includes all Gram-positive bacteria tested, including food pathogens such as Listeria monocytogenes and Staphylococcus aureus and food spoilage micro-organisms such as Clostridium tyrobutyricum (Ryan et al., 1996; Galvin et al., 1999). The native lactococcal plasmid, pMRC01, encoding lacticin 3147 production and immunity has been sequenced completely (Dougherty et al., 1998). Ten genes are involved in lacticin 3147 production and immunity and are expressed from divergent promoters which control two clusters, namely ltnA1A2M1TM2D and ltnRIFE. The putative functions of some of these genes have been confirmed using a series of knock-out deletions in single or multiple genes. Investigation of the regulation of the lacticin 3147 gene revealed that the promoter controlling biosynthesis (Pbac) appears to be constitutive (McAuliffe et al., 2001). Characterisation of a downstream region revealed a stem-loop structure within the ltnM1 gene which may act as a rho-independent terminator, functioning as a signal for processing of the ltnA1A2M1TM2D transcript. Furthermore, the promoter of the ltnRIFE operon (Pimm) was shown to be regulated by the repressor LtnR. A very useful general feature of the bacteriocins of starter cultures is that they are frequently encoded on mobilisable genetic elements. This has been advantageous for the transfer of nisin to different starters.

However, a general feature of Nis strains is that they are not rapid acidifiers of milk, a quality necessary for successful cheese manufacture. The genetic determinants for lactacin 3147 can also be readily transferred between strains. In this case, the 60 kb self-transmissible pMRC01 plasmid-encoding lacticin 3147 has been transferred to more than 25 different lactococcal hosts, many of which are commonly used lactococcal starters in the cheese industry (Coakley et al., 1997; O’Sullivan et al., 1998; Fenelon et al., 1999). The transconjugants are lacticin 3147 producers and can be substituted for the parent strains in commercial applications. The genetics of bacteriocins, such as lacticin 3147 and nisin, has also become important tools for the manipulation of starter bacteria. The best example of this is the generation of the nisin-controlled expression (NICE) system (de Ruyter et al., 1996). It is based on the promoter for the structural gene for the antimicrobial peptide, nisin (Fig. 3). The nisA promoter is autoregulated in response to nisin through the nisPR gene products. The system is very sensitive to nisin concentration and by varying the nisin level in the medium, very subtle control of genes cloned downstream of the nisA promoter can be achieved. Bacteriophage

The interaction of starters and bacteriophage is covered elsewhere (see ‘Starter Cultures: Bacteriophage’, Volume 1) but one particular aspect relates specifically to the genetics of starter cultures, namely the effects of prophage and other phage sequences in the bacterial chromosome. Phage-related starter failure remains a problem in the dairy industry, and recent studies indicate that the chromosomal phage sequences contribute


to the evolution of new phage (Bouchard and Moineau, 2000; Durmaz and Klaenhammer, 2000). The rapid appearance of new recombinant phage is a constant problem in combating phage infections. The new phage can bypass the natural phage defence mechanisms employed to protect the starter cultures. Durmaz and Klaenhammer (2000) demonstrated that the appearance of recombinant phages is dependent on chromosomally encoded phage sequences. Therefore, sequences encoded by the lactococcal chromosome may have an injurious effect on the strain in the cheesemaking environment. However, from an evolutionary point, chromosomal phage DNA must confer some advantage or the lysogenic cells would not be maintained in the population. An obvious advantage is the exclusion from super-infection. Another is the potential of the mobile phage to exchange beneficial genes between strains. It is tempting to assume that lactococcal strains without prophage would be more suitable for dairy fermentation processes but this may not be the case. Genetic manipulation

The genetic tools now available allow very sophisticated manipulation of Lactococcus strains. Reliable gene inactivation and expression systems are available. The tools have evolved over three decades of research and reflect the investment in Lactococcus genetics. One of the key factors in the development of the genetic tools was the discovery of efficient transformation protocols for lactococci (Holo and Nes, 1995). The potential of electroporation to introduce DNA into eukaryotic cells was quickly adopted for bacteria. High-voltage electric field pulses result in the permeabilisation of the cell membrane allowing the transient passage of macromolecules into the cell. Other methods for mobilising DNA, such as conjugation and transduction, were crucial in the early development of lactococcal genetics (reviewed by Gasson and Fitzgerald, 1994) but the relative simplicity and reproducibility of electroporation has made it the mechanism of choice for genetic studies. In the case of the indigenous large plasmids, conjugation remains the most efficient mechanism of transfer. The procedure requires that the naturally occurring plasmid possesses a suitable selectable marker for the transconjugant. Plasmidlinked phage resistance has been used in numerous conjugations since the first successful report of improved resistance to a homologous phage as the selection basis (Klaenhammer and Sanozky, 1985). The presence of lactose-fermenting determinants offers an alternative naturally occurring selectable marker. However, interference with the natural lactose-fermenting

capability of the starter in order to select for the introduced plasmid has diminished the usefulness of this approach for strain improvement. As noted above with lacticin 3147, bacteriocin production and immunity have proven to be other suitable targets for selection in natural conjugations. In order to genetically investigate any organism, the tools for cloning and manipulating the genes of the organism must be available. The plasmid vectors currently used to clone lactococcal DNA have evolved from shuttle vectors incorporating non-lactococcal origins to plasmids derived from indigenous lactococcal plasmids. These plasmids have been adapted further as integration vectors encoding expression systems. The isolation of a temperature-sensitive (Ts) pWV01 origin of replication was instrumental in the development of an efficient gene inactivation system for lactococci (Law et al., 1995). An elegant two-plasmid system was developed whereby a fragment of the gene of interest is cloned on a vector encoding the wild-type pWV01 origin of replication, an antibiotic selection marker but no replication protein. The replication protein is supplied by an addition plasmid encoding the Ts version of the pWV01 origin. Once the strain is shifted to a non-permissive temperature, the plasmid encoding the replication protein is lost. The plasmid encoding the homologous DNA is forced to integrate when antibiotic selective pressure is maintained. The system has been employed successfully to generate not only chromosomal mutations but has recently been adapted to study genes encoded by the native plasmids (Cotter et al., 2003). With the availability of sophisticated genetic tools, complex metabolic engineering of Lactococcus is feasible and attractive due to the largely independent catabolic and anabolic pathways in Lactococcus (Hols et al., 1999). A number of innovative studies have demonstrated the potential to manipulate the metabolic pathways of Lactococcus and exploit these bacteria as cell factories (Hugenholtz and Smid, 2002). However, with respect to lactococcal starter cultures, their direct incorporation into food products means that the recombinant DNA technologies required for metabolic engineering cannot be used in strain development. New food-grade technologies are being developed but for the foreseeable future traditional mutagenesis and natural selection of mutants with high throughput screening facilities hold more potential for the development of improved starter bacteria. Leuconostoc

Leuconostocs are heterofermentative LAB that function as starters for fermented dairy products only in association with lactococci. Their major role is to


metabolise citrate to CO2 (eye formation) and diacetyl, an important flavour component of cultured buttermilk, cottage cheese, sour cream and ripened cream butter. As yet, the genetics of Leuconostoc is at an early stage and the importance of citrate metabolism has provided the focus for most of the genetic studies of Leuconostoc metabolism. Like their mesophilic partners, the lactococci, the technologically important genes encoding citrate and lactose utilisation are plasmid-encoded. The gene encoding the citrate permease, citP, shares almost complete identity with the gene from Lactococcus (Vaughan et al., 1995). This level of homology suggests that the gene may have been acquired by recent horizontal transfer between the two LAB species. The regulation of the citrate transport genes encoded by the citMCDEFGRP multienzymatic complex has also been investigated in L. paramesenteroides (Martin et al., 2000). The results demonstrated that the operon was induced by citrate independently of the pH of the growth medium and that a divergently transcribed gene, citI, upstream of the operon is involved in regulation.

Genetics of Thermophilic Starters The cultures that are regarded as thermophilic starters consist of bacteria with an optimum growth temperature of ⬃45 °C. The ability to tolerate higher temperatures is related to their use in the manufacture of Swiss and Italian cheeses, that are cooked to a much higher temperature (50–55 °C), and yoghurt. There has been a rapid growth in recent years of genetic information regarding the bacteria that constitute the thermophilic starter cultures, namely Streptococcus thermophilus, Lb. delbrueckii subsp. bulgaricus, Lb. delbrueckii subsp. lactis and Lb. helveticus. Lactobacillus

Many Lactobacillus species are used in the dairy industry and some have been subject to detailed investigation. In fact, the quantity of genetic information regarding the various lactobacilli is superseded only by Lactococcus. In addition, the probiotic potential of many Lactobacillus species has accelerated the accumulation of genetic data. However, most of the lactobacilli are used as adjuncts and for the purposes of this review only the Lb. delbrueckii subspecies and Lb. helveticus will be discussed in detail. Chromosome

The genomes of many lactobacilli are currently being sequenced including that of Lb. delbrueckii and Lb. helveticus strains. The analysis of one Lactobacillus genome, Lb. plantarum WCSF1, has been completed

and published (Kleerebezem et al., 2003). Lb. plantarum differs from the thermophilic starters in its flexible and adaptive behaviour and is encountered in many different environmental niches, ranging from some dairy fermentations to the human gastrointestinal tract. The genes encoding the genetic machinery for growth in milk, including sugar transporters, EMP and phosphoketolase pathways and peptidase were identified although no proteinase homologue was found. A relatively large number of pyruvate-dissipating enzymes with a remarkable degree of redundancy were observed. Lc. lactis also displays some redundancy especially in its lactate dehydrogenases but the pyruvate-dissipating potential in Lb. plantarum is much greater. However, by far the largest class of proteins in the large (3.3 Mb) genome is represented by transport proteins, including many PTSs for sugar uptake. A large number of PTS systems have already been reported for Lb. casei (Klaenhammer et al., 2002) and it will be interesting to determine the range of pyruvate-dissipating enzymes and transport mechanisms encoded by the smaller (1.8–2.4 Mb) genomes of the strains that remain primarily associated with milk fermentation. Important traits

The genes for lactose utilisation in Lb. delbrueckii and Lb. helveticus comprise a lactose antiport permease (lacS), a regulatory gene (lacR) and a -galactosidase for hydrolysis of lactose to glucose and galactose. In Lb. helveticus, the lacLM genes encoding -galactosidase are divergently transcribed from lacR and lacS, which are separated by 2 kb of DNA (Fortina et al., 2003). Transcription studies confirmed the regulatory role of LacR. In Lb. delbrueckii subsp. lactis and Lb. delbrueckii subsp. bulgaricus, the -galactosidase (lacZ) and permease occur in the order lacSZ. Recent investigation of the regulation of the lactose operon in Lb. delbrueckii has resulted in some interesting observations (Lapierre et al., 2002). The lacSZ genes in Lb. delbrueckii subsp. lactis are regulated by lacR, whereas the L. delbrueckii subsp. bulgaricus, genes were known to be constitutive and unstable due to the presence of insertion elements. Comparison of the lactose metabolism systems of both species revealed that the presence of the insertion elements alone was not sufficient to deregulate the operon, and mutation in the lacR gene must have occurred to make expression constitutive in Lb. delbrueckii subsp. bulgaricus. The proteolytic system of the thermophilic lactobacilli has been investigated extensively. Cell envelopeassociated proteinases have been identified in both Lb. delbrueckii and Lb. helveticus, and Lb. helveticus CNRZ32 may encode more than one proteinase (Pederson et al., 1999). In addition, 18 peptidases from thermophilic


lactobacilli have been described (Christensen et al., 1999). The mechanism of regulation has not been determined for the peptidase genes with the exception of pepQ. A homologue of the CcpA (Central regulator of C metabolism) catabolite regulator has been found upstream of all LAB pepQ genes investigated and shown to regulate the expression of pepQ in Lb. delbrueckii subsp. lactis.

a third strain, LMD-9, is near completion (Klaenhammer et al., 2002). Both completed genomes contain ⬃1.8 mb of sequence encoding about 1800 open reading frames. They are organised as a single circular chromosome and show 95% identity at the nucleotide level. Analysis of the sequence also revealed several metabolic features found in common with pathogenic streptococci even though Sc. thermophilus is characterised as a GRAS (Generally Regarded As Safe) organism.

Genetic manipulation

The genetic interrogation of Lb. helveticus strains is far advanced than Lb. delbrueckii. A major obstacle with Lb. delbrueckii strains was inadequate transformation protocols, a problem that has only recently been resolved (Serror et al., 2002). In contrast, the early resolution of an efficient electrotransformation protocol for laboratory strains of Lb. helveticus allowed the development of gene replacement technologies (Bhowmik et al., 1993). Lactobacillus species harbour many native plasmids (Wang and Lee, 1997) but the tools for manipulating the strains are derived from lactococcal studies. The ability to generate mutants was instrumental in characterising the proteolytic system of Lb. helveticus. In addition, it has facilitated the first metabolic engineering studies. Inactivation of the ldhD gene, which encodes D-lactate dehydrogenase responsible for the production of the D-lactate isomer, resulted in strains that produced the more desirable L-lactate isomer only (Kyla-Nikkila et al., 2000). Similar studies on Lb. delbrueckii await the development of the necessary genetic tools. Moreover, both species lack proper gene expression systems. Streptococcus thermophilus

Streptococcus thermophilus is used in combination with other starter bacteria for the manufacture of Swiss and Italian cheese varieties (with Lb. helveticus or Lb. delbrueckii subsp. lactis) and yoghurt (with Lb. delbrueckii subsp. bulgaricus). The use of Sc. thermophilus has increased significantly during the past two decades because of the increase in the consumption of yoghurt and Mozzarella cheese. Sc. thermophilus, like other LAB, is responsible for producing lactic acid but it can also synthesise exopolysaccharides (EPSs) that typically impart a desirable ‘ropy’ or viscous texture and viscosity to fermented milk products. EPS-producing cultures are particularly important in yoghurt manufacture and have recently been shown to improve the functional properties of low-fat or part-skim Mozarella cheese (Broadbent et al., 2003). Chromosome

The sequence of two strains of Sc. thermophilus, LMG18311 and CNRZ1066, has been completed, while

Important traits

Genes coding for metabolic pathways involved in lactose metabolism, protein and peptide utilisation, and polysaccharide production have been sequenced and characterised. Sc. thermophilus appears to be especially well-adapted for growth in milk with its preference for lactose as a sugar source for glycolysis (van den Bogaard et al., 2000). This contrasts with other LAB that show a preference for glucose. Lactose metabolism is initiated by the uptake of the sugar across the cell membrane via a permease belonging to the glycoside-pentoside-hexuronide-cation symporter family (Poolman et al., 1996). The gene for the transporter (lacS) and -galactosidase (lacZ), required to hydrolyse lactose to glucose and galactose, are organised in an operon with the gene order lacSZ. The glucose is metabolised to lactic acid whereas in most strains galactose cannot be metabolised and is expelled into the external medium (Fig. 2; Gunnewijk and Poolman, 2000). However, Sc. thermophilus does encode the genes (galKTEM) for galactose metabolism upstream of the lacSZ. The inability to metabolise the galactose appears to be related to poor expression of galK (Vaillancourt et al., 2002). Transcription of the lac and the gal genes is governed by the sugar present in the medium and a homologue of the global regulator, CcpA. This regulator acts to repress the expression of lacSZ, probably to match an overcapacity for lactose uptake with rate-limiting glycolytic flux (van den Bogaard et al., 2000). The genetic components of the Sc. thermophilus proteolytic system have not been investigated as extensively as those of Lactococcus or some Lactobacillus species. As a rule, the thermophilic lactobacilli have greater proteolytic activity than Sc. thermophilus, and this is one of the facets of their symbiotic relationship. Although most Sc. thermophilus strains either do not express or express very low levels of CEP activity, a proteinase gene (prtS) has been cloned and sequenced (Fernandez-Espla et al., 2000). The product bears similarities to the CEPs from other LAB being a multi-domain protein belonging to the subtilase family. Sc. thermophilus has also been shown to contain at least 14 different peptidases, two of which


possess biochemical activities not observed in Lactococcus (Rul and Monnet, 1997). The genes for a limited number of these peptidases have been cloned and characterised, including two aminopeptidases (pepN and pepC), an endopeptdiase (pepO), a X-prolyldipeptidyl (pepX) gene and a unique aminopeptidase (pepS) (Anastasiou et al., 2002). Analysis of the genome sequence data should identify the remaining genes corresponding to the activities detected biochemically for Sc. thermophilus. The important contribution of EPSs to the texture and rheological properties of fermented milk products, especially yoghurt, has prompted genetic characterisation of the large eps gene clusters. Four distinct clusters consisting of at least nine genes have been sequenced to date although the function of the majority of gene products can only be inferred from sequence or structural homologies (Broadbent et al., 2003). In general, the genes in the 5 region of the clusters appear to encode regulators of EPS synthesis, chain length determination and membrane translocation. These open reading frames are followed by genes, most likely encoding the glycosyl-1-phosphate transferase glycosyltransferase for assembly of the polysaccharide repeating unit and enzymes involved in repeat unit polymerisation. The remaining genes are probably required for membrane translocation of the polymer subunits and production of sugar nucleotide precursors. More definitive data exists for only five of the thirteen-gene Sc. thermophilus strain Sfi6 eps cluster (Stingele et al., 1999). The epsE, F, G, H and I genes were cloned and over-expressed in E. coli and the data demonstrated that EpsE catalyses the first step in the biosynthesis of the EPS-repeating unit. It exhibits phosphogalactosyltransferase activity and transfers galactose to the lipophilic carrier. The second step is performed by EpsG, which transfers an -N-acetylgalactosamine to the first -galactoside. The activity of EpsF was investigated by characterising the EPS produced by an Sc. thermophilus epsF deletion mutant, which suggested that epsF codes for the branching galactosyltransferase. The epsI gene probably codes for the -1,3-glucosyltransferase, since it is the only glycosyltransferase for which no gene has been assigned and it exhibits similarity to other -glycosyltransferases. These studies improve our understanding of EPS biosynthesis and will be important for potentially novel applications likely to emerge inside and outside the dairy industry for polysaccharides and EPS cultures. Genetic manipulation

Sc. thermophilus has very few native plasmids but this has not greatly hampered the development of genetic tools for the manipulation of these bacteria. Transform-

ation protocols have been established, and tools employed in studying Lactococcus have been readily adapted to Sc. thermophilus. For example, the twoplasmid integration system described above, based on a temperature sensitive pWV01 origin of replication, has been successfully adapted for Sc. thermophilus (Labarre et al., 2001). There is a requirement for a controlled expression system analogous to the NICE system of Lactococcus but the genomics approach is likely to provide the basis for improved molecular tools for the genetic investigation of Sc. thermophilus.

Genomics of Starter Bacteria The genetics of starter bacteria and indeed biology as a discipline are undergoing a revolution. Developments in high throughput sequencing technologies have facilitated the progression to genome-scale sequencing projects. According to the Genomes Online Database (http://wit.integratedgenomics.com/GOLD/), there are 717 prokaryotic and eukaryotic genome-sequencing projects ongoing or completed at the time of writing ( June 2003). These include three Lc. lactis strains, ten different Lactobacillus species and strains, four Sc. thermophilus strains, two Oenococcus oeni strains and one strain each of Leuconostoc mesenteroides and Pediococcus pentosaceus. The availability of this information will radically alter our understanding of starter bacteria. In addition to delineating the genetic complement of each of the species, comparative genomics will allow the identification of the unique genetic traits encoded by each of the bacterial strains. Moreover, the genomic data opens the door for microarray and proteomic technologies. Microarrays, or DNA chips, are essentially glass slides with a representative sample of every gene in a genome spotted onto the surface. They can be used to detect which genes in the genome are expressed under a specific set of conditions. Proteomics refers to new, rapid protein identification systems coupled with improved separation techniques that can individually identify and quantify the proteins present in a cell. Both microarray and proteomic technologies rely on sequence data to produce genome-wide transcription and protein expression profiles. They provide real-time data on RNA expression, protein expression and protein interactions. The genomics approach ultimately provides a comprehensive global prospective on the bacterium, its metabolism and response to the environment. High throughput screening technologies based on these techniques probably represent the future of starter genetics research. These technologies have the potential to produce cultures that can be used readily in industrial fermentations and avoid the need for recombinant DNA technologies which are unlikely to


become acceptable for food production processes in the near future.

Conclusion As we enter the post-genomics age of starter research, it is important to appreciate the landmark discoveries that have enabled our detailed understanding of these industrially relevant bacteria. These have included the elucidation of important traits such as lactose utilisation and casein breakdown and also the development of sophisticated genetic tools. From the genetic point of view, given the level of complex manipulation routinely used with Lc. lactis, it can be considered the E. coli of the LAB, i.e., the genetic workhorse. However, tools are rapidly being developed or adapted and applied to the other LAB. This, allied to the exponential increase in sequence data among the group, will undoubtedly lead to the development of more reliable and valuable strains. These enhanced strains will likely include those engineered to produce nutritional compounds such as folate for improved human health.

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Starter Cultures: Bacteriophage S. McGrath, National Food Biotechnology Centre, Department of Microbiology, University College, Cork, Ireland G.F. Fitzgerald, National Food Biotechnology Centre, Departments of Microbiology and Food and Nutritional Sciences, University College, Cork, Ireland D. van Sinderen, Department of Microbiology, University College, Cork, Ireland

Bacteriophage Bacteriophages (or phage) are viruses that attack bacteria. Unlike prokaryotic and eukaryotic cells, viruses are acellular and are composed of a nucleic acid core surrounded by a proteinaceous coat, and in some cases, a lipid-containing envelope. Viruses are obligate parasites and outside the host cells they are essentially nonliving organic molecules, whereas within host cells, they exhibit various functions that are characteristic of living systems. Viruses are not included in the recognised kingdoms of living organisms and they have been divided into three large groups depending on the host utilised – animal viruses (viruses that replicate in animals), plant viruses (viruses that replicate within plants) and bacteriophage (viruses that replicate within bacterial cells). Twort (1915) and d’Hérelle (1917) were the first to recognise viruses that infect bacteria, and d’Herelle coined the term ‘bacteriophages’, literally meaning ‘eaters of bacteria’.

Bacteriophage Multiplication Depending on the type of life cycle employed, bacteriophage may be differentiated into two groups: lytic or temperate. Infection of a bacterial cell by a lytic (or virulent) bacteriophage ultimately leads to the death and lysis of that cell. In addition to eliciting a lytic cycle, temperate (or lysogenic) bacteriophages are also capable of entering into a life cycle that does not result in the death of the host cell. This non-lethal life cycle is accomplished through the ability of the bacteriophage to integrate its genome in a stable manner into that of the host cell chromosome. This integrated prophage DNA is faithfully replicated in situ by the host cell’s DNA replication apparatus during chromosomal replication, and all progeny cells will therefore receive a copy of it. Integrated prophages may, in response to specific stressful environmental conditions, excise from the host genome and enter into the lytic

cycle (Fig. 1). The specific stages of the phage life cycle will be discussed later.

Bacteriophage of Lactic Acid Bacteria Members of the lactic acid bacteria (LAB) such as Lactococcus lactis, Streptococcus thermophilus, and Lactobacillus, Leuconostoc and Pediococcus spp. are commonly used on an industrial scale in the dairy industry for the production of fermented milk products such as cheese, buttermilk and yoghurt. Bacteriophages are associated with most bacterial species and are therefore ubiquitous in environments where their bacterial hosts are encountered. Bacteriophages infecting Lactococcus were first identified by Whitehead and Cox (1935) and have since been recognised as the major cause of disruption in dairy fermentations. In the modern dairy industry, the disruption of lactic acid fermentations by bacteriophages can lead to serious economic losses. Although technological advances in fermentation processes in conjunction with stringent sanitisation regimes may have reduced the incidence of bacteriophage infection, it has certainly not eliminated it. The persistent problem of bacteriophage infection has focused research on developing phage-resistant starter strains. These studies have involved analysis of phage–host interactions and the characterisation of the genetic processes essential for the phage life cycle. Research initially concentrated on phages that infect lactococcal spp., but more recently interest has expanded to other LAB phages, such as those that infect Lactobacillus spp. and Sc. thermophilus. The advent of molecular biology research tools, such as automated DNA sequencers and bioinformatics, has enabled the complete sequence determination of a still growing number of LAB bacteriophage genomes. This research has led not only to the improvement of the bacterial strains used in the dairy industry and the development of phage-resistance systems, but at a more fundamental level, has provided a detailed understanding of the



164 Starter Cultures: Bacteriophage

A1

C1

A2

C2

A3

B1

B2

B3

C3

Figure 1 Morphological classification of bacteriophages, based on the classification schemes of Ackermann and DuBow (1987). A, Myoviridae; B, Siphoviridae; C, Podoviridae; 1, small isometric head; 2, prolate head; 3, elongated head.

phage life cycle and an insight into the evolution of these organisms.

Control of Bacteriophage in the Dairy Plant Bacteriophage infection remains the largest cause of fermentation disruption in the dairy industry. The continual use of a single starter strain (or the same multiple-strain culture) can allow phage numbers in a cheese factory to rise to detrimental levels. This can result in the reduction in starter viability with a subsequent reduction in the overall rate of acid production, longer manufacturing times and poor flavour-development. The commercial consequences of phage infection include disruption of production schedules, reduction in product quality (and reduction in commercial value) and, in the most severe cases, abandonment of production. Since lactococcal phages were first identified, a number of strategies for reducing their impact in dairy fermentations have been developed. The modern dairy plant is specifically designed to reduce the incidence of phage infection. The area for starter culture preparation is generally physically separated from the production area, with restricted access in order to avoid cross-contamination by personnel. The maintenance of a slight positive pressure in the starter room also reduces the risk of contamination with phage from the factory environment. Further measures include heating of the bulk starter medium (90 °C for at least 20 min) and the filtration of cooling air using high efficiency particulate air (HEPA) filters. Closed fermentation vats have been introduced and equipment coming into contact with the milk is sanitised either by steaming or by

cold disinfection with chlorine and peracetic acid (Cogan and Hill, 1994; Limsowtin et al., 1996; Stanley, 1998). Traditionally, bulk starter preparation involved several scale-up steps from the mother culture through intermediate cultures to the final bulk starter. This process can be time-consuming and may offer the problematic phage a chance to multiply. The finding that the majority of lactococcal phages have an absolute dependence on calcium ions for successful infection (Reiter, 1956) has facilitated the development of phage-inhibitory media, incorporating Ca2 chelating agents, such as phosphate or citrate. Various other steps may be taken to minimise the risk of phage infection and have been reviewed elsewhere (Cogan and Hill, 1994; Limsowtin et al., 1996; Stanley, 1998). The process of bulk starter preparation may be circumvented through the use of commercially available frozen or freeze-dried concentrated cultures. Various types are available and may be used either to inoculate the bulk starter or the milk in the cheese vat directly (direct-set) (Limsowtin et al., 1996). Starter cultures used in the production of Cheddar cheese may be divided into two main groups: mixedstrain cultures and defined-strain cultures. Mixed-strain cultures contain not only different species of bacteria but also different strains of the same species. Approximately 90% of the bacteria in the culture contribute to acid production whereas the other 10% are involved in the production of flavour compounds (Cogan and Hill, 1994). The so-called P cultures (P for practice) that are widely used in the Netherlands are an example of the use of mixed-strain starters in the modern dairy industry. These cultures, which are permanently

Starter Cultures: Bacteriophage 165

contaminated with non-disturbing phage, are used continuously without the need for rotation with phageunrelated cultures (Neve, 1996). The use of defined-strain starter cultures with different phage–host spectra within a carefully designed rotation scheme has proved to be an effective means of controlling phage proliferation. Defined-strain starters (DSS) are usually blends of two or more phage-unrelated strains, which may be used on a rotating basis for cheesemaking (Cogan and Hill, 1994; Neve, 1996). This minimises their exposure to environmental phage in the plant and consequently curtails the accumulation of a specific problematic phage. Heap and Lawrence (1976) developed an elegant protocol for the identification of phage-resistant starter strains. This method involves the sequential culturing of strains in the presence of a cocktail of bacteriophage under conditions similar to those used for cheese manufacture. Cultures identified in this manner have been used extensively in the production of Cheddar cheese. The discovery that lactococci contain plasmids (Cords et al., 1974) enabled researchers to begin unravelling the genetic basis for phage resistance in these bacteria, in earnest. For the dairy industry, a significant outcome of this research has been the development of new and improved phage-resistant strains with desirable fermentative traits. This research has been the focus of several reviews (Klaenhammer and Fitzgerald, 1994; Dinsmore and Klaenhammer, 1995; Garvey et al., 1995a; Allison and Klaenhammer, 1998; Forde and Fitzgerald, 1999; Coffey and Ross, 2002). The continuous development of novel products coupled with increasing production in the dairy industry will undoubtedly pose new challenges for plant engineers, food technologists and microbiologists in the prevention of phage infection.

Classification of LAB Bacteriophage A great deal of research on LAB bacteriophage has focussed on the development of a coherent classification scheme that would accurately reflect the evolutionary relationships between phages. These classification schemes originally relied on the morphological and serological properties of phage, phage–host interactions and protein content. In more recent times, DNA:DNA hybridisation and DNA sequence information have been used.

The original classification was developed by Bradley (1967) and current classification is based on the work of Ackermann and DuBow (1987). Three distinct morphotypes are defined – Myoviridae (Bradley group A) which exhibit contractile tails, Siphoviridae (B) which have long non-contractile tails and Podoviridae (C) which have short non-contractile tails (Ackermann and DuBow, 1987). Almost all phages that infect LAB identified to date belong to the Siphoviridae family, although some lactococcal phage belong to the Podoviridae family (Jarvis et al., 1991). Members of the Siphoviridae have been further divided into three subgroups on the basis of head morphology (Bradley, 1967; Ackermann and DuBow, 1987). Group B1 members have a small isometric head, group B2 members have a prolate head, and group B3 have an elongated head. Podoviridae bacteriophages are similarly subdivided into three groups (C1, C2 and C3) on the basis of head morphology (Fig. 1). The vast majority of phages that infect lactococcal and Lactobacillus species belong to the Siphoviridae morphotype B1 or B2. However, some exceptions have been noted, i.e., a number of Podoviridae phages that infect Lactococcus have been identified (Saxelin et al., 1979, 1986; Braun et al., 1989). Sechaud et al. (1992) identified and classified a number of morphologically distinct phages that infect Lb. helveticus, which have an isometric head and a contractile tail of variable length. All Sc. thermophilus phages reported to date are of the B1 morphotype of the Siphoviridae family. Host range

Classification schemes for LAB phage based on host range are particularly relevant to the dairy fermentation industry. However, these schemes rarely agree with those based on other criteria. For example, in one study it was found that Sc. thermophilus phage with similar host ranges exhibited limited DNA homology, whereas phage with similar DNA restriction profiles exhibited a completely different lytic spectrum (Mata and Ritzenthaler, 1988). Similar observations were made for phages of Lactococcus (Relano et al., 1987). These observations indicate that whatever the advantages these classification schemes may have from an applied viewpoint, they are of little taxonomic value.

Morphology

Serology

This is the classical method for viral classification and relies on direct microscopic examination to characterise the physical shape of the virus particle (Fig. 1).

Several attempts have been made to classify Sc. thermophilus and lactococcal bacteriophage on the basis of their serological properties (Kivi et al., 1987;


Mata and Ritzenthaler, 1988; Jarvis, 1989; Brüssow et al., 1994a). However, a serious drawback of this method is that it is based on differences in antigenic properties exposed on the external structure of the virion and is therefore indicative of the expression of only a small part of the genome. Structural protein profiles

Protein profile analysis has been commonly used to classify Sc. thermophilus bacteriophage and has been found to generally agree with other schemes. To date, three different types of protein profiles have been described for phage that infects Sc. thermophilus. Kivi et al. (1987) described phage containing four major proteins, while several reports have described Sc. thermophilus phage with either two or three major structural proteins (Neve et al., 1989; Prévots et al., 1989; Benbadis et al., 1990; Fayard et al., 1993; Le Marrec et al., 1997; Stanley et al., 1997). There is a correlation between the method of DNA packaging and the number and type of structural proteins, for both Sc. thermophilus and Lb. delbrueckii phages (Forsman and Alatossava, 1991; Le Marrec et al., 1997). Lactococcal phages are generally found to have between one and three major structural proteins together with a varying number of minor structural proteins (Arendt et al., 1994; Johnsen et al., 1996; Van Sinderen et al., 1996). Neve (1996) reported that lactococcal phages of different morphotypes tend to have different protein profiles. DNA homology

Classification of LAB phage based on DNA homology will evaluate the entire phage genome as opposed to a specific portion, which may encode, for example, the structural genes. On the basis of DNA:DNA hybridisation studies, twelve genetically distinct lactococcal

phage species have been defined (Jarvis et al., 1991) (Table 1). Phages that infect Sc. thermophilus appear to be more closely related to one another than those that infect Lc. lactis and they probably belong to a single homology group (Neve et al., 1989; Benbadis et al., 1990; Fayard et al., 1993; Brüssow et al., 1994a,b; Le Marrec et al., 1997). However, the degree of homology does vary and a number of subgroups have been proposed (Neve et al., 1989; Prévots et al., 1989; Benbadis et al., 1990; Fayard et al., 1993). Four different homology groups have been defined for phage of Lb. delbruekii, with the majority of phages belonging to one specific group, designated ‘A’ (Mata et al., 1986; Lahbib-Mansais et al., 1988; Sechaud et al., 1988; Forsman and Alatossava, 1991; Forsman, 1993). However, Lahbib-Mansais et al. (1988) have also described a second homology group, distinct from the Lb. delbrueckii phage group. This is comprised of five members, all of which infect Lb. delbrueckii.

LAB Bacteriophage Epidemiology Of the twelve lactococcal phage species described by Jarvis et al. (1991), phage of three species, c2, 936 and P335, represent the majority of industrial isolates. Most of LAB phages are classified as Siphoviridae, with a noncontractile tail and a small isometric head, such as members of the 936 and P335 species (morphotype B1), whereas c2 type phages have a non-contractile tail with a prolate head and are classified as B2 morphotypes (Ackermann and DuBow, 1987). In a survey of Canadian dairy plants, Moineau et al. (1992) found that members of the c2 species were isolated with the highest frequency whereas, in a later study conducted in the United States, 80% of the phages identified were

Table 1 Lactococcal phage species, type phages and members (adapted from Jarvis et al., 1991) Family

Morphotype species

Phage type

Phage

Members

Siphoviridae

B1 B1

936 P335

P008 P335

B1 B1 B1 B1 B1 B1 B2 C2 C3

P107 1483 P087 1358 BK5-T 949 c2 P034 KSY1

P107 1483 P087 1358 BK5-T 949 c6A P034 KSY1

P008, F4-1, sk1, bIL41, bIL66, US3 P335, LC3, r1t, Tuc2009, TP901-1, 31, 50, Q30, Q33, ul36 P107 1483 P087 1358 BK5-Ta 949 c2, bIL67, vML3, 197, P001 P034 KSY1

Podoviridae

a It has been proposed that BK5-T should be assigned to the P335 species (Labrie and Moineau, 2002).


classified as representatives of the 936 species (Moineau et al., 1996). Similarly, Jarvis et al. (1991) reported that the majority of phages identified in New Zealand, the United States and Ireland were of the 936 species. In the last 10 years or so, P335 type phage has been encountered with increasing frequency and it has been proposed that members of this species represent an emerging dominant phage type in industrial environments (Alatossava and Klaenhammer, 1991; Moineau et al., 1992, 1996; Durmaz and Klaenhammer, 2000).

the assumed origin of replication and both package their DNA utilising a cos site. Each has a relatively small genome, with the complete sequence of bIL67 being 22 195 bp, compared to 22 163 bp for c2. c2 and bIL67 share about 80% of the overall nucleotide sequence identity (Lubbers et al., 1995). However, this is not evenly distributed along the entire genome, with some regions sharing more than 90% identity and others less than 40% (Lubbers et al., 1995). The early region of c2 encompasses approximately 7 kb of DNA, which harbours 22 putative ORFs. Similarityderived functions have been assigned to some of these and they include a DNA polymerase, a recombination protein, a sigma factor and a transcriptional regulator ( Jarvis et al., 1995; Lubbers et al., 1995). The late region spans around 16 kb, and 17 ORFs have been identified in this section of the genome. N-terminal sequence analysis identified three major and eight

Prolate-headed phage

The two sequenced Lc. lactis prolate-headed phage, bIL67 (Schouler et al., 1994) and c2 (Lubbers et al., 1995), display a very similar genetic organisation (Fig. 2). The genomes of both phages are divided into two divergently oriented clusters consisting of the ‘early’ and ‘late’ transcribed regions. The divergent clusters are separated by

(A) c2 (c2) Early

Late

ori

cos Replication

cos Packaging

Morphogenesis

Lysis

Morphogenesis

Lysis

(B) 936 (sk1)

Early

Middle

ori

Late

cos

Replication

Packaging

Morphogenesis

Lysis

(C) P335 (TP901-1) Lysogenic

Lytic early

Integration

Lytic late

pac/cos

genetic switch

attP

Lytic middle

Replication

Packaging

Morphogenesis

Lysis

Figure 2 Schematic representation of the genomic arrangements of the three main lactococcal phage groups: (A) c2, prolate headed; (B) 936, small isometric headed; (c) P335, small isometric headed. Blocks represent genomic regions containing genes involved in the bacteriophage life cycle.


minor structural proteins (many of which appear to be post-translationally processed) (Lubbers et al., 1995). In addition, two putative holins and a lysin were identified ( Jarvis et al., 1995; Lubbers et al., 1995). Similarly, the early region of bIL67 harbours 21 ORFs spanning approximately 7 kb while the late region consists of about 15 kb of DNA, containing 16 putative ORFs. A holin, a terminase subunit, a minor tail subunit, lysin, DNA polymerase and a protein involved in recombination were tentatively identified on the genome (Schouler et al., 1994). Small isometric-headed phage

All remaining LAB phage for which the entire genome sequence has been determined have a small isometric head. They include six Sc. thermophilus, three Lactobacillus and thirteen lactococcal phages (Table 2). Of the thirteen completely sequenced small isometricheaded lactococcal phages, bIL170 and sk1 are mem-

bers of the 936 spp. Six of the remaining eleven phages in this group were identified as prophages on the Lc. lactis IL1403 genome, and bioinformatic analysis revealed that three belong to the P335 group of temperate phage, whereas the remaining three are most probably satellites relying on helper phage(s) for multiplication (Chopin et al., 2001). The remaining five members are P335-type phages (Table 2). Three consecutive phases of transcription (early, middle and late) were apparent for the 936-type phage, sk1 (Chandry et al., 1997) (Fig. 2). The remaining sequenced small isometric LAB phages, include lactococcal P335 spp., and Sc. thermophilus and Lactobacillus phages, all of which share a very similar genetic organisation. The genomes of the lysogenic phages are arranged in two divergent clusters separated on one side by the attachment site and on the other by an intergenic region involved in the genetic switch (Fig. 2). The genome of the lytic Sc. thermophilus, Lactobacillus and lactococcal P335 phage is, for the most part, transcribed in one

Table 2 List of bacteriophages infecting LAB for which the genome has been completely sequenced

Size/bp

Small isometric/ prolate headed, lytic/temperature

Number of putative ORFs

Reference

cos cos cos cos cos pac pac cos pac

22 195 22 163 28 451 31 754 33 350 38 347 36 798 40 003 36 667 35 538 41 834 36 949 14 957 14 510 15 179

Prolate, lytic Prolate, lytic S.Ia, lytic (936) S.I, lytic (936) S.I, temperate (P335) S.I, temperate (P335) S.I, temperate (P335) S.I, temperate (BK5-T) S.I, temperate (P335) temperate (P335) temperate (P335) temperate (P335) temperate temperate temperate

37 39 54 64 50 56 59 63 56 62 61 56 28 21 27

Schouler et al. (1994) Lubbers et al. (1995) Chandry et al. (1997) Crutz-Le Coq et al. (2002) Van Sinderen et al. (1996) Proux et al. (2002) Labrie and Moineau (2002) Mahanivong et al. (2001) Brondsted et al. (2001) Chopin et al. (2001) Chopin et al. (2001) Chopin et al. (2001) Chopin et al. (2001) Chopin et al. (2001) Chopin et al. (2001)

Lactobacillus LL-H (Lb. delbrueckii ) g1e (Lb. plantarum) adh (Lb. gasseri )

pac pac cos

34 657 42 259 43 785

S.I, lytic, S.I, temperate S.I, temperate

52 62 62

Mikkonen et al. (1996) Kodaira et al. (1997) Altermann et al. (1999)

Sc. thermophilus O1205 7201 DT1 Sfi19 Sfi21 Sfi11

pac cos cos cos cos pac

43 075 35 466 34 820 37 392 40 739 39 807

S.I, temperate S.I, lytic S.I, lytic S.I, lytic S.I, temperate S.I, lytic

57 44 46 44 53 53

Stanley et al. (1997) Proux et al. (2002) Tremblay and Moineau (1999) Desiere et al. (1998) Desiere et al. (1998) Lucchini et al. (1998)

Phage Lactococcus bIL67 c2 sk1 biL170 r1-t Tuc2009 ul36 BK5-T TP901-1 bIL285b bIL286b bIL309b bIL310b bIL311b bIL312b

Cos/pac site

a S.I, small isometric. b Prophage identified on the chromosome of Lc. lactis IL1403.


direction. However, transcription of these lytic genes may be controlled in a temporal manner. For example, transcriptional analysis of the P335-type phage, TP901-1, revealed that genes involved in the lytic cycle are transcribed in three distinct temporal phases, early, middle and late, with maximal transcript concentrations present after 10, 30 and 40 min post-infection, respectively (Fig. 2). 936 phage

The first complete 936-type phage genome sequence available was that of sk1 (Chandry et al., 1997). Several of its 54 putative ORFs show sequence similarity to both the small isometric- and prolate-headed morphotypes. From a transcriptional point of view, the genome is organised into three segments, represented by the early (30 ORFs), the middle (4 ORFs) and the late (20 ORFs) transcribed regions. These regions are separated from each other by intergenic regions containing the cos site (middle and late), a transcription terminator (late and early) and divergent promoters (early and middle). The early region consists of ORFs thought to specify replication functions (the DNA polymerase subunits). The middle region is composed of four small ORFs just upstream of the cos site. No function has been assigned to these ORFs. The late region of the genome putatively encodes various structural proteins, proteins involved in the DNA packaging and the lysis functions of the phage (Chandry et al., 1997). The complete genome sequence of bIL170 has been published recently (Crutz-Le Coq et al., 2002). Sixty-four ORFs were identified and the function of 16 of them was assigned by significant homology to proteins in databases. Comparison of the bIL170 genome to that of sk1 showed that insertion/deletion events involving one or two ORFs were the main source of divergence in the early gene clusters. It was proposed that such events resulted in the replacement of a direct repeat-containing genomic fragment in bIL170, resulting in the acquisition of a new functional origin of replication by this phage (Crutz-Le Coq et al., 2002). Partial sequence information is available for other 936-type phages, including F4-1 (Chung et al., 1991; Kim and Batt, 1991a), bIL41 (Parreira et al., 1996) and bIL66 (Bidnenko et al., 1995). Sequence comparisons indicate that these 936-type phages are very closely related. For example, over 2 kb of the genome of sk1 (including most of the middle region) shows 84.9% identity to the expressed middle region of bIL66 (Chandry et al., 1997). Also, a 10.2-kb segment from the late region of bIL41 displays between 69 and 98% identity to an homologous segment from sk1 (Parreira et al., 1996; Chandry et al., 1997).

P335 phage

Members of the P335 phage species are small, isometric-headed with a genome ranging from 30 to 42 kb. Bacteriophages included in the P335 species are heterogeneous, with phages utilising both cos and pac sites for DNA packaging, and this is the only lactococcal phage species that includes both virulent and temperate members. The first complete P335 phage genome sequence published was that of r1t (Van Sinderen et al., 1996). This is a temperate phage and its genome is arranged in two divergent clusters of 3 and 47 ORFs. This appears to be a life-cycle-specific orientation, i.e., the orientation of the ORFs believed to be involved in the lysogenic life cycle is opposite to the remaining ORFs, which are associated with the lytic life cycle. It appears, therefore, that the r1t genome is arranged such that all of the ORFs involved in the lytic life cycle are grouped in one large contiguous gene cluster. Such a lytic cluster appears to be composed of a number of functional modules which are transcribed and organised in a way that reflects the chronological order of the life cycle itself, i.e., those ORFs required for replication are transcribed first and are located at the proximal end of the gene cluster, followed by modules involved in DNA packaging, morphogenesis and, finally, cell lysis (Fig. 3). All other temperate P335-type phages, the genome of which have been sequenced, appear to exhibit this type of gene organisation. The most recent complete P335 phage genome available, that of ul36, also represents the first virulent P335 member to be completely sequenced (Labrie and Moineau, 2002). Interestingly, the genome of this lytic phage appears to be arranged in two divergent clusters of 6 and 53 ORFs. The former represents a cryptic lysogeny module containing genes, the deduced protein products of which display similarities to phage integrases, repressors and a Cro protein. An incomplete lysogeny module was identified in another lytic P335 phage, 31 (Madsen et al., 2001). These observations indicate that some virulent P335-type phages are derived from the temperate phage. Sc. thermophilus phage

The five completely sequenced Sc. thermophilus phages appear to have a genetic arrangement very similar to that of the P335-type species of lactococcal phage, i.e., they have a modular arrangement made up of replication, packaging, morphogenesis and cell lysis components. It has been speculated that O1205 is closely related to the P335 group of lactococcal phage (Stanley et al., 1997). Sequence comparisons of Sc. thermophilus phage revealed a substantial amount of homology between them (Bruttin et al., 1997; Desiere et al., 1998,


Phage attachment to host cell

Phage DNA injection

Adsorption interference

DNA injection blocking

DNA replication and synthesis of phage proteins

DNA packaging and morphogenesis

Cell lysis and release of progeny phage

A

Early R/M systems

Abortive infection

Late

B

Insertional mutagenesis

Recombinant superinfection exclusion

Antisense

Triggered

Per

Figure 3 Schematic representation of LAB bacteriophage lytic life cycle showing main steps in phage development. (A) Stages at which naturally occurring phage resistance mechanisms arrest phage development; (B) Stages at which engineered phage resistance mechanisms arrest phage development.

1999, 2002; Lucchini et al., 1998, 1999a; Neve et al., 1998; Proux et al., 2002). Lactobacillus phage

The genomes of the Lactobacillus phages, gle (Kodaira et al., 1997), adh (Altermann et al., 1999) and LL-H (Mikkonen et al., 1996) have been completely sequenced and display distinct similarities to the genomes of the P335 group of lactococcal bacteriophage, as well as those that infect Sc. thermophilus. The putative ORFs on the genome of these phages are clustered into a number of functional modules. Interestingly, although LL-H is a lytic phage, remnants of an integrase and an attachment site (similar to that of mv4 (Auvray et al., 1997)) can be discerned, indicative of a direct evolutionary relationship between these two Lb. delbrueckii phages (Mikkonen et al., 1996). Furthermore, the genes encoding some of the structural proteins of these two phages were shown to be highly conserved (Vasala et al., 1993).

Genome Organisation and Evolution of LAB Bacteriophage All genomes of phages that infect LAB analysed to date consist of a double-stranded, linear DNA molecule with a G C content consistent with that of the host chromosome (37% for lactococcal phages to ⬃48% for phages of Lb. casei) (Forde and Fitzgerald, 1999). The

majority of chromosomes analysed are 18–40 kb in length, although larger sizes, up to 134 kb, have been reported (Prévots et al., 1990; Moineau et al., 1992). Two distinct genome types have been identified, based on the means by which the phage packages its DNA. Some phage genomes have cohesive ends consisting of single-stranded 3 overhangs of variable length, while others are said to be ‘circularly permuted’ and have ‘terminal redundancy’ (Black, 1989). Botstein (1980) has put forward ‘a theory of modular evolution for bacteriophage’ in which he proposed that the product of evolution is not a given virus but a family of interchangeable genetic elements (modules), each of which carries out a particular biological function. Furthermore, he proposed that evolution does not act primarily at the level of an intact virus, but at the level of individual functional units (modules). This theory is supported by analysis of available bacteriophage DNA sequences, where the genomes of phage are found to be organised in a life-cycle-specific manner, with modules containing genes coding for integration/excision, replication, structural proteins, assembly, DNA packaging and host cell lysis (Fig. 2). Homologous functions may be fulfilled by a number of distinct DNA segments that lack any sequence similarity. Particular modules may be exchanged through recombination among phages belonging to an interbreeding phage population. Hendrix et al. (1999) have further developed this theory and proposed


a model for the genetic structure dynamics of the global phage population in which all double-stranded (ds) DNA phage genomes are mosaics with access, by horizontal exchange, to a large common genetic pool but in which access to the gene pool is not uniform for all phages. Brüssow and Desiere (2001) have discussed the evolution of phages of the Siphoviridae family using LAB phages as a model, and have proposed the establishment of a super-group of Siphoviridae based on structural gene synteny. They also discuss the role of both vertical and horizontal evolutions in relation to these phages. The evolution of new lytic LAB bacteriophage due to the acquisition of genes and/or entire DNA modules, both of which are most probably derived from prophage sequences located on host cell chromosomes, has been reported in phages that infect Lactococcus (O’Sullivan et al., 1993; Moineau et al., 1994; Bouchard and Moineau, 2000; Durmaz and Klaenhammer, 2000). This finding is increasingly common, is likely to be a response to the selective pressure applied by phage resistance systems, and demonstrates the evolutionary flexibility of phage.

encoded membrane-associated protein, called Phage Infection Protein (PIP), from Lc. lactis subsp. lactis c2 has been identified as being necessary for the adsorption and subsequent DNA injection of a number of phages that infect this strain (Valyasevi et al., 1991, 1994; Geller et al., 1993; Monteville et al., 1994). Further studies demonstrated that although the PIP protein is essential for infection by a number of prolateheaded phages, PIP-mutants were still susceptible to infection by phages of the 936 and P335 species (Kraus and Geller, 1998). Genes homologous to pip have been identified in all strains of Lc. lactis tested (Garbutt et al., 1997). Analysis of the PIP protein reveals that it possesses a putative N-terminal signal peptide and six putative transmembrane-spanning domains (Geller et al., 1993). Other studies have indicated that another 32-kDa membrane-associated protein is also necessary for phage infection of Lc. lactis subsp. lactis c2 (Valyasevi et al., 1991). Lucchini et al. (2000) have recently identified a chromosomally encoded protein analogous to PIP in Sc. thermophilus. Insertional inactivation of this gene conferred a phage resistance phenotype on Sc. thermophilus Sfi11 against all fifteen phages used in the study (Lucchini et al., 2000).

Life Cycle of LAB Bacteriophage The individual steps that make up the bacteriophage life cycle will be discussed, with specific reference to phages of LAB (Fig. 4). Lytic life cycle Phage adsorption/ DNA injection

The exact molecular mechanisms by which LAB phage attach to cells and inject their DNA are still not understood completely. However, studies undertaken so far indicate that the processes involved are consistent with those of the better-characterised Gram-negative phage, such as the T-even phage (Dreiseikelmann, 1994). Lactococcal phages appear to attach to the host cell at specific receptor sites which may or may not be evenly distributed over the cell surface (Budde-Niekiel and Teuber, 1987). The majority of phages attach in a tail-first orientation to a carbohydrate moiety of the cell wall. The sugars, galactose and/or rhamnose, have been implicated in most cases (Keogh and Pettinghill, 1983; Valyasevi et al., 1990; Monteville et al., 1994); however, more complex polysaccharide components and cell membrane lipoproteins have also been indicated (Oram, 1971; Schafer et al., 1991). This initial phage ‘docking’ is usually reversible and phages can detach following addition of sugars such as those mentioned above. This initial reversible phase of phage adsorption is followed by an irreversible phase. A chromosomally

Lysogenic/lytic switch

Regulatory regions involved in the control of the lysis–lysogeny decision of temperate lactococcal bacteriophage (Boyce et al., 1995; Nauta et al., 1997; Madsen et al., 1999), temperate Lactobacillus phage (Kodaira et al., 1997; Ladero et al., 1998, 1999) and temperate Sc. thermophilus phage (Stanley et al., 1997; Neve et al., 1998; Lucchini et al., 1999b) have been described. It has become apparent that the general mechanism for life cycle decision is similar to that of bacteriophage , where CI- and Cro-like repressors play opposing roles in a genetic switch mechanism (Ptashne, 1986). CI prevents transcription of the lytic genes (and positively regulates its own expression) by binding to specific DNA sequences (operators) located within the immunity region, whilst Cro prevents transcription of the genes involved in the establishment of lysogenic growth by binding the same operator sites with different affinities (Ptashne, 1986). Cro is a small protein consisting of 66 amino acids within which a helix-turn-helix motif can be discerned. Cro-like proteins in LAB phages do not exhibit a great deal of similarity, but many have been putatively identified based on their relative genome position and the criteria outlined above (Lucchini et al., 1999b). DNA replication

For initiation of DNA replication to occur, a specific starting point of replication must be identified where


Bacterial cell

Host chromosome Phage particle

Attachment to host cell and injection of DNA

DNA replication and synthesis of phage proteins

Integration of DNA into the host chromosome

Induction event

DNA packaging and phage morphogenesis

Cell division

Cell lysis and release of progeny phage Integrated phage DNA replicates along with the host chromosome

Lysogenic life cycle

Lytic life cycle

Figure 4 Lytic and lysogenic life cycles of bacteriophage.

opening of the double-stranded DNA double helix takes place prior to the recruitment of the replication machinery. The DNA region representing the initiation point for (phage) DNA replication, also referred to as the origin or replication (ori) is characterised by the

presence of two or more direct repeats, which facilitate binding of a sequence-specific duplex DNA-binding protein. Such a nucleoprotein-binding complex generally consists of 150–250 bp of DNA and multiple copies of a replication-specific DNA-binding protein.


Formation of the nucleoprotein complex invokes denaturation of an A T-rich region of DNA directly adjacent to the ori, the single-stranded status of which is further promoted by single-stranded binding protein. By so marking the origin and providing a single-stranded DNA region, the replication fork proteins can be recruited to the correct initiation point, and nascent strand synthesis can ensue. Examples of this type of DNA replication initiation include the well-characterised chromosomal replication systems employed by phage and E. coli (Marians, 1992). Analysis of replication modules of LAB phage has so far been largely at the level of database searches, with putative functions being assigned to individual genes on the basis of similarities to genes of known function. However, the study of phage oris and the exploitation of the interactions between phage/hostencoded replication proteins and their cognate phage oris has been quite rewarding. The first LAB phage ori to be described was that of the lactococcal phage, 50, followed by that of another lactococcal phage, 31 (Hill et al., 1990a; O’Sullivan et al., 1993). In both cases, it was shown that the copy number of an ori-containing plasmid drastically increases following infection by a phage utilising the same ori for replication. Furthermore, these plasmids also conferred a phage-resistance phenotype on the lactococcal host. This phenotype was termed per for phage-encoded resistance. These observations led to the conclusion that the phage ori sequences on the plasmid vectors were titrating essential replication functions away from the phage DNA which were in turn driving plasmid amplification. A putative origin of replication for the lactococcal phage, Tuc2009, designated ori2009, has also been identified (McGrath et al., 1999). The ori2009 sequence is located within the gene coding for the putative replisome organiser protein (rep2009), and its encoded protein specifically interacts with the ori2009 DNA (McGrath et al., 1999). In a further study, genes highly homologous to rep2009, that contained sequences identical to ori2009, were identified in two other lactococcal phages, Q30 and Q33 (McGrath et al., 2001), while a third rep2009 homologue was identified in another closely related phage, ul36 (Bouchard and Moineau, 2000). The ability of plasmids harbouring ori sequences to confer a phage-resistance phenotype was also used to identify oris in the Sc. thermophilus phages Sfi21, 1205 and 7201, and the Lb. casei phage A2 (Foley et al., 1998; Moscoso and Suarez, 2000; Stanley et al., 2000). Interestingly, it was found that 7201 appears to contain two oris, each of which is capable of independently mediating a per phenotype (Stanley et al., 2000). Phages infecting Sc. thermo-

philus have been classified into two groups on the basis of their replication module. Members of replication group I employ an ori similar to that of 1205, while members of replication group II use an ori similar to one or both oris of 7201 (Stanley et al., 2000). Another method used to identify phage oris is based on their ability to act as bona fide origins of replication for plasmids. A 611-bp intergenic region located between the early and the late gene regions of the lactococcal phage c2 was identified as an ori in this manner (Waterfield et al., 1996). The presence of this DNA fragment was sufficient to drive plasmid replication in Lactococcus strains but not in E. coli. The absence of any ORFs within the 611-bp fragment suggests that replication of this plasmid requires only host-encoded factors. The c2 ori contains an A/T-rich region (78% A/T), which has several small perfect and imperfect inverted and direct repeats, a phenomenon characteristic of origins of replication. Highly similar sequences were also identified in two other lactococcal phages, bIL67 and 197 (Schouler et al., 1994). Phage replication module genes likely to code for topoisomerases, single-stranded DNA-binding proteins, replisome organisers, DNA helicase/primases and helicase loader proteins have been putatively identified on the basis of similarity to sequences in the databases. Replication functions for phages infecting Lactococcus, Streptococcus and Lactobacillus have been identified in this manner (Forde and Fitzgerald, 1999). DNA packaging

For many phages, intracellular DNA replication results in the formation of large concatameric DNA molecules consisting of several phage genome complements (Black, 1989). Bacteriophages may employ one of the two mechanisms of packaging their genome into the (pro)-capsid prior to assembly and release of mature phage particles from the cell. Phages whose genome contains a pac site employ a so-called headful mechanism of DNA packaging. Here, the phage DNA is initially cut at the pac site, with each subsequent cut occurring when the prohead has been filled with DNA. This mechanism results in phage containing DNA molecules that are circularly permuted and terminally redundant, i.e., coding for more than one unit length of genome (Streisinger et al., 1967; Tye et al., 1974). Alternatively, phage genomes may contain a cos site. Cutting of the concatameric DNA molecules at these specific cos sites results in single genomic units with 3 overhangs on the DNA which are self-complementary (cohesive ends) (Murialdo, 1991). It has been demonstrated that the actual DNA translocation into the prohead requires the action of several proteins – the terminase complex, portal protein and the


expanded major head protein (Black, 1989). Packaging is initiated when the terminase binds to the specific pac or cos site. The terminase is composed of two subunits. The small subunit which binds to, and hydrolyses ATP, is also thought to interact with the phage DNA, while the large subunit appears to bind to the prohead, and may be involved in cutting the concatameric DNA molecules. One of the structural elements of the phage, the portal protein, plays a role in forming the entrance to the phage head. The portal protein has also been implicated in conjunction with the terminase in initiating DNA packaging, DNA translocation and in determining the amount of DNA to be packaged in phages utilising the headful mechanism. The major capsid protein also hydrolyses ATP during the translocation of DNA (Black, 1989). DNA sequence analysis of the regions surrounding cos sites has revealed the presence of several conserved regions, which have been determined as being essential for binding of phage terminases (Chandry et al., 1994; Herrero et al., 1994; Nakashima et al., 1994; Schouler et al., 1994; García et al., 1997). It has also been reported that cos regions have a high G C content which is thought to be necessary for stable basepairing of the cos region once the phage genome has entered the cell. The majority of lactococcal phage genomes analysed to date possess cos sites (Klaenhammer and Fitzgerald, 1994); however, pac sites have been identified in the lactococcal phage, Tuc2009 and TP901-1 (Arendt et al., 1994; Christiansen et al., 1994) and the Lactobacillus phages LL-H and Mv4 (Vasala et al., 1993). Le Marrec et al. (1997) have classified a number of Sc. thermophilus phages into two groups, depending on the DNA packaging mechanism employed. Using Southern hybridisation, they demonstrated that all pac-containing phages tested contained homologs of the genes encoding the three major structural proteins of the pac-containing phage O1205, whereas all cos-containing phages tested exhibited homology to the gene specifying one of the structural components of the cos-containing phage 7201. Structural proteins

Structural protein synthesis begins immediately following phage DNA replication. One of the most comprehensive studies of the structural proteins of an LAB phage is that of c2 (Lubbers et al., 1995). Three major structural proteins of 175, 90 and 29 kDa and eight minor proteins of 143, 82, 66, 60, 44, 42, 32 and 28 kDa were identified by SDS polyacrylamide gel electrophoresis (PAGE). The genes coding for these proteins were also identified. Several of the proteins were thought to have undergone post-translational modification by proteolytic cleavage. It was determined that 175, 143, 90, 82 and

66 kDa proteins had the same N-terminal amino acid sequence, which matched the gene product specified by the l5 gene. Similarly, two structural proteins of 29 and 28 kDa, although containing different N-terminal amino acid sequences, were shown to be encoded by the l7 gene. Using immunogold electron microscopy, it was shown that the structural proteins of 175 and 90 kDa represented major head proteins, while the 29- and 60-kDa proteins were the building blocks of the major tail and tail adsorption structures, respectively. Furthermore, the products of the head protein gene, l5, were suggested to be involved in forming covalently linked multimers, including trimers, hexamers and small amounts of pentamers. This type of multimerisation has been proposed to be involved in the formation of the -icosohedral phage head. The techniques mentioned above, i.e., SDS-PAGE, N-terminal amino acid sequencing, immunological analysis, as well as homology searches of sequence databases, have been used to identify structural proteins of many other LAB phages (Hill, 1993; Klaenhammer and Fitzgerald, 1994; Garvey et al., 1995b; Davidson et al., 1996; Forde and Fitzgerald, 1999). Bacteriophage lysis

Lysis of the host cell by infecting bacteriophages results in the release of progeny phage and requires a cell wall-degrading enzyme (lysin). Three classes of lysin have been described to date, and they are differentiated on the basis of the peptidoglycan bond hydrolysed (Rodgers et al., 1980). However, only lysins of the first two classes described have been identified for phages that infect LAB. The first class, termed glycosidases, hydrolyses the glycosidic linkage between the amino sugars of the peptidoglycan and includes endo N-acetylglucosaminidases (or glucosaminidases) and endo N-acetylmuramidases (muramidases or lysozymes). The second class, N-acetyl muramoyl-L-alanine amidases (amidase), hydrolyses the N-acetylmuramoylL-amide linkage between the glycan strand and the cross-linking peptide. The third class, endopeptidases, break the peptide chain of the peptidoglycan. It has been proposed that lysin proteins consist of two separate modules, with the N-terminus determining the lytic activity and the C-terminal domain specifying cell wall-binding (García et al., 1990). In support of this theory, a chimeric lysin protein has been constructed by fusing the N-terminal half of the lactococcal phage Tuc2009 lysin to the C-terminal domain of the major pneumococcal autolysin (Sheehan et al., 1996). This novel enzyme exhibited a glycosidase activity capable of hydrolysing cholinecontaining pneumococcal cell walls. It is noteworthy that some lysin-encoding genes employ atypical


start codons, and it has been suggested that these act as a control mechanism to prevent premature lysis of the host (Shearman et al., 1994). The second part of the LAB lysis cassette is the holin. Holins are small membrane-associated proteins, which cause non-specific lesions in the cytoplasmic membrane, thus allowing the lysin access to the cell wall (Young and Bläsi, 1995). Holins have several welldefined characteristics, although they exhibit little similarity in either amino acid or DNA sequences. They generally contain a hydrophilic and charge-rich C-terminus, 2–3 hydrophobic, possible membranespanning regions separated by a -turn linker region and a hydrophilic N-terminus (Young and Bläsi, 1995). It has been suggested that the expression of active holin is controlled at the level of transcription, with a methionine dual start motif identified in many holinencoding genes. This facilitates the synthesis of two gene products of slightly different size, one of which acts as an inhibitor of the other (Bläsi and Young, 1996). Three distinct holin groups have been described (Young and Bläsi, 1995). Type I holins are at least 87 amino acids in length and contain three possible membrane-spanning regions. Type II holins are less than 78 residues in length and contain two possible membrane-spanning regions. Finally, a unique

holin has been identified for the E. coli phage, T4, which was assigned to a separate group, III. Several genes encoding phage lysins and their deduced protein products for LAB phage have been characterised at the molecular and biochemical levels. These include the lysins of the lactococcal phages P001, us3, c2, vML3, LC3, Tuc2009 and r1t, as well as that of the Lactobacillus phage LL-H (Table 3). Sheehan et al. (1999) have described a lysis module contained on the genome of the temperate Sc. thermophilus phage O1205, which contains two putative holin genes and one lysin. Southern blot analysis revealed that at least one or more of these genes were present in 30 other Sc. thermophilus phages examined. Lysogenic life cycle

The phenomenon of lysogeny was first reported in Lactococcus by Reiter (1949). Lysogeny is widespread in LAB, particularly in Lactococcus (Huggins and Sandine, 1977; Jarvis, 1989; Davidson et al., 1990) and Lactobacillus (Sechaud et al., 1988), but much less so in Sc. thermophilus (Fayard et al., 1993; Brüssow et al., 1994b, 1998; Le Marrec et al., 1997). It appears that the mechanisms involved in the maintenance of lysogeny in LAB are similar to that of phage (see above).

Table 3 Identified genes encoding restriction/modification systems in Lc. lactis and Sc. thermophilus R/M system or identified subunits

Type

Location

Reference

Lc. lactis Lla1403I Lld I Unnamed (HsdR, HsdM, HsdS)

I I I

pIL2614 pND861 pAH82

I

Chromosome of Lc. lactis IL1403

Schouler et al. (1998a) Deng et al. (2000) O’Sullivan et al. (2000) O’Sullivan et al. (2001) Bolotin et al. (2001)

I I I I II II II II II II II III

pCIS3 pIL7 pIL103 Chromosome of Lc. lactis IL1403 pTR2030 pHW393 pAW153 pJW563 pKR223 Chromosome of Lc. lactis UC503 Chromosome of Lc. lactis DCH-4 pND801

Seegers et al. (2000) Schouler et al. (1998b) Schouler et al. (1998b) Schouler et al. (1998b) Hill et al. (1989) Madsen and Josephsen (1998a) Madsen and Josephsen (1998b) Nyengaard et al. (1996) Twomey et al. (1998) Twomey et al. (1997) Moineau et al. (1995) Su et al. (1999)

I I I I II

pCI65st pER35 pER16 Chromosome Chromosome

O’Sullivan et al. (1999) Solow and Somkuti (2001) Solow and Somkuti (2001) Lucchini et al. (2000) Burrus et al. (2001)

L0308 (HsdR) L0309 (HsdM) L0310 (HsdS) Unnamed (HsdS) Unnamed (HsdS) Unnamed (HsdS) Unnamed (HsdR, HsdM, HsdS) LlaI LlaDII LlaCI LlaBI LlaKR21 ScrFI LlaDCHI LlaFI Sc. thermophilus Unnamed (HsdS) Unnamed (HsdR, HsdM, HsdS) Unnamed (HsdS) Unnamed (HsdR, HsdM, HsdS) Sth368I


Site-specific recombination

The integration of a prophage genome into the host chromosome is a site-specific integration event between the phage attachment site (attP) and the bacterial attachment site (attB), which is facilitated by a phage-encoded integrase. The first such site-specific integration system described for LAB was that of the lactococcal phage, LC3 (Lillehaug and Birkeland, 1993), and since then, nearidentical systems have been described for three other lactococcal phage, BK5-T, Tuc2009 and r1t, all of which possess integrases belonging to the type I-Int family of site-specific recombinases (Van de Guchte et al., 1994b; Boyce et al., 1995; van Sinderen et al., 1996). The lactococcal phage, TP901-1, has been shown to utilise an integrative system that is significantly different from that of other temperate LAB phage (Christiansen et al., 1994). In this system, the integrase is replaced with a larger resolvase-like protein, whilst the attP and attB sequences are different from those used by other phages (Christiansen et al., 1996). Maintenance of lysogeny

Maintenance of the integrated prophage in the host chromosome requires the repression of transcription of the genes of the lytic life cycle. In , this repression is facilitated through the use of a repressor protein (Ptashne, 1986). Two classes of LAB phage repressors have been identified. Class I repressors consist of polypeptides containing 200–300 amino acids, within which two distinct functional domains can be discerned. The N-terminal domain contains a helix-turn-helix motif, which is assumed to be involved in the binding of the repressor to specific recognition sites on the phage genome. The C-terminal domain is thought to be involved in oligomerisation (co-operative binding of repressor proteins) and has a conserved Ala/Gly motif required for RecA-mediated cleavage of the hinge region between the N- and the C-terminal domains (Little, 1993). A second class of LAB phage repressors has also been identified. This group consists of proteins that are considerably smaller than their Class I counterparts. A helix-turn-helix motif has been identified in most cases, and the absence of the consensus RecA-mediated cleavage site. However, it is possible to induce phages containing class II repressors into the lytic cycle following the SOS response (following treatment with mitomycin C or UV treatment) indicating recA-mediated cleavage of these repressors also (Madsen et al., 1999). The majority of class I phage-repressors belong to lactococcal phage (Van de Guchte et al., 1994a; Boyce et al., 1995; Nauta et al., 1996), but these have also been found in phages that infect Lactobacillus (García et al., 1999); Class II repressors appear to be more common

in Sc. thermophilus phage (Stanley et al., 1997; Neve et al., 1998), although they have been identified in phages that infect Lactobacillus (Kakikawa et al., 2000) and Lactococcus (Madsen and Hammer, 1998). Superinfection exclusion

Expression of the repressor protein from an integrated prophage may also prevent the propagation of superinfecting phage and is said to confer ‘immunity’ on the lysogenised bacterial host. Temperate bacteriophage may also express so-called ‘superinfection exclusion’ activities. The latter differ from phage immunity/ repression systems in that they do not play a role in maintaining the lysogenic state and are not specific for homoimmune phage. Superinfection exclusion systems are well-characterised in temperate phages that infect Gram-negative bacteria such as E. coli and S. typhimurium (Susskind and Botstein, 1978; David et al., 1982; Matz et al., 1982; Yu and Snyder, 1994) and had, until recently, not been identified in LAB phage. Bruttin et al. (1997) characterised the lysogeny module of the Sc. thermophilus phage Sfi21. ORF203 is positioned between the genes encoding the integrase and the repressor, and it was demonstrated that when ORF203 was supplied on a plasmid vector it confers a phage-resistant phenotype against 12 Sc. thermophilus bacteriophages. A superinfection exclusion protein (Sie2009) for the temperate lactococcal bacteriophage Tuc2009 has recently been described (McGrath et al., 2002a). Expression of the Sie2009 protein from a plasmid vector confers a complete phage resistance phenotype on Lc. lactis MG1363 against a number of phages of the 936 species. This phageresistant phenotype was shown to be due to an injectionblocking mechanism, mediated by the Sie2009 protein.

Natural Bacteriophage Resistance Systems in LAB Since bacteriophages were first identified as a major cause of dairy fermentation failure, much research effort has been directed at the development of phageresistance systems for use in the dairy industry. The majority of this research to date has focussed on lactococcal strains, although recently, efforts have also been made with Sc. thermophilus (Moineau, 1999; Coffey and Ross, 2002). Naturally occurring phageresistance systems have been identified in wild-type lactococcal strains. These systems are often encoded on native conjugative plasmids, which has facilitated the generation of novel resistant starter strains through food-grade, gene transfer techniques. These resistance systems have been divided into four main groups on the basis of their mode of action: (1) inhibition


of phage adsorption, (2) blockage of phage DNA injection, (3) restriction/modification and (4) abortive infection (Fig. 3). These systems have been recently reviewed (Dinsmore and Klaenhammer, 1995; Garvey et al., 1995a; Allison and Klaenhammer., 1998; Forde and Fitzgerald, 1999; Coffey and Ross, 2002) and will only be briefly discussed here. Adsorption inhibition

Spontaneous bacteriophage-resistance mutants can be isolated following infection of a bacterial population with a specific phage at a high titre. Analysis of these phage-resistant strains revealed changes in a variety of host-encoded biochemical traits such as carbohydrate composition, masking of cell surface characteristics or assumed changes in specific phage protein receptors that in many instances rendered phage unable to adsorb to the cells (Klaenhammer and Fitzgerald, 1994; Daly et al., 1996). However, these mutants are of only limited value as starter cultures, since their spectrum of resistance tends to be narrow, while their growth characteristics also frequently undergo undesirable alterations. Native plasmid-encoded adsorption inhibition systems have been identified in lactococci and it has been found that these systems can be separated on the basis of the molecular mechanism employed. These plasmids generally direct the synthesis of cell surface antigens or mediate the production of extracellular polysaccharides which shield the host’s phage receptors against phage attachment (Valyasevi et al., 1990, 1994; Schafer et al., 1991; Forde et al., 1999). The genetic basis for adsorption inhibition remains poorly understood and it has been proposed that plasmid-mediated adsorption blocking may not be a true phage defence mechanism but rather a secondary effect of some other cellular function (Forde and Fitzgerald, 1999). Furthermore, because of the instability of plasmids mediating these adsorptioninhibition phenotypes, it is possible that mixed cultures consisting of both phage-sensitive and -resistant cells can develop which may limit the significance of adsorption inhibition as a potent defence mechanism. Injection blocking

Following successful adsorption to the cell wall receptors, an energy-requiring, calcium-dependent, irreversible interaction between the phage and the cytoplasmic membrane occurs, allowing DNA translocation into the cytoplasm (Monteville et al., 1994). As is the case for adsorption inhibition, relatively little is known about injection-blocking mechanisms in LAB. Early reports of such systems in Lc. lactis and Lb. casei did little to elucidate the underlying genetic mechanisms (Marshall and Berridge, 1976; Watanabe et al., 1984). However,

more recently, researchers are beginning to unravel these processes. Garvey et al. (1996) were the first to report the identification of a plasmid-encoded injection-blocking mechanism. They demonstrated that the naturally occurring lactococcal plasmid pNP40 confers an early-acting resistance mechanism against c2. Following infection with c2, no difference in phage adsorption was noted; however, 90% of cells harbouring pNP40 remained viable whereas control strains without pNP40 exhibited essentially no survival. Furthermore, this resistance mechanism could be circumvented by electroporation of phage DNA into resistant host cells, whereby such transfected cells released progeny phages. The authors concluded that the resistance was due to an alteration in a plasma membrane component or components required for c2 infection, but to date the gene or genes responsible for this alteration have not been identified (Garvey et al., 1996). As mentioned earlier, a phage-encoded DNA injectionblocking mechanism acting against a number of 936type phages has recently been reported in Lc. lactis (McGrath et al., 2002a). The sie2009 gene (superinfection exclusion) of the temperate lactococcal phage Tuc2009 is located on the lysogeny module. Data were presented showing that the Sie2009 protein was associated with the cell membrane and its expression left phage adsorption, transfection and plasmid transformation unaffected but prevented plasmid transduction as well as phage DNA replication. The authors also showed that similar prophage genes are widespread not only in lactococcal genomes but also in the genomes of many Gram-positive and -negative bacteria (McGrath et al., 2002a). Restriction/modification

Following successful adsorption and DNA injection, the next obstacle in the LAB phage life cycle is presented by restriction/modification (R/M) systems. First identified in Lc. lactis by Collins (1956), they are found in many bacteria where they act to protect the cell from invading foreign DNA. A R/M system has to exhibit two enzymatic activities, i.e., restriction endonuclease and methylase, and must also be capable of finding its DNA recognition sequence. The methylase modifies the recognition sites on the host’s DNA, thus protecting it from restriction by the endonuclease, whereas unmodified recognition sequences on foreign or invading DNA molecules are specifically digested (Wilson and Murray, 1991). The severity of restriction is dependent on the system and the phage. In general, the efficiency of plaquing (EOP) of the phage decreases logarithmically as the number of sites on the phage DNA molecule increases. To date, four


distinct types of R/M system have been identified (Wilson and Murray, 1991). The majority of R/M systems described in LAB are type II. These systems usually have simple co-factor requirements and molecular organisations, cleaving at or near the recognition site. Furthermore, type II R/M systems are generally composed of two structural genes, the endonuclease (Enase) and the methyltransferase (MTase). The majority of R/M systems that have been characterised in Lactococcus are plasmid-encoded, with ScrFI being a notable exception (Twomey et al., 1997). Another notable exception to the norm is that several lactococcal type II systems contain two methylase enzymes (ScrFI, LlaAI and LlaDCHI) (Moineau et al., 1995; O’Sullivan et al., 1995; Twomey et al., 1997). Little is known about the role of the dual methylases; however, it has been shown that ScrFIBM and ScrFIAM independently confer protection against ScrFI restriction (Twomey et al., 1997). In recent years, several type I R/M systems have been described in LAB. These systems consist of large multimeric proteins consisting of three subunits, HsdR which is responsible for restriction, HsdM which mediates host DNA methylation and HsdS which determines target recognition specificity (Hsd denotes host specificity determinant; Bickle and Kruger, 1993). These types of systems have been identified with increasing frequency in LAB (Table 3) and it has been proposed that more than 50% of lactococcal R/M systems may belong to this category (Schouler et al., 1998b). Schouler et al. (1998a) were the first to describe a type I system in Lactococcus. This system, named Lla14031, was found to be encoded on a native plasmid harboured by Lc. lactis Il1403. Furthermore, introduction of plasmids encoding single HsdS subunits elicited new R/M phenotypes, indicating that these plasmid-encoded HsdS subunits are able to interact with the chromosomally encoded HsdR and HsdM subunits in trans to determine novel R/M specificities. It was proposed that this ‘combinational variation’ may represent a general strategy in which lactococci can acquire R/M systems with novel specificities (Schouler et al., 1998b). O’Sullivan et al. (1999) identified a Type I HsdS subunit-encoding gene on a native plasmid of Sc. thermophilus, NDI-6. It was demonstrated that plasmid-free derivatives of this strain were sensitive to a bacteriophage which displayed no lytic growth on the parent strain, indicating the functional role of this gene in phage resistance. Five chromosomally encoded R/M systems have been identified in Sc. thermophilus, but little is known about their molecular biology (Moineau, 1999). Moineau et al. (1995) have reported that the lactococcal LlaDCHI system, when introduced into Sc. thermophilus, conferred a strong resistance pheno-

type against a number of Sc. thermophilus phages, indicating the possibility of using well-characterised lactococcal systems to protect Sc. thermophilus from phage attack. Chromosomal- and plasmid-encoded R/M systems have also been reported in Lactobacillus strains (Auad et al., 1998; Bourniquel et al., 2002). It is clear that host-encoded R/M systems are an essential component of the cell’s defence systems, thus representing an effective means of protection against phage attack. However, it is possible for phages to breech these defences. In a phage infection of a cell population, a small number of phage genomes may escape restriction and subsequently be modified by the MTase. Such modified phage genomes will be able to propagate to produce a phage population that will be insensitive to the particular R/M system. Furthermore, it has been shown that phages that infect various bacterial species have evolved a number of strategies such as elimination of certain restriction sites from their genomes, modification of bases, production of proteins that inhibit host endonucleases and even the acquisition of methylase genes (Wilson and Murray, 1991), and it is highly likely that LAB bacteriophages have evolved similar tactics. Indeed, Hill et al. (1991) have demonstrated that the lactococcal phage, 50, acquired a functional methylase gene through an in vivo genetic exchange between its genome and the phage resistance-conferring plasmid, pTR2030. This recombinogenic event thus rendered 50 insensitive to pTR2030. Higher levels of phage resistance can be achieved through the combination of two or more R/M systems, or by the combination of an R/M system with other resistance systems such as abortive infection. Abortive infection

Abortive infection (Abi) is a term used to broadly describe any phage resistance mechanism which interferes with intracellular phage development after the phage DNA has entered the cell. Therefore, by definition, Abi systems can interfere with such processes as genome replication, transcription/translation, phage DNA packaging and assembly, and cell lysis. Abis are generally characterised by an attenuated infection due to lower numbers of productive infections and a reduction in the numbers of phage progeny produced (Allison and Klaenhammer, 1998). Abi-mediated resistance typically culminates in the death of the infected cell due to corruption of host functions, as a result of instigation of the defence activity. Many Abi systems have been identified in Lactococcus (Table 4), but the molecular mechanisms underlying many of these systems remain poorly understood. However, reports of studies using phage mutants capable of overcoming Abi systems have provided some valuable


Table 4 Lactococcal abi genes for which DNA sequence data are available

Abi

Location

%GC

Phage species affected

Mechanism

Reference

AbiA AbiB AbiC AbiD* AbiD1* AbiE AbiF* AbiG AbiH AbiI AbiJ AbiK AbiL AbiN AbiO AbiP AbiQ AbiR AbiT AbiU

pTR2030; pCI829 Unspecified; pCI642 pTN20 pBF61 pIL105 pNP40 pNP40, pAJ2074 pCI750 Chromosome pND852 pND859 pSRQ800 pND861 Chromosome pPF144 pIL2614 pSRQ900 pKR223 pED1 pND001

27 27 27 29 26 29, 29 26 29, 27 26 29 30 24 28, 29 31 26 27.5 28 29.8–31.6 33.3, 33.3 26, 25

936, c2, P335 936 936, P335 936 936, c2 936 936 936, c2, P335 936, c2 936, c2 936 936, c2 936, c2 936, c2 936, c2 936 936, c2 c2 936, P335 936, c2, P335

EarlyP335 Late936 Late936 Late936 Latec2 Late936 Early936 Late936/c2/EarlyP335 – Late936 – Late936/EarlyP335 Latec2 – – – Late936/c2 Earlyc2 Late936/P335 –

Hill et al. (1990b) Cluzel et al. (1991) Durmaz et al. (1992) McLandsborough et al. (1995) Anba et al. (1995) Garvey et al. (1995b) Garvey et al. (1997) O’ Connor et al. (1996) Prévots et al. (1996) Su et al. (1997) Deng et al. (1997) Emond et al. (1997) Deng et al. (1999) Prévots et al. (1998) Prévots and Ritzenthaler (1998) Schouler et al. (1998a) Emond et al. (1998) Twomey et al. (2000) Bouchard et al. (2002) Dai et al. (2001)

* AbiD, AbiD1 and AbiF are similar (26–47% identity).

insights into their mode of action (Bidnenko et al., 1995; Dinsmore and Klaenhammer, 1997). It has been proposed that Abi systems may be categorised depending on whether they act prior to or at the level of DNA replication (early) or after replication has occurred (late) (Garvey et al., 1995a). In lactococci, each Abi system appears to be unique in terms of regulation, size and nature of the Abi proteins, number of proteins required for activity and phage affected. Nevertheless, a number of interesting similarities have been noted. Garvey et al. (1995a) reported that all Abi genes tested displayed an atypical G C content of 26–29%, compared to 37% for lactococcal DNA. Furthermore, the proteins encoded by Abi genes have characteristics of cytoplasmic proteins, including the lack of an obvious secretion signal and the presence of hydrophilic, charged residues. To our knowledge, Abi systems in LAB other than lactococci have not been studied, besides a single report of a possible Abi mechanism in Sc. thermophilus (Larbi et al., 1992). Tangney and Fitzgerald (2002) have reported on the introduction of the lactococcal Abi system, AbiA, into Sc. thermophilus. Data were presented which showed that AbiA was effective against six Sc. thermophilus phages at 30 °C and that intracellular phage DNA replication was affected as for phages infecting Lactococcus. However, at 37 or 42 °C, AbiA failed to have any effect on phage propagation, indicating that this system is unsuitable for application in standard fermentations involving Sc. thermophilus.

Engineered Phage Resistance Systems The extensive wealth of knowledge that has been accumulated regarding LAB phage biology has enabled researchers to develop a number of artificial or so-called ‘intelligent’ phage-resistance systems. These utilise specific genes and/or phage or host DNA sequences which are introduced into the cell either on a plasmid vector or by chromosomal integration. The presence of these heterologous DNA sequences or the expression of specific genes may interfere with the phage life cycle, thus providing a level of protection to the host strain (Fig. 3). This topic has recently been extensively reviewed by McGrath et al. (2002b) and will be considered only briefly here. Phage encoded resistance (Per)

Hill et al. (1990a) noted that supplying a specific 50 genomic DNA fragment on a plasmid vector in trans conferred a phage-resistance phenotype on the lactococcal host against 50, and that intracellular phage DNA replication was impeded in strains harbouring this plasmid. DNA sequence analysis revealed that this so-called per-conferring DNA fragment contained a number of direct and inverted repeated sequences, a characteristic of origins of DNA replication. The authors proposed that the per50 fragment was in fact the origin of replication of 50 and that the resistance phenotype conferred was due to the titration of


essential phage DNA replication factors by the plasmidborne oris. The putative ori for the P335-type lactococcal bacteriophage, Tuc2009 (designated ori2009), has also been used in the construction of a per system. The ori2009 sequence is located within a gene coding for a putative replisome organiser protein (rep2009), and a specific protein–DNA interaction between Rep2009 and ori2009 has been demonstrated (McGrath et al., 1999). In a further study, it was shown that cloning multiple copies of the ori2009 sequence on a single plasmid vector increased the level of phage resistance conferred. Furthermore, ori2009-containing plasmids were found to be effective against three other P335-type phages, and DNA sequence analysis confirmed that these three phages utilised oris identical to that of Tuc2009. Other per systems have been constructed for use in lactococci, Sc. thermophilus and Lb. casei (O’Sullivan et al., 1993; Foley et al., 1998; Moscoso and Suarez, 2000). Per systems generally do not confer a complete resistance phenotype and do not represent an insurmountable obstacle to bacteriophage proliferation. The level of resistance conferred has been found to be directly dependent on the copy number of the Perconferring fragments supplied in trans (O’Sullivan et al., 1993; McGrath et al., 2001). Furthermore, the incidence of per-insensitive mutant phage, capable of replicating in Per host strains has been noted by several authors and data have been presented supporting the hypothesis that these mutant phages have, through a recombinant process, acquired new DNA from their host strains (O’Sullivan et al., 1993; Bouchard and Moineau, 2000; McGrath et al., 2001). Antisense mRNA

The utilisation of an antisense mRNA strategy involves cloning of a target gene in the reverse orientation relative to an active promoter. The resulting antisense mRNA produced is assumed to form stable hybrids with the target mRNA, thus inhibiting translation through ineffective ribosome loading, and/or increased sensitivity to RNA-degrading enzymes (Inouye, 1988). Recombinant antisense strategies have been used to successfully control gene expression in animals (Izant and Weintraub, 1984), plants (Ecker and Davis, 1986) and bacteria (Coleman et al., 1984). Kim and Batt (1991b) were the first to describe the use of antisense technology for the control of bacteriophage proliferation in LAB. They cloned a gene of unknown function (gp51C) from 7–9 in the antisense orientation under the control of a constitutive lactococcal pro-

moter. It was found that this plasmid conferred a resistance phenotype on the host lactococcal strain against 7–9 and a number of related phages. Further studies on the application of this technology in lactococci targeted other genes of unknown function, a gene encoding a major coat protein and a transcriptional activator (Chung et al., 1992; Kim et al., 1992a,b; Walker and Klaenhammer, 2000). However, these systems were found to confer only a very modest phage-resistance phenotype. In an attempt to amplify the amount of antisense mRNA generated within the cell, Walker and Klaenhammer (2000) developed the so-called ‘explosive antisense RNA strategy’. In this system, different 31 genes (two middle-expressed and four late-expressed) were cloned between the strong Lactobacillus P6 promoter and the T7 terminator (TT7) in a low-copy number plasmid, containing the putative 31 origin of replication (ori31). Following 31 infection of a cell harbouring this plasmid, ori31 allows for ‘explosive’ plasmid amplification, thereby increasing the levels of antisense transcripts late in the lytic cycle. However, while this strategy significantly increased the concentration of antisense mRNA produced, it had only a minor impact on bacteriophage proliferation, suggesting that the genes targeted were not essential or sufficiently limiting for the 31 life cycle. In a report by McGrath et al. (2001), the effectiveness of targeting a number of different replication module genes was studied. These included genes with putative functions such as a topoisomerase, a single-stranded DNA-binding protein, a replisome organiser protein, a helicase loader, a type II methyltransferase and a Holiday junction resolvase. All constructs tested (except the construct directed at the Holiday junction resolvase) conferred a phage-resistant phenotype on the lactococcal host against Tuc2009. Similar replication module genes were identified in three other phages, Q30, Q33 and ul36, and it was demonstrated that constructs targeting the putative replisome organiser protein and the putative helicase loader provided significant protection against these phages also. Sturino and Klaenhammer (2002) recently developed an antisense system for use in Sc. thermophilus. This system targets a putative helicase gene which is found on the replication module of many Sfi21-type phages and was found to be effective against a number of phages that infect Sc. thermophilus. Gene replacement/insertional mutagenesis

The role of the chromosomally encoded host gene, pip (phage infection protein), the expression of which is required for infection of Lc. lactis subsp. lactis by a


number of phages, has been discussed earlier. A lactococcal strain that is insensitive to attack by c2-type phage has been engineered by replacing the chromosomal pip gene by an allele that had been mutated in vitro (Garbutt et al., 1997). This resulted in the production of a food-grade lactococcal strain that contained no recombinant DNA sequences. This type of engineered phage resistance is advantageous because of its stable, chromosomal location, which obviates the selective pressure required for many plasmid-borne systems. Lucchini et al. (2000) have described a chromosomal gene (orf394) of Sc. thermophilus Sfi11, the expression of which is necessary for infection by all tested phages. A second type of phage-resistant mutant was also isolated which had the phenotypic characteristics of an abortive infection system. In this instance, it was determined that the insertional mutagenesis event occurred adjacent to a hsdR gene encoding the R subunit of a type I R/M system. The authors proposed that this led to the upregulation of the hsdR gene resulting in a more active phage-resistance phenotype (Lucchini et al., 2000). The development of phage-resistant LAB strains by means of chromosomal engineering may represent one of the most promising strategies for the generation of stable, food-grade, strains for industrial use. This approach has advantages over plasmid-borne systems which can be intrinsically unstable and/or may represent a high metabolic load to the cell, leading to deletions within the plasmid or even plasmid loss during non-selective growth.

orf203, which when supplied in trans on a multicopy vector provided resistance to Sc. thermophilus Sfi11 against 12 out of 25 phages tested. The orf203 gene is located between the integrase and the repressorencoding genes on the lysogeny cassette of Sfi21, and while providing protection against heteroimmune phage it does not protect against Sfi21. The deduced protein product of orf203 contains a hydrophobic N-terminus, indicating that this protein may be associated with the host cell membrane; however, the exact mechanism by which this orf mediates a phageresistance phenotype remains to be elucidated. Cruz Martin et al. (2000) recently described the construction of a phage-resistant food-grade strain of Lb. casei. A single copy of the A2 repressor gene was integrated into the Lb. casei chromosome using a site-specific integration vector, with subsequent ‘clearing’ of all non-food-grade DNA by in-trans expression of a -recombinase gene. The resulting strain was completely immune to A2 infection during milk fermentation. It is likely that the naturally occurring phage-resistance phenotypes attributed to some LAB strains are due, at least in part, to the expression of superinfection exclusion and immunity factors by (defective) prophages. The identification and characterisation of such genes may facilitate the development of new broad-range phage-resistance systems for many LAB.

Recombinant superinfection exclusion/immunity systems

A genetically engineered form of abortive infection has been described in Lactococcus (Djordjevic et al., 1997). This system used a phage-inducible promoter in combination with the LlaI restriction/modification system from a lactococcal plasmid. The middle phageinducible promoter (31p) was cloned upstream of the lethal LlaIR restriction cassette so that infection of a cell harbouring this plasmid with 31, causes the lethal gene product of LlaIR to be produced, resulting in death of the host cell before the infecting phage has a chance to reproduce itself. However, as was found for per systems, 31 mutants considerably lesssensitive to the 31p–LlaIR system were isolated when phages were propagated on these strains (Djordjevic and Klaenhammer, 1997). DNA sequence analysis revealed that a mutation had occurred, resulting in a single amino acid transversion in a transcriptional activator of 31p (ORF2). Furthermore, the ability of these mutant phages to induce the native 31p promoter was demonstrated to be reduced, compared to that of the parent 31. Pairing the 31p–LlaIR system with other abortive infection systems, Per31 and AbiA, resulted in a reduction in numbers of 31

The recently described superinfection exclusion gene, sie2009 (McGrath et al., 2001) has been discussed earlier. When cloned under the control of a constitutive promoter on a high-copy number plasmid, sie2009 mediates a phage-resistance phenotype in Lc. lactis against bacteriophages of the 936-type species. Adsorption and electron microscopic analyses demonstrated that bacteriophages adsorbed to cells expressing Sie2009 as readily as they did to a control strain, whilst intracellular phage DNA replication was demonstrated not to occur in Sie2009-expressing strains. Analysis of the deduced Sie2009 amino acid sequence revealed that the protein contains a putative transmembrane-spanning domain while cell fractionation and SDS-PAGE demonstrated that the Sie2009 protein is in fact associated with the cell membrane. Furthermore, plasmid transduction experiments demonstrated that the Sie2009-mediated phage resistance phenotype is due to an injectionblocking mechanism (McGrath et al., 2001). Bruttin et al. (1997) described a gene from the lysogeny module of the Sc. thermophilus phage Sfi21,

Bacteriophage-triggered defence


below detectable limits (Djordjevic and Klaenhammer, 1997). The ongoing research into the natural phage defence mechanisms as well as phages infecting other genera of LAB will undoubtedly pave the way for the development of similar resistance systems. The development of engineered phage-resistance systems in LAB has been the focus of intensive research since the early 1990s. Besides the obvious benefits, such as the development of phage-resistance systems with potential industrial applications, it has also led to an increased understanding of bacteriophage–host relationships, and in turn has stimulated research in other areas. It would appear that the use of single, strong phage-resistance systems is unsuitable for industry, due to the selective pressure applied for the emergence of insensitive phages. More industrially robust strains could be developed by the stacking of two or more such systems in a single strain or by the introduction of engineered-resistance systems into naturally phage-resistant strains. At present, the use of the majority of these engineered-resistance systems is restricted in the dairy industry due to their recombinant origins. However, they will be readily available if and when regulations allow their applied use. Furthermore, some of the systems outlined above may be adapted using food-grade methods for immediate use under current legislative guidelines in certain jurisdictions. Current status and future perspectives

The extensive knowledge that has been accumulated about the physiology and genetics of LAB and their phages has led to a detailed understanding of many aspects of the phage–host relationship. In the past 10 years or so, the advent of modern molecular genetic techniques, such as automated DNA sequencing and the use of bioinformatics has resulted in a wealth of biological information pertaining to these organisms and their infectious parasites. This knowledge has been utilised not only to generate phage-derived systems with the potential to prevent phage infections in dairy fermentations (discussed above), but it was also applied to develop sophisticated genetic tools (Raya and Klaenhammer, 1992; Raya et al., 1992; Nauta et al., 1997; Stoll et al., 2002). Additional biotechnological applications may be envisaged in the light of the recent renaissance of phage therapy, in which complete phage or phage-encoded lytic enzymes may be used to treat certain bacterial infections (Biswas et al., 2002; Schuch et al., 2002; Stone, 2002). It is clear that gene expression from lysogenic prophages on bacterial chromosomes significantly

contributes to the host cell phenotype, from immunity/exclusion systems and lysogenic conversion to virulence (Susskind et al., 1971; Waldor, 1998; Desiere et al., 2002). Indeed, Desiere et al. (2001) published a report on the genome of a highly pathogenic strain of Sc. pyogenes which contains eight prophage elements, two of which harbour genes coding for likely virulence factors as well as sharing extensive DNA sequence homology to two LAB bacteriophages. These findings indicate that the substantial amount of knowledge amassed on phages infecting dairy bacteria may be useful in gaining insights into the molecular mechanisms underlying virulence in certain infectious bacteria. Conversely, it is known that commensal and probiotic bacteria also carry prophages on their genomes, which introduces the intriguing possibility that these prophages may contain functional genes that confer an advantage on these hosts and possibly play a role in probiotic functionality.

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Secondary and Adjunct Cultures J.-F. Chamba, Institut Technique Francais de Fromages, La Roche sur Foron, France F. Irlinger, INRA, Thiverval-Grignon, France

Introduction Two types of cultures are used in cheesemaking: primary and secondary. The primary cultures include all the starter lactic acid bacteria and are involved in acid production during cheese manufacture and in cheese ripening. The secondary and adjunct cultures involved include yeasts, e.g., Geotrichum candidum, Debaryomyces hansenii, moulds, e.g., Penicillum camemberti, P. roqueforti, and bacteria, e.g., Corynebacterium, Staphylococcus, Micrococcus, Propionibacterium sp. and heterofermentative lactobacilli and are involved only in cheese ripening. Except for Propionibacterium and the heterofermentative lactobacilli, the secondary cultures grow mainly on the cheese surface (see ‘Bacterial Surface-ripened Cheeses’, Volume 2). They are called secondary cultures to distinguish them from the primary acid-producing starters and are as important as the primary ones in those cheeses in which they are found. In the past, only a small number of secondary cultures or adjuncts was used, mainly in traditional cheeses made from raw milk. For example, in blue-veined cheeses, P. roqueforti was added to the curd before moulding in the form of grated, mouldy bread. In fact, the use of moulds as adjuncts in cheesemaking pre-dates the commercial use of lactic acid starters. Traditionally, the secondary flora originated in either the milk, the cheesemaking utensils and/or the cheese factory environment. Like the production of traditional smearripened cheese, mature cheeses were smeared, i.e., washed with dilute solutions of NaCl, which may also contain some of the surface microflora (see ‘Bacterial Surface-ripened Cheeses’, Volume 2), before young ones. Therefore, the cheese surface microorganisms were transferred from the old to the young cheeses. Since then, improvement in the microbiological quality of raw milk, the use of thermisation and bactofugation of milk, the high level of hygiene and practice modifications in cheese factories have reduced the sources of the indigenous secondary flora. Cheese has become more bland in taste and therefore cheese factory personnel became aware of the decisive role played by the secondary flora in producing good quality cheese and this, in turn, has increased the demand for secondary starters. Today, this need is true of most cheese varieties. In the present

chapter, the most important groups of secondary flora, the species found in cheeses, the properties used in their selection, and the form and use of these cultures as adjuncts will be described. Information on how these cultures are produced is very difficult to obtain as it is mainly propriety to the institution producing the culture. Consequently, it cannot be reviewed in any detail.

Yeast Yeasts are encountered and used as culture adjuncts in many cheeses. They are used mainly in mould and bacterial surface-ripened cheeses because they promote the growth of other microorganisms. For example, yeasts are used as adjuncts in the French cheeses, Brie, Camembert, Pont l’Evêque, Maroilles and Reblochon, in the Belgian cheeses, Herve and Limburger, and in the Italian cheese, Tallegio. Yeasts are also used in blueveined cheeses such as Danablu (Denmark), Cabrales (Spain), Fourme d’Ambert (France), Gorgonzola (Italy) and Stilton (UK). Species found in cheeses

The species of yeasts isolated most frequently from cheeses are listed in Table 1. Geotrichum sp. are often described as intermediate between mould and yeast and is now recognised as a yeast (Barnett et al., 1990). Yeasts colonise numerous cheeses, particularly their surfaces. They can grow during the early stages of cheesemaking, e.g., during whey draining after moulding and before salting. Commonly, their population reaches 106–108 cfu cm 2 of cheese surface during the first 5 days and remains at this level throughout ripening. Generally, their number in the interior of the cheese is 100 or 1000 times lower. In traditional cheeses, the source of the yeasts is raw milk, utensils, cheese factory environment, brine and/or use of natural whey starters used in production (Zambonelli et al., 1996). However, Baroiller and Schmidt (1990) have shown that the great diversity of yeast species in milk for Camembert cheese was drastically reduced by the selective action of processing. Today, the use of yeasts as adjuncts is a common practice in modern cheese factories; they are added to the cheese milk and/or are used in the smear preparation.



192 Secondary and Adjunct Cultures

Table 1 Main yeast species encountered in/on the surface of cheese Perfect form

Imperfect form

Galactomyces geotrichum Debaryomyces hansenii Kluyveromyces marxianus var. lactis Kluyveromyces marxianus var. marxianus Pichia membranifaciens Pichia fermentans Sacchoromyces cerevisiae Sacchoromyces dairensis Torulospora delbrueckii Yarrowia lipolytica Zygosaccharomyces rouxii

Geotrichum candidum Candida famata Candida sphaerica Candida kefyr Candida valida Candida lambica Candida robusta Candida dairensis Candida colliculosa Candida lipolytica Candida mogii

Other minor species: Candida catenulata, Candida intermedia, Candida rugosa, Candida sake, Candida vini, Candida zeylanoides. From Nunez et al., 1981; Baroiller and Schmidt, 1990; Nahabieh and Schmidt, 1990; Bârtschi et al., 1994; Eliskases-Lechner and Ginzinger, 1995b.

Useful properties in selecting yeast adjuncts Effect on appearance of cheese surface

The yeast flora contribute directly or indirectly to the appearance of cheese. For example, G. candidum varies considerably from slimy cream to velvet mould-like depending on the strain. Consequently, the growth behaviour of G. candidum is of great importance in choosing the correct strain for the type of cheese being produced. Utilisation of residual sugars and lactate de-acidification activity

The yeasts encountered on the surface of cheese show varied abilities to metabolise sugars, lactate and citrate (Eliskases-Lechner and Ginzinger, 1995b). Because Kluyveromyces marxianus and Debaryomyces hansenii are able to ferment lactose, their use as adjuncts is very common. G. candidum assimilates galactose and lactate which is of paramount importance in the ripening of mould and bacterial smear-ripened cheese. The degradation of lactate results in de-acidification at the cheese surface and the increased pH, in turn, stimulates the growth of moulds and corynebacteria. Consequently, the de-acidification activity is always taken into account in selecting yeast strains as culture adjuncts for soft cheese. Proteolytic activity

Yeasts show a large diversity in proteolytic activity between species and strains of the same species. They have caseinolytic, aminopeptidase and carboxypeptidase

activities (Schmidt and Lenoir, 1980; Schmidt et al., 1993). It is generally recognised that Yarrowia lipolytica, Saccharomyces cerevisiae and K. marxianus subsp. marxianus are more proteolytic than D. hansenii (Vannini et al., 2001). In addition, G. candidum has higher aminopeptidase activity than P. camemberti (Molimard et al., 1994). Naturally, proteolytic activity is also used in the selection of strains by suppliers of adjunct cultures. The high tyrosinase activity of some strains of Y. lipolytica is thought to be responsible for the production of brown pigments below the cheese surface (Van den Tempel and Jakobsen, 2000; Carreira et al., 2001) and consequently this activity is also assessed in commercial strains. Lipolytic activity

Yeasts contribute to lipolysis in cheeses, and Y. lipolytica has the highest lipase activity of all yeast found in cheese (Schmidt et al., 1993). In particular, Y. lipolytica is much more lipolytic than D. hansenii and S. cerevisiae (Van den Tempel and Jakobsen, 2000). However, it is not used commercially. G. candidum lipase preferentially releases oleic acid from milk fat (Gripon, 1993). Although the contribution of Penicillium, Staphylococcus and Corynebacterium spp. to lipolysis in mould, smeared and blue-veined cheese is greater than that of yeasts, this activity is generally used as a criterion in the selection of yeast by culture suppliers. Production of aroma

Yeasts produce aroma compounds. They can produce ethanol, aldehydes and esters and they degrade amino acids to ammonia and the corresponding keto acid. Further metabolism provides numerous compounds such as alcohols, esters, methyl ketones and carbonyl compounds. G. candidum produces much more aromatic compounds from methionine than other yeasts tested, including D. hansenii and K. lactis (Demarigny et al., 2000; Spinnler et al., 2001). Nevertheless, the relationship between compounds identified by GC–MS and the sensorial characteristics of cheese curd inoculated with selected yeast were not very consistent (Martin et al., 2001). Thus, ‘the lack of progress in describing cheese flavour in precise chemical terms’, emphasised by Fox et al. (1993), remains topical in this area. Starter suppliers frequently use sensorial analysis of model cheeses instead of chemical analysis to select strains of the surface flora. However, the Degussa Company use both methods to characterise their strains of surface adjuncts. Interactions with other microorganisms

Generally, yeasts promote the growth of G. candidum, which, in turn, reduces the occurrence of undesirable

Secondary and Adjunct Cultures 193

moulds, e.g., Aspergillus, Mucor and Penicillium spp. Therefore, the ability to inhibit Mucor spp., which produces thin strands of mycelial growth, the so-called ‘cat hair’ defect on cheese (in French ‘poil de chat’), is one of the criteria used in the selection of Geotrichum strains (Gueguen and Schmidt, 1994). Hansen and Jakobsen (1998) have shown that the growth of P. roqueforti is not affected by D. hansenii, that its growth and sporulation are inhibited by D. marxianus subsp. marxianus and that its growth rate and colour formation are increased by S. cerevisiae. The interaction between yeast and other surface microorganisms, such as moulds and bacteria, is taken in to consideration by suppliers in the selection of yeast strains. Moreover, G. candidum can inhibit Listeria monocytogenes by the production of two components: D-3phenyllactic acid and D-3-indolelactic acid (Dieuleveux et al., 1998). This property offers an interesting criterion for the selection of cheese adjuncts to increase the safety of mould and smeared cheeses. Forms and use of adjunct culture

For G. candidum, the diversity in the forms of adjunct produced by culture suppliers is generally large. Besides appearance (slimy to mould-like, and colour), several other activities are taken into account, including de-acidification, proteolysis, lipolysis, aroma production (analytical and/or sensorial), sensitivity to NaCl and ability to inhibit Mucor spp. G. candidum cultures are sold by several companies such as Clerici-Sacco (freezedried), Degussa and Chr. Hansen (liquid), Rhodia Food (liquid, freeze-dried), Standa Industries (liquid) and Wiesby (liquid, freeze-dried). Moreover, some Dairy Research or Technical Centres provide Geotrichum cultures for use in their respective countries, e.g., Switzerland. Some major cheese companies also produce ‘in-house’ cultures. These cultures can be added directly in the cheese milk or sprayed on the cheeses, generally after salting. The manufacturer’s recommendations are about 105 cfu ml 1 of cheese milk or 106–107 cfu ml 1 of suspended cells for spraying. Only a few species of yeasts are available commercially and this does not reflect the diversity of yeast found on the surface of cheese. In fact, three yeast species, D. hansenii, K. marxianus and S. cerevisiae, are much more frequently sold than others. Torulospora delbrueckii (Candida valida) is used occasionally and each yeast is sold by a different company. Sometimes, yeasts are available as mixed cultures of yeasts and as other surface microorganisms such as G. candidum or Brevibacterium linens. S. cerevisiae is recommended for blue-veined cheese because high CO2 production by it from lactose pro-

duces a more open textured cheese. These cultures are added to the cheese milk and the ‘smear’ solutions at levels of about 105–106 cfu ml 1

Moulds Moulds are used mainly as adjuncts in two types of cheese, viz., mould surface-ripened soft cheese, e.g., Brie, Camembert or goats’ milk cheeses (France), and blue-veined cheeses, e.g., Bavarian Blue (Germany), Bleu d’Auvergne, Bleu des Causses and Roquefort (France), Cabrales (Spain), Gorgonzola (Italy), Danablu (Denmark) and Stilton (UK). Moreover, a few varieties of semi-hard cheeses, e.g., Tomme (France and Switzerland) and Toma (Italy) are also surface-ripened with moulds. Species found in cheeses

The white mould, P. camemberti, and the blue-green mould, P. roqueforti, are the two main species of mould used as adjuncts. Previously, P. camemberti was called P. caseicolum Bainier or P. candidum, for strains which remain white during growth and P. album for strains which develop a grey-green colour. Today, these two phenotypic forms have been amalgamated into one species, P. camemberti Thom (Pitt, 1979). Other Penicillium spp. growing on cheese also have a white mycelium; P. thonii, P. nalglovensis and P. verrucosum. P. roqueforti strains can exhibit variations in colour from yellowish-green (called viride in Italy) to dark green. Sometimes, it can be confused with a similarly coloured mould, P. verrucosum var. cyclopium; however, the latter species produces a strong musty odour. This spoilage mould can contaminate P. roqueforti cultures. Without the use of a selected mould adjunct, many other Penicillium spp. may be found in hard, semi-hard and semi-soft cheeses such as Cheddar, Danbo, Port Salut or Bel Paese. P. commune and P. nalgiovensis are the most common (Lund et al., 1995). The appearance and properties of P. caseifulvum allow its use as a cheese adjunct instead of P. album. Other moulds can grow on cheeses but they are generally undesirable. Nevertheless, some species occur spontaneously and are desirable on the surface of certain cheeses, e.g., Chrysosporum sulfureum is responsible for yellow spot formation on St. Nectaire cheese. In some cases, these moulds are produced as adjuncts, e.g., P. nalgiovensis, P. commune, Trichothecium domesticum (Cylindrocarpon sp.) and Verticillium lecanii. C. sulfureum and Sporendonema casei (red-orange spot) encountered on some semi-hard cheeses are no longer produced by starter companies (Ratomahenina et al., 1995). Rhizomucor spp. are generally considered to be spoilage moulds, producing the ‘cat


hair defect’ in most cheeses; however, it is a desirable mould on Tomme de Savoie and St Nectaire cheese. Consequently, three species R. fuscus, R. plumbeus and R. sinensis are produced by ITFF, a cheese technical centre in France. Useful properties for which to select moulds as adjuncts

olism of Fatty Acids in Cheese’, Volume 1). These compounds contribute to the typical flavour of mouldripened cheeses (Cerning et al., 1987). Consequently, the lipolytic activity of P. camemberti and P. roqueforti is an important criterion in their selection, and this activity is always indicated in the product specification of the supplying companies.

Appearance of mould on/in cheeses

Production of aroma

Moulds contribute directly to the appearance of the cheese surface or, in the case of blue-veined cheeses, to the appearance of the cheese body. The growth behaviour of P. camemberti varies according to the strain. The colour and length, and density of the mycelium are very important criteria in choosing a strain to be used as an adjunct. The colour of P. roqueforti is also of paramount importance in selecting strains. Generally, strains showing light blue or yellowish colours are used for Gorgonzola cheese whereas dark green strains are used in Danablu, Bleu des Causses, Roquefort and Stilton cheese. Naturally, this diversity in the appearance of Penicillium strains is indicated in the catalogues of mould suppliers.

Besides methyl ketones and secondary alcohols, many esters, aldehydes, volatile amines and ammonia also contribute to the aroma of mould-ripened cheeses. The typical mushroom note of Brie and Camembert flavour produced by P. camemberti is mainly due to 1-octen-3-ol (Gripon, 1993). Like yeasts, mould suppliers frequently use sensorial analysis of cheese models to characterise their strains and the aromatic notes or profiles produced are given in the product sheets.

De-acidification activity

P. camemberti and P. roqueforti are able to utilise lactic acid as a carbon source. Therefore, their growth leads to an increase in pH and proteolysis of cheese and consequently causes the cheese to soften. This property is also indicated by culture suppliers in their product sheets. Proteolytic activity

Both P. camemberti and P. roqueforti have endopeptidase and exopeptidase activities which make a major contribution to proteolysis in cheese. Consequently, the ripening process begins on the surface of the mould-ripened cheese (Gripon, 1993). The extracellular proteolytic systems of these two moulds are somewhat similar and they hydrolyse s1-, - and -caseins. Moreover, their peptidases release free amino acids and have debittering activity. Amino acids are catabolised with the production of ammonia and other volatile compounds (Cerning et al., 1987). Of course, these proteolytic activities are considered by mould culture suppliers but, generally, methods used in this characterisation are only indicative. Azocasein has been suggested as a substrate for determining proteolytic activity (Larsen et al., 1998). Lipolytic activity

Lipolysis is much more extensive in mould-ripened cheeses than in other varieties, especially blue-veined cheeses and the main agents are Penicillium spp. Their lipolytic activity varies greatly according to the strain. Methyl ketones and their corresponding secondary alcohols are produced by -oxidation of free fatty acids, produced by lipolysis (see ‘Lipolysis and Catab-

Interactions with other microorganisms

Besides the interaction with yeast described above, Hansen and Jakobsen (1997) have shown positive and negative interactions between 20 strains of P. roquefortii and 15 strains of Leuconostoc, Lactococcus, Lactobacillus and Streptococcus spp., many of which were strainspecific. The stimulation of P. roqueforti is mainly due to the release of amino acids, like arginine and leucine, by the lactic acid bacteria. Fast growth of P. camemberti prevents the establishment of Rhizomucor spp. which produce a ‘cat hair’ defect on the cheese surface. This property is also used by mould suppliers in the selection of suitable strains. The elevated pH of the cheese surface induced by the growth of P. camemberti, in turn, promotes the growth of coryneform bacteria. Moulds produce tyramine, histamine and tryptamine by decarboxylation of the corresponding amino acid but these biogenic amines are metabolised by coryneform bacteria such as B. linens which possess deaminase activity (Leuschner and Hammes, 1998). Production of mycotoxins

P. roquefort produces several mycotoxins whereas P. camemberti produces only one, chloplazonic acid, but there is little risk to human health because the toxins are present in mould-ripened cheeses at very low levels (Gripon, 1993; ‘Toxins in Cheese’, Volume 1). Since this production is strain-specific it must be taken into account in the selection of moulds for use as cheese adjuncts. Form and use of mould adjuncts

The number of strains of P. camemberti available from suppliers varies from 3 to 16, with the largest number


being available from Rhodia Foods. Other P. camemberti suppliers are Clerici-Sacco, Chr. Hansen and Degussa. The colour and the length and density of the mycelium are always shown on product specification sheets. Growth rates and de-acidifying, proteolytic, lipolytic and anti-Rhizomucor activities are also given. These are sold as liquid or freeze-dried cultures. For P. roqueforti, 2–7 strains are available, depending on suppliers, which differ in colour, growth rate, NaCl and temperature sensitivities, proteolytic and lipolytic activities and their effects on the texture and aroma of cheese. Companies producing P. roqueforti cultures are Clericci-Sacco, CSL, Chr Hansen, Rhodia Food and Wiesby. The latter company also has a white mutant of P. roqueforti, which produces the typical flavour of Blue cheese without the blue colour. These are sold as liquid, dried or freeze-dried cultures. Moreover, some technical centres such as LIP, Aurillac, France, produce mould cultures, especially P. roqueforti for private users. Other moulds, e.g., P. album, P. nalgiovensis, Trichothecium domestimum (cylindrocarpon) and Verticillium lecanii are produced by Rhodia Foods. The oldest method in which grated mouldy bread is used to inoculate the curd of blue-veined cheese remains topical in few cases. Traditional methods using small units on agar surface or other solid medium to produce Penicillium conidiaphores are still used. Nevertheless, spore production by submerged cultivation with sufficient oxygen supply has been developed for P. camemberti (Bockelmann et al., 1999). Submerged batch fermentation provides high spore yields, short fermentation intervals and automation. P. camemberti cultures can be added directly to the cheese milk and/or sprayed on the cheese, generally after salting. The general recommendation is ⬃106–107 spores L 1. P. roqueforti cultures are generally inoculated directly into the cheese milk at a level of ⬃107 spores L 1.

Coryneform Bacteria and Staphylococci These bacteria are present on the surface of many cheeses. They are used as adjuncts mainly on smeared soft- and semi-hard cheeses, e.g., Epoisse, Livarot, Morbier and Munster in France, Limburger in Belgium, Bel Paese and Tallegio in Italy, Romadour and Tilsit in Germany, Raclette and Appenzeller in Switzerland, Brick and Monterey in the USA (Table 2). Some cultures, especially B. linens or Staphylococcus spp. are used as an ‘enzyme bag’ in ripened cheese without surface microorganisms. Generally, these bacteria are isolated from soft or semi-soft cheeses such as Camembert, Munster, Livarot or Gubbeen, from blue-veined cheeses but also from hard cheeses such as Gruyère, Beaufort and Comté (Piton-Malleret and Gorrieri, 1992). Generally, counts

of 107–109 cfu cm 2 are reached on soft smear cheeses within the first 2 weeks of ripening, and 1013 cfu g 1 of rind in Gruyère cheese within the first 3 weeks of ripening. The bacterial populations remain constant thereafter until the cheese is consumed (Reps, 1993; Eliskases-Lechner and Ginzinger, 1995a). The flora is composed of two principal Gram-positive groups: coryneform bacteria (irregularly shaped, catalase-positive rods) and staphylococci (catalase-positive cocci). These two groups have certain physiological properties which permit their growth on the cheese surface; they are aerobic, alkalophilic, mesophilic and salt-tolerant and cannot grow under acid conditions (see ‘Bacterial Surface-ripened Cheeses’, Volume 2). It has been observed that the cultivation methods used in the laboratory are always selective and the classification of coryneform and staphylococci groups is equivocal and confusing, because it is based on phenotypic characteristics (Seiler, 1986; Kämpfer et al., 1993; Irlinger et al., 1997). In recent years, molecular approaches, such as ribotyping, amplified fragment polymorphism and randomly amplified polymorphic DNA, have been developed and could give significant insight into specific isolates and dominant microbial populations during cheese manufacture. However, these techniques are laborious and time-consuming for monitoring population dynamics and have not been used to any great extent in classifying the microorganisms. Coryneform bacteria

Coryneform bacteria include organisms from the genera Arthrobacter, Brevibacterium, Corynebacterium, Microbacterium. The term has no taxonomic significance except that bacteria in these genera are generally small irregularly shaped rods. For a long time, B. linens was considered to be the typical, orange-red pigmented, red smear-cheese bacterium due to its role in colouring the surface of the cheese and its ability to produce typical flavours. For that reason, B. linens is the main adjunct culture available and used for smear-cheeses. Nevertheless, some authors have cast doubt on the exclusive importance of B. linens in the cheese smear. The maximum proportion of B. linens found in Tilsit cheese ranges from 0 to 15% (Eliskases-Lechner and Ginzinger, 1995a). This species was not isolated from the surface of Gubbeen, an Irish smear cheese, even though the cheese surface was deliberately smeared with this species at the beginning of ripening (Brennan et al., 2002); however, 9.3% of isolates were B. linens-like but had different Pulse-Field Gel Electrophoresis patterns than the deliberately inoculated strain. Moreover, none of the other brevibacteria isolated from cheese, e.g., B. casei, have been found on Tilsit, Brick and other German cheeses or on

196 Table 2 Diversity and identification of the smear bacteria flora from different cheeses Number of isolates

Characterisation methods

Identification of Gram-positive bacteria

Limburger, Romadour, Weinkäse, Handkäse, Munster, Camembert, Appenzeller

372

Phenotypic from Seiler (1986)

61% Yellow and white colonies of which: – 29.5% Corynebacterium: mainly C. ammoniagenes, C. variabilis and unidentified – 33% Arthrobacter: A. nicotianae and unidentified – 1% Rhodococcus sp. 31% Brevibacterium: B. linens 8% Yellow and white non-clustered isolates unidentified

Seiler (1986)

Tilsit

386


45% Arthrobacter: mainly A. globiformis, A. citreus, A. nicotianae and some unidentified 24.5% Corynebacterium: mainly C. ammoniagenes, C. variabilis and unidentified 21% Brevibacterium: mainly B. linens 5% Curtobacterium: mainly Cu. poinsettiae, Cu. betae, Cu. oxydans and Cu. helvolum 2% Microbacterium: mainly Mb. imperiale 2% Clavibacter: mainly Cl. insidiosum 0.5% unidentified

Eliskases-Lechner and Ginzinger (1995a)

Brick cheeses: Limburger, Romadur, Weinkäse, Munster, Harzer, Tilsit

195


41% Corynebacterium: mainly C. ammoniagenes, C. variabilis and unidentified 24%: Arthrobacter: mainly A. nicotianae and unidentified 17% Brevibacterium: mainly B. linens 10% Rhodococcus: mainly R. fascians 5% Microbacterium: mainly Mb. imperiale, Mb. oxydans and unidentified 2% Cellulomonas: C. cellulans

Valdès-Stauber et al. (1997a)

Tilsit

unpublished

Phenotypic from Bergey (1986) 16s rRNA sequencing (some isolates)

5–15% Staphylococcus: mainly S. equorum, small numbers of S. saprophyticus and S. sciuri 75–95% Coryneform bacteria: mainly unidentified, B. linens and Arthrobacter sp.

Bockelmann et al. (1997a)

Gubbeen

400

Molecular identification methods: RAPD-PCR, PFGE

2.5% Staphylococcus sp. 49% Corynebacterium casei 25.5% Corynebacterium mooreparkense 12.5% Microbacterium gubbeenense 1.25% Corynebacterium flavescens 9.25% unidentified coryneforms

Brennan et al. (2002)

Cheese

Authors


Gubbeen (Seiler, 1986; Eliskases-Lechner and Ginzinger, 1995a; Bockelmann et al., 1997a; Brennan et al., 2002). The occurrence of yellow-pigmented Arthrobacter strains, especially A. nicotianae, on surface-ripened cheeses has been reported (Marcellino and Benson, 1992; Valdès-Stauber et al., 1997). Yellow coryneform isolates from several varieties of Austrian cheese were classified as A. globiformis (Eliskases-Lechner and Ginzinger, 1995a). This was confirmed by Bockelmann et al. (1997a), who identified several yellowpigmented strains as A. globiformis. Some corynebacteria are major components of the microflora of surface-ripened cheese. The dominant genus differs depending on the type of cheese studied. In Comté, only 12% of the surface flora was assigned to the genus Corynebacterium (Piton, 1988; Piton and Fontanier, 1990) while in Brick cheeses it was the dominant genus (nearly 50% of isolates) (Valdès-Stauber et al., 1997). In cheese smears, C. ammoniagenes (previously named Brevibacterium ammoniagenes) and C. variabilis (previously named Caseobacter polymorphus) were reported when the key of Seiler (1986) was used to identify the isolates. Two newly described species, C. mooreparkense and C. casei, were isolated from the surface of an Irish smear-ripened cheese. C. ammoniagenes and C. variabile were their nearest known phylogenetic neighbours (Brennan et al., 2001a, 2002). The genus, Brachybacterium, including three new species isolated from milk and Beaufort, Gruyère and Camembert cheeses (Brachybacterium nesternkovii, B. alimentarium and B. tyrofermentans; Gvozdiak et al., 1992; Schubert et al., 1996; Lefresne, 2000), may be present on other smear cheeses. Brachybacteria are highly salt-tolerant and yellow in colour. However, the incidence of Brachybacteium on cheeses has not been studied systematically. Microbacterium sp. are widely distributed in various environments and three species have been isolated from milk products or cheese, M. lacticium (Collins et al., 1986), M. liquefaciens (Collins et al., 1983) and M. gubbeenense (Brennan et al., 2001b). Moreover, Eliskases-Lechner and Ginzinger (1995a), ValdèsStauber et al. (1997) and Brennan et al. (2002) isolated 8, 7 and 50 Microbacterium strains from Tilsit, Brick and Gubbeen cheeses, respectively. Brennan et al. (2002) showed that, generally, M. gubbeenense was isolated more frequently late in ripening. Until the mid-1970s, micrococci were erroneously considered to constitute a major portion of the secondary flora of cheeses and to be important for flavour development. The Micrococcus strains isolated from various cheeses were most probably misclassified staphylococci. However, some reports confirm that micrococci, especially Kocuria varians and Micrococcus

luteus, play a role in goats’ milk cheese, but compared to staphylococci their numbers are not very high and they decrease rapidly during ripening (Michaux, 1983; Massa and Turtura, 1989; Vernozy-Rozand et al., 1996; Caceres et al., 1997). Staphylococcus

Many investigators have noted the predominance of novobiocin-resistant and coagulase-negative staphylococci in cheeses, particularly in hard varieties, made from ewes’ or goats’ milk (Delarras and Laban, 1981; Garcia et al., 1988; Massa and Turtura, 1989). Coagulasenegative staphylococci are found mainly in high cell numbers early in ripening and they make up 5–25% of total surface cell counts (Bockelmann et al., 1997a). Staphylococcus spp. are replaced by coryneform bacteria after about 15 days ripening (Brennan et al., 2002). The prevailing species in soft, smeared cheeses are Staph. equorum, Staph. vitulinus and Staph. xylosus (Irlinger et al., 1997). This last species is commercially available as an adjunct and, is also used as a starter in fermented sausage. A new coagulase-negative and novobiocinresistant Staphylococcus, S. fleuretti, has been isolated from raw goats’ milk cheese (Vernozy-Rozand et al., 2000). In addition, a new subspecies, Staph. succinus subsp. casei, isolated from a Swiss surface-ripened cheese, has been described (Place et al., 2002). This species has been used as a starter component in typical Swiss cheeses. Naturally, it is necessary to characterise these strains carefully and to confirm that they are food grade. Useful properties for selecting surface bacteria as adjuncts Growth

All species encountered on the cheese surface are salt tolerant, e.g., B. linens tolerates up to 15% of NaCl (Ferchichi et al., 1985; Collin and Law, 1989). Their minimal pH for growth and their sensitivity to the ripening temperature are the main determinants in colonising the cheese surface. The growth rate at pH 5.8 differs significantly between strains of B. linens. Staphylococci promote the growth of other smear bacteria at the beginning of ripening because they grow rapidly at pH 5.5 and below (Bockelmann et al., 1997a). Consequently, sensitivity to pH and temperature are criteria used in the selection of surface bacteria strains. Effect on the colour of cheese surface

The colour of the cheese surface is an important characteristic of smear-ripened cheeses. The contribution of the surface bacteria varies according to genus, species and strain. Most strains of B. linens produce distinctive red-orange carotenoid-type pigments but numerous


strains are non-pigmented. Corynebacterium spp. are similar. Arthrobacter are generally yellow-pigmented and produce the typical red-brown colour of smearcheeses by conversion of yellow pigments (Bockelmann et al., 1997a). Brachybacterium spp. found in cheeses are yellow-pigmented and staphylococci produce orange pigments. Of course, the colour exhibited by surface bacteria is a major criterion used in the screening and in the selection of strains as adjuncts. Several tests are used, the most relevant being the development of colour on cheese models which takes into account the interactions between surface microorganisms. Proteolysis, peptidolysis and amino acid catabolism

Although the caseinolytic activity varies greatly between species and strains, this property of surface bacteria has only a small additional effect on cheese proteolysis. However, their peptidase activities and amino acid catabolism are much more important for the production of aroma compounds (Gobbetti et al., 2001; Curtin et al., 2002). Their demethiolase activity produces sulphur compounds, particularly methanthiol from methionine (Brennan et al., 2002). Their deaminase activity produces ammonia and degrades biogenic amines (Leuschner and Hammes, 1998). Moreover, proteolysis plays a role in the production of the typical colour of the cheese surface (Bockelmann et al., 1997b). Naturally, these properties are taken into consideration by culture suppliers in the process of strains selection. Lipolysis

Staphylococci have higher lipolytic activity than other surface bacteria (Bergère and Tourneur, 1992; Curtin et al., 2002). However, this property is sometimes shown in the product sheets of suppliers, but the methods used to characterise it are not indicated. Antimicrobial activities

B. linens produces antimicrobial substances which inhibit the growth of many Gram-positive food-poisoning bacteria as well as several yeasts and moulds (Maisnier-Patinaud and Richard, 1995; Motta and Brandelli, 2002). Some isolates of M. lacticum show anti-listerial activity (Carminati et al., 1999). Kocuria varians NCC 1482 produces variacin, an antibiotic of the same class of antimicrobial peptides as nisin. It inhibits food-borne pathogens such as species of Enterococcus sp., Staphylococcus aureus, Bacillus cereus and Clostridium botulinum (Pridmore et al., 1996; O’Mahony et al., 2001). Staphylococci produce many anti-bacterial substances such as antibiotics (Brennan

et al., 2002). A strain of Staph. equorum was found to produce a macrocyclic peptide antibiotic, micrococcin P1, on soft cheese and to inhibit Listeria monocytogenes (Carnio et al., 2000). Naturally, these antimicrobial activities provide interesting criteria for the selection of strains in order to control the safety of smear-ripened cheeses using specific adjuncts. Form and use of adjunct culture

Like yeasts, only a few species and strains of these bacteria are commercially available and this does not reflect the complexity of the cheese surface bacteria described previously. Of course, several strains of B. linens are often marketed by the main suppliers (Crerici-Sacco, Degussa, Chr Hansen, Rhodia Food and Wiesby). In contrast, A. nicotianae, A. globiformis and C. flavescens are available from only two companies (Degussa and Rhodia Food). Some Dairy Research or Technical Centres may provide coryneform cultures for their domestic cheese producers. For staphylococci, S. xyloasus or S. carnosus is recommended by Chr. Hansen and Rhodia Food, whereas other companies (Bioprox, Degussa and Wiesby) do not indicate the identity of the staphylococcal adjuncts in their product sheets. These surface bacterial cultures are always available in freeze-dried form but Wiesby market their cultures in three forms, liquid, frozen and freeze-dried. Several mixed-cultures are supplied by Degussa, Rhodia Food and Wiesby, which contain mixed bacterial species or mixtures of yeasts and bacteria. In using adjunct cultures, the manufacturers recommend direct inoculation of the cheese milk to obtain 5.104–105 cfu ml 1 or spraying a smear solution on the cheese surface.

Propionic Acid Bacteria Propionic acid bacteria (PAB) are used mainly as adjuncts in cheeses with eyes which are also called Swiss-type cheeses, particularly Emmental, Jarlsberg and Maasdam (see ‘Cheese With Propionic Acid Fermentation’, Volume 2). They could also be used in other hard- or semi-hard cheeses for their protective effects and their contribution to taste and aroma. Species found in cheeses and characteristics

The bacteria that produce propionic acid were named Propionibacterium by Orla-Jensen in 1898 and their fermentation was studied by Pasteur and Fitz. The PAB were seriously classified only in 1928 by Van Niel but most of the current classification keys emerged from the work of Cummins and Johnson (1981). The ‘classical’ or ‘dairy’ PAB should be differentiated from the


cutaneous ones which occur on the human skin. Four species are currently classified as ‘dairy’ PAB: P. acidopropionici, P. freudenreichii, P. jensenii and P. thoenii. The other two ‘classical’ species, P. cyclohexanicum and P. microaerophilus, were not isolated from dairy products (Kusano et al., 1997; Koussemon et al., 2001). P. freudenreichii is the most common species found in cheese and, consequently, is the one used as an adjunct. Of course, the number of PAB is high around 109 cfu g l in hard cooked cheeses such as Emmental which are ripened in a warm room. However, significant numbers of PAB, between 107 and 109 cfu g l, are also found in semi-hard cheeses, e.g., Abondance, Appenzell, Gouda, Maasdam, Morbier, Tomme de Savoie and some ewes’ milk cheeses. This is not surprising because PAB are part of the natural flora of raw milk and they can grow at a low temperature. Propionic acid bacteria are Gram positive, nonmotile, non-sporing, small rods (0.5–0.8 m 1 to 5 m). PAB are pleomorphic, paired small rods or coccoid-shaped cells, often in clumps with Chinese characters; sometimes filamentous shapes are seen. Their genome size is between 1.6 and 3.1 kb, their G C content ranges from 65 to 67% and plasmids occur frequently (1–3). PAB are anaerobic to aerotolerant mesophiles and many strains are able to grow slowly at a temperature as low as 3 °C. They are able to metabolise many different carbon sources: sugars (lactose, glucose, galactose, fructose), alcohols (glycerols, erythritol, adonitol), organic acids (lactate, citrate, aspartate). Biotin and pantothenic acid are essential factors for their growth (Cummins and Johnson, 1981). Optimum growth occurs in the pH range 6.5–7, and pH 5.0–5.2 is the lowest limit for the growth of most strains. Low temperatures and high sodium chloride concentrations (up to 3%) enhance the inhibitory effect of low pH on the growth of PAB (Hettinga and Reinbold, 1972a,b,c).

have a more complex lactate metabolism. Recently, the use of in vivo 13C-NMR was used to follow more precisely carbon metabolism in PAB and indicated the presence of six other minor pathways (Deborde et al., 1999; Deborde and Boyaval, 2000). In hard cheese, the rate of the propionic acid fermentation and CO2 production is decisive for the successful formation of the desirable round, shiny eyes. A fast lactate fermentation is needed to accumulate CO2 in the cheese (there must be a balance between CO2 production in the cheese and CO2 diffusion from the cheese). Consequently, the growth rates of PAB under conditions encountered in cheese during the ripening (pH 5.2, 1–2% NaCl, 18–22 °C) is the first criterion in the selection of PAB. For this purpose, the use of minicheesemaking as described by Richoux and Kerjean (1995) is better than in vitro studies. Acetic and propionic acids also contribute to the preservation and the taste of cheese. Proteolytic activities and amino acid catabolism

Orla-Jensen was the pioneer researcher on the propionic acid fermentation in Emmental cheese. He was the first to show the relationship between this fermentation and eye formation (von Freudenreich and Orla-Jensen, 1906). Already in 1878, Fitz had established the well-known stoichiometric equation:

The caseinolytic potential of PAB is estimated to be 5–15 times less than that of lactococci (El Soda et al., 1992; Dupuis et al., 1995). Propionic acid bacteria contain numerous peptidases, including a wide spectrum of general aminopeptidase activities and many activities towards proline-containing peptides, e.g., proline aminopeptidase, X-prolyl-dipeptidyl aminopeptidase, prolinase and prolidase (Lemée et al., 1998; Gagnaire et al., 1999; Stepaniak, 2000). They are mainly intracellular but, unfortunately, autolysis of PAB in cheese is limited and slow. It is less important than autolysis of lactic acid bacteria (Sahlstrom et al., 1989; Lemée et al., 1995; Valence et al., 1998; see Ostlie et al., 1999). Consequently, these proteolytic activities may be omitted in the selection of PAB as secondary cultures. Propionibacteria are able to catabolise amino acids, especially aspartic acid, asparagine, alanine, valine, serine, tyrosine, glutamic acid, arginine, cysteine and methionine, to different flavour compounds (Keenan and Bills, 1968; Brendenhaug and Langsrud, 1985). But today, the production of aroma compounds by PAB remains measurable only in real cheeses. However, their ability to metabolise aspartate is easily checked in the laboratory. This aspartate deamination pathway leads to high CO2 production which can provoke a late blowing defect in aged Emmental cheese (Fröhlich-Wyder et al., 2002).

3 lactate : 2 propionate 1 acetate 1 CO2 H2O.

Lipolysis

Nevertheless, this fermentation balance is often different in cheese where strong propionic acid fermentation occurs, such as Emmental. This suggests that PAB

Propionic acid bacteria are well known for their high lipolytic activity and have 10–100 times more activity than lactic acid bacteria (Oterholm et al., 1970; Dupuis et al., 1993). In vitro studies, as well as data

Useful properties for selecting PAB as adjuncts Lactate metabolism


from experimental cheeses, have shown that PAB release FFAs in cheese (Chamba and Perreard, 2002). P. freudenreichii exhibits the highest lipolytic activity but it is strain-dependant. This activity on lipids in cheese produce aroma compounds through the release of free fatty acids and their subsequent catabolism. This is an important property in choosing strains as adjuncts. Unfortunately, the available laboratory methods to characterise the lipolytic activity of PAB do not correlate well with their lipolysis in cheese (Kerjean et al., 2000). In order to use the lipolytic activity as a screening criterion for PAB, improvements in analytical methods are needed. Probiotic properties

Propionic acid bacteria can survive in the digestive tract and reach and maintain high populations in the human intestine (Bouglé et al., 1999; Jan et al., 2002). They are able to inhibit undesirable intestinal bacteria, and have a growth-promoting effect on bifidobacteria (Kaneko et al., 1994). Some dairy strains produce nitric oxide with a positive effect on intestinal peristalsis. To date, it seems that probiotic properties have not been taken into consideration in selecting strains to be used as cheese adjuncts. Form and use of adjunct cultures

Swiss cheesemakers were the pioneers in the use of PAB as adjuncts; their Dairy Research Station at Liebefeld-Berne began to produce selected propionibacteria cultures as early as 1926. The use of PAB culture is justified for three main reasons: • to replace the low level of PAB in the cheese milk (natural or after treatment) which often is unable to reach the necessary level to obtain a satisfactory propionic acid fermentation; • to increase the rate of propionic acid fermentation in order to obtain the desirable openings or eyes (the balance between CO2 production and CO2 diffusion); • to better control propionic acid fermentation and cheese quality. Generally, PAB are not cultivated in the cheese factories because their cultivation is laborious, timeconsuming and sensitive to microbial contamination. In the past, liquid cultures were the normal form; today, concentrated frozen or freeze-dried cultures are commonly supplied by specialised companies or Dairy Research Centres. These cultures are added directly to the milk in the cheese vat. Production of PAB is done in a sterile fermenter using complex media containing lactate. After the desired number of bacteria has been

attained (⬃109 cfu ml 1), the culture is concentrated by centrifugation or microfiltration. Then, the biomass is frozen or freeze-dried after addition of a cryoprotectant(s). The bacterial concentration of commercially freeze-dried cultures is about 1010 cfu g 1. The main commercial suppliers are Clerici Sacco, Centro Sperimentale del Latte (CSL), CSK, Chr-Hansen, Rhodhia Foods, Standa Industrie and Wiesby. Most commercial strains are P. freudenreichii and, generally, the number available is limited (1–3). In contrast, the portfolio of Standa Industrie contains a large choice of cultures (⬃15) with various technological abilities. In addition, the frequent and strong interaction between PAB and lactic acid bacteria must be taken into consideration in the choice of the starters associations (Chamba, 1994; Kerjean et al., 2000). The manufacturer’s recommendations for propionibacteria are to use ⬃103 cfu ml 1 of milk for Emmental-type cheese and between 105 and 106 cfu/ml for other cheeses. Below this amount, the risks of a slow, propionic acid fermentation and several defects, e.g., brown spots, and blind cheese, are high.

Heterofermentative Lactobacilli In spite of not being added deliberately to the milk, heterofermentative lactobacilli grow to high numbers (⬃108 cfu g 1) in many hard and semi-hard cheeses, especially in the major ripened cheeses produced such as Cheddar and Emmental. Their use as adjuncts is in its infancy. Very few culture producers produce heterofermentative lactobacilli as adjuncts. However, their use could increase in the future. Species found in cheeses

It is generally recognised that Lactobacillus paracasei subsp. paracasei, Lb. rhamnosus, Lb. plantarum and Lb. curvatus are the main species of facultative heterofermentative lactobacilli (FHL) in cheese (Jordan and Cogan, 1993; Lindberg et al., 1996; Coppola et al., 1997; Bouton et al., 1998; Crow et al., 2001). They are Gram positive, non-motile and catalase negative, their G C content ranges from 44 to 47%. Under the microscope, FHL appear as short rods. They are aerotolerant and mesophilic and are able to grow at 15 °C; Lb. paracasei subsp. paracasei and Lb. rhamnosus can grow at 45 °C but Lb. plantarum cannot. Many sugars, such as lactose, glucose, galactose, fructose and especially ribose, are fermented with production of L- or DL-lactate, but without CO2 production, and some strains metabolise lactate, citrate, amino acids and glycolipids (Williams et al., 2000). The fermentation of pentoses results in production of lactic and acetic acids. The heat resistance of


heterofermentative lactobacilli varies according to the species and strain. Lb. paracasei and Lb. rhamnosus are more resistant than Lb. plantarum. They can survive pasteurisation at 72–75 °C for 15 s (Jordan and Cogan, 1999). Likewise, the two former species resist the cooking temperature, 50–55 °C for at least 1 h, used in hard cheeses such as Emmental and Grana. Lb. brevis, Lb. buchneri and Lb. fermentum are the main obligatory heterofermentative lactobacilli encountered in cheese. The C G content of Lb. fermentum, at 52–54%, differs from other lactobacilli. These three species produce DL lactate, ethanol and CO2 from glucose. Gluconate is also fermented and arginine is metabolised with the production of NH3. Lb. brevis and Lb. buchnerii can grow at 15 °C but Lb. fermentum cannot; Lb. brevis does not grow at 45 °C, but Lb. buchneri and Lb. fermentum can. The obligatory heterofermentative lactobacilli have been considered as a spoilage bacteria in cheese for a long time, but this opinion should be revised. Generally, FHL are present in cheese in much higher numbers than obligate heterofermenting lactobacilli. Heterofermentative lactobacilli are regarded as an adventitious flora in cheese and they originate in the raw milk and factory environment. This flora can reach 108 cfu g 1 in most, if not all, ripened cheeses. In spite of this observation, which was made over 30 years ago, the role of heterofermenting lactobacilli in flavour formation in cheese is still unclear compared to the homofermentative starter lactobacilli. The use of heterofermentative lactobacilli as cheese adjunct culture is still at an early stage. Useful properties to select heterofermentative lactobacilli as adjuncts

Heterofermentative lactobacilli exhibit a large diversity of properties and their effects on cheese characteristics vary from negative to no effect to positive effects. These properties are strongly strain-dependant and offer several ways for their selection as adjuncts. Proteolysis and amino acids catabolism

The proteinase activity of heterofermentative lactobacilli seems to be lower than that of homofermentative lactobacilli and their contribution to casein hydrolysis during ripening of Cheddar cheese appears to be relatively small (Lynch et al., 1997). In contrast, the peptidase activities, at least in certain strains, contribute to the hydrolysis of bitter peptides to non-bitter peptides with the release of free amino acids (Gagnaire et al., 2001). This is the case for Lb. casei subsp. casei LLG which has an active aminopeptidase and a proline-specific peptidase with debittering activity (Park et al., 1995). Likewise, Lb. curvatus DPC 2024 has a

PepN-like aminopeptidase, which shows broad substrate specificity (Magboul and McSweeney, 1999). Amino acid catabolism and the production of aroma compounds by heterofermentative lactobacilli, especially their glutamate dehydrogenase activity, appear to be one of the determinant properties for their use as cheese adjuncts. This activity also provides -ketoglutarate for transamination of other amino acids to produce aroma compounds (Rijnen et al., 2000). This activity is strain-dependant; about half of Lb. plantarum and Lb. paracasei strains have glutamate dehydrogenase activity (Tanous et al., 2002). Moreover, Lb. fermentum and Lb. reuteuri produce aroma compounds from sulphur amino acids but Lb. brevis, Lb. paracasei and Lb. curvatus do not (de Angelis et al., 2002). Consequently, measurement of aminopeptidase activity and the ability to catabolise amino acids should be taken into account in their selection. Formation of biogenic amines

Amino acid decarboxylase activity of obligately heterofermentative lactobacilli, particularly Lb. buchneri, has been implicated in biogenic amine production in Swiss cheeses and outbreaks of food poisoning (Sumner et al., 1985; Joosten and Northolt, 1987). It is certainly an exceptional case since the use of Lb. fermentum and Lb. buchnerii as adjuncts in Emmental cheese produced less than 10 mg kg 1 of histamine and 50 mg kg 1 of tyramine (Chamba, 2000). However, Crow et al. (2001) take the ability to form biogenic amines into consideration in screening heterofermentative lactobacilli as cheese adjuncts. Lipolytic activities

Like other lactic acid bacteria, heterofermentative lactobacilli are generally considered to be weakly lipolytic in comparison with other microorganisms, such as PAB, corynebacteria, yeast and moulds in cheese (Knaut and Mazurek, 1974; Khalid and Marth, 1990; Fox et al., 1993; Gobbetti et al., 1997). Therefore, lipolytic activity can be omitted in the selection of heterofermentative lactobacilli as adjuncts. Antagonistic activities

The production of inhibitory metabolites and bacteriocins by heterofermentative lactobacilli is also problematic in cheese. The first one was observed against PAB in Swiss cheese. Lb. casei and Lb. rhamnosus produce acetate, formate and small amounts of diacetyl from citrate in cheese and interfere negatively with the growth of P. freudenreichii (Jimeno et al., 1995). Swiss researchers use this ability to prevent secondary fermentation, which causes cracks and splits in Emmental cheese. Antimicrobial activities of lactobacilli have been known and recognised for more than 14 years. Many


heterofermentative lactobacilli such as Lb. brevis, Lb. curvatus, Lb. fermentum, Lb. plantarum and Lb. rhamnosus produce bacteriocins. Generally, these inhibit several Gram-positive bacteria, including enterococci, clostridia, S. aureus and Listeria spp., but, unfortunately, lactic acid bacteria are also frequently inhibited (Klaenhammer et al., 1994; Malik et al., 1994). It is possible that this property will become important in the future use of heterofermentative lactobacilli as cheese adjuncts. An interesting inhibitory activity against the spoilage bacterium, Clostridium tyrobutyricum, is produced by Lb. rhamnosus LC705 (DSM7051). This was patented and is commercially available (Maÿra-Mäkinen and Suomalainen, 1996). This adjunct was tested successfully in Emmental and Gouda cheeses. In Gouda cheese, Lb. rhamnosus LC705 provides an efficient substitute for nitrate. Probiotic properties

Some strains of heterofermentative lactobacilli, Lb. casei Shirota, Lb. plantarum DSM9843, Lb. rhamnosus GG, have shown probiotic capabilities such as the prevention and treatment of diarrhoeal disease, intestinal inflammation or permeability disorders, immunomodulation and tumour prevention (Huis In’t Veld and Marteau, 1997). Their ability to survive in the gastro-intestinal tract and colonise the intestine, especially exopolysaccharideproducing strains, could be useful in the development of probiotic dairy products (Chabot et al., 2001). In this way, Gardiner et al. (1998) have shown that cheese is a better vector than fermented milk for increasing the numbers of lactobacilli in the intestines of piglets. The use of heterofermentative lactobacilli selected for their probiotic properties as cheese adjuncts could be a possibility in the future. Form and use of adjunct cultures

The statement of Fox et al. (1993) ‘The contribution of NSLAB to cheese ripening and quality is a vexed question’ remains topical. The main reason for this statement is certainly the high versatility of heterofermentative lactobacilli. Their effects are strain-specific, strongly affected by the technological context and probably by the interactions with other microorganism in cheese. This is probably why the supply of heterofermentative lactobacilli as adjuncts is still limited. Nowadays, in modern cheese factories, cheesemaking conditions are better and more clearly understood and good hygiene is practised. The bulk milk is of good quality; the cheese milk is standardised (for fat and protein), clarified and thermised. Technological and environmental parameters of cheesemaking and ripening are under control. Moreover, efficient starters are used. Nevertheless, NSLAB, especially heterofermentative lacto-

bacilli, remain an uncontrolled part of the cheese microbial ecosystem. For that reason, their use as adjuncts should increase in the next years in order to: • Overcome the probable negative effect encountered by the growth of the indigenous flora. For this purpose, useful strains of heterofermentative lactobacilli must be able to grow faster than the indigenous NSLAB without affecting the characteristics of the cheese. • Improve cheese quality by using adjuncts with desirable properties. In Switzerland, the use of FHL as adjuncts is common for Emmental cheese to prevent late blowing. This adjunct is supplied by the Federal Dairy Research Institute, Liebefeld-Berne, and the recommended level of inoculation is around 1 104 cfu ml 1 of cheese milk. For Cheddar cheese, Crow et al. (2001) provide a very good description of the screening process to select NSLAB as starter adjuncts which are used at levels of 300–1000 cfu ml 1 of vat milk. They maintain that ‘NSLAB adjuncts are required for improved flavour control in aged cheeses such as mature Cheddar as uncontrolled adventitious strains of NSLAB can cause defects’. From this sentence, can we understand that the use of NSLAB as cheese adjuncts is now a common practice in New Zealand? Today, the portfolios of heterofermentative lactobacilli usable as cheese adjunct from starter suppliers are small. Heterofermentative lactobacilli are frequently marketed as probiotics, for example by Clericii-Sacco, CSL, DSM Food Specialities, Chr. Hansen or Rhodia Foods. Only a few cultures are available and often these are sold as mixed cultures with other lactic acid bacteria or with surface-ripening microorganisms. Wiesby market Lb. rhamnosus strain LC 705 which inhibits Cl. tyrobutyricum. Generally, these cultures are in freeze-dried form for direct inoculation of the vat milk or sometimes for bulk starter preparation.

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Salt in Cheese: Physical, Chemical and Biological Aspects T.P. Guinee and P.F. Fox, Dairy Products Research Centre, Moorepark, Fermoy, Ireland. Department of Food and Nutritional Sciences, University College, Cork, Ireland

Introduction The use of salt (NaCl) as a food preservative dates from pre-historic times and, together with fermentation and dehydration (air/sun), is one of the classical methods of food preservation. So useful and widespread was the use of salt as a food preservative in Classical and Medieval times that it was a major item of trade and was used as a form of currency in exchange for goods and labour. It is perhaps a little surprising that Man discovered the application of salt in food preservation so early in civilization since, in contrast to fermentation and dehydration, salting is not a ‘natural event’ in foods but requires a conscious act. It is interesting that the three classical methods of food preservation, i.e., fermentation, dehydration and salting, are exploited in cheese manufacture and, in fact, are interdependent. The fourth common method for food preservation, i.e, use of high and/or low temperatures, was less widespread than the others because the exploitation of low temperatures was confined to relatively few areas until the development of mechanical refrigeration about 1870 and, although heating was probably used to extend the shelf-life of foods throughout civilization, its controlled use dates from the work of Nicolas Appert (1809) and Louis Pasteur (1860–1864). In modern cheese technology, temperature control complements the other three methods of food preservation. The level (%, w/w) of salt in cheese ranges from ⬃0.7 in Swiss to ⬃6 for Domiati (see Table 1). Salt, together with the desired pH, water activity and redox potential, contributes to minimization of spoilage and prevention of the growth of pathogens in cheese (see Naguib et al., 1979; Russell and Gould, 1991; Eckner et al., 1994; Guraya et al., 1998; Bolton and Frank, 1999; Erkmen, 2001). In addition to its preservative effect, NaCl plays two other important roles in foods. Man requires ⬃2.4 g Na, i.e., ⬃6 g NaCl, per day (Kaplan, 2000) and although this requirement can be met through the indigenous Na content of foods, added NaCl is a major source in modern western diets. In fact, western diets contain approximately two to three times more Na than is necessary and excessive intakes of Na have toxic, or at least undesirable, physiological effects, the most significant of

which are hypertension and increased calcium excretion which may lead to osteoporosis (see Abernethy, 1979; Anonymous, 1980, 1983; Moses, 1980; Schroeder et al., 1988; Midgley et al., 1996; Beard et al., 1997; McCarron, 1997; Beilin, 1999; Cutler, 1999; Feldman and Schmidt, 1999; Korhonen et al., 1999, 2000; Cappuccio et al., 2000; Kaplan, 2000; McCarron and Reusser, 2000). Cheese, even when consumed in large amounts (see ‘Cheese: An Overview’, Volume 1), as in France and Switzerland, makes a relatively small contribution to dietary Na intake, although it may be a major contributor in individual cases where large amounts of highsalt cheese, e.g., Blue, Feta or Domiati, are consumed. Nevertheless, there is interest in many western countries in the production of low-Na cheese, for at least certain sectors in the population, but, as discussed in ‘Reduced Sodium Cheese’, this has significant repercussions in cheese manufacture. The most common approach at present is to replace some or all of the NaCl by KCl, but apart from cost, this practice adversely affects the taste of cheese since the taste of KCl is distinctly different from that of NaCl and a bitter flavour (not due to abnormal proteolysis) is detectable in cheese containing 1%, w/w, KCl (see ‘Reduced Sodium Cheese’ for discussion on lowsodium cheese). The third major feature of the use of NaCl in foods is its direct contribution to flavour. The taste of salt is highly appreciated by many and saltiness is regarded as one of the four basic flavours. Presumably, the characteristic taste of NaCl resides in the Na moiety since KCl has a distinctly different flavour sensation. At least part of the desirability of salt flavour is acquired but while one can easily adjust to the flavour of foods without added salt, the flavour of salt-free cheese is insipid and ‘watery’, even to somebody not ‘addicted’ to salt; the use of 0.8%, w/w, NaCl is probably sufficient to overcome the insipid taste (Schroeder et al., 1988). In this chapter, we will concentrate on the significance of NaCl in cheese ripening rather than on its dietary and direct flavour effects. NaCl influences cheese ripening principally through its effects on



208 Salt in Cheese: Physical, Chemical and Biological Aspects

Table 1 Typical composition of major cheeses (from various sources) Cheese

Moisture (%, w/w)

Salt (%, w/w)

S/M a (%, w/w)

pH

Blue Brick Bulgarian White Camembert Cheddar Edam Emmental Gouda Grana (Parmesan) Gruyère Limburger Muenster Provolone Pecorino Romano Roquefort Domiati Feta

42 40 32 52 37 43 35 41 31 33 45 43 42 23 40 55 53

4.5 1.9 3.5 2.5 1.5 2.0 0.7 2.0 2.6 1.1 2.0 1.8 3.0 5.5 3.5 6.0 3.0

10.5 4.8 10.9 4.8 4.1 4.7 2.0 4.9 8.4 3.3 4.4 4.2 7.1 23.9 8.8 10.9 5.7

6.5 6.4 5.0 6.9 5.5 5.7 5.6 5.8 5.4 5.7 6.8 6.2 5.4 5.4 6.4 4.6 4.5

a S/M salt-in-moisture.

water activity but it probably has some more specific effects also. Among the principal effects of salt are: • control of microbial growth and activity; • control of the various enzyme activities in cheese; • syneresis of the curd and thus in a reduction in cheese moisture, which also influences the above; • physical changes in cheese proteins which influence cheese texture, protein solubility and probably protein conformation.

Control of Microbial Growth Probably the most extreme example of the use of NaCl for this purpose in cheese is in the manufacture of Domiati cheese from milk to which 12–15%, w/w, NaCl is added to inhibit bacterial growth and thus maintain milk quality (Naguib et al., 1979; Sußmuth, 1998; ‘Cheese Varieties Ripened in Brine’, Volume 2). For all other major varieties, NaCl is added after curd formation but nevertheless it plays a major role in regulating and controlling cheese microflora. The simplest example of this is the contribution of NaCl to the regulation of cheese pH, which in turn influences cheese ripening and texture. The pH of cheese may be regulated by: • reducing the amount of residual lactose in the curds by washing the curds with water, as practised in the manufacture of Dutch-type, Tallegio and Cottage cheeses; • the natural buffering capacity of the cheese and the toxic effect of the lactate anion which establishes a natural lower limit to pH (⬃4.5), e.g., Blue, Camembert, hard Italian varieties; • addition of salt.

The use of salt, together with buffering capacity, to regulate the final pH appears to be confined almost exclusively to British-type cheeses, i.e., dry-salted varieties such as Cheddar, Cheshire and Stilton. The curds for most, if not all, non-British cheeses are placed in moulds while the pH is still high (6.0) and acid development continues during pressing. Since a level of NaCl 1.5%, w/w, inhibits starter activity, such cheeses are salted by immersion in brine or by surface application of dry salt. In British cheeses, the pH has almost reached its ultimate value at hooping and salt is added to maintain the pH at that desired value. One could probably argue that the method of salting cheese that predominates in a certain region reflects the form of salt available locally; in regions where salt deposits occur, dry salt was readily available and thus permitted the manufacture of cheese in which dry salt was added to the curd or to the surface of the cheese; in regions where salt was prepared by evaporation of sea water, it would have been more convenient to salt the cheese by immersion in concentrated brine rather than wait for crystallization. Curd for Cheddar and similar varieties contains ⬃0.6–1.0%, w/w, lactose at hooping (Turner and Thomas, 1980); this is fermented during the early stages of ripening by continued starter activity but this depends strongly on the salt-in-moisture (S/M) level in the curd and the salt tolerance of the starter. Irvine and Price (1961) showed that acid development by six commercial lactic acid cultures in reconstituted 10%, w/v, skim milk powder (RSM) was either stimulated or not affected by a low level (1%, w/w) of NaCl but was strongly inhibited by 2.5%, w/w, NaCl. However, even at 5%, w/w, NaCl, acid was produced by all starters to

Salt in Cheese: Physical, Chemical and Biological Aspects 209

a level ⬃45–55% of the maximum. In the same study, portions (454 g) of curd at pH ⬃6.05 were taken after whey drainage and placed in brine containing 0–5%, w/w, NaCl at ⬃38 °C for 2 h; a sample of the curd held in the cheese whey was used as a control sample (it is assumed that the S/M equilibrium between the brine and curd moisture was rapid because of the high temperature and the open structure of the mass of curd particles). The pH decreased to a minimum of 5.53 at 2%, w/w, NaCl brine but did not fall below 5.9 at 5%, w/w, NaCl brine; the pH of curd held in 4%, w/w, NaCl brine was similar to that of curd held in water (0%, w/w NaCl brine) (Fig. 1). The pH decreased to 5.65, 5.53, 5.62 and 5.90 at 0, 2, 4 and 5%, w/w, NaCl brine, respectively (Fig. 1). These results suggest that starter activity is stimulated by 2%, w/w, NaCl. Overall, the experiments of Irvine and Price (1961) suggest that the growth of lactococci in Cheddar curd is generally not inhibited by #4%, w/w, S/M and that the inhibitory effect of NaCl is less in curd than in RSM. This conclusion is supported by the results of Schroeder et al. (1988) who found that varying S/M level from 0.18 to 4.1%, w/w, had little effect on the starter population in 1-day-old Cheddar cheese made with a six-strain culture of Lactococcus lactis supsp. cremoris. The pH at which salt was added to RSM and curd (6.7 and 6.05, respectively) in the study of Irvine and Price (1961) was much higher than that (⬃5.25–5.35) at which full-fat Cheddar is salted in practice and, therefore, may not reflect the full combined inhibitory effect of salt and low pH. However, the pH of half-fat Cheddar which was salted at pH ⬃5.75, rather than at 5.3, to increase the moisture level (Guinee et al., 1998; Fenelon

et al., 1999), decreased to 5.2 at 1 day, i.e., similar to that of half-fat Cheddar salted at pH 5.3. This observation confirms the findings of Irvine and Price (1961), i.e., that S/M #4%, w/w, has little inhibitory effect on starter lactococci in Cheddar curd, and suggests that the pH of Cheddar is controlled by a combination of salting and buffering capacity. Cheshire cheese has a considerably lower pH than Cheddar (e.g., typically 4.7–4.9 compared to 5.1–5.3). This low pH is probably primarily due to a high level of starter (⬃4%) and, consequently, a very rapid rate of acidification, which causes extensive solubilization of colloidal calcium phosphate and a reduction in buffering capacity (Lucey and Fox, 1993). As a result, although Cheshire is salted at a level and at a curd acidity similar to that for Cheddar (Robinson and Wilbey, 1998), the pH of the former is lower, because of the lower buffering capacity, a higher moisture level, a higher lactate content and, hence, a higher lactateto-buffering ratio. This importance of S/M in controlling the pH of Cheddar curd is also evident from the data of O’Connor (1974). Curd (presumably at ⬃pH 5.3) was salted at a varying level in the range 0.5–6%, w/w (Fig. 2). The pH decreased after salting, presumably due to the action of starter, at S/M levels 5%, w/w, but starter activity decreased abruptly at higher values of S/M, and the pH remained high or increased. The grade assigned to the cheese also decreased sharply at S/M levels 5%, w/w. The control of pH and lactose metabolism by S/M concentration in commercial Cheddar cheese, produced with a linear S/M gradient of 4–6%, w/w, within a single cheese, was clearly demonstrated (Fig. 3) by Thomas and Pearce (1981).

5.9 5.85

Cheese pH

5.8 5.75 5.7 5.65 5.6 5.55 5.5 0

1

2

3

4

5

Concentration of brine, %, w/w, NaCl Figure 1 Influence of NaCl concentration on the pH of Cheddar cheese curds after holding in the brine for 2 h at 37.7 °C; the pH of the mass of curd particles at the time of placing in the brine was 6.05 (redrawn from Irvine and Price, 1961).


Total grade score (maximum 30 points)

28

26

24

22

20 5.5 5.4

pH

5.3 5.2 5.1 5.0 0

1

2 3 4 5 6 7 Salt-in-moisture, %, w/w

8

9

Figure 2 Relationship between the salt-in-moisture (S/M) level and the pH () at eight weeks, and between the S/M and the total grade score (maximum 30) (●) of cheeses made from the same vat but salted at different levels (drawn from data of O’Connor, 1974, from Lawrence and Gilles, 1982).

5.4

1

5.3

0.6 5.2

pH

Lactose, g/100 g cheese

0.8

0.4

5.1 0.2

0 4.0

4.5

5.0

5.5

5.0 6.0

Salt-in-moisture, g/100 g Figure 3 Effect of salt-in-moisture concentration on lactose concentration () and pH () within a single block of Cheddar analysed at 14 days after manufacture (redrawn from Thomas and Pearce, 1981).

The above studies show that inhibition of starter occurs within quite a narrow S/M range (Fig. 2), emphasizing the importance of precise control of S/M level. However, since the sensitivity of starter cultures to salt varies, the influence of NaCl concentration on post-salting acid production in cheese obviously depends on the starter used and a general value for S/M cannot be definitely stated. At pH 5.3, Lactococcus lactis subsp. lactis strains are generally more salttolerant than strains of Lc. lactis subsp. cremoris but there is also considerable variation in salt sensitivity between strains of Lc. lactis subsp. cremoris (Martley and Lawrence, 1972; Turner and Thomas, 1980). If starter activity is inhibited after manufacture, residual lactose will be metabolized by non-starter lactic acid bacteria (NSLAB). However, the number of NSLAB present, which is influenced by the level of contamination at salting, level of S/M, NSLAB strain, rapidity with which pressed curd is cooled and ripening temperature (Fryer, 1982; Jordan and Cogan, 1993; Bechaz et al., 1998), is usually insufficient (e.g., #1000 cfu/g) to cause significant lactose metabolism for several days and, consequently, the pH falls slowly. In the study by Turner and Thomas (1980), NSLAB, mainly Pediococcus, were more salt-tolerant than starter bacteria and metabolized the lactose with the production of DL-lactate and the racemization of L-lactate. Non-starter lactic acid bacteria grew in all cheeses but their growth was markedly dependent on temperature and they had little influence on lactose or lactate concentration until numbers exceeded 106–107 cfu/ml. The high salt tolerance of NSLAB was confirmed by Jordan and Cogan (1993) who found that ⬃90% of NSLAB strains (Lactobacillus casei, Lb. plantarum and Lb. curvatus) isolated from commercial Cheddar grew in the presence of 6%, w/w, NaCl while 58% grew in the presence of 8%, w/w, NaCl. Similarly, Lane et al. (1997) reported that ⬃6%, w/w, S/M was required to retard the growth of NSLAB in Cheddar cheese and NSLAB numbers after ripening for 6 months were approximately equal at all S/M levels (2.8–6.1%, w/w). The greater salt tolerance of NSLAB was clearly apparent from the study of Thomas and Pearce (1981), which showed that the fermentation of lactose to D-lactate and the racemization of L-lactate in cheeses with 6%, w/w, S/M occurred relatively late (90–180 days) during ripening. However, the results of Bechaz et al. (1998), which showed significantly higher populations of NSLAB in reduced-salt Cheddar (1.0%, w/w) than in the control (1.8%, w/w), suggest that salt level has a major effect on the growth of NSLAB. The salt resistance of lactococci and other bacterial species isolated from African cheeses was studied in detail by Sußmuth (1998).


Although acid production can be uncoupled from cell growth, it is likely that acid production at low salt levels will be accompanied by high cell numbers which tend to lead to bitterness (Lowrie and Lawrence, 1972). Not surprisingly, bitterness in Cheddar cheese is markedly influenced by S/M level over a very narrow range; Lc. lactis subsp. cremoris HP generally yielded bitter cheese at S/M levels 4.3%, w/w, but rarely at 4.9%, w/w (Lawrence and Gilles, 1969). In the foregoing discussion on the influence of NaCl on the fermentation of residual lactose in cheese curd by starter microorganisms, it has been assumed that the NaCl is distributed throughout the cheese within a very short period after salting. However, this is not so. Cheddar cheese curd is usually milled into quite large particles (chips) of cross-section 2 cm 2 cm or larger. Obviously, dry salt applied to the surface of such chips requires a considerable period of time to diffuse to the centre of the chips and to attain an inhibitory level throughout (see ‘Factors that influence salt diffusion in cheese during salting’). Consequently, starter bacteria will continue to grow and produce acid at the centre of a chip for a considerable period after growth at the surface has ceased. Experimental support for this is provided by the experiments of Hoecker and Hammer (1944) who measured the levels of salt and moisture and pH at the surface and centre of individual chips, prised from a block of Cheddar cheese, over a 72 h period after salting and pressing. Their data showed that the pH fell faster and to a lower value at the centre, where NaCl concentration was lower, than at the surface. In one experiment, the difference in pH persisted for 72 h but in a duplicate experiment the difference in pH had essentially disappeared after 48 h. Thomas and Pearce (1981) showed that a higher level of added salt is required to inhibit lactose metabolism when the curd is milled into large chips than smaller ones. In surface-salted Meshanger cheese, Noomen (1977) showed considerable zonal variations in lactose and pH throughout the cheese in response to variations in S/M concentration. Similarly, Pavia et al. (1999) showed that a decreasing S/M gradient from the surface (⬃9%, w/w) to the centre (⬃0.2%, w/w) of freshly brinesalted Manchego cheese was paralleled by a pH gradient in the same direction and a lactate gradient in the opposite direction, indicating inhibition of the starter culture. Streptococcus salivarius subsp. thermophilus is considerably less salt-tolerant than Lc. lactis subsp. lactis (Rüegg and Blanc, 1981); its critical NaCl concentration is 0.4 M (2.34%, w/w), corresponding to an aw of 0.984, compared with 1.1 M NaCl (aw 0.965) for Lc. lactis subsp. lactis; Lb. delbrueckii subsp. helveticus

and Lb. lactis subsp. lactis were also less salt tolerant, being inhibited by 0.95 M and 0.90 M NaCl, respectively. Data on the sensitivity of propionibacteria to NaCl appear to be variable: Orla-Jensen (1931) reported that concentrations of NaCl as low as 0.5%, w/w, are sufficient to reduce the growth of Propionibacterium in a medium containing calcium lactate. However, Antila (1954) reported that 3%, w/w, NaCl is necessary to reduce growth. In fact, salt tolerance appears to be strain- and pH-dependent (Rollman and Sjostrom, 1946); in a lactate medium, 6%, w/w, NaCl was required to inhibit the growth of a fast-growing strain of Propionibacterium at pH 7.0 and 3%, w/w, at pH 5.2, whereas a slow-growing strain was more salt-tolerant at pH 5.2 than at pH 7.0. The data of Rüegg and Blanc (1981) show that P. shermanii was the most salt tolerant of the starter species investigated; its critical NaCl concentration was 1.15 M (⬃6.7%, w/w; aw 0.955). Boyaval et al. (1999) studied the effect of varying NaCl concentration (0–0.8 M; ⬃0.0–4.8%, w/w, S/M) on the growth of P. freudenreichii subsp. shermanii CIP 103027 in a chemically-defined medium (modified M63), Yeast Extract-Lactate medium (YEL), or 10% (w/v) reconstituted skim milk at pH 7.0 and at 30 °C under quiescent conditions. Increasing NaCl concentration in modified M63 resulted in a progressive decrease in cell growth, with a 50% reduction at 0.3 M and almost complete inhibition at 0.7 M. Growth inhibition was due to the osmotic effect rather than NaCl per se, as reflected by the similar inhibition of growth on substitution of NaCl by KCl or sucrose. In YEL or milk, 0.79 and 0.25 M NaCl, respectively, were needed to double the generation time, and concentrations of 1.5 M and 1.0 M to effectively inhibit growth completely. Differences in the effect of salt on growth rate in the three media were attributed to the presence of different types and levels of osmoprotective compounds, such as choline and glycine-betaine in YEL and various choline derivatives and carnitine in milk. While some inhibition of P. shermanii is expected in Emmental cheese, the aqueous phase of which has an osmolarity ⬃0.7 M NaCl (Salvat-Brunaud et al., 1997), the presence of osmoprotective compounds in milk assists growth (Boyaval et al., 1999). Interestingly, Emmental cheese, which contains ⬃0.7%, w/w, is the least heavily salted among major cheese varieties. Blue cheeses are among the most heavily salted varieties, with 3–5%, w/w, NaCl (Stilton 3%, w/w). Ripening in these varieties is dominated by Penicillium roqueforti and consequently good growth of this mould is paramount. Germination of P. roqueforti spores is stimulated by 1%, w/w, NaCl but inhibited by 3–6%, w/w, NaCl, depending on strain. However, the growth of germinated spores on malt extract agar or in cheese curd is less dependent on NaCl concentration than is


germination, and some strains grow in cheese curd containing 10%, w/w, NaCl, although growth is retarded compared to that in curd containing less NaCl (Godinho and Fox, 1981a,b). Morris (1981) reported that it is fairly common commercial practice to add 1%, w/w, NaCl to Blue cheese curd before hooping, possibly to stimulate spore germination, although it also serves to give the cheese a more open structure which facilitates mould growth. Since most Blue cheeses are surfacesalted, a salt gradient from the surface to the centre exists for a considerable period after manufacture; a high initial level of salt in the outside zone of the cheese may inhibit spore germination at a critical time and a mould-free zone at the outside is a common defect in Blue cheeses (Godinho and Fox, 1981b). Growth of P. camemberti is also stimulated by low levels of NaCl; 0.8%, w/w, NaCl, mould growth on Camembert cheese is poor and patchy (O’Nulain, 1986).

Influence of NaCl on Enzyme Activity in Cheese Coagulant

With the exception of Emmental and similar highcooked cheeses, the initial proteolysis in cheese is catalysed by residual coagulant. Polyacrylamide gel electrophoresis of cheese during ripening has shown that in hard and semi-hard, bacterially-ripened cheeses, s1-casein undergoes considerable proteolysis but -casein remains unchanged until an advanced stage of ripening (Ledford et al., 1966; Phelan et al., 1973; Creamer, 1975; Visser and de Groot-Mostert, 1977; Yun et al., 1995; Kristiansen et al., 1999; Fenelon and Guinee, 2000; Feeney et al., 2001; ‘Proteolysis in Cheese during Ripening’, Volume 1). A similar pattern is evident during the early phases of mould-ripened cheeses, when the coagulant is the principal ripening agent (Godinho and Fox, 1982; Hewedi and Fox, 1984) but fungal proteinases dominate in these cheeses during the later phases of ripening (see ‘Surface Mould-ripened Cheeses’ and ‘Blue Cheese’, Volume 2). The hydrolysis of s1-casein by milk clotting enzymes is greatly influenced by the concentration of NaCl. The proteolytic activity of chymosin, pepsins, Rhizomucor miehei and Cryphonectria parasitica rennets on dilute casein fractions is stimulated by increasing NaCl concentration to an optimum at ⬃6%, w/w (Fox and Walley, 1971; Gouda, 1987). Activity is inhibited at higher NaCl levels, but limited proteolysis of s1-casein occurs up to 20%, w/w, NaCl (Fox and Walley, 1971; Gouda, 1987). However, degradation of s1-casein is retarded by very low levels of salt in Cheddar (Phelan et al., 1973; Thomas and Pearce, 1981; Kelly et al., 1996; Mistry and

Kasperson, 1998) and on salting at 1.36%, w/w (S/M 2.55%, w/w) in Mozzarella (Guo et al., 1997). The inverse relationship between casein degradation and salt concentration in cheese is mirrored by the reduction in the level of pH 4.6-soluble N and/or water-soluble N (as %, w/w, total N), and/or NPN in Blue (Godinho and Fox, 1982), Camembert (O’Nulain, 1986; Table 2), Cheddar (Thakur et al., 1975; Kelly et al., 1996), Danbo (Kristiansen et al., 1999), Ragusano (Licitra et al., 2000), Romano (Guinee and Fox, 1984; Fox and Guinee, 1987), Feta (Pappas et al., 1996) and other cheeses (Wisniewska et al., 1990). In contrast to the above trends, the level of water-soluble N in serum expressed on centrifugation of unsalted low-moisture Mozzarella cheese at its unadjusted pH is significantly lower than that from salted cheese (Guo et al., 1997). However, the higher water-soluble N in the former is due to an increase in casein hydration as a result of a salting-in-effect at a S/M level of ⬃2.6%, w/w (see ‘Effect of NaCl on casein hydration in model systems and in cheese’) rather than to proteolysis which is very low in low-moisture Mozzarella. The latter effect is somewhat similar to the large increase in soluble N obtained on adding sodium citrate or sodium phosphate emulsifying salts during the manufacture of processed cheese, even though the level of pH 4.6-soluble N remains essentially constant (see ‘Pasta-Filata Cheeses’, ‘Pasteurized Processed Cheese and Substitute/Imitation Cheese Products’ and ‘Cheese as an Ingredient’, Volume 2). In contrast to the trends noted for s1-casein, proteolysis of -casein in dilute solution by chymosin or pepsins is strongly inhibited by 5%, w/w, and completely inhibited by 10%, w/w, NaCl (Fox and Walley, 1971; Lane and Fox, 1999). Sucrose and glycerol selectively inhibit proteolysis of -casein by chymosin and pepsins (Creamer, 1971; Al-Mzaien, 1985). KCl, LiCl, NH4Cl and CaCl2 are as effective as NaCl in inhibiting the proteolysis of -casein (O’Nulain, 1986). Since the inhibitory effect of solutes is substrate- rather than enzyme-specific, it appears that NaCl and similar solutes cause some conformational changes in -casein (Barford et al., 1988) which render its chymosin (pepsin)susceptible bonds less accessible to the enzyme. The nature of these conformational changes does not appear to have been investigated but may arise from the strongly hydrophobic nature of -casein. -Casein undergoes significantly less breakdown than s1-casein in most cheese varieties. The resistance of -casein in cheese to proteolysis is not dependent solely on the salt concentration since it is also quite resistant to proteolysis in salt-free, and low S/M (e.g., 2.7%, w/w) cheese (Phelan et al., 1973; Kelly et al., 1996), suggesting that a high protein concentration is sufficient to induce the necessary conformational


Table 2 Influence of NaCl on pH and proteolysis in Camembert cheese (4-weeks old)a

NaCl (%, w/w)

Zoneb

pH

0.20

I O I O I O I O I O

5.5 6.4 5.3 6.1 5.2 6.0 5.2 6.2 5.1 6.4 5.0 5.1 5.0 6.3

0.70 0.93 1.14 1.73

2.4

I O

Water-soluble N (% total N)

pH 4.6-soluble N (% total N)

70% ethanol-soluble N (% total N)

5% PTA-soluble N (% of total N) c

36.0 100.0 28.7 100.0 17.9 100.0 22.5 93.7 26.6 85.3

43.3 54.4 29.1 39.2 17.3 49.5 23.8 43.4 28.3 37.1

23.7 35.7 15.8 28.7 13.3 32.8 15.8 28.4 15.8 22.7

16.8 18.9 10.4 15.5 12.1 15.2 8.1 10.4 8.8 10.1

22.2 63.2

23.1 29.8

18.0 26.1

8.3 9.3

a Compiled from data of O’Nulain (1986). b I and O correspond to the inner and outer portions of the cheese. c PTA phosphotungstic acid.

change(s). A level of S/M 4.9%, w/w, is necessary to prevent the development of bitterness in cheese (Lawrence and Gilles, 1969). The inhibitory effect of NaCl on proteolysis of sodium caseinate, s1-casein and -casein is pHdependent, with the extent of inhibition generally decreasing with pH in the range 6.6–5.4 (Fox and Walley, 1971; Mulvihill and Fox, 1980; Lane and Fox, 1999). At low pH, NaCl also alters the proteolytic specificity of chymosin and pepsins: NaCl (2.5%, w/w) inhibits the formation of -III but promotes the formation of -IV and -V (Mulvihill and Fox, 1978). Formation of the s1-casein peptides, s1-VII and s1-VIII, in solution is stimulated by NaCl (5%, w/w) and these peptides are also formed in cheese (Mulvihill and Fox, 1980). The proteolytic activity of R. miehei and C. parasitica rennets on -casein is less strongly inhibited by NaCl than that of chymosin or pepsins (Phelan, 1985; Gouda, 1987). Milk proteinase

Milk contains several indigenous proteinases, the most significant, alkaline milk proteinase (plasmin), is almost exclusively associated with the casein micelles at the normal pH of milk, but dissociates from the micelles as the pH is reduced (Humbert and Alais, 1979; Fox, 1981; Visser, 1981; Reimerdes, 1982; Grufferty and Fox, 1988a; Sousa et al., 2001; Nielsen, 2002; Visser and van den Berg, 2002). Richardson and Elston (1984) reported that the dissociation of plasmin from the casein micelles is pH- and time-dependent and that it occurs at pH 5.7

and possibly higher, but Grufferty and Fox (1988a) found no dissociation on holding at a pH 4.9 for 4 h. This implies that all the plasmin in milk should be present in the curd for most rennet-coagulated cheese varieties. However, the concentration of plasmin in Swiss-type cheese is two to three times that in Cheddar (Richardson and Pearce, 1981; Lawrence et al., 1983) while the activity in Cheshire cheese is very low (Lawrence et al., 1983), suggesting that the plasmin content of cheese may be influenced by the pH at hooping (Lawrence et al., 1983). The difference in plasmin level between Cheddar and Swiss cheeses is considered unlikely to be due to pH dependent dissociation of the enzyme as the pH of both cheeses is 6.1 and 6.4 at whey drainage (Grufferty and Fox, 1988b). The differences may to be due to different rates of plasminogen activation in the two cheeses due to different processing conditions, especially cooking temperature (Ollikainen and Nyberg, 1988; Farkye and Fox, 1990), and possibly the higher pH in Swiss cheese during ripening (Grufferty and Fox, 1988b). The increase in pH in Swiss-type cheese during ripening is paralleled by a large increase in plasmin activity (Ollikainen and Nyberg, 1988). Owing to the relatively high buffering capacity of Swiss-type cheese (as affected by the retention of colloidal calcium phosphate due to the relatively high pitching pH (i.e., ⬃6.4 compared to ⬃6.1 for Cheddar)), its relatively high protein level (i.e., ⬃29 compared to 24% for Cheddar) and the propionic acid fermentation, during which lactic acid is converted to the weaker propionic and acetic acids, the pH


of Swiss does not fall as low as, and rises more rapidly than, that of Cheddar. The role of plasmin in cheese ripening has not been studied extensively but the presence of -caseins in most cheese suggests at least some activity (see Farkye and Fox, 1992). Plasmin appears to make a significant contribution to the maturation of Gouda (Creamer, 1976; Visser and de Groot-Mostert, 1977), possibly because of the removal of proteinase inhibitors by washing during curd manufacture, and in Romano-type cheese (Guinee and Fox, 1984) and in Swiss (Richardson and Pearce, 1981; Sweeney, 1984; Ollikainen and Nyberg, 1988; Ollikainen and Kivelä, 1989) in which the coagulant is extensively denatured by the high cooking temperature (Matheson, 1981). However, it has only a limited role in the ripening of Cheddar (Green and Foster, 1974; Creamer, 1976; Fenelon and Guinee, 2000; Kubis et al., 2001) and soft Meshanger-type cheese (Noomen, 1978). The coagulant is also extensively denatured in lowmoisture Mozzarella because of plasticization of the curd at ⬃58–60 °C (Feeney et al., 2001; see ‘Pasta-Filata Cheeses’, Volume 2), but plasmin makes little contribution to proteolysis, as reflected by the low levels of -casein (Yun et al., 1993a; Feeney et al., 2001) except where C. parasitica, a rennet substitute with a high proteolytic activity on -casein, is used (Yun et al., 1993b). However, degradation of -casein is very substantial in Mozzarella made from milk pre-acidified to ⬃5.6 prior to rennet addition (Feeney et al., 2002; Guinee et al., 2002). This effect concurs with the findings of Grufferty and Fox (1988b) who reported no dissociation of plasmin from the casein micelles in milk at pH 4.9. Noomen (1978) suggested that plasmin may make a significant contribution to proteolysis in soft cheeses with a surface flora, in which the pH rises markedly during ripening to a value more favourable for plasmin activity. The presence of -caseins in Camembert-type cheese, Cooleeney (Sousa and McSweeney, 2001) and blueveined cheeses, including Stilton, Danablu, Cashel, Chetwynd and Gorgonzola (Zarmpoutis et al., 1998), suggests a high level of plasmin activity, which is expected in view of the high pH of these cheeses. The addition of plasmin to milk, at levels which increased the activity by 3- to 4-fold the indigenous level normally found in the cheese, resulted in increased degradation of -casein and level of pH 4.6-soluble N (Farkye and Fox, 1992); the organoleptic quality of the plasmin-enriched cheese was superior to that of the control and the ripening rate was accelerated considerably. Noomen (1978) showed that the activity of alkaline milk proteinase in simulated cheese was stimulated by concentrations of NaCl up to a maximum at 2%, w/w, but was inhibited by higher concentrations of NaCl, although some activity remained at 8%, w/w, NaCl.

Milk also contains an acid proteinase, cathepsin D, which apparently has a specificity similar to chymosin (Kaminogawa and Yamauchi, 1972; Kaminogawa et al., 1980; Larsen and Petersen, 1995; Hurley et al., 2000a). About 80% of cathepsin D is in the serum and although it partially survives pasteurization (Larsen et al., 2000), its contribution to proteolysis in most cheeses is probably low. However, it has been claimed to make a contribution to proteolysis in Feta coagulated with GDL (Wium et al., 1998) or in Quarg (Hurley et al., 2000b). To our knowledge, the influence of NaCl on the activity of acid milk proteinases has not been investigated. Microbial enzymes

There appears to be relatively little information on the influence of NaCl on microbial enzymes in cheese. Indirect evidence, e.g., in relation to bitterness in cheese (Lawrence and Gilles, 1969; Sullivan and Jago, 1972; Stadhouders and Hup, 1975; Thomas and Pearce, 1981) suggests that the activity of starter proteinase is inhibited by a moderately high level of NaCl. P. roqueforti lipases (Morris and Jezeski, 1953) and proteinases (Madkor, 1985) are inhibited by NaCl concentrations 6%, w/w. Vafopoulou-Matrojiannaki (1999) found that an increase in S/M from 3 to 6%, w/w, reduced the activity of intracellular aminopeptidase, dipeptidylaminopeptidase and carboxypeptidase, but had little effect on the intracellular esterase activity of Leuconostoc mesenteroides subsp. mesenteroides strain K1G8. Gobbetti et al. (1999a) studied the interactive effects of pH (5.5–7.0), S/M (0.0–7.5%, w/w) and temperature (4–16 °C), under conditions designed to simulate the cheese environment, on the peptidase activities (aminopeptidases N and A, and proline iminopeptidase) of 11 strains of NSLAB bacteria isolated from cheese: Lb. casei subsp. casei 2107, 2756, 2788; Lb. plantarum 2788, 2789, 2741; Lb. casei subsp. pseudoplantarum 2745 and 2742; and Lb. curvatus 2771 and 2770. A low pH and a high S/M level markedly inhibited the peptidases of Lb. casei subsp. pseudoplantarum and Lb. curvatus. In contrast, the peptidases of Lb. casei subsp. casei and Lb. plantarum were quite insensitive to pH and not very sensitive to NaCl. The aminopeptidase activities (especially A) of the latter strains were less sensitive than the proline iminopeptidases to the combined effects of salt, temperature and pH. In a subsequent study, Gobbetti et al. (1999b) investigated the effects of S/M (2.5 to 7.5%, w/w), pH (5.0–5.7) and aw on the proteolytic and lipolytic activities of starter and NSLAB, including Lb. delbrueckii subsp. bulgaricus, Lc. lactis subsp. lactis T12 and Lb. plantarum 2739. The effect of S/M was both enzyme- and species-specific. These authors concluded


that interactions between these three variables were mainly responsible for changes in enzyme activity under conditions simulating cheesemaking. The combined effects of NaCl and pH did not significantly influence the lipase/esterase activity of Lb. plantarum 2739 and it was suggested that strains like 2739 might be responsible for a moderate level of lipolysis during long-term ripening of cheese. The cell envelope-associated proteinase (lactocepin) of Lc. lactis subsp. lactis BN1 and Lc. lactis subsp. cremoris SK11 was stabilized by NaCl (5%, w/v), especially at pH 5.2 (i.e., cheese-like conditions) and by the humectants, polyethylene glycol and sorbitol (Reid and Coolbear, 1998, 1999). The specificity of both proteinases on s1-, - and -caseins was changed considerably by NaCl level and water activity.

Influence of NaCl on the Water Activity (aw) of Cheese The preservative action of NaCl is due to its effect on the water activity (aw) of the medium: aw

p po

where p and po are the vapour pressure of the water in a system and of pure water, respectively. If the system is at equilibrium with its gaseous atmosphere, then aw ERH/100, where ERH is the equilibrium relative humidity. Due to the presence of various solutes in foods, the vapour pressure of water in a food system is always less than that of pure water, i.e., aw 1.0. The relationship between aw and the moisture content of food is shown in Fig. 4. Three zones are usually evident: • Zone I represents monolayer water that is tightly bound to polar groups in the food, e.g., the 9OH I

II

III

group of carbohydrates, or the 9NH 3 and 9COO groups of proteins; • Zone II consists of multilayer water in addition to the monolayer water; • Zone III contains bulk phase water in addition to monolayer and multilayer water.

Comprehensive discussions on the general concept of water activity in relation to foods are provided by Duckworth (1975), Rockland and Stewart (1981), Simatos and Multon (1985), Rockland and Beuchat (1987) and Fennema (1996). More specific aspects in relation to dairy products are discussed by Kinsella and Fox (1986) and Roos (1997). The aw of food depends on its moisture content and the concentration of low molecular mass solutes (Russell and Gould, 1991). The aw of young cheese is determined almost entirely by the concentration of NaCl in the aqueous phase: aw 1 0.033 [NaClm] 1 0.00565 [NaCl] where [NaClm] is the molality of NaCl, i.e., moles NaCl per litre of H2O and [NaCl] is the concentration of NaCl as g/100 g cheese moisture (Marcos, 1993). This equation was used to construct the nomograph shown in Fig. 5, which facilitates the calculation of aw. The salt content of cheese varies from ⬃0.7%, w/w, for Emmental to ⬃5%, w/w, for Domiati (Table 1). Other compounds, including lactic and other acids, amino acids, very small peptides and calcium phosphate, in addition to NaCl, contribute to the depression 60

0

1.00

55

0.99

50

0.98

1

g water/g dry matter

2

45

0.97

40

0.96 3

% H2O 0

0.25

0.5

0.75

1

Water activity, aw Figure 4 Idealized relationship between the water activity (aw) of food and its water content.

aw

% NaCl

Figure 5 Nomograph for estimation of water activity (aw) of fresh cheese from the percentages of moisture and salt. Examples: for respective salt and moisture levels (%, w/w) of 57 and 1.5, or 44.5 and 2.0, respectively, then aw 0.95 or 0.974, respectively (from Marcos and Esteban, 1982).


of aw, especially in extra-mature cheeses. Salt increases the osmotic pressure of the aqueous phase of foods, causing dehydration of bacterial cells, killing them or, at least, preventing their growth. The minimum water activity for the growth of various microorganisms in foods is shown in Table 3. Typical values for the aw of some cheese varieties are shown in Table 4. It will be apparent from Table 4 that the aw of most cheese varieties is not low enough to prevent the growth of yeasts and moulds and many bacteria but in combination with a low pH and low temperature, is quite effective in controlling microbial growth. Measurement of the salt content of cheese is an important quality control step in cheese production. As

described above, the aw of cheese can be calculated from its composition but can also be determined experimentally (see Marcos, 1993). The concentration and distribution of salt in cheese have a major influence on various aspects of cheese quality, as discussed in ‘Introduction’, ‘Control of Microbial Growth’, ‘Influence of NaCl on Enzyme Activity in Cheese’, ‘Influence of NaCl on the Water Activity (aw) of Cheese’ and ‘Overall Influence of NaCl on Cheese Ripening and Quality’. The aw of cheese, factors that affect it and related aspects have been reviewed by Acker (1969), Rüegg and Blanc (1977, 1981), Streit et al. (1979), Rockland and Nishi (1980), Marcos et al. (1981), Rüegg (1985), Fernandez-Salguero et al. (1986), Larsen and Anon (1989a,b, 1990), Marcos (1993) and Hardy (2000).

Table 3 Water activity (aw) of some cheese varieties* aw

Cheese

1.00 0.99 0.98

Fresh cheese curd, Ricotta Beaumont, Cottage, Fresh, Quarg Belle des Champs, Münster, Pyrénées, Processed, Taleggio Brie, Camembert, Emmental, Fontina, Limburger, Saint Paulin, Serra da Estrêla Appenzeller, Chaumes, Edam, Fontal, Havarti, Mimolette, Norvegia, Samsø, Tilsit Bleu de Bresse, Cheddar, Gorgonzola, Gouda, Gruyère, Manchego Idiazábal, Majorero, Mozzarella, Norzola, Raclette, Romano, Sbrinz, Stilton Danablu, Edelpilzkäse, Normanna, Torta del Casar Castellano, Parmesan, Roncal, Zamorano Provolone, Roquefort Cabrales, Gamalost, Gudbrandsdalsost, Primost

0.97 0.96 0.95 0.94 0.93 0.92 0.91 0.90

* Compiled from various sources.

Table 4 Minimum water activity (aw) for microbial growth in foods* Pathogen

Minimum aw

Shigella spp. Yersinia enterocolitica Vibrio parahaemolyticus Pseudomonas spp. E. coli Clostridium botulinum Salmonella spp. Listeria monocytogenes Micrococcus spp. Staphylococcus aureus (aerobic) Most yeasts and moulds Osmophilic yeasts and moulds

0.96 0.96 0.94 0.95 0.95 0.94 0.94 0.92 0.87 0.86 0.80 0.55

* Compiled from various sources.

Overall Influence of NaCl on Cheese Ripening and Quality Cheddar cheese

The influence of salt-in-cheese moisture (S/M) on lactose metabolism in young Cheddar cheese has already been discussed. There appears to be little information available on the influence of %, w/w, S/M on lipolysis in Cheddar and other cheeses. However, Thakur et al. (1975) compared lipolysis in salted (1.48–1.79%, w/w, NaCl) and unsalted Cheddar; the concentration of volatile acids was significantly higher in the unsalted than in the salted cheese mainly due to acetic acid, which is presumably a product of lactose metabolism. The concentrations of all individual fatty acids, except linoleic and linolenic (at certain ages), were also higher in the unsalted cheese than in the control; the authors did not comment on the markedly lower levels of linoleic acid in the unsalted cheese. However, Lindsay et al. (1982) found little difference between the level of free fatty acids in cheeses with low (3.5%, w/w) or intermediate (4–2%, w/w) S/M levels except for myristic and palmitic acids which were considerably higher in the higher-salt cheese. Reduced-sodium cheeses will be discussed in more detail in ‘Reduced Sodium Cheese’. Proteolysis is considerably more extensive in unsalted than in salted Cheddar cheese and consequently the body of the former is less firm (Thakur et al., 1975; Thomas and Pearce, 1981; Schroeder et al., 1988; Kelly et al., 1996). Wisniewska et al. (1990) reported that the salt content Cheddar, Gouda, Tilsit, Roquefort and Camembert was inversely related to the levels of primary and secondary proteolysis and directly to the time required to attain proper ‘organoleptic’ characteristics; the authors suggested that reducing the salt


content may offer a possible means of accelerating cheese ripening. However, as discussed below, a low level of NaCl has been found to adversely affect the quality of Cheddar cheese and a relatively narrow desirable range has been prescribed for premium quality (see also ‘Cheddar Cheese and Related Dry-salted Cheese Varieties’, Volume 2). A linear relationship between the extent of degradation of both s1- and -caseins in young (1 month) cheese and %, w/w, S/M is apparent from the data of Thomas and Pearce (1981) and Kelly et al. (1996). During the normal ripening of Cheddar cheese, sl-casein is the principal substrate for proteolysis with little degradation of -casein (see ‘Coagulant’); proteolysis of -casein is more extensive at low salt levels (Phelan et al., 1973; Kelly et al., 1996). However, Thomas and Pearce (1981) noted that while the normal products of -casein degradation (-CNf1-192, -CNf1-189 and -CNf1-165 produced by rennets, and -caseins by milk proteinase) were not apparent in their studies, the concentration of unhydrolysed -casein decreased, suggesting that proteolysis of -casein in low-salt cheese may be due to bacterial proteinases. Kelly et al. (1996) noted that cleavage of Leu192–193 in -casein and Leu101–Lys102 in s1-casein was particularly sensitive to the salt concentration in Cheddar cheese. In contrast to primary proteolysis, the level of secondary proteolysis, as measured by the level of 5% (w/v) phosphotungstic acid-soluble N, tended to be higher in salted (2.7–5.7%, w/w, S/M) than in unsalted Cheddar at 12 and 24 weeks; no effect of S/M was apparent at 5 weeks. At least five studies (O’Connor, 1971; Gilles and Lawrence, 1973; Fox, 1975; Pearce and Gilles, 1979; Lelievre and Gilles, 1982) have attempted to relate the quality of Cheddar cheese to its composition. While these authors agree that the moisture content, %, w/w, S/M and pH are the key determinants of cheese quality, they disagree as to the relative importance of these three parameters. In a study of 300 Scottish Cheddar cheeses, O’Connor (1971) found that flavour and aroma, texture and total score were not correlated with moisture content but were significantly correlated with %, w/w, NaCl and particularly with pH. Salt content and pH were themselves strongly correlated, as were salt and moisture; a very wide variation in composition was noted. Based on analysis of cheese made at the New Zealand Dairy Research Institute over many years and also by commercial cheese factories in New Zealand, Gilles and Lawrence (1973) proposed a grading scheme for young (14-day-old) Cheddar cheese. The influence of cheese composition on quality and compositional grading of Cheddar cheese is discussed in ‘Factors that

Affect the Quality of Cheese’, Volume 1 and ‘Cheddar Cheese and Related Dry-salted Cheese Varieties’, Volume 2; suffice it to record here that the S/M specified for premium and First Grade Cheddar in New Zealand are 4.0–6.0 and 4.7–5.7, respectively (Lawrence et al., 1993). Fox (1975) assessed the influence of moisture, salt and pH on the grade of 123, 10-week-old Irish Cheddar cheeses (70 high quality and 53 ‘rejects’) from six factories and 27 extra-mature, high-quality Cheddars. The composition of the cheeses varied widely and while the correlations between grade and any of the compositional factors were poor, a high percentage of cheeses with compositional extremes was downgraded, especially those with low salt (1.4%, w/w), high moisture (39%, w/w) or high pH (pH 5.4). In the samples studied, salt concentration seemed to exercise the strongest influence on cheese quality and the lowest percentage of downgraded cheeses can be expected in the salt range 1.6–1.8%, w/w (S/M range, 4.0–4.9%, w/w). The composition of high quality extramature cheeses also varied widely but less than that of the young cheeses. Although the mean salt level was identical for both groups of cheeses, the spread was much narrower for the mature cheeses and only three had 1.7%, w/w, NaCl. The mean moisture content of the mature cheeses was 1%, w/w, lower than that of the regular cheeses. The grading ratio (ratio of high to low grading cheeses) for 486 14-day-old cheeses produced at the New Zealand Dairy Research Institute was most highly correlated with the percentage of moisture in nonfat-substances (MNFS) and second best with the percentage of salt (Pearce and Gilles, 1979). The optimum compositional ranges were: MNFS 52–54%, w/w; S/M 4.2–5.2%, w/w; pH 4.95–5.15. Cheese with an S/M of 3.1%, w/w, received the highest grade in a study by Knox (1978) although there was little difference in grade in the S/M range 3.1–5.2%; quality declined markedly at S/M 6.4%, w/w. A very extensive study of the relationship of the grade and composition of nearly 10 000 cheeses produced in five commercial New Zealand factories was undertaken by Lelievre and Gilles (1982). As in previous studies, considerable compositional variation was evident but the variation was considerably less for some factories than others. While the precise relationship between grade and composition varied from plant to plant, certain generalizations emerged: • within the compositional ranges suggested by Gilles and Lawrence (1973) for ‘premium’ quality cheese, composition does not have a decisive influence on grade, which falls off outside this range;


• composition alone does not provide a basis for grading as currently acceptable to the dairy industry (New Zealand); • MNFS was again found to be the dominant factor influencing quality; • within the recommended compositional bands, grades declined marginally as MNFS increased from 51 to 55%, w/w, increased slightly as S/M decreased from 6 to 4%, w/w, while pH had no consistent effect within the range 4.9–5.2 and FDM had no influence in the range 50–57%, w/w. The authors stress that since specific inter-plant relationships exist between grade and composition, each plant should determine the optimum compositional parameters pertinent to that plant. Apart from the acid flavour associated with low-salt cheese, bitterness has been reported consistently as a flavour defect in such cheeses. A complex correlation exists between the propensity of a cheese to develop bitterness and starter culture, pH, rate of acid development and %, w/w, S/M. There is still some controversy on the development of bitterness (see Lowrie and Lawrence, 1972; Mills and Thomas, 1980; Stadhouders et al., 1983; Gomez et al., 1997; McSweeney, 1997; Smit et al., 1998, 2002; Kirin, 2001; Morales et al., 2001; Broadbent et al., 2002), but the subject will not be reviewed here. From the compositional viewpoint, S/M, %, w/w, appears to be the most important factor influencing bitterness (Lawrence and Gilles, 1969). The probability of bitterness developing is greatly increased at S/M 4.9%, w/w; pH, in the normal range encountered for Cheddar, i.e., 4.9–5.3, where para-casein is most soluble (Creamer, 1985) and therefore most susceptible to proteolysis, has little effect except at low S/M values, i.e., 4.9%, w/w. Rennet has maximum activity on paracasein in salt solutions between 2.5 and 4%, w/w (Stadhouders, 1962). The bitterness of peptides is strongly correlated with hydrophobicity (Guigoz and Solms, 1976; Bumberger and Bleitz, 1993). The bitter peptides in cheese appear to arise primarily from -casein (see Hill et al., 1974; Visser et al., 1983a,b; Dinakar et al., 1989; Vandeweghe, 1994; Casal and Gomez, 1999; Frister et al., 2000) which might be expected since -casein is the most hydrophobic casein (Swaisgood, 2003); however, peptides from s1- and s2-caseins, especially those containing proline, probably also contribute to bitterness in cheese (Lee and Warthesen, 1995; Kai Ping Lee, 1996; Frister et al., 2000). The effectiveness of NaCl in preventing bitterness is very likely due to the strong inhibition of -casein hydrolysis by NaCl (Fox and Walley, 1971; Phelan et al., 1973; Mulvihill and Fox, 1978; Pearce,

1982; Stadhouders et al., 1983; Gouda, 1987; Banks et al., 1993; Kelly et al., 1996; Mistry and Kasperson, 1998). However, Laan et al. (1998) found that the addition of salt (4%, w/w) and Ca (120 mM), at levels to simulate those in cheese, increased the aminopeptidase activity of starter lactococci and non-starter lactobacilli isolated from Cheddar cheese. Such aminopeptidase activities, which debitter -casein hydrolysates (Kai Ping Lee, 1996; Parra et al., 1999; Barry et al., 2000; Bouchier et al., 2001), are considered important in reducing the risk of bitterness in cheese. The protein matrix in young cheese appears to consist of s1-casein molecules linked through hydrophobic interactions between their amino-terminal regions; the primary site for rennet action on s1-casein is Phe239Phe24 (Hill et al., 1974) or Phe249Val25 (Creamer and Richardson, 1974), hydrolysis of which weakens the matrix. This specific cleavage is considered to be primarily responsible for the loss of firmness during the early stages of ripening (de Jong, 1976; Creamer and Olson, 1982; Fenelon and Guinee, 2000; ‘Cheese as an Ingredient’, Volume 2). Hard and semi-hard cheese, such as Cheddar, becomes shorter also during maturation (Visser, 1991; Fenelon and Guinee, 2000). Luyten (1988) found that increased s1-casein breakdown in Gouda cheese had little effect on shortness (which may be best described as the inverse of fracture strain). Indeed, the increase in shortness of Gouda cheese on ripening was attributed more to in-depth proteolysis (e.g., NPN formation) than to gross proteolysis. The increase in shortness with maturation may arise as a result of an upward shift in pH away from pH 5.2–5.35 where casein hydration as a function of pH in the range 4.6–6.0 (Creamer, 1985) and fracture strain (Luyten et al., 1987; Visser, 1991) are maximal (see ‘Cheese as an Ingredient’, Volume 2). Indeed, this seems highly probable when one considers the production of pastafilata-type cheeses such as Mozzarella and Kashkaval; the cheeses flow and stretch over a narrow pH range, 5.2–5.35, outside which flow is very restricted unless some processing changes, such as reduction of calcium level or plasticizing in hot dilute brine (so as to partially solubilize the casein), are implemented (see ‘Pasta-Filata Cheeses’ and ‘Cheese as an Ingredient’, Volume 2). During the time required for the pH to fall from ⬃6.1 at pitching to ⬃5.2 at stretching, little or no degradation of s1-casein occurs. It is probable that both mechanisms (i.e., NPN formation with consequent movement of pH from the point of maximum fracture strain, and hydrolysis of s1-casein) contribute to the age-related rheological/textural changes to different extents depending on the variety and the ratio of primary-to-secondary proteolysis. Owing to its


effects on primary and secondary proteolysis, the salt content of cheese has a major influence on its rheological properties, as discussed in ‘Effect of NaCl on cheese rheology’. Blue cheese

The influence of NaCl concentration on the principal ripening events in Blue cheese was studied by Godinho and Fox (1981a,b,c, 1982). Proteolysis, as measured by polyacrylamide gel electrophoresis and the formation of 12% TCA-soluble N, was invariably lower in the outer (high salt) region than in the middle or centre (lower salt) zones; the differences were apparent both before visible mould growth (during the first two weeks when the coagulant is the principal proteolytic agent) and during the mould phase (after two weeks) (Godinho and Fox, 1982; Hewedi and Fox, 1984). There was a strong negative correlation between salt concentration and TCA-soluble N. Unfortunately, the formation of amino acid N (e.g., PTAsoluble N) or other more detailed characterizations of proteolysis were not investigated. With a few exceptions, the pH increased faster at the centre than in the outer region of the cheese, indicating that the catabolism of amino acids or lactic acid is also influenced by NaCl concentration. Lipolysis in Blue cheese is also influenced by salt concentration, with maximum activity occurring at 4–6%, w/w, NaCl (Godinho and Fox, 1981c). However, the concentration of methyl ketones was relatively independent of salt concentration. Camembert cheese

The ripening of the surface mould-ripened cheeses, Camembert and Brie, is characterized by a very marked softening, almost liquefaction, of the body from the surface to the centre. This ripening pattern is mainly due to the combination of s1-casein hydrolysis and the decreasing pH gradient from the surface to the centre, due to the production of ammonia by the surface mould, P. camemberti, and its inward diffusion, and the catabolism of lactic acid, and outward diffusion of calcium (Le Graet et al., 1983; Noomen, 1983; Karahadian and Lindsay, 1987). Proteolysis by the coagulant and starter proteinases is also important and although the proteinases excreted by P. camemberti undergo only very limited diffusion in the cheese (Noomen, 1983), peptides produced by them do, apparently, diffuse into the cheese (see ‘Surface Mould-ripened Cheeses’, Volume 2 and ‘Biochemistry of Cheese Ripening: Introduction and Overview’, ‘Metabolism of Residual Lactose and of Lactate and Citrate’, ‘Lipolysis and Catabolism of Fatty Acids in

Cheese’, ‘Proteolysis in Cheese during Ripening’, ‘Catabolism of Amino Acids in Cheese During Ripening’, Volume 1). In this variety also, NaCl concentration has a major influence on proteolysis and pH changes, as well as on surface mould growth (Table 2). Other cheeses

Feta and Domiati are special in the sense that they are stored in brine, containing typically 6–8%, w/w, NaCl, after manufacture. The high level of salt strongly affects the microflora, enzymology and ripening of these cheeses (see ‘Cheese Varieties Ripened in Brine’, Volume 2). In addition to the inward migration of salt, outward diffusion of low molecular mass water-soluble compounds (e.g., small peptides, amino acids, lactate, volatile water-soluble acids, and minerals) occurs and these accumulate in the brine. Studies on the diffusion of these molecules are lacking. Pappas et al. (1996) studied the effects of S/M level (4.3–5.8%, w/w, S/M) in Feta cheese, by altering the duration of dry-salting prior to storage in 7–8%, w/w, brine. Increasing S/M level reduced the moisture content and the levels of pH 4.6-soluble N and lipolysis (as measured by acid degree value), or organoleptic characteristics. El-Sissi and Neamat-Allah (1996) studied the effect of different salt levels in cheesemilk (5, 7, 9 and 12%, w/w, added NaCl) on the ripening and quality of Domiati; the corresponding S/M levels in the cheese were ⬃6.0. 8.3, 9.5 and 12.7%, w/w. Increasing the salt level increased yield (2.9–3.5 kg/100 kg), moisture (⬃61–69%, w/w), pH (⬃5.3–6.5) and reduced the level of pH 4.6-soluble N (% total N) and of total volatile fatty acids; these effects were most dramatic as the S/M level was increased from 9.5 to 12.8%, w/w. The development of the desired flavour and texture/body characteristics was prevented at 12.8%, w/w, S/M (even after 4 weeks storage in pasteurized whey at 14 °C) and delayed at 9.5%, w/w, S/M, compared to cheese with a lower S/M level. The authors recommended a S/M level of 5–9%, w/w, for accelerating the ripening of Domiati. Nájera et al. (1994) reported that the concentrations of individual (C4–C18 and C18:1) and total FFAs in Idiazabal cheese increased on increasing the brining time from 12 to 24 or 36 h; however, no details on cheese composition were given. Kaya et al. (1999) studied the effect of storage time in brines of different NaCl concentration on the ripening and quality of a Turkish white pickled cheese, Gaziantep, the manufacture of which does not involve the addition of a starter culture. Increasing the S/M level in the experimental Gaziantep cheese from 8.7 to 24%, w/w,


resulted in significant reductions in moisture content and the level of FFAs, and increases the firmness and peroxide value. Sensory analysis showed that increasing the S/M level to 13.2%, w/w, increases the intensity of off-flavours, metallic, oxidized, rancid and bitterness. Kristiansen et al. (1999) investigated the effects of varying S/M on proteolysis in Danbo-type cheese brine-salted for different times. Increasing S/M, in the range 0.1–6.4%, w/w, significantly reduced the level of MNFS, the degradation of -casein and the levels of pH 4.6 and 5% (w/v) phosphotungstic acid-soluble N over the 10-week ripening period. In contrast, higher salt levels had little effect on the primary breakdown of s1-casein. Capillary gel electrophoresis showed that the hydrolysis of -casein in dilute solution (0.5%, w/v, in sodium phosphate buffer, pH 7.0) by chymosin or bovine pepsin was strongly inhibited by 5%, w/w, S/M, while that by plasmin was not (Kristiansen et al., 1999); the opposite trend was noted for s1-casein. These results concur with those of Lane and Fox (1999) who studied the effects of salt (0–10%, w/w, S/M) and pH on the proteolysis of -casein and sodium caseinate in dilute (0.5%, w/v) solutions. The negative effect of salt on proteolysis is associated with its effect on ionic strength, which affects casein hydration and conformation (see ‘Effect of NaCl on Casein Hydration and the Physical Properties of Cheese’), and the concomitant reductions in the level of MNFS and aw (Creamer, 1971; Lawrence and Gilles, 1980; Rüegg and Blanc, 1981; van den Berg and Bruin, 1981). A higher S/M level (i.e., 3.0%, w/w) in accoustically brined Mahon cheese, compared to the conventionally brined control cheese (2.8%, w/w) gave higher levels of all individual FFAs analysed (apart from caprylic and stearic acids) and an increase (9%) in the concentration of total FFAs (Sánchez et al., 2001). This trend is similar to that noted by Nájera et al. (1994) for Idiazabal cheeses but disagrees with that reported by Thakur et al. (1975) and Lindsay et al. (1982) for Cheddar (who reported that increasing the salt level from 0.03 to 1.78, and from 1.25 to 1.5%, w/w, respectively, led to reductions in the levels of individual and/or total FFAs) and to that of Freitas and Malcata (1996) for Picante, a hard Portuguese cheese.

Effect of NaCl on Casein Hydration and the Physical Properties of Cheese The extent of the hydration and aggregation of casein has a major impact on the formation and textural/functional characteristics of dairy products, including cheeses (see Fox and McSweeney, 2003). Indeed, the

manufacture of many protein-based products and ingredients, such as cheese, yoghurt and casein, is based on a limited destabilization and aggregation of the casein micelles. The extent of casein aggregation, or hydration, affects the microstructure and nature of attractions between protein molecules within the protein phase of dairy products containing protein. Consequently, it has a major influence on several aspects of product quality: rheology, texture and cooking characteristics of cheese (see ‘Rheology and Texture of Cheese’, Volume 1 and ‘Pasta-Filata Cheeses’ and ‘Cheese as an Ingredient’, Volume 2); texture and mouth-feel of yoghurt; and re-hydration characteristics of casein in food formulation (see Fox and McSweeney, 2003). Apart from Domiati, all cheeses are salted after rennet coagulation and curd formation, at a level ranging from ⬃2.0%, w/w, in Emmental to ⬃12%, w/w, in Feta. The practice of adding salt to the curd, rather than to the milk, has been deliberate as the early cheesemakers would have soon discovered that its addition prior to renneting severely impaired or prevented the coagulation of milk (Fig. 6) and curd syneresis (Cheeseman, 1962; Grufferty and Fox, 1985; Walstra et al., 1985; Pearse and Mackinlay, 1989; Abou-El-Nour, 1998). The adverse effects of salt at the concentrations used in cheese on curd formation are probably a consequence of the solubilization of colloidal calcium phosphate as a result of a sodium–calcium interchange, and the positive effect of salt on casein hydration, which impairs casein aggregation. In Domiati cheese, where 5–15%, w/w, NaCl is added to the milk (Abou-El-Nour, 1998), the effects of NaCl in curd formation are off-set by the use of water buffalo milk, which has a higher casein content than bovine milk (see Kosikowski and Mistry, 1997), or by the fortification of milk with skim milk powder, and/or the addition of CaCl2 (M.M. Hewedi, personal communication). Owing to the importance of casein hydration on cheese quality, the effects of NaCl on hydration are discussed below. Effect of NaCl on casein hydration in model systems and in cheese

The significance of casein hydration, as affected by NaCl, on the physical properties of cheese has been demonstrated using dilute model systems. Creamer (1985) studied the effect of NaCl on casein hydration in rennet-treated skim milk at pH values in the range 4.6–6.6, by measuring the levels of moisture and protein in the para-casein pellet obtained on ultracentrifugation at 81 000 g for 2 h. The addition of 5%, w/w, NaCl to the milk increased the levels of serum Ca and casein hydration at all pH values, with a maximum in the pH range 5.2–5.3 (Fig. 7). The increase in


80

70

Elastic shear modulus, G′, Pa

60

50

40

30

20

10

0 0

1000

2000

3000

4000

5000

6000

Time from renent addition, s

Supernatent para-casein, % of total para-casein

Figure 6 Influence of added salt on the firmness, as measured by the elastic shear modulus (G), of reduced-fat milk (2.0%, w/w, fat; 3.3%, w/w, protein; 4.7%, w/w, lactose). Salt was added at levels of 0 (), 1 (), 2 (), 4, 6, 8 or 12%, w/w, and stirred into the milk for 30 min prior to measurement of G using dynamic low amplitude oscillation strain rheometry; no coagulation was detected at salt levels 4%, w/w. The pH of the milks at the different salt levels was 6.66, 6.48, 6.42, 6.38, 6.35, 6.25 and 6.27, respectively (T.P. Guinee, unpublished results).

80 70 60 50 40 30 20 10 0 4.7

5.0

5.3

5.6

5.9 6.2 pH

6.5

6.8

7.1

7.4

Figure 7 Effect of salt, added at 5 %, w/w, on the solubility of a 5%, w/w, dispersion of rennet casein (80%, w/w, protein; 7.1%, w/w, ash). The rennet casein was thoroughly mixed with water () or 5%, w/w, NaCl () and the pH adjusted to different values using 20%, w/v, lactic acid. The dispersions were held at 15 °C for 22 h and then centrifuged at 3000 g for 30 min at 15 °C. The protein content of the supernatant was measured and expressed as a percentage of the total protein (T.P. Guinee and B.T. O’Kennedy unpublished results).

casein hydration with NaCl may be attributed to the binding of Na by the casein (Gál and Bánky, 1971; Hardy and Steinberg, 1984) and the displacement of calcium or calcium phosphate from the para-casein by the Na. In effect, the addition of NaCl appears to create a sodium–calcium ion exchange effect with the para-casein, somewhat similar to that observed between emulsifying salts (sodium phosphates and sodium citrates) and the casein matrix during the manufacture of processed cheese products (see ‘Pasteurized Processed Cheese and Substitute/Imitation Cheese Products’, Volume 2). Indeed, an inverse relationship between casein hydration and casein-bound calcium in model systems has been reported by many investigators. In these studies, casein hydration was measured directly or indirectly by determining the levels of water and protein in pellets obtained on ultracentrifugation of milk (Sood et al., 1979, 1980; Guillaume et al., 2002) or the uptake of water by casein(ate)s with different levels of added calcium on exposure to environments with different aw values in the range 0.58–0.95 (Rüegg and Moor, 1986). Interestingly, a study by Pastorino et al. (2003b) showed that the addition of calcium, at a level of 0–1.4%, w/w,


to Mozzarella cheese by high pressure injection of a 40% (w/w) CaCl2 solution resulted in marked ‘weeping’ and water loss, a reduction in pH and a more aggregated para-casein matrix interspersed with large voids containing free water; the reduction in moisture was ⬃12%, w/w, at 1.4%, w/w, Ca. These findings indicate that the addition of calcium increases casein– casein interactions and thereby reduces casein hydration. Conversely, the levels of moisture and non-expressible serum, which is an index of protein hydration, in Mozzarella cheese increase as the calcium content is reduced (Guinee et al., 2002). In Mozzarella cheese, the effect of salt on casein hydration has been measured by determining the level of expressible serum (ES), i.e., the serum released on centrifugation of the cheese at ⬃12 500 g at 25 °C, and the level of serum protein (Guo et al., 1997). The level of ES in brine-salted Mozzarella (49.5%, w/w, moisture; 1.4%, w/w, NaCl; 2.8%, w/w, salt-in-moisture) decreased from ⬃16 to 1 g/100 g cheese over the first 10 days of ripening, indicating an increase in the water-binding capacity of the protein (Guo et al., 1997); the level of water-soluble protein (WSP) in the serum increased from ⬃5 to 10%, w/w, over the same time period. In contrast, the levels of ES and WSP for the unsalted cheese (0.13%, w/w, NaCl) changed from ⬃19 to 14 g/100 g, and from ⬃3 to 5%, w/w, respectively, during storage. However, the level of pH 4.6-soluble protein in the sera from both the salted and unsalted cheeses was similar, indicating that the differences in WSP were due to the solubilization of intact casein by the added NaCl rather than to differences in proteolysis of the casein. Thus, electrophoretic analysis of the sera showed that the levels of intact s1-, s2-, - and para-casein in the ES of brine-salted Mozzarella were higher than those for the unsalted cheese at all times over a 10-day storage period. A similar trend was reported for fat-free Mozzarella for which the curd was dry-salted to different levels (0, 0.5 or 1.0%, w/w) prior to stretching in hot water containing 0, 5 or 10%, w/w, NaCl (Paulson et al., 1998). At all times throughout the 24-day storage period, the level of ES in cheeses from curds stretched in hot water decreased as the level of dry-salting prior to stretching was increased to give a salt level of 0.14–0.68%, w/w (0.2–1.1%, w/w, S/M) in the cheese. No ES was obtained at any stage from cheeses made from curds stretched in hot solutions containing 5 or 10%, w/w, NaCl and with salt levels of 0.85–2.18%, w/w (i.e., 1.4–3.5%, w/w, S/M). These results indicate a higher water-binding capacity of the protein matrix of cheeses as the S/M level increased to 0.85%, w/w. Dry-salting of cheese (by mixing milled curd pieces and salt prior to moulding or further curd treatments)

offers several advantages over brine-salting, including savings in plant space (occupied by brine tanks) and labour, more uniform salt-in-moisture distribution (at least initially), less zonal variations in texture, melting properties and quality (see ‘Effect of salt content on cooking properties’ and ‘Attainment of salt and moisture equilibria after salting’). Consequently, the effect of dry-salting Mozzarella cheese prior to plasticization in hot water as an alternative to brining the plasticized curd in cold brine has been investigated (Fernandez and Kosikowski, 1986; Paulson et al., 1998; Guinee et al., 2000). Plasticization of curd in hot salt solutions containing 5 or 10 (Paulson et al., 1998) or 18 (Piacquadio et al., 2001) %, w/w, NaCl has also been investigated; this form of salting is similar to plasticizing dry-salted curd in hot-water. A combination of dry-salting (e.g., 1%, w/w) and plasticizing in hot dilute brine (e.g., 5%, w/w, NaCl) has also been examined (Paulson et al., 1998; Guinee et al., 2000). The use of dry salting at 4.6%, w/w, or a combination of dry salting (to 1%, w/w, NaCl) and plasticizing in hot dilute brine (i.e., 5%, w/w, NaCl) gave the desired S/M level (e.g., 2.3%, w/w) and a moisture content which was higher (by ⬃2–3%, w/w) than that of conventionally brine-salted Mozzarella (Paulson et al., 1998; Guinee et al., 2000). The higher moisture content suggests a salting-in of the casein and a concomitant increase in the degree of para-casein hydration in the cheeses which were dry-salted or drysalted and platicized in hot dilute brine. In contrast to the above, Fernandez and Kosikowski (1986) reported that stretching in hot brine (10%, w/w, NaCl) resulted in a marked decrease in moisture content (3–4.5%, w/w) in low-moisture Mozzarella. The above findings for Mozzarella are consistent with those from the model systems described in this section which showed that a low concentration of NaCl (⬃5–6%, w/w, S/M) enhances the solubilization of casein or para-casein (Hardy and Steinberg, 1984; Creamer, 1985). Such a trend is expected as the protein matrix of Mozzarella, and all rennet-curd cheeses, may be considered as highly concentrated hydrated para-casein. The influence of low salt concentrations on casein hydration in cheese is also apparent from: • the higher level of 5% salt-soluble N than watersoluble N in Cheddar and other cheese varieties, e.g., 93 and 26% total N for 6-month-old Cheddar (Reville and Fox, 1978); • the uptake of water by cheese in dilute brines, especially if calcium is absent (Geurts et al., 1972; Guinee and Fox, 1986a, 1993). On salting in brines of typical composition (e.g., 18–22%, w/w, NaCl and 0.5%, w/w, CaCl2), cheese looses water during brining, resulting in a net weight


loss, as discussed in ‘Moisture level’. In contrast, salting in freshly prepared dilute brine (e.g., #12%, w/w, NaCl) without added calcium has been found to increase the moisture content in the rind region of Gouda (Fig. 8) and in Romano cheese slices (Fig. 9; Guinee and Fox, 1986a). This phenomenon can lead to a defect known as soft-rind or rind rot (soft, slimy, sticky surface) in Gouda and other cheeses which are salted in freshly prepared brines without calcium (Geurts et al., 1972), unless casein hydration is reduced by adding CaCl2 and reducing the pH to ⬃4.8–5.0. The problem rarely occurs in mature (stable composition and pH) well-used brine because of the accumulation of soluble calcium which migrates from the cheese with the moisture. Effect of NaCl on cheese microstructure

Owing to its effect on protein hydration, salt has a major influence on the microstructure of cheese. Scanning electron microscopy has shown that the protein matrix of salted non-fat Mozzarella (⬃0.25 or 3.5%, w/w, S/M) or Muenster (⬃1.2, 3.6, or 6.7%, w/w, S/M) cheeses is more swollen, homogeneous and continuous, and has a higher specific surface area than their unsalted counterparts (Paulson et al., 1998; Pastorino et al., 2003a). Moreover, the unsalted cheeses had large

44

24

42 40 16 38 12

36 34

Moisture, %, w/w

Salt-in-cheese moisture, %, w/w

20

8 32 4 30 0

0

1

2

3

4

5

6

28

Distance from cheese surface, cm Figure 8 Moisture content (open symbols) and salt-in-moisture concentration (closed symbols) in Gouda cheese as a function of distance from the salting surface after unidimensional brinesalting for 4 days at 20 °C in 5 ( , ), 12 (,), 20 (,) or 24.8 (,●) %, w/w, NaCl solution (without calcium) (redrawn from Guinee, 1985)

open channels with free serum (whey pockets) distributed throughout the matrix, which acted as light scattering surfaces and gave the cheese an opaque white colour, compared to a translucent, waxy appearance in the salted cheeses in which such fissures were fewer and smaller. These observations suggest that salt increases casein hydration and are consistent with the positive relationships between the NaCl content (at low levels, i.e., ⬃6%, w/w, S/M) and casein hydration in dilute casein systems and in cheese (see ‘Effect of casein hydration in model systems and in cheese’). Effect of NaCl on cheese rheology

The influence of NaCl on cheese texture is most obvious on comparing salt-free cheeses with their salted counterparts; the former is generally weak, soft, pasty and/or adhesive depending on age. In contrast, high salt concentrations lead to shortness, crumbliness, dryness and hardness, as observed for curds held in brine for an excessively long time. The relationship between cheese rheology/texture and salt level is also evident on visual examination of brine-salted cheese on completion of salting; the outside rind region is hard, brittle, dry and white while the inside is more pliable, waxy, softer and more translucent. The influence of NaCl on cheese texture is probably due to its effects on composition (moisture content, MNFS), para-casein hydration/solubility and conformation, agerelated effects on pH (see Table 2; Furtado et al., 1982) and proteolysis. The effects of salt are discussed in more detail below. Numerous investigators have studied the effects of salt concentration, or salt-in-moisture (S/M), on rheological properties such as firmness (max, e.g., force required to attain a given compression, or to push a probe to a given depth into cheese), fracture stress (f), fracture strain ( f) and/or sensory hardness. These studies have shown that increases in S/M within the range 0.4–12%, w/w, result in increased firmness, max, f, and sensory hardness for Camembert (Pagana and Hardy, 1986; Lesage et al., 1992), Cheddar (⬃0.2–6%, w/w, S/M; Thakur et al., 1975; Schroeder et al., 1988), reduced-fat Cheddar (2.7–4.5%, w/w, S/M; Mistry and Kasperson, 1998), Feta (6.7–10.5%, w/w, S/M; Prasad and Alvarez, 1999; 2.0–5.5%, w/w, S/M; Pappas et al., 1996), Gaziantep cheese (Kaya, 2002), Mozzarella (0.5 and 5.14%, w/w, S/M; Cervantes et al., 1983), Muenster (0.3–7.45%, S/M; Pastorino et al., 2003a), and low pH model cheese made from ultrafiltered skim milk retentate (2.0 and 4.7%, w/w, S/M; Euston et al., 2002). The increase in max and f may be attributed in part to the concomitant changes in composition, e.g., reduction


g NaCl gained per 100 g cheese

12 10 8

8

6

6

4

4

2

2

0

0 0

100 240 0

100 240 0 100 200 0

100 200 0

100 200

g water lost per 100 g cheese

14

–2

Brining time, min Figure 9 Moisture loss (open symbols) and salt uptake (closed symbols) by Romano-type cheese slices (0.5 cm thick; 7 cm diameter) brine-salted in 6.5 ( , ), 10.7 (,), 14.8 (,), 18.9 (,) or 24.9 (,), %, w/w, NaCl solution (without calcium) at 20 °C, as a function of time in the brine (redrawn from Guinee and Fox, 1986a).

in moisture level and increase in protein, and the effects of salt on proteolysis (see ‘Cheese as an Ingredient’, Volume 2). The effect of salt on composition is very evident in brine-salted cheeses, in which there is generally an increasing moisture gradient from the surface to the centre, and a salt gradient in the opposite direction (see ‘Salt Absorption and Diffusion into Cheese’) at the end of salting. However, even in cheeses where the other compositional parameters were relatively constant (Visser, 1991; Euston et al., 2002), salt had a major effect on cheese rheology. This suggests that in addition to its indirect effects on rheology via its influence on gross composition, salt also exerts more direct effects on rheology, e.g., by promoting changes in the degree of casein hydration and aggregation which alters the ratio of viscous to elastic character in the cheese. Thus, Euston et al. (2002) noted an effect of the interaction between salt level and pH on the rheology of model cheeses with similar gross composition. Pagana and Hardy (1986) reported an inverse linear relationship between the brittleness of Camembert cheese, as measured by fracture strain, and S/M level in the range ⬃3–21%, w/w. Visser (1991) noted that the fracture strain of model Gouda cheeses increased monotonically with S/M in the range 0 to ⬃4.5%, w/w, then decreased sharply to a value which was about half the maximum at 5.5%, w/w, S/M and remained relatively constant thereafter as the S/M was increased to 11.3%, w/w. Similar to f and max, the effect of salt on

f is probably also attributable to a salting-in effect with a concomitant increase in para-casein hydration as S/M increases to ⬃5%, w/w, and a salting-out effect with a

concomitant loss in casein hydration at higher S/M levels (Geurts et al., 1972; Guinee and Fox, 1986a; ‘Cheese as an Ingredient’, Volume 2). An increase in casein hydration would impart a more viscous (and less elastic) character to the cheese and a transition from elastic fracture behaviour to plastic fracture behaviour, which would necessitate a higher strain for fracture (see ‘Rheology and Texture of Cheese’, Volume 1). Conversely, a lower degree of casein hydration at the higher S/M (5%, w/w) would favour a more elastic casein matrix and an elastic fracture behaviour, i.e., a shorter, firmer, more brittle cheese. Effect of salt content on cooking properties

The development of acceptable cooking characteristics in cheese, such as Mozzarella and Cheddar, generally requires an ageing period (see ‘Pasta-Filata Cheeses’ and ‘Cheese as an Ingredient’, Volume 2), the duration of which depends, inter alia, on the cheese type, manufacturing conditions, ripening conditions and the specifications set by the customer. Age-related biochemical and microstructural changes which contribute to the development of the desired cooking characteristics include: increases in proteolysis, casein hydration and non-globular fat; a concomitant swelling of the protein matrix; and decreases in the levels of aggregation and continuity of the para-casein matrix. The increase in casein hydration leads to an enhanced water-binding capacity of the protein matrix, improved moisture retention during cooking, easier displacement of neighbouring planes of the protein matrix in the heated cheese mass, and improved (higher)


flowability and stretchability of the heated cheese. Moreover, the heated aged cheese is generally more moist and less prone to blistering and charring than fresh cheese. Salt level has a marked influence on the cooking properties of low-moisture part-skim Mozzarella (Apostolopoulos et al., 1994) and non-fat Mozzarella cheese (Paulson et al., 1998), a trend expected because of its influence on casein hydration, as discussed in ‘Effect of NaCl on casein hydration in model systems and in cheese’. In these studies, salt level was varied by brining in salt solutions of different concentration, or by dry salting at 0.0, 0.5 or 1.0%, w/w, NaCl prior to stretching the curd in hot water containing 0, 5 or 10%, w/w, NaCl (Paulson et al., 1998). The flowability of melted cheeses increased fairly linearly with salt level in the range 0.1–0.5%, w/w (S/M ⬇ 0.2–1.2%, w/w) and changed only slightly as the salt level was further increased to ⬃2.2%, w/w (S/M ⬇ 0.2 to 3.3%, w/w). The increase in flowability coincided with an increase in free oil and water-binding capacity of the para-casein matrix, as reflected by a decrease in the level of ES. Moreover, the protein matrix of the salted cheese was more swollen and uniform than that of unsalted cheese which had fissures thought to be pockets of free unbound water (Paulson et al., 1998). Thus, Kindstedt and co-workers (Kindstedt, 1990, 1995; Kindstedt et al., 1992) found that zonal variations in salt level, which occur in brine-salted Mozzarella, lead to variations in cooking properties at different locations in the cheese throughout a 16-day ageing period. Melted cheese from the high-salt (⬃3.04%, w/w, at day 2, and 1.7%, w/w, at day 16) surface region had a higher apparent viscosity and a low level of free oil, and was tough and chewy. In contrast, cheese from the low-salt interior (⬃0.38%, w/w, at day 2, and 0.9%, w/w, at day 16) had a markedly lower apparent viscosity and was smooth, soft, fluid and gelatinous. After 16 days, the melted interior sample had become excessively soft while the exterior sample still remained unacceptably tough and chewy. The above trends suggest a salting-in effect of the para-casein matrix with a concomitant increase in hydration at low S/M levels (e.g., 1.5%, w/w) and a salting-out effect and decrease in paracasein hydration at the high salt levels (especially at 6.3%, w/w, S/M). Consequently, Kindstedt and Guo (1997) concluded that, in addition to proteolysis, the increase in casein hydration, at 3–4%, w/w, S/M is a major factor contributing to the development of the desirable cooking properties in low-moisture part-skim Mozzarella during storage. In contrast to the above studies, Pastorino et al. (2003a) reported no effect of salt in the range 0.14–2.8%, w/w (0.34–7.4%, w/w, S/M) on the degrees of melt and flowability of 40-day-old

Muenster cheese in which salt level was varied by the number of injections of brine.

Reduced Sodium Cheese While the physiological requirement of Na as a dietary constituent is universally accepted, there is growing concern that an excess (⬃2.4 g/day for healthy adults) induces physiological defects, including hypertension (see Beard et al., 1997; Beilin, 1999; Kaplan, 2000). Such concern has led to a recommendation for a reduced dietary intake of Na, classification of foods (high, medium, low) according to sodium level, declaration of sodium level on the food labels and an increased demand for reduced-sodium foods, including cheese (see Demott, 1985; Petik, 1987; Schroeder et al., 1988; Morris and Dillon, 1992; Narhinen et al., 1998). In addition to its preservative effect, salt in cheese exerts a major influence on cheese composition, microflora, ripening rate, texture, flavour and quality (see ‘Control of Microbial Growth’, ‘Influence of NaCl on Enzyme Activity in Cheese’, ‘Influence of NaCl on the Water Activity (aw) of Cheese’, ‘Overall Influence of NaCl on Cheese Ripening and Quality’ and ‘Effect of Salt on Cheese Composition’). The sodium level (%, w/w) in cheese ranges from ⬃0.26, 0.62 and 2.6, respectively, for Swiss, Cheddar and Domiati (United States Department of Agriculture, 1976; Volume 2 Kindstedt and Kosikowski, 1988). Approaches to reduce the Na level in cheese include: • reducing the level of added salt per se (Kosikowski, 1983; Wyatt, 1983; Lindsay et al., 1985); • partial or complete substitution of NaCl by other salts such as KCl, MgCl2 or CaCl2 (Fitzgerald and Buckley, 1985; Demott et al., 1986; Lefier et al., 1987; Anonymous, 1993; Iwanczak et al., 1996; Katsiari et al., 1997; Salem and Abeid, 1997; Abou-ElNour, 1998); • a reduced salt level in combination with flavourenhancing substances such as autolysed yeast extract (Karahadian and Lindsay, 1984; Demott et al., 1986); • the use of ultrafiltration and reverse osmosis retentate supplemented milks to alter the mineral level in the cheese (Kosikowski, 1983, 1985; Kindstedt and Kosikowski, 1984b; Lindsay et al., 1985); • alterations of cheesemaking procedure, e.g., washing curd at a low temperature (⬃20 °C; to remove lactose) and heating moulded curd to a core temperature curd of 85 °C (Drews, 1991); or high intensity centrifugation of milk and re-incorporation of the bacteria-rich portion after sterilization followed by normal cheese manufacture apart from a shorter brining time (Wessanen, 1983).


Attempts to produce low-sodium processed cheese products include the partial substitution of sodium phosphates with the corresponding potassium phosphates, the use of flavour enhancers (e.g., monosodium glutamate, glucono-delta-lactone, enzyme-modified cheese and cheese pastes) (Karahadian and Lindsay, 1984) and the use of selected cheese blends and dairy ingredients in the production of emulsifying salt-free processed cheese foods and spreads. Cheddar cheese

Schroeder et al. (1988) showed that it was possible to reduce the salt content of Cheddar to 1.12%, w/w, without ill-effects on quality; cheese with 0.73%, w/w, NaCl was also acceptable. Fitzgerald and Buckley (1985) studied the influence of KCl, MgCl2, CaCl2 and 1:1 mixtures of these salts with NaCl on the quality of Cheddar cheese salted to give an ionic strength similar to the control (i.e., 1.44%, w/w, NaCl in the cheese) ripened at 4 $C over a 4-month period. The use of KCl, MgCl2 or CaCl2 alone resulted in oversoft cheeses with very bitter and unacceptable flavours. These defects may be attributed to the higher moisture level and greater proteolysis and lipolysis in the case of MgCl2 and CaCl2 but not in the case of KCl, where the moisture and water-soluble N levels were similar to, or slightly higher than, respectively, the controls. Similarly, the cheeses with salt mixtures generally had higher levels of proteolysis and lipolysis than the control but the blends had little effect on the hardness or firmness, except in the case of the NaCl/MgCl2 blend which gave softer cheese than the control. Both flavour and texture scores for the CaCl2/NaCl- and MgCl2/NaCl-salted cheeses were significantly lower than the controls. A KCl/NaCl (1:1) mixture gave cheese at 16 weeks which was not significantly different from the control in terms of proteolysis, texture flavour and acceptability. Similar results with NaCl/KCl (1:1) mixtures were observed for Cheddar (Lindsay et al., 1982), and Camembert, Gouda and Camping type cheeses (Iwanczak et al., 1996). Moreover, both of these studies reported that reduction of salt (i.e., NaCl or 1:1 NaCl/KCl mixtures) by ⬃30% in these cheeses resulted in no major differences in flavour, texture or acceptability scores (Lindsay et al., 1982) or improved them (Iwanczak et al., 1996). In Cheddar cheese (⬃34%, w/w, moisture) salted with NaCl/KCl mixtures, free fatty acid levels were higher and grading scores were somewhat lower due to a slight bitterness (Lindsay et al., 1982). Reddy and Marth (1995) studied the starter and NSLAB populations in Cheddar cheeses made with different starters and salted at a level of

1.5–1.75%, w/w, with NaCl, KCl or mixtures of NaCl/KCl (2:1, 1:1, 1:2 and 3:4). For a given starter system, the type of salt did not effect the predominant bacterial species in the cheese. Kosikowski (1983, 1985) found that in Cheddar cheeses with a reduced level of NaCl (i.e., 1.05%; ⬃3.0%, w/w, S/M), increasing the protein content of the milk, from ⬃3.36 to 6.26%, w/w, prior to renneting, by supplementing the cheese milk with increasing amounts of ultrafiltered milk (4.5:1 retentate), was paralleled by a decrease in moisture and increases in the Ca (from ⬃590 to 730 mg/100 g) and P (from ⬃470 to 556 mg/100 g) levels and in the scores for flavour, body and texture during ripening at 10 °C over 4 months. Grading scores for flavour and texture increased to an optimum at a milk protein concentration of 4.97–6.26% in the supplemented milks; at the lower milk protein level, the cheeses become progressively more acidic, bitter, pasty and devoid of cheese flavour. The enhancing effect of increased milk protein level on grading score was attributed to the increased buffering capacity which prevented a rapid decline in pH (in the absence of a normal salt level) during moulding and pressing and hence excessive loss of calcium and phosphorus which influence cheese structure and rate of proteolysis (Kosikowski, 1983; Kindstedt and Kosikowski, 1984a,b, 1986). However, the results of Kosikowski (1983) could not be confirmed under practically identical conditions by Lindsay et al. (1985). Contrary to the results of Kosikowski (1983), the latter group found that: • the calcium level in low-sodium (1%, w/w, NaCl) Cheddar made from control milk was not significantly lower than that made from milk supplemented with retentate; • the grading scores of cheeses made from supplemented milk were of the same magnitude as, or slightly lower than, those of the control ‘nonsupplemented’ low-salt Cheddar. Moreover, the former cheeses were generally softer and had a less intense cheese flavour. The inclusion of reverse osmosis (RO) retentate in UF retentate-supplemented milk gave low-salt Cheddar (1%, w/w NaCl) with grading scores similar to the control (Lindsay et al., 1985); such cheeses had a unique sharpness which could be used to enhance the flavour of other cheese products, such as processed cheese. Undoubtedly, the quality of commercial reducedsodium cheese depends on many factors, including pitching pH, the type and amount of residual coagulant in the cheese, types and counts of starter and non-starter bacteria, composition and ripening temperature. Ranges of compositional parameters for


good quality (New Zealand) Cheddar as proposed by Lelievre and Gilles (1982) are: 4.0–6.0%, w/w, S/M; 50–57%, w/w, FDM; 50–56%, w/w, MNFS and pH 5.0–5.4; outside these ranges quality deteriorates sharply. With the modern continuous production methods for Cheddar, in combination with rapid cooling of blocks, it may be possible (though somewhat more expensive) to produce consistently highquality Cheddar by reducing the MNFS, keeping the pH close to 5.1, avoiding the use of bitter starters and microbial rennets, and ripening at a low temperature (i.e., 5 °C). Cottage cheese

Because of its relatively large serving size (⬃112 g compared to ⬃66 g for other cheeses), Cottage cheese has been viewed as a potentially high source of dietary sodium (Marsh et al., 1980). Hence, much interest has focused on various ways of reducing the salt level of Cottage cheese. Wyatt (1983) evaluated preference scores for Cottage cheeses in which the NaCl content was reduced stepwise from 1%, w/w, (control commercial cheese) to 0.25%, w/w, NaCl. It was concluded that a 35% reduction in salt did not influence consumer response to the cheese compared to the control; however, reduction by 50% or greater resulted in significantly lower scores. Demott et al. (1986) also evaluated consumer reactions to lowsodium Cottage cheese salted with various mixtures of KCl and NaCl and found that the sodium level could be reduced by 50% (by adding 1.26%, w/w, of an NaCl/KCl mixture instead of 1.26%, w/w, NaCl) without affecting grading scores. Reducing the sodium level by more than 50% resulted in a significant reduction in score (Demott et al., 1984, 1986). Lindsay et al. (1985) also found that a 50% reduction caused no significant changes in consumer acceptability. However, the use of substitutes, i.e., KCl or KCl/NaCl, to reduce the level of sodium by 50% gave a significant reduction in quality. Other cheeses

Martens et al. (1976) reported the successful manufacture of low-sodium Gouda cheese using mixtures of NaCl and KCl in curd manufacture and brining. While the Na and K levels (mg %) in the control cheese were ⬃830–650 and 120, those of the reduced-sodium cheese were 200 and 200, respectively. However, reduction of salt in dry matter (SDM) in Gouda cheese by ⬃20% is claimed to increase significantly the susceptibility to butyric acid fermentation (van den Berg et al., 1986). To prevent such undesirable fermentation in cheeses at salt levels 3.8%, w/w, SDM requires

process modifications such as bactofugation of the milk and reduction of the cheese moisture level. Lefier et al. (1987) reported the production of low-sodium Gruyère (⬃45 mg Na/100 g compared to 272 mg/100 g in the control) by replacing NaCl by MgCl2. While the degree of proteolysis and the concentrations of free fatty acids were similar in both cheeses, the cheese containing MgCl2 had a more bitter taste and a softer body than the control, but was organoleptically acceptable. Partial replacement (50%) of NaCl in brines with CaCl2, MgCl2, KCl, or a mixture of CaCl2, MgCl2 and KCl did not significantly affect the level of proteolysis, as measured by pH 4.6-soluble N (percentage total N) and NPN in Turkish White cheese (Güven and Karaca, 2001). However, the pH 4.6soluble N (percentage total N) was numerically highest for the cheeses salted in the NaCl brine and lowest in those salted in NaCl:CaCl2 KCl MgCl2 brines during most of the ripening period. Katsiari et al. (1997) reported that the sodium content of Feta cheeses was successfully reduced by 50%, by partial replacement of NaCl with KCl, without affecting gross composition, water activity, lipolysis (Katsiari et al., 2000b), proteolyis (Katsiari et al., 2000a), or sensory or textural properties. Similarly, a 50% reduction in the sodium content of Kefalograviera cheese, by substituting KCl for NaCl, had no effect on lipolysis or proteolysis (Katsiari et al., 2001a,b). Processed cheese products contain a relatively high level of Na (1.0–1.5%, w/w; USDA, 1976) because of the inclusion of sodium phosphate emulsifying salts in their formulation. Karahadian and Lindsay (1984) produced acceptable low-sodium processed cheese (75% reduction, 0.34%, w/w, Na in product) by using reduced-sodium Cheddar cheese and/or various combinations of potassium-based emulsifying salts (citrates, phosphates). A similar process for reducedsodium processed cheese, based on the selective use of sodium-, potassium- and calcium-based phosphates, was patented by Henson (1999). The most efficient means for reducing Na in processed cheese products is by eliminating the emulsifying salts, i.e., as in emulsifying salt-free processed cheese foods and spreads which have been commercially available since 1988. The production of such products requires careful blending of cheeses (i.e., high and low calcium cheeses, cheeses with varying pH and degree of proteolysis) and alteration of processing conditions so as to obtain a stable emulsion (McAuliffe and O’Mullane, 1989; Guinee, 1991). In the latter products, lack of saltiness is easily overcome by adding ingredients such as monosodium glutamate, autolysed yeast extract, ‘high-cured cheese’, cheese powders, enzyme-modified cheese, cheese pastes and/or acidulants.


Brine-salted cheese

concluded that the penetration of salt into cheese and the concomitant outward migration of water could be described as an impeded diffusion process, i.e., NaCl and H2O molecules move in response to their respective concentration gradients but their diffusion rates are much lower than those in pure solution (Georgakis, 1973; Resmini et al., 1974) due to a variety of impeding factors. The diffusion coefficient (D) for NaCl in cheese moisture is typically ⬃0.2 cm2/d, though it varies from ⬃0.1 to 0.45 cm2/d with cheese composition and brining conditions (Geurts et al., 1974b; Guinee and Fox, 1983a; Guinee, 1985; Turhan and Kaletunç, 1992; Turhan and Gunasekaran, 1999; Simal et al., 2001), compared to 1.0 cm2/d for NaCl in pure H2O at 12.5 °C. Geurts et al. (1974b) used the term ‘pseudo-diffusion coefficient’ in relation to the movement of NaCl in cheese moisture since the value of the observed coefficient depended on the net effect of many interfering factors on true diffusion. The discrepancies between the true- and pseudo-diffusion coefficients, i.e., D and D*, respectively, were explained by a simplified model of cheese structure consisting of moisture and discrete spherical fat globules dispersed in a protein matrix comprised of discrete spherical protein particles and 15% (w/v) bound water. Based on theoretical considerations, the impedance of various compositional and structural features intrinsic to the model structure was formulated and their effects on D quantified. The principal factors responsible for impedance of NaCl diffusion in cheese, as postulated by Geurts et al. (1974b), are:

When cheese is placed in brine there is a net movement of NaCl molecules, as Na and Cl , from the brine into the cheese as a consequence of the osmotic pressure difference between the cheese moisture and the brine. Consequently, the water in the cheese diffuses out through the cheese matrix so as to restore osmotic pressure equilibrium. Gels, including cheese, consist of a three-dimensional network of strands of fused para-casein micelles in clusters, which gives the mass its structure and a certain degree of rigidity and elasticity; the properties of the inter-penetrating fluid are generally not appreciably different from those of corresponding solutions. It would appear, therefore, that NaCl molecules diffusing in cheese moisture, while having a longer distance to travel than in solution (diffusing ions must travel a circuitous route to by-pass obstructing protein strands and fat globules through which they cannot penetrate), would not be appreciably affected otherwise. However, based on the mobilities of NaCl and H2O in Gouda-type cheeses brine-salted under model conditions to obey Fick’s law for unidimensional brine flow, Geurts et al. (1974b)

1. The larger outward migration of water, compared to inward flux of NaCl, during brining. The pores (estimated to be ⬃2.5 nm wide) of the protein matrix exert a sieving effect on both the inwarddiffusing NaCl molecules and outward-moving H2O molecules but the effect is more pronounced on the former because of their greater effective diffusion radius, which is approximately twice that of the H2O molecules. Hence, during brining, the H2O flux is approximately twice the NaCl flux. The net outflow of H2O during brining causes the plane of zero mass transfer (a plane where the average flux of all diffusing species is zero) to recede from the cheese/brine interface into the brine and hence reduces the apparent rate of NaCl diffusion due to the additional path length through which the NaCl molecules must migrate. The interference, which is most pronounced when moisture loss is high, e.g., when using concentrated brines or high brining temperature, was estimated to reduce the diffusion coefficient by 20%; hence D* ⬇0.8D.

Salt Absorption and Diffusion into Cheese Methods of salting

There are three principal methods of salting cheese curd: • direct addition and mixing of dry salt crystals to broken or milled curd pieces at the end of manufacture, e.g., Cheddar and Cottage; • rubbing of dry salt or a salt slurry to the surface of the moulded curds, e.g., blue-veined cheeses; • immersion of moulded cheese in a brine solution, e.g., Edam, Gouda, Saint Paulin, Provolone, etc. Sometimes, a combination of the above methods is used, e.g., Emmental, Parmesan, Romano and Brick. Other methods which have been used less frequently, mainly in experimental studies, include: brine injection under pressure, e.g., 17 MPa (Lee et al., 1978; Pastorino et al., 2003a); high pressure brining, e.g., at isostatic pressures up to 500 MPa (Messens et al., 1999), acoustic brining in an experimental scale (28 l) brine vat with an acoustic system generating high intensity (300 W) ultrasonic (30 kHz) waves (Sánchez et al., 2000); vacuum impregnation brining at a vacuum of 3.7 kPa, absolute (Pavia et al., 1999). In high pressure brining, cheese portions were placed in high-density polythene containers, which were filled with brine, closed, placed in an isostatic press and pressurized. Mechanism of salt absorption and diffusion in cheese


2. The high relative viscosity of the aqueous phase of cheese. The viscosity of cheese moisture (juice) is about 1.27 times that of pure water at 12.5 °C due to the presence of dissolved materials, e.g., acids, salts and nitrogenous compounds. When the NaCl molecules enter the cheese, they encounter collisions/obstructions with the dissolved substances, an occurrence which retards movement. Moreover, the charge fields of these substances also affect the movement of the diffusing ions. Both of these factors reduce NaCl mobility and contribute to the reduction of the pseudo-diffusion coefficient by a factor of 1/rel. 3. Obstructions of fat globules and globular protein particles. On proceeding from one parallel plane to another within the cheese, the diffusing molecules must travel by a circuitous route to bypass obstructing particles. Hence, the real distance travelled by diffusing molecule proceeding from the rind (cheese–brine interface) to a point x cm inside the rind is x cm rather than x cm (apparent distance), where is the tortuosity factor. The ratio of the real to the apparent distance travelled is a measure of the obstructions caused by fat globules (f) and protein particles (p). These reduce the diffusion coefficient by a factor of 1/(fp). Theoretically, f can vary from /2 for a close-pack arrangement to 1 for a very low-fat system, e.g., skim milk cheese. Typical values of f and p are 1.32 and 1.35, respectively, for a Gouda cheese containing 29% fat and 43% moisture (Geurts et al., 1974b). However, the values of f and p in experimental Gouda cheese vary markedly with composition (Tables 5 and 6). 4. The mechanical sieving effect of the pores of the protein matrix on migrating ions. The relatively narrow pore width of the protein matrix exerts a frictional effect on the diffusing NaCl and H2O, analogous to the restriction of movement of a sphere through a pipe with a diameter which varies somewhat and which at its smallest is comparable to that the sphere. In cheese, the pore width, estimated to be typically ⬃2.5 nm, is not much larger than the combined diameters of the Na and Cl , estimated to be ⬃0.6 nm (see Geurts et al., 1974b); moreover, consideration of cheese microstructure suggests that pores are not uniform in diameter or in orientation (aspect). The sieving effect (S) reduces the relative diffusion rates of the diffusing NaCl and water from 1 in pure solution to ⬃0.5 and 0.75, respectively, in cheese moisture. As the effective pore restriction on the diffusion of NaCl in cheese moisture is determined by its effect on the larger molecule, i.e., NaCl, the pseudodiffusion coefficient was estimated to be reduced by a factor of S 0.5.

5. Frictional effects of protein-bound water. Water binding in cheese (0.1–0.15 g H2O/g para-casein; Geurts et al., 1974a) accounts for 10–15% of the total cheese moisture. The bound water increases the effective diameter of protein particles of which the matrix is composed, and, thereby, reduces the relative pore width of the protein matrix. This in turn increases the sieving effect on the migrating ions, as discussed in 4, and the tortuosity factor (p) as discussed in 3. Beginning from a simplified model of cheese structure and considering the relative effects of the interfering factors discussed, Geurts et al. (1974b) postulated a theoretical ‘pseudo-diffusion’ coefficient, D*

0.8 DS r!rel

While the model cheese structure adopted by Geurts et al. (1974b) is simplified in view of the results of electronmicroscopic examinations of cheese structure (Kimber et al., 1974; de Jong, 1978; Kaláb, 1995; Guinee et al., 1998; Auty et al., 2001), the calculated impedance derived from it was sufficient to explain the very low diffusion coefficient of NaCl in cheese moisture and the variations of D* with variations in cheese composition and brining conditions. A number of later studies (Minarik, 1985; Luna and Bressan, 1986, 1987; Luna and Chavez, 1992; Payne and Morison, 1999) on the modelling of salt and water diffusion in semi-hard cheeses have verified the low diffusivity of salt in cheese moisture. Using developed prediction models, these studies found a close correlation between the experimental data from earlier studies (Geurts et al., 1974b; Guinee and Fox, 1983a) and predicted salt and moisture levels in the cheese. Direct mixing of salt with milled curd

When dry salt is distributed over the surface of milled curd or curd granules, some NaCl dissolves in the surface moisture and diffuses slowly inwards a short distance (Breene et al., 1965; Sutherland, 1974). This causes a counterflow of whey from the curd to the surface which dissolves the remaining salt crystals and, in effect, creates a supersaturated brine solution around each particle, provided mixing of curd and salt is adequate. However, because of the relatively large surface area to volume ratio of the curd as a whole, salt uptake occurs from many surfaces simultaneously and less time is required for uptake of an adequate amount of salt in dry-salting milled curd (10–20 min) than in brining whole cheeses (0.5–5 days depending on the

230 Table 5 Influence of cheese composition on salt diffusion in cheese moisture Properties of unsalted cheese

Cheese code

Fat (%)

Moisture (%)

Solids not-fat (%)

Fat-in-dry matter (%)

Moisture in-fat-free cheese (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14

0.00 10.88 19.88 26.25 0.00 10.00 18.25 30.28 18.18 20.00 21.00 21.00 29.00 27.66

53.00 49.00 45.93 43.48 52.90 48.93 44.90 40.50 49.20 47.94 45.02 44.44 41.11 44.02

47.00 40.12 34.19 30.27 47.10 41.07 36.85 29.22 32.62 32.06 37.98 34.60 28.89 28.32

0.00 21.33 36.77 46.44 0.00 19.58 33.12 50.89 37.59 38.42 38.20 37.77 49.24 49.41

53.00 54.98 57.33 58.96 52.90 54.37 54.92 58.09 60.13 59.93 56.99 56.25 57.90 60.85

Obstructing factors

Diffusion coefficients

Volume fraction of fat phase (f)

Volume fraction of protein matrix (p)

Relative pore width of protein matrix (y/dp)

Fat tortuosity (f)

Protein tortuosity (p)

D* (cm2/day)

D* f (cm2/day)

0.000 0.127 0.227 0.297 0.000 0.117 0.207 0.340 0.207 0.227 0.240 0.240 0.326 0.310

0.466 0.442 0.413 0.394 0.467 0.449 0.442 0.404 0.380 0.382 0.417 0.427 0.406 0.371

0.132 0.152 0.178 0.198 0.132 0.146 0.152 0.187 0.213 0.211 0.174 0.165 0.185 0.223

1.000 1.117 1.229 1.296 1.000 1.105 1.208 1.333 1.208 1.229 1.242 1.242 1.322 1.308

1.425 1.409 1.389 1.378 1.425 1.414 1.409 1.383 1.365 1.366 1.392 1.400 1.384 1.358

0.136 0.153 0.203 0.227 0.140 0.202 0.205 0.236 0.295 0.263 0.207 0.176 0.216 0.247

0.136 0.171 0.249 0.294 0.140 0.223 0.248 0.315 0.356 0.323 0.257 0.218 0.285 0.324

For calculation of f and p it was assumed that: (a) cheese moisture contained 5% dissolved solids, density 1 g/ml; (b) the protein matrix consisted of protein 15% water bound and had a specific gravity of 1.25; (c) the specific gravity of fat 0.93. Cheeses were from four trials, i.e cheeses 1–4, from trial 1; 5–8, from trial 2; 9–12, from trial 3; and 13, 14 from trial 4. All cheeses were salted in 18.5% NaCl at 20 °C for 3–4 days (from Guinee, 1985).

Table 6 Experimentally determined diffusion coefficient (D*) of salt in moisture in cheese varying in properties and brined under different conditions. Calculation of diffusion coefficient in moisture (D*v) and of relative pore width of the protein matrix (y/d)e in fat-free cheese

Brine (g NaCl/ 100 g H2O)

g fat in 100 g DM

19.7 19.0 19.5 20.2 19.8 20.5 20.0 19.6 20.4 34.8 14.0 19.7 13.8 20.0 13.8 20.1 19.4 20.0 20.1

12 12 12 22 50 50 50 50 50 50 50 50 50 50 50 50 50 62 62

Diffusion coefficient (cm2/day)

Calculation of factors

Properties of non-salted cheese

pH

Fat content (%)

Moisture content (%)

v

v

e

(y/d)e

D*

D* v

5.00 5.01 4.99 5.00 5.10 4.79 5.42 5.09 5.02 5.09 5.10 5.07 5.18 5.64 4.98 4.92 5.09 5.00 4.98

5.3 4.9 5.0 10.9 33.0 31.2 30.9 30.4 29.9 29.9 30.2 29.2 29.5 28.9 26.9 26.6 25.8 39.1 37.1

53.0 54.0 55.0 50.2 36.2 41.1 41.5 42.2 42.5 42.5 42.5 42.9 43.0 43.4 48.0 49.0 50.1 38.5 40.8

0.06 0.06 0.06 0.13 0.37 0.35 0.34 0.34 0.33 0.33 0.34 0.33 0.33 0.32 0.30 0.29 0.29 0.43 0.41

1.04 1.04 1.04 1.12 1.36 1.34 1.33 1.33 1.32 1.32 1.33 1.32 1.32 1.32 1.30 1.29 1.29 1.40 1.39

0.42 0.41 0.40 0.42 0.44 0.37 0.37 0.37 0.36 0.36 0.36 0.37 0.36 0.36 0.31 0.30 0.29 0.34 0.32

0.171 0.181 0.191 0.171 0.153 0.225 0.225 0.225 0.238 0.238 0.238 0.225 0.238 0.238 0.312 0.330 0.349 0.265 0.295

0.164 0.185 0.162 0.152 0.100 0.160 0.160 0.172 0.185 0.148 0.194 0.187 0.177 0.168 0.235 0.258 0.239 0.179 0.224

0.170 0.192 0.168 0.171 0.136 0.215 0.213 0.229 0.245 0.196 0.258 0.248 0.234 0.221 0.305 0.333 0.309 0.251 0.311

v volume fraction of fat in cheese, calculated from fat content. v tortuosity factor of fat, a function of v. e volume fraction of protein matrix in fat-free cheese. (y/d)e relative pore width of this matrix. D* pseudo-diffusion coefficient in moisture in cheese. In calculating v and e it is assumed that cheese moisture contains 5% dissolved substances (density of solution 1), that the protein matrix consists of protein 15% water (density 1.25), and that the density of the fat is 0.93. All experiments were carried out at about 12.5 °C (from Geurts et al., 1974b).

231


0.21 Diffusion coefficeint, D*, cm2/d

dimensions). Some of the ‘brine’ on the surface of curd particles drains away through the curd mass while more is physically expelled from the curd particles during pressing and is lost in the ‘press whey’. As the salt/surface area ratio is usually low, and the period of contact of the curd surface with the concentrated brine layer is relatively short (i.e., 20 min mellowing period), little localized surface protein contraction occurs compared to that in dry-salted, moulded curds (Sutherland, 1974).

0.2 0.19 0.18 0.17 0.16

Dry surface-salting of moulded pressed cheese curd

Factors influencing salt absorption by cheese

The only pre-requisite for salt absorption by cheese is the existence of a salt-in-moisture gradient between the cheese and the salting medium. However, the quantity of salt absorbed depends on the intrinsic properties of the cheese, the conditions of salting and the duration of salting. As the different procedures of salting all involve salt absorption via an impeded diffusion process, the general factors affecting salt uptake by cheese apply equally to granules or milled curd pieces on mixing with dry salt and to moulded cheeses which are brined and/or dry salted. Certain peculiarities of the salting of milled curd pieces, as in Cheddar, which affect salt absorption will be discussed separately. Brine concentration and concentration gradient

It is generally accepted that an increase in brine concentration results in greater salt absorption and an increased salt-in-moisture level in the cheese (Figs 8, 9); Breene et al., 1965; Geurts et al., 1980; Godinho and Fox, 1981b; Guinee and Fox, 1986a; Apostolopoulos et al., 1994; Pappas et al., 1996; Kaya et al., 1999). While the rate of NaCl diffusion is scarcely affected by brine concentration in the range 5–20% (Guinee, 1985; Geurts et al., 1974b; Fig. 10), the rate of uptake increases at a diminishing rate with increasing brine

0

5

10

15

20

25

Brine concentration, %, w/w, NaCl Figure 10 The effect of brine concentration on the diffusion coefficient, D*, of salt in the moisture phase of experimental Gouda cheese (43.6%, w/w, moisture; 49.1%, w/w, fat-in-dry matter) salted for 4 days at 20 °C (redrawn from Guinee, 1985).

concentration in the range 5–25%, w/w (Fig. 11; Breene et al., 1965; Sutherland, 1974; Guinee, 1985; Guinee and Fox, 1986a; Chamba, 1988; Kaya et al., 1999; Prasad and Alvarez, 1999; Melilli et al., 2003). This is due to the reduction in the concentration gradient of S/M between the cheese moisture and the brine. Hence, in model brining experiments, in which cheese slices of different thickness were completely submerged in brine, there was a sharp decrease in the rate of salt absorption (per unit weight) as the difference between the NaCl concentration in the cheese moisture and the brine decreased, especially when the

7

6 Salt in cheese, %, w/w

A block of curd can be regarded as a very large particle and solution of dry salt in the surface moisture layer is a pre-requisite for salt absorption in this method also. The counter flow of moisture from the cheese creates a supersaturated brine layer on the cheese surface and salt uptake then occurs by an impeded diffusion process. Because the surface is in contact with a concentrated brine for a long time (several days), there is considerable contraction of the curd surface (salting out of protein) and this probably leads to relatively high moisture losses from the surface region and hence a reduction in the inward mobility of NaCl which accounts for the lower rate of salt uptake in this method than in brining (Godinho and Fox, 1981b; Melilli et al., 2003).

5

4

3

2

1 5

10

15

20

25

Brine concentration, %, w/w, NaCl Figure 11 Salt level in Romano-type cheese slices (0.5 cm thick; 7 cm diameter) as a function of brine concentration after salting for 50 (), 100 () or 200 (●) min in 20%, w/w, NaCl brine containing 0.5%, w/w, Ca (redrawn from Guinee, 1985).


g NaCl absorbed per 100 g cheese per min

initial difference was large (Fig. 12; Guinee and Fox, 1986a). A somewhat similar situation applies to drysalted cheese: the increase in salt-in-moisture level in Cheddar curd is not proportional to the increase in the level of dry salt added to the milled curd (O’Connor, 1974; Gilles, 1976). This is attributed to increased salt losses with increased salting level, which reflects the decreasing effect of the driving force (concentration gradient) in raising the quantity of salt absorbed as the salt-in-moisture level in the cheese approaches that of the brine. While increasing the NaCl concentration in the brine from ⬃19%, w/w, to 25% or 31%, w/w, at 20 °C, results in an increase in the level of salt absorbed by brine-salted Gouda cheeses (Geurts et al., 1980; Guinee, 1985), Romano cheese slices (Guinee and Fox, 1986a), brine-salted Cheddar cheese cubes (Breene et al., 1965), the diffusion coefficient, D*, and hence, the depth of penetration of salt into the cheese, decreases sharply (Fig. 10). In contrast, Melilli et al. (2003) reported a markedly lower mean salt content (⬃35%) in Ragusano cheese, at day 8, on raising the NaCl concentration in the brine from 18 to 30%, w/w. The discrepancy between the results of Melilli et al. (2003) and previous studies (Breene et al., 1965; Geurts et al., 1980; Guinee, 1985; Guinee and Fox, 1986a) may be related to differences in the lactate content of the cheese, which is likely to be lower in Ragusano than in Gouda, Cheddar or Romano, because of the loss of lactate during plasticization of the Ragusano curd in hot water at a curd-

0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 0

5

10

15

20

Δ NaCl level (%, w/w) between cheese moisture and brine Figure 12 Influence of NaCl concentration gradient between the cheese moisture and the brine on salt uptake by Romanotype cheese slices (0.5 cm thick; 7 cm diameter) salted in 20%, w/w, NaCl brine containing 0.5%, w/w, Ca (redrawn from Guinee and Fox, 1986a).

to-water ratio of ⬃1:3. Owing to the higher concentration of lactate in the moisture phase of cheeses than in the brine, an outward migration of lactate form the cheese to the brine and a counter-flow of water from the brine to cheese would be expected in all cheeses during brine-salting. We are not aware of studies on this but the data of Pavia et al. (1999) show lower concentrations of lactate in the surface than in the centre of Manchego cheese. The quantity of lactate lost and water gained as a result of this mutual lactate-water diffusion process depends on the lactate concentration in the cheese moisture prior to salting, with a higher lactate concentration leading to a higher water uptake by the cheese. Hence, all other factors being equal, a low level of lactate-in-moisture before brine-salting is expected to result in a greater net loss of water from the cheese that occurs as a result of the mutual saltwater diffusion process during brining. Consequently, salting of Ragusano cheese, for which the lactate level is expected to be lower than that of Gouda and other non-plasticized cheeses, in high-NaCl brine (e.g., 25–30%, w/w) probably leads to a relatively large water loss, especially from the surface layer of the cheese. A high level of dehydration in the surface layer would impede salt absorption because of the concomitant increases in protein content and the consequent reduction in pore width of the protein matrix (see ‘Moisture content of cheese’). Method of brine-salting

High pressure (HP) treatment of cheese during brining at 100–500 MPa, at 20 °C for 15–30 min, did not significantly affect salt uptake in Gouda cheese but in some cases reduced moisture loss (Messens et al., 1999). Similarly, vacuum impregnation brining at 3.7 kPa, absolute, did not effect the mean S/M level in Manchego-type cheese (Pavia et al., 1999). However, in contrast to observations with HP-brining (Messens et al., 1999), vacuum impregnation brining gave a more uniform S/M distribution immediately after brining than conventional brine-salting. The quantity of salt taken up by Mozzarella on brine injection under pressure was directly proportional to the number of injections for salt levels of 0–3%, w/w (Pastorino et al., 2003a); the results of Lee et al. (1978) suggest that salt retention on pressure injection is proportional to the square root of pressure and decreases with the diameter of the jet orifice. Cheese geometry

It is generally agreed that the rate of salt absorption increases with increasing surface area to volume ratio of the cheese (Breene et al., 1965; Gilles, 1976; Guinee


and Fox, 1986a). This is most readily observed on comparing the rate of salt uptake by milled curd (e.g., Cheddar) and whole moulded cheeses (Brick, Emmental, Romano or Blue-type cheeses) in brine; in the former, salt absorption occurs from many surfaces simultaneously, and the time required to attain a fixed level of salt is very much less than for brine-salted moulded cheeses. While at first sight it may appear that smaller cheeses would have a higher mean salt content than larger ones after brining for equal intervals, this applies only to cheeses of the same shape and relative dimensions since salt uptake is linearly related to the surface area to volume ratio of the cheese (Geurts et al., 1980; Guinee and Fox, 1986a,b). In addition to its influence on the surface area to volume ratio, cheese shape also affects the rate of salt absorption via its effect on: (i) the number of directions of salt penetration from the salting medium into the cheese and (ii) the ratio of planar to curved surface area of the cheese (Geurts et al., 1980; Guinee and Fox, 1983b, 1986a,b). Geurts et al. (1980) found that on brining Edam-type cheese, the quantity of NaCl absorbed per cm2 cheese surface was greater for an infinite slab than for a sphere, and the relative reduction in salt uptake through curved surfaces increased with brining time and with the degree of curvature. In Romano-type cheeses with approximately equal surface area to volume ratios, the rate of salt absorption by rectangular-shaped cheeses (volume: 4000 cm3; three effective directions of salt penetration) was higher than that by cylindrical cheese (volume: 3400 cm3; two effective directions of salt penetration at any time during a 9-day brining period (Fig. 13; Guinee and Fox, 1986b). For cylindrical, spherical or rectangular cheeses with a volume 1000 cm3, the surface area is

Salting time

It is well established that the quantity of salt absorbed increases with salting time (Byers and Price, 1937; Hoecker and Hammer, 1944; Breene et al., 1965; Sutherland, 1974; Geurts et al., 1980; Godinho and Fox, 1981b; Luna and Bressan, 1986; Guinee and Fox, 1986a,b; Turhan and Kaletunç, 1992; Nájera et al., 1994; Kristiansen et al., 1999; Messens et al., 1999; Melilli et al., 2003). However, the rate of salt absorption decreases with time due to a decrease in the NaCl concentration gradient between the cheese moisture and the brine (Geurts et al., 1980; Guinee and Fox, 1986a,b; Melilli et al., 2003). Indeed, the quantity of salt taken up by a cheese is proportional to the square root of brining time, √t (Geurts et al., 1980; Guinee and Fox, 1986a; Messens et al., 1999). However, as the curvature of the cheese surface increases, the proportionality of salt uptake with √t is lost and the relative reduction of salt uptake per unit area of cheese surface increases with increasing degree of curvature, and with time (Geurts et al., 1980). This implies that for cheeses with an equal volume and composition, brined under the same conditions, the rate of salt absorption per unit surface area (and hence the cheese as a whole) would be in the order: rectangular cylindrical spherical (Guinee and Fox, 1986b); however, aspects of cheese other than geometry affect the mean salt level, as discussed above. Geurts et al. (1980) derived a theoretical 0.18

(a) Water loss, g/cm2 cheese surface

0.12 Salt uptake, g/cm2 cheese surface

607, 483 and 600 cm2, respectively, i.e., surface area to volume ratios of 0.607, 0.483 and 0.600, respectively. As discussed below, the rate of salt uptake is affected by whether the surface is flat or curved; for the above cheeses, the area of the curved surface is 275, 483 and 0 cm2, respectively.

0.1 0.08 0.06 0.04 0.02 0

(b)

0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0

0

3

6

Brining time, days

0

3

6

9

Brining time, days

Figure 13 Influence of cheese shape on salt uptake (,) and moisture loss (,) by Romano-type cheese during salting in 19.5%, w/w, NaCl brine at 23 °C. Rectangular cheeses (,), cylindrical cheeses (,) (redrawn from Guinee and Fox, 1986c).


relationship for the quantity of salt absorbed through a flat surface as a function of brining time: Mt 2(C Co) (D*t/ )1/2 Vw

(1)

where Mt quantity of salt absorbed over time, g NaCl/ cm2, C salt content of brine, g NaCl/ml, Co original salt content of the cheese, g/ml, t duration of the salting period, days, D* pseudo-diffusion coefficient, cm2/day, Vw average water content throughout the cheese at time t, g/g. Applying this theoretical relationship to their model brining experiments on cylindrical Gouda cheeses brinesalted by unidimensional diffusion through one of the planar surfaces in contact with the brine (Geurts et al., 1974b), Geurts et al. (1980) found that the predicted values for the quantity of salt absorbed per cm2 planar surface (Mp) were in close agreement with the experimental values (Mt) over a three-day brining period: Mt 0.98Mp. The strong relationship between Mt and Mp confirms the accuracy of the experimentally determined D* values. Temperature of curd and brine

In model brining experiments, Breene et al. (1965) studied the effect of curd temperature (27, 32, 38, 43 °C) on salt uptake by Cheddar curd cubes (1 cm3) in brines (20 or 25%, w/w, NaCl) at the same temperature as the curd. Salt uptake was lowest at 32 °C, similar at 27 and 38 °C, and highest at 43 °C. The reduction in salt content on changing the brining temperature from either 27 or 38 °C to 32 °C was ⬃6.5%. The low salt uptake at 32 °C was attributed to a layer of exuded fat on the surface of the curd particles which impeded salt uptake; less fat was exuded at lower temperatures while at temperatures 32 °C, exuded fat was liquid and dispersed in the brine. Increasing brine temperature increases the mobility of NaCl and salt absorption in Gouda (Geurts et al., 1974b; Guinee, 1985), Emmental (Chamba, 1988), Romano-type (Guinee, 1985; Guinee and Fox, 1986a) and Turkish White (Turhan and Kaletunç, 1992) cheeses partly due to an increase in true diffusion and partly to an increase in the effective pore width of the protein matrix as non-solvent water decreases with increasing temperature (Geurts et al., 1974b). The increase in D* for Romano and Gouda cheeses in the range 5–25 °C in 19.2% NaCl brine was similar at ⬃0.0083 cm2/day (Guinee, 1985). Geurts et al. (1974b) reported that D* for Gouda cheeses salted in ⬃20% NaCl increased by ⬃40–50% on increasing the temperature from 12.5 to 25 °C, compared to an increase in D in pure water of ⬃20% over the same temperature range.

Initial salt-in-moisture level of curd and pre-salting

Brine-salting of cheese can be an expensive process in terms of space, maintenance cost and corrosiveness of the brine. Consequently, pre-salting of cheese as a means of reducing the brining time of Gouda (Guinee, 1985) and Ragusano (Melilli et al., 2003) cheeses has been investigated. Guinee (1985) mixed Gouda curds, immediately after whey drainage, with varying quantities of dry salt to give an S/M level ranging from ⬃0.25 (control, unsalted curd) to ⬃14%, w/w, in the (pre-salted) curd. The salted curds were left undisturbed for ⬃5 min and then moulded and pressed in the usual manner; the moulded cheeses were stored at the brine temperature (15 °C) for 1 day, covered with wax on three sides and the unwaxed side placed in contact with 19.2%, w/w, NaCl for 3 days. On completion of brine-salting, the S/M levels decreased with distance from the cheese–brine interface until it approached that of the pre-salted curd while moisture showed the opposite trend (Fig. 14a). Increasing the level of presalting and, hence, the S/M level in the curd prior to brine-salting, increased the levels of salt and S/M and reduced the level of moisture in the final cheese. A similar trend was noted for Ragusano cheese which was pre-salted by adding dry salt at a level of 4%, w/w, prior to plasticization in dilute brine (4.5%, w/w), and brine-salted for 1–24 days in 18%, w/w, NaCl at 18 °C (Melilli et al., 2003). In agreement with earlier studies, which showed that the difference in salt-inmoisture gradient between the cheese moisture and the brine was a major determinant of the quantity of salt absorbed (see ‘Brine concentration and concentration gradient’; Equation 1; ‘Salting time’), the magnitude of the increase in S/M decreased with the level of pre-salting (Fig. 14b) indicating a decrease in the quantity of salt absorbed per unit surface area of cheese (Guinee, 1985). However, the S/M level increased in all cheeses and approached closer to that of the brine with the level of pre-salting prior to brining. A similar trend was noted for Feta cheese stored in brines of different salt concentration (Prasad and Alvarez, 1999). Indeed, the S/M level in the outer (rind; 5 mm thick) layer of cheeses pre-salted to 6%, w/w, S/M exceeded the NaCl concentration in the brine at the end of the 3-day-brining period, to a degree which increased with the level of pre-salting (Fig. 14a). This occurrence was attributed to an intense ‘salting-out’ and shrinkage of the protein matrix in the rind layer of these cheeses because of the very high level of S/M (⬃19–22%, w/w) (Guinee, 1985). Hence, the moisture level in rind of the brinesalted cheese decreased markedly on pre-salting to 6%, w/w, S/M (Fig. 14b).


(a)

(b) 44

22.00 20.00

40

16.00

Moisture, %, w/w

Salt-in-moisture, %, w/w

18.00

14.00 12.00 10.00 8.00 6.00

36

32

4.00 2.00 0.00

28 0

1

2

3

4

5

6

Distance from cheese surface, cm

0

1

2

3

4

5

6

7

Distance from cheese surface, cm

Figure 14 Salt-in-moisture concentration (a) and moisture (b) as a function of distance from cheese/brine interface in experimental Gouda-type cheeses which were pre-salted to 0.5 ( ), 4.8 (), 6.0 (), 8.4 (), 9.5 (), 10.5 () or 12.5 (●) %, w/w, NaCl before brine-salting at 15 °C in 19.2%, w/w, NaCl brine with ⬃0.5% %, w/w, Ca. The drained curds were thoroughly mixed with dry salt, added at the desired quantity; the salted curds were held at room temperature for 5 min, then pressed, and brine-salted; initial NaCl concentration in brine (redrawn from Guinee, 1985).

Initial moisture content of the curd

Geurts et al. (1974b) showed that the quantity of salt absorbed by experimental Edam and Gouda-type cheeses during brine-salting generally increased as the initial moisture content of the curd increased, with the effect becoming more pronounced with the duration of brining. Similar results were obtained by Byers and Price (1937) for brine-salted Brick cheese. This increased salt uptake by the experimental cheeses concurs with the linear increase in predicted salt uptake through a flat cheese surface as the water content increases (see ‘Cheese geometry’). The increased salt uptake undoubtedly reflects the concomitant increase in D* as moisture level increases. A high moisture content leads to a lower protein level, a lower volume fraction of the protein matrix occluding the moisture through which NaCl diffuse, an increase in the relative pore width of the protein matrix and hence a reduced frictional effect on the inward-diffusing Na and Cl . On dry-salting milled Cheddar curd, the reverse situation occurs: as the initial moisture level increases, the rate of salt absorption decreases giving lower salt and S/M values in the cheese for a fixed salting level (Sutherland, 1974; Gilles, 1976). Such decreases were attributed to greater whey and salt losses from the high-moisture curds; an increase in curd moisture content from 39.1 to 43.4%, w/w, caused a 30% increase in the amount of whey drained off and a decrease in salt

retention from 59 to 43%, w/w, of the amount applied (Sutherland, 1974). Thus, while the extent of salt penetration within each granule increases, there is less salt available for uptake as the initial curd moisture increases (salt causes loss of moisture from the curd and at the same time is itself removed). The apparent discrepancy between the effects of moisture content on salt uptake in brined-salted and dry-salted cheeses may be due to differences in the degree of contact between the salting medium and the curd, and in the length of contact time between curd and salting medium. On increasing the moisture content of dry-salted cheeses, the higher outflow of moisture may result in ‘excess brine’ which percolates through the spaces between the chips, drains away, loses contact with the chips and thereby lowers the effective amount of salt available for uptake. This does not happen when cheese is submerged in brine, as in brine-salting. Moreover, there is sufficient time for a lactate/water (brine) mutual diffusion process during brine-salting whereas this is not expected during drysalting as the water ‘drawn out’ of the cheese by the applied dry salt quickly drains away through the curd bed and loses contact with most of the curd. As the lactate level in cheese increases with moisture content, a greater influx of water as a result of the lactate/water diffusion process is expected. Consequently, an increase in the moisture level in cheese prior to brine-salting is expected to reduce the net water loss per given weight


of cheese and increase salt penetration and uptake during subsequent brine-salting. This hypothesis concurs with the increase in D* (see ‘Factors that influence salt diffusion in cheese during salting’), and the tendency of the flux ratio (i.e., the ratio of water lost to salt absorbed during brining) to decrease (Geurts et al., 1980) as the moisture level of Gouda cheese prebrining is increased. pH of curd and brine

While D* for Gouda cheese was not influenced by cheese pH in the range 4.7–5.7 (Geurts et al., 1974b), the rate of salt uptake was higher at pH 4.7 than at 5.7 even though the initial moisture content of the cheeses was similar (Geurts et al., 1980). This finding was consistent with that of previous studies, which showed higher NaCl uptake at low, than at high, cheese pH (Geurts et al., 1980). The higher salt uptake at lower cheese pH coincides with a lower water loss during brining (per unit weight of salt gained), which may be attributed to a higher lactate level in the low-pH cheese (Geurts et al., 1980). A higher lactate level pre-brining would reduce the net loss of water during brining (see ‘Brine concentration and concentration gradient’ and ‘Initial moisture content of the curd’). A number of investigators have examined the effect of titratable acidity at salting on salt retention by Cheddar cheese curd. However, Cheddar curd dry-salted at low acidity retained more salt than more acidic cheeses (Lawrence and Gilles, 1969, 1982; Gilles, 1976). Since low-acid curd normally contains more moisture than high-acid curd, one might expect more syneresis and higher salt losses, less available salt for absorption, and therefore less salt uptake in the low-acid curd. However, for a given salt availability, the rate of salt diffusion and salt uptake would be expected to increase as the level of moisture in the curd increases, as discussed in ‘Initial moisture content of the curd’). Lawrence and Gilles (1969) suggested that the observed difference in salt retention may be due to the higher degree of curd hydration at the higher pH values (i.e., ⬃5.3), which may effect a higher retention of salt by the curd structure per se (see Dolby, 1941; Creamer, 1985; ‘Effect of NaCl on casein hydration in model systems and in cheese’). In practice, the pH of brine is adjusted to ⬃5.0–5.3, which is close to that of most brine-salted cheeses. Acidification of the brine has a preservative effect and also minimizes the risk of surface defects (e.g., velvety, non-drying rind) associated with an imbalance in [H] which affects the level of casein hydration (see ‘Effect of NaCl on casein hydration in model systems and in cheese’). While little information is available on

the effect of brine pH on salt uptake (Geurts et al., 1980), it is conceivable that excessive lowering of the pH (e.g., 4.6) would lead to protein precipitation and a high loss of water at the cheese surface, which in turn would reduce salt uptake. Factors that affect salt uptake in Cheddar curd Method of salting

Breene et al. (1965) showed that salting of milled Cheddar curd by brining gives a higher rate of salt absorption and a higher level of salt-in-moisture in the pressed curd than dry salting. Differences in absorption rate were explained on the basis of availability of salt at the surfaces of the curd. When dry salt is placed on freshly milled curd, a portion dissolves in the surface moisture, creating a very thin layer of supersaturated brine. The salt-in-moisture gradient between the brine and the cheese moisture results in mutual movements of salt and water in opposite directions in response to their respective concentration gradients. Some water is also ‘squeezed out’ of the curd due to localized surface contraction (salting-out of the protein matrix) as a result of contact with the super-saturated brine. The level of moisture in the curd, which influences whey release, affects the rate of solution of surface salt. When curd is placed in brine, salt absorption begins immediately through all surfaces. Release of whey occurs, as in dry salting, but its extent is not a limiting factor (Sutherland, 1974). Level of salting

As expected, an increase in salting level (especially when the level is low) increases the rate of salt absorption by, and whey drainage from, cheese, thus giving higher levels of salt and salt-in-moisture and a lower level of moisture in the cheese after salting for a fixed time (Breene et al., 1965; O’Connor, 1970, 1971, 1973b, 1974; Gilles, 1976; Guinee, 1985; Kelly et al., 1996). However, the relationship is curvilinear (Fig. 15; O’Connor, 1973b), i.e., the increase in the salt and salt-moisture levels in the cheese is not proportional to the level of salt added, especially at the higher salting levels, because of higher salt losses at increased salting levels. Although these principles are probably generally applicable, the precise relationship between salt loss and retention depends on the pH and moisture content of the curd and the period of time allowed for salt diffusion into the curd. These inter-relationships have been studied by Sutherland (1974) and Gilles (1976). Sutherland (1974) showed that the volume of whey released from the curd and the percentage of added salt lost increased linearly with the level of salt added (over a narrower range than that used by O’Connor) while the percentage


9.0

6.0

Salt in cheese, %, w/w

4.5

3.0

3.0

1.5

1.5

0.0

Salt-in-moisture, %, w/w

7.5

0.0 0

2

4

6

Salting level, g/100 g Figure 15 Relationship between salting level of curd and the concentration of salt (●) and salt-in-moisture () in Cheddar cheeses, prepared from batches of curd from the same vat (redrawn from Lawrence and Gilles (1982) using the data of O’Connor (1974)).

of moisture decreased and the percentage of salt, saltin-moisture and pH of the cheeses increased in a curvilinear fashion as the level of added salt was increased. The level of salt added had no significant effect on the loss of fat (⬃0.25 kg/100 kg curd). Kelly et al. (1996) reported a linear increase in salt and S/M and a linear decrease in moisture with salting level in the range 0–3.0%, w/w. Curd temperature

Increasing the temperature of Cheddar curd chips from 24 to 41 °C resulted in a marked increase in the percentage of added salt lost during holding (mellowing), a decrease in the level lost during pressing, and a slight increase in the percentage lost overall during holding and pressing (Sutherland, 1974). Consequently, the levels of salt and S/M decreased by ⬃11% on raising the curd temperature from 24 to 41 °C and the percentage of fat lost increased markedly, e.g., by ⬃0.6 kg fat/tonne curd per 1 °C rise in temperature in the normal working temperature range of 29–35 °C. The pH and moisture level in the finished cheese were essentially unaffected by salting temperature. Degree of mixing of salt and curd

Extending the stirring time of salted Cheddar curd from 20 s to 6 min caused a significant increase in salt and S/M levels, i.e., from 1.53 to 1.97%, and 4.41

to 5.71%, respectively (Sutherland, 1974). Undoubtedly, better mixing leads to salt absorption from more faces and hence there is less ‘free’ salt to be lost in the press whey. Hence, salt losses decreased with the duration of stirring time even though there was little effect on the volume of whey released (Sutherland, 1974). For a given mixing period, increasing the surface area of the curd by reducing the size of the curd chips results in a significant increase in salt level (Gilles, 1976). Increased mixing time resulted in a higher loss of fat, probably as a consequence of shrinkage at the surface of the curd chips and a concomitant increase in the loss of fat in the salt/water (Sutherland, 1974). Mechanical salting procedures give more uniform distribution of salt in Cheddar cheese than hand or semi-automated salting systems (O’Connor, 1968, 1970, 1973b; Fox, 1974; Knox, 1978). Because of the significance of salt level and distribution in relation to cheese quality, salting of Cheddar curd at factory level is a carefully controlled operation. It is performed on enclosed inclined, perforated (to allow whey drainage) belts where a single, or twin, oscillating boom distributes the salt, delivered from an overhead metering device according to a sensor which measures the bed depth, onto the moving curd bed and overhead stirrers continuously mix the curds. Improved means of salting cheese curds, e.g., trommel salt mixers, are being developed (Zahlus, 1986; Cosentino et al., 1987; Ryskowski et al., 1989; ‘General Aspects of Cheese Technology’, Volume 2). Holding time between salting and pressing (mellowing period)

Extending the holding time between salting and pressing increases the salt and S/M levels in the pressed Cheddar cheese (Breene et al., 1965; Sutherland, 1974; Gilles, 1976), e.g., by ⬃0.3%, w/w, on increasing time from 15 to 30 min. The increase is attributed to a higher total absorption and hence a reduction in the physical loss of salt. Curd depth during holding

When the depth of salted Cheddar curd during holding was increased from 12.7 to 68.0 cm, the moisture, salt and S/M levels decreased from 35.1 to 34.9%, 1.81 to 1.68%, and 5.1 to 4.8%, respectively (Sutherland, 1974). Moisture content of the curd

Sutherland (1974) and Gilles (1976) studied the effect of moisture content, which was varied by altering agitation speed, degree of whey removal at half-whey-off stage, and/or the level of dry-stirring, on salt uptake in Cheddar. Increasing the moisture


content of the curd before salting from 37 to 45%, w/w, resulted in reductions (⬃42%) in the levels of salt and S/M, and in pH, and an increase in moisture content. These changes coincided with an increase in the level of whey released and a reduction in salt retention, e.g., from ⬃59 to 39% of total salt on increasing the moisture level from 39 to 43.5%, w/w (Sutherland, 1974).

Manufacturing conditions

Cheese dimensions at salting

Curd acidity at salting

Shape

Surface area to volume ratio

Size

Other factors

As well as confirming the work of Sutherland (1974), Gilles (1976) showed that salt particle size has little effect on salt retention, milling the curd to smaller particles increases salt retention, and extensive flow and development of a fibrous structure during cheddaring leads to increased variation in S/M levels. Indeed, Gilles (1976) maintained that the best way to regulate the salt content of cheese is to control its moisture content (which can be best done by dry stirring). Regulating moisture content by altering the level of salt added was considered undesirable because of the influence of several factors on salt retention and the effects of salt concentration on cheese quality. The interaction of some factors influencing salt uptake in Cheddar-type curd and brine- or dry-salted cheeses is summarized in Figs 16 and 17, respectively. Factors that influence salt diffusion in cheese during salting

While it is well established that the diffusion coefficient of salt in cheese moisture is much lower than that in pure water (see ‘Mechanism of salt absorption and diffusion in cheese’), there is relatively little information on the factors which influence the movement of NaCl in cheese during salting. The first such study was made by Georgakis (1973), who related the diffusion of NaCl in Greek Feta to cheese surface area, Manufacturing conditions

Curd acidity at salting

Moisture content of curd

Salt uptake

Salt-in-moisture

Curd particle size at milling Salting rate Extent of mixing Method of salting

Dry salting

Moisture content of curd Salt uptake

Brining conditions (e.g. temperature, % NaCl)

Salt-in-moisture

Figure 17 Principal factors that affect the uptake of salt by brine-salted cheeses (from Fox, P.F., ed., Cheese: Chemistry, Physics and Microbiology, 2nd edn, Chapman & Hall, p. 281).

duration of salting, brine concentration and the fat and moisture contents of the cheese. In model brining experiments, Geurts et al. (1974b) quantified the influence of variations in cheese composition and brining conditions on the pseudo-diffusion coefficient (D*) of NaCl in the moisture phase of Gouda cheese. The factors which affect the movement of salt in cheese during brining presumably also apply to the cheese after brining and hence have a decisive effect on the rate of attainment of equilibrium of S/M and of moisture; under normal brining conditions (e.g., 18–20%, w/w, NaCl; 0.2% Ca; 10–20 °C), moisture and salt move in opposite directions as a consequence of diffusion (Geurts et al., 1974b; Guinee and Fox, 1983b, 1986a,b). Although continuing physico-chemical and structural changes during ripening may alter the situation somewhat, it is worth noting that the diffusion coefficient for NaCl in the moisture phase of a dry-salted, 12-week-old Cheddar (50% FDM, 37.9% H2O) (Sutherland, 1974) corresponded well with that found by Geurts et al. (1974b) for brine-salted Gouda cheese of similar composition. The influence of the various factors on NaCl diffusion in Gouda cheese has been studied by Geurts et al. (1974b), Guinee (1985), and Turhan and Kaletunç (1992). Concentration gradient

Brine salting

Figure 16 Principal factors that affect the uptake of salt by Cheddar curd (from Fox, P.F., ed., Cheese: Chemistry, Physics and Microbiology, 2nd edn, Chapman & Hall, p. 281).

The concentration gradient between cheese and brine is determined by the difference in the level of salt in the brine and the S/M of the cheese, while the concentration gradient between neighbouring regions within


a cheese loaf is determined by the difference in S/M level between the two regions. The gradient changes with time either for cheeses which are matured after removal from the brine (as for most cheeses) or matured in the brine (e.g., Feta, Gaziantep) until S/M equilibrium is attained (Godinho and Fox, 1981b; Guinee and Fox, 1986a,b,c; Pappas et al., 1996; Kaya et al., 1999; Messens et al., 1999; Pavia et al., 1999; Prasad and Alvarez, 1999; Melilli et al., 2003). While the concentration gradient is a major determinant of the rate of salt absorption by cheese during salting, it scarcely affects the mobility of the diffusing species at concentrations in the range 5 to 20%, w/w, NaCl (Geurts et al., 1974b; Guinee, 1985). However, a sharp drop (⬃18%) in D* for Gouda-type cheese occurred when the salt content of the brine was increased from 20 to 24.8%, w/w, at 20 °C, i.e., from ⬃0.205 to 0.17 cm2/day (Guinee, 1985). Similarly, increasing the brine concentration from 15 to 20%, w/w, NaCl reduced the moisture diffusivity in Turkish white cheese, with the reduction at 20 °C (⬃17%) being more pronounced than that at 4 or 12.5 °C (⬃8%) (Turhan and Gunasekaran, 1999). While the apparent D* decreases on increasing NaCl level in the brine to 25%, w/w, especially at high temperatures, the true value would be somewhat higher if allowance was made for relatively high water loss which in effect causes the plane of zero mass transfer of all diffusing species to recede further from the cheese/brine interface into the brine. However, since S/M seldom reaches 20%, w/w, in cheese, except in the rind layer, the large inter-zonal variations in S/M level at the end of brining should not significantly alter the rate of attainment of S/M equilibrium within a given cheese loaf or between loaves of the same variety. Temperature of brine and cheese

Increasing brine (and curd) temperature is paralleled by increasing diffusion mobilities of NaCl and H2O in cheese (Geurts et al., 1974b; Turhan and Kaletunç, 1992); an increase of ⬃0.008 cm2/day/°C was found for Gouda-type cheeses for brine temperature in the range 5–25 °C (Guinee, 1985). This increase was attributed (Geurts et al., 1974b) to an increase in true diffusion and to some effect on diffusion-interfering factors, i.e., possible decreases in the relative viscosity of cheese moisture and the amount of protein-bound water which effects an increase in the relative pore width of the protein matrix (in cheese, water non-solvent for sugars decreases with increasing temperature; Geurts et al., 1974a). Because of the large effect of temperature on D*, the higher the storage temperature, the shorter should be the time required for the equilibration of salt and moisture levels within the cheese mass after salting.

Concentration of calcium in the brine

Preparation of brine requires the addition of calcium (normally added as a CaCl2 solution) so as to minimize moisture uptake by the exterior of the cheese (Figs 8, 9) and the associated risk of defects such as rind rot and soft rind, especially where the NaCl concentration in the brine is relatively low, e.g., 15%, w/w (Geurts et al., 1972; Guinee and Fox, 1986a). The latter defects are due to diffusion of calcium from the cheese moisture to the brine which ultimately results in the solubilization, and loss, of protein-bound calcium to an extent depending, inter alia, on the level of calcium in the cheese and brining conditions (pH, temperature, NaCl level, duration, size of cheese relative to brine volume). The depletion of cheese calcium leads to hydration of the para-casein and associated moisture uptake and softening (see ‘Effect of NaCl on casein hydration in model systems and in cheese’). Typical levels of CaCl2 added give a calcium level in the brine (e.g., 0.2–0.3%, w/w) which is probably somewhat lower than that of some semi-hard brinesalted cheeses, such as Gouda; however, these levels are adequate to prevent the occurrence of the above defects with typical NaCl level (19–21%, w/w) and brine temperature (⬃12 °C). For hard and semi-hard cheeses, the percentage of total cheese calcium which is soluble increases with decreasing pH, e.g., from ⬃20% for commercial low-moisture part-skim Mozzarella (mean pH ⬃5.5) to ⬃37% in Cheddar at pH 5.2 (Guinee et al., 2000). Hence, the concentration of calcium in the serum phase is ⬃0.72 and 0.34%, w/w, for Cheddar and Mozzarella, respectively. The level of serum calcium for other hard and semi-hard cheeses such as Gouda, Masdammer and Emmental, with a pH of ⬃5.3 to 5.4 before brining is probably similar to that for Mozzarella; the level for low-calcium, soft cheeses with a low pH (e.g., 4.6–5.0) before salting, e.g., Camembert, Cheshire, Brie and Blue-types, are undoubtedly much lower than 0.34%, w/w. Typical levels of total calcium in Emmental, Mozzarella, Camembert, Blue and Stilton are 1000, 710, 530, 475 and 400 mg/100g, respectively. Hence, the minimum calcium level required in the brine depends on the type of cheese being salted. Increasing the concentration of Ca in freshly prepared brine (19%, w/w, NaCl; 15 °C) from 0 to 1.8%, w/w, reduced the moisture level (⬃3%, w/w) and increased the salt content (⬃1.5%, w/w) of the outer 0.5 cm layer of Gouda cheese (Guinee, 1985). However, the increase in calcium had little effect otherwise on cheese composition or on D* (⬃4% reduction). Consequently, the levels of total calcium and serum calcium probably have little influence on the rate at which equilibrium of S/M is attained post-brining.


Moisture content of cheese

It is generally accepted that the moisture content of cheese affects the rates of salt absorption and/or diffusion (McDowall and Whelan, 1933; Byers and Price, 1937; Georgakis, 1973). However, from calculations of diffusion coefficients it has been shown (Geurts et al., 1974b; Guinee, 1985) that for two cheeses of the same variety, the rate of diffusion is not necessarily higher in the higher moisture cheese; the diffusion coefficient depends on the ratios of fat to solids-not-fat (SNF) and moisture to SNF. These factors affect the volume fractions of the fat and protein phases, which in turn determine the impedance to the diffusing molecules (see ‘Brine-salted cheese’). The results in Table 5 indicate the importance of cheese composition, and hence structure, in salt diffusion. The diffusion coefficient for NaCl in the cheese moisture (D*) increased with increasing FDM when the percentage of SNF (and hence protein tortuosity, p) decreased and the relative pore width of the protein matrix (/dp) increased (e.g., cheeses 1–8, Table 5) but decreased when both the FDM and SNF levels increased (cheeses 9 and 10). In some instances (e.g., cheeses 2, 6 and 9 with ⬃49%, w/w, H2O or cheeses 12 and 14 which had ⬃44%, w/w, H2O) the

moisture contents were approximately equal but the D*-values differed considerably due to differences in fat (and hence fat tortuosity, f) and protein (SNF). In contrast, D* was almost equal for other cheeses (e.g., 11 and 13) which differed appreciably in moisture level. Similar results, shown in Table 6, were obtained by Geurts et al. (1974b). Therefore, while it is difficult to elucidate the effect of moisture, or indeed of any one compositional parameter separately, on salt flux, the effect of moisture is discussed separately below. Within a series of cheeses of the same variety with equal FDM, D* increased curvilinearly with moisture content (Fig. 18; Geurts et al., 1974b). Considering cheeses 9–12, Table 5, it is apparent that the contribution of the decreasing fat tortuosity (f) (with increasing moisture content) to the increase in D* was small (D* and D* f increased by a factor of 1.7 and 1.6, respectively, when the moisture content increased from 44.5 to 49.2%). The principal factor affecting the increase in salt flux was the reduced frictional effect on the diffusing molecules as the volume fraction of the protein matrix (p) decreased; hence the relative pore width (y/dp) increased concomitantly with increasing moisture content (see Tables 5 and 6).

0.4

D* (cm2/day)

0.3

60

50

40

30

20 10

0.2

0.1 30

40

50

60

Moisture content of cheese (%) Figure 18 Diffusion coefficient of NaCl in cheese moisture (D*) as a function of the initial moisture content of the cheese. Parameter is g fat/100 g DM in unsalted cheese. The solid lines are experimental values, broken lines are extrapolations (from Geurts et al., 1974b).


The relationship between the diffusion coefficient in the fat-free cheese, D* f, and the relative pore width of the protein matrix is seen in Fig. 19 (Geurts et al., 1974b); D* f can be considered as the ‘theoretical’ value of D* for a system with the same structural features as cheese but from which the impedance to salt diffusion, due to the physical presence of fat globules, has been eliminated. While the decrease in the protein tortuosity (p) contributes to the increase in D* associated with increasing moisture content (e.g., cheeses 9–12, Table 5), its effect is small as it varies little within the range of p values encountered. The role of moisture as the main compositional factor affecting salt flux has been confirmed by Morris et al. (1985) who found that the D* values for different commercial cheese varieties (⬃37.3–49% H2O; ⬃23.5–27.5% fat; ⬃40.5–49.5% FDM; ⬃28–35% SNF) were directly related to the moisture content of the unsalted cheeses (Fig. 20). Of the variation in D* which could be attributed to compositional factors (⬃70% of total variation) in the latter cheeses, ⬃49, 29 and 22% could be attributed to variations in the relative pore width of the

protein matrix, the protein tortuosity and fat tortuosity, respectively (Guinee, 1985). The strong positive correlation between moisture content of the cheese and D* suggests that S/M equilibrium in brine-salted cheese is attained more rapidly with increasing moisture content. This is indeed the case as reflected by the much shorter time required for attainment of equilibrium in soft cheeses compared to hard cheeses, e.g., 3 days in Camembert (Hardy, 2000), ⬃45 days in a 2.2 kg Blue cheese (mean moisture, ⬃40%, w/w; Godinho and Fox, 1981b), and ⬃60 days for a 3 kg cylindrical Romano-type cheese (mean moisture, ⬃40%, w/w; Guinee and Fox, 1986c). However, the rate of attainment of equilibrium, as discussed later, is very dependent on cheese dimensions and weight. While D* is strongly dependent on the composition and structure of the unsalted cheese, especially the moisture content, it is, surprisingly, scarcely affected by variations in composition along the different planes of a cheese resulting from salt uptake and moisture loss during brine salting. This is reflected by: (i) the consistency of D* over the region of salt and water movement

D*λ f (cm2/day)

0.3

0.2

0.1

0.1

0.2

0.3

Relative pore width of protein matrix (y/dp) Figure 19 Diffusion coefficient of NaCl in the moisture in fat-free cheese (D * f), as a function of the relative pore width of the protein matrix (y/dp). Brine concentration, 19–20 g NaCl/100 g H2O; temperature, 12.5 °C; 50%, w/w, fat in DM; pH 5, unless stated otherwise. g fat/100 g DM: ●, 12; , 22; , 62; , pH 4.79; , pH 5.50; brine concentration, 14 g NaCl/100 g H2O (from Geurts et al., 1974b).


0.23 A B

C

0.21

gradient within the cheese during salting or postsalting should have little effect on salt diffusion in cheese moisture. Fat content of cheese

D ∗, cm2/day

D 0.19

E

0.17 F 0.15

G

H I

0.13 35

39

43

47

51

Moisture, g/100 g Figure 20 Dependence of the pseudo-diffusion coefficient of salt in cheese moisture (D*) on the initial moisture content of cheese salted in ⬃20%, w/w, NaCl brine at 15–16 °C. Blue cheese (A, B), Gouda (C, D), Romano-type cheese (E), Jarlsberg (F), Emmental (G, I), unsalted milled Cheddar (H) (from Morris et al., 1985).

and with time (Geurts et al., 1974b; Guinee and Fox, 1983a) and (ii) the almost-constant D* value for brine concentrations in the range of 5–20% NaCl (Geurts et al., 1974b; Guinee, 1985). Thus, while pre-salting the curd to different S/M levels prior to brine-salting (as described in ‘Initial salt-in-moisture level of curd and pre-salting’) reduced the level of salt absorbed, it had little effect on D*, which increased from 0.18 to 0.19 cm2/day on increasing the S/M level from 0.3 to 4.7%, w/w, or the penetration depth of the salt during subsequent brine-salting (Fig. 14a) (Guinee, 1985). These results confirm that compositional changes accompanying salt uptake have little, or no, effect on D*. Consideration of the physico-chemical changes in cheese associated with the physical presence of salt per se and ageing may provide a tentative explanation (Geurts et al., 1974a). Salting and ageing of cheese are paralleled by a reduction in the amount of protein-bound water (i.e., water unavailable as a solvent) and a decrease in the mean diameter of the protein particles (Geurts et al., 1974a,b) and hence an increase in the effective moisture concentration and relative pore width of the protein network. Such changes possibly offset the impeding effects of moisture loss during brining on salt flux, and hence D* remains essentially constant during brining. Moreover, as concentration gradient in the range 0–20% S/M has no effect on D* (see ‘Concentration gradient’), the variations in salt

As discussed in ‘Mechanism of salt absorption and diffusion in cheese’, the diffusion coefficient of NaCl in cheese moisture is much lower than that in pure solution, i.e., ⬃0.2 cm2/day compared to ⬃1.0 cm2/day at 12.5 °C. This is because salt diffusion in cheese takes place in moisture held in a matrix which is comprised of protein aggregates and occludes fat globules, both of which obstruct the movement of diffusing molecules and increase the real distance travelled by a salt molecule on proceeding from one parallel plane to another (see ‘Brine-salted cheese’). Therefore, the physical presence of fat per se reduces the apparent D-value due to the inverse of its tortuosity, i.e., 1/f. However, D* increases with fat content in cheeses with equal moisture content (Fig. 18). In unidimensional brine salting experiments, Geurts et al. (1974b) observed that for Gouda cheese with 50% moisture, but with 11 or 26% fat, the D* values were 0.15 and 0.25 cm2/day, respectively. While the fat tortuosity factor increased with fat level, i.e., 1.12 and 1.29 at 11 and 26% fat, respectively, the relative pore width of the protein matrix also increased (i.e., 0.17 and 0.35 at 11 and 26% fat, respectively). Hence, the increase in D* with fat content is not due to fat per se (which actually reduces D* by a factor of 1/f) but rather to the concomitant decrease in the protein volume fraction and, hence, the increase in the relative pore width of the protein matrix. The reduction in the sieve-effect of the protein matrix on the salt molecules overrides the increased obstruction caused by increasing fat levels and, hence, D* increases. Indeed, for cheeses of equal moisture content in fat-free cheese (i.e., cheeses with equal protein volume fractions), D* is always higher in cheese with the lower fat content (Geurts et al., 1974b). However, in practice, reducing the fat level of cheese, while increasing the moisture percentage per se, results in a reduction in the level of moisture in non-fat substances and an increase in protein level (see Fenelon and Guinee, 1999). Hence, the concomitant increases in p and p and reduction in relative pore width would be expected to reduce D* significantly, unless the reduction in fat content is small (e.g., 1–3%, w/w) and the cheesemaking process modified to prevent a reduction in the MNFS. From the foregoing, it is apparent that the effect of varying any cheese compositional parameter on salt mobility depends on the concomitant changes it causes in the cheese structure (i.e., the ratios of fat to solids-not-fat, and solids-to-moisture). Since increasing


the fat level in cheese reduces syneresis (Whitehead, 1948; Marshall, 1982; Walstra et al., 1985; van Dijk and Walstra, 1986; Pearse and Mackinlay, 1989), D* should generally increase with increasing fat content due to the concomitant decrease in p.

44

20 42

Cheese geometry

16

12 38

8 36

4 34

32 0

Attainment of salt and moisture equilibria after salting Brine- and surface dry-salted cheeses

In cheeses which are salted by immersion in brine and/or by surface application of dry salt there is a large decreasing salt gradient from the surface to the centre and a decreasing moisture gradient in the opposite direction at the completion of salting (see Fig. 21; Guinee, 1985; Guinee and Fox, 1986a, 1993; Turhan and Kaletunç, 1992; Messens et al., 1999; Pavia et al., 1999; Turhan and Gunasekaran, 1999; Melilli et al., 2003). Due to the slow diffusion of salt from the rind inwards, these gradients disappear slowly and equilibrium of S/M is practically reached at some stage of ripening, depending on cheese type, size of cheese and curing conditions (Fig. 22; Guinee and Fox, 1986b; Messens et al., 1999; Pavia et al., 1999). Salt absorption is a relatively rapid event, varying from 15–30 min for salt uptake by Cheddar-type curd chips (Sutherland, 1977, 2002) and ⬃7.5 h (Camembert) to ⬃15 days (e.g., Parmesan). In contrast, diffusion of salt and moisture post-salting, and hence the rate of attainment of S/M equilibrium throughout the cheese mass, is a slow process, e.g., 10–12 days for Limburger (McDowall and Whelan, 1933; Kelly and Marqurdt, 1939), 8–12 weeks for Gouda (Morris, 1961), Brick (Beyers and Price, 1937), Blue (Godinho and Fox, 1981b) and Romano-type cheeses (Guinee and Fox, 1983b, 1986b), ⬃40 days for Feta (Georgakis, 1973) and ⬃10 months for Parmesan (Resmini et al., 1974).

Moisture, %, w/w

40 Salt-in-moisture, %, w/w

Cheese geometry influences the rate of attainment of salt-in-moisture equilibrium via its effect on the relative dimensions of the cheese. Guinee and Fox (1986b) working with commercial Romano-type cheeses of different shapes, showed that at any time during storage, the net difference in S/M concentration along layers of the cheeses increased with layer length. This observation is consistent with the fact that the depth of salt penetration during brining is proportional to the square root of brining time (Geurts et al., 1974b; Guinee and Fox, 1986a). Using differently shaped cheeses, it was found that the rate of attainment of S/M equilibrium is not necessarily directly proportional to the volume when comparing cheeses of the same variety (Guinee and Fox, 1986c).

1

5 2 4 3 7 6 Distance from cheese surface, cm

Figure 21 Moisture content (open symbols) and salt-in-moisture (g NaCl/100 g H2O) (closed symbols) in Gouda cheese (initial fat-in-dry matter 49.1%, w/w; moisture, 43.64%, w/w; pH, 5.26) as a function of distance from the salting surface after unidimensional brine salting (20.3%, w/w, NaCl) for 1 (,), 2 (,●), 3 (,) and 4 ( , ) days at 15 °C (redrawn from Guinee, 1985).

Sutherland (2002) reported 7–10 days for Camembert (0.25 kg, flat disc; 55%, w/w, moisture), 4–6 weeks for Edam (2.5 kg sphere; 46%, w/w, moisture), 7–9 weeks for Gouda (10 kg wheel; 42%, w/w, moisture) and 4 months for Emmental (60–130 kg wheel; 36%, w/w, moisture). However, on consideration of these data and times reported elsewhere (see ‘Moisture content of cheese’), it is clear that intra- and inter-variety differences occur as a result of variations in cheese dimensions, surface area-to-volume ratio, composition and brining conditions (which may affect cheese composition and distribution of salt and moisture at the end of brining). Though the importance of the mean, and the uniformity of, S/M levels in cheese in relation to quality have received much attention (see ‘Overall Influences of NaCl on Cheese Ripening and Quality’ and ‘Effect of NaCl on Casein Hydration and the Physical Properties of Cheese’), the factors that affect the diffusion of NaCl and moisture in cheese after salting and hence the rate of attainment of equilibrium have received little study. However, as for diffusion during the brine/dry-salting


Cheddar and dry-salted varieties

22 Top surface layer, A1 Centre layer, I1, I2

20

Bottom surface layer, A2

18

Salt-in-moisture, g/100 g

16

14

12

10

8

6

4

2

0 –5

–4

–3

–2

–1

0

1

2

3

4

5

Distance from cheese surface, cm Figure 22 The mean salt-in-moisture levels in discs A1/A2, B1/B2, C1/C2 etc. (as indicated) of cylindrical Romano-type cheese salted in 19.5% NaCl brine at 23 °C for 1 (●), 3 (), or 5 (■) days or salted for 5 days and stored wrapped at 10 °C for 30 () or 83 () days (redrawn from Guinee and Fox, 1986b).

process per se, the salt diffusion during ripening/storage, and thus the rate of attainment of S/M equilibrium within a cheese loaf, is undoubtedly similarly affected by: • cheese dimensions and shape, which determine the distance between the high salt exterior zones and low salt interior zones at the end of salting (Guinee and Fox, 1986c); • temperature; • concentration gradient between different zones; • cheese composition (see ‘Factors that influence salt diffusion in cheese during salting’). Though not investigated to date, conditions of relative humidity, rate of air circulation, and frequency of turning the cheese during storage possibly alter the rate of attainment of salt and moisture equilibria as a result of alterations in cheese moisture.

Salt is fairly uniformly distributed in Cheddar-type cheese initially, as salt is mixed with the milled curd. However, in contrast to brine-salting or surface dry-salting, complete equilibrium is slow and rarely, if ever, reached (McDowall and Whelan, 1933; Morris, 1961; O’Connor, 1968, 1971, 1973a; Sutherland, 1977; Thomas and Pearce, 1981; Morris et al., 1985; Wiles and Baldwin, 1996). Thus, significant intra- and interblock variations in salt concentration occur in mature commercial Cheddar cheese, giving rise to considerable variations in the rate of ripening and grading quality (O’Connor, 1971, 1973b, 1974; Fox, 1975; Thakur et al., 1975; Sutherland, 1977; Thomas and Pearce, 1981; Lawrence et al., 1984; Kelly et al., 1996; ‘Cheddar Cheese and Related Dry-salted Cheese Varieties’, Volume 2). A preliminary investigation of brine-salting Cheddar curd chips (Breene et al., 1965) showed that adequate uptake of salt could be accomplished by placing chips (3/8 in 3/8 in 2 in) in 25% brine for 5 min and holding for 15 min after removal from the brine before pressing. Considering the problems encountered in controlling salt uniformity using the drysalting procedures, brine-salting of Cheddar could be potentially useful and appears to warrant further investigation. O’Connor (1968) assessed the uniformity of salt distribution in cheeses salted by hand, semi-automatic or fully automatic systems; one plug from every 10th cheese per vat was analysed. While the range of mean salt content was relatively narrow, there was very considerable intra-vat and inter-vat variation in salt content, with greatest variation in the hand-salted cheese and least with the fully automated system; an inverse correlation between salt and moisture contents was apparent. Further evidence of high within-vat variation in salt distribution is provided by O’Connor (1973b). The findings of O’Connor (1968, 1973b) were confirmed and extended by Fox (1974) who showed that, in general, mechanical salting systems gave more uniform salt distribution than hand-salting systems or a semiautomatic system. Considerable within-block variability (12 samples per 20 kg block) in salt concentration was also demonstrated. Morris (1961) also found very large differences in the salt content of blocks from the same vat (spread of 0.6%, w/w, on a mean of 1.38%, w/w). All the foregoing investigators stress the importance of inter- and intra-block variations in salt content, which is inversely related to moisture content. Since it is generally agreed that the quality of cheese is strongly dependent on moisture, S/M and pH (which was not reported in any of the above studies) (see


‘Cheddar Cheese and Related Dry-salted Cheese Varieties’, Volume 2), it might reasonably be expected that the quality of cheese also varies between blocks from the same vat and even within the same block. It is normal cheese-grading practice to grade a vat of cheese on the basis of 1–3 plugs taken from a single cheese per vat, at any one time; obviously, the quality of this sample may not be representative of the vat. For similar reasons, calculation of mass balances in cheese factories on the basis of a few plug samples per vat may be very inaccurate. In practice, salting of Cheddar is performed mechanically on salting belts where the salt is delivered by oscillating booms in proportion to curd depth and the salted curd is mixed by rotating peg mixers mounted over the belt. The method of salt application used in Cheddar cheese manufacture would appear to be particularly amenable to ensuring accurate control of salt concentration with respect to both level and uniformity. However, in commercial practice, it has been difficult to achieve uniform distribution (Morris, 1961; Fox, 1974), possibly because of the many factors which influence salt uptake by curd (Fig. 16) and the design of salting equipment capable of giving adequate mixing in the time allowed. Undoubtedly, a more consistent salt level and distribution could be obtained by: • the production of curd with more consistent composition, – e.g., via standardization of milk protein level (i.e., by ultrafiltration or addition of milk ingredients) and protein-to-fat ratio; – a more defined cheesemaking process where stages/operations are regulated (e.g., levels of rennet and starter in proportion to casein level; pH at set, drain and milling; firmness at cut; faster curd/whey emptying, especially from larger vats; washing to constant lactose level in the curd); • more thorough mixing of salt and curd, e.g., via the use of curd/salt mixers or tumblers (Sutherland, 2002; ‘General Aspects of Cheese Technology’, Volume 2); • better control over the temperature/humidity conditions of salt storage and salting room to ensure uniform delivery to salt application device; • optimum performance of the curd mill by regular maintenance to ensure uniform chip size. Salt and moisture equilibration during storage

Although salt is fairly well distributed in Cheddar cheese during the initial salting, in contrast to brine and/or dry salted cheese, full equilibrium is approached slowly. Sutherland (1977), who prepared Cheddar cheeses (9.5 kg) with regions of high and low salt,

found that equilibria of salt, moisture, and hence, S/M, were not established after 25 weeks at 13 °C. Samples situated 7.6 cm apart, which showed an initial difference in S/M concentration of 4.27%, still showed a difference of 1.56% at the end of the 25-week period. As zones of high and low salt within commercial cheese blocks (⬃20 kg, ripened at 4–7 °C) are likely to be more widely separated, it was concluded that equilibrium of S/M within such cheeses is unlikely. A similar study by Thomas and Pearce (1981) showed that there was only a very slight shift towards equilibrium of S/M during a 6-month ripening period in Cheddar cheeses prepared with an approximately linear S/M gradient diagonally across the blocks. Equilibration of NaCl in Cheddar cheese intentionally prepared with poor salt distribution was studied by Morris et al. (1985). Salt and moisture analyses were performed on samples taken from 32 selected locations in 20 kg blocks (stored at 10 °C) over a 24-week ripening period (a similar sampling pattern was used on each of six occasions); the results indicated that there was only a slight equilibration of salt over the 24-week period. Hoecker and Hammer (1944), who measured the salt and moisture levels at the surface and centre of individual chips, prized from a block of Cheddar, over a 72 h period after pressing, found that salt and moisture equilibria were established within individual chips 48 and 24 h after hooping, respectively (a comparable study by Morris et al. (1985) gave almost identical results). However, analysis of two 4-month-old cheeses showed significant intra- and inter-block variation in both variables. Hence, while salt and moisture equilibria are attained relatively rapidly within chips because of the short distance over which NaCl molecules have to diffuse from the surface to the centre, the variations throughout the block, as a result of the different quantities of salt absorbed by individual chips, do not disappear during normal ripening. The foregoing observations suggest that movement of salt and water across the chip boundaries, and hence the cheese mass as a whole, even where a concentration gradient exists, is inhibited because: • the contracted protein layers (salting-out of protein at chip surfaces possibly occurs because of the high initial S/M concentration before equilibrium is established) at the surface of individual chips; • and/or microspaces between milled curd (chips) junctions which break the continuity of the interpenetrating gel fluid/moisture (in which salt is dissolved); • lack of a continuous S/M gradient in combination with an impeded diffusion process.


2.5 (a)

(b) NaCl, %(w/w)

Indeed, milling results in the development of a ‘skin’ which has fewer fat globules and hence a denser protein matrix than the interior of the chip (Brooker, 1979). Moreover, light microscopy studies (Rammell, 1960; Kaláb et al., 1982; Lowrie et al., 1982) show that the ‘skin’ at milled curd junctions appears much thicker than that of the curd granules. Observations by Morris (1961) on salt diffusion in Cheddar cheese lend support to the view that the milled curd pieces ‘trap’ absorbed salt; the spread in salt level within individual cheeses at 3 weeks was the same as that observed immediately after hooping. Morris et al. (1985), who also studied salt diffusion in model Cheddar cheese systems, found that equilibrium was established rapidly in cheeses prepared from alternate discs (2 cm thick) of salted and unsalted, unmilled curd, but not in model cheeses prepared from alternate layers (2 cm thick) of salted and unsalted chips. In agreement with the results of a similar experiment by McDowall and Whelan (1933), NaCl diffusion across the interface formed between the salted and unsalted layers of milled curd was very slow (Fig. 23). Morris et al. (1985) suggested that the fragmented structure of Cheddar cheese (due to its construction from chips) may retard salt diffusion but a further experiment, the results of which showed that the diffusion coefficient for NaCl in the moisture phase of a brine-salted block of Cheddar prepared from unsalted chips at 0.15 cm2/day was as expected from its moisture content (see Fig. 20), could not verify this. Thus, it appears that the slow diffusion of NaCl in the moisture phase of Cheddar is due to contracted protein layers between salted chips, which possibly offer a very tight screening effect on the diffusing molecules, and thereby override the effect of low discontinuous gradients in various directions in commercial Cheddar or even at interfaces between salted and unsalted regions where the concentration gradient is high (see ‘Concentration gradient’). The absence of a continuous salt-in-moisture gradient and the fact that salt diffusion in moisture is impeded, as discussed in ‘Mechanism of salt absorption and diffusion in cheese’ and ‘Factors that influence salt diffusion in cheese during salting’, also reduces the tendency for salt and moisture equilibrium, especially between locations in a block which are far apart. The surface of chips in unsalted milled Cheddar would not be as dense as those in dry-salted milled Cheddar due to their higher moisture and fat contents. Hence, the sieve-effect of the matrix on the diffusing molecules would be much lower than in the latter. Indeed, the impedance on the salt molecules penetrating the surface layers of milled Cheddar chips during brining is possibly similar to that encountered on pen-

2.0

1.5

Days 1 3 14 28 56

0.5

4

3

2

1

1

2

3

4

Distance from interface between (a) unsalted and (b) salted curds, cm Figure 23 Distribution of salt (NaCl) throughout a 7.5-cm Cheddar ‘cheese’ prepared from half-salted curd and half-unsalted curd at 1, 3, 14, 28 and 56 days after manufacture. The preparation of the ‘cheese’ involved half-filling aluminium cans with unsalted chips (2 2 2 cm) of Cheddar curd and pressing lightly; filling the remainder of the can with salted (4%, w/w, NaCl) chips of Cheddar curd; pressing the cheese in the can overnight; storing cheeses in cans at 5 °C (from Morris et al., 1985).

etrating the surface of curd granules (no light microscopy studies have been reported on unsalted, milled Cheddar curd).

Effect of Salt on Cheese Composition In the light of studies (O’Connor, 1968, 1971, 1974; Sutherland, 1974; Gilles, 1976; Morris et al., 1985; Kelly et al., 1996) showing that varying salting levels in Cheddar cheese manufacture are associated with large compositional variations in the cheese, the effect of salt on the gross composition of cheese merits brief discussion. Moisture level

The moisture content of cheese curd is influenced primarily by syneresis of the cheese curd during manufacture


roughly half of the whey is released during mellowing and the remainder during pressing. Although other factors, e.g., curd temperature, stirring time after salting, depth of curd, degree of mixing of salt and curd, and duration of mellowing period influence the ratio of whey released during mellowing to that released on pressing, the overall level of whey released was not significantly influenced by these factors (Sutherland, 1974). The moisture content of the cheese was inversely related to salting level (Sutherland, 1974). Geurts et al. (1974b) expressed the relative fluxes of NaCl and H2O during the unidimensional brine-salting of Gouda-type cheese in terms of the flux ratio, p: Wx ⬇ pSx where W and S are the changes (from the unsalted cheese) in the g H2O and g NaCl, respectively, per 100 g cheese solids-not-salt in planes of cheese x cm from the cheese/brine interface; the minus sign indicates the loss of water from the cheese to the brine. Experimental values for W and S are shown in Fig. 24, together with theoretical curves calculated for various values of p. The experimental curve for W approximated the theoretical curve for p 2 (i.e., when the amount of H2O leaving the cheese is twice that of the NaCl entering) but varied from 1.5 at the salt front to 2.34 at the

20

44 4 3 2

16

40

–1 12

36

8

32

4

28

Moisture content of cheese (%)

Salt in moisture (g NaCl/ 100 g H2O)

which is, in turn, influenced by the composition of the cheese milk, i.e., fat, protein and calcium levels, the level of rennet used, firmness of the gel at cutting, and curd treatments during manufacture, i.e., size of curd cut, degree of curd agitation, cooking rate, cooking temperature, rate of acid development, extent of drystirring of curd and depth of curd during cheddaring and size of pressed cheese (Whitehead, 1948; Emmons et al., 1959; Lawrence, 1959a,b; Aiyar and Wallace, 1970; Lelievre, 1977; Geurts, 1978; Marshall, 1982; Walstra et al., 1985; van Dijk and Walstra, 1986; Fenelon and Guinee, 1999; ‘Cheddar Cheese and Related Drysalted Cheese Varieties’, Volume 2). Further syneresis occurs on addition of salt after milling (e.g., for Cheddar and Cheshire), during pressing and brine and/or dry salting. It is generally accepted that there is an inverse relationship between the levels of salt and moisture in cheese. This is most readily observed in brine and/or dry salted moulded cheeses during, or immediately after, salting, where a decreasing salt gradient from surface to the centre is accompanied by a decreasing moisture gradient in the opposite direction (see Fig. 21; McDowall and Whelan, 1933; Beyers and Price, 1937; Geurts et al., 1972, 1974b; Godinho and Fox, 1981b; Guinee and Fox, 1983a,b, 1986a,b; Turhan and Kaletunç, 1992; Messens et al., 1999; Licitra et al., 2000; Melilli et al., 2003). The inverse relationship between salt and moisture levels is also observed in cheeses where the mean salt level is varied by brine- or surface dry-salting for different times (Guinee and Fox, 1986b; Freitas and Malcata, 1996; Kristiansen et al., 1999) or for similar times in brines of different NaCl levels (Guinee and Fox, 1986a; Pappas et al., 1996; Kaya et al., 1999). A similar trend was observed in brineinjected Muenster cheese for which the salt content increased linearly with the number of injections, applied 24 h apart (Pastorino et al., 2003a). O’Connor (1971) found that there is a negative correlation between the salt and moisture concentrations in commercial Scottish Cheddar cheeses. Although there was considerable scatter, the data of Fox (1975) show an inverse correlation between the percentage of moisture and percentage of NaCl in 123 commercial Irish Cheddar cheeses. Direct evidence of this relationship is also apparent from the work of O’Connor (1970, 1973a,b) and Kelly et al. (1996) for Cheddar cheeses from the same batch of curd salted at different levels. An inverse correlation between %, w/w, moisture and %, w/w, NaCl in Cheddar cheese is not surprising since a considerable volume of whey is released from Cheddar curd following salting and during pressing (Sutherland, 1974). The amount of whey released is directly related to the amount of salt added to the curd;

0 0

10

20

30

40

50

60

70

Distance from cheese surface (mm) Figure 24 Moisture (●) and salt () content of a full-cream Gouda cheese after 8 days of brining, as a function of penetration depth; pH 5.64, brine concentration 20.5 g NaCl/100 g H2O, temperature 12.6 °C. Experimental moisture values (●); (1) moisture content calculated from salt content and a flux ratio (g water:g salt) p 2.5; (2) the same, but p varies from 1.7 at the cheese–brine interface to 2.9 in the cheese surface; (3) the same, but p 1; (4) the same, but p 0 (from Geurts et al., 1974b).


brine/cheese interface and was always 1. While a similar trend in p values was observed by Guinee and Fox (1983a) for commercial Romano-type cheese (salted for 9 days in 19.3% NaCl brine), the value of p varied more, i.e., from 3.75 at the rind to 1 in a region between the rind and the salt front. Guinee (1985) concluded that the value of p at a particular location within the region of salt and water movement depends on the concentrations of NaCl and H2O at the location; indeed, this is possibly the reason why p decreases from the rind inwards (Geurts et al., 1974b; Guinee and Fox, 1983a), along which significant variations of salt and moisture occur as a result of salt uptake. Indeed, changes in cheese texture and appearance corresponding to the changes in p which occur in the region of high salt and moisture movement are visible when a brined cheese is cut parallel to the direction of brine movement, during or shortly after brining (Geurts et al., 1974b; Guinee and Fox, 1983a; Bochtler, 1987). In the outermost region (0.3–1.3 cm depending on the duration of brining) bordering the brine, where the S/M level is high, e.g., 12%, w/w, the cheese is hard, brittle, dry and white (indicative of salting-out), whereas further removed from the interface, where percentage of S/M 3% and 10%, the cheese is soft, yellowish and somewhat waxy translucent (indicative of protein hydration and swelling); between the ‘waxy’ layer and the salt front, the cheese had a uniform appearance and resembled the unsalted cheese (Guinee, 1985). It is noteworthy that pockets of free serum in unsalted non-fat Mozzarella provide light scattering surfaces and thereby contribute to opaqueness of the cheese (Paulson et al., 1998); in contrast, the increase in protein hydration in salted Mozzarella led to a significant reduction in the level of free serum and a more translucent appearance. The extent of the outer dry white layer in brine-salted cheeses is augmented by a low cheese moisture and a low brine pH, i.e., 4.6 (Bochtler, 1987); such factors contribute further to protein insolubility. Since the average flux ratio over the region of salt and water movement is 1, there is a net outflow of water which accounts for the commonly observed volume reduction in cheese during brining and/or dry salting. Perhaps unexpectedly, salt uptake during brining is sometimes accompanied by an increase in moisture content in the vicinity of the cheese–brine interface (see ‘Effect of NaCl on casein hydration in model systems and in cheese’ and ‘Concentration of calcium in the brine’), especially in weak brines (10%, w/v, NaCl) without calcium (see Figs 8, 9). Such an effect is associated with the ‘soft rind’ defect and swelling in cheese and is attributed to a salting-in of the protein matrix in low percentage NaCl brines which results in increased

protein solubility. There are several reports on the composition and maintenance of brines and brining times in relation to cheese quality (Jakubowski, 1968; Geurts et al., 1972; de Vries, 1979; Brazhnikov et al., 1986; Blanchard, 1987; Cohen-Maurel, 1987; Chamba, 1988; Schaegis, 1988). Salt content

Higher salt levels in Cheddar cheese usually coincide with increased fat content (O’Connor, 1971, 1974; Thakur et al., 1975), probably due to the greater loss of water than salt uptake during salting. Thus, it is noteworthy that the moisture content of Cheddar is inversely related to the salt content (O’Connor, 1971; Sutherland, 1974; Jordan and Cogan, 1993; Kelly et al., 1996). However, on considering the findings of Breene et al. (1965), the fat content may decrease, especially at high salting levels if the curd temperature at salting exceeds 32 °C. Lactose content and pH

As discussed in ‘Control of Microbial Growth’, the lactose content and pH of cheese are strongly influenced by the level and time of salt application (Fox et al., 1990).

Conclusion Clearly, salt plays a multi-faceted role in cheese ripening with an influence on the physical, chemical and microbiological attributes of the mature cheese. While a considerable amount of information is currently available on many aspects of the significance of salt in cheese and on salt diffusion in cheese curd, many gaps persist, e.g., its effects on individual enzymes, protein–protein interactions (and its consequences in hydrolysis, rheology and functionality), effect on the growth of pathogens, interaction with pH and other hurdles in controlling cheese microbiology.

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Application of Membrane Separation Technology to Cheese Production V.V. Mistry, Dairy Science Department, South Dakota State University, Brookings, USA J.-L. Maubois, Laboratoire de Recherches Laitières, Institut National de la Recherche Agronomique, Rennes Cedex, France

Introduction Membrane processing has revolutionized the dairy industry in many interesting ways and has led to significant new process and product development. This revolution has been in the making for the past 34 years and has encompassed not only ultrafiltration and nanofiltration but, more recently, microfiltration as well. In 2001, more than 450 000 tonnes of cheese were made using ultrafiltration technology. The history of cheesemaking using membranes commenced in the late 1960s with the invention of the MMV process (Maubois et al., 1969; Maubois and Mocquot, 1971, 1975). This process, named after its inventors (Maubois, Mocquot and Vassal), opened up new avenues for significant advances in cheesemaking, including improvements in plant efficiency, increases in cheese yield, development of a continuous process and possibilities for creating new cheese varieties. As a result, numerous plants all over the world, but mainly Europe, now use this process to manufacture a wide range of cheeses (Korolczuk et al., 1986; Maubois, 2002). Since the introduction of the MMV process, commercial applications of membranes in the cheese industry, as well as research efforts aimed at developing new applications and understanding and improving current applications, have expanded all over the world. For instance, it was reported by Kosikowski (1986a) that during the period 1979–1983, a total of 213 scientific papers were published dealing with membrane separations in food processing. Of these, publications dealing with cheese formed the largest category at 25%. Publications dealing with cheese and whey, combined, represented 50% of the total. In a more recent literature search, it was found that more than 1200 publications on the application of membranes in food processing appeared between 1995 and 2002. These data clearly illustrate the magnitude of effort that has been invested in developing and understanding applications of membranes in the food industry. Since 1969, cheese-related applications of membranes have expanded into numerous areas, including the manufacture of fresh, soft, semi-hard and hard cheeses from the milk of cows, goats, ewes or water

buffaloes, production of milk powders with good cheesemaking properties (Maubois et al., 1973), restoration of the rennet coagulation properties of ultrahigh temperature (UHT)-treated milk (Maubois et al., 1972; Ferron-Baumy et al., 1991), on-farm concentration of milk (Maubois, 1979), removal of somatic cells (Le Squeren and Canteri, 1995; Maubois, 2002) and bacteria from cheese milk by microfiltration (Trouvé et al., 1991; Saboya and Maubois, 2000) and casein enrichment of cheese milk by microfiltration (Fauquant et al., 1988; Maubois et al., 2001). These developments were catalysed by improvements in membrane components such as the development of mineral and ceramic membranes, studies on physico-chemical equilibria of UF retentates, characterization of the rheological behaviour of protein-enriched milks, studies on the growth and activity of starter bacteria in liquid pre-cheeses and in the resulting cheeses, and more importantly by the generation of new ideas and the acceptance of new cheesemaking concepts in laboratories and in cheese plants around the world. In this chapter, cheesemaking using UF, reverse osmosis and microfiltration will be discussed as well as other cheese-related applications using these processes. Initially, some membrane terms, membrane design and configuration will be discussed briefly.

Membrane Design and Configuration Membrane technology is a broad term that encompasses several molecular separation processes. Each process requires its own specialized equipment and has its own characteristics that make it suitable for some applications but not for others. Reverse osmosis and ultrafiltration are two of the most commonly used membrane processes in the dairy industry (Glover, 1985; Van der Horst and Hanemaaijer, 1990) but nanofiltration and microfiltration have emerged and have demonstrated tremendous potential for dairy applications for the future (Gregory, 1987; Eriksson, 1988; Fauquant et al., 1988; O’Shea, 1990; Kelly et al., 1992; Saboya and Maubois, 2000; Maubois, 2002).



262 Application of Membrane Separation Technology to Cheese Production

Definitions Ultrafiltration (UF)

Ultrafiltration is a process which selectively separates macromolecules having a molecular weight of 1000–200 000 Da from solvent and dissolved solutes. With cross-flow over a membrane surface at a relatively low pressure (less than 1000 kPa), UF produces from milk a permeate (also called ultrafiltrate) containing water, lactose, soluble minerals, non-protein nitrogen and water-soluble vitamins, and a retentate in which the proteins, fat and colloidal salts are increased in proportion to the amount of permeate removed (Maubois et al., 1969; Maubois and Mocquot, 1971; Glover, 1985).

(Maubois and Brulé, 1982; Cheryan, 1998). The characteristics of each of these configurations are described below. Tubular

Reverse osmosis (sometimes known as hyperfiltration) is a dewatering process which operates at pressures at least five to ten times higher than those used for UF (Glover et al., 1978; Glover, 1985; Dziezak, 1990). Reverse osmosis membranes separate solutes with a molecular weight of approximately 150 Da or less. Hence, fat, proteins, lactose and all undissociated minerals are retained and concentrated by the membrane, and only water and some ionized minerals pass through.

In this configuration, feed flows through a tube, 85–600 cm long and 3–25 mm inside-diameter. The inside wall of the tube is lined with the membrane and the outside consists of support material. Several tubes may be connected in series or in parallel as a bundle and are housed in a stainless steel cartridge. A multichannel tubular geometry was developed in France (Gillot and Garcera, 1986) and in the USA (Renner and Abd El-Salam, 1991) for MF and UF mineral membranes (Fig. 1). Tubular membranes are easy to clean and allow recirculation of liquids with a high level of solids and viscosity (Mahaut et al., 1982; Maubois and Brulé, 1982; Cheryan, 1998). However, they have the lowest surface area-to-volume ratio and therefore require a high feed flow rate and consequently, a high running energy (0.6–1.0 kW/m2) (Maubois and Brulé, 1982). Reverse osmosis operations are conducted at high pressures; hence tubular membranes for RO require additional support material to withstand the high pressures.

Microfiltration (MF)

Hollow fibre

Microfiltration is a process which selectively separates particles with a molecular weight greater than 200 000 Da. According to the membrane pore size, milk materials removed by MF include somatic cells, fat globules, bacteria, casein micelles (Saboya and Maubois, 2000), aggregated whey components (Maubois et al., 2001), -casein (Terré et al., 1987) and -lactoglobulin (Maubois, 1988; Léonil et al., 1997).

Hollow fibre membranes can be thought of as the tubular-type except that they are self-supporting. Hollow fibres also have a much smaller diameter than the tubular-type membranes. The diameter of each fibre ranges from 0.19 to 1.25 mm (Cheryan, 1998). In a see-through casing, 50–3000 such fibres may be bundled together in parallel. Each such unit is referred to as a cartridge. Hollow fibre membranes have a very high surface area-to-volume ratio, providing for very low floor space requirements. As in the tubular design, feed flows through the inside of the fibres, and permeate is collected outside in the casing. A disadvantage with this system is that even if only one fibre fails, the entire cartridge must be replaced. Replacement costs of membranes are, therefore, high. On the other hand, since hollow fibres are self-supporting, operating pressures are low (Maubois and Brulé, 1982). Transmembrane pressure is limited to 170–270 kPa. While this is an advantage in terms of energy consumption (0.2 kW/m2), this configuration may not be suitable for applications requiring high pressures. One of the greatest advantages of hollow fibre membranes, as bundled tubular membranes, is the ability to backflush. This aids in cleaning the membrane as well as in preventing a build-up of fouling material on the surface. Hollow fibres such as those described above are used for UF and MF applications. For RO applications,

Reverse osmosis (RO)

Nanofiltration (NF)

Nanofiltration, also known as loose-RO (Horton, 1986), falls between RO and UF. It separates low molecular weight compounds (200 and 1000 Da) from larger molecules. Small ionized molecules, such as dissolved mineral salts, are removed, at a rate inversely proportional to their valency, along with water, whereas other materials such as lactose, proteins and fat are retained, making it suitable for desalting cheese whey (Daufin et al., 1998a), for recovering of water generated during thermal concentration of milk or whey or for recycling cleaning solutions (Daufin et al., 1998b). Membrane configuration

Four basic configurations are currently available for UF, RO, MF and NF applications: (1) tubular, (2) hollow fibre, (3) plate and frame and (4) spiral-wound

Application of Membrane Separation Technology to Cheese Production 263

Figure 1 An arrangement of multi-channel geometry ceramic membranes (courtesy of GEA filtration, Hudson, WI, USA). (See Colour plate 1.)

even smaller fibres, known as hollow fine fibres, are used (Cheryan, 1998). In these fibres, feed flows from the outside of the fibre to the inside. Plate and frame

This configuration consists of a stack of plates and flat sheet membranes, much like a filter press arrangement (Fig. 2). The flat sheet membrane and its support are sandwiched together in large numbers to form a module. Feed flows parallel to the membrane surface, and permeate is chanelled out of the module. Plate and

frame configurations are available in horizontal as well as vertical designs. The surface area-to-volume ratio is between hollow fibre and tubular designs. The required pumping energy is around 0.5–0.7 kW/m2 (Maubois and Brulé, 1982). Spiral-wound

This configuration is the most widely used in the dairy and food industries and is also the most inexpensive (Fig. 3). Spiral-wound membranes consist of two flat sheet membranes along with spacers wrapped around

Figure 2 Plate and frame UF system (courtesy of GEA filtration, Hudson, WI, USA). (See Colour plate 2.)


Figure 3 Spiral-wound UF membranes (courtesy of GEA filtration, Hudson, WI, USA). (See Colour plate 3.)

a perforated permeate-collecting tube (Cheryan, 1998). As feed passes over the membrane surface, permeate spirals its way to the centre of the tube. Spacers are included in the assembly to promote turbulence, thereby minimizing fouling, but sometimes cleaning and sanitizing difficulties are experienced, especially when highly viscous retentates are recirculated. Spiralwound membranes are available for UF, RO, MF and NF applications. The nature of the membrane support and the general design permit operation at high transmembrane pressures without damaging the membrane. Vibrating membrane system

Developed by the Pall Corporation (Boston, MA), this is a relatively new concept in which vibration energy is used to reduce the thickness of the fouling layer and consequently to improve the flux rate. This is accomplished by vibrating a disc filter stack at 50–60 Hz on the vertical axis to generate shear rates of 100 000– 150 000 s 1 on the membrane surface. Membranes are bonded to both sides of stainless steel discs, which are then arranged in a stack to obtain a total membrane surface area of up to 40 m2 (Fig. 4). With this system, it has been possible to separate milk proteins effectively. While commercial applications have not been

Figure 4 Vibrating membrane system (courtesy of Pall Corporation, Portsmouth, UK). (See Colour plate 4.)


developed yet, there is potential for the future (Hurwitz and Brantley, 2000). Good quality membrane material is critical for the proper operation of UF, RO, MF or NF plants. Cellulose acetate was the most common material for UF and RO membranes but these have now been almost completely replaced by polysulphone membranes, especially for UF applications. Numerous other materials have been assessed, e.g., polyamide, polyimide, polyvinylidene fluoride, etc. Mineral membranes, specifically zirconium oxide, or titanium oxide supported by carbon or by alumina and ceramic membranes are now being used increasingly for UF, MF and even NF. These materials have high mechanical strength and tolerate wider pH and temperature ranges than polymeric membranes. They are more expensive but have a substantially longer life than organic membranes (at least 5 years compared to 18 months). The above overview is concerned only with membrane processes used in cheese applications. Other membrane processes, such as dialysis and electrodialysis and details of UF, RO, MF and NF, such as flux rates, thermodynamics of operation, fouling and concentration polarization have been discussed in depth elsewhere (Cheryan, 1998; Meireles-Masbernat et al., 1998) and will not be addressed here.

Membrane Applications in Cheesemaking Ultrafiltration is the most widely used membrane process for cheesemaking and is fairly well-advanced. Microfiltration techniques for the removal of bacteria, separation of milk fat globules and enrichment of micellar casein have been developed and have already entered industrial operations. To successfully make cheese by UF or MF, specific properties of the protein-enriched products must be well understood because they strongly determine the quality of the end products, as well as economy of the use of the membrane technology. Properties of UF retentates Buffering capacity

If milk is ultrafiltrated at its normal pH (6.6–6.8), mineral salts (Ca, Mg, P) bound to casein micelles are concentrated in the same proportion as proteins. This results in an increase in the buffering capacity of UF retentates which will consequently modify the basic parameters of the cheesemaking process – acidification kinetics by lactic acid bacteria, ultimate pH value, rennet coagulation kinetics and rheological characteristics of the curd, activity of ripening enzymes, lysis of mesophilic lactic acid bacteria during ripening (Saboya et al., 2001), growth and rate of survival of spoilage

flora (Rash and Kosikowski, 1982) and water-holding capacity of the cheese mass during ripening. A similar increase in buffering capacity is also observed when milk is specifically enriched in micellar casein by the use of 0.1 m MF membranes. According to the volumetric concentration factor (F) (ratio of the volumes of milk and retentate), higher production of lactic acid by lactic starter bacteria is required to obtain optimum pH in cheese, usually 5.2 in hard cheeses and 4.6 in soft and fresh cheeses. For the latter category, the increase in required lactic acid production was quantified by Brulé et al. (1974) and was expressed as: QL 4.4 F 1.5 where QL is g of lactic acid per kg of pH 6.7 UF retentate. Consequently, for most cheese varieties, use of pH 6.7 retentates results in acid-tasting products (Maubois, 1979). On the other hand, a large quantity of calcium salts is released into the aqueous phase of cheese curd during acidification. Ionic strength is strongly increased and casein micelle aggregation is modified. Cheese texture is crumbly or sandy (Brulé et al., 1975), and spreadability and stretching properties are poor (Green and Grandison, 1987). The buffering effect of pH 6.7 UF retentates leads to higher numbers of lactic starter bacteria in curd and resulting cheese than in non-UF curd and cheese (Mistry and Kosikowski, 1985; Goudédranche et al., 1986). While this may result in bitterness in some cheeses, such as Quarg (Mortensen, 1985), it has been used to advantage in the development of a new bulk starter (Mistry and Kosikowski, 1986a). This starter is manufactured by fermenting a whole or skim milk retentate containing 12% protein with a mesophilic lactic culture at 22 °C for 12–15 h (Fig. 5). It has a built-in internal pH control mechanism due to the buffering capacity of the UF retentate, which maintains the pH of the starter steady at 5–5.2. It, therefore, has a greater activity than traditional bulk starters made from pasteurized milk and maintains its activity for 10–12 h at room temperature. The UF retentate starter also has a high protein content which contributes to increased cheese yield (Mistry and Kosikowski, 1986b; Mistry, 1990), making it suitable for either traditional or UF cheesemaking. The increased buffering capacity of pH 6.7 UF milk could also offer a favourable environment for the growth and survival of certain bacteria such as enteropathogenic E. coli (Rash and Kosikowski, 1982). This underscores the importance of adjusting the mineral content of UF retentates to avoid the unfavourable consequences in cheesemaking due to the increase in buffering capacity of pH 6.7 UF milk. This aspect was


(Brulé et al., 1974). An increase of ionic strength also lowers the isoelectric pH of casein, which may offer an increased security margin to the cheesemaker for handling acidified UF retentate. Addition of NaCl to milk or reduction of the pH by acidification reduces UF flux because of increased membrane fouling but it is obvious that any cheesemaker will prefer to have satisfactory cheeses even at the expense of reduced performance of UF equipment rather than defective cheeses resulting from a process involving the highest UF flux. It must also be remembered that milk having a pH lower than 5.0, when ultrafiltered, leads to a higher UF flux than that observed at pH 6.7 because of the weak texture of the polarization layer at the isoelectric pH of casein (Mahaut et al., 1982). Rheological behaviour of UF retentates

Figure 5 Flow diagram for the production of retentate starter (derived from Mistry and Kosikowski, 1986a).

emphasized by Brulé et al. (1974), who suggested several suitable ways for adjusting the mineral salts content of UF retentates, which is specific for each cheese variety. The first method involves a reduction in milk pH before or during ultrafiltration by the growth of a lactic starter or by any approved acidifying agent (glucono--lacatone or organic acids in some countries). Acidification leads to solubilization of colloidal calcium and magnesium phosphate salts, which pass into the permeate. Reduction of milk pH from 6.6 to 6.0 and 5.6 increases the Ca content of UF permeate from 0.38 to 0.50 and 0.80 g per kg, respectively. Consequently, a 5X UF retentate obtained at pH 5.6 has a Ca content 2.6 times that in milk instead of 3.8 times for the 5X UF retentate prepared at pH 6.6 (Brulé et al., 1974). The second method, which eventually can be combined with the first one, is the addition of NaCl (0.5–0.9%, w/w) to UF retentate during or after ultrafiltration. The increase of ionic strength resulting from NaCl addition reduces the ionization of casein phosphoseryl groups and consequently leads to solubilization of colloidal calcium in the permeate or in the aqueous phase of UF retentate (up to 15–18%, w/w, depending on the pH and amount of NaCl added)

Milk can be considered as a Newtonian liquid, while UF retentates behave differently. The higher the protein content and/or the lower the temperature, the more pseudoplastic is their behaviour (Culioli et al., 1974). Such a rheological behaviour must be taken into consideration in the design and in the operating parameters of UF equipment (for example, in the restarting procedure after an electrical failure to avoid a hydraulic ram). The viscosity of UF retentates increases markedly with an increase in their protein content. At 30 °C, at a shear rate of 437.4 s 1, the observed viscosity is 45 cP at a protein content of 19.6%, w/w, and 370 cP at a protein content of 20.6%, w/w (Culioli et al., 1974; Goudédranche et al., 1980). The manufacture of semihard or related cheese from these highly viscous UF retentates requires removal of all dissolved gases which are entrapped in the liquid and the use of special mixing devices (static and dynamic) to enable thorough blending of rennet and lactic starters (Maubois, 1987). If dissolved gases are not removed by application of a vacuum, a spongy curd is obtained and the appearance and the taste of the cheese are poor. If rennet is not mixed satisfactorily, the resulting curd will be flaky due to localized coagulation. Rennet coagulation

If the same amount of rennet is added to equal volumes of milk or UF retentate, the rennet-clotting time is not affected by the increase in protein content (% P) but the time from clotting to cutting is reduced (Maubois and Mocquot, 1971; Garnot et al., 1982; Lucisano et al., 1985; Garnot, 1988; Maubois, 1989). This is the net result of numerous phenomena – there is an increase in the velocity of the enzyme reaction as the protein content is increased (Garnot, 1988) but


the degree of proteolysis at gelation decreases as % P increases. At pH 6.6 and at the normal casein content of milk, coagulation occurs when 80–90% of the -casein has been hydrolysed. However, in a 4X UF retentate, hydrolysis of only 50% is necessary for curd formation (Dalgleish, 1980). Because the secondary phase of rennet action is a diffusion-controlled process, an increase in protein content leads to a sharp increase in the rate of aggregation (Garnot, 1988). The final firmness of rennet-induced coagulum is generally directly related to the casein content (Maubois and Mocquot, 1971, 1975; Maubois et al., 1972, 1973; Korolczuk et al., 1986; Kosikowski, 1986b; Ferron-Baumy et al., 1991). This is of particular importance when low-concentrated retentates (LCR) are used to make cheese because traditional equipment is employed. This would require stronger knives and agitators to handle the firmer, stronger coagulum (Kosikowski et al., 1985b). If the primary phase of -casein hydrolysis by rennet is slightly affected in UHT milk (Ferron-Baumy et al., 1991), coagulation does not occur owing to the increased electronegativity of the casein micelles resulting from the covalent binding of -lactoglobulin with -casein (Dalgleish, 1990). Erdem (2000) has suggested that a decrease in the surface hydrophobicity of proteins due to UF is the cause of the unique renneting properties of UF milk. Increasing the protein content by UF before or after UHT treatment restores curdforming ability (Maubois et al., 1972). According to Ferron-Baumy et al. (1991), such a phenomenon would result from lowering the zeta potential of casein micelles on UF. This hypothesis, which must be confirmed by direct observations, agrees with the fact that UF retentates coagulate at a lower degree of -casein hydrolysis than normal milk. Applications of UF in cheesemaking

Cheesemaking using UF can be divided into three main categories: 1. protein-standardized milk; 2. intermediate or medium concentrated retentates; 3. liquid pre-cheeses, i.e., UF retentates having the composition of the cheese variety to be made. Protein-standardized milk

The protein content of milk collected by dairy plants varies according to season due to multiple factors – stage of lactation, weather, feeding and breed of lactating cows. Such a variation in the composition of the incoming milk requires adjustment of processing parameters by cheesemakers. Moreover, at a low protein content the rennet-induced coagulum is weak and leads to relatively high losses of caseins as fines in

whey. A slight increase in the protein content by UF eliminates these difficulties. In many cheese plants, generally those using highly mechanized equipment, the protein content of cheese milk is increased to 3.7–4.5% throughout the year (Korolczuk et al., 1986; Mietton, 1990). Protein-standardized milk is used in Europe for the manufacture of Camembert cheese (Korolczuk et al., 1986) using an Alpma coagulator or similar equipment. It is also used for semi-hard and hard, cooked cheese. In the USA, the acronym, LCR (lowconcentrated retentates), was proposed (Kosikowski, 1986b) to characterize this use of UF in cheesemaking. Several pilot plant and industrial studies have reported on the use of the LCR concept for Cheddar and other hard cheeses using either direct concentration or supplementation (Chapman et al., 1974; Kealey and Kosikowski, 1985; Kosikowski et al., 1985b; Sharma et al., 1989). These studies concluded that the optimum degree of concentration for making these cheese varieties is between 1.7:1 and 1.8:1. In the LCR or protein standardization process, cheese is made using conventional equipment and a cheese plant can easily adapt this application of UF. Manufacturers of UF equipment have now proposed specially designed ultrafiltration systems that are equipped with in-line protein and fat sensors (Friis, 1985). This will make it possible to determine the fat and protein content of the incoming milk and to standardize the cheese milk for fat and protein simultaneously. The cost of UF for this application is balanced by a slight increase in manufacturing efficiency due to increased production of cheese per vat per day, reduced rennet requirements, improved quality of cheese (Kosikowski, 1986b) and a slight increase in yield (generally less than 1% for most varieties). This increase in yield results from reduced losses of fat and casein particles in whey and better retention of whey proteins in the aqueous phase of cheese. The increase in retention of whey proteins with LCR is relatively small compared to the MMV process. The effect of this on cheese yield can be estimated according to the formula proposed by Vandeweghe (2000). Another advantage is the possible added value of the resulting whey, which has an increased content of protein/total solids. However, it must be said that in industrial situations these advantages are minor. Therefore, it is somewhat surprising that a large number of UF plants have been installed for protein standardization in Europe. An indirect but important advantage of the LCR/protein standardization concept is the utilization of permeate to reduce the protein content of fluid UHT milk to the minimum required by law – 2.8%, w/w, in most EC countries but 3.15%, w/w, in France. Such a practice, which is forbidden in the EU since


1997, had led to considerable profit for many UHT milk processors. The payback of UF investment was less than six months. While it is possible to detect dilution of milk with water, it is impossible to detect dilution with permeate. A number of cheese varieties have been made using the LCR concept. These include Cottage (Mattews et al., 1976; Athar et al., 1983; Kosikowski et al., 1985a; Kealey and Kosikowski, 1986b; Zall and Chen, 1986), Mozzarella (Fernandez and Kosikowski, 1986a,b) and Saint Paulin (Abrahamsen, 1986). Industrial and pilotplant trials with Cottage cheese indicate that a concentration ratio in the range of 1.2:1.7 is optimum for yield, flavour and body characteristics. Above these levels, the texture becomes firm and the cheese has a flat flavour. Thermization of milk (74 °C for 10 s) prior to UF gives the highest increase in Cottage cheese yield compared with thermization after UF or no thermization (Zall and Chen, 1986). Good-quality low-moisture Mozzarella cheese with excellent stretching and melting properties can be produced from low-concentration retentates at 1.75:1.0 ratios (Fernandez and Kosikowski, 1986a,b). Cheese from higher concentrates was firmer and had greater fat losses in the brine. Using LCR, it is possible to produce both starter-acidified and directly acidified Mozzarella. The LCR concept has also been applied to Brick and Colby cheese (Bush et al., 1983). LCR Brick cheese had a lower pH and higher fat losses in whey than in controls. The cheese was firmer and more mealy and scored lower in overall preference than control cheese. For Colby, the use of UF made it possible to eliminate the curdwashing step. Sensory scores were similar to those of controls. Reduction in cooking time and rennet usage was reported. In studies on Edam cheese by Pahkala et al. (1985), 2:1, 4:1 and 6:1 retentates were used. LCR (2:1 concentration) produced the best cheese with the fewest defects. In this cheese, the rate of proteolysis of s1-casein was similar to that in control cheese but that of -casein was slower. With UF Danbo cheese, made from 2:1 diafiltered UF milk, a slight increase in yield, a 50% reduction in rennet requirements, and a 40% increase in the cheesemaking capacity of vats was possible (Qvist et al., 1985). The LCR concept for cheese appears to have been well-accepted commercially for cheeses such as Cheddar, mainly because of increased yield without the need for additional equipment. Its application in correcting the effects of seasonal variation in milk composition on Cheddar cheesemaking has been studied (Broome et al., 1998a,b). It was suggested that when milk was ultrafiltered up to 4.5%, w/w, protein, the moisture content of Cheddar cheese was optimal

and the yield increased. A recent study demonstrated the possibility to further enhance the impact of UF on cheesemaking by homogenizing the cream (Oommen et al., 2000). Cream containing 35%, w/w, fat was homogenized and mixed with skim milk and UF milk to obtain 6%, w/w, protein for Cheddar cheesemaking. Fat recovery in the cheese was 96.8% compared with 94.7% for the control cheese. It was possible to also improve the meltability of cheese as well as its texture. The LCR concept has been used to improve the quality of low-fat cheeses. Ultrafiltered sweet buttermilk has been used to manufacture low-fat Cheddar (Mistry et al., 1996; Turcot et al., 2002), Mozzarella (Poduval and Mistry, 1999) and Processed cheese (Raval and Mistry, 1999). The UF of buttermilk allows for the selective concentration of its phospholipid content, which may play a role in developing the texture of cheese. Medium or intermediate concentrated retentates

Numerous cheese varieties, ranging from soft to hard, have been made from medium-concentrated retentates. In this approach, cheese is made by using specially designed equipment able to cut and handle the firm gel resulting from the coagulation of 2:1–5:1 concentrated retentates, eventually diafiltrated with pure, salted or acidified water. The main application, which is in industrial operation, is the manufacture of structured Feta cheese (Hansen, 1985). APV-sirocurd process. An Australian dairy research

team, in collaboration with the APV firm, developed this process. Commercial plants were installed in Australia and USA but the process has been discontinued for technical and economic reasons. The details of the process have been discussed by Mistry and Maubois (1993). This process had the potential to increase cheese yield by 6–8% under continuous and automated conditions. This approach in making Feta-like cheese was developed in Denmark in response to consumer demand from many Mediterranean countries where people desired cheeses having an appearance and texture (presence of mechanical holes) similar to those of traditional products, characteristics they did not find in UF Feta-like cheese made from liquid pre-cheeses (Mortensen, 1985). Pasteurized, fat-standardized milk, generally homogenized at 18 MPa and 60 °C, is ultrafiltered at 50 °C. The final concentrate contains 28.5%, w/w, TS, which corresponds to a concentration factor of 3:1. Lipase, starter culture or glucono--lactone are added to the UF retentate, previously homogenized at 5 MPa at 65 °C, heat-treated to 80 °C for 60 s and cooled to 34 °C.

Structured Feta-like cheese.


After a short storage period in a tank, the UF retentate is pumped to specially designed Alfa-Laval Alcurd or Pasilac equipment. Rennet is added in-line. In both types of equipment, rennet is mixed thoroughly and the UF retentate coagulates in tubes. The resulting coagulum is removed from the tubes, cut into cubes, moulded and drained (16–24 h at 10–14 °C) until the pH has decreased to 4.8 (Skovhauge, 1987). The product of this process is virtually indistinguishable from the traditional product. A yield increase of about 14% on a solids basis is claimed over the traditional process (Mortensen, 1985), a far smaller value than that obtained with the process using liquid pre-cheese (30%). Such a difference explains why only a few plants produce structured Feta-like cheese. Other cheeses produced from medium concentration retentates. Several experiments on the use of UF

retentates of up to 5:1 concentration have been reported for making Havarti, a semi-soft cheese of Danish origin, containing approximately 26%, w/w, fat and 56%, w/w, solids (Bundgaard et al., 1972; Qvist et al., 1986, 1987; Qvist, 1987). Cheese milk used in these experiments was not pre-acidified, and diafiltration was not performed with acidified water. Consequently, the buffering capacity of the UF retentates was high and it was reported that more starter was required than with the traditional process or the use of specially selected cultures (Skovhauge, 1987). However, the taste and the flavour of UF cheeses were similar to the traditional product. The texture was, nevertheless, softer and the melting properties poorer. A 10% saving in the cost of skim milk cheese manufacture was claimed, resulting in a net profit of US$42 000 per year for a production of 600 tonnes of cheese (Skovhauge, 1987). In experiments with Gouda cheese (Spangler et al., 1989, 1990), whole milk was first ultrafiltrated to 3.3:1 and then diafiltered to 3.6:1–5:1 concentration. Gouda cheese produced from 5:1 retentate was similar in moisture, hardness and proteolysis to controls produced from non-UF milk. A savings of 33% in the cost of rennet was reported. Flavour development in UF Gouda cheese could be accelerated by using a combination of liposome-entrapped enzyme and freezeshocked Lactobacillus helveticus cells. Attempts have also been made to manufacture Blue cheese from UF milk (Mahaut and Maubois, 1978; Maubois, 1979; Abd El-Salam et al., 1988). According to French studies (Mahaut and Maubois, 1978), the organoleptic qualities of cheese made from UF retentates with a protein content ranging from 3.2 to 10% and treated in traditional cheese vats were similar to reference cheese. Above 12%, w/w, protein, modifications of cheesemaking parameters and new cutting

and handling equipment were required to produce satisfactory Blue cheese. That was successfully accomplished in 1996 by the French Guilloteau Society which produced the award-winning ‘La Roche’ variety. Egyptian workers describe the use of recombined ultrafiltered milk for making Blue cheese (Abd El-Salam et al., 1988; Abdou et al., 1988; Dawood et al., 1988). General considerations on the use of intermediate UF retentates. Benefits accruing from the use of

intermediate UF retentates for making any cheese variety must be substantial enough to justify substitution of traditional cheesemaking technology. Moreover, the organoleptic quality must be acceptable to the consumer. Investments involve not only UF equipment, as in the LCR concept, but also additional equipment, such as curd makers. Increased cheese yield is strongly related to the volume concentration factor (F) and to the difference between the composition of the UF retentate and the final cheese (Jacobsen, 1985). The saving of skim milk increases logarithmically as the difference becomes smaller but UF-operating costs also increase logarithmically with F. The economic study to be made by cheese plants to assess investment must also take into consideration the potential value of the two by-products obtained – drained whey, which contains more protein and fat than normal whey, and permeate; each is of interest to a different downstream industrial network. Minor benefits also accrue from reduced rennet consumption and reduced requirement in volume and floor space. Liquid pre-cheeses (LPC concept)

In this approach, cheese milk is concentrated by UF to the composition of the drained curd being made before addition of rennet. There is very little whey drainage, and there is no need for cheese vats (Maubois et al., 1969; Maubois and Mocquot, 1971). This principle was first applied to Camembert cheese (Maubois et al., 1969; Maubois, 1979) but many applications have been developed successfully for the manufacture of other cheese varieties, ranging from ‘fromages frais’ or Quarg to semi-hard cheeses, such as Saint Paulin. Fresh unripened cheeses. In early attempts to apply

UF for the manufacture of cheese varieties belonging to this category, milk was preconcentrated prior to starter and rennet addition. The cheese produced had a highly acid and metallic taste, frequently associated with bitterness. These defects were attributable to the high mineral content of the curd and consequently its high buffering capacity (Brulé et al., 1975; Mahaut et al., 1982; Lawrence, 1987). Some reduction of acid flavour was observed when pre-acidified (pH 6.0) milk


was ultrafiltered or when milk was concentrated to a higher degree than necessary and subsequently diluted with water (Brulé et al., 1975). Introduction of new membranes, such as mineral membranes and specially designed membrane supports that permit the UF of high-viscosity products (Maubois, 1979; Herbertz, 1984a), has made it possible to solve this organoleptic defect completely by using the process initially proposed by Stenne (1973), i.e., first fermenting the milk to pH 4.6 with a conventional mesophilic culture, adding rennet and then ultrafiltering to remove lactose and mineral salts but retaining whey proteins. A high initial flux rate and a decrease in flux rate with concentration were observed when pH 4.6 milk was ultrafiltered (Mahaut et al., 1982), both phenomena being attributable to the highly porous structure of the polarization layer (Maubois, 1979). Because of the relatively high protein content (12%), this use of UF was successfully developed for the manufacture of Quarg, a German cheese variety (Baurle et al., 1984; Anon, 1984b,c; Herbertz, 1984b, 1985; Patel et al., 1986; Koch International GmbH, 1987). For application to similar French cheeses, which contain much less protein, it was necessary to develop specially designed UF equipment, which minimized the mechanical shear stress applied to the retentate (Fig. 6). The viscosity of pH 4.6-acidified curds decreases markedly with the increase of mechanical treatment imposed during centrifugal drainage or UF concentration (Mahaut, 1990). Thanks to UF technology, it was possible to make fresh unripened cheeses from buttermilk. Interest in using this new starting material, which has poor ren-

neting characteristics, because of the high heat treatment generally applied to cream before churning, is due to its high content in phospholipids, which give a very unctuous texture. Many other fresh unripened cheese varieties are now made according to the LPC approach or the MMV process. Some examples include Ricotta (Maubois and Kosikowski, 1978; Skovhauge, 1988), Cream cheese (Covacevich and Kosikowski, 1977; Resmini et al., 1984; Dos Santos Neves and Ducruet, 1988) and Mascarpone (Resmini et al., 1984; Sordi, 1984). The manufacture of Ricotta presents special problems because of the complexity of precipitation and requirements for suitable texture and flavour (Maubois and Kosikowski, 1978; Kosikowski and Mistry, 1997). In one UF process (Maubois and Kosikowski, 1978), whole milk is acidified to pH 5.9 with lactic starter, acid whey powder or food-grade acid and ultrafiltered at 55–60 °C to 12%, w/w, protein. The acidified liquid pre-cheese is heated in a scraped-surface heat exchanger at 80 °C and filled directly into packages. In another process (Skovhauge, 1988), milk or whey is pasteurized, acidified to pH 6.3 and ultrafiltered to 30%, w/w, solids at 50 °C. The retentate is heated to 90 °C at a pressure of 1–1.5 bar, following which the pressure is reduced to atmospheric to aid curd formation. The product is cooled to 70 °C, packaged and chilled to 10 °C. No whey drainage occurs. Another interesting application of UF for the manufacture of cheese varieties belonging to this high-moisture category is the procedure developed for the production of ‘Faisselles’ or ‘country cheese’. Traditionally,

Figure 6 Commercial UF system for the production of fresh cheese from pH 4.6 milk (courtesy of TIA, Bollene, France). (See Colour plate 5.)


this cheese is made from whole milk inoculated at 18–22 °C with a mesophilic starter and rennet. After overnight cooling to 12–16 °C, pieces of coagulum are scooped by hand into moulds for slight whey drainage. The drained curd is then removed, always by hand, from the moulds and gently laid down in the retail cups. This production was disappearing because of the increasing cost of labour. Use of UF has allowed the draining step to be eliminated and consequently labour requirements are reduced substantially. The production of this cheese variety has now reached its former maximum level (Maubois, 1985). Soft cheese. Camembert, a French surface-moulded cheese variety, was the first cheese to be made according to the MMV principle (Maubois et al., 1969; Maubois and Mocquot, 1971). Several recipes were proposed (Maubois and Mocquot, 1975; Maubois, 1979, 1989) to optimize the use of UF with industrial cheesemaking constraints (24-h production of UF retentate with moulding for 16 h) and to obtain the very delicate equilibrium in calcium salts in the curd required to obtain texture and flavour similar to traditional Camembert cheese. The procedure used with the continuous moulding and demoulding equipment, ‘Camatic’, developed by Alfa-Laval (Hansen, 1981; Gutter, 1984; Korolczuk et al., 1986), is the following – HTST-pasteurized milk is ultrafiltered at 50 °C to a pre-cheese concentration of 5:1 and a total solids content of 35%, w/w. The resulting pre-cheese is cooled to 30 °C, and 2%, w/w, mesophilic lactic starter and 0.75%, w/w, NaCl are added. Then, the mixture is allowed to acidify to pH 5.5 and is automatically filled into forms with online inoculation with rennet. Curd wheels develop rapidly and are continuously and gently moved in the Camatic equipment for 45 min. After being inverted once, a continuous electric current is applied to each cheese between the air-exposed surface in contact with a carbon electrode and the stainless steel cup holding the cheese. Limited electrolysis of whey occurs and the use of an air injector allows perfect demoulding of the wheels onto cheese trays. Then, the fresh Camembert cheeses are brined for about 30 min, removed, sprayed with Penicillium camemberti spores and held for 12 days at 11–12 °C to permit development of the white mould covering. A yield increase between 12 and 15% is obtained. Several units of Camatic equipment have been sold, mainly in Germany. In France, UF Camembert cheeses have encountered ‘psycho-commercial’ difficulties. The organoleptic qualities of UF Camembert were indistinguishable from those of traditional cheese but the bulk density of the UF cheese paste is much higher than

that of traditional cheese because there are no mechanical openings. Since French consumers are accustomed to buying Camembert cheese by the piece and not by weight, they are conscious of the volume of this cheese variety and get the impression that they receive less cheese for their money when buying UF Camembert (Qvist et al., 1985; Maubois, 1987). The commercial failure of UF Camembert has led French cheesemakers to develop new varieties, most of which have achieved a very rapidly growing production. For example, ‘Pavé d’Affinois’ was developed in 1982 and reached a production of 5000 tonnes in 2000 (Fig. 7). This cheese is made from 4:1 UF whole milk retentate fermented with a thermophilic lactic starter. Rennet is added and the mixture is poured into rectangular plastic trays that are 5 cm high. The trays are set in an incubator at 43 °C for 6 h to allow acidification and coagulation to occur. After cooling to room temperature, the cheese slabs are removed from the trays and cut with an automatic dividing knife to 96 pieces, each having the size and form of a small rectangular paving stone (approximately 7 5 5 cm). The resulting fresh cheeses are ripened for 10 days, as for Camembert (Korolczuk et al., 1986). Several other soft cheese varieties (bacterial and mould surfaceripened, Blue) have been developed according to the same principles and have achieved commercial success (Maubois, 2002). The greatest success worldwide of the MMV process is unquestionably the manufacture of Feta-like cheese (Fig. 8) (Hansen, 1980, 1984; Mortensen, 1985; Lawrence, 1987). Until the EU regulations changed market dynamics in 1997, Feta accounted for 35% of all cheese produced in Denmark and more than 90% of it was produced by UF. Most of this cheese was manufactured for the Iran market but UF Feta cheese has been produced in Iran itself for several years (Ziabary and Hoffmann, 2001). The LPC concept for making Feta has made feasible an old dream of cheesemakers – to make the cheese in its retail package. Yield increases of 30% were reported, higher than could be expected from the retention of whey proteins in the cheese (22% at most). The difference must be related not only to the total elimination of curd particle and fat losses arising from the coagulation and curd cutting inside the retail tins but also to the retention in the curd of all the -caseinomacropeptide (4%, w/w, of the casein content of the LPC). A similar concept was applied to the manufacture of Domiati, an Egyptian cheese variety – 5:1 UF whole milk retentate was homogenized, 5%, w/w, NaCl, 2%, w/w, lactic starter and lipase–rennet mixture were added prior to pouring into 18-kg tins (Al Khamy, 1988) or Tetra Pak packages.


Figure 7 A selection of cheeses made by UF using the liquid pre-cheese concept. (See Colour plate 6.)

UF processes for Mozzarella have been reported since the mid-1970s (Covacevich and Kosikowski, 1978). In one of the first attempts to use the MMV principle for Mozzarella cheese (Covacevich and Kosikowski, 1978), retentates were adjusted to 33.6% solids with freeze-dried retentate, and then blended with 69% fat cream to 45–50% solids. This mixture was fermented, and rennet curd was prepared. It was concluded that diafiltration is required to produce good flavour, stretch and melting characteristics. In a subse-

quent continuous process for low-moisture Mozzarella (Resmini et al., 1984; Kosikowski, 1986b), pasteurized skim milk was pre-acidified to pH 6.0 to reduce its calcium content. It was then ultrafiltered/diafiltered at 54 °C to approximately 10:1 concentration. The retentate was blended with cream to obtain 20%, w/w, fat, 28%, w/w, protein, and then dosed with starter and rennet. Coagulation occurred continuously, followed by conventional stretching and moulding at pH 5.2. Some problems encountered with this process included poor

Figure 8 A UF plant for producing Feta cheese by the MMV process (courtesy of TIA, Bollene, France). (See Colour plate 7.)


stretching characteristics of the cheese. Lack of proper stretching of UF Mozzarella cheese may be attributed to the incorporation of large quantities of whey proteins and their denaturation during cooking, an improper calcium:protein ratio (Hansen, 1987; Lawrence, 1987), inadequate removal of dissolved gases and incomplete blending of rennet (Maubois, 1987). Semi-hard cheeses. Saint Paulin is a bacterial surface-ripened, semi-hard cheese of French origin and contains approximately 47%, w/w, moisture and 2.5%, w/w, salt (Kosikowski and Mistry 1997). In the manufacture of this cheese by UF, it is necessary to obtain at least 21%, w/w, protein (45%, w/w, solids) (Maubois, 1979). This is more easily attainable with mineral membranes (Goudédranche et al., 1980) than with polymeric membranes. Procedures have been developed for both brine-salted and dry-salted cheese. Increases in cheese yield up to 19% may be realized, with 85% savings in rennet (Goudédranche et al., 1980). Acid flavour and slow ripening of UF Saint Paulin cheese can be controlled by reducing the lactose and ash content of the retentate to less than 1.9% each (Delbeke, 1987). The flavour of UF Saint Paulin may be improved by adding lysozyme at 0.5–1.0 g/l (Goudédranche et al., 1986), which increases the proteolytic count and reduces the mesophilic count of the cheese or by adding broken lactococci cells (Saboya et al., 2001). A new cheese variety, with propionic bacteria fermentation, has been studied in France (Ducruet et al., 1981; Maubois, 1987). The procedure includes the preparation of a 7.5:1 retentate in two steps – first with continuous diafiltration at 3.0:1 concentration regulated by a refractometric sensor inserted in the permeate line, followed by heat treatment at 4.0:1 concentration, and second, with continuous ultrafiltration to 7.5:1 using specially designed equipment (short cartridges and positive displacement recirculation pumps) for handling highly viscous products. Original mixing devices for starters and rennet addition were used. The moulding equipment includes a vacuum step for removing dissolved gases and a special injection head for pouring renneted LPC into two-part spherical or cylindrical moulds. Other applications of the LPC concept. Jolly and Kosikowski (1975) pioneered an original application of UF in processed cheesemaking by proposing the substitution of UF skim milk retentate previously incubated with blue mould spores for aged cheeses. Some interesting results were reported by Sood and Kosikowski (1979) for the replacement of Cheddar cheese – fully acceptable processed cheese was obtained by substituting 40% of aged Cheddar cheese by enzyme-treated UF retentate containing up to 30%,

w/w, solids. On the other hand, cheese base made by UF can satisfactorily replace the young cheese component in the manufacture of conventional processed cheese. An Australian process (Ernstrom et al., 1980) was commercialized in the USA (Kosikowski, 1986b). In this process, whole milk or whole milk acidified to pH 5.7 was ultrafiltered to 40% of its original weight and then diafiltered to 20% of its original weight. The product was fermented with a lactic starter for 16 h at 30 °C and then vacuum-evaporated to 64%, w/w, solids. A similar process was developed in Denmark (Madsen and Bjerre, 1981b). Spraying pre-fermented LPC onto the surface of fresh cheese curd instead of using hand or mechanical washing can also represent an interesting improvement of the process for making bacterial surfaceripened varieties because it allows for a concentrated layer of lactic acid bacteria on the surface of the cheese and thereby avoids generalized contamination by undesirable bacteria such as Listeria or Pseudomonas and accelerates ripening. By means of ultrafiltration and drying, a pre-cheese powder can be produced for subsequent reconstitution and conversion into cheese (Maubois et al., 1973; Glover, 1985). The primary use is for export to countries with low milk production or where the milk supply is very seasonal. In the importing country, the user needs only to add water, starter and rennet to make cheese (Maubois and Fauconneau, 1977; Madsen and Bjerre, 1981a). Such powders could also be used in dairy countries for home-cheesemaking (Maubois et al., 1973; Le Graet and Maubois, 1979). Pre-cheese powders offer many advantages to both exporting and importing countries – cheesemaking characteristics are better than those of even low-heat normal milk powders (Maubois et al., 1973; Le Graet and Maubois, 1979; Lablée, 1982; Mahaut and Maubois, 1988), cheesemakers in the importing country have no whey problem, and economy of production is favourable for both countries since both spray drying and transport costs are cheaper than those for normal milk powder (Maubois and Fauconneau, 1977). However, this application of UF has found very few uses, mainly because of the regulations of dairy-exporting countries, such as the USA and the EC, which subsidize the export of milk solids regardless of the amount of liquid milk used to make 1 kg of these milk solids (Maubois and Fauconneau, 1977). Cheese quality Texture. Although UF cheeses offer moderate to sig-

nificant yield benefits and have been well-accepted by consumers, they possess some inherent characteristics that make them unique with respect to composition,


ripening characteristics and texture qualities. It has even been suggested (Lawrence, 1987; Lawrence et al., 1987) that separate standards of identity for UF cheeses would be advisable, and that a new range of cheese varieties should be developed rather than duplicating traditional varieties. Most texture defects of UF cheeses, such as sandiness, firmness or crumbliness, are caused by the higher content of Ca salts if UF retentates of pH 6.7 are used for making cheese. Mineralization of the drained curd (total Ca and repartitioning of Ca between the casein matrix and the soluble phase) play an essential role in the rheology of the cheese variety (Maubois and Kosikowski, 1978; Kindstedt and Guo, 1998). The mineral content may be adjusted as described above under Properties of UF Retentates. One of the most notable characteristics of UF cheese is the incorporation of whey proteins in the cheese. The quantity of whey proteins retained depends on the variety and the degree of UF concentration. If all the whey proteins of milk are retained, they will represent approximately 20% of the total protein in the cheese. Lower quantities will be retained when the LCR method is used. Part of the casein is replaced by whey proteins, which act as an inert filler and may soften the cheese (De Koning et al., 1981). On the other hand, the water-binding capacity of whey proteins is much higher than that of casein, and UF cheeses are less susceptible to drying during retailing than traditional cheeses. UF cheeses also contain more Ca phosphate salts and -GMP, both components with interesting nutritional and nutraceutical properties (Maubois et al., 2001). Proteolysis and ripening characteristics. It has been commonly observed that UF cheese ripens more slowly than traditional cheese (De Koning et al., 1981; Hickey et al., 1983; Creamer et al., 1987; Lawrence et al., 1987, Furtado and Partridge, 1988; Harper et al., 1989; Guinee et al., 1994, Broome et al., 1998a,b). Generally, the larger the amount of whey proteins incorporated, the slower the flavour development. Large variations in the flavour quality of UF cheese have also been observed and these have been attributed to the varying levels of immunoglobulin and proteosepeptones in the whey proteins (Lawrence et al., 1987). The effect of whey proteins on flavour development is less pronounced in LCR cheeses due to the smaller quantities of whey proteins present but is more significant in cheeses made from higher retentate concentrations and those that are ripened for long periods. The retarded maturation could be due to several reasons. The high content of -lactoglobulin in UF cheeses could inhibit to some extent the general prote-

olytic activity of rennet (Creamer et al., 1987) and plasmin (Visser, 1981). Undenatured whey proteins found in UF cheeses are resistant to proteolysis by these proteases, as well as by starter-derived enzymes. The high buffering capacity of UF cheeses prepared from pH 6.7 ultrafiltered milk is the most probable cause. Indeed, it retards or even completely inhibits the rate of autolysis of mesophilic lactic starter (Goudédranche et al., 1986; Saboya et al., 2001) and consequently hydrolysis of the casein network. The rate of s1-casein breakdown, as well as of -casein, has been found to be reduced in UF cheese (Creamer et al., 1987). The rate of flavour development of UF cheese may be improved by adding flavour-producing enzymes (Spangler et al., 1990) or cell extracts (Saboya et al., 2001) which are totally retained in the retentate, contrary to what happens in traditional cheesemaking where 80–90% of the added enzyme is lost in the whey. Non-starter bacteria or slow-acidifying lactic micro-organisms may be used for their proteolytic and flavour production potential because UF cheesemaking permits separate management of acidification, drainage and ripening flora. Functionality. When cheeses are used as ingredients, various characteristics become important. These include melting behaviour, shredding ability, viscosity and stretchability (Kosikowski and Mistry, 1997). The ultrafiltration of milk prior to cheesemaking alters the physico-chemical properties of cheese in a way that has a distinct impact on some of these functional properties. Acharya and Mistry (2002) reported that the meltability of processed cheese manufactured from Cheddar cheese base made from ultrafiltered milk (up to 6% protein) is lower than that from control (regular) Cheddar. These cheeses also had the highest calcium content. These observations were also true for the base Cheddar cheese; Cheddar cheese made from UF milk containing 6% protein had a melting value of 62 mm compared to 77 mm for the control. Likewise, the viscosity of molten processed cheese at 80 °C was significantly higher for the UF cheese (1043 cP) than for the control (557 cP). Methods to reduce the calcium content of cheese, such as pre-acidification, should help improve functionally. Madsen and Qvist (1998) attributed the impaired melting of UF Mozzarella cheese to the presence of whey proteins and suggested the use of proteolytic enzymes to accelerate the degradation of casein to improve melting. The primary cause of the altered functionality appears to be the difference in the calcium equilibrium and its relationship with the casein structure but the texture and the proteolysis characteristics discussed earlier are also involved. For example, the reduction in


the rate of proteolysis and the presence of whey proteins impact directly on the melting characteristics of cheese. The consumer is the final judge of cheese quality and the success of any new cheese or traditional cheese made with new technology, such as membrane separations, will depend to a large extent on acceptance by consumers.

acceptance in cheesemaking. However, there are several plants in the USA that already use thermal evaporators to pre-concentrate cheese milk (Sandfort, 1983; Honer, 1984). Excess lactose and minerals in the curd are removed by washing with water. Benefits of the overall process must therefore be examined closely.

Reverse osmosis in cheesemaking

On-farm concentration

Richardson (1929) proposed the use of evaporated milk in cheesemaking. This idea was revived some 30 years later by Stenne (1964), but within the framework of an original combination with the observations of Berridge (1951) on the separation of the primary and secondary phases of rennet action at a low temperature. The same basic idea lies behind increasing the solids content of cheese milk by adding dried milk or by concentration using reverse osmosis. For Cheddar cheesemaking, reverse osmosis was proposed for preconcentrating whole milk to 20–25% solids (Agbevavi et al., 1983; Barbano and Bynum, 1984; Bynum and Barbano, 1985; Mayes, 1985; Schmidt et al., 1986). Cheesemaking is conducted in traditional equipment, and the gross composition of the resulting cheese is identical to that of cheese from unconcentrated milk. The amount of starter and rennet required are reduced by 50 and 60%, respectively (Agbevavi et al., 1983), and with a 20% milk volume reduction by RO, a 2–3% increase in cheese yield can be expected (Barbano and Bynum, 1984). However, fat losses in the whey increase with increasing solids concentration due to a partial homogenization effect during processing. A sudden release of pressure during RO can induce lipolysis in the milk and cheese. A 15% volume reduction, representing 1.8:1 concentration, has been reported to be optimal (Barbano et al., 1983). For Cottage cheese, a 5% increase in yield can be realized with an 8% skim milk volume reduction by RO (Barbano, 1986). Such yield increases result from the entrapment of concentrated whey within the network formed by calcium paracaseinate in the cheese. Depending on the degree of concentration, the same consequences for cheese quality result from a greater retention of minerals as described for UF retentates. In RO cheese, there is a high concentration of residual lactose, which may lead to a resumption of lactic acid fermentation after several days in the ripening room, when sufficient lactic acid has been consumed by ripening micro-organisms. This is almost always very detrimental to organoleptic qualities (Richard et al., 2000). Reverse osmosis is widely used for processing whey but it is doubtful whether it will find widespread

Interest in on-farm concentration of milk started in 1974 in France (Maubois, 1979), and in 1977, the AlfaLaval company developed an on-farm UF-processing unit (Kosikowski, 1985). It was believed at that time that on-farm concentration of milk by UF would reduce milk transportation costs due to reduction in milk volume. An increase in cheese yield was also anticipated. At four French farms, milk was concentrated to 2:1 and then delivered to a cheese plant for the manufacture of Emmental and St Paulin cheeses (Anon, 1984a). Permeate produced at the farms was fed to cows, resulting in savings in feed. The French on-farm operation thermized the 2:1 UF milk prior to cooling and delivery. The microbiology of on-farm UF milk was favourable (Benard et al., 1981). Slack et al. (1982a,b) studied the economics of on-farm ultrafiltration in the US by using milk concentrated 2:1–3:1. Economic advantages were possible when the on-farm concentration concept was used on farms with 100–1000 cows. In a year-long study in California, a 900-cow herd was used to study the feasibility of on-farm UF (Zall, 1987). Retentate from this farm was delivered to cheese plants for cheesemaking. The regulatory aspect of this operation was not fully resolved. The use of RO to concentrate milk on the farm has been evaluated in Australia (Cox and Langdon, 1985; Cox et al., 1985). Another approach to treating milk by UF and thermization at the producer level was studied in France (Kosikowski, 1985). During two years, milk collected from 22 farms located on an island south of Brittany was concentrated 2:1 every second day. The retentate was HTST pasteurized and cooled to 2 °C. Pooled retentates were shipped to a dairy plant twice a week. The mesophilic flora of the UF retentate was on average 7700 cfu/ml. A net benefit of 0.0482 FF/litre of milk was achieved. While concentration of milk on the farm showed promise in its early years, the idea was abandoned after various large-scale attempts because of unfavourable economics (capital investment and membrane-replacement costs relative to returns), safety of the process on the farm and regulatory considerations. These problems have apparently been overcome, and the North American Milk Products Company has been successfully using the process since 1997 (Fig. 9) in Texas, California and New


Figure 9 Farm with a UF facility in Dexter, New Mexico, USA (courtesy of North American Milk Products, LLC, St Louis, Missouri, USA). (See Colour plate 8.)

Mexico in the USA (Fassbender, 2001). In this process, raw milk is ultrafiltered at 8 °C to approximately 3.5X (28%, w/w, total solids and 10%, w/w, true protein). The total bacterial count of the product is less than 300 000 per ml and is classified as a Grade A product according to the US Food and Drug Administration. The concentrate is then shipped by truck at 4 °C to several cheese plants in other parts of the country where it is used to standardize milk to 13.5–15%, w/w, total solids for making several varieties of cheeses (Cheddar, Monterey Jack, Mozzarella). In total, an excess of 1 million kilograms of raw milk is processed per day. Permeate is used for animal feeding or spread on land; other applications are being developed. Applications of microfiltration in cheesemaking

Microfiltration, curiously often referred to as crossflow microfiltration whereas the terms ‘cross-flow UF’ and ‘cross-flow RO’ are never used, is a relatively new processing technique in the dairy industry. Introduction really started with the development of mineral MF membranes made from alumina (Gillot and Garcera, 1986) or from zirconium oxide supported on carbon (Cacciola and Leung, 1980). In 1990, the total area installed in the world dairy industry was less than 750 m2 (Van der Horst and Hanemaaijer, 1990), but studies have projected a potential market six to seven times higher than that for UF. In dairying, MF applications have gained increasing attention because of the wide range of available pore size, which makes it possible to separate and fractionate all milk particles.

Microbial epuration of raw milk by MF

Decontamination of raw milk is generally achieved through heat treatment. Various combinations of time– temperature treatments can be used, depending on the desired bacteriocidal effect. While heat treatment is necessary to ensure the safety of milk and milk products, it almost always induces irreversible modifications of milk components, alters physio-chemical equilibria and also adversely affects the organoleptic quality and cheesemaking properties. As with bactofugation, MF allows the heat treatment for decontaminating milk to be minimized, but MF appears to be more efficient than bactofugation. Holm et al. (1986) and Piot et al. (1987) were the first to suggest the use of MF for the removal of bacteria from milk. Initially, permeation of milk components and retention of bacteria were very satisfactory, but serious fouling of the MF membrane occurred rapidly. To overcome this, a new hydraulic concept, developed by Sandblom (1974), could be applied because of the development of new MF ceramic membranes with a highly permeable structure and a multichannel geometry (Gillot and Garcera, 1986). It includes a recirculation loop of micro-filtrate, which permits a constant and low transmembrane pressure all along the MF tubular membrane in spite of a high retentate recirculation velocity (7 m/s). Commercialized equipment using this concept for the removal of bacteria, named ‘Bactocatch’, is used as follows (Holm et al., 1986; Maubois, 1990); raw skim milk is microfiltered continuously using 1.4 m pore size membranes at a temperature between 35 and 50 °C. Retentate flow from the corresponding


loop generally represents 5% of the entering milk flow but it can be reduced to 0.5% by using a second MF equipment in cascade. This retentate, which contains the bacteria and somatic cells in the milk, may be used for animal feed after heat treament, or it may be blended continuously with cream for fat standardization. The cream–retentate mixture is subjected to a moderate UHT treatment (115–120 °C for 3 s), cooled and incorporated into the microfiltrate (Fig. 10). Because of the high content of thermostable enzymes (present in the retained bacteria and somatic cells) in the MF retentate, such a practice could have negative effects on cheese quality and is inadvisable. Fat standardization must be done only with heat-treated cream. MF fluxes ranging from 500 to 700 l/h m2 are obtained for 10 h, with an average bacterial removal of 99.6% (Malmberg and Holm, 1988; Vincens and Tabard, 1988; Meersohn, 1989; Olesen and Jensen, 1989; Trouvé et al., 1991) regardless of the initial count in the raw skim milk. Improvement of the degree of bacterial removal to 99.96% was obtained by using double-layer MF membranes (Saboya and Maubois, 2000). Morphology of bacterial cells and cellular volume slightly influence membrane retention. High retention levels (greater than 99.98%; 99.998% with the double layer MF membrane) observed for spore-forming bacteria, such as Bacillus cereus (Olesen and Jensen, 1989) or Clostridium

tyrobutyricum (Trouvé et al., 1991), are likely due to binding of bacterial spores to a part of the cell wall, consequently resulting in an apparently larger cell size. Retention of Listeria monocytogenes, Brucella abortus, Salmonella typhimurium and Mycobacterium tuberculosis during the Bactocatch process, using double-layer MF membranes, shows a decimal reduction of 3.4, 4.0, 3.5 and 3.7, respectively (Madec et al., 1992; Saboya and Maubois, 2000). Such results mean that MF cheese skim milk will contain less than 1 cfu/l of these pathogenic bacteria, bearing in mind the usual level of contamination at the farm. Such results have led French regulatory authorities to permit the provisional use of MF milk for the making of Protected Designation of Origin (PDO) raw milk cheeses (CNA, 2002). While treatment of milk by the Bactocatch process tremendously improves the hygienic quality and shelflife of manufactured dairy products (Malmberg and Holm, 1988; Meersohn, 1989; Kosikowski and Mistry, 1990; Maubois, 1990; Saboya and Maubois, 2000), it also raises the problem of how to make good quality cheese from ultra-clean cheese milk, often described by cheesemakers as ‘dead milk’. Extensive research must be conducted to understand fully the growth of lactic starters in these ultra-clean cheese milks. It appears from French studies (Maubois, 1990) that each cheese variety requires an independent study. For example, satisfactory distribution of eyes in Emmental cheese made from Bactocatch-treated milk requires the incorporation of specific non-starter lactic acid bacteria, such as heterolactic strains, along with mesophilic lactic, thermophilic and propionic starters added to this milk at the start of cheesemaking (Maubois, 1990, 2002). The development of typical flavour in Camembert cheese requires the addition of Hafnia alvei, a bacterial species able to produce volatile sulphur compounds from methionine in the cheese (Cousin, 1994) Casein enrichment of cheese milk by MF

Figure 10 Flow diagram for the treatment of raw milk by the Bactocatch process (Maubois, 1990).

Microfiltration of skim milk with 0.1 m pore size membranes enables the selective separation of micellar casein, i.e., native calcium phosphocaseinate (Fauquant et al., 1988; Maubois et al., 2001). Depending on the amount of microfiltrate (MMF) removed (referred to by Maubois as ‘ideal whey’ because of its sterility and composition), the casein content of the retentate increases. Consequently, applications of MF retentates, such as those aforementioned for UF retentates in cheesemaking, can be done but prior removal of microfiltrate from the cheese milk offers the cheesemaker a method for optimizing both the cheese process and the value of derived co-products (Maubois et al., 2001). Rennet coagulation of a 20% casein-enriched cheesemilk is improved (Saint-Gelais


et al., 1998; Maubois et al., 2001), leading to a significant decrease in curd fines and fat in the whey and consequently an increase in cheese yield of 2–4% (Daviau, 2000). MMF is easily transformed by UF and diafiltration to a highly purified whey protein isolate (WPI) (protein/total solids ratio0.975) with very interesting functional properties such as gelling, foaming and solubility (Bacher and Konigsfeldt, 2000). This WPI is also a convenient starting material for the purification of major and minor whey proteins (Maubois et al., 2001). Partial removal of -lactoglobulin from milk through this use of 0.1 m MF membranes according to the process of Quiblier et al. (1992) eliminates the detrimental effects of the heat treatments caused in drying and consequently allows the production of new powders showing very interesting cheesemaking characteristics (Garem et al., 2000). Microfiltration membranes of 0.1 m pore size combined with diafiltration with water allows the preparation of a new dairy product. This product, called micellar casein powder (Schuck et al., 1994), has potential either for fortifying yoghurt and cheese milks with proteins or Ca, for replacing cheese curd in processed cheese formulations or for preparing individual caseins or -casein glycomacropeptide (Maubois et al., 2001). These are examples of strategies to produce nutraceutical derivatives from milk proteins (Maubois and Ollivier, 1997). Casein enrichment by MF has been used for Mozzarella and Cheddar cheeses. For Mozzarella (Brandsma and Rizvi, 1999), concentrations of 17.9% were achieved with 0.2 m membranes. Calcium content was reduced by approximately 20% by lowering the pH of the retentate to 6. For Cheddar cheesemaking, milk was microfiltered 2-fold (4.2% casein) such that casein was 87% of true protein (Neocleous et al., 2002a,b). Fat recovery in cheese was not affected but protein recovery increased. Selective fractionation of globular milk fat

Separation of milk fat into small and large globules was proposed by Goudédranche et al. (2000) through the use of special ceramic MF membranes under hydraulic conditions which cause no damage to the native fat globule membrane (FGM). Using a patented process, cheese made from milk with small globules had a higher yield and a smoother and finer texture, probably because of the interaction of FGM with the cheese casein matrix, and the differences in triglycerides content of the fat globules according to their size. Modifications of ␣s /␤-casein ratio by MF

Hydrophobic binding entraps most of the -casein in micelles. On cooling milk or a caseinate suspension to a temperature lower than 5 °C, -casein is solubilized. This soluble -casein can be separated by 0.2 m pore

size MF membranes (Terré et al., 1987) or by 100 000 Da cut-off UF membranes (Murphy and Fox, 1991a). The retentates have a casein content with an increased s/casein ratio and the microfiltrate is a solution of almost pure -casein (Terré et al., 1987). Such a separation process may allow in the future, if the economics are favourable, cheesemaking from milk with a variable s/-casein content (Terré et al., 1987; Murphy and Fox, 1991b). Existing knowledge on cheese made from goats’ or ewes’ milk and on the role played by the degradation of each individual caseins in the development of cheese flavour suggests that a large range of new cheese varieties might be possible from -caseinadjusted milks. Recent developments in ceramic membrane technology allow the MF permeate recirculation loop to be removed, and thus reduce the investment cost per m2 of installed equipment with a saving of consumed energy. The first system, known as ‘Membralox GP®’, was developed by Garcera and Toujas (1998). The required counter-pressure on the permeate side is obtained by a continuous variation of porosity of the membrane support. In the second system, named ‘Isoflux®’, the required UTP (uniform transmembrane pressure) is obtained by a continuous gradient of membrane thickness. Both developments should be used for well-defined applications, i.e., a product and a selective separation. Milk protein concentrates

Milk protein concentrates (MPC) have emerged over the past decade utilizing UF, MF or a combination of various concentration technologies and have become important products for cheesemaking (Mistry, 2002). They present interesting new technical possibilities in cheesemaking and have also initiated intense discussion concerning their impact on trade and local milk production in some countries, particularly the US. Since there are no specific standards of identity in any country for MPC, they, like whey protein concentrates (WPC), cover a wide range of compositional parameters (in the dried product, the milk protein content may range from 35 to over 85%) and functional characteristics. Unlike WPCs, MPCs contain both major milk protein groups in proportions similar to milk. Manufacturing technologies for MPC include UF, diafiltration and spray drying, if the end product is to be in the dry form (Mistry and Hassan, 1991). It is essential to have raw milk of good quality (low total and spore counts). Skim milk is ultrafiltered and diafiltered to approximately 21% total protein. With diafiltration, the lactose content is reduced such that the


final product contains less than 0.5% lactose. The diafiltered product is then spray-dried to less than 5% moisture. This dried or liquid product can be used to supplement milk for cheesemaking using techniques described earlier. For example, in experiments with Gouda cheese, it was shown that fortification of milk with 1% MPC increased cheese yield (Mistry and Pulgar, 1996). This was attributed to reduced losses of casein and better retention of whey proteins. Milk protein concentrates have introduced new possibilities in cheesemaking, as demonstrated by recent patents (Bhaskar et al., 2001; Blazey et al., 2001; Moran et al., 2001a,b). Moran et al. (2001a) developed a continuous method for manufacturing Process cheese in which acidified milk is ultrafiltered and diafiltered to a concentration factor of 4–7, followed by evaporation up to 70% solids to give reduced-calcium pre-cheese. Process cheese is made from this pre-cheese in the traditional manner using flavouring agents and emulsifiers. No cheese base is required, but, instead, high protein concentrates are used as the base material along with flavouring agents. Concentrates with a high micellar casein content have been developed using microfiltration (Schuck et al., 1994). The casein content of such powder is approximately 90% and forms almost 96% of total protein (Saboya and Maubois, 2000). When used in cheesemaking, increases in cheese yield have been reported (Caron et al., 1997). Such powders also provide flexibility in usage regardless of the extent of heat treatment because the -casein–-lactoglobulin complex typically found in NDM does not exist due to the removal of the -lactoglobulin during microfiltration.

Concluding Remarks Membrane technologies have, during the last 20 years, opened new avenues for improving traditional cheesemaking procedures and consequently improved not only the overall quality of a number of cheese varieties but also increased net profit resulting from this transformation of milk. Membrane processing has truly evolved from processes requiring highly specialized equipment to those that have now become an integral part of the cheesemaking operation. They have also allowed the survival of cheese varieties that require unacceptable and tedious manual labour by the traditional process. In addition, they have led to the creation of new cheeses in response to consumer demand. On the other hand, because of difficulties encountered in making cheeses with satisfactory organoleptic qualities through membrane technologies, an impressive amount of knowledge has been acquired in numerous fields of dairy science – protein biochemistry and

physiochemistry, inter-relationships between protein and minerals, dairy microbiology, rheology, etc. The future of the use of these technologies in the world dairy industry is very promising. Many new cheese varieties might be prepared by combining the properties of mineral-adjusted UF retentates and enzymic abilities of lactic starters. Microfiltration has opened new and much diversified avenues for research and technology. Some have already quickly penetrated the cheese industry. In the future, numerous ideas for cheese scientists and technologists may also originate from microfiltration applications. For example, somatic cells are the only milk components which contain all the genome of the producing animal. Their specific separation by MF and the use of molecular genetics could be the starting point for determining the origin (producing cows) of all dairy products except those made from MF milk. Removal of the entire contaminating flora by MF also offers a means to study precisely how each type of starter bacteria added to the cheese milk will act on ripening of different cheese varieties.

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The Microbiology of Cheese Ripening T. Beresford, Dairy Products Research Centre, Teagasc, Moorepark, Fermoy, Cork, Ireland A. Williams, CHARIS Food Research, Hannah Research Institute, Scotland, UK

Introduction Micro-organisms, including bacteria, yeast and moulds, are present in cheese throughout ripening and contribute, in a positive manner, to the maturation process either directly through their metabolic activity or indirectly through the release of enzymes into the cheese matrix through autolysis. The cheesemaker encourages the growth of such organisms; however, other micro-organisms, such as food-borne pathogens, have a negative impact on cheese quality, and thus technologies to remove or prevent their entry to cheese are required. In this chapter we will review the major groups of those micro-organisms which contribute in a positive manner to cheese ripening. The microflora associated with cheese ripening is extremely diverse; however, it may be conveniently divided into two groups – the starter lactic acid bacteria (LAB) and the secondary microflora. Starter bacteria are primarily responsible for acid production during manufacture and, thus, need to be capable of producing sufficient acid to reduce the pH of milk rapidly; a useful rule of thumb is a pH5.3 in milk in 6 h at 30–37 °C, depending on the cheese variety. The secondary microflora do not play any active role during cheese manufacture but are involved with the starter bacteria in the ripening process. Using this approach, Lactococcus, Streptococcus thermophilus, Lactobacillus delbrueckii and Lb. helveticus are regarded as starter bacteria. Enterococcus has been regarded as a starter by some researchers; however, as most strains isolated from cheese are not significant acid-producers (Cogan et al., 1997), they will be regarded as part of the secondary microflora within this review. The secondary microflora may be divided into a number of primary groups including: (i) non-starter lactic acid bacteria (NSLAB) consisting of non-starter lactobacilli, Pediococcus, Enterococcus and Leuconostoc, (ii) propionic acid bacteria (PAB), (iii) moulds and (iv) bacteria and yeast, which grow on the surface of smearripened cheeses.

Techniques used to Study Micro-organisms in Cheese When studying the micro-organisms in cheese it is important that the complete flora is monitored and that the individual components are accurately identified and characterised. Approaches used to achieve these objectives include methods that: (1) depend on cultivation followed by phenotypic characterisation, (2) depend on cultivation followed by molecular characterisation and (3) are culture-independent methods. These approaches and their associated advantages and disadvantages were reviewed recently (Beresford et al., 2001). Useful media are discussed in Cogan and Beresford (2002).

Source of Micro-organisms in Cheese Micro-organisms gain entry into the cheese either by deliberate addition as part of the starter culture or are naturally associated with the ingredients used in cheese production. Thus, the manufacturing technology is central to defining the biodiversity of the cheese flora. Milk in the udder of healthy animals is essentially sterile; however, during milking and storage, opportunities for contamination occur. Milk extracted from the udder at farm level under hygienic milking conditions can routinely contain 5 103 cfu ml 1 (Fox et al., 2000). The rapidity and degree of milk cooling postmilking has a significant impact on the microbial flora. Milk cooled to 15–21 °C is dominated by mesophilic micro-organisms, particularly Lactococcus and Enterobacter species (Bramley and McKinnon, 1990). Cooling milk to 4 °C will greatly retard the growth of most micro-organisms, but psychrotrophic bacteria, such as Pseudomonas, Flavobacterium and Acinetobacter will continue to grow slowly and dominate the flora. Pasteurisation, which is part of the manufacturing process for most commercial cheeses kills ⬃99.9% of the bacteria found in raw milk. However, Bacillus and Clostridium spores and thermoduric organisms, e.g., Micrococcus, Microbacterium and Enterococcus, will survive pasteurisation and gain entry into the cheese.



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The most prevalent micro-organisms in cheese, particularly early in ripening, are the starter bacteria (see ‘Starter Cultures: General Aspects’, Volume 1). Other ingredients used in cheese manufacture include rennet and salt. During preparation, rennets undergo a series of extraction and purification steps, and 15–20% NaCl is added to them to inhibit microbial growth during storage. Little information is available regarding the microbial flora of commercial rennet. However, it is generally considered that they add little to the microbial load of cheese. Salt may be added either (i) directly to the milled cheese curd, (ii) rubbed to the surface of moulded curds as a dry salt or salt slurry or (iii) by immersion of moulded cheese in a brine solution containing 15–23% NaCl. While rubbing dry salt to the cheese surface aids transfer of micro-organisms from the cheesemaker’s hands and the environment to the cheese surface, it is unlikely that salt is directly responsible for the addition of any flora. Industrial brines are used repeatedly and are pasteurised infrequently. While the relatively high salt content of brine inhibits the growth of most micro-organisms, leaching of proteins and other nitrogenous compounds from the cheese into the brine may enhance the survival of micro-organisms that gain access to the brine. A number of studies have indicated that the microflora of commercial brines include LAB, e.g., Lb. paracasei, Lb. casei and Lb. plantarum (Bintsis et al., 2000) and yeast, e.g., Debaromyces hansenii and Candida versatilis (Seiler and Busse, 1990). The presence of such microbes in the brine contributes subsequently to the surface flora developing on the cheese; however, it is unlikely to add to the internal flora. Complex microbial communities composed of bacteria, yeast and mould develop on the surface of smear- and mould-ripened cheeses during ripening (‘Bacterial Surface-ripened Cheeses’, Volume 2). The smear micro-organisms may evolve due to natural contamination from the milk or the ripening room environment or result from deliberate inoculation of the surface. Traditionally in the production of some smearcheeses, a process referred to as ‘old-young’ smearing is used to promote the development of the smear. Mixtures of defined strains are also being developed for direct application to the cheese surface (Bockelmann, 2002). Deliberate addition of specific strains of Penicillium roqueforti or P. camemberti is now common in large-scale production of mould-ripened cheeses. Traditional Swiss Emmental cheese is made from raw milk and the propionic acid fermentation depends on the presence of ‘wild’ PAB in the milk. However, in Emmental made from pasteurised milk, PAB are added to the milk at the beginning of manufacture to ensure that they are present at ⬃105 cfu g 1 of milk.

Factors Influencing Growth of Micro-organisms in Cheese The manufacturing process plays a major role in defining the final environmental conditions of the cheese. This environment is highly selective and exerts a major impact on growth and survival of micro-organisms during processing and ripening. The manufacture of most cheese varieties involves coagulation at temperatures of 30–37 °C, followed by cooking to 37–54 °C. The coagulation temperature will facilitate the growth of most micro-organisms; however, the temperature achieved during cooking has the potential to inhibit the growth of some organisms. For example, Swiss-type cheese is cooked to 52–54 °C and is maintained above 50 °C for up to 5 h. This heat treatment is considered to play an important role in controlling the growth of starters and undesirable micro-organisms (Steffen et al., 1993). The manufacturing process also influences the gross composition of the cheese which is best defined by the four parameters – salt-in-moisture, moisture in non-fat substance, fat in dry-matter and pH (Gilles and Lawrence, 1973). These parameters in turn influence the environment in which the micro-organisms proliferate. The primary environmental factors controlling growth of micro-organisms in cheese include water and salt content, pH, presence of organic acids and nitrate, redox potential and ripening temperature, and were reviewed recently (Beresford et al., 2001).

Starter Bacteria The primary function of starter bacteria is to produce sufficient acid during cheese manufacture to reduce the pH of milk to the desired level. However, they also contribute to cheese ripening since their enzymes are involved in proteolysis, lipolysis and conversion of amino acids to flavour compounds (Fox and Wallace, 1997). Starter cultures are reviewed in ‘Starter Cultures: General Aspects’, Volume 1; thus, discussion on starters in this chapter will be limited to their behaviour in cheese during ripening. Starters provide the most significant contribution to the microbial biomass in young curd, typically attaining densities of 108 cfu g 1 within one day of manufacture. This biomass represents considerable biocatalytic potential for cheese-ripening reactions. However, the majority of the starter enzymes are intracellular and do not have immediate access to the cheese matrix. During cheese ripening, many starters loose viability and release their intracellular enzymes due to autolysis. Feirtag and McKay (1987) first reported this phenomenon for lactococci, and observed that some strains lost viability when incubated at 40 °C due to lysis by thermo-inducible

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Population size (log cfu g–1)

phage. Interest was further stimulated when it was demonstrated that cheese manufactured with autolytic strains was more flavoursome. The pathways of autolysis have been studied extensively and two main mechanisms are currently recognised involving induction of lysogenic phage or defects in cell wall synthesis. The relationship between lysogeny and lysis of lactococci in Cheddar cheese has recently been demonstrated for a large number of lactococcal strains (O’Sullivan et al., 2000). Bacterial cell wall synthesis is a complex process. Modifications to the specificity of the enzymes involved or their levels of production can result in defective cell walls. Muraminidase is the major autolytic enzyme in lactococci (Niskasaari, 1989). Wilkinson et al. (1994) studied starter autolysis by assaying cell viability and release of intracellular enzymes in Cheddar cheese and concluded that Lc. lactis subsp. cremoris strains had different autolytic patterns (Fig. 1). Levels of proteolysis, as measured by free amino acids in the cheese, correlated with the autolytic phenotype, being higher in the most autolytic strain (AM2) and lowest in the least autolytic strain (HP). It has been reported (Crow et al., 1995) that intact cells ferment lactose, remove oxygen and initiate a number of flavour reactions, while autolysed cells accelerate peptidolytic processes. Bacteriocin production is common among lactococci, and some bacteriocins induce lysis in susceptible starter strains. This phenomenon was used to induce lysis of starter lactococci during cheese ripening and resulted in cheese with improved flavour (Morgan et al., 1997). Autolysis has also been reported for Lb. helveticus in Grana (Botazzi et al., 1992), in Swiss-type (Gagnaire et al., 1998; Valence et al., 1998) and Cheddar cheeses (Kiernan et al., 2000). The extent of autolysis varied between strains (5–7-fold) and had a direct impact on the degree of proteolysis in cheese (Valence et al., 2000). The mechanism of autolysis in Lb. helveticus has not been fully elucidated; however, many strains are lysogenic (Carminati et al., 1997). Six of eight

lysogenic strains grew during the manufacture of Swiss cheese, exhausted the galactose and lysed extensively early in ripening (Deutsch et al., 2002). Phage was detected in four of the cheeses at day 1, strongly implicating the role of phage induction in autolysis. Little investigation has occurred regarding autolysis in Lb. delbrueckii (Kang et al., 1998). Autolysis of Sc. thermophilus in cheese has received little attention. Autolysis was reported in a number of strains at the end of growth in laboratory media (Sandholm and Sarimo, 1981; Thomas and Crow, 1983a; Husson-Kao et al., 1999). Prophage-induction triggered by environmental signals, e.g., low pH, may contribute to autolysis of Sc. thermophilus (Husson-Kao et al., 2000).

Non-starter Bacteria Non-starter lactic acid bacteria are a significant proportion of the microbial population of, probably, all ripened cheese varieties. Except for leuconostocs, NSLAB are not deliberately added as part of the starter culture or as secondary adjunct cultures but are adventitious contaminants, which grow during ripening. They do not contribute to acid production during cheese manufacture, but impact on flavour development in the ripening cheese. The principal bacterial groups involved are non-starter lactobacilli, leuconostocs, pedicocci and enterococci. Non-starter lactobacilli

Non-starter lactobacilli constitute the majority of the NSLAB population in most cheese varieties during ripening (Beresford et al., 2001). They grow at 2–53 °C and are acid-tolerant with an optimal pH for growth of 5.5–6.2. They have been subdivided into three groups, viz., obligate homofermenters, facultative heterofermenters or obligate heterofermenters (Kandler and

10 9 8 7 6 5 4 3 2 1 0 0

10

20

30

40

50

60

70

Ripening period (days) Figure 1 Changes in the populations of three lactococcal starters during ripening of Cheddar cheese. G11/C25 (), HP () and AM2 ( ) (redrawn from Wilkinson et al. (1994)).


Weiss, 1986). The obligate homofermenters include the starter bacteria Lb. delbrueckii and Lb. helveticus. The non-starter lactobacilli frequently recovered from cheese are facultative heterofermenters and are often referred to as the facultatively heterofermentative lactobacilli (FHL). The obligate heterofermenters are detected less frequently in cheese. Information on the non-starter Lactobacillus population of over 50 different cheese varieties is summarised in Table 1. Studies on non-starter lactobacilli occurring in Cheddar cheese produced in various countries consistently report the dominance of Lb. paracasei and Lb. plantarum. Other species that are frequently detected as minor components of the population include Lb. curvatus, Lb. casei, Lb. brevis and Lb. rhamnosus. Several other species, including Lb. bifermentans, Lb. buchneri, Lb. collinoides, Lb. farciminis, Lb. fermentum, Lb. kefiri, Lb. parabuchneri and Lb. paraplantarum are occasionally reported in commercially manufactured Cheddar cheese (Williams and Banks, 1997; Fitzsimons et al., 1999; Chandry et al., 2002). Lb. paracasei and Lb. plantarum are also prevalent in many other cheese varieties (Table 1). Non-starter lactobacilli were present in 19 of 35 European artisanal cheeses and were a major component of the microflora of Kasseri, Feta, Serra da Estrela, Gredos and Majorero cheeses (Cogan et al., 1997). The range of species detected is almost identical to that described for Cheddar cheese. The limited range of species reported suggests that few species of Lactobacillus are capable of surviving the environmental conditions that pertain in cheese. The factors required to facilitate proliferation in cheese have not been determined, although an ability to utilise the available growth substrates and an inherent resistance to the adverse pH and salinity are imperative. Pediococci

Although pediococci have been used as adjunct cultures to improve the flavour of Cheddar and Feta cheeses, they also occur along with, and on occasions can predominate, the non-starter population (Law et al., 1976; Bhowmik et al., 1990; Vafopoulou-Mastrojiannaki et al., 1990; Bhowmik and Marth, 1990a). Pediococcus acidilactici and Pd. pentosaceus are isolated most frequently from cheese. The presence of pediococci in Cheddar cheese was first reported by Dacre (1958) who found that they comprised ⬃25% of the bacterial population after 6 months of maturation. Pediococci have subsequently been reported in the non-starter flora of Cheddar cheese manufactured in the UK, Canada and the USA (Fryer and Sharpe, 1966; Elliott and Mulligan, 1968; LitopoulouTzanetaki et al., 1989), in Manchego and Serra da Estrela cheeses (Nunez, 1976; Tavaria and Malcata, 1998), Parmigiano Reggiano and Sicilian artisanal cheeses (Coppola

et al., 1997; Randazzo et al., 2002), Comté (Bouton et al., 1998) and Feta and other white-brined cheeses (Tzanetakis and Litopoulou-Tzanetaki, 1989, 1992; Bintsis and Papademas, 2002; Hayaloglu et al., 2002). Leuconostoc spp.

Many leuconostocs produce diacetyl and acetoin from citrate and are widely used in mixed-strain L and DL cultures (Dellaglio et al., 1995). The CO2 produced is responsible for eye formation in Dutch-type cheese. The identity of strains in starters has not always been established, although the application of molecular techniques has indicated that dairy starters were principally Leuc. lactis and the three subspecies of Leuc. mesenteroides (Morea et al., 1999; Server-Busson et al., 1999). While isolation of leuconostocs is not restricted to the cheeses produced with leuconstoc-containing starters, their apparent infrequent occurrence may, in part, be due to their poor growth on the selective media used (Mathot et al., 1994). Leuconostoc spp. have been isolated from artisanal cheese produced from raw milk and white-brined cheese varieties (Aran, 1998; Bintsis and Papademas, 2002; Hayaloglu et al., 2002) and from French (Cibik et al., 2000), Greek (Litopoulou-Tzanetaki, 1990) and Italian (Coppola et al., 1988, 2001; Morea et al., 1999; Randazzo et al., 2002) cheeses. Several cheeses produced from ovine and/or caprine milks on the Iberian peninsula contain Leuconostoc spp. (Poullet et al., 1993; Garcia et al., 1995; Macedo et al., 1995; Centeno et al., 1996a; Arizcun et al., 1997a; Estepar et al., 1999; Dahl et al., 2000; Freitas and Malcata, 2000; Fontan et al., 2001; Menendez et al., 2001; Pérez et al., 2002). Enterococci

Enterococci occur widely in the environment but are associated principally with the gastrointestinal tract and, because of this, their presence in food products is often perceived as an indicator of poor hygiene. However, enterococci have a history of safe use in dairy products and additionally may exhibit probiotic characteristics or produce bacteriocins (Franz et al., 1999). Conversely, they are regarded as emerging nosocomial pathogens of humans and have been implicated in the aetiology of bacteraemia, endocarditis and in infections of the urinary tract, the central nervous system, the pelvis, the abdomen and the neonate. Many exhibit resistance to vancomycin and other antibiotics and also possess recognised virulence factors (Franz et al., 1999; Giraffa, 2002). Gene transfer mechanisms in enterococci have been described, and the intra- and inter-species transfer of antibiotic resistance genes has been reported (Noble et al., 1992).

Table 1 Occurrence of non-starter lactobacilli in different cheese varieties Dominant non-starter lactobacilli* Cheese variety

Country of origin

Cheddar

Australia

ace

aci

ali

bre

cur

cyp

far

fer

pca

New Zealand Ireland

pen

pla

rha

UK

Cheddar

Cheddar Cheddar

USA

Jarlberg, Norvegia, Greve, Gouda Herrgård

Norway/ Sweden

Sweden

France

Emmental (Swiss type) Vacherin Mont d’Or Afuega’l Pita Armada

Spain

Comté

Artisanal

ppl

Cheddar

Comté Comté

pub

Cheddar

Cheddar

hil

Cheddar Cheddar Cheddar

cas

Reference Broome et al. (1990a) Chandry et al. (2002) Crow et al. (2001) Jordan and Cogan (1993) Fitzsimons et al. (1999) Fitzsimons et al. (2001) Naylor and Sharpe (1958a,b) Williams and Banks (1997) Williams et al. (2002a) Swearingen et al. (2001) Lindberg et al. (1996)

Antonsson et al. (2001) Bouton et al. (1998) Grappin et al. (1999) Berthier et al. (2001) Thierry et al. (1998) Sozzi and Maret (1973) Cuesta et al. (1996) Tornadijo et al. (1995) López and Mayo (1997)

291

continued

292 Table 1 continued Dominant non-starter lactobacilli* Cheese variety

Country of origin

ace

aci

ali

bre

cas

cur

cyp

far

fer

hil

pub

pca

ppl

pen

Arzua Cabrales Casar de Caceres Idiazabal

pla

Manchego Penamellera

Roncal Vadeon Azeitas

Portugal

Evora Picante da Beira Baixa Serra da Estrela

Serra da Estrela

Serra da Estrela Caciocavallo Pugliese Caciocavallo Silano

Italy

Centeno et al. (1996a) Nunez (1978) Poullet et al. (1993)

Reference

La Serena Leon Mahon Majorero

rha

Elortondo et al. (1998) Freitas and Malcata (2000) Medina et al. (1995) Ramos et al. (1982) Fontecha et al. (1990) Freitas and Malcata (2000) Estepar et al. (1999) Freitas and Malcata (2000) López-Díaz et al. (2000) Freitas and Malcata (2000) Freitas and Malcata (2000) Freitas et al. (1996) Macedo et al. (1995) Roserio and Barbosa (1996) Tavaria and Malcata (1998) Gobbetti et al. (2002) Corsetti et al. (2001b)

Canestrato Pugliese Casu Axedu Ewe milk

Fiore Sardo Fontina

Fossa (Pit)

Mozzarella Parmigiano Reggiano Pecorino Sardo Pecorino Toscano Ricotta Forte Scamorza Altamurana Toma

Greece

Feta

Feta Teleme

Kefalotyri Turkey

Beyaz Peynir Kashar Domiati

Domiati

Beyaz

Montasio

Batzos

Egypt

Albenzio et al. (2001) Ledda (1996) De Angelis et al. (2001) Mannu et al. (2000) Cocconcelli et al. (1996a) Gobbetti et al. (1999b) Lombardi et al. (1995) Morea et al. (1998) Coppola et al. (1997) Mannu et al. (2002) Bizzarro et al. (2000) Baruzzi et al. (2000) Baruzzi et al. (2002) Cocconcelli et al. (1996b) Nikolaou et al. (2002) Bintsis and Papademas (2002) Bintsis and Papademas (2002) Tzanetakis and LitopoulouTzanetaki (1992) LitopoulouTzanetaki (1990) Durlu-Ozkaya et al. (2001) Hayaloglu et al. (2002) Aran (1998) El Zayat et al. (1995) continued

293

294 Table 1 continued Dominant non-starter lactobacilli* Cheese variety

Country of origin

Halloumi

Cyprus

Pichtogalo Chanion Ragusano

Crete

Tenerife caprine milk

Tenerife

Sicily

ace

aci

ali

bre

cas

cur

cyp

far

fer

hil

pub

pca

ppl

pen

pla

rha

Reference Papademas and Robinson (2000) Papageorgiou et al. (1998) Randazzo et al. (2002) Zárate et al. (1997)

* The abbreviations used for the non-starter lactobacilli are as follows: ace, Lb. acetotolerans; aci, Lb. acidophilus; ali, Lb. alimentarius; bre, Lb. brevis; cas, Lb. casei; cur, Lb. curvatus; cyp, Lb. cypricasei; far, Lb. farciminis; fer, Lb. fermentum; hil, Lb. hilgardii; pbu, Lb. parabuchneri; pca, Lb. paracasei; ppl, Lb. paraplantarum; pen, Lb. pentosus; pla, Lb. plantarum; rha, Lb. rhamnosus.


Enterococci are a major component of the bacterial population of cheeses produced in Italy (Senini et al., 1997; Suzzi et al., 2000; Andrighetto et al., 2001), France (Bouton et al., 1998), Spain, Portugal (Freitas and Malcata, 2000), Greece (Papageorgiou et al., 1998; Nikolaou et al., 2002), Turkey, The Balkans (Bintsis and Papademas, 2002; Hayaloglu et al., 2002) and Egypt (Hemati et al., 1998). Their numbers at the end of ripening range from 105 to 107 cfu g 1, although numbers vary with cheese type (Fig. 2A). The species

isolated most frequently are Ec. faecalis, Ec. faecium and Ec. durans. Source of NSLAB

Non-starter lactic acid bacteria are present in cheeses made from both raw and heat-treated milk. Hygienically produced raw milk may contain ⬃102 lactobacilli ml 1 and it is probable that the milk is the principal source of organisms in cheeses made from raw milk.

8 7 6 5 4 3 2 1 0

A 0

20

40

60

80

100

120

140

160

180

200

Population size (log cfu g–1 )

10 9 8 7 6 5 4 3 2 1

B

0 0

20

40

60

0

20

40

60

80

100

120

140

160

180

200

10 9 8 7 6 5 4 3 2 1 0

C 80

100

120

140

160

180

200

Ripening period (days) Figure 2 Changes in the populations of (A) enterococci, (B) non-starter lactobacilli and (C) Leuconostoc spp. during ripening of Afuega’l Pitu (; Cuesta et al., 1996), Armada ( ; Tornadijo et al., 1995), Cabrales (; Nunez, 1978), Canestrato Pugliese ( ; Albenzio et al., 2001), Cheddar (×; Dasen et al., 2003), Fossa Pit ( ; Avellini et al., 1999), La Serena (; Fernandez del Pozo et al., 1988), Penamellera (; Estepar et al., 1999), Serra da Estrela (; Dahl et al., 2000), Swiss-type (; Beuvier et al., 1997 and Demarigny et al., 1996) and Tenerife caprine (; Zárate et al., 1997) cheeses.


The diversity of the non-starter population is greater in cheeses made from raw than from pasteurised milk, and Berthier et al. (2001) were able to demonstrate, using molecular techniques, that most of the nonstarter lactobacilli in Comté originated from the milk. The presence of high numbers of enterococci in artisanal cheeses is usually associated with poor hygienic practices (Franz et al., 1999), although bovine faeces was not considered to be the source of enterococci in the farm-house raw milk Cheddar type cheese studied by Gelsomino et al. (2002). Although some lactobacilli are inactivated by pasteurisation (Turner et al., 1986), other strains may survive the heat treatment and proliferate in the cheese during ripening (McSweeney et al., 1994; Jordan and Cogan, 1999). Enterococci are also likely to survive pasteurisation. The production of natural milk cultures from pasteurised milk that is incubated at 42–44 °C for 12–15 h inevitably promotes the selection of heattolerant LAB, including enterococci (Giraffa et al., 1997). It has been suggested (Martley and Crow, 1993) that milk is not the principal source of NSLAB in cheeses made from pasteurised milk and that the manufacturing equipment was the more probable source. Serological typing methods confirmed that air-borne lactobacilli in the plant during cheesemaking are recovered from the cheese (Naylor and Sharpe, 1958b). Non-starter lactic acid bacteria have been isolated from the floor and drains in the dairy environment and from the surfaces of equipment used in cheese manufacture and vacuum packaging (Somers et al., 2001). Lactobacilli are able to form and persist in biofilms on cheesemaking equipment and could be re-isolated from batches of cheese produced after the plant had been cleaned, implying that they survive cleaning and sanitising treatments (Somers et al., 2001). The source of enterococci in cheese milk has been identified as the milking equipment (Gelsomino et al., 2002). The proposition that a factory-specific flora could impart distinctive flavour characteristics to the cheese produced in a given plant (Chapman and Sharpe, 1981) implies that contamination during manufacture is a critical component of the process. However, studies of the non-starter Lactobacillus populations of cheeses made in a single plant over an extended time period failed to detect the recurrence of specific strains (Fitzsimons et al., 2001; Williams et al., 2002a), indicating that, if the contamination was from within the plant, the source was either intermittent or at a low level. Factors affecting growth and survival of NSLAB Environmental conditions

Non-starter lactic acid bacteria, in particular nonstarter lactobacilli and enterococci, are not adversely affected by environmental conditions in the cheese

curd and are able to proliferate during maturation. Non-starter lactobacilli have a generation time of approximately 8.5 days in cheese ripened at 6 °C (Jordan and Cogan, 1993) and viable cells can be recovered from cheese stored at 10 °C for 3 years. The growth rate and final population density of non-starter lactobacilli and enterococci are not affected significantly over the pH range, salt and moisture levels that normally occur in the curd during Cheddar cheese manufacture (Lane et al., 1997). Their growth rate is temperature-dependent but ripening temperature had little influence on the final numbers of lactobacilli in the cheese. Rapid block cooling and ripening at low temperatures reduces their growth rates (Folkertsma et al., 1996). In cheese ripened at 1 °C, the non-starter lactobacillus population was 3 log cycles lower than in a cheese ripened at 8 °C (Shakeel-Ur-Rehman et al., 2000). Nutrient availability

Non-starter lactic acid bacteria require an energy source for growth. The level of residual lactose in fresh curd is usually low but nevertheless some is likely to be present when the non-starter Lactobacillus population is becoming established in the cheese. However, the subsequent increase in the non-starter Lactobacillus population is likely to occur after the lactose has been utilised, indicating that it is not the sole energy source (Turner and Thomas, 1980). Waldron (1997) showed that growth of mesophilic lactobacilli was independent of the lactose content of the cheese. Mesophilic lactobacilli possess glycoside hydrolases (Williams and Banks, 1997) and can utilise sugars derived from the glycomacropeptide of casein and the glycoproteins of the milk-fat globule membrane (Fox et al., 1998; Diggin et al., 1999; Williams et al., 2000). In addition, starter culture autolysis during ripening releases sugars, e.g., ribose (Thomas, 1987; Rapposch et al., 1999). However, studies by Lane et al. (1997) on the growth rate of non-starter lactobacilli in cheeses made with fast- and slow-lysing starter cultures indicated that cell lysate was not a major source of growth substrates. Citrate is present in small amounts (⬃8 mmol kg 1) in unripened Cheddar cheese but is not used as an energy source by non-starter lactobacilli (Palles et al., 1998; Williams et al., 2000); high numbers of lactobacilli also develop in cheese in which there has been no significant citrate utilisation (Jordan and Cogan, 1993). Lipids and bacterial catabolites are not effective substrates but peptides and amino acids are catabolised by lactobacilli, provided that a keto acid acceptor is present to facilitate the aminotransferase involved (Tammam et al., 2000; Williams et al., 2000,


2001). Proteolytic products formed during ripening stimulate the growth of Lb. casei (Nath and Ledford, 1972), whilst Laht et al. (2002) concluded that arginine was one of the main energy sources for non-starter lactobacilli in Swiss-type cheese. The ATP available from arginine metabolism is theoretically sufficient to support growth to 108 cfu g 1. Interactions

The microbial flora of cheese are complex and it is inevitable that interactions between members of the population will occur. The complex nature of cheese ecosystems complicates the interpretation of these interactions; however, Martley and Crow (1993) were able to demonstrate interactions between NSLAB during ripening. It has also been reported that Lb. casei, Lb. rhamnosus and Lb. plantarum inhibit PAB and enterococci in cheese as a result of competition for limiting nutrients (Jimeno et al., 1995; Lynch et al., 1996). In addition to the inter-species competition for nutrients, some of the metabolic products formed by NSLAB, e.g., lactate, acetate and formate, may interfere with the growth of other species (Lindgren and Dobrogosz, 1990; Vandenbergh, 1993; Ouwehand, 1998). In addition, many NSLAB are able to produce bacteriocins with broad or narrow spectra of activity (Stiles, 1994; Ouwehand, 1998; Franz et al., 1999). The inclusion of bacteriocin-producing starter LAB offers a means of suppressing the growth of undesirable bacteria. This approach has been used to control the growth of both food-borne pathogens and NSLAB in cheese (Ryan et al., 1996; Buyong et al., 1998; Benkerroum et al., 2000; Benech et al., 2002), and as a means of manipulating the ripening process (Fenelon et al., 1999; Oumer et al., 2001; Garde et al., 2002). Non-starter lactic acid bacteria may undergo autolysis during ripening. However, Kiernan et al. (2000) were unable to find evidence for the autolysis of mesophilic lactobacilli during the ripening of Cheddar cheese. The ability of Leuconostoc spp. to autolyse is strain-dependent (Cibik and Chapot-Chartier, 2000). Population dynamics

Although the population size remains relatively stable from 3 months to the end of maturation (Fig. 2), the population is not static but is in a dynamic state as the balance of the species and the strains change. Cheddar cheese

The initial non-starter Lactobacillus level in commercially produced Cheddar curd is ⬃102 cfu g 1 which increases to ⬃107 cfu g 1 within 3 months of manufacture and remains at this level throughout the remainder of maturation (Peterson and Marshall,

1990; Fox et al., 1998). However, the heterogeneity of the population of non-starter lactobacilli in Cheddar cheese decreases during ripening. While several species are detected in young cheeses, they are replaced, as the cheese ages, by strains of Lb. paracasei which dominate throughout the remainder of the ripening period (Fitzsimons et al., 2001; Williams et al., 2002a). Similar dynamics was observed in New Zealand Cheddar except that the dominant species were Lb. paracasei and Lb. rhamnosus (Crow et al., 2001). Population shifts at strain level also occur during the ripening of Cheddar cheese. Although some strains may persist throughout ripening while others may recur in the latter stages of ripening, having been undetected in the intervening period, the overall trend is for the number of strains present to decline during maturation. Most mature cheeses are dominated by no more than six strains (Crow et al., 2001; Fitzsimons et al., 2001; Williams et al., 2002a; Dasen et al., 2003). Swiss-type cheeses

The number of non-starter lactobacilli and enterococci are higher in Swiss-type cheese made from raw milk than in cheese made from pasteurised milk but the diversity of non-starter lactobacilli declines during ripening (Beuvier et al., 1997). The population of young cheese was comprised of Lb. paracasei, Lb. plantarum and Lb. brevis but as the cheese matured Lb. paracasei dominated (Demarigny et al., 1996). Similar results were obtained in Emmental cheese made from thermised milk (Thierry et al., 1998). The non-starter Lactobacillus population in Comté cheese was dominated by Lb. paracasei, although Lb. plantarum and Lb. rhamnosus were also present (Bouton et al., 1998; Grappin et al., 1999). Enterococci remained at low levels throughout ripening (Fig. 2A). Berthier et al. (2001), using PCR methods, detected fewer strains in the mature cheese than at other stages throughout ripening. Spanish artisanal cheeses

There is very little information available on changes in the species profiles in Spanish cheeses during ripening. Most data are restricted to counts of the dominant genera at various times during maturation, and significant variation occurs between the different groups of bacteria in different cheeses (Fig. 2A–C). This variation may be due to the artisinal nature of many of these cheeses. Lb. plantarum and Lb. brevis dominated, and the population remained constant at ⬃108 cfu g 1 throughout the 60-day ripening of Afuega’l Pitu cheese but the numbers of leuconostoc and enterococci decreased during ripening (Cuesta et al., 1996). In San Simon cheese, enterococcal numbers were maximal


during the first week of ripening, and stayed constant throughout the remainder of the 6-week maturation period (Fontan et al., 2001). The numbers of leuconostoc, lactobacilli and enterococci varied little during the ripening of Penamellera cheese (Estepar et al., 1999), but leuconostocs were one of the major groups at the drying room stage in the maturation of Cabrales cheese (Nunez, 1978). Lb. plantarum was dominant in the cheese’s interior during the cave-ripening stage. Non-starter lactobacilli and leuconostocs varied little during the ripening of La Serena and ovine milk cheeses, coagulated with Cyanara extracts (Fernandez del Pozo et al., 1988; Vioque et al., 2000), and the enterococcal population declined; in contrast, the proportion of enterococci in Manchego cheese increased throughout ripening (Ordoñez et al., 1978). Variations in the individual populations during the ripening of caprine milk cheeses were cheesespecific. The development of the LAB population of Valdeón, a hand-made blue cheese, exhibited an initial dominance of lactococci and enterococci. However, from the drying stage, lactobacilli and leuconostoc replaced the lactococci, and the mature cheese was dominated by enterococci (López-Díaz et al., 2000). The population of 1-week old Armada cheese was dominated by lactococci whereas Lb. casei and Lb. plantarum were the most abundant during the latter stages of ripening (Tornadijo et al., 1995). The numbers of enterococci, leuconostocs and lactobacilli declined during ripening in summer-made cheese, but the latter two microbial groups remained at ⬃108 cfu g 1 throughout the 16-week period of ripening in autumn-made cheese. Leuconostoc and enterococcal numbers remained at ⬃107 cfu g 1 during the 60-day ripening period of Tenerife cheese whereas the numbers of lactobacilli increased from 105 cfu g 1 after 2 days to 107 cfu g 1 at the end of ripening. Lb. plantarum was dominant in the young cheese whereas in the mature cheese Lb. paracasei was predominant among the lactobacilli (Zárate et al., 1997). Portuguese cheese varieties

The microbiology of Appélation d’Origine Protegée (AOP) Portuguese cheeses was reviewed by Freitas and Malcata (2000). The more dominant LAB in 9-day-old Picante da Beira Baixa cheese were Leuc. mesenteroides (19%), Lb. plantarum (15%), Lb. paracasei (15%) and Ec. faecalis (8%). Leuconostoc spp. were not detectable after 40 days, with Lb. plantarum and Lb. paracasei persisting throughout ripening (Freitas et al., 1996). Lb. brevis and Lb. fermentum were also detected in the mature cheese. The geographical location and season of manufacture influence the micro-

bial population of Serra da Estrela (Tavaria and Malcata, 2000). The numbers of LAB are maximal after a ripening period of 7 days, and, in this population, Leuc. lactis and enterococci are the most abundant (Macedo et al., 1995; Dahl et al., 2000). The proportion of Leuc. mesenteroides and Lb. plantarum in the population tended to increase throughout ripening, whilst that of Ec. faecium, Ec. faecalis and Lb. pentosus declined (Tavaria and Malcata, 1998). Italian cheese varieties

Ricotta forte is produced by ripening cottage ricotta cheese for 12 months, during which time the curds are mixed regularly to prevent mould growth. At the end of ripening, the dominant lactobacilli are Lb. paracasei, Lb. acetotolerans, Lb. alimentarius and Lb. brevis, and of these only Lb. paracasei was detected early in ripening. Lb. kefiri, Lb. gasseri, Lb. hilgardii, Lb. plantarum, Lb. paraplantarum and Lb. zeae were detected transiently (Baruzzi et al., 2000). Major shifts in species profile also occurred during the ripening of the pasta-filata cheese, Caciocavallo Pugliese. The two dominant species at the end of the 60-day ripening period were Lb. parabuchneri and Lb. paracasei, while Lb. fermentum was the dominant species in the young cheese (Gobbetti et al., 2002). The proportion of Ec. faecalis and Ec. durans in the population decreased from ~6% to 0.1% during ripening whilst that of Pd. pentosaceus increased. The Lactobacillus community involved in traditional Mozzarella production has been investigated (Morea et al., 1998). Lb. fermentum, which was dominant in the natural whey starter, was not detected during manufacture or ripening. Following heat treatment associated with the stretching process the complexity of the population decreased from 11 to 5 strains of Lb. casei, Lb. fermentum, Lb. plantarum and Weissella hellenica. Only Lb. plantarum was detected during ripening. The most numerous non-starter lactobacilli in Scamorza Altamurana cheese ripened for 6 days were Lb. fermentum and Lb. paracasei (Baruzzi et al., 2002). Lb. paracasei was not detected in the whey or curd whereas Lb. fermentum was present at all stages in the manufacturing process. Lb. gasseri and W. viridescens were also detected in the whey but not in the ripening curd whereas Ec. durans was only detected in the mature cheese. Dramatic changes in the diversity of the microbial communities of the Sicilian artisanal pastafilata-type cheese, Ragusano, during the manufacturing process were revealed by classical and cultureindependent PCR and density gradient gel electrophoresis techniques (Randazzo et al., 2002). Mesophilic LAB, including Leuconostoc spp. and Lc. lactis, dominated the raw milk population but disappeared during cooking


and fermentation of the curd. However, Lb. delbrueckii and Lb. fermentum grew during ripening, and enterococci were also present in reasonable numbers as the microbial population stabilised in 15- and 30-day-old cheeses. Marked shifts in the species profiles have also been observed in cheeses ripened for longer periods of time. Non-starter lactobacilli and enterococci increased during the 60-day ripening period of Pecorino Sardo ovine milk cheese, though significant differences occurred between batches (Mannu et al., 2002). Lb. casei numbers were constant throughout the ripening of one batch of cheese, appeared after 60 days in a second batch whereas, in a third batch, although they were present in high numbers during ripening, they only became dominant when the starter LAB population declined. The numbers of non-starter lactobacilli in traditional farm-house Fiore Sardo cheese increased from 105 cfu g 1 in 1-day-old cheese to 108 cfu g 1 after ripening for 30 days; the population then decreased slowly, and by 7 months the level was reduced to 104 cfu g 1 (Mannu et al., 2000). Lb. plantarum decreased dramatically during maturation whereas Lb. paracasei, when present, dominated the cheese microflora. Lb. paracasei was also dominant in Montasio cheese, and although it was not detectable immediately after manufacture its population increased to 107 cfu g 1 during the first month of ripening and remained at that level up to 120 days (Lombardi et al., 1995). Enterococcal numbers decreased by one to two orders of magnitude during the 9-week ripening of Canestrato Pugliese cheese (Albenzio et al., 2001), whilst the non-starter Lactobacillus population increased to 28 days and remained at that level for the remainder of maturation (Fig. 2A,B). The population in cheese made from raw milk (108 cfu g 1) was 3 log cycles higher than that in cheeses made from thermised or pasteurised milk. A similar population developed during the 60-day maturation period of Fossa (pit) cheese; the numbers of nonstarter lactobacilli then declined by two orders of magnitude during the 3-month aging process (Avellini et al., 1999). Prolonged ripening times are a feature of Parmigiano Reggiano cheese. During ripening the number of non-starter lactobacilli decreased from 108 cfu g 1 after 5 months to approximately 104 cfu g 1 at 24 months (Coppola et al., 1997). Lb. paracasei/Lb. casei and Lb. rhamnosus, which persisted throughout the 24-month maturation, dominated the population, and Pd. acidilactici was present for 22 months. Enterococci disappeared during ripening of Parmesan for 14 months (Thompson and Marth, 1986).

Greek and eastern European cheeses

Kefalotyri is a hard, salted cheese, which traditionally is produced without starters. Lactobacilli and enterococci are present throughout the 120-day ripening period. Lactobacilli predominate for 30 days when enterococci became increasingly dominant (LitopoulouTzanetaki, 1990). The proportion of Lb. plantarum, Ec. faecium, Ec. durans and Pediococcus spp. recovered increased during ripening whereas the proportion of Lb. casei remained at 16–20% throughout the period. Lb. brevis, Lb. buchneri and Leuconostoc spp. decreased during ripening and were absent in 120-day-old cheese. Ripening-related changes in the LAB population of two other Greek ovine milk soft cheeses, Feta and Teleme, have also been studied (Tzanetakis and LitopoulouTzanetaki, 1992). Lb. plantarum was the dominant isolate recovered from both cheeses. Phenotypic characterisation of isolates indicated that different strains dominate at different stages of ripening in Feta cheese (Xanthopoulos et al., 2000). The proportion of enterococci and pediococci in Feta after ripening for 90 days was lower than that in the original curd. After 180 days of ripening, the proportion of enterococci in Teleme cheese had increased from the levels in the curd whereas that of the Leuconostoc spp. present was similar at both stages after a transient increase between 30 and 60 days. In Turkish white cheeses, species of Enterococcus, Lactobacillus, Leuconostoc and Pediococcus dominate during ripening (Bintsis and Papademas, 2002; Hayaloglu et al., 2002). Enterococci numbers remained constant during the ripening of Turkish Kashar cheese, with Ec. faecium and Ec. durans being the most frequently isolated, while Lb. casei, Lb. plantarum and Lb. rhamnosus were the dominant non-starter lactobacilli in the mature cheese (Aran, 1998). Significance of NSLAB in cheese manufacture Influence of non-starter lactobacilli on cheese quality

Non-starter lactobacilli can impact on cheese quality in both beneficial and detrimental ways; however, an increasing number of studies have shown that selected adjunct strains of Lactobacillus spp. positively influence cheese quality (Fox et al., 1998; Table 2). Cheddar cheese produced under controlled bacteriological conditions in aseptic vats can develop full mature flavour in the absence of non-starter lactobacilli, although non-starter lactobacilli are believed to add desirable flavour notes and reduce harshness and bitterness associated with some starter cultures (McSweeney et al., 1994; Shakeel-Ur-Rehman et al., 2000). The presence of non-starter lactobacilli in commercial cheese is associated with the development of

300 Table 2 Examples of the observed effects of adjunct non-starter lactobacilli on cheese characteristics Cheese variety

Adjunct cultures

Flavour/texture effects

Reference

Arzua-Ulloa

Lb. casei, Lb. casei pseudoplantarum (syn Lb. paracasei), Lb. plantarum

Menendez et al. (2000)

Cheddar

Lb. casei

Cheddar

Cheddar Cheddar

Lb. brevis, Lb. fermentum, Lb. casei, Lb. casei pseudoplantarum (syn Lb. paracasei) Lb. casei Lb. casei, Lb. casei pseudoplantarum (syn Lb. paracasei), Lb. curvatus, Lb. plantarum Lb. casei, Lb. casei pseudoplantarum (syn Lb. paracasei), Lb. curvatus, Lb. plantarum Lb. casei Lb. paracasei, Lb. plantarum

Improved acidification during ripening; s1-casein degradation increased; -casein degradation decreased; soluble nitrogen, volatile fatty acid, diacetyl and acetoin levels increased; less bitter/astringent, more acid flavour; texture improved Increased peptide degradation; enhanced flavour development and intensity Fruity flavours, open texture, late-gassing defects Prevented late-gassing

Cheddar Edam (reduced fat)

Lb. paracasei, Lb. rhamnosus Lb. reuteri

Emmental

Lb. casei

Herrgå rd-type

Lb. casei pseudoplantarum (syn Lb. paracasei), Lb. plantarum Lb. casei

Cheddar Cheddar Cheddar

Mozzarella (reduced fat) Norvegia

Lb. paracasei

Spanish bovine milk semi-hard

Lb. plantarum

St Paulin type

Lb. plantarum

Increased flavour intensity Higher free amino acid levels; increased flavour intensity and acceptability Higher free amino acid levels; different free amino acid profiles; increased flavour acceptability and cheese quality Sensory characteristics, flavour and texture influenced Increased free amino acid levels; significant differences in some important sensory attributes; apparent acceleration of ripening as assessed by effects on aroma/flavour intensity, perceived maturity and creamy/milky flavour notes Improved flavour quality and increased maturation rates Proteolysis increased; free amino acid levels higher; texture quality improved; no flavour benefits detected Concentrations of ethanol, propan-1-ol, 2-methylbutanol, 3-methylbutanol, 2,3-pentandione, 2-methylbutanal and 3-methylbutanal affected. No significant impact on flavour Overall quality and texture improved; flavour quality and intensity improved; off-flavour intensity reduced Cheese stretch reduced at 1–7 days of ripening; melt not affected; cook colour increased pH, free amino acid, -aminobutyric acid and cysteine concentrations reduced; lactate level increased; more rapid decline in starter numbers Lower pH, reduced casein degradation and soluble nitrogen during early stages. Reduced bitterness. Higher elastic modulus, breaking force and hardness values. Negative impact on flavour Free amino acid levels increased and peptide profiles affected

Broome et al. (1990b) Laleye et al. (1990)

Tré panier et al. (1991a,b) McSweeney et al. (1994) Lynch et al. (1996)

Muir et al. (1996) Lynch et al. (1999)

Crow et al. (2001) Tungjaroenchai et al. (2001) Rychlik et al. (1997)

Antonsson et al. (2002) Merrill et al. (1996) Skeie et al. (2001)

Gomez et al. (1996)

Hynes et al. (2001)


more intense Cheddar flavour in a shorter time (Reiter et al., 1967). Obligate heterofermentative lactobacilli that occur in the latter stages of ripening have been associated with the occurrence of undesirable flavours and textures in Cheddar (Dacre, 1953; Laleye et al., 1987; Khalid and Marth, 1990) and flavours in Gouda (Kleter, 1977). The racemisation of L-lactate to D-lactate by non-starter lactobacilli can result in the occurrence of a surface white spot defect as a consequence of calcium D-lactate crystal deposition in the mature cheese (Thomas and Crow, 1983b). However, Sherwood (1939) observed that Lb. casei and Lb. plantarum could improve Cheddar flavour, and although the inclusion of non-starter lactobacilli accelerated flavour development, Law et al. (1976) were unable to attribute the improved flavour obtained with a curd-derived whole reference flora to any specific non-starter group. Later investigations by Puchades et al. (1989), Broome et al. (1990b) and Lee et al. (1990a,b) established that cheeses containing adjuncts of Lb. casei and Lb. plantarum developed higher levels of free amino acids and received higher flavour intensity scores than control cheeses; Lb. brevis-containing cheese had an inferior flavour. Typically, the inclusion of adjunct strains of nonstarter lactobacilli results in improved flavour intensity, increased aroma and accelerated ripening. Although primary proteolysis was not affected by the adjunct cultures, the levels of small peptides and free amino acids were higher than in the control cheese (Table 2). The same volatiles tend to be present in both control and adjunct-containing cheeses but their relative concentrations differ significantly (Dasen et al., 1999). An alternative strategy to accelerate cheese ripening is the use of attenuated cultures (El Soda et al., 2000). Cheddar cheese made with attenuated adjunct strains of Lb. casei had improved sensory and textural characteristics (Trépanier et al., 1992; Madkor et al., 2000). The presence of adventitious NSLAB introduces variability into the ripening process that cannot be easily controlled by the cheesemaker. The species and strain composition of the non-starter Lactobacillus population exhibits not only inter-factory differences (Williams and Banks, 1997; Fitzsimons et al., 1999; Antonsson et al., 2001; Berthier et al., 2001; Crow et al., 2001; De Angelis et al., 2001), but also differences in cheeses produced at the same factory on different days and in cheeses from different vats on the same day (Naylor and Sharpe, 1958b; Fitzsimons et al., 2001; Williams et al., 2002a). The relationship of these population differences to between-batch variations in the quality of the cheese has not been established.

Use of other NSLAB as adjunct cultures

Pediococci enhanced the flavour of Feta (VafopoulouMastrojiannaki et al., 1990) and low-fat Cheddar cheeses (Bhowmik et al., 1990). In contrast, Law et al. (1976) observed that pediococci alone had no impact on flavour development in Cheddar cheese, but were effective in combination with other starter bacteria. There are contradictory reports on the role of enterococci. Although unsuitable as starters due to their low milk-acidifying ability and poor extracellular proteolytic activity, many strains have beneficial metabolic traits (Sarantinopoulos et al., 2001; Delgado et al., 2002). Enterococci impart desirable flavours to a number of cheeses, including Cheddar (Jensen et al., 1975; Gardiner et al., 1999b), Manchego (Ordoñez et al., 1978) and Cebreiro (Centeno et al., 1999). In contrast, high numbers of enterococci resulted in the deterioration of the sensory properties of Parmesan and a Spanish blue cheese (Thompson and Marth, 1986; López-Díaz et al., 1995). Recent results show that three strains of Ec. faecalis, two of Ec. faecium, one of Ec. casseliflavus and one of Ec. durans had no effect on the flavour of Cheddar cheese (Rea and Cogan, unpublished). The perceived beneficial role of enterococci on flavour has resulted in their inclusion in defined-strain starter cultures for Mozzarella (Coppola et al., 1988; Parente et al., 1989), Feta (Litopoulou-Tzanetaki et al., 1993), Venaco (Casalta and Zennaro, 1997) and Cebreiro (Centeno et al., 1996b) cheeses. The use of enterococci as adjuncts will also depend on the resolution of outstanding safety issues. Adjunct NSLAB as probiotics

Lactic acid bacteria have a long history of safe use in foods and there is now considerable interest in their probiotic potential (Mattila-Sandholm et al., 1999). Cheese is effective as a functional food and as a convenient vehicle for the introduction of probiotic cultures into the diet because, in comparison with yoghurt and other fermented milk products, cheese has a solid matrix, and a higher pH, buffering capacity and fat content, which help protect the probiotic strain during intestinal transit to the site of action (Ross et al., 2002). Cheddar cheese is an effective carrier for probiotic Lb. paracasei (Gardiner et al., 1998), Ec. faecium (Gardiner et al., 1999a,b) and two bifidobacteria (McBrearty et al., 2001). The probiotic Lb. paracasei adjuncts had no adverse effects on the flavour and sensory characteristics of the cheese (Gardiner et al., 1998), whereas after ripening for 6 months, Cheddar containing the Ec. faecium adjunct exhibited improved flavour over the control (Gardiner et al., 1999b). Bulgarian yellow cheese and Argentinian Fresco cheese have also been


used for the delivery of probiotic strains (Vinderola et al., 2000; Atanassova et al., 2001).

The range and extent of the activities of the principal NSLAB, which establish during ripening, determine their overall impact on cheese quality. The use of NSLAB to manipulate or accelerate cheese flavour development requires effective pre-screening to identify isolates that have suitable metabolic capabilities. Historically, the choice of adjunct strains for evaluation in cheesemaking trials has been random and not based on biochemical criteria, and consequently the effects of adjuncts were often inconclusive. The use of strains with defined metabolic attributes as adjuncts offers the potential to specifically manipulate cheese flavour development (Williams et al., 2000; Tanous et al., 2002).

enzymes (Fox and McSweeney, 1996). Starter bacteria make a greater contribution to protein breakdown than NSLAB (Lane and Fox, 1996; Lynch et al., 1996, 1997), whose principal contribution appears to be peptidolysis and the release of free amino acids (Williams and Banks, 1997; Muehlenkamp-Ulate and Warthesen, 1999). Peptides are hydrolysed intracellularly by a variety of endopeptidases, aminopeptidases, dipeptidases and tripeptidases, some of which are proline-specific and some of which have overlapping specificities (Christensen et al., 1999). Wide-ranging peptidolytic activities have been detected in non-starter lactobacilli (Khalid and Marth, 1990; Williams and Banks, 1997; Williams et al., 1998), pediococci (Bhowmik and Marth, 1990b; Vafopoulou-Mastrojiannaki et al., 1994) and Leuconostoc spp. (El Shafei et al., 1990), although activities in enterococci, isolated from cheese, are generally low (Arizcun et al., 1997b; Hemati et al., 1998; Sarantinopoulos et al., 2001).

Citrate utilisation

Amino acid catabolism

Although citrate is present at low levels in milk, it is the precursor of diacetyl and acetate, important flavour components of some cheese varieties; the CO2 produced is responsible for eye formation in Dutch cheeses and can affect the texture of other varieties. Other products of citrate metabolism, acetoin and 2,3-butanediol, do not impart flavour. It is probable that citrate is catabolised by the adventitious lactobacilli, although it is not used as an energy source (Palles et al., 1998; Williams et al., 2000). Leuconostoc spp. also have the ability to co-metabolise sugar(s) and citrate but the excess pyruvate produced is reduced to D-lactate (Hugenholtz, 1993). Enterococci also metabolise citrate and can form acetalydehyde, acetoin and diacetyl, metabolic capabilities that have resulted in the inclusion of enterococci in starter cultures for Cebreiro (Centeno et al., 1996b), Feta (Litopoulou-Tzanetaki et al., 1993) and Mozzarella cheeses (Coppola et al., 1988; Parente et al., 1989). The production of diacetyl from glucose by pediococci has also been reported (Ray, 1995).

Although peptides and amino acids contribute to cheese flavour, attempts to increase their formation by over-expression of enzyme activity (Christensen et al., 1995) or the addition of free amino acids to the curd at the manufacturing stage (Wallace and Fox, 1997) have not been successful in enhancing flavour. The implication, therefore, is that the transformation of amino acids rather than their release is the rate-limiting step in flavour formation. The catabolism of amino acids can result in the formation of many compounds that contribute to cheese flavour (McSweeney and Sousa, 2000). Degradative mechanisms potentially include deamination, decarboxylation, desulphuration, oxidation and reduction reactions resulting in the formation of amines, aldehydes, alcohols, indoles, carboxylic acids and sulphur-containing moieties (Yvon and Rijnen, 2001; see ‘Catabolism of Amino Acids in Cheese during Ripening’, Volume 1). The range of amino acid converting enzymes in cheese lactobacilli is restricted (Yvon and Rijnen, 2001). Amino acid breakdown by LAB is initiated by an -ketoglutarate-dependent transaminase. The resultant -keto acids are subjected to further enzymatic or chemical reactions to hydroxyacids, aldehydes, alcohols and carboxylic acids (Yvon and Rijnen, 2001). Cell-free extracts (Groot and De Bont, 1998; Klein et al., 2001) and non-proliferating suspensions of lactobacilli (Kieronczyk et al., 2001), leuconostoc and enterococci (Tavaria et al., 2002) generate important cheese flavour compounds from amino acids. Branched-chain and aromatic amino acid aminotransferase activities have been detected in non-starter lactobacilli (Gummalla and Broadbent, 1999, 2001; Curtin et al., 2001; Hansen

Biochemical activities of NSLAB that are important in cheese ripening

Proteolysis

Lactic acid bacteria possess a complex, well-characterised, proteolytic system, which enables them to meet their amino acid requirements from the hydrolysis of milk proteins (Christensen et al., 1999). The peptides and the amino acids released also contribute to, and act as precursors for, flavour development in cheese. In Cheddar cheese, primary proteolysis is effected by the added chymosin and endogenous milk enzymes, whilst small peptides and free amino acids are released from the primary products by the action of the LAB proteolytic


et al., 2001; Williams et al., 2001, 2002b). The degradation of sulphur-containing amino acids proceeds via an aminotransferase (Dias and Weimer, 1998; Amarita et al., 2001) or cystathionine lyase-mediated pathway (Smacchi and Gobbetti, 1998). The addition of -ketoglutaric acid to cheese curd enhances the conversion of amino acids into aroma compounds (Yvon et al., 1998; Banks et al., 2001). Lipolysis


Screening studies using natural substrates, triglycerides and synthetic chromogenic substrates have confirmed the presence of lipase and esterase activities in non-starter lactobacilli (Khalid and Marth, 1990). In general, the enzymes are intracellular and activities are strain-specific. In the majority of strains, activities increased as the carbon chain length of the fatty acid decreased. A 65-kDa intracellular lipase from Lb. plantarum has been purified (Gobbetti et al., 1996). Intracellular esterases have also been purified and characterised from Lb. plantarum (Andersen et al., 1995; Gobbetti et al., 1997a), Lb. casei (Castillo et al., 1999) and Lb. fermentum (Gobbetti et al., 1997b). They are all serinedependent enzymes with an estimated molecular mass ranging from 70 to 105 kDa (subunit mass 25–40 kDa). The response of esterase activity to the effects of salt, temperature and pH is strain-dependent (Gobbetti et al., 1999a) but retention of this activity during ripening is important for flavour formation both from lipolysis and ester formation. The beneficial effect of enterococci in cheesemaking has been attributed to the hydrolysis of milk fat by esterases (Tsakalidou et al., 1993). The released fatty acids can be further converted into methyl ketones and thioesters which have been implicated as cheese flavour compounds. A survey confirmed that food isolates were lipolytic and hydrolysed all triglycerides from tributyrin to tristearin with decreasing efficiency as the carbon chain length of the fatty acid increased (Sarantinopoulos et al., 2001). Multiple esterase activities were present

and whereas Ec. faecalis strains were the most lipolytic, Ec. faecium strains were more esterolytic. Pediococci and some Leuconostoc spp. are also actively esterolytic (Bhowmik and Marth, 1989; VafopoulouMastrojiannaki et al., 1994; Katz et al., 2002).

Propionic Acid Bacteria Propionic acid bacteria are usually found in Swiss-type cheeses where they grow during ripening and contribute to the characteristic flavour and appearance of these cheeses. Their primary contribution is their ability to metabolise lactic acid present in the cheese curd: 3 Lactate : 2 Propionate Acetate CO2 H2O The CO2 produced is responsible for formation of large eyes that are a feature of these cheeses and the acetic and propionic acids contribute to flavour development. Propionic acid bacteria in the cheese milk survive the relatively high cooking temperature, ⬃54 °C, used in the manufacture of these cheeses and their growth is stimulated by increasing the ripening temperature to 18–22 °C (Fig. 3). Propionic acid bacteria will typically attain levels of 108–109 cfu g 1 cheese after a few weeks, at which time the cheese is cooled to limit further growth (Steffen et al., 1993). Studies on autolysis of PAB are limited and while spontaneous autolysis of P. freudenreichii occurs in synthetic media (Lemée et al., 1995), no evidence of its autolysis was detected during cheese ripening (Valence et al., 1998). Propionic acid bacteria have been implicated in late blowing of Grana cheese. Scanning electron microscopy demonstrated the presence of damaged cells of P. freudenreichii, suggesting that autolysis did occur in Grana cheese (Cappa et al., 1997). Bacteriophage infection of P. freudenreichii

10 9

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Ripening period (days) Figure 3 Changes in the population of PAB during ripening of Swiss-type cheese (redrawn from Turner et al. (1983)).


occurs in Swiss-type cheese and may contribute to PAB lysis during cheese ripening (Gautier et al., 1995). Interactions between PAB and other bacteria are important during cheese ripening. Propionic acid bacteria do not grow well in milk-based media; however, proteolysis of casein by rennet and starter bacteria stimulates growth (Baer, 1995). Further, Piveteau et al. (2000) demonstrated that growth of PAB in milk or whey did not occur unless the initial cell density was 106 cfu ml 1. Growth inhibition appeared to be due to a heat-stable inhibitor(s) present in the whey. Pre-growth of some LAB, used as starter cultures in Swiss-type cheese manufacture, in milk removed the inhibition. Antagonistic interactions between PAB and various LAB were reported by Alekseeva et al. (1983). Nine of twenty-two strains of LAB tested were antagonistic for PAB; Lc. lactis subsp. lactis had the greatest inhibitory effect, while Lc. lactis subsp. cremoris, Sc. thermophilus and Lb. helveticus were compatible with P. freudenreichii and P. shermanii. Inhibition of PAB by Lb. rhamnosus and Lb. casei has also been reported by Jimeno et al. (1995). Interactions between 14 LAB, including strains of Lb. helveticus, Lb. acidophilus, Lb. lactis, Sc. thermophilus and Lc. lactis and P. freudenreichii or P. acidipropionici in whey were investigated by Piveteau et al. (1995). No inhibition was observed, and Lb. helveticus and Sc. thermophilus stimulated the growth of the PAB.

Micrococcus and Staphylococcus Micrococci and staphylococci have traditionally been placed in the family Micrococcaceae; however, phylogenetically they are not closely related. Micrococci have a high GC content and are related to the actinomycetes whereas staphylococci have a low GC content and are found in the clostridal branch of the eubacteria. Most micrococci and staphylococci grow in 5% NaCl and are considered by some authors to contribute to the ripening process. Many of the media used for the isolation of micrococci and staphylococci from cheese are not very selective and do not distinguish between them. Confirmation of genus requires further biochemical or molecular analysis. As many of the reported studies do not include such characterisation, care must be taken when drawing conclusions regarding the type of bacteria isolated (Fig. 4). Micrococci

Micrococci are obligate aerobes with optimum growth temperatures of 25–37 °C. The genus Micrococcus has been recently divided into Micrococcus, Kocuria, Nesterenkonia, Kytococcus and Dermacoccus based on a phylogenetic and chemotaxonomic study (Stackebrandt

et al., 1995). They have been isolated from a variety of cheeses, including Cheddar, Iberian and whitebrined cheeses (Bhowmik and Marth, 1990a; Freitas and Malcata, 2000; Bintsis and Papademas, 2002). However, the ripening temperature and the absence of oxygen internally in most cheese varieties inhibit their growth and thus they are present at lower numbers than the other microbial groups. Micrococci possess a range of hydrolytic enzymes that could contribute to cheese ripening (Bhowmik and Marth, 1990a). The population in Tenerife cheese during ripening ranged from 106 to 108 cfu g 1 and it was proposed that their lipolytic activity could have contributed to flavour development (Zárate et al., 1997). Micrococci are also believed to contribute positively to the maturation of surface-ripened Taleggio cheese (Gobbetti et al., 1997c). However, attempts to improve the flavour of low-fat Cheddar with a Micrococcus sp. adjunct were not successful (Bhowmik et al., 1990). The adjunct-containing cheese contained higher levels of acetate and, although the sulphur volatiles were not affected, an intense off-flavour developed. Staphylococci

Staphylococci are facultative anaerobes, but growth is more rapid and abundant under aerobic conditions. Most strains grow in the presence of 15% NaCl and between 18 and 40 °C. They have been isolated from a number of cheese varieties and form a significant portion of the surface flora of some cheeses (Cuesta et al., 1996; Aran, 1998; Avellini et al., 1999; Albenzio et al., 2001; Corsetti et al., 2001a). Batch, geographical location, year and season of manufacture affect their numbers in Serra da Estrela and Caciocavallo Silano cheeses (Tavaria and Malcata, 2000; Corsetti et al., 2001b). In Serra da Estrela cheese the major staphylococci are Staph. xylosus, Staph. aureus and Staph. epidermidis, with Staph. xylosus predominating at the end of ripening (Macedo et al., 1995). Lower numbers of Staph. simulans and Staph. hominis were present. Similar species have been identified in other ovine and caprine milk cheeses (Fernandez del Pozo et al., 1988; Freitas and Malcata, 2000). Staph. aureus is a recognised food-borne pathogen and, although present during the initial ripening stages of Serra da Estrela cheese, it showed a tendency to disappear during maturation (Fernandez del Pozo et al., 1988; Macedo et al., 1995). The contribution of staphylococci to flavour development in cheese has not been clearly defined.

Moulds Moulds contribute to ripening of many cheeses, particularly surface mould-ripened cheeses like Camembert and Brie, which depend on growth of P. camemberti


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Ripening period (days) Figure 4 Changes in the population of micrococci and/or staphylococci during ripening of: (A) Tenerife ( ), Afuega’l Pitu (), La Serena (), Penamellera (surface) (), Penamellera (interior) () and Fossa Pit (); (B) Kashar (; Aran, 1998)), Cabrales (surface) (), Cabrales (interior) (), Armada ( ), Taleggio (surface) (; Gobbetti et al., 1997c) and Taleggio (interior) (; Gobbetti et al., 1997c) and (C) Swiss type (a) (), Swiss type (b) (), Picante de Beira Baixa (; Freitas et al., 1996), Serra da Estrela () and Canestrato Pugliese ( ). Where not indicated data were collected from sources as outlined for Fig. 2.

on the cheese surface, and blue-veined cheeses, like Roquefort, Gorgonzola, Stilton and Danish Blue which depend on the growth of P. roqueforti within the cheese matrix. In Camembert and Brie, P. camemberti develops on the cheese surface 6–7 days post-manufacture. Once

fully grown, the surface is covered with a white ‘mat’ of mould hyphae. P. camemberti metabolises lactate to CO2 and H2O and contributes to proteolysis, resulting in production of NH3. This results in deacidification of the cheese surface within 3 weeks and the establishment of a pH gradient from the surface (basic) to the interior


(acidic). The increase in pH and breakdown of s1-casein by rennet are responsible for the softening of the curd which gradually extends towards the centre, and is visible in a cross-section of the cheese (see ‘Metabolism of Residual Lactose and of Lactate and Citrate’, Volume 1; ‘Surface Mould-ripened Cheeses’, Volume 2). During the production of most blue-veined cheeses, a water suspension of P. roqueforti spores is added to the milk prior to setting, or spores are dusted onto the curd. Following whey drainage and salting, the cheese is pierced, which facilitates the diffusion of oxygen into the interior of the cheese and growth of P. roqueforti. Gas production by heterofermentative LAB and yeasts, results in curd-openness, which further aids the diffusion of oxygen (Devoyod et al., 1972). The production of methyl ketones by P. roqueforti is inhibitory to further mould growth, and may be a factor in preventing excessive mould development in blue-veined cheese (Girolami and Knight, 1955; see ‘Blue Cheese’, Volume 2). Moulds are associated with a range of other cheese varieties; however, the moulds involved and their impact on ripening are less well understood. A complex fungal flora comprising Penicillium, Mucor, Cladosporium, Geotrichum, Epicoccum and Sporotrichum develop on the surface of the French cheeses, St Nectaire and Tome de Savoie, while Penicillium, and Rhizomucor, have been reported on the surface of the Italian cheese, Taleggio and Geotrichum on that of Robiola (Gripon, 1993). The surface of the Norwegian cheese, Gammelost, is sprayed with Rhizomucor (Oterholm, 1984), while P. roqueforti is sometimes introduced to the cheese interior after piercing (Gripon, 1993).

Yeast Yeasts occur naturally in many cheeses, but particularly in those made from raw milk. The low pH, moisture content, temperature and high salinity, favour the growth of yeast, and numbers on the surface can reach 105–108 cfu g 1 (Fleet, 1990). Their role in deacidification and the formation of metabolites such as ethanol, acetaldehyde and CO2 is beneficial. However, they can also cause spoilage. Fruity and bitter offflavours, gassy and open texture have been attributed to yeast activity. There is considerable diversity in the yeast flora although Debaromyces hansenii is the dominant one on smear- and surface-ripened cheeses such as Limburger, Tilsit, St Nectaire, Roquefort, Camembert and Cabrales (Fox et al., 2000), Danish Blue (van den Tempel and Jakobsen, 1998), white-brined cheeses (Bintsis and Papademas, 2002) and various Spanish and Portuguese AOP cheeses (Freitas and

Malcata, 2000). Many commercial smear preparations include Candida utilis, Geotrichum candidum and Kluyveromyces lactis together with D. hansenii. Other yeasts frequently found include Candida, Geotrichum, Kluyveromyces, Pichia, Rhodotorula, Saccharomyces, Trichosporon, Torulospora, Yarrowia and Zygosaccharomyces spp. Changes in the total yeast population over the maturation period have been monitored for several cheese varieties (Fig. 5), and there is evidence that the yeast population of the traditional Greek cheese, Anevato, was affected by the season of manufacture (Hatzikamari et al., 1999). Although there is considerable information on the population size and species composition there is very little information on the changes in species and strain profiles throughout ripening. van den Tempel and Jakobsen (1998) reported that D. hansenii, C. rugosa, Y. lipolytica and Zygosaccharomyces spp. were the dominant species in Danish Blue cheese ripened for 1 or 14 days, but after 28 days only D. hansenii and C. rugosa were found. D. hansenii was the dominant species throughout the ripening of Danbo, whereas Trichosporon, Rhodotorula and Candida spp. were detected in the initial stages (Petersen et al., 2002). Restriction fragment length polymorphism of mitochondrial DNA confirmed that several strains of D. hansenii were present from the beginning of ripening, and a succession of strains occurred during maturation. A sequential appearance of yeasts on the surface of the ripening curd of St Nectaire cheese over a 2-month period has been observed (Marcellino and Benson, 1992). The surface was initially colonised by Debaromyces and Torulopsis spp. but within 4 days rapid growth of G. candidum and filamentous fungi occurred and rind thickening continued up to 2 months as the fungal hyphae penetrated into the curd. The involvement of yeasts in the maturation process necessitates that further insights into their population changes during ripening be sought. Yeasts are located not only on the cheese surface but are also found within the curd. Yeast levels in the curd of Camembert are 1 log lower compared to the surface (Schmidt and Lenoir, 1980). Most studies on the microflora of Cheddar cheese neglect to monitor the presence of yeasts although a high proportion of Australian and South African cheeses sampled contained yeasts (Fleet and Mian, 1987; Lues et al., 1999; Welthagen and Vijoen, 1999). In these studies, the population in the majority of cheeses exceeded 105 cfu g 1 at some stage during maturation, a level at which the population can impact on flavour development. The yeast population declined from 105 cfu g 1 to 103 cfu g 1 over a 3-month ripening period in one trial in cheeses manufactured in open vats, whilst in a


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Ripening period (days) Figure 5 Changes in the population of yeast during ripening of: (A) Tenerife ( ), Afuega’l Pitu (), La Serena (), Penamellera (surface) () and Penamellera (interior) (); (B) Kashar (), Cabrales (surface) (), Cabrales (interior) () and Armada () and (C) Ovine (ewes’) milk cheese (; Vioque et al., 2000), Swiss type (), Picante Beira () and Serra da Estrela (). Where not indicated data were collected from sources as outlined for Figs 2 and 3.

different cheese production (Welthagen and Vijoen, 1999) the number increased transiently from 102 to 106 cfu g 1 over the first 40 days of ripening before declining. The involvement of yeast in the ripening process of Cheddar cheese is uncertain. Yeasts possess proteolytic and lipolytic enzymes (van den Tempel and Jakobsen, 2000; Klein et al., 2002), form volatile sulphur compounds (Bonnarme

et al., 2001) and are able to develop appropriate flavour and aroma notes in cheese curd (Martin et al., 1999; Wyder and Puhan, 1999b).

Surface Smear Micro-organisms Many European cheeses are characterised by a complex surface ‘smear’ flora that consists of yeasts


and Gram-positive cocci, mainly Staphylococcus, and irregular rod-shaped coryneform bacteria that are classified within families of the Actinobacteria including, Arthrobacter, Brevibacterium, Corynebacterium and Microbacterium (Bockelmann and Hoppe-Seyler, 2001; Brennan et al., 2002). The surface smear may develop from the deliberate inoculation, after salting, with combinations of specific strains or a defined starter culture (Bockelmann, 2002), but more traditionally the growth of the surface microflora is initiated by ‘old-young’ smearing. The microbiology of the smear is complex and not fully characterised. Ripening conditions (12–16 °C; RH 90%) and repeated smearing result in rapid smear development. It is believed that yeasts develop initially, oxidise the lactate to CO2 and water and release ammonia by deamination of amino acids. This results in the pH on the surface increasing to a level favourable for bacterial growth. A number of yeast genera have been isolated from the smear population (Eliskases-Lechner and Ginzinger, 1995; Wyder and Puhan, 1999a; Corsetti et al., 2001a). Studies on the evolution of yeast indicate that the highest numbers of 108–109 cfu g 1 are reached after about 7 days of ripening (EliskasesLechner and Ginzinger, 1995). The population is not static; however, and a succession of species and strains occurs during ripening (Petersen et al., 2002). Coryneform bacteria are dominant in the surface flora for most of the ripening period and although there are reports of the isolation and identification of species from many different genera, there is little information on changes that occur in the population profile during ripening (Beresford et al., 2001). In smears developed from defined starters, the initial high numbers of A. nicotianae declined in aged cheese whilst the numbers of B. linens were generally variable and low throughout ripening (Bockelmann, 2002). A recent study (Brennan et al., 2002) in which the bacteria in the smear population were identified, using a polyphasic approach, at four different stages during ripening, found little evidence for microbial succession. The absence of species progression during ripening may reflect the manufacturing processes used as the cheese surface was washed frequently with the resultant disruption of microcolonies and widespread distribution of the released cells over the cheese surface. The interactions between and within the yeast and the bacterial populations are essential for smear development and cheese ripening. The progression of microbial growth on the cheese surface is a consequence of these interactions. This development, depicted schematically by Bockelmann (2002), results in the establishment of a complex stable smear population and the development of the typical colour, texture and flavour of the cheese variety. In view of the impact that different species may

exert during ripening, a systematic assessment of the dynamics of the bacterial smear population during maturation is warranted (see ‘Bacterial Surface-ripened Cheeses’, Volume 2).

Summary A major diversity of microbial flora is associated with cheese ripening. This flora may result from deliberate addition or through adventitious colonisation. The application of molecular techniques to the study of cheese microbiology is providing a valuable insight into the behaviour of individual strains and populations during ripening. Further characterisation of metabolic potential of the cheese flora is required to elucidate the methods by which these micro-organisms influence cheese quality. The diversity of the flora at species and strain level provides a major biotechnological resource that offers the potential for manipulation in the development of new and innovative cheese products.

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on ripening changes of Feta cheese. Microbiol. Alim. Nutr. 8, 53–62. Vafopoulou-Mastrojiannaki, A., Litopoulou-Tzanetaki, E. and Tzanetakis, N. (1994). Proteinase, peptidase and esterase activity of crude cell-free extracts of Pediococcus pentosaceus isolated from cheese. Lebensm. Wiss. Technol. 27, 342–346. Valence, F., Richoux, R., Thierry, A., Palva, A. and Lortal, S. (1998). Autolysis of Lactobacillus helveticus and Propionibacterium freudenreichii in Swiss cheeses: first evidence by using species-specific lysis markers. J. Dairy Res. 65, 609–620. Valence, F., Deutsch, S.-M., Richoux, R., Gagnaire, V. and Lortal, S. (2000). Autolysis and related proteolysis in Swiss cheese for two Lactobacillus helveticus strains. J. Dairy Res. 67, 261–271. Vandenbergh, P.A. (1993). Lactic acid bacteria, their metabolic products and interference with microbial growth. FEMS Microbiol. Rev. 12, 221–238. van den Tempel, T. and Jakobsen, M. (1998). Yeasts associated with Danblu. Int. Dairy J. 8, 25–31. van den Tempel, T. and Jakobsen, M. (2000). The technological characteristics of Debaromyces hansenii and Yarrowia lipolytica and their potential as starter cultures for production of Danblu. Int. Dairy J. 10, 263–270. Vinderola, C.G., Prosello, W., Ghiberto, G. and Reinheimer, J.A. (2000). Viability of probiotic (Bifidobacterium, Lactobacillus acidophilus, Lactobacillus casei) and nonprobiotic microflora in Argentinian Fresco cheese. J. Dairy Sci. 83, 1905–1911. Vioque, M., Gómez, R., Sanchez, E., Mata, C., Tejada, L. and Fernández-Salguero, J. (2000). Chemical and microbiological characteristics of ewes’ milk cheese manufactured with extracts from flowers of Cyanara cardunculus and Cyanara humilis as coagulants. J. Agric. Food Chem. 48, 451–456. Waldron, D.S. (1997). Effect of Lactose Concentration on the Quality of Cheddar Cheese. MSc Thesis, National University of Ireland, University College, Cork. Wallace, J.M. and Fox, P.F. (1997). Effect of adding free amino acids to Cheddar cheese curd on proteolysis, flavour and texture development. Int. Dairy J. 7, 157–167. Welthagen, J.J. and Vijoen, B.C. (1999). The isolation and identification of yeasts obtained during the maturation and ripening of Cheddar cheese. Food Microbiol. 16, 63–73. Wilkinson, M.G., Guinee, T.P., O’Callaghan, D.M. and Fox, P.F. (1994). Autolysis and proteolysis in different strains of starter bacteria during Cheddar cheese ripening. J. Dairy Res. 61, 249–262.

Williams, A.G. and Banks, J.M. (1997). Proteolytic and other hydrolytic enzyme activities in non-starter lactic acid bacteria (NSLAB) isolated from Cheddar cheese manufactured in the United Kingdom. Int. Dairy J. 7, 763–774. Williams, A.G., Felipe, X. and Banks, J.M. (1998). Aminopeptidase and dipeptidyl peptidase activity of Lactobacillus spp. and non-starter lactic acid bacteria (NSLAB) isolated from Cheddar cheese. Int. Dairy J. 8, 255–266. Williams, A.G., Withers, S.E. and Banks, J.M. (2000). Energy sources of non-starter lactic acid bacteria isolated from Cheddar cheese. Int. Dairy J. 10, 17–23. Williams, A.G., Noble, J. and Banks, J.M. (2001). Catabolism of amino acids by lactic acid bacteria isolated from Cheddar cheese. Int. Dairy J. 11, 203–215. Williams, A.G., Choi, S.-C. and Banks, J.M. (2002a). Variability of the species and strain phenotype composition of the non-starter lactic acid bacterial population of Cheddar cheese manufactured in a commercial creamery. Food Res. Int. 35, 483–493. Williams, A.G., Noble, J., Tammam, J., Lloyd, D. and Banks, J.M. (2002b). Factors affecting the activity of enzymes involved in peptide and amino acid catabolism in nonstarter lactic acid bacteria isolated from Cheddar cheese. Int. Dairy J. 12, 841–852. Wyder, M.-T. and Puhan, Z. (1999a). Investigation of the yeast flora in smear ripened cheeses. Milchwissenschaft 54, 330–333. Wyder, M.-T. and Puhan, Z. (1999b). Role of selected yeasts in cheese ripening: an evaluation in aseptic cheese curd slurries. Int. Dairy J. 9, 117–124. Xanthopoulos,V., Hatzikamari, M., Adamidio, T., Tsakalidou, E., Tzanetakis, N. and Litopoulou-Tzanetaki, E. (2000). Heterogeneity of Lactobacillus plantarum isolates from Feta cheese throughout ripening. Int. J. Food Microbiol. 88, 1056–1064. Yvon, M. and Rijnen, L. (2001). Cheese flavour formation by amino acid catabolism. Int. Dairy J. 11, 185–201. Yvon, M., Berthelot, S. and Gripon, J.C. (1998). Adding -ketoglutarate to semi-hard cheese curd highly enhances the conversion of amino acids to aroma compounds. Int. Dairy J. 8, 889–898. Zárate, V., Belda, F., Pérez, C. and Cardell, E. (1997). Changes in the microbial flora of Tenerife goats’ milk cheese during ripening. Int. Dairy J. 7, 635–641.


Raw Milk Cheeses E. Beuvier and S. Buchin, Station de Recherches en Technologie et Analyses Laitières, Institut National de La Recherche Agronomique, Poligny, France

Introduction About 700 000 tonnes of raw milk cheeses (R cheeses) are produced annually in Europe, particularly in France, Italy and Switzerland, and they represent a significant proportion of the cheese produced (approximately 10% of the total cheese production in the European Union and Switzerland) (Grappin and Beuvier, 1997). Due to large-scale production and widespread areas of production, R cheeses have a true social and economic importance in several European countries (Cogan and Rea, 1996; Grappin, 1997). However, contrasting situations exist in Europe concerning R cheese production; for example, 191 000 tonnes per year (20% of the total ripened cheese production) was produced in France in 1999 (CNIEL, 2002) and close to 5 000 tonnes (1.5% of the total cheese production) in Spain in 2001 (ValentinGamazo, personal communication). These cheeses represent many years of tradition, constitute the product of a specific territory, evolve from a rural civilisation and could be considered a type of handcraft. Moreover, in Europe, some traditional R cheeses have a protected designation of origin (PDO) (Bertozzi and Panari, 1994). Cheese manufacture is constantly evolving and there is a tendency to consolidate numerous small units into larger ones for most varieties of cheese. This implies changes in milk production, with consequences for the quality of the milk. In particular, milk collection has changed; milk is collected over a wider area, resulting in co-mingled milks, and increased transport and storage time before processing. This induces the development of microbial populations which are different from those present in milk at the farm (Mocquot, 1986). One of the consequences of this is the need for milk that is more and more microbiologically ‘clean’ due to the improvement in hygiene on the farm, which, in turn, is enforced particularly by European microbial standards (Directive 92/46/EEC) (Odet, 1999). For example, at present, it is common to find in France, and in particular in the provinces of FrancheComté and Rhône-Alpes, raw milk with a total count on the farm of less than 5 000 cfu ml 1 (Bouton; Michel, personal communication). Another consequence is the modification of the cheese manufacturing practices. In order to destroy pathogens and standardise the milk

microflora, pasteurisation of milk has become widespread. The use of raw milk and ‘wild’ starters requires constant adaptation of the technological conditions to ensure a good-quality product. In contrast, the use of selected starters, however unspecific, is now in general use. Combined with the standardisation of milk and the general use of a secondary microflora, this leads to cheeses with a more constant and uniform quality (Mocquot, 1986). Faced with this change in milk production and cheese manufacture, there is a need for knowledge of the natural biodiversity of microorganisms, their role, and the need and the way of preserving it. This is the reason why, over the last decade, much experimental work has been carried out, mainly in Europe, in order to demonstrate the specific characteristics of R cheeses. This has led to numerous publications from different laboratories in which R cheeses have been compared with pasteurised (P cheese) and/or microfiltered milk cheeses (MF cheese), in which most of the natural microflora is removed. In contrast to pasteurisation, microfiltration of milk eliminates a great part of the indigenous microflora without concomitant heatinduced changes in enzymes, except for the cream which is heat-treated (by pasteurisation or higher heat treatment); microfiltration is applied to skim milk. Moreover, if heat treatment leads to a selection of the microorganisms according to heat sensitivity, microfiltration reduces the level of microorganisms more or less according to their morphology and volume (Grappin and Beuvier, 1997; Saboya and Maubois, 2000). Otherwise, heat treatment also acts on natural milk enzymes; this effect is more marked in P cheese than in MF cheese in which only cream (including sometimes retentate) is heat-treated. In this chapter, the knowledge of the contribution of raw milk to the development of the biochemical and sensory characteristics of R cheeses is summarised. Other studies (not direct comparisons) dealing with R and P cheeses are also considered when necessary. Moreover, recent work is used to give some examples of the diversity of the microflora of R cheeses. This chapter focuses particularly on biochemical and sensory aspects, with data on the microbiology of cheeses. Only a little information on pathogenic aspects



320 Raw Milk Cheeses

of R cheeses is given because pathogens are covered in ‘Growth and Survival of Microbial Pathogens in Cheese’, Volume 1.

14 °C

18 °C

9 8 7

Levels of microorganisms

Generally, there is an opposite trend in numbers between starter lactic acid bacteria (LAB) and nonstarter lactic acid bacteria (NSLAB) during cheese maturation. Starter counts are high, usually 108–109 cfu g 1, at the beginning of ripening, and decrease regularly by two or more log cycles during ageing, whereas adventitious microorganisms, which are initially at a level often 103–104 cfu g 1, grow during ripening, generally reaching higher counts than the initial numbers of starter in mature cheese. McSweeney et al. (1993) and Beuvier et al. (1997), working on Cheddar and Swiss-type cheeses, respectively, found that the decline in mesophilic or thermophilic starter bacteria was slower in R cheeses than in P or MF cheeses, with a difference between R and P and MF cheeses at the end of ripening of one or two log cycles for the streptococci or lactobacilli (Beuvier et al., 1997). Adventitious bacteria in R, P or MF cheeses generally had similar growth curves but in R cheeses, they reached a plateau more rapidly, because of their high number at the beginning of ripening. Non-starter lactic acid bacteria (NSLAB), such as mesophilic lactobacilli, grow regularly during ripening, reaching 107–108 cfu g 1 in R cheeses and one or two log cycles lower in MF or P cheeses at the end of ripening (Fig. 1) (McSweeney et al., 1993; Beuvier et al., 1997; Buchin et al., 1998; Eliskases-Lechner et al., 1999; Xanthopoulos et al., 2000; Rehman et al., 2000c; Albenzio et al., 2001; Buffa et al., 2001b). Other groups of microorganisms, such as enterococci and propionibacteria, which occur naturally in raw milk, are found at higher numbers in R Raclette cheese (Klantschitsch et al., 2000) and Swiss-type cheese (Beuvier et al., 1997) than in P and MF cheeses. At the end of ripening, enterococci counts ranged between 106 and 107 cfu g 1 in R Raclette and 106 cfu g 1 in R Swiss-type cheeses. In corresponding cheeses made from P or MF milk, enterococcal counts were four or five logs lower than in Raclette, and one to three logs lower, in Swiss-type cheeses. Likewise, propionibacteria reached 107–108 cfu g 1 in R Raclette cheese, at ripening temperatures of 17 and 20 °C, and 10 8 cfu g 1 in R Swiss-type cheese, whilst propionibacteria counts did not exceed 102 cfu g 1 in P and MF Raclette cheeses (Klantschitsch et al., 2000), and 104 cfu g 1 in P Swiss-type cheese. On average, 107 cfu g 1 was found in MF Swiss-type cheese, probably due to

log cfu g–1

Microbiological Aspects

6 Raw Microfiltered Pasteurised

5 4 3 2 1 0 1

15

29

43

57

71

Days of ripening Figure 1 Evolution of mesophilic lactobacilli during the ripening of Swiss-type cheese made from raw, pasteurised or microfiltered milk (from Beuvier et al., 1997).

good growth of residual propionibacteria during ripening (Beuvier et al., 1997). Enterococcal counts were found to be 3.5 logs higher in R semi-hard (Morbiertype) cheese than in P cheese (Buchin et al., 1998) and on average 1.5 logs higher in 25 R Castellano cheeses (a hard Spanish cheese made from ewes’ milk) than in 25 P Castellano cheeses, collected at the market (Román-Blanco et al., 1999). Coliforms, Enterobacteriaceae and Gram- bacteria were generally found at higher levels during ripening of R than in P or MF cheeses; the differences depended on the cheese variety (McSweeney et al., 1993; Buchin et al., 1998; Xanthopoulos et al., 2000; Albenzio et al., 2001). There was no difference in coliform populations between R and P Swiss-type cheeses, as coliforms do not survive the manufacturing process (Beuvier et al., 1997; Eliskases-Lechner et al., 1999). Diversity of microorganisms

Generally, in studies comparing R and P cheeses, R cheeses are characterised by a natural highly variable, ‘rich’ microflora; this microbial diversity is not found in P cheeses (Grappin and Beuvier, 1997). Indeed, McSweeney et al. (1993) showed that strains of Lactobacillus spp., which dominated the non-starter microflora in Cheddar cheese, were much more heterogeneous in the R than in the P cheese. Compared to raw milk, combined pasteurisation and microfiltration of milk resulted in a significant alteration of the Lactobacillus species and strain profile in the cheese (elimination of most of the Lb. paracasei strains and all the Lb. plantarum strains (Dasen et al., 2003). In Canestrato Pugliese cheese (a hard Italian cheese), Polymerase chain reaction-Randomly Amplified Polymorphic DNA

Raw Milk Cheeses 321

(RAPD) revealed a greater diversity analysis in Lb. plantarum strains in R cheese than in P cheese (Albenzio et al., 2001). De Angelis et al. (2001) found that, in general, commercial Italian cheeses produced from raw ewes’ milk contained a larger number of more diverse strains of NSLAB, especially mesophilic lactobacilli, than commercial Italian cheeses produced from pasteurised milk. The LAB populations found in farmhouse-produced Pecorino Sardo (an Italian semi-cooked ewes’ milk cheese), made with raw milk and without the addition of a starter culture, were different from those found in the industrially manufactured cheese made from thermised or pasteurised milk inoculated with autochthonous thermophilic whey starters. Moreover, the intra-species and intra-genus genetic diversity in the LAB population is higher in the former (58–80%) than in the latter (20–28%) (Mannu et al., 2002). These authors concluded that the use of raw milk in the farmhouse cheese leads to the preservation of its wild microbiota and, consequently, the microbial diversity of natural LAB in cheese. New molecular techniques, such as denaturing gel gradient electrophoresis (DGGE) and temporal temperature gel electrophoresis (TTGE), based on the analysis of nucleic acids, without cultivation of microorganisms, were carried out to study the complexity of the microbial communities in cheese. Coppola et al. (2001) observed that artisanal Mozzarella cheeses made from raw milk were distinguished from industrial ones, made with pasteurised milk, by the appearance of a greater number of bands in the DNA profiles, reflecting a higher species diversity, in the former than in the latter cheese. Similar results were obtained by Ogier et al. (2002) in Camembert cheese. R Camembert cheese showed the most complex profiles; eight bands were detected in it, compared to five for P Camembert cheese. The results obtained with this approach showed that it could be possible to discriminate traditional products from industrial ones using culture-independent methods. In order to prevent the loss of microbial biodiversity in traditional R cheeses, several studies have been carried out in the past few years to increase information on the natural microflora of R cheeses. Raw milk cheeses have a very diverse microflora at the genus level and in species within the same genus (Estepar et al., 1999; Hatzikamari et al., 1999; Mannu et al., 2000; Callon et al., 2001; Prodromou et al., 2001; Garcia et al., 2002; Fortina et al., 2003). For example, in Avenato cheese, a traditional spreadable Greek cheese made from raw goats’ or ewes’ milk, 12 different species of LAB were isolated and identified throughout manufacture and storage: three species of Lactococcus (Lc. lactis, Lc. garviae and Lc. raffinolactis), three

species of Leuconostoc (Ln. mesenteroides, with the two subspecies mesenteroides and dextranicum, Ln. paramesenteroides and Ln. lactis) and six species of Lactobacillus (Lb. plantarum, Lb. coryneformis, Lb. paracasei, Lb. brevis, Lb. bifermentans and Lb. viridescens) (Hatzikamari et al., 1999). Likewise, among cocci isolated from Piemontese Toma cheese, an Italian semi-cooked cheese, two species of Lactococcus, six species of Streptococcus and three species of Enterococcus were identified throughout manufacture and ripening (Fortina et al., 2003). Raw milk cheeses also have a large diversity in terms of strains within the same species (Berthier et al., 2001; Andrighetto et al., 2002; Bouton et al., 2002). For example, in two individual Comté cheeses, made in two factories called ‘fruitières’, known to produce cheeses with different sensory properties, 13 and 15 different strains of Lb. paracasei and Lb. rhamnosus species were detected throughout cheesemaking and ripening (Berthier et al., 2001). Those strains isolated during ripening originated, for the most part, from the raw milk. A recent study of Marcellino et al. (2001) reported great genetic diversity in the PCR–RAPD patterns of Geotrichum candidum strains isolated from traditional R cheeses, from seven regions of France; 48 different strains among the 64 isolates tested were found. All this microbial diversity contributes to the diversity in the flavour of R cheeses. Hygienic aspects

Table 1 indicates the microbiological standards set by EEC directive 92/46 for the four pathogens, Listeria monocytogenes, Staphylococcus aureus, Salmonella spp., and pathogenic E. coli, that constitute a major threat to the safety of cheese. Overall, dairy products and cheese, in particular, have a remarkably good safety record (Alterkruse et al., 1998; Neaves, 2000). According to the data available, milk and milk products were implicated in 5% of the total 3839 bacterial outbreaks reported in France from 1988 to 1997 and in 1–5% of the total bacterial outbreaks in six other countries: USA, Finland, The Netherlands, England and Wales, Germany and Poland (De Buyser et al., 2001). In all those countries, R cheeses were as frequently involved as P cheeses (30% each) in outbreaks reported between 1983 and 1997 (surveillance period varied according to the country). For the other 40%, milk treatment was unspecified (De Buyser et al., 2001). Analysis of unpublished data on food-borne disease outbreaks, listeriosis excluded, collected by the coordinator of the French surveillance system from 1992 to 1997, revealed 69 documented outbreaks involving milk and milk products as the


Table 1 Microbiological criteria for European raw and heattreated milk cheeses (Directive 92/46 EEC) Microorganism

ma

Listeria monocytogenes Salmonella

No L. monocytogenes in 25 g No Salmonella in 25 g 103 cfu g 1 104 cfu g 1

Staphylococcus aureus Escherichia coli

Mb

nc

cd

5

0

5

0

104 cfu g 1

5

2

105 cfu g 1

5

2

a m: threshold value for the number of bacteria; the result is considered satisfactory if the number of bacteria in all sample units does not exceed ‘m ’. b M: maximum value for the number of bacteria, the result is considered unsatisfactory if one or more sample units contains ‘M ’ or more. c n: number of sample units comprising the sample. d c: number of sample units where the bacterial count may be between ‘m ’ and ‘M ’; the sample is still considered acceptable if the bacterial count of the other sample units is ‘m ’ or less.

vehicle confirmed by the isolation of the etiological agent. Thirty R cheeses and thirty unspecified milk cheeses were involved. In these cheeses, the etiologic agents were S. aureus (88%), Salmonella (8%) and E. coli (3%) (De Buyser et al., 2001). Finally, according to the results mentioned above and the recent review by Donnelly (2001), it has never been clearly demonstrated that R cheeses are less safe than those made from pasteurised milk. As noted by Grappin and Beuvier (1997), the growth of milk pathogens in R cheese is highly dependent on the variety of cheese and on the technology involved. It is well documented that pathogens will grow more easily in cheese with high moisture, high pH and low salt content, than in cooked, long-ripened cheeses. The occurrence of pathogenic bacteria has been reported in certain soft cheeses (high moisture and high pH) made from raw milk (Ryser and Marth, 1987; Centeno et al., 1994b; Eppert et al., 1995; Freitas and Malcata, 2000; De Buyser et al., 2001; Menendez et al., 2001). Generally, surface-ripened cheeses represent a greater risk for the transmission of pathogens than other cheeses (Johnson et al., 1990a). Indeed, Maher and Murphy (2000) demonstrated that the rind of two Irish smear-ripened, farmhouse cheeses produced from raw milk may provide favourable conditions for the growth of many undesirable pathogenic microorganisms such as E. coli and S. aureus. However, the occurrence of pathogens such as L. monocytogenes is not solely a problem for R cheeses. Rudolf and Scherer (2001) have reported higher incidences of L. monocytogenes in red smear-ripened cheeses made from pasteurised milk

(8.0%) than in cheeses made from raw milk (4.8%). In another study (Loncarevic et al., 1995), however, L. monocytogenes was found less frequently in soft and semi-soft cheeses made from heat-treated milk than in R cheeses. In the case of cheeses made from pasteurised milk, post-contamination is involved, as demonstrated, for example, by Canillac and Mourey (1993). They found that contamination by L. monocytogenes was principally due to the washing with brushing machines. Although S. aureus can be found in R cheeses, and, in particular, in semi-hard cheeses, only a small percentage (7%) of S. aureus strains isolated from different varieties of French R cheeses were able to produce enterotoxin(s) at a detectable level in vitro (Meyrand and Vernozy-Rozand, 1999; Lamprell et al., 2003). If one considers that the conditions in vitro optimise enterotoxin synthesis, one can reasonably conclude that the risk of enterotoxin production in cheese is low. Studies on cheeses made from artificially inoculated milk have been carried out to show the behaviour (growth, survival or inhibition) of pathogens in these products. Bachmann and Spahr (1995) studied the behaviour of E. coli, S. typhimurium, S. aureus and L. monocytogenes, during the manufacture of an Emmental-type, hard cooked cheese, made from raw milk. They inoculated milk with pathogens at levels between 104 and 106 cfu ml 1. One week after manufacture, the pathogens were not detectable in the cheese. Panari et al. (2001) did a similar study on ParmigianoReggiano, an Italian hard cooked cheese made from raw milk. None of the inoculated pathogens were detected 24 h after manufacture. The rapid disappearance of pathogens depends on the specific technology of these varieties of cheese: a high cooking temperature (53–55 °C) for a long time period (45–60 min) associated with the fast growth of thermophilic LAB which lowers the pH to 5.2–5.0 within a few hours and prevents the development of the other bacteria. Yousef and Marth (1990) also demonstrated that Parmesan cheese was not a favourable medium for the survival of L. monocytogenes. These results show that hard cooked cheeses are hygienically safe. This is not necessarily valid for the surface of these cheeses, which is considered to be part of the packing (Bachmann and Spahr, 1995). Neaves (2000) indicated that E. coli and Salmonella die slowly during the maturation of hard cheeses and, for these products, the use of a minimum maturation time forms a requirement of hazard analysis critical control point (HACCP) plans. It also forms the basis of US legislation that requires that all imported cheeses made from unpasteurised milk be matured for at least 60 days, so that Salmonella and E. coli will be presumed to have died before the cheese is consumed.


Bachmann and Spahr (1995) demonstrated that Swiss semi-hard R cheese (Tilsiter-type) inoculated with 104 and 106 cfu ml 1 of E. coli, S. typhimurium, S. aureus and L. monocytogenes, was except for L. monocytogenes, free of these pathogens at the end of ripening (90 days). These authors concluded that potentially pathogenic bacteria, except L. monocytogenes, do not survive the manufacture of this cheese produced with good manufacturing practices, because of the synergistic effect of active antimicrobial enzyme systems in the fresh milk, the antagonistic starter culture flora, fast acidification and antimicrobial activities of LAB, combined with the specific brining and ripening conditions.

Biochemical Aspects Cheese is a biochemically dynamic product and undergoes significant changes during ripening (McSweeney and Sousa, 2000). Proteins, carbohydrates and fat are metabolised by both microbial activities (starter and non-starter microorganisms), and by the action of indigenous milk enzymes and residual coagulant. Proteolysis (degradation of the casein matrix to a range of peptides and free amino acids (FAAs)) has a direct and indirect role in the formation of texture and flavour of cheeses. Other biochemical reactions such as lipolysis (liberation of free fatty acids (FFAs)), metabolism of residual lactose, lactate and citrate, and the formation of volatile compounds are also extremely important in the development of flavour compounds (Grappin and Beuvier, 1997; McSweeney and Sousa, 2000).

Proteolysis

According to Grappin et al. (1985) and Rank et al. (1985), proteolysis can be considered as two phenomena. Primary proteolysis represents the extent of breakdown of the native casein and is estimated by electrophoresis. Secondary proteolysis is the further degradation that leads to the formation of peptides and FAAs; the global amount of these compounds is measured by nitrogen fractionation and their proportions by high performance liquid chromatography (HPLC). Primary proteolysis

In most cheese varieties, primary hydrolysis of caseins is caused by the residual coagulant, plasmin, and perhaps by cathepsin D (acid protease) and other somatic cell proteinases. Although LAB are weakly proteolytic, they possess proteinases that also degrade casein. Traditional secondary starters, e.g., Penicillium roqueforti, P. camemberti and Brevibacterium linens, have extracellular proteinases which act on s1- and -caseins (Grappin and Beuvier, 1997; McSweeney and Sousa, 2000). Likewise, yeasts might also exhibit some endopeptidase activity on both s- and -caseins (Xanthopoulos et al., 2000). Table 2 summarises the data from different studies on the degradation of s1- and -caseins in R, P and MF cheeses. The more pronounced degradation of s1-casein in R Swiss-type, including Bergkäse, Raclette and Anevato cheeses, may be attributed to enzymes of the raw milk microflora, which is almost totally eliminated by pasteurisation. Mesophilic lactobacilli, which are part of the

Table 2 Primary proteolysis: levels of native casein in raw (R), pasteurised (P) and microfiltered (MF) milk cheeses Cheese

s1-Casein

-Casein

Reference

Swiss-type Swiss-type (Bergkäse) Canestrato Pugliese Cheddar Cheddar Cheddar Raclette St Paulin Morbier-type Manchego Manchego Idiazabal Arzua (soft cheese) Avenato Travnicki (Feta type)

R MF P RP RP RP R P MF RP RP RP RP RP RP R P ( s-casein) 90 da RP RP RP

R P MF RP RP RP R P MF RP RP RP RP RP RP R P 180 d RP RP RP

Beuvier et al. (1997) Ginzinger et al. (1999b) Albenzio et al. (2001) Lau et al. (1991) McSweeney et al. (1993) Rehman et al. (2000c) Gallmann and Puhan (1982) Beuvier (1990) Buchin et al. (1998) Gaya et al. (1990) Gomez et al. (1999) Mendia et al. (2000) Centeno et al. (1994) Xanthopoulos et al. (2000) Saric et al. (2002)

a 90d: 90 days of ripening.


indigenous milk microflora in Swiss-type, and other cheeses (Demarigny et al., 1996; Eliskases-Lechner et al., 1999), and yeasts in Avenato cheese, are able to hydrolyse s1-casein significantly (Khalid and Marth, 1990; Xanthopoulos et al., 2000). The possible inactivation of cathepsin D by pasteurisation may also be involved, explaining the similar degradation of s1-casein in R and MF Swiss-type cheeses, which was higher than in the corresponding P cheese (Beuvier et al., 1997). On the other hand, the lower level of s1-casein found in P Manchego, St Paulin, Arzua and Travnicki cheeses can be explained by greater retention of rennet in the curd (Grappin and Beuvier, 1997). The higher level of -casein observed in R Swisstype cheese, including Bergkäse, and in Manchego, Idiazabal and Canestrato Pugliese cheeses, than in P cheeses, is explained by a lower plasmin activity in the former cheeses, since pasteurisation of milk increases plasmin activity due to inactivation of plasmin inhibitors and/or increases in the activity of plasminogen activators (Fox and Stepaniak, 1993). This activation is confirmed by the higher levels of -caseins found in P cheeses (Beuvier et al., 1997; Ginzinger et al., 1999b). On the other hand, the lower levels of -casein in R Raclette, St Paulin, Arzua and Avenato cheeses may be explained by the higher proteolytic activity of the indigenous microflora. Gallmann and Puhan (1982), Centeno et al. (1994a, 1996, 1999) and Sarantinopoulos et al. (2002) clearly demonstrated that raw milk microorganisms, such as Pseudomonas fluorescens, micrococci and enterococci, can hydrolyse -casein. Lau et al. (1991) suggested that interactions between denatured whey proteins and caseins, which reduced the accessibility of the caseins to proteases, affected the degradation of -casein in P Cheddar cheese. These results suggest that the breakdown of s1and -caseins is highly dependent on the cheese variety and reflects different manufacturing parameters (cooking temperature, pH at drainage) and different physico-chemical characteristics (pH, salt-in-moisture, moisture), and their effect on the activity of enzymes. Secondary proteolysis

Nitrogen (N) fractionation is commonly used to quantify the extent of proteolysis in cheese. Table 3 summarises the results of different investigations on nitrogen fractions from different cheese varieties. Generally, the proportion of total N soluble in 12% TCA or in PTA, and the amount of free amino acids (FAAs) is higher in R cheeses than in P cheeses, reflecting more extensive proteolysis due to aminopeptidases of indigenous milk microorganisms. However, sometimes a discrepancy occurs and the R cheese shows less secondary proteolysis, for reason(s) which are not clear.

In most of the studies where the RP–HPLC peptide profiles were analysed, P and MF cheeses were close, but differed significantly from R cheeses. In R cheeses, some peaks present in the hydrophobic zone were smaller in Cheddar (Lau et al., 1991; McSweeney et al., 1993) and in Swiss-type cheeses (Ginzinger et al., 1999b) than in P and MF cheeses; larger peaks corresponding to hydrophilic peptides (Lau et al., 1991; McSweeney et al., 1993) or to peptides containing phenylalanine or tryptophan residues (Ginzinger et al., 1999b) have been observed in cheeses made from P or MF milk. In contrast, some peaks containing hydrophobic peptides were larger in P than in R Canestrato Pugliese cheese (Albenzio et al., 2001). In the study by Beuvier et al. (1997) on Swiss-type cheeses, some peaks present in the medium hydrophobicity zone were smaller in the R cheeses. The composition of the FAAs also varied significantly with the milk treatment in all varieties of cheese and differed from one study to another. Nevertheless, some amino acids appeared to be characteristic of the treatment, maybe because of some typical metabolic pathways due to the activity of specific enzymes. Hard Spanish cheeses, such as Idiazabal or Mahon, asparagine and glutamine, as well as taurine were characteristic of P cheeses (Frau et al., 1997; Ordonez et al., 1999; Mendia et al., 2000). According to Frau et al. (1997), asparaginase and glutaminase would be denaturated by pasteurisation. Aspartic acid was higher in R than in P cheeses (Frau et al., 1997; Ordonez et al., 1999; Albenzio et al., 2001). Serine and tyrosine were also found preferentially in P milk cheeses of these varieties, as well as in P or MF Swiss-type cheeses (Skie and Ardö, 2000), whereas -aminobutyric acid seems to be characteristic of R cheeses (Ordonez et al., 1999; Mendia et al., 2000; Skie and Ardö, 2000). According to the latter authors, serine would be degraded to pyruvate by lactobacilli and decarboxylation of glutamine, tyrosine and histidine would result in -aminobutyric acid, tyramine and histamine, respectively. The influence of the indigenous microflora on the decarboxylation of amino acids was confirmed in some studies by the measurement of biogenic amines in ripened cheeses. In general, R cheeses contained more biogenic amines than P cheeses, e.g., in semi-soft (Schneller et al., 1997) Manchego (Inigo et al., 1986) and Swiss-type cheeses (Ginzinger et al., 1999b). Inigo et al. (1986) related this to the staphylococci and micrococci, whereas Schneller et al. (1997) concluded that enterococci, and Enterobacteriaceae are able to produce biogenic amines in cheese. Overall, the differences between R and P or MF cheeses concern mainly secondary proteolysis.


Table 3 Secondary proteolysis: levels of N fractions of raw (R), pasteurised (P) and microfiltered (MF) milk cheeses (soluble N as a % total N) high

molecular weight

Cheese

Water b

Swiss-type

R MF

R MF

Swiss-type Swiss-type Bergkäse (Swiss-type) Canestrato Pugliese Cheddar

R MF R P MF RP

R MF R P MF RP

R P

12% TCAd

RP

PTAe

R P

R MF P RP RP

Cheddar Cheddar Cheddar Raclette

R

Morbier-type Manchego Idiazabal (ovine)

RP

Idiazabal (ovine)

RP 90–180 d

a b c d e f

pH 4.6 c

RP RP

R P MF 60 d R P MF 90 d RP RP

R P 240 da R P 470 d R P MF

RP R P or R#P depending on starter R P 180 d

RP RP

low FAAfs

Reference

RP

Bouton and Grappin (1995) Demarigny et al. (1997) Beuvier et al. (1997) Ginzinger et al. (1999b)

R P

Albenzio et al. (2001)

R MF P

Mc Sweeney et al. (1993) Lau et al. (1991) Rosenberg et al. (1995) Rehman et al. (2000c) Klantschitsch et al. (2000)

P

R P or RP depending on starter RP 90–180 d

Buchin et al. (1998) Gaya et al. (1990) Ordonez et al. (1999)

Mendia et al. (2000)

240 d: 240 days of ripening. Water: water-soluble nitrogen. pH 4.6, pH 4.6 soluble nitrogen. 12% TCA: 12% trichloroacetic acid-soluble nitrogen. PTA: nitrogen soluble in 5% phosphotungstic acid. FAAs: free amino acids.

Lipolysis

The level of lipolysis and its impact on cheese quality depend on the cheese variety. It is limited in Swiss-type and semi-hard cheeses, where a level higher than 0.25–1.5% (Cheddar, depending on the commercial quality), 1.5% (Gouda), 1% (Emmental) or 2% (Comté), is considered to induce flavour defects such as rancid (Choisy et al., 1997). In contrast, lipolysis is much more extensive in some hard Italian, mould-ripened (5–20%), blue-veined (18–25%) and goat-milk cheeses, where lipolysis is essential for typical flavour formation. Lipolysis in cheese is due to the activity of indigenous milk lipoprotein lipase (LPL), to lipases or esterases of starter bacteria or the native microflora, or to added pregastric esterases (Deeth and Fitz-Gerald, 1983; Gripon, 1993). Table 4 summarises the differences between FFAs obtained in R, P or MF cheeses.

In semi-hard and hard, uncooked cheeses, the total amount of FFAs appears to be lower in P than in R cheeses (Table 4); more than 50% lower in Cheddar (McSweeney et al., 1993; Shakeel-Ur-Rehman et al., 2000b,c) and 38% lower in Manchego (Gaya et al., 1990). This was attributed to the inactivation of LPL by pasteurisation. This inactivation varies according to the severity of the heat treatment since heating for 15 s at 70 or 75 °C results in a residual activity of 2% and 0%, respectively (Andrews et al., 1987). In the study of McSweeney et al. (1993), lipolysis in MF Cheddar cheeses was comparable to that in P cheeses; so it is likely that lipases of the native microflora (NSLAB in this case) could also play a role. According to Choisy et al. (1997), LPL would have a lesser impact on the lipolysis of R cheeses than microbial or technological enzymes. Some fatty acids do not originate from lipolysis, e.g., only fatty acids with six or more carbon atoms

326

Table 4 Total amounts and composition of free fatty acids in raw (R), pasteurised (P) and microfiltered (MF) milk cheeses Total free fatty acids Cheese

Age

Method (units)

R

P

Cheddar

120 days

13

7

Cheddar

180 days 8 °C

Global: CSM1 (g palmitate/g cheese) Global: CSM (g palmitate/g cheese)

216

142

135

103

Cheddar

180 days 1 °C 180 days

Cheddar

27 weeks

Morbier-type semi-hard cheese Manchego

5 weeks

Idiazabal

Canestrato Pugliese Goat pressed cheese Serra Ovine soft cheese

24 h 4 months 8 °C 4 months 16 °C 180 days winter/ spring 180 days summer 63 days 60 days Fresh

Global: CSM (meq palmitate/kg cheese)

Global: titration (meq/100 g fat)

C29C18:3: GC (mmol/kg cheese)

C4:09C18:3: HPLC (mg/kg cheese) C4:09C18:2: GC (mg/g fat) C4:09C18:3: HPLC (mg/kgcheese)

68 days Goat soft cheese

Fresh (2 weeks 4 °C) 10 days 12 °C 4 weeks 4 °C 10 days 12 °C 8 weeks 4 °C

1 CSM: copper soap method.

C4:0—C20:4: GC (g/100 g fat)

4.7

2.2

12.86

5.86

11.09 16.21 15.5/8.3

6.95 9.87 10.8/6.5

11.4

13.8

1673

1397

8

6

3538

3002

6517

8115

1.5

1.4

9.2

7.2

12.9

10.3

MF

2.1

Individual free fatty acids

Reference

C49C59C69C79C8: R P, C99C109C10:19C119C129C149C14:19C169C16:1: R P

Shakeel-UrRehman et al. (2000c) Shakeel-UrRehman et al. (2000b)

C49C59C69C79C89C99C109C10:19C119C129C149 C14:19C159C16: R P C16:1: R P C69C89C109C129C149C169C18: R P/MF

Shakeel-UrRehman et al. (2000a) McSweeney et al. (1993)

C5: R P C49C69C79C89C9: R P

Buchin et al. (1998) Gaya et al. (1990)

C49C69C89C109C16:19C189C18:1: R P, C129C149C169C18:29C18:3: R P

Chavarri et al. (2000)

C69C109C18:2: R P, C49C89C129C149C169C189C18:1—C18:3: R P C49C69C109C129C149C169C189C18:1—C18:2: R P, C8: R P

Albenzio et al. (2001) Buffa et al. (2001a) Sousa and Malcata (1997)

C49C69C89C10: P R C129C149C169C189C18:1- C18:29C18:3: R P Morgan et al. (2001)


come exclusively from lipolysis. The proportion of short-chain fatty acids in total FFAs varies according to the extent of lipolysis of the cheeses; it can reach 50–80% (mol/mol) in hard-cooked cheeses. Thus, the validity of global measurement to estimate lipolysis in such cheeses is questionable, and it can be concluded that the examination of individual FFAs is more informative. The higher level of lipolysis in R compared with P cheeses affects different fatty acids: all FFAs from C6 to C16 in Cheddar cheeses (Shakeel-Ur-Rehman et al., 2000a), all the major fatty acids in a goats’ milk hard cheese (Buffa et al., 2001a), only short-chain (C4–C8) fatty acids in Cheddar (Shakeel-Ur-Rehman et al., 2000c) and only C6, C8 and C18:1 in Canestrato Pugliese cheese (Albenzio et al., 2001). All the major fatty acids in Cheddar were affected to the same extent by pasteurisation or MF (McSweeney et al., 1993). In the study by Chavarri et al. (2000), the amounts of short-chain FFAs (C4 to C10) and of C16:1, C18:0, C18:1 in Idiazabal were affected by pasteurisation. But, the effect of pasteurisation varied according to the season; amounts of FFA were higher in R cheeses made in winter and spring, but lower in those made in summer. In the study by Buchin et al. (1998) on R and P Morbier-type, semi-hard cheeses, no differences were observed in the levels of C6 to C9 fatty acids. The attribution of the observed differences in lipolysis, either to LPL or to microbial lipases, is difficult. Inactivation of LPL was suggested by Gaya et al. (1990). Chavarri et al. (1998) found that depending on the period of the trial, LPL was inactivated by 73–95%, by pasteurisation of milk for the manufacture of Idiazabal. Short-chain fatty acids were released preferentially during ripening, showing microbial participation in lipolysis, but the balance between the different FFAs was not affected by pasteurisation, indicating that microbial lipolysis was not significantly affected by pasteurisation (Chavarri et al., 2000). Albenzio et al. (2001) attributed the slight differences in lipolysis between R and P cheeses to differences in lipase and esterase activities of the milk microflora (NSLAB). Shakeel-Ur-Rehman et al. (2000a) also attributed the differences observed in Cheddar to the activity of NSLAB, because NSLAB were highest in the most highly lipolysed cheeses. However, agreement between NSLAB counts and FFA levels did not occur in all cheeses, so the authors supposed that not only number but also species or strains play a role. In hard cheeses, such as Cheddar, where moulds are not present, LAB could be the main cause of lipolysis (Choisy et al., 1997). In particular, starter LAB may contribute to the release of fatty acids, because they have lipase and esterase activities (Deeth and Fitz-Gerald, 1983).

The role of starter LAB in the observed differences is also questionable; modification of their activity due to interaction with the natural microflora cannot be excluded. Such differences could be expected in soft cheeses, but the studies on this type of cheese show contradictory results. In goats’ milk mould-ripened cheeses, Morgan et al. (2001) found higher lipolysis, determined by a global method, in R cheeses than in P cheeses. The extent of lipolysis in mature cheeses was related to that of the milk. Pasteurisation was supposed to inactivate LPL. But, in this study, pasteurisation also induced lower moisture cheeses, which could reduce the activity of the lipolytic surface microflora. In contrast, Sousa and Malcata (1997) found higher lipolysis in P cheeses made from ovine milk (Serra) than in R cheeses, despite higher levels in the fresh cheese. However comparison between the two types of cheese was made difficult by a higher fat retention in P than in R cheeses, and the amounts of FFAs were expressed per weight of cheese. Thus, in both studies, interpretation of the results is difficult because of the difficulties in controlling the cheese composition. In Swiss-type cheeses, the level of C6 could be a good indicator of the extent of lipolysis, as suggested by Kuszdal-Savoie (Choisy et al., 1997). In the studies of Bouton and Grappin (1995), Beuvier et al. (1997) and Demarigny et al. (1997), the variations in C4 followed those of C6, which indicates that, in these cases, C4 was mainly from lipolysis, and not from the growth of clostridia on lactate. Bouton and Grappin (1995) and Beuvier et al. (1997) found no differences in these two acids in cheeses (R, P, MF or P with the addition of the natural microflora), whereas Demarigny et al. (1997) found that R cheeses tended to be more lipolysed than MF cheeses, depending on the season of the year and the age of the cheese. In the latter cheeses, lipolysis may have been of microbial origin. On the one hand, the heating of the milk in the vat, performed in this type of technology, would inactivate the LPL (Chamba and Perreard, 2002), which would explain why no differences were observed between P and MF cheeses (Beuvier et al., 1997). On the other hand, thermophilic starter LAB and propionibacteria have lipolytic activity in Swisstype cheeses (Deeth and Fitz-Gerald, 1983). According to Chamba and Perreard (2002), the lipolytic activity of propionibacteria is far greater than that of starter bacteria. Moreover, it could be strain-dependant, which could also explain the variability in the above results. Volatile compounds

Comparisons of the volatile compounds in R and P or MF cheeses, have been undertaken only recently and are not very numerous. The same volatile compounds


were present in each type of cheese within the same study, whether the microflora was present or removed from the milk. Differences were observed in the levels of many volatile compounds, R cheeses generally having higher levels than P or MF cheeses, with exceptions in certain chemical families. Volatile fatty acids (VFAs)

Volatile fatty acids in cheese are the products of various metabolic pathways, mostly microbial. Acetic acid may have different origins, e.g., from the catabolism of lactose, citrate, amino acids, or the propionic fermentation (PF). Propionic acid is one of the end products of lactose or lactate fermentation by propionibacteria. Butyric acid can originate from the catabolism of triglycerides, but also from lactate fermentation by clostridia. Hexanoic acid (C6) is found only in triglycerides and is liberated by lipolysis (its behaviour is described under Lipolysis and catabolism of fatty acids in cheese. Branched-chain VFAs result from catabolism of the amino acids, e.g., 2-methyl propanoic (isobutyric), 2-methyl butanoic, 3-methyl butanoic (isovaleric) acids from valine, isoleucine and leucine, respectively. These compounds are major volatile compounds in cheese, and participate significantly in the flavour of many cheese varieties, as suggested by results from olfactometry: acetic acid has a characteristic vinegar flavour, propionic acid a pungent, fruity one, butyric, isobutyric and isovaleric acids have similar flavours, associated with cheesy, sweaty, rancid and sour notes (Yvon and Rijnen, 2001; Curioni and Bosset, 2002). Table 5 summarises the differences between VFAs obtained in R, P or MF cheeses. The PF is one of the major fermentations that occur in Swiss-type cheeses (Curioni and Bosset, 2002). In the studies of Bouton and Grappin (1995), Beuvier et al. (1997), Demarigny et al. (1997) and Eliskases-Lechner et al. (1999), the presence of the raw milk microflora was associated with higher levels of acetic and propionic acids and lower levels of lactic acid in the ripened cheeses. These results showed a greater PF in R cheeses, due to the presence of the native propionibacteria, which are eliminated by pasteurisation or microfiltration. It should be noted that no propionibacteria were added to the cheeses studied, as required in the manufacture of Bergkäse or Comté. In contrast, in a hard cheese such as Emmental, in which propionibacteria are used as starters to ensure a high level of PF, the effect of native populations of propionibacteria seems to disappear. Buchin et al. (2002) compared R and MF Emmental-type cheeses: the total population of propionibacteria tended to be lower in R cheeses from midripening, with a subsequent lower level of propionic acid at the end of ripening.

Generally, acetic acid is more important in Swisstype cheeses made from R milk than in those from P or MF milk (Bouton and Grappin, 1995; Demarigny et al., 1997; Eliskases-Lechner et al., 1999). This was related to the presence of propionibacteria and facultatively heterofermentative lactobacilli (FHL) in the R milk. In Swiss-type cheeses, the level of butyric acid tended to be higher in R than in MF cheeses (Demarigny et al., 1997). Because no butyric acid bacteria were found in these cheeses, the origin of this compound could not be butyric fermentation. It is likely that it was liberated from triglycerides by the lipolytic activity of the microorganisms present in the cheese, particularly propionibacteria (Chamba and Perreard, 2002). Caproic acid (C6) followed the same trend as butyric acid, but the differences between R and MF cheeses were less marked (see ‘Lipolysis’). In contrast, Bouton and Grappin (1995) and Beuvier et al. (1997) observed no differences between R, P, MF or P cheeses with added native microflora. The level of isovaleric acid was higher in R cheeses than in P or MF cheeses (Beuvier et al., 1997; Demarigny et al., 1997). The occurrence of this compound was correlated with FHL, propionibacteria or enterococci populations, and at least the former two populations may be involved (Langsrud and Reinbold, 1973; Paulsen et al., 1980; Thierry and Maillard, 2002). However, Buchin et al. (2002) found that the production of isovaleric acid was positively correlated with the level of propionibacteria, but negatively with the levels of starter LAB, native mesophilic lactobacilli or enterococci. The influence of the native microflora on VFA production was also observed in other cheese varieties, e.g., Cheddar (Shakeel-Ur-Rehman et al., 2000a,c), Raclette (Klantschitsch et al., 2000) and Morbier-type cheese (Buchin et al., 1998). In Cheddar, several VFAs, e.g., acetic, propionic, butyric, valeric and caproic acids, were found at higher levels in R than in P cheeses. Moreover, the levels of all these compounds increased at a ripening temperature of 8 °C, compared with 1 °C, and the levels in cheeses of the different acids, except caproic, increased with the proportion of raw milk in several blends of R and P milk. The authors attributed all these differences to the presence of NSLAB in the raw milk, because their presence and growth in cheese were closely related to the production of fatty acids. NSLAB were mostly eliminated by pasteurisation, and the higher the R:P milk ratio or the higher the ripening temperature, the higher was the number of these bacteria in cheese, at all times during ripening. The influence of the presence of the native microflora on the production of acetic and propionic acids in a semi-hard, Morbier-type cheese was confirmed by Buchin et al. (1998) (comparison of R and P cheeses), by Klantschitsch et al. (2000) in Raclette (comparison of R, P and MF cheeses) and by

Table 5 Volatile fatty acids in raw (R), pasteurised (P) and microfiltered (MF) milk cheeses

Cheese

Formic

Acetic

Propanoic

Swiss-type

RP

RP

RP

Swiss-type

R MF

R MF

Swiss-type

R MF varb RPM RPM F F

Swiss-type Emmental

2-Methyl propanoic (isobutyric)

Butanoic (C4)

2-Methyl butanoic

3-Methyl butanoic (isovaleric)

Pentanoic (C5)

n.d.a

R MF

R MF

var

R MF

var

n.d.

R P MF

n.d.

Nonanoic (C9)

RP

RP

RP

RP

RP

RP

n.d.

RP

RP

RP

RP

n.d.

n.d.

n.d.

n.d.

n.d.

Cheddar

RP

R (1 °C) RP R (8 °C) P

n.d.

R (1 °C) R (8°C) P

RP

Morbier-type (semi-hard cheese) Raclette

RP

R (8 °C) R (1 °C) P RP

n.d.

n.d.

n.d.

RP

Roncal

RP (240 d)

n.d.

Octanoic (C8)

R MF

RP

a n.d.: no difference. b var: variable.

RP

Heptanoic (C7)

R MF

n.d.

RP MF (90 d, 17 °C) RP (240 d)

Hexanoic (C6)

n.d.

Cheddar

R P, MF

4-Methyl pentanoic (isocaproic)

n.d.

Reference EliskasesLechner et al. (1999) Bouton and Grappin (1995) Demarigny et al. (1997) Beuvier et al. (1997) Buchin et al. (unpublished) Shakeel-UrRehman et al. (2000c) Shakeel-UrRehman et al. (2000a) Buchin et al. (1998) Klantschitsch et al. (2000)

n.d.

n.d.

n.d.

Ortigosa et al. (2001)

329


Ortigosa et al. (2001) in ovine Roncal cheese (comparison of R, P and P Lb. casei). In Cheddar cheese, the variations in VFAs originating from amino acid catabolism were not the same as those of other VFAs. In a study by Shakeel-Ur-Rehman et al. (2000c), 2- and 3-methyl butanoic acids were found at higher levels in P cheeses than in R cheeses, and in another of their studies (Shakeel-Ur-Rehman et al., 2000a), 2-methyl propanoic and 3-methyl butanoic acids were found at higher levels in cheeses ripened at low temperature (1 °C compared to 8 °C). These compounds correlated negatively with the number of NSLAB in the cheeses, so the authors hypothesised that NSLAB have a minor role in the formation of these compounds or that NSLAB broke them down further. Carbonyl compounds

Tables 6 and 7 summarise the differences between ketones and aldehydes found in R, P or MF cheeses. Diacetyl (2,3-butanedione) and acetoin (3-hydroxy2-butanone) are products of the metabolism of citrate by Lc. lactis spp. lactis bv. diacetylactis, Leuconostoc spp. or some Lactobacillus species (McSweeney and Sousa, 2000). They are important aroma compounds in numerous varieties of cheese (Curioni and Bosset, 2002). Nevertheless, they tend to decrease during ripening. In 6-month-old Cheddar, diacetyl is present in quantities too low to participate in flavour (Urbach, 1997). In cheese, diacetyl can be converted into acetoin, then 2,3butanediol and 2-butanone and finally 2-butanol (Urbach, 1993). It is notable that 2-butanone and 2-butanol, in contrast to diacetyl and acetoin, generally increase during ripening, like other methyl ketones and secondary alcohols (Bosset and Liardon, 1985; Barlow et al., 1989; Urbach, 1993; Carbonell et al., 2002). Therefore, it is likely that the transformation of diacetyl into the more reduced compounds progresses throughout ripening due to the enzymatic activities of microorganisms, so the raw milk microflora can be expected to influence these compounds. Indeed, diacetyl and acetoin were found in lower quantities in R cheeses than in P or MF cheeses in semi-hard (Buchin et al., 1998; Shakeel-Ur-Rehman et al., 2000a,c; Ortigosa et al., 2001) or Swiss-type cheeses (Buchin et al., unpublished). The contrasting result was observed in only one study on Morbier-type cheese (Buchin et al., unpublished). The quantities of 2-butanone in ripened cheese were affected differently by the presence of the raw milk microflora. Ortigosa et al. (2001) found less 2-butanone in Roncal cheese made from raw ewes’ milk than in cheese made from P milk, but the opposite was true for the Morbiertype cheeses studied by Buchin et al. (1998). These contradictory results could be expected because the amount of 2-butanone in a ripened cheese arises from

the balance between its formation and its reduction to 2-butanol. It is difficult to establish a correlation between the presence of microbial populations and the level of 2-butanone at a given time of ripening, because of the continuous production and degradation of this compound. To follow the evolution of microbial growth and the level of 2-butanone throughout the ripening period may provide valuable information. In contrast, as expected, 2-butanol was more abundant in R cheeses than in P cheeses in all studies (Buchin et al., 1998; Shakeel-Ur-Rehman et al., 2000c; Ortigosa et al., 2001). The other carbonyl compounds found in cheeses are methyl ketones and aldehydes. Their characteristics have been summarised in a recent review by Curioni and Bosset (2002). Methyl ketones originate from the -oxidation of fatty acids by microorganisms. Their aromatic impact is of primary importance in blue and surface mould-ripened cheeses, but they are also likely to have an influence on the flavour of other varieties, e.g., 2-heptanone in Emmental, Gruyère and Grana Padano. This ketone is typical of blue cheese flavour, whereas the others have fruity, floral or musty notes. Straight-chain aldehydes originate from the oxidation of unsaturated fatty acids. They are characterised by green, fatty odours. The branched-chain aldehydes with four or five carbon atoms originate from the catabolism of the amino acids, valine (2-methyl propanal), leucine (2-methyl butanal) and isoleucine (3-methyl butanal). In cheese, they have green or malty notes. As previously noted, the variations of these compounds in the presence of the raw milk microflora differed according to the study. In the studies of ShakeelUr-Rehman et al. (2000c) and Buchin et al. (1998) on Cheddar and Morbier-type cheese, respectively, R cheeses contained less of the different methyl ketones and aldehydes than P cheeses. This is in contrast with the results of the studies of Shakeel-Ur-Rehman et al. (2000a) comparing R and P Cheddar, and Buchin et al. (2002) comparing R and MF Morbier-type cheeses. The results of Buchin et al. (2002) showed less ketones but more aldehydes in R Swiss-type cheeses compared to MF ones. The explanation for these contrasting results may be the same as that for the products of pyruvate metabolism. Methyl ketones and aldehydes are intermediate products in the degradation of fatty acids or amino acids. Due to the enzymatic activities of microorganisms in cheese, methyl ketones are progressively reduced to 2-alkanols, aldehydes are oxidised to acids or reduced to n-alkanols. Therefore, their levels depend on the balance between production and degradation, which is linked to the degree of maturity of the cheese. The maturity of the cheeses for each of the studies may be


Table 6 Ketones in raw (R), pasteurised (P) and microfiltered (MF) milk cheeses Cheese

Emmental

Cheddar

Cheddar

Semi-hard

Semi-hard

Roncal

Propanone 3-Hydroxy-2butanone (acetoin) 2,3-Butanedione (diacetyl) 2-Butanone

R MF R MF

RP

RP

n.d.a RP

n.d. R MF

n.d. n.d.

R MF

RP

n.d.

R MF

RP

R MF

RP (120 d)c RP (120 d)

3-Methyl 2butanone 2-Pentanone 2,3-Pentanedione 4-Methyl 2pentanone 3-Methyl 2pentanone Cyclopentanone 2-Hexanone 4-Methyl cyclohexanone Cyclohexanone 2-Heptanone 3-Methyl 2heptanone 5-Methyl 2heptanone 6-Methyl 2heptanone 6-Methyl-5-hepten2-one 2-Octanone 2-Nonanone 8-Nonen-2-one 2-Decanone 2-Undecanone 2-Dodecanone 2-Tridecanone 2-Pentadecanone Acetophenone Reference

a b c d

n.d. R MF

RP

n.d.

R MF

RP

R MF

RP (240 d)d n.d. n.d.

RP R MF n.d.

n.d.

RP

R MF

RP

RP

n.d. n.d. n.d. n.d. RP n.d.

n.d. R MF

n.d.

R MF

n.d.

varb

R MF n.d. n.d. R MF R MF

n.d.

n.d. Buchin et al. (unpublished)

n.d. RP RP RP RP RP RP n.d. R

RP RP PR n.d. RP RP RP RP

Shakeel-UrRehman et al. (2000c)

Shakeel-UrRehman et al. (2000a)

n.d. n.d.

n.d.

MF Buchin et al. (1998)

Buchin et al. (unpublished)


n.d.: no difference. var: variable. 120 d: 120 days of ripening. 240 d: 240 days of ripening.

different, which could explain the different results from one study to another, even using the same technology. Alcohols

Table 8 summarises the differences between alcohols obtained in R, P or MF cheeses. The observation of the levels of alcohols in the cheeses confirms the previous hypothesis. The presence of the native microflora in milk has a major influence on

the production of alcohols in cheeses. All studies agreed that R cheeses contained greater amounts of the different alcohols, compared to P or MF cheeses (Buchin et al., 1998; Shakeel-Ur-Rehman et al., 2000a,c; Ortigosa et al., 2001; Buchin et al., 2002). This was valid for n-alkanols as well as 2-alkanols. Because alcohols result from the reduction of previously formed compounds, it can be concluded that the raw milk microflora induced faster cheese ripening.


Table 7 Aldehydes in raw (R), pasteurised (P) and microfiltered (MF) milk cheeses Cheese

Emmental

Acetaldehyde Propanal 2-Methyl propanal Butanal 3-Methyl butanal 2-Methyl butanal 2-Methyl butenal Pentanal Hexanal Heptanal 2,4-Heptadienal Octanal Nonanal Decanal Undecanal Dodecanal Tetradecanal Hexadecanal Furancarboxaldehyde Benzaldehyde Phenylacetaldehyde

R MF

n.d.

R MF

R MF

Reference


a b c d

Cheddar

Cheddar

R MF R MF n.d. n.d.

n.d. R MF R MF

n.d.

Semi-hard

n.d. RP n.d. n.d. n.d.

n.d. PR PR

n.d. n.d.

RP

n.d.

n.d. varb RP RP var

RP RP var RP RP



n.d. n.d. n.d. n.d.

Semi-hard

R MF

Roncal

n.d. var P R (240 d)c n.d. R P (120 d)d

n.d. n.d. R MF n.d. R MF R MF

R P (240 d) R P (240 d)

n.d. Buchin et al. (1998)



n.d.: no difference. var: variable. 240 d: 240 days of ripening. 120 d: 120 days of ripening.

Esters

Esters are formed by the condensation of an acid and an alcohol. In cheese, this reaction may be spontaneous, or may be mediated by microbial esterases. According to Urbach (1995), esters are not likely to be formed by the starter culture, although Yvon and Rijnen (2001) reported that esterification reactions can be mediated by various LAB, including lactococci, lactobacilli, Sc. thermophilus, leuconostocs and pediococci. This ability is highly strain-dependent. Esters generally have fruity odours and may also influence cheese flavour. They are particularly numerous in hard cheeses such as Swiss Emmental and Parmesan, in which they play an important role in flavour (Urbach, 1997). In contrast, their presence in Cheddar cheese is limited; in fact, fruity flavour in this cheese is a defect. Table 9 summarises the differences between esters obtained in R, P or MF cheeses. In all studies (Buchin et al., 1998, 2002; Shakeel-UrRehman et al., 2000a,c; Ortigosa et al., 2001), whatever the cheese variety, the presence of the raw milk microflora was linked to a greater formation of esters. As expected, ethyl esters were the most important, in rela-

tion to the levels of ethanol, and they were more diversified in Swiss-type cheese (Buchin et al., unpublished). Sulphur compounds

Sulphur compounds contribute to cheese flavour. They are numerous in mould- or smear-surface cheeses, and provide typical cabbage or garlic flavours (Urbach, 1997; Yvon and Rijnen, 2001). Hydrogen sulphide, methional, methanethiol, dimethyldisulphide and dimethyltrisulphide are related to Cheddar flavour (McSweeney and Sousa, 2000); methional, methanethiol and dimethyltrisulphide are key flavour compounds in Emmental cheese (Rychlik and Bosset, 2001), 3-methylthio-1propanol is present in premium quality Cheddar cheese, ethyl 3-methylthiopropanoate in Parmesan cheese, while methanethiol is related to unpleasant odours in Grana cheese (Urbach, 1997). The sulphur compounds in cheese derive from the sulphur amino acids. Several mechanisms are involved in their formation. In the reducing environment of cheese, purely chemical decomposition of methionine or cysteine could occur to produce compounds such as methanethiol or H2S. A negative redox potential is a necessary condition for the production of volatile


Table 8 Alcohols in raw (R), pasteurised (P) or microfiltered (MF) milk cheeses Cheese

Emmental

Ethanol 1-Propanol 2-Methyl propanol 1-Butanol 3-Methyl butanol 1-Pentanol 1-Hexanol 1-Heptanol 1-Octanol 1-Nonanol 1-Decanol 2-Ethyl 1-decanol 2-Propanol 2-Propen-1-ol 2-Butanol 3-Methyl 2-butanol 2-Pentanol 2-Hexanol 2-Heptanol 2-Octanol 2-Nonanol 2-Decanol 3-Methyl 3-buten-1-ol 3-Methyl 2-buten-1-ol 3-Penten-2-ol 2-Methyl 3-pentanol 2,3-Butanediol 1,3-Butanediol Furan methanol Phenol Phenethyl alcohol

R MF R MF

Reference

a b c d

R MF R MF

R MF n.d. R MF R MF R MF

R MF

Cheddar

PR10d P PR10 P PR10 P RP PR10 P n.d. PR10 P PR10 P PR10 P

Cheddar

R RP n.dc RP n.d. RP RP n.d.

Semi-hard

Semi-hard

Roncal

RP RP

n.d. R MF

RP n.d. n.d. RP

R MF R MF

R P (240 d)a R P (120 d)b P (240 d) n.d. R P (240 d)

n.d.

n.d.

n.d. n.d. R MF

R P (240 d) R P (120 d)

n.d.

R P (120 d)

n.d. RP PR10 P RP PR10 P RP PR10 P PR10 P PR10 P

RP

RP RP RP

R MF

n.d.

R MF

n.d. n.d. n.d. n.d. PR10 P

n.d.

RP

n.d.




n.d. n.d. n.d.

n.d. Buchin et al. (1998)



240 d: 240 days of ripening. 120 d: 120 days of ripening. n.d.: no difference. PR10: mix of 90% pasteurised milk with 10% of raw milk.

sulphydryl compounds in cheese, but enzymatic reactions may also be involved (Urbach, 1997). On the one hand, native milk enzymes may produce disulphide linkages as precursors of sulphydryl groups, and heating of milk stops the production of H2S and reduces the production of methanethiol by inactivating these enzymes (Urbach, 1995). On the other hand, the surface microorganisms of smear cheeses are high producers of sulphur compounds, like methanethiol or methylthioesters. Lactic acid bacteria may also contribute to the production of sulphur compounds. Cheese lactobacilli can produce H2S and Lc. lactis has the ability to cleave methionine

and produce methanethiol (Yvon and Rijnen, 2001), and starters may contribute by providing a reducing environment. The further formation of dimethyldisulphide or dimethyltrisulphide and of most of the methyl thioesters from methanethiol is due to chemical rather than biological reactions. Table 10 summarises the differences in sulphur compounds between R, P or MF cheeses. As expected, comparisons of R and P cheeses showed higher levels of sulphur compounds in the R cheeses (Buchin et al., 1998; Shakeel-Ur-Rehman et al., 2000c; Ortigosa et al., 2001), except in the study by


Table 9 Esters in raw (R), pasteurised (P) and microfiltered (MF) milk cheeses Cheese

Emmental

Methyl acetate Methyl propanoate Methyl butanoate Methyl hexanoate Methyl octanoate Ethyl methanoate Ethyl acetate Ethyl propanoate Ethyl butanoate Ethyl hexanoate Ethyl heptanoate Ethyl octanoate Ethyl decanoate Ethyl dodecanoate Ethyl tetradecanoate Propyl acetate Propyl propanoate Propyl butanoate Butyl acetate Butyl propanoate Pentyl acetate 1-Methyl-propyl acetate 2-Methyl-propyl propanoate 2-Methyl-propyl butanoate 3-Methyl-butyl acetate 2-Methyl-butyl butanoate 3-Methyl-butyl butanoate

R MF n.d.a n.d. n.d.

R MF R MF n.d. n.d. n.d.

R MF R MF R MF R MF

R MF R MF n.d. R MF

Reference

a b c d

R MF

Cheddar

Cheddar

RP

RP

RP PR10d P PR10 P RP

RP RP RP RP

n.d. n.d. n.d. R MF n.d.

Semi-hard

RP n.d.

Semi-hard

Roncal

n.d. R P (120 d)b R P (240 d)c n.d.

R MF n.d.

n.d.

R MF R MF R MF n.d. n.d. R MF

R MF R MF

R MF

R MF R MF

R MF n.d.

n.d.

n.d.




Buchin et al. (1998)



n.d.: no difference. 120 d: 120 days of ripening. 240 d: 240 days of ripening. PR10: mix of 90% pasteurised milk with 10% of raw milk.

Shakeel-Ur-Rehman et al. (2000a), where dimethyldisulphide and dimethyltrisulphide were absent from R cheeses. In comparisons of R and MF cheeses, Buchin et al. (2002) found no differences in Morbier-type cheeses, and only a higher level of dimethyldisulphide in raw milk Swiss-type cheeses. This would indicate that the inactivation of native enzymes by heating the milk may be a major event in the diminution of sulphur compound formation, compared to the elimination of the native flora. It is noteworthy that in all these studies, the diversity of the sulphur compounds reported was very poor. Lactones, hydrocarbons

Table 11 summarises the differences in lactones between R and P cheeses.

Lactones are the result of spontaneous cyclisation of the hydroxy-acids naturally present in milk fat. In the studies by Shakeel-Ur-Rehman et al. (2000a,c), heattreatment of the milk influenced the levels of some lactones, but the results were inconsistent. Their occurrence in cheese may also be linked to feeding (Urbach, 1997). Whether aliphatic or aromatic, the levels of hydrocarbons in cheeses do not seem to be influenced by the presence of the native microflora in milk. In conclusion, the presence of the native microflora in R cheeses is of primary importance for the formation of most volatile compounds. Nevertheless, considering the present state of knowledge, it is difficult to establish precisely the role of this microflora. This role can be direct, in transforming the milk constituents into volatile


Table 10 Sulphur compounds in raw (R), pasteurised (P) and microfiltered (MF) milk cheeses Cheese

Emmental

Carbon sulphide Carbon disulphide Dimethyl sulphide Dimethyl disulphide Dimethyl trisulphide Methional Methane sulfonylbis

n.d.a

n.d.

n.d. R MF

n.d. n.d.

n.d. R P (240 d)b n.d.



Reference

Cheddar

n.d. n.d. RP

Cheddar

RP RP RP

Semi-hard

RP RP

Semi-hard

Roncal




Buchin et al. (1998)

a n.d: no difference. b 240 d: 240 days of ripening.

compounds, or indirect, by modifying the composition of the cheese, with the production of precursors of volatile compounds or of molecules that influence chemical reactions or the activity of other microorganisms. In particular, the activities of the indigenous populations can interfere with those of the starter bacteria. Within the complexity of the native milk microflora, it is difficult presently to establish the role of each population, at the species and at the strain level. It is likely that many of the metabolic pathways producing volatile compounds are strain-dependant (Yvon and Rijnen, 2001), which would make their elucidation all the more difficult. The development of molecular techniques for the discrimination of microbial populations at the strain level could be very beneficial to such studies. This situation underlines the importance of maintaining a high diversity of strains in the milk, to retain the diversity of the molecules produced.

Table 11 Lactones in raw (R) and pasteurised (P) milk cheeses Cheese

Cheddar

Cheddar

-Octanolactone -Decanolactone -Dodecanolactone -Hexadecanolactone -Decanolactone -Dodecanolactone -Dodecenolactone

PR10b PR n.d. n.d. n.d. RP RP

n.d.a RP RP n.d. n.d. RP RP

Reference



a n.d.: no difference. b PR10: mix of 90% pasteurised milk with 10% of raw milk.

Sensory Aspects In order to avoid any ambiguity, due to the different use of the same terms by different authors, we have chosen to define sensory perceptions as follows: odour is perceived by the nose, with no introduction of the food into the mouth, while flavour is the perception of the food during mastication, either retronasaly or by the tongue (five basic tastes: sweet, acid, bitter, salty, umami). Flavour/odour

Table 12 summarises the differences between the flavour and odour attributes reported for R, P or MF cheeses. Raw milk cheeses ripen faster than cheeses made from milk, the microflora of which has been removed. As a consequence, R cheeses tend to develop a stronger odour/flavour at the same age than those made from P or MF milk (Johnson et al., 1990b; Lau et al., 1991). This has been observed in all types of cheese studied: Cheddar (McSweeney et al., 1993; Shakeel-Ur-Rehman et al., 2000a,b), Manchego (Gaya et al., 1990; FernandezGarcia et al., 2002; Gomez-Ruiz et al., 2002), Raclette (Gallmann and Puhan, 1982), other hard and semi-hard cheeses (Lau et al., 1991; Van den Berg and Exterkate, 1993; Buchin et al., 1998; Skie and Ardö, 2000), Bergkäse (Ginzinger et al., 1999a), Swiss-type cheeses (Bouton and Grappin, 1995; Beuvier et al., 1997; Demarigny et al., 1997) and soft goats’ milk cheese (Morgan et al., 2001). In all cases, this phenomenon seems to be directly linked to the activity of the indigenous microflora of the milk. In Cheddar, it has been attributed, in part, to the presence of NSLAB (composed mainly of lactobacilli, but also of pediococci and micrococci) in the raw milk, which are the major part of the natural microflora of this variety of cheese


Table 12 Characteristic flavour and odour attributes of raw (R), pasteurised (P) and microfiltered (MF) milk cheeses Cheese variety

Raw milk

Bergkäse

Odour: intense Flavour: intense Flavour: intense, typical, acid, pungent Flavour: intense, pungent, salty Odour: intense, creamy/milky, fruity/sweet, acid/sharp, pungent Flavour: intense, sour/ acid, sulphur/eggy, bitter, rancid, unclean Only cheeses ripened at 8 °C (vs 1 °C) Odour: intense, acid Flavour: intense, sour Odour: intense, fruity/ sweet, pungent Flavour: sour/acid Flavour: intense, of acid milk, of rind, Flavour: of fresh milk, fruity, of garlic, spicy, animal, chemical, rancid, bitter, pungent Flavour: intense, animal, spicy, sour

Swiss-type Swiss-type Cheddar

Cheddar

Cheddar

Semi-hard cheese, Morbier-type

Semi-hard round-eyed cheese Roncal

Idiazabal

Idiazabal

Odour: intense (120 d), animal (240 d) Flavour: characteristic, pungent (240 d), animal Aftertaste:intense Odour: characteristic, pungent, sour Flavour: characteristic, pungent, salty Aftertaste: characteristic, pungent Flavour: characteristic, creamy, pungent, acid

Pasteurised milk

Microfiltered milk

Flavour: bitter Flavour: bitter Flavour: acid, bitter, salty Odour: musty

Reference Ginzinger et al. (1999a) Bouton and Grappin (1995) Beuvier et al. (1997) Shakeel-UrRehman et al. (2000a)

Shakeel-UrRehman et al. (2000b) Shakeel-UrRehman et al. (2000c) Buchin et al. (1998)

Skie and Ardö (2000) Odour: animal (120 d)


Flavour: torrefied (240 d) Odour: sweet

Mendia et al. (1999)

Flavour: sweet, bitter, sour Aftertaste: bitter, sour Flavour: sweet

Ordonez et al. (1999)

Odour: sweet

(McSweeney et al., 1993). In Swiss-type cheeses (Beuvier et al., 1997), flavour intensity was correlated with counts of FHL, propionibacteria and enterococci, which occur naturally in the raw milk. In pasteurised milk cheeses, denaturation of enzymes and whey proteins by the heat treatment may also be involved; the aggregation of whey proteins on the surface of the caseins micelles also prevents proteolysis of the caseins. This difference in maturity is enhanced by the temperature of ripening and depends on the age of the cheese (Klantschitsch et al., 2000; Shakeel-Ur-Rehman et al., 2000b).

Besides the intensity of flavour, differences in the flavour profile of cheese can be observed. The flavour of the ripened cheese is richer and more complex when the indigenous microflora is present in the milk to be processed. Some observations are constant from one study to another, whereas others vary. In almost all studies comparing cheeses made from raw milk and raw milk after elimination of the microflora, the R cheeses received a higher score for the pungent attribute. Similarly, acid, sour or rancid characteristics were also generally higher in these cheeses. It is likely that these sensory attributes are related to the presence of volatile and FFAs (Curioni and Bosset, 2002;


Gomez-Ruiz et al., 2002). In general, R cheeses are characterised by more ‘strong’ attributes, such as animal, garlic, spicy, sulphur and unclean. All these characteristics of R cheeses are linked to the notion of higher maturity, expressed from a sensory point of view, but also revealed by physico-chemical patterns, i.e., a greater degree of proteolysis, a higher content of most volatile compounds, and sometimes greater lipolysis (Fig. 2). The distribution of milder attributes, such as fruit, milk or sweet, differs with the study; they can be characteristic of cheeses made either from R or P milk. In Idiazabal cheese, Ordonez et al. (1999) found a relationship between the sweet taste and the amounts of free proline and asparagine, which were higher in P cheeses. Fruity notes may be linked to some methyl ketones such as 2-nonanone (Gomez-Ruiz et al., 2002) or esters (Ortigosa et al., 2001; Gomez-Ruiz et al., 2002). Milky notes are characteristic of diacetyl and acetoin (GomezRuiz et al., 2002). The relationship between bitterness and the presence of the microflora depends on the variety of cheese. When differences were observed in relation to the milk treatment, R semi-hard cheeses were more bitter than P (Buchin et al., 1998; Shakeel-Ur-Rehman et al., 2000a) or MF cheeses (Buchin et al., 2002). In contrast, hard cheeses made from R milk were less bitter than those made from MF or P milk (Bouton and Grappin, 1995; Beuvier et al., 1997; Mendia et al., 1999; Ginzinger et al., 1999a). On the one hand, it seems that the presence of the indigenous microflora is involved,

Axis 3 14 %

Acid milk diMe trisulphide 3Me 2butanol Garlic Animal 1propanol diMe disulphide 2butanol Bitter C2 Intense 2pentanol 1hexanol C3 2butanone Chemical Et hexanoate Rancid ethanol Pungent 1butanol C5

isopentanone Fruit 2heptanone 2,3pentanedione Axis 1 diacetyl 43 % acetoin 2pentanone 3me butanal 3me 2pentanone heptane Fresh milk

Figure 2 Distribution of volatile compounds and flavour attributes (additional variables, italicized and boldfaced) within R ( ) and P () semi-hard Morbier-type cheese using principal component analysis C2, C3, C5: acetic, propionic, valeric acids (from Buchin et al., 1998).

because P and MF cheeses were similar and differed from R cheeses. On the other hand, the heat treatment is likely to play a role. In the study by Beuvier et al. (1997), where R, P, MF and P indigenous microflora (PR) milks were processed, the most bitter cheeses were P and PR. Bitterness is attributed mainly to the presence of hydrophobic peptides, resulting from the hydrolysis of caseins, mostly s1- and -. Bitterness in cheese results from the balance between the production of bitter peptides by the action of rennet (preferentially in semi-hard cheeses), plasmin (preferentially in hardcooked cheeses), bacterial proteinases and peptidases, and their further degradation by bacterial peptidases. The role of the respective proteolytic systems and their interactions are known to differ according to the cheese variety (Bergère and Lenoir, 1997). In Bergkäse, a Swisstype cheese, Ginzinger et al. (1999a) found more hydrophobic peptides in P cheeses, which were also more bitter than R cheeses. This distribution of peptides was confirmed in Cheddar by Lau et al. (1991). According to Gomez et al. (1997), bitterness of peptide origin is more likely to be masked by other flavour components in R than in P cheeses. Bergère and Lenoir (1997) pointed out that other components such as indole, amino acids, amines, amides, long-chain ketones or monoglycerides could contribute to the bitter taste of cheese. Thus, Ordonez et al. (1999) found a relationship between bitterness and the amounts of arginine and aromatic amino acids in Idiazabal, though no differences in bitterness were found between R and P cheeses. It is likely that the presence of the R milk microflora affects the flavour characteristics in two ways: on the one hand, acceleration of ripening by faster metabolic pathways, and, on the other hand, the occurrence of a greater variety of metabolic pathways, specific to particular strains of bacteria, which is also influenced by the microbial diversity. The acceptability of R cheeses is also dependent on the cheese variety. Cheddar cheese made from raw milk is, in general, of lower quality than that made from pasteurised milk ( Johnson et al., 1990b; McSweeney et al., 1993). In the study of Shakeel-Ur-Rehman et al. (2000b), R Cheddar cheese received higher flavour scores than P cheeses, but this was dependent on the ripening temperature, as higher temperatures (8 °C instead of 1 °C) led to defects in R cheeses after 6 months. In the study by Morgan et al. (2001), soft goat’s milk cheeses had more flavour defects when made from raw than from pasteurised milk, in relationship to microflora and lipolysis levels. Moreover, although the ‘goat’ flavour of these cheeses is linked to the liberation of particular fatty acids (Le Quéré et al., 1996), no differences were found in this attribute.


According to Klantschitsch et al. (2000), the quality of Raclette cheese in relation to raw milk is related to ripening temperature and time; to avoid flavour and openness defects, R cheeses should be ripened for less than 90 days at 11 °C or 60 days at 14 °C, whereas P or MF milk cheeses can be ripened at 17 °C for 90 days. These differences in acceptability are of course related to the speed of ripening, since the presence of the native microflora accelerates biochemical transformations in the cheese. The difference in maturity, and hence in the occurrence of defects, is more perceptible in soft or semi-hard cheeses, because of their high moisture content; biochemical activities are favoured by the presence of water. Thus, besides the elimination of pathogens, pasteurisation is useful in this type of cheese to obtain a longer shelf-life by slowing the ripening and delaying the occurrence of flavour defects. The consumer of these cheeses is used to the milder flavour provided by pasteurised milk, and may regard the stronger flavour of R cheeses as a defect. It is likely that these varieties of cheese made from raw milk would be appreciated mostly by ‘connoisseurs’. Conversely, Swiss-type cheeses, hard Italian cheeses ( Johnson et al., 1990b; Bouton and Grappin, 1995), or hard Spanish ovine cheeses, like Idiazabal (Ordonez et al., 1999; Chavarri et al., 2000), are preferred when made from raw milk. Because of their low moisture content, hard cheeses ripen more slowly than soft or semi-hard ones. The presence of the natural microflora in the raw milk may have a lesser influence on the speed of ripening and on the shelf-life of these cheeses. The use of raw milk does not induce defects, and may even reduce some, e.g., bitterness. Moreover, the more complex flavour provided by raw milk may be appreciated by the consumer of hard cheeses. The loss of microflora and, to a lesser extent, of native milk enzyme activities, in pasteurised milk, affects the typical flavour of these cheeses. In Idiazabal cheese, the level of the sensory scores was related to the level of lipolysis, the less lipolysed cheeses being rated ‘rather mild’, suggesting that this cheese requires a minimum level of lipolysis to develop its characteristic flavour (Chavarri et al., 2000). In hard Italian cheeses such as Romano, Parmesan or Asiago, the inhibition of milk lipase (LPL) in pasteurised milk may be detrimental to the development of typical flavour (Johnson et al., 1990b). In goats’ milk cheeses, the preservation of LPL activity can be important for the development of the ‘goat’ flavour, linked to the liberation of typical goat-flavoured fatty acids from glycerides (Le Quéré et al., 1996). According to Ordonez et al. (1999) and Chavarri et al. (2000), the characteristic Idiazabal flavour is related to the extent of proteolysis. In Swiss-type cheeses, Bouton and Grappin (1995) found a relationship between the extent of primary proteolysis

and the flavour intensity, whereas the typical flavour was related to the concentration of propionic acid. The presence of the raw milk microflora contributes to the sensory diversity of raw milk cheeses. This has been supposed by Shakeel-Ur-Rehman et al. (2000b) for Cheddar cheese, and shown in Swiss-type cheese models by Beuvier et al. (1997) and Demarigny et al. (1997). There is a higher heterogeneity in the sensory characteristics of cheeses when the native microflora was retained than when it was removed from milk (Fig. 3). The diversity of R cheeses is likely to depend on the level but also on the nature of the strains present in the microflora. Whether the strains in themselves have different metabolic potentialities or interfere by affecting the activity of starter bacteria has not yet been elucidated. Nevertheless, Bouton and Grappin (1995) have shown an interaction between the composition of starter mixtures and the raw milk microflora in the biochemical transformations and sensory characteristics of Swiss-type cheeses. Thus, whatever the mechanisms involved, the preservation of the microbial diversity in raw milk seems to contribute to the diversity of cheeses such as Swiss-type cheeses, particularly Comté. This diversity in the sensory characteristics is a point of major interest in the production of PDO cheeses. Texture

The texture of cheeses is the macroscopic expression of the structure of the cheese matrix, i.e., its composition and organisation. The texture is formed during two

Axis 2 17 %

R

MF

Elastic

P Bitter Acid

Ferm Entero Granular MesoLb PAB pH Axis 1 iC5 55 % PTA C3

P + bact Pungent αs1-CN γ-CN

Salted Aroma intensity NaCl

C2

Figure 3 Distribution of physico-chemical, microbiological and flavour criteria (additional variables, italicized and boldfaced) within raw (R), pasteurised (P), microfiltered (MF) and pasteurised microorganisms contained in retentate (P Bact) milk using principal component analysis. MesoLb: mesophilic lactobacilli; Entero:enterococci; PAB: propionibacteria; C2: acetic acid; C3: propionic acid; iC5: isoavaleric acid; PTA: PTA-soluble N (from Beuvier et al., 1997).


stages of cheese processing: manufacturing and ripening. The events that occur during these two steps are different in nature. During manufacture, the cheese matrix is formed. It begins with coagulation of the milk, where the caseins organise themselves into a network, entrapping fat globules, water pockets and gas bubbles. The initial structure of the network is thus determined by the composition of the milk, and also by the technological conditions of coagulation: renneting parameters and work in the vat which influence the moisture content of the curd. The network is then modified by acidification due to the fermentation of lactose, that begins in the vat and continues in the mould. Acidification influences the extent of mineralisation of the caseins, and thus their hydration as well as their interactions. During ripening, changes occur in the matrix through the influence of the loss of water and proteolysis. Proteolysis begins with coagulation in the vat; this is essentially primary proteolysis, i.e., internal hydrolysis of casein molecules, by the coagulant or indigenous enzymes of milk, such as plasmin. Secondary proteolysis occurs essentially during ripening, by the action of peptidases of microorganisms. Proteolysis weakens the structure of the casein matrix. It can be easily supposed that removal of the native microflora from raw milk may alter the texture of subsequent cheeses by two major mechanisms. On the one hand, the heat treatment of the milk used to destroy the microflora may alter the structure of the casein matrix by denaturation of whey proteins or the loss of water, or modify the proteolysis patterns by denaturation, activation or modified retention of enzymes. On the other hand, the elimination of most of the indigenous microflora, either by heating or microfiltration, may modify the biochemical changes in cheeses, in particular proteolysis (Grappin and Beuvier, 1997). Among all the articles in which R, P or MF cheeses were compared, few deal with cheese texture. Some work resulted in no differences related to the treatment of milk: no clear differences between R/P/MF milks (McSweeney et al., 1993) and R/P milks (Shakeel-UrRehman et al., 1999) in Cheddar cheese, no sensory textural differences between R/MF milk Swiss-type cheeses (Bouton and Grappin, 1995) or rheological differences between R/P Bergkäse cheese (Ginzinger et al., 1999a). The comparison of R and P cheeses from a texture point of view is difficult, because of the differences in the behaviour of milk during the coagulation step, due to the heat treatment. Depending on the cheesemaking procedures, contradictory findings have been reported, in terms of moisture, on the compositional differences of

cheeses (Lau et al., 1990; Buffa et al., 2001b), or pH (Buffa et al., 2001b). Texture differences are thus difficult to interpret. The results of Beuvier et al. (1997) seem to indicate that in Swiss-type cheeses, sensory texture characteristics of R cheeses are influenced by both the heat treatment of milk and the activity of the indigenous microflora. They showed in a comparison of R, P, MF and P cheeses to which the indigenous microflora contained in microfiltration retentate had been added, that R cheeses had a firmer and more granular texture. Proteolysis appears to be the main factor responsible for differences in texture between R and P cheeses. According to Shakeel-Ur-Rehman et al. (2000a), the increase in chymosin retention and in plasmin activity by heat-treatment of milk is the major cause of texture differences between R and P Cheddar cheeses ripened at 1 or 8 °C. While the temperature influenced all texture descriptors, milk treatment influenced only rubberiness (P R) and graininess (R P). They attributed the texture characteristics mostly to differences in water-soluble N (WSN) (Shakeel-Ur-Rehman et al., 2000b), in which enzymes such as chymosin or plasmin have more influence than the activity of the indigenous microflora. Gaya et al. (1990) found a lower fracturability, elasticity and hardness in Manchego cheese made from R ewes’ milk than in P cheeses, whatever the ripening time (2 or 4 months) and the ripening temperature (between 8 and 16 °C). They attributed these differences to higher secondary proteolysis in R cheeses, measured by pH 4.6-, TCA- and PTA- soluble N. Buffa et al. (2001b) studied the rheological characteristics of goats’ milk semi-hard cheeses made from R or P milk. R cheeses were firmer, less fracturable, and more cohesive than P ones. These characteristics were attributed to the levels of moisture and WSN: the lower the moisture and more intact the caseins, the less the fracturability and deformability. Fracture stress was higher for R cheeses, i.e., a lower fracturability than the P cheeses. This parameter was correlated with the levels of moisture and WSN: the less the moisture and more intact the caseins, the less the fracturability. Fracture strain, which describes the deformability of cheese, was higher for R cheeses, but only at one day. It could be due to the higher pH of these cheeses at this stage, water being partly absorbed to hydrate the negative charges formed in caseins with high pH values. This parameter has the same correlation with moisture and WSN as previously – deformability decreases when the hydration of proteins decreases and when elastic structural elements disappear. The microstructure of R cheeses was more regular, with a closed protein matrix, and smaller and more uniform fat globules, whereas


P cheeses had an open structure with irregular cavities. As a consequence, differences in colour were observed. Rosenberg et al. (1995) measured the viscoelastic characteristics, G (storage modulus) and G (loss modulus), of Cheddar cheeses. These parameters were higher in R cheeses than in P cheeses ripened for 8 months. In P cheeses, they were found to be related to the extent of proteolysis; a higher G signified a higher elastic behaviour of the matrix with the accumulation of proteolysis products. The authors explained this observation by the binding of water by the ionic groups liberated by the cleavage of peptide bounds. This relation with the extent of proteolysis was not observed in R cheeses, maybe because of different proteolytic activities during the ripening of these cheeses, as revealed by differences in peptide composition. Mendia et al. (1999) found more graininess and firmness and less creaminess and elasticity in R ewes’ milk Idiazabal cheeses than in P cheeses. These differences were attributed to the slower maturation of P cheeses. This was confirmed by the fact that the differences diminished with increases in ripening time, and were thought to be linked to the moisture content. For certain types of cheese consumed mainly in a melted form, such as Raclette, it is more interesting to evaluate the texture characteristics of the cheese after melting. Melting properties were evaluated in Raclette cheeses made from R, P or MF milk and mixtures of the three types of milk in different proportions (Klantschitsch et al., 2000). R cheeses had a longer consistency than P/MF cheeses after 90 days ripening. According to the authors, this is related to the proteolysis patterns, proteolysis ‘in width’, pH 4.6 N/TN (lower in MF) leading to longer consistency and higher viscosity, proteolysis ‘in depth’ (NPN/TN) leading to shorter consistency. The viscosity did not differ between the cheeses. The firmness of melted cheese was also higher in R than in P/MF cheeses after 90 days ripening, with a score indicating insufficient melting quality. Fat separation increased more rapidly with ripening time in R than in P cheeses. Softening and dropping points were in the range for good melting quality in all cheeses ripened at 11 or 14 °C, but only in the MF cheeses ripened at 17 °C. The effect of the microflora on the melting quality of Raclette is dependant on the ripening temperature and time; a high temperature (17 °C) is detrimental when using raw milk, whereas, in the case of microfiltered milk, it is useful to accelerate ripening. In all these studies, the lack of microbial investigations made it difficult to establish a relationship between the microbial populations, whether of indigenous or starter origin, and the characteristics of texture. Nevertheless, when the observed differences were

attributed to the secondary proteolysis, microbial activity was involved.

Conclusion Microbial communities play an essential role in the control of sensory qualities of cheese. They are more diverse and complex in R cheeses for which milk undergoes no treatment to reduce the microflora. They contribute to the development of a typical cheese taste and flavour. Diversity of the sensory qualities is a specific feature of R cheese. Elimination of the raw milk microflora by pasteurisation or microfiltration definitively leads to different cheeses from a sensorial point of view. Still, is it necessary to have raw milk that is sufficiently rich in terms of quantity and diversity of microorganisms? As outlined at the beginning, the improvement in hygienic practices on farms has led to a ‘clean’ raw milk, with low microbial counts (Odet, 1999). Raw milk with a low level of microbes could induce a reduction in sensorial diversity of cheese due to a reduction of microbial diversity. Indeed, Dasen et al. (2003) have observed that the strain diversity of mesophilic lactobacilli in raw milk experimental Cheddar cheese was close to that observed in industrial Cheddar cheese manufactured with pasteurised milk. The former was made from raw milk with a total of around 10 000 cfu ml 1. The fact that raw milk tends to be more and more microbiologically ‘clean’ implies that there is a risk that the sensorial differences between R and P cheeses will be erased. Some experiments in progress, particularly in France, aim to evaluate dairy farming practices, including milking practices, on the raw milk microflora in terms of quantity and diversity. Recently, Michel et al. (2001) observed links between milking practices and the bacteriological quality of milk, showing that it is possible to manage the microbial quality of milk on the farm to promote the technologically ‘useful’ microflora, while maintaining pathogens at a low level. This is a good way to keep the natural microflora in R cheese production, in terms of quantity and diversity, in order to preserve their sensorial diversity. To add selected microorganisms could enhance the aroma of cheese, but the cheese would have a more uniform flavour, a characteristic which is not sought by both the producers and the consumers, because diversity of flavour is considered a special feature of traditional R cheeses (Grappin and Beuvier, 1997). Otherwise, according to Montel (2002), microbial communities may play a key role in the microbiological safety of R cheese. This potential role is supported by several studies in which cheeses or milk, with a more complex microflora, were less contaminated by


L. monocytogenes than those with a less diversified flora (Brouillaud-Delattre et al., 1997; Eppert et al., 1997). Thus, well-monitored microbial diversity, from farm to cheese, by acting as a barrier against pathogens, may be a trump card for cheese safety (Montel, 2002). According to Stanton et al. (1998), cheeses, because of their high fat content and their texture, could offer protection to the living microorganisms contained within them, especially at the moment of their passage into the gastrointestinal tract of the consumer. More and more studies demonstrate the beneficial effects on health of strains of microorganisms and give hope for other discoveries in R cheeses, which are rich in microorganisms (Bouton, 2001; Moreau and Vuitton, 2002). The preservation of the microbial diversity in raw milk, essential to obtain cheeses with greater sensorial diversity, more and more appreciated by (European) consumers, potentially useful to fight against pathogens and potentially useful for health, is a challenge for milk and cheese producers, and researchers, to take up over the next years.

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Biochemistry of Cheese Ripening: Introduction and Overview P.L.H. McSweeney, Department of Food and Nutritional Sciences, University College, Cork, Ireland

Introduction As discussed in ‘Cheese: An Overview’, Volume 1, rennetcoagulated cheeses are ripened (matured) for a period ranging from 2 weeks (e.g., Mozzarella) to 2 or more years (e.g., Parmigiano Reggiano or extra-mature Cheddar) during which the flavour and texture characteristic of the variety develop. Ripening usually involves changes to the microflora of the cheese, including death and lysis of the starter cells, development of an adventitious non-starter microflora and, in many cheeses, growth of a secondary microflora (e.g., Propionibacterium freudenreichii subsp. shermanii in Swiss cheese, moulds in mould-ripened varieties and a complex Gram-positive bacterial microflora on the surface of smear-ripened cheeses). The metabolic activity of the secondary microflora often dominates flavour development, and in some cases, e.g., whitemould cheeses, the texture, of varieties in which they grow. The microbiology of cheese during ripening is discussed in ‘The Microbiology of Cheese Ripening’, Volume 1. As discussed in ‘Rheology and Texture of Cheese’, Volume 1, ripening usually involves the softening of cheese texture, as a consequence of the hydrolysis of the casein matrix, changes in the waterbinding ability of the curd and changes in pH (which may cause other changes such as the migration and precipitation of calcium phosphate). The flavour of cheese curd immediately after manufacture is rather bland and indeed it can be difficult to differentiate the flavours of certain varieties at this stage. During ripening, cheese flavour develops due to the production of a wide range of sapid compounds by the biochemical pathways described below. Volatile flavour compounds are of particular importance to cheese flavour and are discussed in ‘Sensory Character of Cheese and its Evaluation’, Volume 1. Quantification of the volatile flavour compounds of cheese are described in ‘Instrumental Techniques’, Volume 1. Biochemical reactions which occur in cheese during ripening are usually grouped into four major categories: (1) glycolysis of residual lactose and catabolism of lactate, (2) catabolism of citrate, which is very important in certain varieties, (3) lipolysis and the

catabolism of free fatty acids and (4) proteolysis and the catabolism of amino acids (Fig. 1). These reactions are discussed in ‘Metabolism of Residual Lactose and of Lactate and Citrate’, ‘Lipolysis and Catabolism of Fatty Acids in Cheese’, ‘Proteolysis in Cheese during Ripening’, ‘Catabolism of Amino Acids in Cheese during Ripening’, Volume 1. Since the biochemistry of cheese ripening is complex, the purpose of this chapter is to present an overview of the principal biochemical pathways which contribute to cheese ripening and to discuss the role of the principal ripening agents in cheese and the acceleration of cheese ripening. Aspects of cheese ripening common to many varieties are discussed in ‘Metabolism of Residual Lactose and of Lactate and Citrate’, ‘Lipolysis and Catabolism of Fatty Acids in Cheese’, ‘Proteolysis in Cheese during Ripening’, ‘Catabolism of Amino Acids in Cheese during Ripening’, Volume 1; ripening of specific varieties is discussed in the relevant chapters in Volume 2.

Glycolysis of Residual Lactose and Catabolism of Lactate Since cheeses are fermented dairy products, the metabolism of lactose to lactate is essential in the manufacture of all varieties. Cheese curd contains a low level of residual lactose which is metabolised rapidly early in ripening to lactate which may be catabolised subsequently via a range of pathways. Catabolism of lactate probably occurs in all cheeses and is particularly important in surface mould-ripened varieties (e.g., Camembert) and in Swiss cheese. These reactions were reviewed by Fox et al. (1990, 1993) and McSweeney and Sousa (2000) and are discussed in detail in ‘Metabolism of Residual Lactose and of Lactate and Citrate’, Volume 1. The pathway through which lactose is metabolised depends on the starter type (see ‘Starter Cultures: General Aspects’, Volume 1; Cogan and Hill, 1993; Fox et al., 2000; McSweeney and Sousa, 2000; Broome et al., 2003). The final step in the glycolysis of lactose is the conversion of pyruvate to lactate which is catalysed by lactate dehydrogenase (LDH). Depending on



348 Biochemistry of Cheese Ripening: Introduction and Overview

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Figure 1 General overview of the biochemical pathways which operate in cheese during ripening.

the type of LDH (D- or L-LDH) in the cell, D- (e.g., Lb. delbrueckii subsp. bulgaricus), L- (e.g., Lactococcus, Sc. thermophilus) or D/L- (e.g., Lb. helveticus) lactate is the end product of glycolysis which converts 1 mol of lactose to 4 mol of lactate with the production of 4 mol of ATP. Unlike most lactic acid bacteria (LAB), Leuconostoc spp. use the phosphoketolase pathway to metabolise lactose; the end products of this pathway are lactate, ethanol and CO2 and thus differ from that of the glycolytic pathway. Although essential for cheese manufacture, the metabolism of lactose to lactate is essentially complete at the end of manufacture or during the early stages of ripening. Most lactose in milk is lost in the whey and that which is retained in the curd is metabolised rapidly after drainage. However, the activity of the starter is greatly reduced at the end of manufacture or soon thereafter due to the combination of low pH, high NaCl and lack of a fermentable carbohydrate. The inhibition of acid production is particularly abrupt in dry-salted varieties (e.g., Cheddar) where NaCl concentration reaches equilibrium much faster than in brine-salted cheeses. Fresh cheese curd contains a low level of lactose which, in the case of Cheddar cheese, is reduced to trace levels within one

month of ripening by the (albeit reduced) activity of the starter or by the action of the non-starter lactic acid bacteria (NSLAB). Lactate contributes to the flavour of cheese, particularly early during maturation, but the major effect of acidification on flavour development is indirect since, together with the buffering capacity of the curd, it influences pH and thus the growth of the secondary flora and the activity of ripening enzymes. Lactate is an important substrate for a range of reactions which contribute positively or negatively to cheese ripening. L-Lactate, produced by Lactococcus, can be racemised to DL-lactate by the NSLAB flora in Cheddar and Dutch-type cheeses. DL-Lactate is less soluble than L-lactate, resulting in the formation of Ca-D-lactate crystals which appear as white specks on the surface of the mature cheese. Lactate can also be metabolised to acetate and CO2 by some members of the NSLAB flora, although this oxidative pathway is relatively minor in cheese due to its low oxidation– reduction (redox) potential (c. 250 mV) and is limited by the availability of O2. Late gas blowing is a defect in certain hard and semi-hard varieties caused by the anaerobic catabolism of lactate to butyrate and H2

Biochemistry of Cheese Ripening: Introduction and Overview 349

by Clostridium tyrobutyricum. This problem can be overcome by good hygiene, addition of NaNO3 or lysozyme or by the physical removal of the spores by bactofugation or microfiltration. However, catabolism of lactate is particularly important in Swiss and surface mould-ripened cheeses. In the former, lactate is catabolised by Propionibacterium freudenreichii subsp. shermanii to propionate, acetate, H2O and CO2. Propionate and acetate contribute to the flavour of Swiss cheese; CO2 migrates through the curd to points of weakness where it collects to form the large eyes characteristic of Swiss-type cheese. The oxidative catabolism of lactate to H2O and CO2 by Penicillium camemberti at the surface of Camembert and Brie-type cheeses is of great indirect importance to their ripening. The catabolism of lactic acid causes a large increase in the pH of the surface of these cheeses which leads to a pH gradient from the surface to the core and to the migration of lactate towards the surface. The high pH at the surface causes precipitation of calcium phosphate, which, in turn, causes the migration of calcium and phosphate to the cheese surface. These changes lead to the characteristic softening of surface mould-ripened cheese which, when mature, have an almost liquid-like consistency. Oxidative metabolism of lactate is also of significance at the surface of smear-ripened cheeses (e.g., Tilst or Limburger) where, early in ripening, yeasts deacidify the surface which encourages the growth of the Gram-positive bacteria characteristic of the surface. Oxidative metabolism of lactate probably also occurs in Blue cheese but its effect is less important than in surface mouldripened cheese since P. roqueforti is distributed throughout the cheese and thus gradients do not develop across the cheese mass.

Lipolysis and Metabolism of Fatty Acids Studies in which milk fat was replaced with other lipids have demonstrated that milk fat is essential for the development of the flavour of Cheddar and probably all other ripened cheeses. As in all high-fat foods, lipids present in cheese can undergo hydrolytic or oxidative degradation; the latter is generally considered not to be important in cheese, primarily due to its low redox potential. Lipolysis in cheese during ripening is discussed in detail in ‘Lipolysis and Catabolism of Fatty Acids in Cheese’, Volume 1. As discussed by McSweeney and Sousa (2000) and Collins et al. (2003a), lipases in cheese originate from a number of sources. Milk contains an indigenous lipoprotein lipase (LPL), which contributes to lipolysis in cheese during ripening. Lipoprotein lipase activity is more important in cheese made from raw milk than

in that made from pasteurised milk since the enzyme is extensively inactivated by pasteurisation. Rennet paste, used as coagulant in certain Italian cheese varieties, contains a potent lipase, pregastric esterase, which is responsible for lipolysis in cheeses such as Provolone and the Pecorino varieties. Lactic acid bacteria are weakly lipolytic, but their enzymes have been shown to contribute to the low level of lipolysis characteristic of Cheddar cheese (Collins et al., 2003b). Likewise, Pr. freudenreichii subsp. shermanii possesses a lipase which, together with enzymes from the thermophilic starter organisms, contributes to the low level of lipolysis in Swiss cheese. Penicillium roqueforti produces potent extracellular lipases which are responsible for the extensive lipolysis characteristic of Blue cheese. P. camemberti and the complex Gram-positive surface microflora of smear cheeses also produce extracellular lipases which contribute to lipolysis in surface-bacterial or white mould-ripened varieties. The level of lipolysis in cheese is determined using various non-specific techniques (e.g., solvent extraction and titration of the fatty acids with alcoholic KOH or by the formation of coloured Cu soaps) or by quantitation of individual fatty acids, usually by gas chromatography (see Collins et al., 2003a). Fatty acids have a direct impact on the flavour of many cheese varieties. In particular, C4–C10 acids are strongly flavoured. Levels of fatty acids vary considerably between varieties. Many internal bacterially ripened varieties (e.g., Edam, Swiss and Cheddar) contain low levels of fatty acids (c. 200–1000 mg kg 1). Very high levels of fatty acids are found in Blue cheese (c. 30 000 mg kg 1). In addition to their direct role in cheese flavour, fatty acids are important precursors for the production of other volatile flavour compounds during ripening (Fig. 2). Fatty acid esters are produced by reaction of fatty acids with an alcohol; ethyl esters are most common in cheese. Thioesters are formed by reaction of a fatty acid with a thiol compound formed via the catabolism of sulphur-containing amino acids. Fatty acid lactones are cyclic compounds formed by the intramolecular esterification of hydroxyacids; - and -lactones contribute to the flavour of a number of cheese varieties. The principal class of volatile flavour compounds in Blue cheese is n-methyl ketones (alkan2-ones) which are produced from fatty acids by partial -oxidation. n-Methyl ketones may be reduced to the corresponding secondary alcohols. Fatty acid catabolism is summarised in Fig. 2 and is discussed in detail in ‘Lipolysis and Catabolism of Fatty Acids in Cheese’, Volume 1. Volatile flavour compounds in cheese, including those derived from fatty acids, are usually quantified using gas chromatography–mass spectrometry (GC–MS; see ‘Instrumental Techniques’, Volume 1).


Triglyceride

O C O

O C O

O C O

e

as

OH

p Li

O C

OH

γ- or δ-hydroxy fatty acids Partial glycerides H 2O O C O

γ- or δ-lactones

O C

OH

Fatty acids R-SH

Thiols

CH3CH2OH

Ethanol

CO2

O C

Thioesters

Partial β-oxidation

S-R

O C

O C

OCH2CH3

CH3

Alkan-2-ones

Ethyl esters

OH CH CH3

Alkan-2-ols

Figure 2 Pathways for the production of flavour compounds from fatty acids during cheese ripening.

Proteolysis and Catabolism of Amino Acids Proteolysis is the most complex, and in most varieties, the most important biochemical event which occurs during cheese ripening. Proteolysis has been discussed in reviews by Grappin et al. (1985), Rank et al. (1985), Fox (1989), Fox et al. (1993, 1994, 1995), Fox and McSweeney (1996), McSweeney and Sousa (2000) and Sousa et al. (2001) and is covered in detail in ‘Proteolysis in Cheese during Ripening’, Volume 1. Proteolysis is very important for cheese texture by hydrolysing the para-casein matrix which gives cheese its structure and by increasing the water-binding capacity of the curd (i.e., to the new -carboxylic and -amino groups produced on cleavage of peptide bonds). Proteolysis may indirectly affect texture by increasing pH through the production of NH3 following amino acid catabolism.

Peptides may have a direct impact on cheese flavour (some are bitter) or they may provide a brothy background flavour to cheese. However, recent research has indicated that the major role of proteolysis in cheese flavour is in the production of amino acids which act as precursors for a range of catabolic reactions which produce many important volatile flavour compounds (see McSweeney and Sousa, 2000; Yvon and Rijnen, 2001). In most cheese varieties, the initial hydrolysis of caseins is caused by the coagulant and to a lesser extent by plasmin and perhaps somatic cell proteinases (e.g., cathepsin D) which result in the formation of large (water-insoluble) and intermediate-sized (watersoluble) peptides which are subsequently hydrolysed by the coagulant and enzymes from the starter and non-starter flora of the cheese. The production of


small peptides and amino acids is caused by the action of microbial proteinases and peptidases, respectively. Preparations of selected aspartyl proteinases are used to coagulate milk. Chymosin (EC 3.4.23.4) is the principal proteinase (88–94%) in traditional calf rennets, the remainder being pepsin (EC 3.4.23.1) (Rothe et al., 1977). Although, the principal role of the coagulant in cheesemaking is to coagulate milk, some activity is retained in the curd, depending on factors such as coagulant type, cooking temperature and pH at drainage, and contributes to proteolysis in many varieties (Creamer et al., 1985). Plasmin (fibrinolysin; EC 3.4.21.7) is the dominant indigenous proteinase in milk and is produced from its inactive precursor, plasminogen, by a system of plasminogen activators (PA). Inhibitors of plasmin and of PA are also present in milk. Plasmin, which is optimally active at pH 7.5 and 37 °C, is most active in high-cook cheeses due to denaturation of inhibitors and increased activation of plasmin and in cheeses in which the pH increases during ripening (e.g., Blue cheese or the surfaces of white-mould and smearripened varieties). Plasmin is most active on -casein, hydrolysing it at three sites to produce the -caseins and some proteose peptones. Milk contains somatic (white blood) cells, which contain lysosomes, which in turn, contain many proteolytic enzymes. To date, cathepsin D (see review by Hurley et al., 2000) and cathepsin B (Magboul et al., 2001) have been confirmed in milk. Lactic acid bacteria (Lactococcus, Lactobacillus, Streptococcus) possess very comprehensive proteolytic systems that have been studied extensively and reviewed (e.g., Fox and McSweeney, 1996; Kunji et al., 1996; Law and Haandrikman, 1997; Christensen et al., 1999). Lactic acid bacteria possess a cell envelope-associated proteinase (PrtP or lactocepin), 3–4 intracellular proteinases, intracellular oligoendopeptidases (PepO, PepF), a number of aminopeptidases (PepN, PepC, PepG, PepA, PepL), a pyrolidone carboxyl peptidase (PCP), a dipeptidylaminopeptidase (PepX), a proline iminopeptidase (PepI), an aminopeptidase P (PepP), a prolinase (PepR), a prolidase (PepQ), general dipeptidases (PepV, PepD, PepDA) and a general tripeptidase (PepT). They also possess oligopeptide, di/tripeptide and amino acid transport systems (Fig. 3). This proteolytic system is necessary to enable the LAB to grow to high numbers in milk (109–1010 cfu ml 1), which contains only low levels of small peptides and amino acids. PrtP contributes to the formation of small peptides in cheese, probably by hydrolysing larger peptides produced from s1-casein by chymosin or from -casein by plasmin, whereas the aminopeptidases, dipeptidases and tripeptidases (which are intra-

Intracellular proteinases Endopeptidases

Cell envelopeassociated proteinase (CEP, PrtP, Lactocepin)

(PepO, PepF)

Aminopeptidases (PepN, PepC, PepA, PCP, PepL)

Proline-specific peptidases (PepX, PepI, PepR, PepQ, PepP)

Dipeptidase

Caseins

(PepV, PepD, PepDA)

Tripeptidase

LYSIS

(PepT) Amino acids

Transport systems

Di, tripeptides Oligopeptides

No carboxypeptidase

CHEESE

Figure 3 Summary of the proteolytic system of Lactococcus. The proteolytic systems of other lactic acid bacteria are generally similar.

cellular) are responsible for the release of free amino acids after the cells have lysed. Non-starter lactic acid bacteria, although present initially at low numbers (50 cfu g 1 in Cheddar made from pasteurised milk, and probably in other cheeses), grow at a rate largely governed by ripening temperature to reach ⬃107 cfu g 1 within 4 weeks and remain relatively constant thereafter. The activity of the NSLAB appears to supplement the proteolytic action of the starter. Non-starter lactic acid bacteria in cheese are discussed in ‘The Microbiology of Cheese Ripening’, Volume 1. In many cheese varieties, a secondary microflora (secondary starter) is added intentionally and/or encouraged to grow by environmental conditions and has a diverse range of functions, depending on the organisms used. A number of different LAB, e.g., strains of Lactobacillus, have been added to Cheddar cheese with the objective of improving flavour or accelerating ripening. These have proteolytic systems similar to those of other species of LAB. Brevibacterium linens is the best-studied smear microorganism; it secretes an extracellular proteinase and aminopeptidase, and possesses a number of intracellular peptidases, which contribute to proteolysis at the surface of smear-ripened cheeses (see Rattray and Fox, 1999). Penicillium roqueforti produces potent extracellular aspartyl and metalloproteinases and various peptidases which are major contributors to the extensive proteolysis found in Blue cheese. P. camemberti secretes active metallo and aspartyl proteinases which contribute to proteolysis in Camembert and Brie-type cheeses. The final products of proteolysis are amino acids, the concentration of which depends on the cheese variety. The concentration of amino acids in cheese at a given stage of ripening is the net result of the liberation of amino acids from the caseins by proteolysis and


an -keto acid (usually -ketoglutarate) with the production of the corresponding amino acid and an -keto acid corresponding to the amino acid substrate (cf. Fig. 5 for leucine). The second pathway, which is initiated by elimination reactions, is particularly important in the production of volatile sulphur compounds from the side chain of methionine. In addition, decarboxylases remove the carboxylic acid group of amino acids to produce amines, some of which have physiological effects (see ‘Toxins in Cheese’, Volume 1). Decarboxylases may also act on -keto acids to produce aldehydes, which in turn may be oxidised to carboxylic acids or reduced to primary alcohols. The -amino group of amino acids may be removed by the action of deaminases, with the formation of a carboxylic acid and ammonia. In addition, the side chains of amino acids may be degraded by the action of various lysases (see ‘Catabolism of Amino Acids in Cheese during Ripening’, Volume 1).

their catabolism or transformation into other amino acids by the cheese microflora. The principal amino acids in Cheddar cheese are Glu, Leu, Arg, Lys, Phe and Ser. Medium and small peptides contribute to a brothy background flavour in many cheese varieties; short, hydrophobic peptides are bitter. Amino acids contribute directly to cheese flavour as some amino acids taste sweet (e.g., Gly, Ser, Thr, Ala, Pro), sour (e.g., His, Glu, Asp) or bitter (e.g., Arg, Met, Val, Leu, Phe, Tyr, Ile, Trp). However, research in the last decade has shown that accelerating proteolysis does not necessarily accelerate flavour development, suggesting that the production of amino acids is not the rate-limiting step in the development of cheese flavour. It is now generally believed that the principal role of proteolysis in the production of flavour compounds is the liberation of amino acids as precursors for a complex series of catabolic reactions that produce many important volatile flavour compounds. Amino acid catabolism was reviewed by Yvon and Rijnen (2001), and is discussed in detail in ‘Catabolism of Amino Acids in Cheese During Ripening’, Volume 1 and summarised in Fig. 4. Amino acid catabolism appears to proceed via two major pathways – transaminase action and elimination reactions. Transaminases catalyse the transfer of the -amino group from an amino acid to

Casein

Coagulant Plasmin

Ripening Agents in Cheese Agents from five, and possibly six, sources are involved in the ripening of cheese: • enzymes from the coagulant; • indigenous milk enzymes;

Large and small polypeptides Starter proteinases Small peptides Starter and non-starter peptidases

Small peptides

NH3

Amino acids

Deaminases Decarboxylases

Acids

Carbonyls

α-Keto acid 1

Lyases

Transaminases α-Amino acid α-Keto acid 2 Various compounds (e.g., sulphur compounds)

Figure 4 Summary of proteolysis and amino acid catabolism in cheese during ripening.

CO2

Amines


O O

α-Ketoglutarate

C

C

O OH

H2 N

CH

CH2

CH2

CH2

CH2

C

O

C

OH

C

OH

Glutamic acid OH CH2

O

CH2

OH

CH

CO2 O

CH3

O CH3

H2N

CH

C

OH

C

O

C

TRANSAMINASE

H

3-Methylbutanol

CH2

CH2 CH3

CH

CH3

CH3

CH

CH3

Leucine

C

DECARBOXYLASE

CH2 CH

O

OH

CH3

CH3

2-Keto-4-methylpentanoic acid

3-Methylbutanal

O

C

OH

CH2 CH

CH3

CH3

DECARBOXYLASE

CO2

DE

3-Methylbutanoic acid

AM

IN

AS

E

O C

OH

NH3 CH2 CH2

NH2 CH

CH3

CH2 CH3 CH2 CH

CH3

4-Methylpentanoic acid

CH3

3-Methylbutylamine

Figure 5 Catabolism of leucine initiated by transaminase, deaminase or decarboxylase action and volatile flavour compounds which may be formed from this amino acid. Similar catabolic pathways operate for the other branched-chain aliphatic amino acids (isoleucine and valine).

• starter bacteria and their enzymes, which are released after the cells have died and lysed; • enzymes from secondary starters (e.g., Propionibacterium freudenreichii subsp. shermanii, Gram-positive bacteria on the surface of smear-ripened cheese, yeasts and moulds, such as Penicillium roqueforti and P. camemberti), which are of major importance in some varieties; • non-starter bacteria, i.e., organisms that either survive pasteurisation of the cheese milk or gain access to the pasteurised milk or curd during manufacture; and, in certain circumstances, • exogenous enzymes added to accelerate cheese ripening. There has been interest for about 40 years in developing model systems in which to quantify the contribution of each of these agents to cheese ripening. The techniques developed eliminate one or more of the above agents, thereby enabling its role to be assessed, directly or indirectly.

Non-starter bacteria may be eliminated by using an aseptic bucket milking technique, developed by Perry and McGillivray (1964); the teat cups and clusters were chemically sterilised and the bucket steam-sterilised. Cows were screened for the bacteriological quality of their milk and animals with counts 100 cfu ml%l selected; prior to milking, their udders were cleaned with a quaternary ammonium solution. An essentially similar approach was used by O’Keeffe et al. (1976a), who obtained milk with a total bacterial count 500 cfu ml%1. Kleter and de Vries (1974) included a cooling coil between the cluster and the bucket and succeeded in achieving counts averaging 46 cfu ml%1. This approach was also used by Visser (1976). Reiter et al. (1969) withdrew milk aseptically by means of a teat cannula, but the quantities obtained (l l) were sufficient to produce cheeses of only about 100 g. More recently, it has been our experience (McSweeney et al., 1994; Lynch et al., 1996, 1997) that special precautions for milking are unnecessary; good quality raw milk pasteurised at 78 °C for 15 s is suitable for aseptic cheesemaking.


Having collected low-count milk, a heating step is usually used to reduce bacterial counts further. Perry and McGillivray (1964) used batch pasteurisation (68 °C 5 min) in a steam-jacketed cheese vat. Chapman et al. (1966), who did not use an aseptic milking technique, used HTST pasteurisation (71.6 °C 17 s) to produce low-count milk. Reiter et al. (1967), Kleter and de Vries (1974) and Visser (1976, 1977a) also used HTST pasteurisation. An LTLT regime (63 °C 30 min) was used by Reiter et al. (1969) and O’Keeffe et al. (1976a). Le Bars et al. (1975) used a UHT treatment and offset the ill-effects of the high heat treatment on the rennetability of milk by using a higher rennet concentration, a higher setting temperature and adding CaCl2. Tyndallisation (three successive cycles of heating at 75 °C 5 min) or treatment with H2O2 followed by catalase was used by Roberts et al. (1995) to treat milk for the production of aseptic cheese curd. Both treatments were successful, although H2O2 caused the development of oxidised off-flavours. Cheese with a controlled microflora must be manufactured under aseptic conditions. Enclosed vats equipped with integral rubber gauntlets were used by Mabbitt et al. (1959) and modified by Perry and McGillivray (1964) to include pressurised or sterile air. Chapman et al. (1966) and Reiter et al. (1967, 1969) used a similar technique. Le Bars et al. (1975) made cheese in an aseptic room (5 3 m) with a filtered air supply and the cheesemakers were clothed in sterile garments. O’Keeffe et al. (1975, 1976a,b), McSweeney et al. (1994) and Lynch et al. (1996, 1997) made cheese in 20-l vats set in thermostatically controlled water baths in a laminar air-flow unit. If the cheese curd is to be acidified chemically, antibiotics should be added to the cheesemilk to inhibit the growth of any surviving (or contaminating) bacteria. Nisin, penicillin and streptomycin were used by Le Bars et al. (1975) and O’Keeffe et al. (1976a). Addition of antibiotics is probably necessary to achieve aseptic starter-free cheese. Acidification of cheese curd to ⬃pH 5, which is an essential element of cheese manufacture, is normally achieved by in situ production of lactic acid by a culture of LAB (starter). If the contribution of starter to cheese ripening is to be assessed, the use of starter must be avoided and acidification is then accomplished by pre-formed acid or acidogen. Early workers used dilute acid for direct acidification but encountered difficulties in controlling the pH. Mabbitt et al. (1955) largely overcame this problem by using an acidogen, gluconic acid--lactone (GDL), which hydrolyses to gluconic acid at a predictable rate in aqueous solutions. O’Keeffe et al. (1975) found that GDL, used as recommended by Mabbitt et al. (1955), caused

excessively rapid acidification, leading to extensive demineralisation of the casein micelles. Demineralisation was considered to be responsible for the excessively rapid rate of proteolysis observed in chemically acidified cheese but this may have been due to increased retention or activity of rennet in over-acid curd (Creamer et al., 1985). O’Keeffe et al. (1975) overcame excessively rapid acidification by using incremental addition of lactic acid to mimic the pH drop during cooking, followed by the addition of GDL to the curd after whey drainage. Roberts et al. (1995) developed an aseptic system for making cheese curd which was then used to produce slurries. The system consisted of a 1L cylindrical polypropylene ‘vat’ fitted with a lid and a stainless steel cutter/stirrer. The milk was coagulated, cut, cooked and stirred in the sealed ‘vat’. At the end of cooking, the lid was replaced by a steel mesh screen through which the whey was drained off. Role of rennet in cheese ripening

The manufacture of rennet-free cheese is necessary if the contribution of rennet to ripening is to be assessed. Since rennet must be used to form a para-casein curd, the approach usually adopted is to inactivate the rennet after it has completed the first stage of rennetinduced coagulation. Four techniques have been developed to achieve this objective. Visser (1976) used cheesemilk which had been depleted of Ca and Mg by treatment with an ion-exchange resin; at the reduced Ca concentration, the enzymatic phase of renneting could be completed without coagulation. The rennet was subsequently inactivated by heat treatment (72 °C 15 s), the milk cooled to 5 °C and CaCl2 added. To induce coagulation, the renneted milk was heated dielectrically to avoid agitation. Cheesemaking was then completed in aseptic vats. This technique was used by Visser (1977a,b,c) and Visser and de Groot-Mostert (1977). Porcine pepsin is very unstable at pH values near or above neutrality. O’Keeffe et al. (1977) used porcine pepsin as coagulant; after the gel had formed, it was cut and the pH of the curds–whey mixture raised to ⬃7 using NaOH; this technique has been used subsequently by Lane et al. (1997) with satisfactory results. Mulvihill et al. (1979) demonstrated the potential of piglet chymosin for the manufacture of rennet-free cheese; this enzyme hydrolyses bovine -casein but appears to be inactive on s1- or -caseins or to be inactivated rapidly during the early stages of cheesemaking. Its use in cheesemaking was demonstrated in small-scale experiments. Meinardi et al. (1998) developed methodology and equipment for the production of rennet-free cheese


using pH-inactivated pepsin. Pasteurised milk supplemented with CaCl2 was cooled to 6 °C in a cylindrical glass vessel (15 l) equipped with a series of glass tubes through which water could be circulated to control the temperature and to heat the contents of the vessel without stirring. Porcine pepsin was added and the first stage of rennet action allowed to progress. The coagulant was inactivated by titrating the milk to pH 7.8 using NaOH and holding at this pH for 45 min. The milk was adjusted to pH 7.0 using HCl; starter was then added and addition of HCl continued until the milk reached pH 6.5. Shakeel-Ur-Rehman et al. (1999) added pepstatin A (isovaleryl-Val-Val-statine-Ala-statine), a potent inhibitor of aspartyl proteinases, to the curds–whey mixture during cooking; results indicated that this compound very effectively inhibited chymosin action in cheese during ripening. Immobilised rennets have been suggested as another approach to making rennet-free curd (e.g., Fox et al., 1993) but leaching of the enzyme from the solid support makes this technique unsuitable. Furthermore, properly immobilised rennets are unable to coagulate milk as the Phe9Met bond of -casein may not be able to reach the active site of immobilised chymosin and the rate of diffusion of large casein micelles to immobilised rennet is very slow compared to the rate of diffusion of the small chymosin molecule towards the casein micelle (Beeby, 1979). Plasmin

Eliminating the proteolytic activity of plasmin presents more difficulties than eliminating the action of the coagulant. The contribution of plasmin to proteolysis in cheese has been assessed indirectly, i.e., in cheese from which all other agents have been eliminated (e.g., Visser and de Groot-Mostert, 1977). Plasmin is inhibited by soybean trypsin inhibitor which should be suitable for the inhibition of plasmin activity in cheese but no studies to evaluate this approach have been reported. The high heat stability of plasmin and the finding that its activity is increased by high cooking temperatures (Farkye and Fox, 1990) suggest that a model system could be developed in which aseptic curd is produced, the rennet denatured by a suitable cooking temperature and the curd acidified by GDL; such a system would allow plasmin to act in isolation. 6-Aminohexanoic acid (AHA) is an inhibitor of plasmin and/or plasminogen activators but does not inhibit chymosin or bacterial peptidases. It was used by Farkye and Fox (1991) to assess the role of plasmin in Cheddar cheese made without aseptic precautions and with a normal lactic acid starter. -Casein bands on electrophoretograms were less intense in cheeses con-

taining AHA than in the control, suggesting that plasmin plays a role in Cheddar cheese ripening. It was necessary to use a high concentration of AHA to inhibit the plasmin in cheese curd and this appeared to cause increased syneresis and consequently reduced the moisture content of the cheese. Further, since AHA contains N, the background level of soluble N was increased greatly. Several specific irreversible inhibitors of serine proteinases were described by Harper et al. (1985) who recommended dichloroisocoumarin; as far as we are aware, none of these inhibitors have been used in studies on cheese. Since most of the potential plasmin activity in cheese is in the form of its inactive precursor, plasminogen, it is possible to increase plasmin activity in cheese by activation of plasminogen to plasmin using exogenous plasmin inhibitors. Barrett et al. (1999) used urokinase to activate plasminogen to plasmin, while Upadhyay et al. (unpublished) used streptokinase, a plasminogen activator produced by the mastitis pathogen, Streptococcus uberis. In both studies, the rate of proteolysis was accelerated on activation of plasminogen to plasmin. Since plasmin associates with the casein micelles, most exogenous plasmin added to milk is retained in the curd, unlike many exogenous enzymes added to cheese milk, which are lost in the whey. Studies in which exogenous plasmin was added to milk include Farkye and Fox (1992), Farkye and Landkammer (1992) and O’Farrell et al. (2002). Increasing the level of plasmin in milk increased the rate of primary proteolysis, but did not greatly increase the production of secondary proteolysis products. Cathepsin D

Cathepsin D is a lysosomal proteinase found at low levels in milk and which has a very similar specificity on the caseins to chymosin (McSweeney et al., 1995). Thus, it is difficult to assess the role of cathepsin D in proteolysis in rennet-coagulated cheeses due to the presence of the much greater amount of chymosin (or other coagulant). The hydrolysis of s1-casein to s1-CN (f24-199) in ‘rennet-free’ cheeses (e.g., Lane et al., 1997) and in high-cook varieties such as Swiss (in which much of the rennet is inactivated; see for example Cooney et al., 2000) has been attributed to the action of cathepsin D. However, it is also possible that the production of s1-CN (f24-199) in these cheeses was due to a low level of residual chymosin activity rather than cathepsin D which is largely inactivated on pasteurisation (Hayes et al., 2000). However, clear evidence for a minor role for cathepsin D in proteolysis in cheese during ripening has emerged from the study of Quarg, an acid-curd cheese (Hurley et al., 2000) and


a pickled Feta-type cheese made from ultrafiltered milk in which no rennet was used in manufacture (Wium et al., 1998). Other indigenous enzymes

As discussed by Fox (2003), milk contains about 60 indigenous enzymes, of which about 20 have been isolated and characterised in detail. However, the contributions of only plasmin, cathepsin D and lipoprotein lipase (see Proteolysis in Cheese during Ripening and ‘Lipolysis and Catabolism of Fatty Acids in Cheese’, Volume 1) to cheese ripening have been investigated. It is possible that other indigenous enzymes, e.g., xanthine oxidase, sulphydryl oxidase or acid phosphatase may contribute to cheese ripening, although these enzymes have not been studied in this context. Starter enzymes

Advances in the genetics of LAB have permitted study of the roles of specific bacterial enzymes in cheese during ripening. The first enzyme to be studied in this way was lactocepin (cell envelope-associated proteinase, PrtP) of Lactococcus. The gene for this enzyme is plasmid-encoded, and therefore it is easy to produce PrtP mutants. The role of this enzyme has been studied by comparison of cheese made with PrtP or PrtP strains (e.g., Farkye et al., 1990; Law et al., 1993; Lane and Fox, 1997; Broadbent et al., 2002) or cheeses made with control strains and starters with enhanced PrtP activities (e.g., Law et al., 1993). The principal role of PrtP during cheese ripening appears to be the degradation of intermediate-sized peptides produced from the caseins by the action of chymosin or plasmin. The genes for peptidases are chromosomally encoded and therefore more sophisticated techniques are required to prepare mutants with different peptidase genes than those that were used to produce PrtP strains. Strains deficient in specific peptidases (e.g., Christensen et al., 1995; Meyer and Spahni, 1998), strains which overproduce specific peptidases (e.g., McGarry et al., 1994; Christensen et al., 1995) or strains which express peptidase genes from other organisms (e.g., Wegmann et al., 1999; Luoma et al., 2001; Courtin et al., 2002; Joutsjoki et al., 2002) have been developed. Although the objective of many of these studies has been to study the role of peptidases in nitrogen metabolism in LAB in milk, some (e.g., McGarry et al., 1994; Christensen et al., 1995; Courtin et al., 2002) have made cheeses using these mutant strains and thus information is available on the roles of peptidases in cheese during ripening which appears to degrade polypeptides to shorter peptides, usually with the release of amino acids.

Recent research has suggested that the principal role of proteolytic enzymes is the production of amino acids as precursors for a range of catabolic reactions which produce volatile flavour compounds. The genetics of enzymes involved in amino acid catabolism is now an active area of research and it is expected that mutant strains of LAB which are deficient in, or overproduce, specific amino acid catabolic enzymes will be produced in the near future. Cheese made using such strains or their wild types as starters would give very clear insights into the role of specific amino acid catabolic enzymes in the development of cheese flavour. NSLAB and their enzymes

Non-starter lactic acid bacteria affect cheese quality and almost certainly contribute to the intensity of flavour, although sometimes they may cause offflavours in cheese. The role of NSLAB in cheese ripening has been studied actively, principally with a view to explaining the differences observed between cheese made from raw or pasteurised milk. Comparison of raw and pasteurised milk cheese (e.g., Lau et al., 1990, 1991; McSweeney et al., 1993; Shakeel-Ur-Rehman et al., 1999) generally showed that raw milk cheese ripens more quickly and develops a stronger flavour than pasteurised milk cheese. Descriptive sensory analysis of the flavour profiles of raw and pasteurised milk Cheddar cheese (Muir et al., 1997) showed that raw milk cheese is more intensely flavoured than pasteurised milk cheese but has higher ratings for certain atypical flavours and is more variable. The role of NSLAB in cheese has been studied by physically removing them by microfiltration (e.g., McSweeney et al., 1993; Bouton and Grappin, 1995; Beuvier et al., 1997; Roy et al., 1997) or by inhibiting their growth by the addition of antibiotics at salting (e.g., Walsh et al., 1996; Shakeel-Ur-Rehman et al., 1999) or by the use of bacteriocin-producing starters (Fenelon et al., 1999). Several investigators have added selected strains of NSLAB to pasteurised milk as adjuncts (e.g., Broome et al., 1990; Muir et al., 1996). Cheese has been made under controlled bacteriological conditions in an attempt to prevent NSLAB gaining access to the cheese from the environment (e.g., McSweeney et al., 1994; Lynch et al., 1996, 1997). Raw milk (Shakeel-Ur-Rehman et al., 2000c) or MF retentate from raw skim milk (Beauvier et al., 1997) has been added to pasteurised cheesemilk as an inoculum of NSLAB. Blends of as little as 1% raw milk with 99% pasteurised milk influence the quality of cheese (Shakeel-Ur-Rehman et al., 2000c). Since the rate of growth of NSLAB is strongly affected by temperature (see Folkertsma et al., 1996), Shakeel-Ur-Rehman


et al. (2000a,b) successfully prevented the growth of NSLAB in raw milk Cheddar by ripening at 1 °C. The results of these studies suggest that the differences observed between cheese made from raw and pasteurised milk are due principally to heat-induced changes to the NSLAB microflora, although pasteurisation largely inactivates the indigenous lipoprotein lipase, which results in a reduced level of lipolysis in pasteurised milk cheese. Since the proteolytic systems of NSLAB are generally similar to those of other LAB, they appear to contribute to proteolysis in a similar way to the starter, but to a lesser extent since maximum NSLAB numbers in cheese (often c. 107–108 cfu g 1) are lower than maximum numbers of starter (c. 109–1010 cfu g 1).

Acceleration of Cheese Ripening Cheese ripening is a slow, and consequently an expensive, process. The expense of cheese ripening arises principally from the inventory cost associated with holding a large amount of cheese in storage and the capital cost of providing a ripening facility adequate to hold sufficient cheese during ripening. The temperature and, in certain cases, the relative humidity of ripening rooms must be controlled, adding to the cost of cheese ripening. Thus, acceleration of cheese ripening has received considerable attention in the scientific literature. This topic has been reviewed by Fox (1988/89), El Soda and Pandian (1991), Wilkinson (1993), Fox et al. (1996) and Upadhyay and McSweeney (2003). Various approaches have been used to accelerate the ripening of cheese, including the use of an elevated ripening temperature, addition of exogenous enzymes or attenuated starters, use of adjunct cultures, use of genetically modified starter bacteria and high-pressure treatments. As novel processing technologies become available, it is likely that they will find an application to accelerate cheese ripening. Certain approaches (in particular the use of attenuated starters and adjunct cultures) are also used commercially to intensify the flavour of hard cheese and low-fat variants thereof, without necessarily reducing ripening time. Enzyme-modified cheeses are products in which a cheese-like flavour develops rapidly in a base material of young cheese (or sometimes caseinates) using cocktails of exogenous enzymes (and sometimes LAB) (see Kilcawley et al., 1998; ‘Cheese as an Ingredient’, Volume 2). The simplest and the most successful approach to accelerate ripening studied to date is an elevated ripening temperature (e.g., Folkertsma et al., 1996). Modification of the ripening temperature is used to control the rate of flavour development in hard cheese,

and ripening at an elevated temperature (e.g., c. 16 °C) results in the rapid development of flavour, although problems can occur with texture but this is not a serious drawback if the cheese is to be used in certain ingredient applications. Recent advances in the genetics of LAB and a greater understanding of the role of specific enzymes in the generation of volatile flavour compounds in cheese during ripening will facilitate the development of genetically modified starter strains to enhance flavour development.

Acknowledgements The author wishes to express his sincere thanks to Ms Niamh O’Sullivan, Ms. Patricia O’Connell and Ms Anne Cahalane for their assistance in preparing the typescripts of this and the other sub-chapters on cheese ripening.

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opment in Gouda cheese. 1. Description of cheese and aseptic cheesemaking techniques. Neth. Milk Dairy J. 31, 120–133. Visser, F.M.W. (1977b). Contribution of enzymes from rennet, starter bacteria and milk to proteolysis and flavour development in Gouda cheese. 2. Development of bitterness and cheese flavour. Neth. Milk Dairy J. 31, 188–209. Visser, F.M.W. (1977c). Contribution of enzymes from rennet, starter bacteria and milk to proteolysis and flavour development in Gouda cheese. 3. Protein breakdown: analysis of the soluble nitrogen and amino nitrogen fractions. Neth. Milk Dairy J. 31, 210–239. Visser, F.M.W. and de Groot-Mostert, A.E.A. (1977). Contribution of enzymes from rennet, starter bacteria and milk to proteolysis and flavour development in Gouda cheese. 4. Protein breakdown: a gel electrophoretical study. Neth. Milk Dairy J. 31, 247–264. Walsh, E.M., McSweeney, P.L.H. and Fox, P.F. (1996). Inhibition of the growth of non-starter lactic acid bacteria in Cheddar cheese using antibiotics. Int. Dairy J. 6, 425–431. Wegmann, U., Klein, J.R., Drumm, I., Kuipers, O.P. and Henrich, B. (1999). Introduction of peptidase genes from Lactobacillus delbrueckii subsp. lactis into Lactococcus lactis and controlled expression. Appl. Environ. Microbiol. 65, 4729–4733. Wilkinson, M.G. (1993). Acceleration of cheese ripening, in, Cheese: Chemistry, Physics and Microbiology, 2nd edn, P.F. Fox, ed., Chapman & Hall, London. pp. 523–556. Wium, H., Kristiansen, K.R. and Qvist, K.B. (1998). Proteolysis and its role in relation to texture of Feta cheese made from ultrafiltered milk with different amounts of rennet. J. Dairy Res. 65, 665–674. Yvon, M. and Rijnen, L. (2001). Cheese flavour formation by amino acid catabolism. Int. Dairy J. 11, 185–201.

Metabolism of Residual Lactose and of Lactate and Citrate P.L.H. McSweeney and P.F. Fox, Department of Food and Nutritional Sciences, University College, Cork, Ireland.

Metabolism of Lactose in Cheese During the manufacture of cheese curd, lactose is converted to lactic acid (mainly the L-isomer) by the starter bacteria (see ‘Starter Cultures: General Aspects’, Volume 1). In the case of Cheddar-type cheeses, most of the lactic acid is produced in the vat before salting and moulding whereas for most other varieties, acidification occurs mainly after the curds have been placed in moulds (see Fox et al., 2000). For many common varieties, the pH of the curd reaches ⬃5.0–5.3 within ⬃12 h from the start of cheesemaking. The rate and extent of acidification has a major impact on cheese texture via demineralization of the casein micelles (see Creamer et al., 1985, 1988; Lawrence et al., 1987; Fox et al., 1990) and on cheese proteolysis owing to the increased susceptibility of demineralized casein micelles to proteolysis (O’Keeffe et al., 1975) and/or greater retention of chymosin at low pH (Holmes et al., 1977; Stadhouders et al., 1977; Visser, 1977; Creamer et al., 1985; Garnot et al., 1987). However, such aspects will not be considered here. Although ⬃98% of the lactose is removed in the whey as lactose or lactate during the manufacture of Cheddar (Huffman and Kristoffersen, 1984), Cheddar cheese curd contains 0.8–1.0% lactose at milling. Under normal circumstances, this residual lactose is metabolized quickly, predominantly to L-lactate, mainly through the activity of the starter bacteria (see ‘Starter Cultures: General Aspects’, Volume 1). The complete and rapid metabolism of the lactose and its constituent monosaccharides in cheese curd is essential for the production of good quality cheese since the presence of a fermentable carbohydrate may lead to the development of an undesirable secondary flora (see Fox et al., 1990, 2000). In Cheddar cheese, the residual lactose is fermented at a rate and to an extent dependent on the salt-in-moisture (S/M) content of the curd (Turner and Thomas, 1980). Lactococcus lactis subsp. cremoris is more salt-sensitive than Lc. lactis subsp. lactis, which in turn is more sensitive than non-starter lactic acid bacteria (NSLAB; Turner and Thomas, 1980). Since some NSLAB are facultatively heterofermentative, the

% S/M may determine the products of lactose fermentation post-manufacture. At low S/M concentrations and low populations of NSLAB, residual lactose is converted mainly to L-lactate by the starter. At high populations of NSLAB, e.g., at a high storage temperature, considerable amounts of D-lactate are formed, partly by fermentation of residual lactose and partly by isomerization of L-lactate (Turner and Thomas, 1980). At high S/M levels (e.g., 6%) or at low NSLAB populations the concentration of lactose falls very slowly and changes in levels of lactate are slight. The quality of cheese is strongly influenced by the fermentation of residual lactose, as is evident from the data of O’Connor (1974). The pH decreases after salting, presumably due to the continued action of the starter at S/M levels 5%, but at higher level of S/M, starter activity decreases abruptly, as indicated by the high level of residual lactose and high pH. The quality of the cheeses also decreases sharply at 5% S/M (O’Connor, 1974). In Cheddar cheese made from milk supplemented with lactose to a concentration of 8% (⬃2.5% lactose in the cheese curd), lactose persisted and the pH continued to fall throughout a 9-month ripening period; the cheese had a harsh, strong over-acid flavour (Waldron, 1997; Shakeel-ur-Rehman and Fox, unpublished). In certain varieties (e.g., Edam, Gouda, Samsoe, Havarti), some whey is removed during curd manufacture and replaced with hot water. Probably, the initial function of this step was to cook the curds (on farms lacking steam-generating facilities and jacketed cheese vats) but it removes some lactose and reduces the ratio of lactose to buffering substances and hence acts to control pH (Walstra et al., 1993). Dutch-type cheese curd contains up to ⬃1.4% lactose at pressing but this decreases to 0.1% after ⬃12 h and to undetectable levels after brining. The lactose is fermented by the starter to L-lactate. The curd for some Cheddar-type cheeses (washedcurd Cheddar and Monterey Jack) is washed to reduce its lactose content. The lactose in washed-curd Cheddar is exhausted rapidly, the pH, which remains higher



362 Metabolism of Residual Lactose and of Lactate and Citrate

than normal for Cheddar, rises during ripening (in common with most varieties) and the cheese has a mild clean flavour (Waldron, 1997; Shakeel-ur-Rehman and Fox, unpublished). The fermentation of lactose in Swiss-type cheeses is quite complicated (see Mocquot, 1979; Turner et al., 1983; Fox et al., 1990). Typically, 30 min after moulding, Emmental contains ⬃1.7% lactose which is metabolized rapidly by Streptococcus thermophilus to a low level within 12 h, with the production of up to 0.8% L-lactate (Turner et al., 1983). Only the glucose moiety of lactose is metabolized by Sc. thermophilus and, consequently, galactose accumulates to a maximum of ⬃0.7% at ⬃10 h. The lactobacilli metabolize lactose and galactose to a mixture of D- and L-lactate, reaching ⬃0.35 and 1.2%, respectively, at day 14, by which time all sugars are normally completely metabolized. Thereafter, the concentrations of L- and D-lactate change little until the cheese is transferred to the hot room, when the propionic acid bacteria begin to grow. Changes in the concentrations of lactose, galactose, glucose, D- and L-lactate and their degradation products in Swiss-type cheese were studied by Turner et al. (1983) and are shown in Fig. 1.

Changes to Lactate During Ripening Lactate is an important substrate for a series of reactions in cheese during ripening (Fig. 2):

• in most cheeses, L-lactate is racemized to D-lactate by the NSLAB flora; • lactate is catabolized in Swiss-type cheese by Propionibacterium freudenreichii subsp. shermanii which is important for the development of characteristic eyes and flavour; • lactate is catabolized to CO2 and H2O by Penicillium camemberti in surface mould-ripened cheeses, such as Camembert and Brie, which is important for texture development; • in the presence of O2, some members of the NSLAB flora, particularly pediococci, can oxidize lactate to formate and acetate; • lactate can be metabolized anaerobically by Clostridium tyrobutyricum leading to defects known as ‘late gas blowing’. Racemization of lactate

The concentrations of lactate in Camembert, Swiss, Cheddar and Dutch-type cheeses are 1.0, 1.4, 1.5 and 1.2%, respectively (Raadsveld, 1957; O’Connor, 1974; Turner and Thomas, 1980; Thomas and Pearce, 1981; Turner et al., 1983; Karahadian and Lindsay, 1987; Walstra et al., 1993). Turner and Thomas (1980), Thomas and Pearce (1981) and Tinson et al. (1982) showed that experimental and commercial Cheddar cheese contains a considerable concentration of D-lactate, which could

Figure 1 Relationship between lactose and lactate metabolism, growth of propionic acid bacteria and production of propionate and acetate in Swiss cheese (Turner et al., 1983).

Metabolism of Residual Lactose and of Lactate and Citrate 363

Butyrate, H2

Non-starter lactic acid bacteria

DL-Lactate

Clo

strid

ium

sp.

Pro

H

2 HO H3C

rium

acte

ib pion

sp.

Propionate, acetate H2O, CO2

C COOH

Lactic acid 4

Formate, acetate, CO2

3

. sp um illi ts nic eas y

Pe

No n ac -sta (e id b rter pe sp ac la dio eci ter cti co ally ia c cc i)

5

1

CO2, H2O

Figure 2 Pathways by which lactate is metabolized in cheese during ripening. (1) racemization, (2) metabolism by Propionibacterium freudenreichii subsp. shermanii in Swiss cheese, (3) oxidative metabolism of lactate, (4) conversion to formate, ethanol and acetate and (5) anaerobic metabolism of lactate to butyrate and H2 which leads to late gas blowing.

be formed from residual lactose by lactobacilli or by racemization of L-lactate. Racemization of L-lactate is likely to occur more rapidly in cheese made from raw milk than in pasteurized milk cheese due to higher numbers of NSLAB and a more diverse nonstarter microflora in the former. Commercial Gouda contains a relatively low proportion of D-lactate, probably due to the short ripening time. The level of L- or D-lactate in Camembert is very low (Gripon, 1993) due to the catabolism of lactate by the mould, as discussed below. Racemization presumably involves oxidation of L-lactate by L-lactate dehydrogenase (LDH) to pyruvate which is then reduced to D-lactate by D-LDH. Except in cases where the post-milling activity of the starter is suppressed, racemization is likely to be the principal mechanism (Thomas and Crow, 1983). Racemization of lactate is a major change in cheese during ripening, transforming up to approximately half the lactate or ⬃0.7% of the cheese mass (Thomas and Crow, 1983).

H

HO H3C

H

C

L(+)-Lactic

C COOH acid

HOOC D(–)-Lactic

OH CH3 acid

Thomas and Crow (1983) showed that pediococci isolated from cheese racemize L-lactate more actively than lactobacilli; all 27 pediococci isolated from Ched-

dar cheese and P. pentosaceus NCDO 1220 were capable of converting L-lactate to D-lactate, eventually producing a racemic mixture, while only 5 of 16 Lactobacillus isolates were capable of racemizing L-lactate, at much slower rates and to a lesser extent than the pediococci. However, pediococci constitute only a small proportion of the microflora of Cheddar cheese ( Jordan and Cogan, 1993; Crow et al., 2001) and thus racemization of lactate in Cheddar and similar cheeses is presumably mainly a consequence of the growth of non-starter lactobacilli. Both lactobacilli and pediococci possess L()-LDH and D( )-LDH, both of which are NAD dependent. Racemization of L-lactate by cell suspensions of both pediococci and lactobacilli is pH dependent (optima: 4.0–5.2 and 4.5–6.0, respectively) and is retarded by an NaCl concentration 5% or 2% for pediococci and lactobacilli, respectively (Thomas and Crow, 1983). Racemization of lactate in a Cheddar cheese inoculated with pediococci was complete after ⬃19 days, while it required ⬃3 months in a control cheese with a much lower number of NSLAB, especially pediococci (Thomas and Crow, 1983). The racemization of L-lactate is probably not significant from the favour viewpoint. However, Ca-lactate may crystallize in cheese, causing undesirable white specks, especially on cut surfaces (Pearce et al., 1973; Severn et al., 1986; Dybing et al., 1988). Such crystals are harmless, but they may cause consumers to reject cheese as being mouldy or containing foreign bodies (Dybing et al., 1988). The solubility of Ca-DL-lactate is lower than that of pure Ca-L-lactate (Thomas and Crow, 1983; Dybing et al., 1988) and hence racemization of lactate favours the development of crystals in cheese. Dybing et al. (1988) calculated that the amount of available lactate in cheese can potentially create enough Ca-lactate pentahydrate to exceed its


solubility only slightly at 0 °C. Thus, crystal formation is favoured if microbial metabolism increases the concentration of D- relative to L-lactate, due to the lower solubility of Ca-DL-lactate. Crystal growth requires nucleation centres which may be bacterial cells, microcrystals of calcium phosphate or undissolved CaCO3. Increased levels of residual lactose, which favour the growth of NSLAB, can facilitate production of Ca-lactate crystals (Pearce et al., 1973; Sutherland and Jameson, 1981). Likewise, factors which increase the release of casein-bound Ca (e.g., low pH or high salt which causes the ion-exchange of Na for Ca2; Dybing et al., 1988) or reduce the solubility of Ca-lactate (e.g., a lower ripening temperature) favour crystal formation. Oxidation of lactate

Lactate can be metabolized by LAB, depending on strain, to acetate, ethanol, formate and CO2 (see Fox et al., 2000). Pediococci, if present in cheese together with high concentrations of O2, produce l mol of acetate and 1 mol of CO2 and consume 1 mol of O2 per mol of lactate utilized (Thomas et al., 1985). The pH optimum for oxidation is 5–6 and depends on the lactate concentration. The concentration of lactate in cheese exceeds that required for optimal oxidation, and lactate is not oxidized until all sugars have been exhausted. However, the oxidation of L-lactate to acetate occurs to a very limited extent in cheese wrapped in film due to the low level of O2 available. The oxidative activity of suspensions of starter and NSLAB isolated from cheese on lactose, lactate, citrate, amino acids and peptides was studied by Thomas (1986). Starter bacteria were active mainly on lactose, with low activity on enzyme-hydrolysed casein; Lb. casei oxidized citrate, while Lb. plantarum, Lb. brevis and P. pentosaceus oxidized lactose, peptides, L- and D-lactate, but not citrate. These results suggest that the oxidation of lactate to acetate in cheese depends on the NSLAB population and on the availability of O2, which is determined by the size of the block and the oxygen permeability of the packaging material (Thomas, 1987). Acetate, which may also be produced by starter bacteria from lactose (Thomas et al., 1979), citrate or from amino acids by starter bacteria and lactobacilli (Nakae and Elliott, 1965), is usually present at high concentrations in most, or all, cheeses and is considered to contribute to cheese flavour, although a high concentration may cause off-flavours (Aston and Dulley, 1982).

Oxidative metabolism of lactate in surface mould-ripened varieties

The catabolism of lactate is very extensive in surface mould-ripened varieties, e.g., Camembert and Brie. The concentration of lactate in these cheeses at day 1 is ⬃1.0%, produced mainly or exclusively by the mesophilic starter, and hence, presumably, is L-lactate. Secondary organisms quickly colonize and dominate the surface of these cheeses (Addis et al., 2001), initially Geotrichum candidum and yeasts (e.g., Kluyveromyces lactis, Debaryomyces hansenii and Saccharomyces cerevisiae; Gripon, 1993), followed by a dense growth of Penicillium camemberti (Mollimard et al., 1995) and, particularly in traditional manufacture, by low numbers of Gram-positive organisms similar to those found on the surface of smear-ripened cheeses, which do not colonize the cheese surface until the pH has increased to 5.8 (see ‘Surface Mould-ripened Cheeses’, Volume 2). G. candidum and P. camemberti rapidly metabolize lactate to CO2 and H2O, causing an increase in pH. Deacidification occurs initially at the surface, resulting in a pH gradient from the surface to the centre and causing lactate to diffuse outwards. When the lactate has been exhausted, P. camemberti metabolizes proteins, producing NH3 which diffuses inwards, further increasing the pH. The concentration of calcium phosphate at the surface exceeds its solubility at the high pH and precipitates as a layer of Ca3(PO4)2 on the surface, thereby causing a calcium phosphate gradient within the cheese, resulting in its outwards diffusion; reduction of the concentration of calcium phosphate in the interior helps to soften the body of the cheese (Fig. 3). In addition to softening the texture, changes to the cheese matrix may influence cheese flavour by changing the rates of migration or release of flavour compounds (Engel et al., 2001). The elevated pH stimulates the action of plasmin, which, together with residual coagulant, is responsible for proteolysis in this cheese rather than proteinases secreted by the surface microorganisms, which, although very potent, diffuse into the cheese to only a very limited extent, although peptides or other low molecular weight compounds produced by them at the surface may diffuse into the body of the cheese (Sousa and McSweeney, 2001; Churchill et al., 2003). The combined action of increased pH, loss of calcium (which affects to the integrity of the protein network) and proteolysis are necessary for the very considerable softening of the body of Brie and Camembert (see Noomen, 1983; Lenoir, 1984; Karahadian and Lindsay, 1987; Sousa and McSweeney, 2001). Changes which occur in Camembert-type cheese during ripening are indicated in Fig. 3.


Growth of Penicillium camemberti at surface

Ammonia produced at surface by proteolysis diffuses into cheese High pH

Low pH

Migration of soluble Ca, PO43– and lactate towards surface

Cheese softens from surface towards score

Direction of migration

pH

Concentration gradient

Lactate metabolized at surface Ca3(PO4)2 precipitates

Figure 3 Schematic representation of the changes which occur in Camembert-type cheese during ripening as a consequence of the growth of Penicillium camemberti at the surface.

Anaerobic metabolism of lactate by Clostridium tyrobutyricum

Gas (CO2 or H2) production by microorganisms may occur in cheese during ripening and may be desirable (e.g., eye production in Swiss and Dutch-type cheeses) or a defect. Organisms responsible for gas production in cheese are summarized in Table 1.

Table 1 Major microbial groups responsible for producing gas in cheese (modified from Mullan, 2000)

Microorganism

Substrate

Gaseous product(s)

Clostridium tyrobutyricum Lactobacillus casei Lactobacillus brevis Streptococcus thermophilus Coliforms Yeasts Citrate-positive lactococci Leuconostoc mesenteroides Leuconostoc dextranicum Propionibacterium freudenreichii subsp. shermanii

Lactate

CO2, H2

Citrate Lactose Urea

CO2 CO2 CO2

Lactose Lactose Citrate

CO2, H2 CO2 CO2

Lactose/citrate

CO2

Lactose/citrate

CO2

Lactate

CO2

Late gas blowing and accompanying off-flavours are defects associated with certain hard cheeses resulting from the anaerobic metabolism of lactate (or glucose) by Clostridium tyrobutyricum (and perhaps other clostridia; see Ingham et al., 1998) to butyrate and H2 (Fox et al., 1995; Klijn et al., 1995; Fig. 4); Cl. tyrobutyricum preferentially utilizes D-lactate in a mixture of both isomers (Huchet et al., 1997). Late gas blowing is a problem only in certain varieties, principally those that are brinesalted, owing to the time lag for NaCl to reach an inhibitory level throughout cheese (Kleter et al., 1984). Cheddar cheese is not susceptible to late gas blowing mainly because it is dry-salted. The combined effects of pH, lactate, glycerol and NaCl on the growth of vegetative cells of Cl. tyrobutyricum were studied by Huchet et al. (1995) who found that the growth of this organism is very sensitive to changes in these parameters within the ranges found during the manufacture of Emmental cheese (pH, 5.3–5.9; NaCl level, 0–0.6%; lactic acid concentrations, 0–1.6%; aw, 0.965–0.99). Late gas blowing may be avoided by minimizing spore numbers in milk by good hygiene and avoiding the feeding of silage (Driehuis and Elferink, 2000). Germination of spores and the growth of the vegetative cells may be inhibited by the use of lysozyme or NO3 or perhaps by biological control (e.g., Carminati et al., 2001, who used co-inocula of Sc. thermophilus), or in the case of processed cheese, by long-chain polyphosphates (Loessner et al., 1997). Spores may be removed from milk by bactofugation or microfiltration (see McSweeney and Sousa, 2000).


H OH H O

HO HO O

H

OH OH

H Glucose

O NAD

2Pi

Butyrate

Pi

H

2ADP

CoA

Acetyl-CoA NADH2

Acetyl-P Acetate

2ATP

ADP

NADH2

O

ATP

CoA

S

NAD OH

O O

2

Butyryl-CoA

2

O Pyruvate

O O Lactate

NAD 2H2 2Fd

NADH2

2 CO2 2 CoA

O

2Fd+H2 S

CoA O

Crotonyl-CoA

2

CoA

S Acetyl-CoA

H2O

CoA OH

O S

O

CoA

O S

L-β-Hydroxybutyryl-CoA

NAD

NADH2

CoA

Acetoacetyl-CoA

Figure 4 Pathway for the formation of butyrate and H2 from glucose, lactose or lactate by Clostridium tyrobutyricum (Fd: ferredoxin) (McSweeney and Sousa, 2000).

Bactofugation of milk, an increased level of NaCl in cheese and a reduced ripening temperature are effective measures for preventing or reducing gas production by Clostridium spp. (Su and Ingham, 2000). Lactate metabolism by Propionibacterium

In Swiss-type cheese, Propionibacterium freudenreichii subsp. shermanii metabolizes lactate to propionate, acetate, CO2 and H2O (Piveteau, 1999). 3 CH3CH(OH)COOH : 2 CH3CH2COOH CH3COOH CO2 H2O Lactic acid propionic acid acetic acid

The CO2 generated migrates through the cheese mass to points of weakness where it accumulates as eyes, a characteristic feature of Swiss cheese. Carbohydrate metabolism in Swiss cheese is summarized in

Fig. 1. Since propionibacteria are very sensitive to NaCl (Richoux et al., 1998), Swiss-type cheeses contain a low level of salt. Eye development in Swiss cheese, which is discussed in ‘Cheese with Propionic acid Fermentation’, Volume 2 and by Steffen et al. (1993) and Polychroniadou (2001), depends mainly on: • Rate and quantity of CO2 production. • Number and size of loci suitable for future eye development. • CO2 pressure and diffusion rate. • Cheese texture and temperature (Steffen et al., 1993). Relatively little of the total amount of CO2 produced by the propionic acid fermentation in Swiss cheese remains trapped in the eyes; in a cheese of c. 80 kg,


120 l CO2 are produced during ripening. Approximately 60 l remain dissolved in the cheese mass, ⬃40 l are lost from the cheese and only c. 20 l remain in the eyes (Steffen et al., 1993). L-Lactate is metabolized preferentially to D-lactate by propionic acid bacteria (Crow, 1986) to reach 0.2% after ⬃20 days in the hot room (Turner et al., 1983). In fact, the concentration of D-lactate continues to increase to ⬃0.4% during the early days in the warm room, before being metabolized by propionic acid bacteria. Increasing the number of starter lactobacilli accelerates sugar metabolism and causes higher concentrations of both D- and L-lactate but suppresses the growth of propionibacteria (due to a lower pH in the cheese) and thus delays the production of propionate and acetate. In the absence of lactobacilli or with Gal lactobacilli, galactose accumulates and no D-lactate is formed. Therefore, the proportion of lactobacilli in the starter probably influences the production of CO2 and volatile acids.

Citrate Metabolism The relatively low concentration of citrate in milk (⬃8 mmol l 1) belies the importance of its metabolism in many cheeses made using a mesophilic culture (for reviews see Cogan, 1985; Cogan and Daly, 1987; Fox et al., 1990; Cogan and Hill, 1993; ‘Starter Cultures: General Aspects’, Volume 1). Approximately 94% of the citrate in milk is soluble and most of it is lost in the whey; however, the concentration of citrate in the aqueous phase of cheese is ⬃3 times that in whey (Fryer et al., 1970), presumably reflecting the concentration of colloidal citrate; Cheddar cheese contains 0.2–0.5% citrate. Citrate is not metabolized by most strains of Lc. lactis subsp. lactis or Lc. lactis subsp. cremoris, but is metabolized, with the production of diacetyl, acetate, acetoin and CO2, by citrate-positive (Cit) strains of lactococci (formerly referred to as Lc. lactis subsp. lactis biovar diacetylactis or Streptococcus diacetilactis) in which the trait is plasmid-encoded, and Leuconostoc mesenteriodes subsp. cremoris and Ln. lactis. It is not metabolized by Sc. thermophilus or by thermophilic lactobacilli (see Fox et al., 2000). Citrate is not used as an energy source by Cit lactococci or Leuconostoc spp., but it is metabolized very rapidly in the presence of a fermentable carbohydrate by the pathway outlined in Fig. 5. Enzymes in this pathway have been characterized (see Cogan and Hill, 1993; O’Sullivan et al., 2001). The CO2 produced on citrate metabolism is responsible for the characteristic eyes of Dutch-type cheese and for the undesirable openness and

floating curd defects in Cheddar and Cottage cheese, respectively. Due mainly to the formation of diacetyl, citrate metabolism is very significant in aroma/flavour development in Cottage cheese (e.g., Antinone et al., 1994), Quarg (Mohr et al., 1997), and many fermented milks, particularly cultured ‘buttermilk’ (Ulberth, 1991; Gaafar, 1992; Laye et al., 1993; Hernandez et al., 1995; Rankin and Bodyfelt, 1995). Diacetyl also contributes to the flavour of Dutch-type and Cheddar cheeses (McGugan, 1975; Manning, 1979a,b; Dacremont and Vickers, 1994; Christensen and Reineccius, 1995; Milo and Reineccius, 1997). However, diacetyl is produced in small amounts (0.11 mmol l 1 in milk); acetoin production is usually 10–50 times greater. According to the pathway shown in Fig. 5, 1 mol acetate should be produced from 1 mol citrate. However, studies suggest that c. 1.2 mol acetate are actually produced per mol citrate used; the excess probably results from small amounts of acetate produced on the metabolism of sugars. Acetate produced from citrate may also contribute to cheese flavour. There are few data on the production of 2,3-butanediol by starters (see Fox et al., 2000). In cheese with a controlled microflora, Fryer et al. (1970) showed that in cheese made using Lc. lactis subsp. cremoris, citrate remained constant at 0.2% up to 3 months, but decreased to 0.1% at 6 months. Cheese made using Lc. lactis subsp. cremoris plus a Cit strain of Lactococcus contained no citrate at 3 months. Although Lb. casei could metabolize citrate in milk, the concentration of citrate in cheese made using Lc. lactis subsp. cremoris and Lb. casei decreased at about the same rate as in cheese made with Lc. lactis subsp. cremoris alone. Although the pathway shown in Fig. 5 is probably the major route for the metabolism of citrate by LAB, the possibility that lactate may be formed from the pyruvate produced from citrate cannot be overlooked. Three out of four strains of Cit Leuconostoc growing on glucose plus citrate produced no diacetyl or acetoin and more lactate than could be accounted for in terms of the amount of glucose used, suggesting that pyruvate derived from citrate was being reduced to lactate (Cogan, 1987). Citrate may be metabolized by some strains of facultatively heterofermentative lactobacilli, which are components of the NSLAB flora, to acetoin, acetate and probably diacetyl (Palles et al., 1998) by the same pathway as Cit lactococci and Leuconostoc spp. (Fig. 5). Thomas (1987) showed that citrate in Cheddar cheese decreased slowly to almost zero at 6 months, presumably as a result of metabolism by lactobacilli which became the major component of the NSLAB flora. Inoculation of cheese milk with Lb. plantarum accelerated the depletion of citrate. Pediococci did not appear to utilize citrate.


OH O

O O

O O

OH OH

H

Citrate

OH

H O H HO

H O HO

H H

O

O H

OH H

H

H

O

Citrate lyase

OH OH

O

Lactose

Acetate O

O O

O O

Oxalacetate Acetolactate decarboxylase OH

NAD+

CO2

O

Lactate

CO2

TPP

O

NADH

O

O

O

TPP O

Lactate dehydrogenase

Acetaldehyde TPP

Pyruvate

Acetolactate synthase OH

Acetyl-CoA

Other metabolic pathways

O

Diacetyl synthase

O

O

CoASH

α-Acetolactate CO2

TPP

Spontaneous? OH

O

Acetoin dehydrogenase O

O

Acetoin NAD(P) NAD(P)H Butanediol dehydrogenase NAD(P)+ OH

+

NAD(P)H

Diacetyl

OH

2,3-Butanediol Figure 5 Pathways for citrate metabolism in citrate-positive strains of Lactococcus and Leuconostoc species (modified from Cogan and Hill, 1993).


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Lipolysis and Catabolism of Fatty Acids in Cheese Y.F. Collins, Teagasc, Dairy Products Research Centre, Ireland P.L.H. McSweeney, Department of Food and Nutritional Sciences, University College, Cork, Ireland M.G. Wilkinson, Department of Life Sciences, University of Limerick, Ireland

Introduction Lipids present in foods may undergo oxidative or hydrolytic degradation (McSweeney and Sousa, 2000). However, lipid oxidation does not occur to a significant extent in cheese, probably due to its low redox potential ( 250 mV) (Fox and Wallace, 1997; Fox et al., 2000; McSweeney and Sousa, 2000). Enzymatic hydrolysis (lipolysis) of triglycerides to fatty acids and glycerol, mono- or di-glycerides is considered to be essential for flavour development in cheese (McSweeney and Sousa, 2000). Lipolysis is an important biochemical event during cheese ripening and has been studied extensively in varieties such as Blue and hard Italian cheeses where extensive lipolysis occurs and is a major pathway for flavour generation. However, in the case of cheeses such as Cheddar and Gouda, in which the level of lipolysis during ripening is low, the contribution of lipolysis to cheese quality and flavour has received relatively little attention. Free fatty acids (FFAs) are important precursors of several volatile compounds which contribute to flavour (McSweeney and Sousa, 2000; Collins et al., 2003a,b).

Lipolytic Agents in Cheese Lipolytic enzymes may be classified as esterases or lipases, which are distinguished according to three main characteristics: length of the hydrolysed acyl ester chain, physico-chemical nature of the substrate (whether emulsified or not) and enzymatic kinetics (esterases have classical Michaelis Menten-type kinetics while lipases, since they are active only at an interface, obey interfacial Michaelis Menten-type kinetics) (Chich et al., 1997; Villeneuve and Foglia, 1997; Deeth and Touch, 2000). Unfortunately, the terms ‘esterases’ and ‘lipases’ are often used interchangeably in the scientific literature. In general, lipolytic enzymes are specific for fatty acids esterified at the sn-1 or sn-3 positions of triglycerides (Deeth and Touch, 2000). Initially, triglycerides are hydrolysed to 1,2- and 2,3-diglycerides and later to

2-monoglycerides; butyrate, as well as the other shortand medium-chain acids, are located mainly at the sn-1 and sn-3 positions in milk lipids and thus are preferentially released by lipolytic enzymes (Parodi, 1971; Christie, 1995; Deeth and Touch, 2000). Lipases in cheese originate from six possible sources: • • • • • •

milk rennet paste starter bacteria secondary starter microorganisms non-starter lactic acid bacteria (NSLAB) exogenous lipase preparations

(Deeth and Fitz-Gerald, 1995; Fox and Wallace, 1997; McSweeney and Sousa, 2000). Milk contains a very potent indigenous lipoprotein lipase (LPL) (Olivecrona and Bengtsson-Olivecrona, 1991; Fox and Stepaniak, 1993; Fox et al., 1993; Olivecrona et al., 2003). The enzyme consists of 450 amino acid residues, with an overall molecular mass of 55 kDa and in milk exists as a dimer of identical subunits. The enzyme has an isoelectric point above pH 9, with a positive charge at physiological pH (Olivecrona and Bengtsson-Olivecrona, 1991). The lipase is of blood origin and is involved in the metabolism of plasma triglycerides; its presence in milk is due to leakage through the mammary cell membrane. Bovine milk contains 10–20 nmol lipase/l which, under optimum conditions (37 °C, pH 7, in the presence of an apolipoprotein activator, apo-CII), could theoretically release sufficient FFAs within 10 s to cause perceptible hydrolytic rancidity in milk (Walstra and Jenness, 1984). This does not occur under normal circumstances as LPL and fat are compartmentalized, c. 90% of the LPL in milk is associated with the casein micelles while the triglycerides in milk occur as globules surrounded by a lipoprotein membrane, the milk fat globule membrane (MFGM). If the MFGM is damaged, e.g., due to agitation, foaming, homogenization, inappropriate milking or milk-handling techniques, significant lipolysis may occur, resulting in off-flavours



374 Lipolysis and Catabolism of Fatty Acids in Cheese

in cheese and other dairy products (Fox et al., 2000). Lipoprotein lipase has a preference for medium-chain triglycerides (MCT) with a 2-fold faster rate of hydrolysis of emulsions containing triC6:0, triC8:0, triC10:0 or triC12:0 compared to long-chain triglyceride (LCT) emulsions containing triC16:0, triC18:0, triC18:1, triC18:2, triC18:3 or triC20:0 (Deckelbaum et al., 1990). The different hydrolysis rates were attributed to higher concentrations of MCT at the emulsion surface due to higher mobility of such triglycerides compared with LCT emulsions (Deckelbaum et al., 1990). Lipoprotein lipase is relatively non-specific for fatty acid type but is specific for the acids at the sn-1 and sn-3 positions of mono-, di- and tri-glycerides (Olivecrona et al., 1992). Therefore, short- and medium-chain fatty acids are released preferentially by LPL. In raw milk cheeses, LPL activity is significant. According to Deeth and Fitz-Gerald (1983), it is generally accepted that high-temperature short-time (HTST) pasteurization (72 °C for 15 s) very extensively inactivates the enzyme. However, it may contribute to lipolysis in pasteurized milk cheese, as heating at 78 °C 10 s is required for its complete inactivation (Driessen, 1989). Commercial rennet extracts are free from lipolytic activity, but rennet paste, used in the manufacture of some hard Italian varieties (e.g., Provolone and Pecorino cheeses), contains a lipase, pregastric esterase (PGE) (Nelson et al., 1977; Hamosh, 1990; Fox, 2003). Although the term ‘esterase’ suggests that this enzyme acts only on water-soluble esters, it is in fact able to hydrolyse water-insoluble triglycerides containing long-chain fatty acids and hence ‘esterase’ is a misnomer as this enzyme is a lipase (Hamosh, 1990). Pregastric esterase is highly specific for short chain acids esterified at the sn-3 position (Nelson et al., 1977; Fox and Stepaniak, 1993; Fox, 2003). Pregastric esterases from different species are optimally active at 32–42 °C, pH 4.8–5.5 in presence of 0.5 M NaCl. The enzyme has an isoelectric point above pH 9 with a positive charge at physiological pH (see Fox, 2003). Suckling stimulates the secretion of PGE by glands at the base of the tongue, and it is washed into the abomasum by the milk. Rennet paste is prepared from the abomasa of calves, kids or lambs slaughtered after suckling. The abomasum is partially dried and ground into a paste, which is slurried in milk before being added to cheesemilk (Fox and Stepaniak, 1993). Some interspecies differences in specificity have been reported for calf, kid and lamb PGEs which result in slight differences in the flavour characteristics of cheese, depending on the source of PGE (Nelson et al., 1977; Fox and Stepaniak, 1993). Due to concerns regarding the hygienic quality of rennet pastes, research has been focused on identifiying exogenous

lipases which could be blended with rennet extracts to produce substitutes for rennet pastes. Most lipases investigated are unsuitable due to incorrect specificity; certain fungal lipases (e.g., a lipase secreted by Rhizomucor miehei) may be acceptable alternatives to rennet paste (Fox, 1988). Lipases and esterases of LAB appear to be the principal lipolytic agents in Cheddar and Dutch-type cheeses made from pasteurized milk (Fox et al., 2000). Evidence for this comes from studies on aseptic starterfree cheeses acidified with gluconic acid--lactone, where very low levels of FFAs are released during ripening (Reiter et al., 1967) and from the relationship between autolysis of starter cells and FFA levels during ripening (Collins et al., 2003a). Lactic acid bacteria possess esterolytic/lipolytic enzymes capable of hydrolysing a range of derivatives of FFAs, tri-, diand mono-glyceride substrates (Holland and Coolbear, 1996; Chich et al., 1997; Fox and Wallace, 1997; Liu et al., 2001). Despite the presence of these enzymes, LAB, especially Lactococcus and Lactobacillus spp. are weakly lipolytic in comparison to species such as Pseudomonas, Acinetobacter and Flavobacterium (Stadhouders and Veringa, 1973; Fox et al., 1993; Chich et al., 1997). However, because they are present at high numbers over an extended ripening period, LAB are responsible for the liberation of significant levels of FFAs in many cheese varieties which do not have a strongly lipolytic secondary flora. Lipases and esterases of LAB appear to be intracellular and a number have been isolated and characterized (Chich et al., 1997; Castillo et al., 1999; Liu et al., 2001). The presence of lipases and esterases has been demonstrated (Piatkiewicz, 1987) in nine strains of lactococci, including Lc. lactis subsp. lactis, citratepositive lactococci and Lc. lactis subsp. cremoris. -Naphthyl laurate (C12:0) and -naphthyl acetate (C2:0) were the substrates used to determine lipase and esterase activities, respectively; esterase activity was higher than lipase activity in all strains. Kamaly et al. (1990) reported the presence of lipases in the cell-free extract of a number of strains of Lc. lactis subsp. lactis and Lc. lactis subsp. cremoris; these lipases were, in general, optimally active at 37 °C and at pH 7–8.5. Lc. lactis subsp. cremoris showed higher lipolytic activity on tributyrin and milk fat emulsions than Lc. lactis subsp. lactis. The activity of all lipases was stimulated by reduced glutathione and low (c. 2%) concentrations of NaCl but inhibited by high NaCl concentrations (c. 20%). Chich et al. (1997) reported esterolytic activity on -naphthyl butyrate by an intracellular extract of Lc. lactis subsp. lactis NCDO 763; the enzyme was active on p-NP esters from C2 to C12, with pH and temperature optima 7.0–8.0 and 55 °C.

Lipolysis and Catabolism of Fatty Acids in Cheese 375

El-Soda et al. (1986) found intracellular esterolytic activities in four species of lactobacilli: Lb. helveticus, Lb. delbrueckii subsp. bulgaricus, Lb. delbrueckii subsp. lactis and Lb. acidophilus. All lactobacilli showed activity on p-nitrophenyl (p-NP) derivatives of fatty acids up to C5:0; Lb. delbrueckii subsp. lactis and Lb. acidophilus showed the highest esterolytic activity. None of the microorganisms tested hydrolysed o- and p-NP derivatives of C6:0 to C14:0. Khalid and Marth (1990) quantified the lipolytic activity of Lb. casei L-7, Lb. casei L-14, Lb. plantarum L-34 and Lb. helveticus L-53 on milk fat, olive oil and tributyrin emulsions; the three substrates were hydrolysed by all lactobacilli with the exception of Lb. casei L-7, which did not hydrolyse olive oil. According to Lee and Lee (1990), esterolytic and lipolytic enzymes are released by lysis of Lb. casei subsp. casei LLG cells. Maximum lipolytic activity was observed at pH 7.2 and 37 °C and activity was inhibited by Ag and Hg2 and stimulated by Mg2 and Ca2 (Lee and Lee, 1990). Lb. fermentum contains a cell surface-associated esterase which is optimally active on C4:0 but which can hydrolyse -naphthyl esters of fatty acids from C2:0 to C10:0 (Gobbetti et al., 1997). Gobbetti et al. (1996) reported the purification of an intracellular lipase from a strain of Lb. plantarum isolated from Cheddar cheese. This enzyme had a molecular mass of 65 kDa and was optimally active at pH 7.5 and 35 °C; it was relatively heat stable at 65 °C but was irreversibly inactivated on heating at 75 °C for 2 min. The enzyme was most active on tributyrin, with less activity on trilaurin and tripalmitin and no activity on triolein. When activity was determined on -naphthyl esters from C2 to C18:1, the enzyme was most active on -naphthyl butyrate, with significant activity on -naphthyl esters of acetate, caproate, caprylate and laurate, and low activity on -naphthyl caprate, myristate, palmitate, stearate and oleate. Liu et al. (2001) identified three intracellular esterases in Streptococcus thermophilus, two of which were purified to homogeneity and designated esterase I and II, with a molecular mass of ⬃34 and 60 kDa, respectively. Differences in substrate specificity between esterases I and II were noted; esterase I hydrolysed p-NP esters of short chain acids from C2 to C8 while esterase II hydrolysed C2–C6 p-NP esters only. Both enzymes had maximum activity on p-NP butyrate. Only esterase I was tested on a range of glyceride substrates; it hydrolysed di- and mono-glycerides containing fatty acids up to C14:0 but tributyrin was the only triglyceride it could hydrolyse. The impact of cheese composition on esterase I activity on p-NP butyrate substrate indicated that activity was reduced by decreasing pH in the range 5.5–8.0, and decreasing water activity in

the range aw 0.99–0.80. Interestingly, esterase activity increased on increasing the NaCl concentration from 3.7 to 7.5%, w/v; this stimulatory effect of NaCl on esterase activity had not been reported previously. The genetic characterization of lipolytic enzymes of LAB by Fernandez et al. (2000) confirmed the intracellular nature of a tributyrin esterase in Lc. lactis subsp. cremoris B1014. These workers showed that the 744 base pair estA gene encoded for a 258 amino acid protein of molecular mass ⬃29 kDa; however, this gene did not encode for a signal sequence required for extracellular secretion. Cloning of the gene and up to 170-fold overproduction of this enzyme was possible using a nisincontrolled expression system which allowed detailed characterization of the specificity and kinetics of the enzyme. The esterase showed highest activity on short-chain p-NP-esters of fatty acids, with highest activity on p-NP hexanoate (C6:0). This enzyme was not active on p-NP esters of fatty acids of chain length longer than C12:0. Tributyrin was readily hydrolysed while activity was also detected on phospholipids. However, increasing the concentration of phospholipids to levels favouring micelle formation did not lead to an increase in enzyme activity through interfacial activation, confirming the esterolytic nature of this enzyme. Since the esterases/lipases of LAB appear to be intracellular, they must be released into the cheese matrix through cell autolysis for maximum efficiency. However to date, few studies have been undertaken to establish whether a relationship exists between the extent of autolysis of LAB and lipolysis in various cheese varieties. Early work by Walker and Keen (1974) showed that Cheddar cheese made with Lc. lactis subsp. cremoris AM2 developed higher levels of oddnumbered C3—C15 methyl ketones than cheese made with Lc. lactis subsp. cremoris HP which indicated that properties of the starter strain may influence the concentrations of these compounds in cheese. However, these workers did not monitor cell viability or autolysis in cheese during ripening. Wilkinson et al. (1994) demonstrated that Lc. lactis subsp. cremoris AM2 is more autolytic than Lc. lactis subsp. cremoris HP and that secondary proteolysis is higher in Cheddar cheese made using the former strain. Collins et al. (2003a) studied the influence of starter autolysis on lipolysis during a 238-day ripening period, as measured by the release of FFAs (C4:0–C18:3) in Cheddar cheese made using Lc. lactis subsp. cremoris AM2 or Lc. lactis subsp. cremoris HP as starter. These workers found that cheese made using the highly autolytic starter, Lc. lactis subsp. cremoris AM2, developed significantly higher levels of caprylate (C8:0), myristate (C14:0), palmitate (C16:0) and stearate (C18:0) during ripening than


cheese made with the less autolytic strain, Lc. lactis subsp. cremoris HP. Cell-free extracts prepared from both strains had generally similar levels of activity on a triolein emulsion (lipase) or a p-NP butyrate (esterase) and these workers suggested that there is a relationship between early autolysis of these starter bacteria and lipolysis, possibly as a result of elevated release of intracellular lipolytic activity in cheese. Freitas et al. (1999) assayed proteolytic and lipolytic activities of Enterococcus faecium, Ec. faecalis, Lb. plantarum and Lb. paracasei and three species of yeasts (Debaryomyces hansenii, Yarrowia lipolytica and Cryptococcus laurentii) isolated from Picante cheese. High lipolytic activity was reported for Y. lipolytica, using tributyrin as substrate; the other species studied were less lipolytic. Brevibacterium linens is a constituent of the microflora of surface smear-ripened cheeses (e.g., Limburger) which undergo a significant level of lipolysis during ripening. Lipolytic activity has been demonstrated in B. linens using emulsified olive oil as substrate (San Clemente and Vadehra, 1967). Sørhaug and Ordal (1974) reported esterolytic and lipolytic activities in five strains of B. linens using tributyrin and olive oil as substrates. Welch Baillargeon et al. (1989) reported lipase activity in three strains of Geotrichum candidum. Emulsified esters of oleic or palmitic acid were used as substrates; optimum pH and temperature for lipolytic activity were pH 7 and 37 °C, respectively. Lipases of different strains of G. candidum may be classified either as type A strains, which are not highly specific for unsaturated substrates with cis-9 double bonds, or type B strains, which are specific for cis-9 18:1 ( Jacobsen and Poulsen, 1995). In mould-ripened cheeses, such as Brie, Camembert and Roquefort, Penicillium spp. are significant agents of lipolysis (Gripon, 1993; McSweeney and Sousa, 2000). P. roqueforti possesses two lipases, one with a pH optimum of 7.5–8, the other with a more alkaline pH optimum (9–9.5) (Morris and Jezeski, 1953; Kman et al., 1966; Niki et al., 1966). The lipase with the lower pH optimum is more active on tricaproin while that with the higher pH optimum is more active on tributyrin (Menassa and Lamberet, 1982). P. camemberti produces an extracellular lipase, which is optimally active on tributyrin at pH 9 and 35 °C (Lamberet and Lenoir, 1976). Propionic acid bacteria are between 10 and 100 times more lipolytic than LAB (Knaut and Mazurek, 1974; Dupuis, 1994). Using emulsified tributyrin as substrate, Oterholm et al. (1970) showed that P. freudenreichii subsp. shermanii possesses an intracellular lipase with pH and temperature optima of 7.2 and 47 °C. The maximum rate of hydrolysis of triglycer-

ides was observed on tripropionin (C3:0), followed in order by tributyrin (C4:0), tricaproin (C6:0) and tricaprylin (C8:0). Hydrolysis of substrates in solution was low in comparison to hydrolysis of emulsified substrates, suggesting that the enzyme is a lipase. Dupuis et al. (1993) screened a number of strains of propionic acid bacteria for both esterolytic and lipolytic activities on actetate, propionate and butyrate esters and tributyrin. Intracellular fractions prepared from strains grown in liquid media showed both esterase and lipase activities. In the study of Dupuis et al. (1993) evidence was provided for the presence of an extracellular esterase; however, the extent to which this activity may have been due to cell lysis was not determined. Kakariari et al. (2000) purified an intracellular esterase from P. freudenreichii subsp. freudenreichii. Cell-free supernatant, cell wall fractions and a sonicated intracellular extract were assayed for esterase activity. However, in contrast to Dupuis et al. (1993), esterase activity was not found in the cell-free medium, or in the cell wall fractions. The enzyme purified by Kakariari et al. (2000) was therefore either cytoplasmic or cell membrane-associated.

Catabolism of Fatty Acids and Other Reactions In cheese, FFAs are precursors of many important flavour and aroma compounds, such as methyl ketones, lactones, esters, alkanes and secondary alcohols. Pathways for fatty acid catabolism are summarized in Fig. 1. Methyl ketones (alkan-2-ones) are the most important flavour components in Blue cheese and are present at very high concentrations. Heptan-2-one and nonan2-one are the predominant methyl ketones in Blue cheese; their concentrations in Gamonedo increased during the early part of ripening to a maximum at 60 days, after which levels began to decrease (Gonzalez de Llano et al., 1990). Methyl ketones are formed in Blue cheese by the action of Penicillium roqueforti (Urbach, 1997). P. camemberti and G. candidum may also produce methyl ketones (Lamberet et al., 1982; Cerning et al., 1987; Molimard and Spinnler, 1996). Penicillium spores, as well as vegetative mycelia, can produce methyl ketones (Chalier and Crouzet, 1998). Metabolism of fatty acids by Penicillium spp. involves four main steps corresponding to the early stages of -oxidation (Fig. 2). Initially, fatty acids are released by lipases, followed by the oxidation of FFA to -ketoacids and decarboxylation to alkan-2-ones, of one less carbon atom than the parent FFA; alkan-2-ones may be reduced to the corresponding secondary alcohol (alkan-2-ol). It has been suggested (Dartey and Kinsella, 1973; Kinsella and Hwang, 1976a) that FFAs are not the


Triglyceride

O C O

O C O

O C O

se

OH

pa

Li

O C

OH

γ - or δ -hydroxy fatty acids Partial glycerides H2 O O C O

γ - or δ -lactones O C

OH

Fatty acids R-SH

Thiols

CH3CH2OH

Ethanol

CO2

O C

Thioesters

β-Oxidation

S-R

O C

O C

OCH2CH3

CH3

Alkan-2-ones

Ethyl esters

OH CH CH3

Alkan-2-ols

Figure 1 Pathways for the catabolism of free fatty acids.

only precursors of methyl ketones; they can also be formed from ketoacids naturally present at low concentrations in milk fat or by oxidation of monounsaturated fatty acids (Kinsella and Hwang, 1976b). The rate of production of methyl ketones in cheese is affected by temperature, pH, physiological state of the mould and the concentration of FFAs. Spores of P. roqueforti can oxidize fatty acids containing 4–12 carbon atoms to methyl ketones, with octanoic acid being most rapidly oxidized. Oxidation of longer chain FFAs containing 16 or 18 carbon atoms has also been reported (Chalier and Crouzet, 1993). Mycelia oxidize FFAs over a wide pH range, with an optimum between pH 5 and 7, which is similar to the pH of mature Blue cheese (Dwivedi and Kinsella, 1974; Gripon, 1993).

Dumont et al. (1974a,b,c) identified 11 methyl ketones in Camembert cheese. Methyl ketones with even-numbered carbon chains appeared late in ripening and were present at low levels, except in very mature cheese. Heptan-2-one is present at significant concentrations in Parmigiano-Reggiano (Meinhart and Schreier, 1986). In full-fat Cheddar cheese, levels of heptan-2-one, nonan-2-one and undecan-2-one increased for approximately 14 weeks, after which levels decreased; concentrations of methyl ketones in low-fat Cheddar cheeses were lower than the levels in full-fat cheese (Dimos, 1992), perhaps due to lower levels of FFAs in the former (Dimos et al., 1996). Engels et al. (1997) compared the volatile compounds in the watersoluble fraction of seven cheese varieties (Gouda,


Saturated fatty acids (C2n) CoA-SH

β-Oxidation, –2H 2 + H2O

Keto acyl-CoA CoA-SH Thiohydrolase

Thiolase

CoA-SH + β-Keto acid

Acetyl-CoA + Acyl (C2n –2 ) -CoA Krebs’ Cycle

β-Ketoacyldecarboxylase

CO2

Methyl ketone (C2n –1 ) + CO2 Reductase Secondary alcohol (C2n –1 )

Figure 2 Pathway for the catabolism of fatty acids by Penicillium spp.

Proosdij, Gruyere, Maasdam, Edam, Parmesan and Cheddar). Nine ketones, mostly methyl ketones, were identified. Dirinck and De Winne (1999) reported levels of heptan-2-one and nonan-2-one ranging from 2903 to 3210 ng g 1 and from 1841 to 1960 ng g 1, respectively, in Gouda cheese; levels varied between 3332 and 3598 ng g 1 and 1768 and 1986 ng g 1, respectively, in Emmental. In another study, pentan-2-one and heptan2-one were the most abundant methyl ketones in aged ewes’ milk cheese (14 samples were analysed, nine of which were Manchego); mean levels were 737 and 368 ~or et al., 2000). g kg 1, respectively (Villasen Secondary alcohols can be formed in cheeses by enzymatic reduction of methyl ketones (Engels et al., 1997). P. roqueforti is responsible for the reduction of methyl ketones to secondary alcohols (e.g., 2-pentanol, 2-heptanol and 2-nonanol) in Blue cheese (Martelli, 1989). Gonzalez de Llano et al. (1990) reported that 2-heptanol and 2-nonanol are the main secondary alcohols in artisanal Gamonedo Blue cheese. Production of 2-propanol from acetone and 2-butanol from butanone has been reported in Cheddar cheese (Urbach, 1993), while Thierry et al. (1999) reported an increase in the concentrations of secondary alcohols in the aqueous phase of Emmental during ripening. A great diversity of esters, formed by the reaction of a FFA with an alcohol, is present in cheese (Molimard and Spinnler, 1996). While methyl, ethyl, propyl and butyl esters of FFAs have been reported in various cheese varieties (Meinhart and Schreier, 1986), ethyl

esters predominate (Arora et al., 1995). Esterification reactions resulting in the production of esters occur between short- to medium-chain fatty acids and ethanol, derived from lactose fermentation or from amino acid catabolism. Ethyl acetate may also arise from esterification of ethanol with acetyl-coenzyme A (Yoshioka and Hashimoto, 1983). Holland et al. (2002) suggested that esters are formed in cheese during ripening by the transesterification of a FFA from partial glycerides to ethanol. Thirty eight esters were identified by Meinhart and Schreier (1986) in Parmigiano-Reggiano cheese; ethyl acetate, ethyl octanoate, ethyl decanoate and methyl hexanoate were the most abundant. Methyl and ethyl esters have been found at high levels in artisanal Blue cheese (Gonzalez de Llano et al., 1990). Fourteen esters were found in Emmental cheese by Imhof and Bosset (1994) and Rychlik et al. (1997). Ethyl esters of fatty acids and smaller quantities of methyl esters have also been ~or et al., identified in Manchego cheese (Villasen 2000). Thioesters are formed when FFAs react with sulphydryl compounds (Molimard and Spinnler, 1996) and may be formed by the action of a wide range of microorganisms associated with cheese (Lamberet et al., 1997). Lactones are cyclic compounds formed by the intramolecular esterification of hydroxy fatty acids through the loss of water to form a ring structure (Christie, 1983; Molimard and Spinnler, 1996) and may be produced by heating their precursor hydroxyacids (Eriksen, 1976). - and -lactones are highly reactive and unstable (Fox and Wallace, 1997) but and -lactones (5- and 6-membered rings, respectively) are stable and have been identified in cheese (Eriksen, 1976). It has been reported that the mammary gland of ruminants has a -oxidation system for fatty acid catabolism (see Fox et al., 2000) which may produce the precursor hydroxy acids for the production of lactones, which may also be formed from keto acids after their reduction to hydroxyacids (Wong et al., 1975). The presence of large amounts of high molecular weight lactones has been reported in rancid Cheddar cheese, which has led to the suggestion that pathways other than the release of hydroxy acids from triglycerides may contribute to the formation of lactones (Wong et al., 1975). Dodecalactone may be formed by P. roqueforti spores and vegetative mycelia from long-chain unsaturated fatty acids (C18:1 and C18:2) (Chalier and Crouzet, 1992). Lactone precursors (hydroxy FA) may also be generated by the action of lipoxygenases and other enzymes present in members of the rumen microflora (Dufossé et al., 1994).


Production of lactone precursors in milk is influenced by several factors, including feed, season, stage of lactation and breed (see Fox et al., 2000). The sweet-flavoured -dodecanolactone and -dodec-Z-6-enolactone occur at much higher levels in milk from grain-fed than from pasture-fed cows (Urbach, 1997). -Dodecalactone and -tetradecalactone are the principal lactones in 75 day-old Blue cheese (Jolly and Kosikowski, 1975). In Cheddar cheese, lactone levels increased most rapidly early in the ripening period and were present at concentrations well above their flavour thresholds (Jolly and Kosikowski, 1975). Wong et al. (1975) reported levels of 1.5, 0.8, 4.9 and 8.9 g g 1 of C10, C12, C12 and C14, respectively, in Cheddar cheese at 14 months. Several lactones have been identified in Parmigiano-Reggiano cheese; quantitatively, the most important lactone is -octalactone (Meinhart and Schreier, 1986). -Decalactone, decalactone, -dodecalactone and -dodecalactone have been found in Camembert cheese (Gallois and Langlois, 1990). Aldehydes may be formed via the catabolism of amino acids (see ‘Catabolism of Amino Acids in Cheese During Ripening’, Volume 1). However, some straightchain aldehydes, e.g., butanal, heptanal and nonanal, may be formed by the -oxidation of unsaturated fatty acids. Gruyere and Parmesan have high levels of FFAs and high concentrations of straight chain aldehydes which have ‘green grass-like’ aromas (Moio et al., 1993).

Contribution of Lipolysis and Catabolism of FFA to Cheese Flavour Lipids play a major role in the quality of cheese: • They affect cheese rheology and texture (see ‘Rheology and Texture of Cheese’, Volume 1). • They influence flavour by: – Acting as a source of fatty acids which in turn may be catabolized to other flavour compounds, e.g., methyl ketones, esters, thioesters and lactones. – Acting as a solvent for sapid compounds produced from lipids or other precursors. • Many reactions occur at the fat–water interface. Reduced-fat cheese lacks typical flavour and contains lower concentrations of FFAs than full-fat cheese, supporting the theory that FFAs are important to cheese flavour (Foda et al., 1974; Olson and Johnson, 1990; Dimos et al., 1996; Wijesundera et al., 1998). Cheddar cheese manufactured from milk in which the fat was replaced by vegetable or mineral oils has also been reported to develop atypical flavours (Foda et al., 1974; Wijesundera and Watkins, 2000). Foda et al.

(1974) found that cheese containing vegetable fats had very little Cheddar flavour, and cheese containing mineral oil had only a slight Cheddar flavour. Cheese containing milk fat gave the best results but its flavour was still inferior to that of cheese made from whole milk. These results suggest that the MFGM, which is replaced on homogenization of the milk fat, may have important enzymes or other factors which play a role in the development of Cheddar flavour. It is also plausible that the interface between the lipid and aqueous phases in the cheese is important for flavour development. However, Wijesundera and Drury (1999) reported no significant difference in Cheddar cheese flavour intensity between cheeses made from whole milk or from milk reconstituted from skim milk and cream or anhydrous milk fat. Long-chain FFAs (12 carbon atoms) are considered to play a minor role in cheese flavour due to their high perception thresholds (Molimard and Spinnler, 1996). Short- and intermediate-chain FFAs (C4:0–C12:0) have considerably lower perception thresholds and each gives a characteristic flavour note. Butanoic acid contributes ‘rancid’ and ‘cheesy’ flavours; hexanoic acid has a ‘pungent’, ‘Blue cheese’ flavour note, while octanoic acid has a purported ‘wax’, ‘soap’, ‘goat’, ‘musty’, ‘rancid’ and ‘fruity’ note. Depending on their concentration and perception thresholds, volatile fatty acids can either contribute positively to the aroma of the cheese or to a rancidity defect. The flavour effect of FFAs in cheese is affected by pH. In cheeses with a high pH, e.g., smear-ripened and Blue cheeses, the flavour impact of fatty acids may be affected due to neutralization of the cheese (Molimard and Spinnler, 1996). In general, the flavour threshold of methyl ketones is quite low. Flavour threshold values determined in water vary widely; for heptan-2-one, ranging from 0.0009 to 3 mg kg 1 and for propan-2-one ranging from 40.9 to 500 mg kg 1 (Molimard and Spinnler, 1996). Octan2-one, nonan-2-one, decan-2-one, undecan-2-one and tridecan-2-one are described as having ‘fruity’, ‘floral’ and ‘musty’ notes, while heptan-2-one has a Blue cheese note (Rothe et al., 1982). The mushroom and musty notes of methyl ketones are important contributors to the flavour of Camembert cheese (Molimard and Spinnler, 1996). According to Eriksen (1976), lactones have a strong flavour; although the aromas of lactones are not cheese-like, they may contribute to overall cheese flavour (see Fox et al., 1993, 2000; Fox and Wallace, 1997) and have been reported to contribute to a buttery character in cheese (Dirinck and De Winne, 1999). -Lactones have low flavour thresholds compared to other volatile flavour compounds (O’Keefe et al., 1969)


and are generally characterized by very pronounced, fruity notes (‘peach’, ‘apricot’ and ‘coconut’) (Dufossé et al., 1994). -Lactones have generally higher detection thresholds than -lactones; thresholds for -octalactone, -decalactone and -dodecalactone are 7–11 g kg 1 in water and are even lower for shorter chain lactones (Dufossé et al., 1994). In a survey of various cheese varieties, Engels et al. (1997) found high concentrations of ethyl butanoate in cheeses with a ‘fruity’ note, e.g., Gruyere, Parmesan and Proosdij. This fruity flavour is considered undesirable in Cheddar cheese (Urbach, 1997; McSweeney and Sousa, 2000). Arora et al. (1995), who analysed the odour-active volatiles in the headspace of Cheddar cheese, found that most of the esters separated had a ‘buttery’/‘fruity’ aroma. However, thioesters formed by the reaction of short-chain fatty acids with methional imparted a characteristic ‘cheesy’ aroma to Cheddar cheese. According to Lamberet et al. (1997), S-methyl thioesters contribute a characteristic flavour to various smear-ripened soft cheeses (e.g., Tilsit, Limburger and Havarti). Secondary alcohols may contribute to cheese flavour (Arora et al., 1995). Propan-2-ol, butan-2-ol, octan-2-ol and nonan-2-ol are present in most soft cheeses and are typical components of the flavour of Blue cheeses (Engels et al., 1997). Moinas et al. (1975) found that heptan-2-ol and nonan-2-ol represented 10–20% and 5–10%, respectively, of all aromatic compounds in Camembert cheese. Oct-1-en-3-ol has the odour of raw mushroom with a perception threshold of 10 g kg 1, and has been proposed as one of the key compounds in the aromatic note of Camembert cheese (Molimard and Spinnler, 1996).

Patterns of Lipolysis in Various Cheese Varieties The level of lipolysis in cheese varies considerably from moderate (e.g., Cheddar, Cheshire, Caerphilly) to extensive (e.g., mould-ripened, hard Italian and surface bacterially-ripened (smear) varieties) (de Llano et al., 1992; McSweeney and Fox, 1993; Fernandez-Garcia et al., 1994; Fox and Wallace, 1997; Fox et al., 2000; McSweeney and Sousa, 2000). Concentrations of FFAs reported for a number of cheese varieties are shown in Table 1. Excessive lipolysis is considered undesirable in many varieties (e.g., Dutch-type cheeses, Cheddar, Emmental) and cheeses containing even a moderate concentration of FFAs may be considered as rancid by some consumers (Fox et al., 1993). However, limited lipolysis is thought to be desirable in these varieties. In Emmental cheese, moderate levels of FFAs, in the range 2–7 g kg 1, are liberated during ripening and

make an important contribution to the characteristic flavour and aroma of both raw and pasteurized milk Emmental cheese (Zerfiridis et al., 1984; Steffen et al., 1993; Chamba and Perreard, 2002). Recent data for lipolysis in Emmental cheese manufactured from raw or microfiltered milk, with or without various strains of propionic acid bacteria, indicate that FFAs are released during ripening by the lipolytic activity of propionic acid bacteria and starter and that the concentration of FFAs produced in the cheese is specific to the strain of Propionibacterium used for cheesemaking (Chamba and Perreard, 2002). The highest levels of lipolysis have been observed in mould-ripened cheeses; 5–10% of total triglycerides are hydrolysed in Camembert and up to 25% in Blue cheeses (Anderson and Day, 1966; Gripon et al., 1991; Gripon, 1993). Extensive lipolysis occurs in mouldripened cheeses without rancidity due to neutralization of fatty acids as the pH increases during ripening (Gripon, 1993). The most important lipolytic agents in mould-ripened cheeses are the enzymes of Penicillium spp., although a high proportion of free oleic acid in Camembert has been attributed to the action of the lipase of Geotrichum candidum (Gripon, 1993). In the manufacture of Danablu cheese, raw milk is separated and the resulting cream is homogenized and held before pasteurization. This process damages its MFGM and activates the LPL; additionally, homogenization reduces fat globule size and increases total fat globule surface area, providing a larger lipid–serum interface for LPL activity (Nielsen, 1993). Extensive lipolysis is also characteristic of many Italian varieties (e.g., Grana Padano and Parmigiano-Reggiano) (Woo and Lindsay, 1984; Bosset and Gauch, 1993). Arnold et al. (1975) found a direct relationship between the flavour intensity of ripened Romano-type cheese and its butyric acid content. Many Italian varieties are manufactured from raw milk (e.g., Parmigiano-Reggiano, Grana Padano, Provolone) which leads to higher levels of lipolysis in the ripened cheese due to the action of LPL. Pregastric esterase is responsible for extensive lipolysis in cheeses made using rennet paste, resulting in the characteristic ‘piccante’ flavour of these varieties (Fox et al., 2000; McSweeney and Sousa, 2000). High levels of lipolysis occur in smear cheeses (e.g., Limburger) (Woo et al., 1984). Brevibacterium linens is a major constituent of the surface microflora of bacterial smear-ripened cheeses and produces active lipolytic enzymes (Reps, 1993).

Measurement of Lipolysis Various quantitative techniques have been developed to monitor release of FFAs in cheese. The ‘copper soaps’ method is a colorimetric method enabling the

Table 1 Concentration (mg kg 1 cheese) of free fatty acids in some selected cheese varieties Cheese type

C6:0

C8:0

C10:0

C12:0

C14:0

C16:0

C18:0

C18:1

C18:2

Rennet coagulated Internal bacterially-ripened Extra-hard Parmesan 1055

451

243

440

439

1540

3896

1171

3471

123

Parmesan Romano

106 843

84 328

158 942

181 428

684 448

1750 785

1890* 1224*

143 191 111 2 33 144a 24 16 25 6 ⬃8

175 159 111 6 38 185a 28 20 27 20 ⬃12

159 175 48 25 67 387a

⬃25

571 619 238 37 68 225a 45 46 46 37 ⬃45

952 746 397 103 183 423a 117 110 120 86 ⬃70

1556 1253 619 285 397 1148a 309 270 299 231 ⬃275

794 508 270 524* 131* 3259a* 131 123 139 81 ⬃130

865

37 43 45 47 56 60 142 115

34 26 35 33 45 44 68 38

22 16 18 25 29 29 73 41

16 29 16 55 22 22 134 49

18 25 13 30 35 35 94 81

38 61 27 59 76 74 175 218

117 202 110 196 216 229 433 503

168* 299* 169* 387* 365* 385* 1377* 172

69 43

105 12

13 6

12 11

15

48

76

61

10

⬃128

⬃490

⬃69

⬃90

⬃171

⬃295

⬃299

59

162

394

132

418

36

12

49 392 358

112 1007 667

275 2243 1553

94 620 372

276 1438 1299

29 100 40

8

Hard Cheddar

C2:0

C4:0

140 1756 476 1587 1270

952 952 794 15 111 308a 10 5 0 0

381

Cheshire

8

12

32

Cheshire Roncal Idiazabal

0 505 285

10 302 118

27 377 65

372 578

C18:3

Total

13697

Method

Reference

GC

de la Feunte et al. (1993) Woo and Lindsay (1984)

GC

2841 1476 667

503 524 496 200 ⬃370

635 413 206

41 40 45 21

467

69

209

36

238 175 111

9492 8254 4842 997 1028 6079a

HPLC

Kilcawley et al. (2001)

GC

Woo et al. (1984)

GC

McNeill and Connolly (1989)

947

GC

450 701 433 832 844 878 2506

GC

Reddy and Marth (1993) Woo and Lindsay (1982)

23 27 26 0

40

Titration GC GC GC GC

8178 5577

GC

Bills and Day (1964) Marsili (1985) Dulley and Grieve (1974) McSweeney et al. (1993) McNeill and Connolly (1989) de la Feunte et al. (1993) continued

382

Table 1 continued Cheese type

C2:0

C4:0

C6:0

C8:0

C10:0

C12:0

C14:0

C16:0

C18:0

C18:1

C18:2

C18:3

Total

Method

Manchego

255

133

110

318

202

443

994

348

816

Manchego

876

704

426

398

340

608

1925

508

1384

106

Semi-hard Caerphilly

18

15

31

57

150

387

127

419

36

Mahon

383

377

274

394

307

810

2107

902

2235

82

8743

GC

Colby Monterey Jack Majorero

81 93 431

9 3 400

33 10 513

49 37 1964

22 20 1378

67 110 2327

196 252 5877

93* 211* 1327

GC

4921

886

356 736 20794

GC

Majorero

216

250

225

723

364

663

1745

528

1400

6144

GC

170 345 392a 112

90 21 135a 45

45 25 150a 71

122 53 381a

208 88 522a 68

311 267 1337a 207

1904 930 4262a 515

1427* 1197* 2664a* 256

4277 2926 9843a

GC

Woo et al. (1984)

GC

1037 120

83 26

6 21

16 44

13 51

58 160

193 475

75* tr

McNeill and Connolly (1989) Woo et al. (1984) Zerfiridis et al. (1984)

60

8

9

14

47

39

122

57*

54 782

7 308

1 81

120 172

12 122

27 120

76 199

66* 334*

35

5

14

35

43

69

270

210*

Cheeses with eyes Swiss-type Swiss

Emmental

Gruyere Gruyere

288

Dutch-type Edam Pasta-Filata cheeses Mozzarella Provolone Mould-ripened Surface mould-ripened Camembert

GC

Reference

842

59

14

50b

7285

13

GC

GC

31

Poveda et al. (1999) FernandezGarcia et al. (1994) McNeill and Connolly (1989) de la Feunte et al. (1993) Woo et al., (1984) de la Feunte et al. (1993) Martin-Hernández and Juarez (1992)

1481

GC GC

356

GC

Woo et al. (1984)

GC

Woo and Lindsay (1984)

GC

Woo et al. (1984)

681

Camembert

208

448

1028

298

622

1442

303

44

74

255

839

1314*

715 707

2104 2280

1403 1295

2632 3185

6452 6230

17404* 2241

777

546

1275

1835

4147

11416

14088*

105

700

815

2135

4460

620

160

235

185

1040

3366

2329

7850

101

58

Camembert

361

287

160

225

Brie

124

9

19

Blue cheese Roquefort Roquefort

992 961

751 626

1146

Blue Blue d’Auvergne (Blue, with conidia) (White, no conidia) Gamonedo blue

937

804

41 1475 72 62 995a 37

4 688 2 27 232a 34

8 24 8 68 446a 33

35.7

34

35

163

102

Concentration/crystallization Gjetost 106

35

Surface-ripened Port Salut Limburger Brick

Serra da Estrela Serra da Estrela Munster

54 50 35 193 1067a 89

19768

1421

9157

GC

1043

6282

896

30434

5066

GC

2678

GC

32453 25969

GC GC

35230

GC

Lesage et al. (1993) de la Feunte et al. (1993) Woo et al. (1984) Woo et al. (1984) de la Feunte et al. (1993) Woo et al. (1984)

GC

Kinderlerer et al. (1996)

75685

GC

de Llano et al. (1992)

700

GC

Woo et al. (1984)

Partidario et al. (1998) Partidario (1999)

33 92 22 153 613a 46

86 602 63 357 1805a 98

275 565 161 952 2835a 198

199* 709* 39* 538* 5977*a 106

220

12

7

GC

97

52

102

201

103

208

11

3

GC

66

154

206

704

2057

833

1412

59

504

GC

de LeonGonzalez et al. (2000)

31

180

49

170

456

631*

GC

Woo et al. (1984)

* C18:0 congeners. a Commercial samples exhibiting distinct flavour defects. b C18:2 C18:3.

402 2350 13970a

4558

383


determination of the total level of FFAs (IDF, 1991). Copper soaps of fatty acids are selectively transferred to an organic phase; a coloured complex is then formed between the copper soaps and sodium diethyl dithiocarbamate, the intensity of which is related to the concentration of FFAs. While the method is sensitive and rapid (IDF, 1991), the total level of FFAs is only estimated. Quantification of the total level of FFAs by extraction and titration of FFAs with alcoholic KOH gives an acid degree value (ADV), defined as the number of milliequivalents of alkali required to neutralize the FFAs in 100 g of fat. Gas chromatography (GC) has been the method most commonly used to quantify levels of individual FFAs in cheese and is the dominant technique for the routine analysis of FFAs; the flame ionization detector is robust with a wide and dynamic range, enabling accurate FFA quantification. In a review of the determination of FFAs in milk and milk products (IDF, 1991) methods for analysis of FFAs by GC were grouped under three headings. In the first group, FFAs are analysed directly and this group includes the method of Nieuwenhof and Hup (1971). Free fatty acids are isolated on an alkaline silica gel column, the eluate is concentrated and FFAs are quantified directly by GC. However, the silicic acid column used by Nieuwenhof and Hup (1971) was later shown to induce hydrolysis of the fat. The method of Woo and Lindsay (1982) is also included in this group; this method involves removal of lactic acid by a partition pre-column, followed by isolation of FFAs on a modified silicic acid-potassium hydroxide arrestant column. Free fatty acids are then separated in formic acidmobilized elutes on a glass column packed with diethylene glycol succinate (DEGS-PS) by GC. This GC procedure enables rapid separation and quantification of FFAs and has been used by others (Woo and Lindsay, 1984; Woo et al., 1984) to quantify individual FFAs in various cheeses. The second group of GC methods includes those of Iyer et al. (1967), McNeill and Connolly (1989) and Fontecha et al. (1990). Solvent is initially used to extract the lipid fraction from the cheese, followed by the preparation of methyl esters of FFAs and the separation and quantification of methyl esters of FFAs by GC. In general, the third group of GC methods quoted in Table 1 involves solvent extraction of the lipid fraction from the cheese, followed by separation of FFAs by, e.g., saponification (Martinez-Castro et al., 1986; Martin-Hernández et al., 1990; MartinHernández and Juarez, 1992; de la Feunte et al., 1993; Poveda et al., 1999), separation using chromatographic columns (Deeth et al., 1983; Lesage et al., 1993), or methylation to fatty acid methyl esters (FAME) (Ha and Lindsay, 1990; Kinderlerer et al., 1996; Partidario

et al., 1998; Partidario, 1999). The method of Dulley and Grieve (1974) uses steam-distillation followed by GC to quantify volatile fatty acids. The method described by de Jong and Badings (1990) is now a commonly used procedure for the quantification of FFA in cheese. Accurate and rapid determination of FFAs from C2:0 to C20:0 is achieved by direct separation of underivatized FFAs using capillary GC. In brief, the method involves extraction of FFA using ether/heptane from a cheese paste containing anhydrous sodium sulphate and H2SO4. Extracted FFAs are then isolated using alumina or an anionexchange method (aminopropyl columns) and separation of FFAs (C2:0–C20:0) is achieved by capillary GC. Few studies have compared the different methods of FFA quantification from the same sample. According to IDF (1991), isolation of FFAs from the sample material is a vital step in any method. FFAs must be isolated from both the aqueous phase and the fat phase. Organic solvents are used in the extraction procedures of the majority of the methods referenced. According to IDF (1991), it is difficult to combine good extraction of short chain FFAs from the aqueous phase together with a good extraction from the fat phase. While these extraction methods commonly involve the use of internal standards of fatty acids dissolved in an organic solvent, this does not guarantee the completeness of the extraction procedure. Chavarri et al. (1997) compared two methods for sample preparation for the determination of FFAs in ewes’ milk cheese by GC. In method 1, after fat extraction, FFAs were separated from triacylglycerides by aminopropyl bonded-phase chromatography. The fraction containing FFAs was then injected directly onto the gas chromatograph. In method 2, extracted fat was treated with tetramethylammonium hydroxide and the methyl ester derivatives were then formed in the injector. It was concluded that FFAs should be separated from the triacylglycerides before derivatization and chromatographic analysis, particularly for samples in which a minor fraction of the triacylglycerides has been hydrolysed to FFAs. Three gas chromatographic methods for the analysis of FFAs in cheese were compared by Ardö and Polychroniadou (1999). Method 1 included the preparation and methylation procedures described by Martinez-Castro et al. (1986) and Martin-Hernández et al. (1988). This involves diethyl ether extraction of fat followed by methylation with tetramethylammonium hydroxide, which results in an upper layer containing methyl esters from glycerides and a lower layer which holds the tetramethylammonium soaps of FFAs. The upper layer may be


used to analyse the fatty acid composition of glycerides. For FFA analysis, the bottom layer is separated, washed with ethyl ether and adjusted to pH 9. The methyl esters are then analysed by programmed GC, as described by Juarez et al. (1992). N2 or He was used as carrier gas; the column was a silica capillary column with silicone, containing 50% phenyl and 50% cyanopropyl groups as the stationary phase. Injecting samples at 300 °C gave the best quantitative results. The derivitisation technique tested resulted in no loss of the volatile components, because the FFAs were injected in the form of soaps. Method 2 involved the extraction of fats with diethyl ether, fixation of FFAs onto an Amberlyst resin, methylation of FFAs followed by quantification of methyl esters of FFAs by GC using an FID detector. Method 3 involves extraction of fat using heptane with the addition of sodium sulphate and sulphuric acid, followed by overnight storage of samples. On the day of analysis, the sample is extracted using a mixture of diethyl ether and heptane, the mixture is then added to a NH2 Sep-Pak Vac cartridge. The cartridge is rinsed using a chloroform/isopropanol mixture, FFAs are eluted using a diethyl ether/formic acid mixture and subsequently analysed by GC. Ardö and Polychroniadou (1999) reported on the accuracy and recovery for method 1 only. The accuracy of method 1 was checked using two types of cheese; one was a fresh cheese with a low FFA content and the other a cheese that had undergone moderate lipolysis. Reproducibility values for the fresh cheese were comparable to those reported by Woo and Lindsay (1982) for Cheddar cheese; however, they were lower than those reported by McNeill and Connolly (1989) and Deeth et al. (1983) also for Cheddar cheese. Accuracy was substantially higher for cheese with a high FFA content and there was an improvement in the overall coefficient of variation. Recovery was examined by adding standard mixtures of FFAs to a sample from a cheese of known FFA content. Recoveries ranged from 91% for butanoic acid (C4:0) to 103% for octadecanoic acid (C18:0). It was concluded that this technique provides a fast and reliable method of FFA analysis in cheeses with a low FFA level and particularly for those with a high FFA level. High performance liquid chromatography (HPLC) may also be used as a method for FFA analysis (Marsili, 1985). High performance liquid chromatography can operate at ambient temperature, ensuring relatively little risk to sensitive functional groups (Christie, 1997). In the method of Kilcawley et al. (2001), C2:0, C3:0 and C4:0 were recovered by steam-distillation and quantified by ligand-exchange, ion-exclusion HPLC. C6:0 to C18:3 are derivatized to bromophenacyl esters follow-

ing solvent extraction and separation is achieved by reversed-phase HPLC. Bills and Day (1964) used a silicic acid column to separate FFAs, which were then quantified by titration with potassium hydroxide, using phenolphthalein as an indicator. Removal of CO2 from the air stream when titrating is vital to maintain the stability of the end point. In this method, the air stream was freed from CO2 by bubbling through 20% KOH, and IDF (1991) recommend titration under nitrogen. It was later shown that the resin used induced fat hydrolysis (McNeill and Connolly, 1989). When using a titration method to quantify FFAs, the eluate of each FFA must be titrated separately and the method is not as accurate as more sophisticated GC or HPLC methods. At present, the most suitable methods for the determination of lipolysis in cheese use GC and HPLC; levels of FFAs and FFA profiles may be determined. There is potential for increased use of established technologies, such as gas chromotography/mass spectroscopy (GC/MS) and emerging technologies, e.g., liquid chromotography/mass spectroscopy, for the measurement of FFAs. GC/MS provides very accurate qualitative and quantitative measurement of FFAs as well as the other volatile components of dairy products (see ‘Instrumental Techniques’, Volume 1).

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Proteolysis in Cheese during Ripening V.K. Upadhyay, P.L.H. McSweeney, A.A.A. Magboul and P.F. Fox, Department of Food and Nutritional Sciences, University College, Cork, Ireland

Introduction As discussed in ‘Biochemistry of Cheese Ripening: Introduction and Overview’, Volume 1, proteolysis is the most complex and, in most varieties, the most important of the three primary biochemical events which occur in cheese during ripening. Because of its importance, the subject has been reviewed extensively (Grappin et al., 1985; Rank et al., 1985; Fox, 1989; Fox and Law, 1991; Fox et al., 1993, 1994, 1995b, 1996; Fox and McSweeney, 1996, 1997; Sousa et al., 2001). Proteolysis contributes to: • The development of cheese texture: – via hydrolysis of the protein matrix of cheese; – via a decrease in aw through changes to water binding by the new carboxylic acid and amino groups liberated on hydrolysis of peptide bonds. These groups are ionized at the pH of cheese and thus bind water; – indirectly via an increase in pH caused by the liberation of ammonia from amino acids produced by proteolysis. • Flavour and perhaps the off-flavour of cheese, – directly by the production of short peptides and amino acids, some of which have flavours; – indirectly by the liberation of amino acids which act as substrates for a range of catabolic reactions which generate important volatile flavour compounds (see ‘Catabolism of Amino Acids in Cheese During Ripening’, Volume 1); – by facilitating the release of sapid compounds from the cheese matrix during mastication. Proteolysis in cheese during ripening is catalysed by proteinases and peptidases from six sources: • The coagulant: The enzymes involved depend on the type of coagulant used (chymosin, pepsin, fungal acid proteinases, plant acid proteinases). Residual coagulant activity retained in the curd is the major source of proteolytic activity in most cheeses except pasta-filata varieties and those with a high cook temperature in which enzymes from this source are denatured extensively.

• The milk: A number of indigenous proteinases are present in milk, the most important of which is plasmin, which is produced from an inactive precursor, plasminogen. The action of plasmin is of particular importance in pasta-filata and high-cook cheeses (since it is a heat-stable enzyme) and in cheeses the pH of which increases during ripening (since the pH optimum of plasmin is c. 7.5). Somatic cells, recruited into milk to flight mastitic infection, contain lysosomes which contain a number of proteinases, including cathepsins D and B. • Starter lactic acid bacteria (LAB): The starter LAB contain a cell envelope-associated proteinase (CEP, lactocepin, PrtP) which contributes to ripening principally by hydrolysing intermediate-sized and short peptides produced from the caseins by the action of chymosin or plasmin. The starter is the principal source of peptidases in cheese, which are responsible for the hydrolysis of short peptides and the liberation of amino acids. • Non-starter lactic acid bacteria (NSLAB): All ripened cheeses contain an adventitious secondary microflora which grows during ripening (see ‘The Microbiology of Cheese Ripening’, Volume 1). The proteinases and peptidases of NSLAB are generally similar to those of starter LAB and contribute to ripening in a similar fashion. • Secondary starter: Many cheese varieties are characterized by the development of a secondary microflora which is added to or deliberately encouraged to grow (e.g., Propionibacterium freudenreichii subsp. shermanii in Swiss-type cheese, Penicillium roqueforti in Blue cheese, P. camemberti in Camembert and Brie-type cheeses and a complex Grampositive bacterial microflora on the surface of smear cheeses). Most of these microorganisms possess potent enzyme activities, including proteinases and peptidases, which contribute greatly to proteolysis in these varieties. • Exogenous proteinases and peptidases: Exogenous proteolytic enzymes have been studied as a means of accelerating ripening, accentuating flavour



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or debittering cheese. With the exception of enzymemodified cheese (see ‘Cheese as an Ingredient’, Volume 2), exogenous proteinases and peptidases are not used widely in cheese manufacture, although the action of these enzymes can dominate patterns of proteolysis in cheeses in which they are used. The use of exogenous proteinases to accelerate cheese ripening was discussed by Upadhyay and McSweeney (2003). The relative importance of enzymes from each of these sources depends on the cheese variety. Techniques used to assess the relative importance of proteolytic enzymes from different sources were discussed by Fox et al. (1993) and are outlined in ‘Biochemistry of Cheese Ripening: Introduction and Overview’, Volume 1. This chapter will focus on the principal proteolytic enzymes found in cheese and their role in cheese during ripening, with particular emphasis on their specificity on the caseins. Most attention will be paid to enzyme systems common to many cheese varieties (i.e., enzymes from the coagulant, milk and starter). Patterns of proteolysis in individual cheese varieties and the enzymes of specific secondary microorganisms are also discussed in some chapters of Volume 2.

Enzymes from the Coagulant As discussed in ‘Rennets: General and Molecular Aspects’, Volume 1, chymosin (EC. 3.4.23.4), the principal proteinase in traditional rennets used for cheesemaking, is an aspartyl proteinase of gastric origin, secreted by young mammals. The principal role of chymosin in cheesemaking is to hydrolyse the Phe1059Met106 bond of the micelle-stabilizing protein, -casein, as a result of which the colloidal stability of the micelles is destroyed, leading to gelation at temperatures ⬃20 °C (see ‘Rennet-induced Coagulation of Milk’, Volume 1). Most of the rennet added to cheesemilk is removed in the whey but some is retained in the curd and plays a major role in the initial proteolysis of caseins in many cheese varieties. From ⬃0 to 30% of the coagulant added to the cheesemilk is retained in the curd, depending on enzyme type, cooking temperature, pH at draining and the moisture content of curd. In Cheddar cheese, ⬃6% of the chymosin added to the milk is retained in the curd, but the amount increases with decreasing pH at whey draining (Holmes et al., 1977; Creamer et al., 1985). Pepsins, especially porcine pepsin, are more pH-sensitive than chymosin and hence the amount of these coagulants retained in cheese curd is very strongly dependent on the pH of the milk at setting and shortly thereafter; in fact, increasing the pH of the curds–whey mixture to ⬃7

after coagulation of milk by porcine pepsin is one of the methods used to produce rennet-free cheese curd (see ‘Biochemistry of Cheese Ripening: Introduction and Overview’, Volume 1). Only 2–3% of Rhizomucor rennets is retained in Cheddar cheese curd and appears to be independent of pH (Creamer et al., 1985). In high-cooked cheeses, e.g., Emmental, chymosin is extensively denatured (Matheson, 1981; Singh and Creamer, 1990; Boudjellab et al., 1994), and makes relatively little contribution to ripening. The action of chymosin on the B-chain of insulin indicates that it is specific for hydrophobic and aromatic amino acid residues (Fish, 1957). Relative to many other proteinases, chymosin is weakly proteolytic; indeed, limited proteolysis is one of the characteristics to be considered when selecting proteinases for use as rennet substitutes (Fox, 1989; Dalgleish, 1993). The primary chymosin cleavage site in the bovine milk protein system is the Phe1059Met106 bond in -casein which is many times more susceptible to chymosin than any other bond in milk proteins. s1-, s2- and -caseins are not hydrolysed during milk coagulation but may be hydrolysed in cheese during ripening. A number of authors (Pelissier et al., 1974; Creamer, 1976; Visser and Slangen, 1977; Carles and Ribadeau-Dumas, 1984) have investigated the hydrolysis of -casein by chymosin. In solution in 0.05 M Na acetate buffer, pH 5.4, chymosin cleaves -casein at seven sites: Leu1929Tyr193 Ala1899Phe190 Leu1659Ser166 Gln1679Ser168 Leu1639Ser164 Leu1399Leu150 Leu1279Thr128 (Visser and Slangen, 1977). The parameters, KM and kcat, for the action of chymosin on the bond Leu1929Tyr193 are 0.075 mM and 1.54 s 1, respectively, for micellar -casein and 0.007 mM and 0.56 s 1 for the monomeric protein (Carles and Ribadeau-Dumas, 1984). NaCl inhibits the hydrolysis of -casein by chymosin to an extent dependent on pH; hydrolysis is strongly inhibited by 5% and completely by 10% NaCl (Mulvihill and Fox, 1978). The primary site of chymosin action on s1-casein is Phe239Phe24 (Hill et al., 1974; Carles and RibadeauDumas, 1985; McSweeney et al., 1993b). Cleavage of this bond is believed to be responsible for the initial softening of cheese texture (de Jong, 1976; Creamer and Olson, 1982) and the small peptide ( s1-CN f1923) is hydrolysed rapidly by starter proteinases. The specificity of chymosin on s1-casein in solution was studied by Pelissier et al. (1974), Mulvihill and Fox (1979a), Pahkala et al. (1989) and McSweeney et al. (1993b). In 0.1 M phosphate buffer, pH 6.5, chymosin cleaves s1-casein at Phe239Phe24, Phe289 Pro29, Leu409Ser41, Leu1499Phe150, Phe1539 Tyr154,

Proteolysis in Cheese during Ripening 393

Leu1569Asp157, Tyr1599Pro160 and Trp1649Tyr165 (McSweeney et al., 1993b). These bonds are also hydrolysed at pH 5.2 in the presence of 5% NaCl (i.e., similar to the conditions in many cheese varieties), and, in addition, Leu119Pro12, Phe329Gly35, Leu1019 Lys102, Leu1429Ala144 and Phe1799Ser180. The rate at which many of these bonds are hydrolysed depends on the ionic strength and pH, particularly Leu1019Lys102 which is cleaved far faster at the lower pH. The kcat and KM for the hydrolysis of Phe239Phe24 bond of s1-casein by chymosin is 0.7 s 1 and 0.37 mM, respectively (Carles and Ribadeau-Dumas, 1985). The hydrolysis of s1-casein by chymosin is influenced by pH and ionic strength (Mulvihill and Fox, 1977, 1980). s2-Casein appears to be relatively resistant to proteolysis by chymosin; cleavage sites are restricted to the hydrophobic regions of the molecule (sequences 90–120 and 160–207), i.e., Phe889Tyr89, Tyr959 Leu96, Gln979Tyr98, Tyr989Leu99, Phe1639Leu164, Phe1749Ala175 and Tyr1799Leu180 (McSweeney et al., 1994b). Although para--casein has several potential chymosin cleavage sites, it does not appear to be hydrolysed either in solution or in cheese (Green and Foster, 1974). Presumably, this reflects the relatively high level of secondary structure in -casein compared to the other caseins (see Swaisgood, 1992); it would be interesting to investigate the hydrolysis of para--casein by chymosin or pepsin in the presence of a high concentration of urea (pepsin is active in 8 M urea). Good quality veal rennet contains about 10% bovine pepsin (EC 3.4.23.1; Rothe et al., 1977) but many ‘calf rennets’ contain up to 50% bovine pepsin. The proteolytic products produced from Na-caseinate by bovine pepsin are similar to those produced by chymosin (Fox, 1969), although, as far as we are aware, the specificity of bovine or porcine pepsins on bovine caseins has not been determined rigorously. However, Mulvihill and Fox (1979b) compared the hydrolysis of bovine, ovine, caprine and porcine -caseins by chymosins and pepsins from these species. The large peptides produced (detectable by urea–polyacrylamide gel electrophoresis) suggested generally similar specificities for chymosins and pepsins although differences were apparent in the short (pH 4.6-soluble) peptides. Pepsins were more proteolytic than the corresponding chymosins. For many years, the supply of calf rennet has been insufficient to meet demand, and much effort has been expended on searching for suitable rennet substitutes for cheesemaking (see Green, 1977; Phelan, 1985). Several proteinases have been assessed but only bovine pepsin and proteinases from Rhizomucor pusillus,

R. miehei and Cryphonectria parasitica have been used extensively in commercial practice (Phelan, 1985; van den Berg, 1992); blends of calf rennet and porcine pepsin (50:50) have been used in the past with generally satisfactory results. Of these rennets, that from R. miehei is the most widely used (under various trade names) and gives generally satisfactory results. The gene for R. miehei proteinase has been cloned in Aspergillus oryzae, resulting in a rennet containing less contaminating proteolytic enzymes. This coagulant was found to be acceptable for the manufacture of Cheddar cheese (Chen et al., 1994). C. parasitica proteinase is considerably more proteolytic than chymosin (Tam and Whitaker, 1972), especially on -casein, and is rarely used for cheesemaking except for high-cooked varieties (e.g., Swiss), in which the proteinase is extensively denatured by the high cook temperature. The specificity of many of these enzymes on the oxidized B-chain of insulin was summarized by Green (1977). Preliminary studies on the hydrolysis of sodium caseinate by fungal rennets were reported by Tam and Whitaker (1972) and later by Phelan (1985). Rea (1997) compared hydrolysates of sodium caseinate by chymosin, R. miehei proteinase and C. parasitica proteinase; the specificities of the three enzymes on the caseins were clearly very different (C. parasitica was particularly active on -casein). However, the bonds cleaved by the fungal proteinases have not been reported (except that the primary cleavage of -casein by C. parasitica proteinase is at Ser1049Phe105, rather than Phe1059Met106 which is cleaved by chymosin and R. miehei proteinase (Drohse and Foltmann (1989)).

Indigenous Proteinases in Milk Plasmin

Milk contains a number of indigenous proteinases, of which plasmin (EC 3.4.21.7) is the most important with respect to cheese ripening. Plasmin is a trypsinlike serine proteinase with pH and temperature optima of ⬃7.5 and 37 °C, respectively. Plasmin is secreted into blood as its inactive zymogen, plasminogen, which is then activated to plasmin, the principal function of which in blood is to degrade fibrin clots. A complex plasminogen/plasmin system exists in blood, comprised of plasmin, plasminogen, plasminogen activators (PA), plasmin inhibitors, and inhibitors of PA, all of which are also present in milk. A diagrammatic representation of the plasmin/plasminogen system in milk is shown in Fig. 1. In milk, plasminogen, plasmin and PA are associated with the casein micelles while plasmin inhibitors and inhibitors of PA are in


Plasminogen activators

Plasminogen

Plasminogen activator inhibitors

Plasmin inhibitors

Plasmin

Casein

Polypeptides

Figure 1 Plasmin/plasminogen system in milk (adapted from Bastian and Brown, 1996).

the serum phase. Plasmin has trypsin-like specificity, showing preference for bonds of the type Lys9X and, to a lesser extent Arg9X, and acts on caseins in the order 艐 s2 >> s1, while -casein appears to be resistant to hydrolysis by plasmin (Bastian and Brown, 1996). Casein has 15–17 (depending on the genetic variant) potential plasmin-susceptible bonds, but only three are hydrolysed at significant rates in milk, Lys289Lys29, Lys1059His106 and Lys1079Glu108, hydrolysis of which results in the release of 1-CN (-CN f29–209), 2-CN (-CN f106–209), 3-CN (-CN f108–209), proteose peptone (PP)8 fast (-CN f1–28), PP8 slow -CN (f29–105) and (f29–107) and PP5 (-CN f1–105 and 1–107) (Fig. 2). In solution, plasmin hydrolyses -casein at Lys979Val98, Lys1139Tyr114, Lys1699Val170, Lys1769Ala177, Arg1839Asp184, Arg2029Gly203 as well as at Lys289Lys29, Lys1059His106 and Lys1079Glu108 (Fox et al., 1994). s2-Casein is also a good substrate for plasmin. In buffered systems, plasmin hydrolyses s2-casein at Lys219Gln22, Lys249Asn25, Lys1499Lys150, Lys1509Thr151, Lys1819Thr182, Lys1889Ala189,

Lys 28 ⎯ Lys

Lys1979Thr198 and Arg1149Asn115 (Le Bars and Gripon, 1989; Visser et al., 1989), but it has not yet been determined whether peptides resulting from cleavage at these sites are produced in milk or dairy products, although this is likely since the concentration of s2-casein, which is a poor substrate for chymosin, decreases in cheese during ripening (Fox and McSweeney, 1997). s1-Casein is less susceptible to hydrolysis by plasmin than -casein (Andrews and Alichanidis, 1983). However, Le Bars and Gripon (1993) identified seven Lys9X and four Arg9X plasminsusceptible bonds in s1-casein while McSweeney et al. (1993c) identified 12 Lys9X and 5 Arg9X plasminsusceptible bonds in s1-casein (Lys39His4, Lys79 His8, Arg229Phe23, Lys349Glu35, Lys369Lys37, Lys589 Gln59, Lys799His80, Arg909Tyr91, Arg1009Leu101, Lys1029Lys103, Lys1039Tyr104, Lys1059Val106, Arg1199 Leu120, Lys1249Glu125, Gln1319Lys132, Arg1519Gln152, Lys1939Thr194), including all those reported by Le Bars and Gripon (1993). It is likely that -caseins originate as a result of hydrolysis of s1-casein by plasmin (Aimutis

Lys105 ⎯His106 Lys113 ⎯Tyr114

29

Arg

183

⎯Asp

184

209

1 Lys107 ⎯ Glu108

Products

γ-Caseins

Proteose peptones Known PP8f PP8s PP8s PP-T PP5 PP5

β-CN f1–28 β-CN f29–105 β-CN f29–107 β-CN f29–113 β-CN f1–105 β-CN f1–107

Probable β-CN β-CN β-CN β-CN β-CN β-CN

f106–113 f108–113 f114–183 f106–183 f108–183 f1–113 (unlikely)

Figure 2 Hydrolysis of -casein by plasmin (adapted from Fox et al., 1994).

γ1-CN γ2-CN γ3-CN γ4-CN γ5-CN

(β-CN f29–209) (β-CN f106–209) (β-CN f108–209) (β-CN f114–209) (β-CN f184–209)


and Eigel, 1982; O’Flaherty, 1997). The resistance of -casein to hydrolysis by plasmin is probably due to the carbohydrate moieties attached to its C-terminal region (Doi et al., 1979). Since they are associated with the casein micelles, plasmin, plasminogen and PA are incorporated into the cheese curd but plasmin inhibitors and inhibitors of PA are present in the cheese whey (Bastian and Brown, 1996). The contribution of plasmin to proteolysis varies depending on cheese type. Farkye and Fox (1990) found that plasmin activity in cheese was in the order EmmentalBlarneyRomano-typeGoudaCheddar Cheshire cheeses. Many factors (e.g., cooking temperature, pH during ripening) contribute to this difference in plasmin activity in cheese. Plasmin is the primary proteolytic agent in Swiss-type cheese due to the fact that these cheeses are cooked at a high temperature (⬃55 °C), at which most of the coagulant, chymosin, is inactivated, while plasmin, which is a heat-stable enzyme, survives cooking. At high cooking temperatures, activation of plasminogen occurs, probably due to heat-induced inactivation of inhibitors of PA and of plasmin (Farkye and Fox, 1990). Hence, differences in plasmin activity between Swiss and Cheddar cheeses are due to differences in the cooking temperature used in the manufacturing protocols. Plasmin also plays a major role in ripening of mould-ripened (e.g., Camembert) and smear-ripened (e.g., Tilsit) cheese varieties. During ripening of Camembert-type cheese, catabolism of lactic acid and deamination of amino acids with the production of NH3 by the mould on the surface of the cheese result in an increase in the pH of the surface layer to ⬃7.0. Gradually, the pH of the interior of the cheese also increases due to the outward migration of lactic acid and the inward migration of NH3 (see ‘Metabolism of Residual Lactose and of Lactate and Citrate’, Volume 1). The increased pH facilitates the action of plasmin, which contributes significantly to proteolysis in these cheeses (Gripon, 1993). The pH of the surface layer of smearripened cheeses also increases during ripening, which facilitates plasmin action on the caseins and influences the quality of these cheese varieties (O’Farrell et al., 2002). Due to the significance of plasmin to proteolysis during the ripening of many cheese varieties, a number of attempts have been made to increase plasmin activity in cheese using different approaches. Farkye and Fox (1992) added exogenous plasmin to cheesemilk for Cheddar cheese. The level of water-soluble N in cheese enriched with plasmin was ⬃20% higher than in the control cheeses. As expected, increased plasmin activity did not increase the level of phosphotungstic acid-soluble N, which is mainly due to free amino acids. The authors claimed superior organolep-

tic quality for the plasmin-enriched cheese. Somers et al. (2002) enriched milk for the manufacture of Mozzarella-type cheese with plasmin; greater hydrolysis of -casein by plasmin was observed in the experimental cheeses compared to the control, but enzyme treatment did not affect the composition or functionality of the cheese. Primary proteolysis (as measured by levels of pH 4.6-soluble N and urea–polyacrylamide gel electrophoresis, PAGE), was accelerated in smearripened cheese made from plasmin-enriched milk (O’Farrell et al., 2002). Farkye and Fox (1991), who added 6-aminohexanoic acid, a plasmin inhibitor, to stirred-curd Cheddar cheese, found slower degradation of -casein compared to control cheese. Successful attempts have been made to accelerate proteolysis in cheese by increasing plasmin activity by the use of exogenous PA (e.g., urokinase) in the manufacture of Cheddar (Barrett et al., 1999), Swiss (Bastian et al., 1997), ultrafiltered Havarti and Saint Paulin (Bastian et al., 1991) cheeses. Bastian et al. (1991) manufactured Havarti and Saint-Paulin cheese by traditional or ultrafiltration (UF) technology and added urokinase and KIO3, individually and in combination, to UF retentate before cheesemaking. Addition of urokinase increased plasmin activity in UF Havarti and Saint-Paulin cheese and increased proteolysis. Swiss cheese manufactured from milk containing 0.05 UL 1 or 0.5 UL 1 urokinase showed increased plasmin activity and faster degradation of -casein than control cheese (Bastian et al., 1997). The use of exogenous urokinase in the manufacture of Cheddar cheese resulted in activation of plasminogen and acceleration of proteolysis compared to control cheese (Barrett et al., 1999). Streptokinase, an extracellular protein secreted by Streptococcus uberis, forms a 1:1 complex with plasminogen, inducing a conformational change in plasminogen, which renders the proteinase active without prior proteolytic cleavage. The active streptokinase–plasminogen complex is then able to convert other plasminogen molecules to plasmin by proteolysis (Johnsen et al., 2000). Streptokinase activated most of the plasminogen in milk and increased plasmin activity in experimental Cheddar cheese compared to controls (V.K. Upadhyay, unpublished). Proteolysis in the cheese was accelerated as indicated by an increased level of pH 4.6-soluble nitrogen, breakdown of -casein and the production of hydrophobic peptides. In an alternative approach, V.K. Upadhyay (unpublished) used a starter strain, which had been genetically modified to produce streptokinase, for the manufacture of Cheddar cheese; plasminogen was activated to plasmin, and proteolysis in the cheese during ripening was accelerated.


All the approaches for increasing plasmin activity in different cheese varieties have accelerated proteolysis. However, plasmin activity is not rate-limiting for flavour development in cheese during ripening as plasmin is responsible for the production of large to intermediate-sized peptides (Farkye and Fox, 1992), which do not contribute directly to flavour. However, plasmin may have an indirect role in cheese flavour by producing intermediate-sized peptides which are hydrolysed further by lactocepin and starter peptidases, ultimately to free amino acids, which are important precursors of volatile flavour compounds (see ‘Catabolism of Amino Acids in Cheese During Ripening’, Volume 1). Other indigenous proteinases

During mastitis, there is an increase in the somatic cell count of milk as a result of increased transfer of leukocytes from blood to milk. Somatic cells contain many active proteinases, including cathepsins B, D, G, H, L and elastase (Kelly and McSweeney, 2003). The presence of cathepsins D and B in milk is confirmed. However, it is likely that other lysosomal proteinases are also present in milk. Cathepsin D is an aspartic proteinase located in the lysosomes of mammalian cells and has pH and temperature optima of 4.0 and 37 °C, respectively. Cathepsin D is synthesized on the rough endoplasmic reticulum as its inactive form, procathepsin D. Procathepsin D is converted autocatalytically to pseudocathepsin D which is then converted to mature active cathepsin D by further proteolysis, mediated by cysteine proteinases. Larsen and Petersen (1995) purified five molecular forms of cathepsin D, having apparent molecular masses of 46, 45, 43, 39 and 31 kDa, from bovine milk; the 46 and 45 kDa forms corresponded to procathepsin D (the reason for this variability in molecular mass is unknown; Hurley et al., 2000a), 43 kDa to pseudocathepsin D and 39 and 31 kDa to mature single-chain and heavychain cathepsin D, respectively (Fig. 3). However, Larsen and Petersen (1995) did not detect a band that corresponding to light chain cathepsin D (14 kDa), probably due to lack of sensitivity of their experimental methods. The concentration of cathepsin D in skimmed milk and whey is estimated to be 0.4 and 0.3 g ml 1, respectively, indicating that it is a serum protein (Larsen et al., 1996). Therefore, little cathepsin D would be expected in cheese. Furthermore, cathepsin D only partially survives heat treatment at 55 °C for 30 min (45% survival) or HTST pasteurization (72 °C 15 s) (8% survival) (Larsen et al., 2000; Hayes et al., 2001). Hydrolysis of the caseins by cathepsin D has been studied in model systems. Kaminogawa et al. (1980) reported that cathepsin D partially purified from milk

1

P1

Procathepsin D

Pseudocathepsin D

46kDa

346

P27 1

43kDa

346

1

39kDa

346

31kDa

346

Cathepsin D (Single chain)

102 Cathepsin D (Heavy chain)

Cathepsin D

1

14 kDa

99

(Light chain)

Figure 3 Molecular forms of cathepsin D present in bovine milk (adapted from Larsen and Petersen, 1995).

hydrolyses s1-casein with the production of a peptide with a molecular mass similar to s1-CN (f24–199), a peptide that is produced from s1-casein by chymosin (Fig. 4a) (McSweeney et al., 1993b). Bonds in s1casein susceptible to the action of cathepsin D include Phe239Phe24, Phe249Val25, Leu989Leu99 and Leu1499 Phe150 (Larsen et al., 1996). Cathepsin D hydrolyses -casein to peptides similar to those produced by chymosin (Fig. 4b); susceptible bonds include Phe529 Ala53, Leu589Val59, Pro819Val82, Ser969Lys97, Leu1259Thr126, Leu1279Thr128, Trp1439Met144, Phe1579Pro158, Ser1619Val162, Leu1659Ser166, Leu1919Leu192, Leu1929Tyr193 and Phe2059Pro206. However, hydrolysates of s2-casein produced by cathepsin D differ markedly from those produced by chymosin and have very few peptides in common when analysed by urea-PAGE and reversed phase-high performance liquid chromatography (RPHPLC) (McSweeney et al., 1995). Cathepsin D-sensitive bonds in s2-casein include Leu999Tyr100, Leu1239 Asn124, Leu1809Lys181 and Thr1829Val183 (Larsen et al., 1996). Cathepsin D hydrolyses -casein at Leu329Ser33, Leu799Ser80 and Phe1059Met106. Two cleavage sites for cathepsin D on -lactalbumin have been identified (Leu529Phe53 and Trp1049Leu105), while -lactoglobulin appears to be resistant to this enzyme (Larsen et al., 1996). Although the specificity of cathepsin D is similar to that of chymosin, it coagulates milk poorly (McSweeney et al., 1995). It is very difficult to assess the contribution of cathepsin D to proteolysis in cheese made with rennet due to the low level of cathepsin D and the masking effect by the much larger level of chymosin in cheese. Wium et al. (1998) reported that s1-CN (f24–199) was produced in a Feta-type cheese made without the addition of rennet from pasteurized, homogenized, ultrafiltered milk, acidified using gluconic acid--lactone (GDL),

(a) Cathepsin G Cathepsin B Elastase RPKHPIKHQGLPQEVLNENLLRFFVAPFPEVFGKEKVNELSKDIGSESTEDQAMEDIKQMEAESISSSEEIVPNSVEQKHIQKEDVPSERYLGYLEQLLRLKKYK 20

40

100

80

60

Plasmin Cathepsin D Chymosin

Cathepsin G Cathepsin B Elastase VPQLEIVPNSAEERLHSMKEGIHAQQKEPMIGVNQELAYFYPELFRQFYQLDAYPSGAWYYVPLGTQYTDAPSFSDIPNPIGSENSEKTTMPLW 120

140

160

180

199

Plasmin Cathepsin D Chymosin Figure 4 Primary structure of (a) bovine s1-casein showing the cleavage sites of elastase (Considine et al., 2000), cathepsin B (Considine et al., 2004), cathepsin G (Considine et al., 2002), plasmin (Le Bars and Gripon, 1993; McSweeney et al., 1993c), cathepsin D (Larsen et al., 1996) and chymosin (McSweeney et al., 1993b) and (b) bovine -casein showing the cleavage sites of elastase (Considine et al., 1999), cathepsin B (Considine et al., 2004), cathepsin G (Considine et al., 2002), plasmin (Eigel et al., 1984; Visser et al., 1989; Singh et al., 1994), cathepsin D (Larsen et al., 1996) and chymosin (Visser and Slangen, 1977).

397

398

(b) Cathepsin G Cathepsin B Elastase RELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQTEDELQDKIHPFAQTQSLVYPFPGPIHNSLPQNIPPLTQTPVVVPPFLQPEVMGVSKVKEAMAPKHKEMPF 20

40

100

80

60

Plasmin Cathepsin D Chymosin

Cathepsin G Cathepsin B Elastase PKYPVEPFTERQSLTLTDVENLHLPLPLLQSWMHQPHQPLPPTVMFPPQSVLSLSQSKVLPVPQKAVPYPQRDMPIQAFLLYQEPVLGPVRGPFPIIV 120

Plasmin Cathepsin D Chymosin Figure 4 continued

140

160

180

200


which was attributed to the action of cathepsin D. Hurley et al. (2000b) made Quarg, from raw skim milk, pasteurized skim milk or raw skim milk with added pepstatin (a potent inhibitor of aspartyl proteinases) by acidification using GDL. Reversed phase–high performance liquid chromatography of the water-soluble fraction of the cheeses made from raw or pasteurized skim milk showed peptides eluting at 46–48 min, but these peptides were absent from the profiles of pepstatin-treated cheese, suggesting indigenous aspartyl proteinase activity in Quarg, presumably cathepsin D. Cathepsin D activity may contribute to a low extent to the degradation of s1-casein in high-cooked cheese varieties (e.g., Swiss-type, Parmigiano-Reggiano) in which most of the chymosin is inactivated. The presence of cysteine proteinase activity in bovine milk has been reported (Suzuki and Katoh, 1990; O’Driscoll et al., 1999). Mammalian cysteine proteinases (cathepsins B, H, L and I) are lysosomal enzymes that are synthesized as proenzymes (MW 37–55 kDa), which are later converted to catalytically active forms (MW 23–30 kDa) (Barrett and Kirschke, 1981; Zeece et al., 1992; Kirschke et al., 1998). Magboul et al. (2001) resolved five fractions (f I–f V) with cysteine proteinase activity by ion-exchange chromatography of acid whey on Q-Sepharose. Fractions fIII and fV were the most active and were capable of hydrolysing s1- and -caseins. Immunoblotting of fIII with antibodies to the bovine lysosomal cysteine proteinase, cathepsin B, indicated the presence of cathepsin B in fIII. Partially purified fIII retained 20% of its original activity when heated at 72 °C 30 s. It is possible that cathepsin B may contribute to cheese ripening but further research is required to determine its role, if any, and the distribution of this enzyme in milk. Cathepsin G is a serine proteinase with a molecular mass of 24–26 kDa. In solution, cathepsin G readily hydrolyses s1- and -casein extensively, including some sites that are very close, or identical, to chymosin cleavage sites; it produces s1-CN (f1–23) by cleavage of the Phe239Phe24 (Fig. 4a) (Considine et al., 2002). Many cathepsin G cleavage sites in s1- and -caseins are also close to those of plasmin. Cathepsin G has broad specificity on s1- and -caseins and hence it is possible that it may contribute to proteolysis in cheese made from high SCC milk; however the presence of this enzyme in milk has not been demonstrated. Elastase, a serine proteinase with a molecular weight of 29.5 kDa, is an important lysosomal enzyme in somatic cells, although its presence in milk has not been demonstrated. Elastase can hydrolyse a wide variety of proteins, including s1- and -caseins and has many cleavage sites in common with chymosin,

plasmin and the lactocepin of Lactococcus (Considine et al., 1999, 2000). Hence, if elastase is present in milk, it may contribute to proteolysis in cheese during ripening. Of all the indigenous enzymes discussed above, only plasmin and, to a lesser extent, cathepsin D, have been studied in detail. Research to establish the presence of the other enzymes in milk, and to elucidate their role in proteolysis in cheese during ripening is needed.

Proteolytic Enzymes of LAB Lactic acid bacteria are fastidious organisms that have complex amino acid requirements (Law et al., 1976; Morishita et al., 1981; Chopin, 1993). The concentrations of amino acids in milk are below the nutritional requirements for the growth of the auxotrophic LAB to high cell populations. When growing in milk, their complex proteolytic system degrades mainly caseins into small peptides and amino acids which fulfil their nutritional requirements and inadvertently contribute to the flavour of fermented dairy products (Law and Mulholland, 1995; Steele, 1995). Many components of the proteolytic system of LAB have been purified and characterized and most of the corresponding genes have been cloned and sequenced (for reviews of the extensive literature on this subject, see Pritchard and Coolbear, 1993; Bockelmann, 1995; Exterkate, 1995; Poolman et al., 1995; Juillard et al., 1996; Kunji et al., 1996; Law and Haandrikman, 1997; Christensen et al., 1999). The best-studied proteolytic system among the LAB is that of Lactococcus followed by themophilic Lactobacillus spp. because of their economic importance as starter cultures in dairy fermentations. The proteolytic systems of mesophilic lactobacilli, which dominate the non-starter microflora of Cheddar, Dutch-type and probably most cheeses during ripening (Jordan and Cogan, 1993; Williams and Banks, 1997; ‘The Microbiology of Cheese Ripening’, Volume 1), have received less attention. The main components of the proteolytic system of LAB are proteinases (mainly the cell envelope-associated proteinase, CEP, or lactocepin; EC 3.4.21.96), although intracellular proteinases have been reported; Muset et al., 1989; Akuzawa et al., 1990), amino acid and peptide transport systems, and a range of intracellular peptidases. During the growth of LAB in milk, the initial step in casein degradation is performed by lactocepin and the short peptides produced are taken up by the cell via peptide transport systems (Juillard et al., 1995). Further degradation to amino acids is catalysed by a number of intracellular peptidases (Kunji et al., 1996; Law and Haandrikman, 1997; Christensen et al., 1999). The starter stops growing in cheese curd soon after the end of manufacture due to the low pH, increasing NaCl


concentration, low temperature and lack of a fermentable carbohydrate substrate (see ‘The Microbiology of Cheese Ripening’, Volume 1). However, its enzymes play a very important role in proteolysis during ripening, particularly when intracellular enzymes are released from the cell following lysis. The rate of secondary proteolysis is higher in cheese made with fast-lysing than that with slow-lysing starter strains (Wilkinson et al., 1994; O’Donovan et al., 1996; Morgan et al., 1997; Martínez-Cuesta et al., 2001; Hannon et al., 2003). Proteinases from LAB

Immunogold-labelling and genetic studies have shown that lactocepin is located outside the lactococcal cell (Hugenholtz et al., 1987; de Vos and Siezen, 1994). Calcium is necessary for stable attachment of lactocepin to the cell envelope; the proteinase is released by incubation of the cells in a calcium-free buffer, a characteristic which is usually exploited as the first step in isolation procedures (Mills and Thomas, 1978; McSweeney et al., 1993a). Lactocepins have a molecular mass of c. 140 kDa and a pH optimum of 5.5–6.5 (Law and Haandrikman, 1997). The lactocepins from a number of Lactococcus strains have been characterized biochemically and genetically; they are homologous with the subtilisin family of serine proteinases, with similar catalytic domains (Pritchard and Coolbear, 1993; Kok and de Vos, 1994; Kunji et al., 1996; Law and Haandrikman, 1997; Siezen, 1999; ‘Starter Cultures: General Aspects’, Volume 1). The lactocepins and subtilisins have a conserved active site triad, consisting of aspartic acid, histidine and serine. A region of 107 residues which includes the active site and the substrate-binding region is highly conserved (Law and Haandrikman, 1997). Lactocepins of Lactococcus were initially classified into two broad groups, PI- and PIII-type proteinases (Tan et al., 1993). PI-Type enzymes (e.g., produced by Lc. lactis subsp. cremoris HP and Wg2) degrade -casein rapidly but act only slowly on s1-casein whereas PIII-type proteinases (e.g., AM1 and SK11) hydrolyse -casein differently to PI-type strains and rapidly hydrolyse s1- and -caseins (Visser et al., 1986; Fox and McSweeney, 1996; Law and Haandrikman, 1997). Although this broad classification scheme remains useful, it soon became apparent that the lactocepin of certain strains of Lactococcus had a specificity intermediate between PI- and PIII-type enzymes. The nucleotide sequence of the gene encoding all lactocepins is very similar (98% homology; Kok et al., 1988; Vos et al., 1989a,b) and alteration of a few amino acid residues in the enzyme can alter its specificity. Exterkate et al. (1993) proposed a classification

scheme for lactocepins based on their specificity on the peptide, s1-CN (f1–23), which is released early in cheese ripening by the action of chymosin (Fig. 5). The specificity of the lactocepins from a number of strains of Lactococcus on s1-, s2-, - and -caseins is summarized in Figs 6–9. The primary role of lactocepin is to degrade the caseins to provide short peptides to support the growth of the lactococcal cells in milk. However, its role in cheese ripening is different. Peptides isolated from Cheddar cheese, the N- or C-terminus of which corresponds to the specificity of lactocepin, do not contain a major chymosin or plasmin cleavage site (Fox and McSweeney, 1996), suggesting that chymosin or plasmin act first and that lactocepin then hydrolyses the resulting intermediate-sized peptides. A number of authors have investigated the action of lactocepins on peptides produced from the caseins by the action of chymosin (Fig. 10). Cell envelope-associated proteinases with properties similar to the lactococcal lactocepins have also been isolated from a number of strains of Lactobacillus (see Kunji et al., 1996; Law and Haandrikman, 1997; Christensen et al., 1999). Although much less well studied than the lactocepins, Lactococcus spp. also possess intracellular proteinases. Muset et al. (1989) isolated an intracellular metalloproteinase from Lc. lactis subsp. lactis NCDO763 which was optimally active at pH 7.5 and 45 °C and exhibited thermolysin-like specificity. Akuzawa et al. (1990) identified four intracellular proteinases with caseinolytic activity. The enzymes with activity on casein ranged in molecular mass from 12 to 160 kDa and were optimally active at pH 5.5–7.0. Two enzymes were metalloproteinases, one had a serine catalytic mechanism and one was a thiol proteinase. The authors also obtained eight fractions with activity on benzgloxycarbonyl-L-Phe-L-Arg-7(4-methyl) coumarylamide but none of the eight fractions was able to hydrolyse casein. Three intracellular proteinases (P1, dimeric, Mr 124 kDa; P2, monomeric, Mr 64 kDa and P3, monomeric, Mr 47 kDa) were demonstrated in the cytoplasmic fraction of the lactocepin-negative strain, Lc. lactis subsp. cremoris MG1363, by Stepaniak et al. (1996). P1 was a metalloproteinase while P2 and P3 were serine proteinases. The enzymes were optimally active at pH 7.0 and 35 °C (P1) or pH 7.5 and 45 °C (P2, P3). Peptidases

While the role of lactocepin when the cell is growing in milk is the degradation of caseins to oligopeptides,

Figure 5 Classification of lactocepins of lactic acid bacteria according to their specificity on s1-CN (f1–23) (from Fox et al., 2000).

401

402 ↓ ↓

[1]

↓

↓

↓

[2] R1 P K H P I K H Q G L P Q E V L N E N L20 L R F F V A P F P E V F G K E K V N E L40 S K D I G S E S T E50

[1] [2]

↓

↓

↓

↓

D Q A M E D I K Q M60 E A E P I P P P E E I V P N S V E Q K H80 I Q K E D V P S E R Y L G Y L E Q L L R100

↓

[1]

↓

↓

↓

↓

↓

[2]

↓ ↓ ↓ L K K Y K V P Q L E I V P N S A E E R L120 H S M K E G I H A Q Q K E P M I G V N Q140 E L A Y P Y P E L F150

[1] [2]

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ R Q F Y Q L D A Y P160 S G A W Y Y V P L G T Q Y T D A P S F S180 D I P N P I G S E N S E K T T M P L W199

αs1-casein [1] Lc. lactis subsp. cremoris SK11 (Reid et al., 1991a) [2] Lc. lactis subsp. lactis NCDO 763 (Monnet et al., 1992) Figure 6 Reported cleavage sites for lactocepins on bovine s1-casein (from Fox et al., 1994).


K N T M E H V S S S E E S I I S Q E T Y20 K Q E K N M A I N P S K E N L C S T F C40 K E V V R N A N E E50 ↓ ↓ E Y S I G S S S E E60 S A E V A T E E V K I T V D D K H Y Q K80 A L N E I N E F Y Q K F P Q Y L Q Y L Y100 Q G P I V L N P W D Q V K R N A V P I T120 P T L N R E Q L S T S E E N S K K T V D140 M E S T E V F T K K150 ↓ ↓ ↓ ↓ ↓ ↓ ↓ T K L T E E E K N R160 L N F L K K I S Q R Y Q K F A L P Q Y L180 K T V Y Q H Q K A M K P W I Q P K T K V200 ↓ I P Y V R Y L207 αs2-casein

Figure 7 Reported cleavage sites for lactocepins on bovine s2-casein (from Monnet et al., 1992).

the hydrolysis of these peptides (after internalization into the cell) to amino acids is catalysed by peptidases. Many different peptidases from LAB have been characterized biochemically and genetically (see Kunji et al., 1996; Law and Haandrikman, 1997; Christensen et al., 1999; Siezen et al., 2002). The biochemical properties of the peptidases from cheese-related bacteria characterized to date are shown in Table 1. While the role of some of these peptidases (e.g., endopeptidases) is the degradation of oligopeptides to shorter peptides, exopeptidases function to release one or two amino acids at a time from short peptides. Based on their substrate specificity, peptidases are classified into different groups, as shown in Fig. 11.

A gene (pepE) encoding a thiol-dependent endopeptidase has been isolated from Lb. helveticus CNRZ32 (Fenster et al., 1997). The deduced amino acid sequence of PepE showed high homology with PepC from Lb. delbrueckii subsp. lactis DSM7290 (Klein et al., 1994a), Lb. helveticus CNRZ32 (Fernandez et al., 1994; Vesanto et al., 1994), Sc. thermophilus CNRZ302 (Chapot-Chartier et al., 1994) and Lc. lactis subsp. cremoris AM2 (Chapot-Chartier et al., 1993). Fenster et al. (1997) isolated and characterized recombinant PepE; the general properties of this enzyme indicated that it was different from the other metallo-endopeptidases characterized from LAB. Di- and tripeptidases

Endopeptidases

Several endopeptidases have been reported in lactococci and lactobacilli (Table 1), most of which are monomeric metallopeptidases. On the basis of substrate specificity, LAB appear to possess three types of endopeptidases (Monnet et al., 1994). PepO is a monomeric metallopeptidase with a molecular mass of ⬃70 kDa. It is capable of efficiently hydrolysing Metenkephalin, bradykinin, substance P, glucagon, oxidized B-chain of insulin and several casein fragments but not di-, tri- or tetra-peptides. PepO was the first endopeptidase for which the gene was sequenced (Mierau et al., 1993). The pepO gene is located immediately downstream of the genes for the oligopeptide transport system, indicating that the two systems are physiologically linked (Tynkkynen et al., 1993). Another oligopeptidase, designated PepF, specifically cleaves the Phe9Ser bond in bradykinin and was purified from Lc. lactis subsp. lactis NDCO 763; its gene (pepF) was cloned and sequenced (Monnet et al., 1994). This enzyme is a monomeric metallopeptidase of ⬃70 kDa and is capable of hydrolysing peptides containing 7–17 amino acids with broad specificity but not smaller or larger peptides. PepF is unable to hydrolyse Metenkephalin, which is a good substrate for PepO.

Tripeptidases (PepT) purified from LAB are generally di- or tri-meric metallopeptidases (Table 1) with broad specificity, capable of hydrolysing tripeptides with acidic, basic or neutral N-terminal amino acid residues. A broad-specificity general dipeptidase, PepV, which hydrolyses only dipeptides, is found in LAB (Kunji et al., 1996; Law and Haandrikman, 1997). A number of dipeptidases with similar properties have been purified and characterized from strains of Lactococcus and Lactobacillus (see Table 1). Most of the dipeptidases isolated from LAB are monomers with a molecular mass in the range 40–55 kDa (Table 1). With the exception of a dipeptidase from Lb. helveticus 53/7, which was reported to have a thiol catalytic mechanism (Vesanto et al., 1996), all the dipeptidases characterized to date are metallopeptidases (Table 1). All dipeptidases of LAB show broad specificity and are capable of hydrolysing all dipeptides except those containing a proline residue. Carboxypeptidases

Carboxypeptidases are exopeptidases which catalyse the hydrolysis of peptides from the C-terminal. No carboxypeptidase activity has been detected in lactococci but some activity towards N-terminal-blocked

404 [1] [2] [3]

[1] [2] [3] [5] [6]

[1] [2] [3] [6]

↓

↓

↓

↓ ↓ R1 E L E E L N V P G E I V E S L S S S E20 E S I T R I N K K I E K F Q S E E Q Q Q40

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ H P F A Q T Q S L V Y60 P F P G P I P N S L P Q N I P P L T Q T80 P V V V P P F L Q P E V M G V S K V K E100 ↓

↓

↓

↓

↓ P L P

[4] [7]

↓

P T V M F P P Q160

↓ ↓ ↓ ↓ ↓ ↓

↓ ↓

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ E S Q S L T L T D V E N L H P L P L L140 Q S W M H Q P H Q ↓ ↓

A M A P K H K E M P F P K Y P V E P F T120

[1] [2] [3] [4] [5] [6] [7]

↓

↓ ↓

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ S V L S L S Q S K V L P V P Q K A V P Y180 P Q R D M P I Q A F L L Y Q E P V L G P200

(↓) ↓ V R G P F P I I V209

β -casein [1] [2] [3] [4]

Lc. lactis Lc. lactis Lc. lactis Lc. lactis

↓ ↓ ↓ ↓ ↓ ↓ T E D E L Q D K I

subsp. cremoris H2 (Reid et al., 1991b) subsp. cremoris SK112 (Reid et al., 1991b) subsp. cremoris AM1 (Visser et al., 1991) subsp. cremoris HP (Visser et al., 1988)

[5] Lc. lactis subsp. cremoris AC1 (Monnet et al., 1989) [6] Lc. lactis subsp. lactis NCDO 763 (Monnet et al., 1989) [7] Lc. lactis subsp. lactis NCDO 763 (Monnet et al., 1986)

Figure 8 Reported cleavage sites for lactocepins on bovine -casein (from Fox et al., 1994).

↓

[1] [2] [3]

↓

E E Q N Q E Q P I R C E K D E R F F S D20 ↓

[1] [2] [3] [4] I N N Q F L P Y P Y60

↓

↓ ↓ ↓ ↓ ↓ ↓ ↓ K I A K T I P I Q Y V L S R Y P S Y G L40 N Y Y Q Q K P V A L50

↓

↓ ↓

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ Y A K P A A V R S P A Q I L Q W Q V L S80 D T V P A K S C Q A Q P T T M A R H P H100

[1] [2] [3] [4]

↓ ↓ ↓ ↓ ↓ ↓ P H L S F M A I P P K K N Q D K T E I P120 T I N T I A S G E P T S T P T I E A V E140 S T V A T L E A S P150

[1] [3] [4]

↓ ↓ ↓ ↓ ↓ ↓↓ ↓↓ ↓ E V I E S P P E I N160 T V Q V T S T A V169

↓

κ-casein [1] Lc. lactis subsp. lactis NCDO 763 (Monnet et al., 1992) [2] Lc. lactis subsp. cremoris H2 (Ried et al., 1994) [3] Lc. lactis subsp. cremoris SK11 (Reid et al., 1994) [4] Lc. lactis subsp. cremoris AM1 (Visser et al., 1994) Figure 9 Reported cleavage sites for lactocepins on bovine -casein (from Fox et al., 1994).

405

406 [1] [2] [3] [4] [5] [6] [7] [8] [9]

↓ ↓ ↓ ↓

↓ ↓ ↓ ↓

R1

Y91

[6] Y165

[5] [4] [5] [8] [9]

↓ ↓↓

↓

↓ ↓ ↓ ↓↓ ↓

↓ ↓ ↓

↓ ↓ ↓ ↓ ↓ ↓ ↓ P K H P I K H Q G L P Q E V L N E N L L R F23

[9]

[4] [5]

↓ ↓ ↓

α s1-CN f1– 23

↓ ↓ ↓ L G Y L E N L L R100

α s1-CN f91– 100

↓ ↓ ↓ ↓ Y V Y P L G T Q Y T D A P S F S D I P N P I G S E N S E K T T M P L W199 ↓ ↓ ↓ ↓ ↓ ↓ ↓ Y193 Q E P V L G P V R G P F P I I V209 ↓ ↓ ↓ ↓ ↓ Y193 Q Q P V L G P V R202 G203 P F P I I V209 ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓↓ ↓ ↓↓ ↓ F1 V N Q H L C G S H L V E A L Y L V C G E R G F F Y T P K A30

[6] [9]

α s1-CN f165– 199

β-CN f193– 209 β -CN f193– 202,

β -Chain of Insulin (Oxidized)

↓ ↓ T G G F M

[8] M106

↓ A I P P K K N Q D K T E I P T I…I E S P P E I N T V Q V T S T A V

[1] Lc. lactis subsp. cremoris H61 (Kaminogawa et al., 1986) [3] Lc. lactis subsp. cremoris AM1 (Exterkate et al., 1991) [5] Lc. lactis subsp. cremoris C13 (Baankreis, 1992) [7] Lc. lactis subsp. cremoris (Yan et al., 1987b) [9] Lc. lactis subsp. cremoris H61 (Yan et al., 1987a)

f203– 209

Met Enkephalin 169

κ -CN f106– 169

[2] Lc. lactis subsp. cremoris HP (Exterkate et al., 1991) [4] Lc. lactis subsp. cremoris HP (Baankreis, 1992) [6] Lc. lactis subsp. lactis MG 1363 (Stepaniak and Fox, 1993) [8] Lc. lactis subsp. lactis NCDO 763 (Monnet et al., 1992)

Figure 10 Reported cleavage sites for lactococcal endopeptidases (4–7, 9) and lactocepins (1–3, 8) on various oligopeptidases (from Fox et al., 1994).

Table 1 Peptidases isolated and characterized from cheese-related bacteria

Microorganism Aminopeptidases PepN** Lc. lactis CNRZ 267* Lc. lactis subsp. lactis AC1 Lc. lactis subsp. cremoris Wg2 Lc. lactis subsp. cremoris MG 1363 Lc. lactis subsp. cremoris HP Lc. lactis subsp. cremoris Wg2 Lc. lactis subsp. cremoris Wg2 Lb. delbrueckii subsp. lactis 1183 Lb. acidophilus R-26 Lb. delbrueckii subsp. bulgaricus CNRZ 397 Lb. helveticus CNRZ 32 Lb. casei subsp. casei LLG Lb. delbrueckii subsp. bulgaricus B14 Lb. helveticus LME-511 Lb. helveticus ITGL1 Lb. delbrueckii subsp. lactis DSM 7290 Lb. delbrueckii subsp. bulgaricus ACA-DC233 Lb. helveticus CNRZ32 Lb. helveticus CNRZ32 Lb. helveticus SBT 2171 Lb. curvatus DPC2024 Lb. plantarum ESB5004 Sc. thermophilus CNRZ1199 Sc. thermophilus CNRZ302 Sc. thermophilus NDCO573 Sc. thermophilus A PepS Sc. thermophilus CNRZ302

407

PepC Lc. lactis subsp. cremoris AM2 Lc. lactis subsp. cremoris AM2 Lb. delbrueckii subsp. lactis DSM 7290 Lb. delbrueckii subsp. bulgaricus B14 Lb. helveticus CNRZ32

Assay substrate

Mol. mass (kDa)

pH optimum

Temperature optimum (°C)

Subunits

Classa

References

M M M/T

M M M

Desmazeaud and Zevaco (1979) Geis et al. (1985) Tan and Konings (1990) van Alen-Boerighter et al. (1991) Exterkate et al. (1992) Strøman (1992) Tan et al. (1992b) Eggimann and Bachmann (1980) Machuga and Ives (1984) Atlan et al. (1989)

Lys-pNA Lys-pNA Lys-pNA – – – – Lys-pNA Lys-pNA Lys-pNA

85 36 95 95 95 95 95 78 38 95

6.5 7.0 7.0 – – – – 6.2–7.2 – –

35 40 40 – – – – 47.5 – –

– 1 1 – – – – 1 – –

Lys-pNA Leu-pNA Lys-pNA

97 87 95

6.5 7 7.0

– 39 50

1 1 1

M M M

Khalid and Marth (1990a) Arora and Lee (1992) Bockelmann et al. (1992)

Lys-pNA Lys-pNA Lys-pNA

92 97 95

7.0 6.5 6.5–7.0

37 50 45–55

1 1 1

M M M

Miyakawa et al. (1992) Blanc et al. (1993) Klein et al. (1993)

Lys-pNA

98

6

40

1

M

Tsakalidou et al. (1993)

– – Lys-pNA Leu-pNA Lys-pNA Lys-pNA Lys-pNA Lys-pNA Lys-AMC

97 97 95 95 70 89 97 96 95

– – 6–8 7 7 6.5 7.0 6.9–7.0 7.0

– – 50 40 37 35 36 35 37

1 1 2 1 1 1 1

M M M M M M M

Varmanen et al. (1994) Christensen et al. (1995) Sasaki et al. (1996) Magboul and McSweeney (1999a) Macedo et al. (2003) Tsakalidou et al. (1993) Rul et al. (1994) Midwinter and Pritchard (1994) Chavagnat et al. (1999)

Arg-pNA

45

7.5–8.5

55

1

M

Fernández-Esplá and Rul (1999)

His-NA – –

300 50 51

7 – –

40 – –

6 – –

T T

Neviani et al. (1989) Chapot-Chartier et al. (1993) Klein et al. (1994a)

Leu-Gly-Gly

220

6.5–7.0

50

4

T

Wohlrab and Bockelmann (1993)

–

–

T

Fernandez et al. (1994)

–

50

– –

–

– – – –

– –

–

continued

408

Table 1 continued

Microorganism

Assay substrate

Mol. mass (kDa)

pH optimum


Classa

References

4

T T T

Vesanto et al. (1994) Fernandez de Palencia et al. (1997) Klein et al. (1997)

4 6

T T

Fernandez de Palencia et al. (2000) Chapot-Chartier et al. (1994)

3 6 ⬃10 6

M M M M M M

Exterkate and de Veer (1987) Niven (1991) Baankreis (1992) Bacon et al. (1994) I’Anson et al. (1995) Rul et al. (1995)

S

Exterkate (1977) Baankreis (1992)

1 2

C C

Klein et al. (1995) Sanz and Toldra (1997) Magboul and McSweeney (1999b)

Subunits

Lb. helveticus 53/7 Lb. casei subsp. casei IFPL 731 Lb. delbrueckii subsp. lactis DSM 7290 Lb. helveticus CNRZ 32 Sc. thermophilus CNRZ 302

– Leu-pNA

51.4 200

– 7.5 7.0

– 55 50–55

Lys-NH-Ph

200 300

6.5 7.0

45 50

PepA Lc. lactis subsp. cremoris HP Lc. lactis subsp. lactis NCDO 712 Lc. lactis subsp. cremoris HP Lc. lactis subsp. cremoris AM2 Lc. lactis subsp. lactis MG 1363 Sc. thermophilus CNRZ 302

Glu-pNA Glu-pNA Glu-pNA Asp-pNA Glu-pNA Asp-pNA

130 245 520 240 – 360

– 8 8 –

50–55 65 50 –

8.5

62

PCP Lc. lactis subsp. cremoris HP Lc. lactis subsp. cremoris HP

Pyr-pNA Pyr-pNA

– 80

– 8.0–8.5

–

Leucyl aminopeptidase (PepL) Lb. debrueckii subsp. lactis DSM7290 Lb. sake IATA115 Lb curvatus DPC2024

Leu-NA Leu-pNA Leu-pNA

35 35–36 64

– 7.5 7

– 37 40

Pro-Gly-Gly

110 33 33

8.5

37

2

M S S

Baankreis and Exterkate (1991) Klein et al. (1994b) Atlan et al. (1994)

Pro-pNA

100

6.0–7.0

40

3

S

Gilbert et al. (1994)

Pro-AMC

70 46

6.5 7.5

35 40

1 1

T/S T

Miyakawa et al. (1994b) Habibi-Najafi and Lee (1995)

bradykinin Leu-Pro-Pro –

43 41 43

8.0 8.5 –

37 – –

1 1 1

M M M

Mars and Monnet (1995) McDonnell et al. (1997) Matos et al. (1998)

150 180 190 117 88

6.0–9.0 7 8.5 6.0–9.0 7.5

– 45–50 40–45 – –

2 2 2 2 2

S S S S S

Lloyd and Pritchard (1991) Kiefer-Partsch et al. (1989) Zevaco et al. (1990) Booth et al. (1990b) Yan et al. (1992)

Proline iminopeptidase (PepI) Lc lactis subsp. cremoris HP Lb. delbrueckii subsp. lactis DSM7290 Lb. delbrueckii subsp. bulgaricus CNRZ 397 Lb. delbrueckii subsp. bulgaricus CNRZ 397 Lb. helveticus LHE-511 Lb. casei subsp. casei LLG Aminopeptidase P (PepP) Lc. lactis subsp. lactis NCDO 763 Lc. lactis subsp. cremoris AM2 Lc. lactis subsp. cremoris

X-Prolyl dipeptidyl aminopeptidase (PepX) Lc. lactis subsp. lactis H1 X-Pro-pNA Lc. lactis subsp. cremoris P8-2-47 X-Pro-pNA Lc. lactis subsp. cremoris NCDO 763 Arg-Pro-pNA Lc. lactis subsp. cremoris AM2 Gly-Pro-AMC Lc. lactis subsp. cremoris nTR –

–

8 –

37

– 2

–

–

Lc. lactis subsp. cremoris Wg2 Lc. lactis subsp. lactis Lb. delbrueckii subsp. lactis Lb. delbrueckii subsp. bulgaricus CNRZ 397 Lb. delbrueckii subsp. bulgaricus B14 Lb. acidophilus 357 Lb. helveticus CNRZ 32 Lb. delbrueckii subsp. bulgaricus LBU-147 Lb. casei subsp. casei LLG Lb. helveticus 53/7 Lb. helveticus ITG LH1 Lb. delbrueckii subsp. bulgaricus DSM7290 Lb. casei subsp. casei IFPL731 Lb. helveticus LHE-511 Lb curvatus DPC2024 Sc. thermophilus Sc. thermophilus ACA-DC 4 Sc. macedonicus ACA-DC 191 Prolinase (PepR) Lb. helveticus CNRZ32 Lb. helveticus 53/7 Lb. helveticus CNRZ32 Lb. rhamnosus 1/6 Prolidase (PepQ) Lc. lactis subsp. cremoris H61 Lc. lactis subsp. cremoris AM2 Lb. delbrueckii subsp. lactis DSM7290 Lb. casei subsp. casei IFPL 731 Lb. delbrueckii subsp. bulgaricus CNRZ 397 Dipeptidases Lc. lactis CNRZ267* Lc. lactis subsp. cremoris H61 Lc. lactis subsp. cremoris Wg2 Lb. delbrueckii subsp. bulgaricus B14 Lb. delbrueckii subsp. lactis DSM7290 Lb. sake L110 Lb. helveticus SBT2171 Lb. helveticus CNRZ32 Lb. helveticus 53/7

– Ala-Pro-pNA Gly-Pro-AMC X-Pro-pNA

– 175 165 82

Ala-Pro-pNA Ala-Pro-pNA X-Pro-pNA Gly-Pro-pNA

–

–

– 2 2

S S

Tan et al. (1992a) Chich et al. (1995) Meyer and Jordi (1987) Atlan et al. (1990)

45 45 40 50

2 2 1 3

S S S S

Bockelmann et al. (1991) Bockelmann et al. (1991) Khalid and Marth (1990b) Miyakawa et al. (1991)

7.0 6.5 7.0 7.0

50 45 40 46–50

1 2 1 1

S S S S

Habibi-Najafi and Lee (1994) Vesanto et al. (1995) Degraeve and Martial-Gros (2003) Meyer-Barton et al. (1993)

67 93–87 200 165 160 167

6.0–7.0 6.5 7.5 6.5–8.2 7.0 7.0

40 50 45 45 50 –

1 1 2 2 2 2

S S S S S S

Fernández-Esplá et al. (1997a) Miyakawa et al. (1994a) Magboul and McSweeney (2000) Meyer and Jordi (1987) Tsakalidou et al. (1998) Georgalaki et al. (2002)

Pro-Leu Pro-Leu Pro-Leu Pro-Leu

35 35 125 34

– – 6.0–6.5 –

– – 45–50 –

T/S T/S S S

Dudley and Steele (1994) Varmanen et al. (1996) Shao et al. (1997) Varmanen et al. (1998)

Leu-Pro Leu-Pro

43 42

6.5–7.5 7.35–9.0

– –

1 1

M M

Leu-Pro

41

6.5–7.5

55

1

M

Kaminogawa et al. (1984) Booth et al. (1990c) Stuckey et al. (1995) Fernández-Esplá et al. (1997b) Morel et al. (1999)

Leu-Leu Leu-Gly dipeptides Leu-Leu

51 100 49 51

7.5–8.0 8.0 8.0 7.0–7.5

– – 50 50

1 – 1 1

M M M M

Desmazeaud and Zevaco (1977) Hwang et al. (1981) van Boven et al. (1988) Wohlrab and Bockelmann (1992)

-Ala-Ala Leu-Gly Leu-leu Leu-Leu Gly-Tyr

52 50 50 54 420

– 7.6–8.0 8.0 – 6.0

– 40–45 55 – 55

–

M M M

Vongerichten et al. (1994) Montel et al. (1995) Tan et al. (1995) Dudley et al. (1996) Vesanto et al. (1996)

– 7.0 7.0

– 50–55 50

170–200 170–200 72 270

6.5 6.5 7.0 6.5

Gly-Pro-pNA Gly-Pro-pNA Gly-Pro-pNA Ala-Pro-pNA

79 165 140 95

Arg-Pro-pNA Gly-Pro-pNA Ala-Pro-pNA Gly-Pro-AMC X-Pro-pNA Gly-Pro-pNA

– –

–

– – 4 –

1 1 – 8

– T

409

continued

410

Table 1 continued

Microorganism

Assay substrate

Mol. mass (kDa)

pH optimum


Subunits

Classa

References

Lb. casei subsp. casei IFPL 731

Leu-Leu

46

7.5

60–75

1

M

Lb. curvatus DPC2024

Leu-Leu

52

8.0

50

1

M

Fernández-Esplá and Martin-Hernandez (1997) Magboul and McSweeney (1999c)

Tripeptides Leu-Leu-Leu Tripeptides Leu-Leu-Leu

75 103–105 105 72

7.0 7.5 8.6 5.8

35 55 – 33

– 2 2 3

M M M T/M

Desmazeaud and Zevaco (1979) Bosman et al. (1990) Bacon et al. (1993) Sahlstrøm et al. (1993)

Leu-Gly-Gly

85

6.0

40

3

M

Bockelmann et al. (1995)

Leu-Gly-Gly

77

6.0

50–55

2

M

Bockelmann et al. (1997)

Ala-Ala Met-Cly-Gly

55 150

7.0 –

40 –

1 3

M M

Sanz et al. (1998) Savijoki and Palva (2000)

peptides s1-CN (f1–23) Met-enkephalin s1-CN (f1–23) s1-CN (f1–23) s1-CN (f1–23) bradykinin bradykinin Met-enkephalin

98 80 70 180 70 70 70 70 70

7–7.5 6.0 6–6.5 8–9 6–7 7.5 6.0 8.0 7.7

40 37 30–38 42 35 40 – 40 47

1 2 1 2 1 1 1 1 1

M M M M N M M M M

Yan et al. (1987a) Yan et al. (1987b) Tan et al. (1991) Baankreis (1992) Baankreis (1992) Stepaniak and Fox (1995) Pritchard et al. (1994) Monnet et al. (1994) Bockelmann et al. (1996)

bradykinin – Met-enkephalin Met-enkephalin s1-CN (f1–23) -CN (f193–209) Met-enkephalin bradykinin bradykinin Ala-Pro-pNA

140 70 350 50 –

8.0 – 7.0 4.5 –

40 – 35 32–37 –

4 1 6–7 1

M M M T

Tobiassen et al. (1997) Mierau et al. (1993) Stepaniak et al. (1998a) Fenster et al. (1997) Christensen et al. (2003a)

70 120 44 84

6.5 6.7–7.5 6.5–8.0 7.0

M M M S

Chavagnat et al. (2000) Stepaniak et al. (1998b) Tobiassen et al. (1996) Fernández-Esplá and Fox (1997)

Tripeptidases Lc. lactis CNRZ267* Lc. lactis subsp. cremoris Wg2 Lc. lactis subsp. cremoris AM2 Lc. lactis subsp. cremoris IMN-C 12 Lb. delbrueckii subsp. bulgaricus B14 Lb. delbrueckii subsp. bulgaricus B14 Lb. sake IATA115 Lb. helveticus Endopeptidases Lc. lactis subsp. cremoris H61 Lc. lactis subsp. cremoris H61 Lc. lactis subsp. cremoris Wg2 Lc. lactis subsp. cremoris HP Lc. lactis subsp. cremoris C13 Lc. lactis subsp. lactis MG 1363 Lc. lactis subsp. cremoris SK11 Lc. lactis subsp. lactis NCDO 763 Lb. delbrueckii spp. bulgaricus B14 Lb. paracasei Lc01 Lc. lactis subsp. cremoris P8-2-47 Lc. lactis subsp. lactis MG1363 Lb. helveticus CNRZ32 Lb. helveticus CNRZ32 Sc. thermophilus A P. freudenreichii ATCC 9614 P. freudenreichii ATCC 9614 P. freudenreichii subsp. shermanii NCDO 853

41 40–50 45–50 40

a Class of enzyme M: metallo and T: thiol-peptidase; NA: -naphthylamide; pNA: p-nitroanilide. * Citrate positive strain. ** Classification of a few of these enzymes as PepN is uncertain.

–

– 1 4 1 1


Endopeptidases (PepO, PepF)

Exopeptidases Aminopeptidases (PepN, PepA, PepC, PepL)

Iminopeptidase (PepI)

P Pyrolidonyl carboxylyl peptidase (PCP)

Pyro-Glu

X-Prolyldipeptidyl aminopeptidase (PepX)

Carboxypeptidase

P Dipeptidases (PepV, PepD)

Prolidase (PepQ)

P

Prolinase (PepR)

P

Tripeptidase (PepT)

Figure 11 Schematic representation of the action of peptidases found in lactic acid bacteria.

peptides has been reported in strains of lactobacilli (Abo-Elnaga and Plapp, 1987; El Soda et al., 1987a,b). There are no reports on the purification and characterization of a carboxypeptidase from Lactobacillus or other LAB. Aminopeptidases

The most thoroughly studied exopeptidase from LAB is the general aminopetidase, PepN. In most strains studied, this enzyme is a monomeric metallopeptidase of 85–98 kDa. PepN is a broad specificity aminopeptidase; in addition to p-nitroanilide (pNA) derivatives of amino acids, the enzyme is capable of hydrolysing a wide range of peptides differing in both size and

amino acid composition (Arora and Lee, 1992; Miyakawa et al., 1992; Tan et al., 1992a,b; Niven et al., 1995; Sasaki et al., 1996). Substrates with a hydrophobic or basic amino acid residue at the N-terminal are hydrolysed preferentially. The ability to hydrolyse peptides containing hydrophobic amino acids suggests its potential as a debittering enzyme. The addition of PepN from Lc. lactis subsp. cremoris Wg2 was found to be effective in reducing the bitterness of tryptic digests of -casein (Tan et al., 1993). The manufacture of cheese using PepN-negative mutants resulted in increased bitterness (Baankreis, 1992). Generally, PepN does not hydrolyse substrates with Glu, Asp or Pro at the N-terminal or dipeptides containing Pro


(Tan et al., 1991; Arora and Lee, 1992; Miayakawa et al., 1992; Tan et al., 1993). However, PepNs from Lb. delbrueckii subsp. bulgaricus B14 (Wohlrab and Bockelmann, 1993) and Lb. helveticus SBT 2171 (Sasaki et al., 1996) hydrolysed Pro-containing substrates. Specificity studies indicated that the PepN from Lc. lactis subsp. cremoris Wg2 was active on oligopeptides with a preference for peptides with six amino acid residues (Niven et al., 1995). PepC in LAB is a metal-independent general aminopeptidase (Kunji et al., 1996; Table 1). PepCs from Lactococcus and Lactobacillus strains characterized so far are multimeric thiol aminopeptidases which are inhibited by p-chloromercuribenzoate and iodoacetamide (Neviani et al., 1989; Wohlrab and Bockelmann, 1993; Fernandez de Palencia et al., 1997). In both cases, the subunit molecular mass of the enzyme is ⬃40–50 kDa. PepC shows broad specificity, with particularly high activity on synthetic substrates containing a hydrophobic amino acid but exhibits little activity on peptides with positively charged amino acid residues (Neviani et al., 1989; Wohlrab and Bockelmann, 1993; Fernandez de Palencia et al., 1997; Mistou and Gripon, 1998). A gene (pepG) encoding a novel cysteine aminopeptidase and with a high degree of similarity to PepC has been identified in Lb. delbrueckii subsp. lactis DSM7290 by Klein et al. (1997). These authors over-expressed the pepG gene in E. coli and compared the enzyme to PepC; although both enzymes were structurally related, they had different substrate specificities. Lactococcal glutamyl/aspartyl aminopeptidase (PepA) is a multimeric metallopeptidase with a subunit molecular mass of 38–43 kDa (Table 1). PepA is a narrow-specificity peptidase which releases only an Nterminal Glu or Asp from di-, tri- and oligo-peptides with up to ten amino acid residues (Exterkate and de Veer, 1987; Niven, 1991; Bacon et al., 1994). Glutamate is a well-recognized flavour enhancer and therefore the role of PepA in the development of flavour in cheese may be of great importance. Studies on mature Cheddar cheese have shown that glutamate is important for Cheddar cheese flavour (McGugan et al., 1979; Aston and Creamer, 1986; Engels and Visser, 1994; Fox et al., 1994). However, the precise role of PepA in the development of cheese flavour is unclear. Under certain conditions, the N-terminal glutamyl residue of a peptide can undergo spontaneous intramolecular cyclization, forming an N-terminal 2-pyrrolidone5-carboxylic acid (PCA; pyroglutamate residue) (Law and Haandrikman, 1997). An N-terminal PCA residue has been found in bitter peptides produced from casein by the lactocepin of Lc. lactis subsp. cremoris HP (Visser et al., 1983). Pyrrolidone carboxylyl peptidase

(PCP) is an aminopeptidase capable of releasing a PCA residue from peptides and proteins (Kunji et al., 1996). This enzyme is present in lactococcal strains and has been partially characterized from Lc. lactis subsp. cremoris HP (Baankreis, 1992). Two serine peptidases with a molecular mass of 25 and 80 kDa and PCAp-nitroanilide hydrolase activity were identified in Lc. lactis subsp. cremoris HP using non-denaturing gel electrophoresis (Baankreis, 1992). The presence of more than one leucyl aminopeptidase in LAB has been reported (Atlan et al., 1989; Blanc et al., 1993; Banks et al., 1998). A gene encoding a specific leucyl aminopeptidase (pepL) in Lb. delbrueckii subsp. lactis DSM 7290 has been cloned and sequenced (Klein et al., 1995). PepL has a molecular mass of 35 kDa (Table 1) and it preferentially hydrolyses dipeptides (and some tripeptides) with an N-terminal leucyl residue. Sequence alignments of PepL with prolinases from Lb. helveticus and B. coagulans and an iminopeptidase from Lb. delbrueckii subsp. lactis and Lb. delbrueckii subsp. bulgaricus showed 46, 21.5, 25.5 and 25.5% homology, respectively. Two aminopeptidases, with characteristics similar to PepL, were purified from Lb. sake IATA115 and Lb. curvatus DPC2024 by Sanz and Toldra (1997) and Magboul and McSweeney (1999b), respectively. The former was a monomer with a molecular mass of 35–36 kDa and maximum activity at pH 7.5 and 37 °C, while the latter was a dimer with a subunit molecular mass of ⬃32 kDa and optimum activity at pH 7.0 and 40 °C. The 20 N-terminal amino acid residues of the PepL from Lb. curvatus DPC2024 showed 50, 80 and 95% homology with PepL from Lb. delbrueckii subsp. lactis DSM 7290 (Klein et al., 1995), the prolinase from Lb. helveticus CNRZ32 (Dudley and Steele, 1994) and the prolinase from Lb. rhamnosus 1/6 (Varmanen et al., 1998), respectively. Proline-specific peptidases

Caseins, the major proteins in bovine milk, are rich in the imino acid, proline. Because of its unique structure, specialized peptidases are required to hydrolyse peptide bonds involving proline, thus making peptides accessible to the action of other peptidases (see review by Cunningham and O’Connor, 1997). Several proline-specific peptidases with distinct substrate specificities have been found in LAB. X-Prolyl dipeptidyl aminopeptidase (PepX) is a peptide hydrolase capable of releasing X-Pro and sometimes X-Ala dipeptides from the N-terminal of oligopeptides. Due to its unique specificity, PepX is the best characterized of the proline-specific peptidases. The enzyme has been demonstrated in several genera of LAB and isolated from a number of strains and


characterized (Table 1). All PepXs purified from LAB have a serine catalytic mechanism and most are dimeric proteins with a native molecular mass of 117–200 kDa (Table 1); however, a high molecular mass endopeptidase (⬃350 kDa) with PepX activity and able to hydrolyse s1-casein was isolated and characterized by Stepaniak et al. (1998a). Increasing the proportion of pepX-negative mutants in a starter culture reduced the organoleptic quality of the resultant cheese but did not increase bitterness (Baankreis, 1992). Meyer and Spahni (1998) studied the role of PepX from Lb. delbrueckii subsp. lactis by using PepXnegative mutants. This enzyme influenced proteolysis and the sensorial characteristics of Gruyere cheese but it was not essential for the growth of the microorganism in milk (Meyer and Spahni, 1998). Proline iminopeptidase (PepI) catalyses the release of an N-terminal proline residue from di-, tri- and oligo-peptides. PepI from Lc. lactis subsp. cremoris HP (Baankreis and Exterkate, 1991) is the only iminopeptidase that has been purified from Lactococcus. This enzyme is a dimeric metallopeptidase with a native molecular mass of 110 kDa (Table 1). In contrast, the iminopeptidases purified from Lb. helveticus LHE-511 (Miyakawa et al., 1994b) and Lb. casei subsp. casei LLG (Habibi-Najafi and Lee, 1995) were monomeric thiol peptidases which were slightly inhibited by the serine protease inhibitor phenylmethyl sulphonyl fluoride. The molecular mass of the enzymes from Lb. helveticus and Lb. casei was estimated as 70 and 46 kDa, respectively. In addition to these two iminopeptidases, a PepI was purified from Lb. delbrueckii subsp. bulgaricus CNRZ 397 by amplification and expression of the gene in E. coli (Gilbert et al., 1994). The purified enzyme was characterized as a trimeric serine peptidase with a subunit molecular mass of 33 kDa (Table 1). Prolinase (PepR) is a specific dipeptidase which hydrolyses dipeptides with the sequence Pro-X. PepR from Lb. helveticus CNRZ32 was purified and biochemically characterized by Shao et al. (1997) and found to have a relatively broad specificity. The PepR from Lb. rhamnosus 1/6 (Varmanen et al., 1998), in addition to its prolinase activity, hydrolysed the aminopeptidase substrates, Pro-NA, Leu-NA and Phe-NA. Prolidase (PepQ) is an X-Pro-specific dipeptidase. With the exception of PepQ from Lb. helveticus CNRZ32, which is a homodimer with a subunit molecular mass of 45 kDa, most PepQs characterized to date are monomeric metallopeptidases with a native molecular mass of ⬃42 kDa. These enzymes hydrolysed most X-Pro dipeptides with the exception of Gly-Pro and Pro-Pro (Kaminogawa et al., 1984; Fernández-Esplá et al., 1997b; Morel et al., 1999). However, PepQs isolated from Lc. lactis subsp. cremoris AM2 (Booth et al., 1990a) and Lb. delbrueckii

subsp. lactis DSM7290 (Stuckey et al., 1995), hydrolysed di- and tripeptides that did not contain Pro, in addition to Pro-X dipeptides. Aminopeptidase P (PepP) is a specific aminopeptidase that catalyses the removal of the N-terminal amino acid from oligopeptides having the sequence X-Pro-Pro-(X)n or X-Pro-(X)n (Kunji et al., 1996). The enzyme has been purified from strains of Lactococcus and is a monomeric metallopeptidase with a molecular mass of 41–43 kDa (Table 1). Provided that the peptide contains the above sequences, PepP is capable of releasing the N-terminal amino acid from oligopeptides up to 11 residues long. This enzyme also hydrolyses peptides with Ala in the penultimate position but at a slower rate (McDonnell et al., 1997).

Enzymes from Secondary Starter Microorganisms Enzymes of LAB play an important role in the secondary proteolysis in internal-ripened cheese varieties, and hence contribute significantly to the development of flavour and aroma. In mould-ripened, smear-ripened and Swiss-type cheeses, microorganisms other than LAB play a pivotal role in the development of characteristic flavour and texture. The ripening of these cheese varieties involves complex biochemical reactions, which are discussed in detail in Volume 2. While the enzymes of LAB have been well studied and characterized, there have been fewer studies on organisms associated with mould-ripened or smear-ripened cheese varieties or on enzymes from Propionibacterium freudenreichii subsp. shermanii. The microbial flora of surface mould-ripened and blue-veined cheese, such as Camembert and Roquefort, includes yeasts (e.g., Kluyveromyces lactis, Saccharomyces spp. and Debaryomyces hansenii), moulds (Geotrichum candidum, Penicillium spp.), lactococci, lactobacilli, micrococci, staphylococci, coryneform bacteria and coliforms. Penicillium spp. are major components of the microflora and their enzymes play an important role in cheese ripening. Proteolytic systems of P. camemberti and P. roqueforti are somewhat similar; both synthesize an aspartyl proteinase, a metalloproteinase, an acid carboxypeptidase and an alkaline aminopeptidase (‘Surface Mould-ripened Cheeses’ and ‘Blue Cheese’, Volume 2). The aspartyl proteinase from P. camemberti hydrolyses s1-casein faster than -casein or -casein (Gripon, 1993). Acid proteinases of P. camemberti and P. roqueforti have similar action on -casein and hydrolyse Lys979Val98, Lys999Glu100 and Lys299Ile30 bonds at a faster rate than other bonds in -casein (Le Bars and Gripon, 1981; Trieu-Cuot et al., 1982). Metalloproteinases of


both species have similar properties and have a pH optimum in the range 5.5–6.0. Chrzanowska et al. (1995) purified an aspartic proteinase from the culture filtrate of P. camemberti by a two-step purification procedure. The proteinase had a molecular mass of 33.5 kDa and an optimum pH of 3.4 on haemoglobin. The enzyme showed specificity towards peptide bonds containing an aromatic or hydrophobic amino acid residue in the B-chain of insulin. Besides these proteinases, P. roqueforti has a carboxypeptidase, which has an optimum pH of 3.5 and releases acidic, basic or hydrophobic amino acids (Gripon, 1993). Geotrichum candidum also synthesizes extracellular and intracellular proteinases, but the contribution of these enzymes to cheese ripening is less than that of enzymes from Penicillium spp. (Gripon, 1993). The bacterial microflora of surface cheeses, such as Tilsit, Limburger, Münster or Taleggio at the beginning of ripening is dominated by yeasts and moulds, which are acid and salt tolerant, but at the end of ripening, bacteria of the genera Brevibacterium, Arthrobacter, Micrococcus, Staphylococcus and Corynebacterium dominate (Eliskases-Lechner and Ginzinger, 1995; Valdés-Stauber et al., 1997; ‘Bacterial Surface-ripened Cheeses’, Volume 2). Growth of B. linens on the cheese surface is thought to play an important role in the development of the characteristic colour, flavour and aroma of smear surface-ripened cheese varieties (Rattray and Fox, 1999) and hence, its enzymes have been characterized. Extracellular enzymes of B. linens include proteinases, aminopeptidases and esterases, the biochemical properties of which vary because of wide inter-strain differences within the species. Brézina et al. (1987) partially purified four extracellular proteinases from B. linens, with pH and temperature optima of 5.0–8.0 and 50 °C, respectively. Hayashi et al. (1990) purified five extracellular proteinases from B. linens F (designated A, B, C, D and E), having a molecular mass of 37, 37, 44, 127 and 325 kDa, respectively, as determined by size exclusion chromatography (SEC). Proteinases A and B were stable at 35 °C for 1 h and had a temperature optimum of 40 °C, while proteinases C, D and E were stable at 45 °C for 1 h and had a temperature optimum of 50 °C. All five proteinases were optimally active at pH 11.0 and were serine proteinases. The production of multiple forms of the extracellular proteinases by B. linens ATCC 9172 is a result of aggregation of subunits and autocatalytic degradation (Buchinger et al., 2001). An extracellular serine proteinase partially purified from a strain of B. linens (Laktoflora 200), had a molecular mass of 52–55 kDa, as determined by SDSPAGE, and pH and temperature optima of 7.0–8.5 and

45 °C, respectively (Juhász and Skárka, 1990). A thermostable proteinase was partially purified from B. linens IDM 376; it had molecular mass of 18.5 kDa and pH and temperature optima of 7.5 and 67.5 °C, respectively, on azocasein (Clancy and O’Sullivan, 1993). An extracellular serine proteinase purified from B. linens ATCC 9174 had a molecular mass of 126 kDa, as determined by SEC and was optimally active at pH 8.5 and 50 °C (Rattray et al., 1995). It hydrolysed s1-casein at His89Gln9, Ser1619Gly162 and either Gln1729Tyr173 or Phe239 Phe24 (Rattray et al., 1996) and -casein at Ser189Ser19, Glu209Glu21, Gln569Ser57, Gln729Asn73, Leu779Thr78, Ala1019 Met102, Phe1199Thr120, Leu1399Leu140, Ser1429Trp143, His1459Gln146, Gln1679Ser168, Gln1759Lys176, Tyr1809Pro181 and Phe1909Leu191 (Rattray et al., 1997). One of the five extracellular enzymes of B. linens ATCC 9172 was purified to homogeneity by Tomaschová et al. (1998) using ion-exchange chromatography and native preparative PAGE. The enzyme had nearly identical properties to the serine proteinase of B. linens ATCC 9174 purified by Rattray et al. (1995). Its molecular mass was estimated to be 56 kDa by SDSPAGE and pH and temperature optima were 8.0 and 50 °C, respectively. B. linens also produces extracellular aminopeptidases, intracellular peptidases and proteinases. Sørhaug (1981) reported the presence of intracellular dipeptidase activity in six strains of B. linens. The presence of three extracellular aminopeptidases in B. linens (Laktoflora 200), having pH and temperature optima of 7.0–9.0 and 30 °C, respectively, was reported by Brézina et al. (1987). Two extracellular aminopeptidases, designated A and B, with a molecular mass of estimated to be 150 and 110 kDa, respectively, and pH and temperature optima of 9.3 and 40 °C, respectively, were purified from B. linens F by Hayashi and Law (1989). Ezzat et al. (1993) reported the presence of cell wall proteinases and dipeptidase activities in B. linens CNRZ 944. The authors partially purified the cell wall proteinase, which had maximum activity at pH 6.5 and 40 °C. An intracellular aminopeptidase from B. linens ATCC 9174, with a molecular mass of 59 kDa, as determined by SDSPAGE, and 69 kDa by SEC, was reported by Rattray and Fox (1997). The enzyme was optimally active at pH 8.5 and 35 °C. Curtin et al. (2002) showed aminopeptidase, dipeptidase and tripeptidase activities in brevibacteria, corynebacteria, staphylococci and brachybacteria, isolated from smear surface-ripened cheeses, Tilsit and Gubeen. Species of the genus Arthrobacter are major components of the microflora of surface mould-ripened cheeses, such as Brie and Camembert and red-smear


cheeses. However, the enzymes of Arthrobacter have not been well studied. Smacchi et al. (1999a) purified two extracellular serine proteinases from A. nicotianae 9458, with molecular masses of about 53–55 and 70–72 kDa, as determined by SDS-PAGE. The enzymes were optimally active at 55–60 and 37 °C, respectively. Both enzymes were optimally active in the pH range of 9.0–9.5 and preferentially hydrolysed -casein over s1-casein. An extracellular PepI from A. nicotianae 9458 with a molecular mass of about 53 kDa, was purified and characterized by Smacchi et al. (1999b). The enzyme was optimally active at 37 °C and 8.0. Some Micrococcus spp. are very proteolytic and produce extracellular proteinases and intracellular proteinases and peptidases (Fox et al., 1993). Nath and Ledford (1972) reported that extracellular proteinases from certain micrococci preferentially hydrolysed s1casein; production of extracellular proteinase was also reported by Garcia de Fernando and Fox (1991). Bhowmik and Marth (1989) purified and characterized an aminopeptidase, with broad substrate specificity, from M. freudenreichii ATCC 407. Propionibacterium spp. are weakly proteolytic, but they are highly peptidolytic, especially on proline-containing peptide bonds, thus contributing to the characteristic flavour of Swiss-type cheeses (see ‘Cheese with Propionic Acid Fermentation’, Volume 2). Biochemical characteristics of peptidases from propionic acid bacteria have been reviewed by Gagnaire et al. (1999). A PepX with a molecular mass of 84 kDa and pH and temperature optima of 7.0 and 40 °C, respectively, was purified and characterized from P. freudenreichii subsp. shermanii NCDO 853 by Fernández-Esplá and Fox (1997). Endopeptidases have been isolated from P. freudenreichii subsp. shermanii and characterized (Table 1) (Tobiassen et al., 1996; Stepaniak et al., 1998b).

Patterns of Proteolysis in Cheese The pattern of proteolysis in many varieties may be summarized as follows: the caseins are hydrolysed initially by residual coagulant activity retained in the curd and by plasmin (and perhaps other indigenous proteolytic enzymes) to a range of large and intermediate-sized peptides which are hydrolysed by proteinases and peptidases from the starter LAB, NSLAB and perhaps secondary microflora to shorter peptides and amino acids. However, the pattern and extent of proteolysis varies considerably between varieties due to differences in manufacturing practices (particularly cooking temperature), which cause differences in moisture content, residual coagulant activity, activation of plasminogen to

plasmin, and possibly the development of a highly proteolytic secondary microflora and ripening time. The extent of proteolysis (i.e., the degree to which the caseins and peptides therefrom are hydrolysed and measured by the development of water- or pH 4.6soluble N) in cheese varies from very limited (e.g., Mozzarella) to very extensive (e.g., Blue) and is summarized for many varieties in Table 2. The pattern of proteolysis (i.e., the relative concentrations of different peptides and amino acids) is very variable and is essentially unique to a particular variety. The differences in soluble N content are due to differences in moisture content, temperature and pH, length of ripening, cooking temperature and pH at draining (Fox and McSweeney, 1996) and is mainly due to the action of chymosin and to a lesser extent of plasmin (Fox and McSweeney, 1997). A short ripening period (⬃3 weeks) and extensive denaturation of chymosin during the high temperature (⬃70 °C) stretching step during the manufacture of Mozzarella cheese explain the low level of soluble N, whereas extensive proteolysis is characteristic of Blue cheese and some smear-ripened varieties, caused by the action of chymosin, plasmin and proteinases from their characteristic secondary microflora. In addition, differences in the action of these proteolytic agents cause differences in peptide profiles. Primary proteolysis is similar during the ripening of most cheeses; chymosin hydrolyses the Phe239Phe24 bond of s1-casein (Hill et al., 1974; Carles and Ribadeau-Dumas, 1985) except in cheeses that are cooked at a high temperature (⬃55 °C, e.g., Swiss cheese), in which plasmin is the principal proteolytic agent. In blue-veined cheeses, after sporulation, enzymes from P. roqueforti hydrolyse s1-CN (f24–199) and other peptides, changing the peptide profile (Gripon, 1993). Analysis of the water-insoluble fraction of various cheeses by urea-PAGE gives insight into the differences in peptide profile between cheeses (Fig. 12). In many cheeses, s1-casein is hydrolysed faster than -casein (Sousa et al., 2001). In Blue-veined cheeses, both s1- and -caseins are completely hydrolysed at the end of ripening. In Swiss-type cheeses, -casein is hydrolysed faster than s1-casein, with concomitant increases in -caseins, indicating a role of plasmin and denaturation of chymosin during cooking. However, s1-CN (f24–199) is produced slowly in Swiss cheese, indicating either the survival of some chymosin during cooking or the activity of indigenous milk acid proteinase, cathepsin D (Gagnaire et al., 2001), which has specificity similar to chymosin (Hurley et al., 2000a). In the case of Camembert-type cheese, about ⬃20% of total N is soluble at pH 4.6 (Khidr, 1995) (Table 2) and the pattern of proteolysis is similar to Cheddar cheese (Fig. 12). During the ripening of Mozzarella


Table 2 Soluble N as % of total nitrogen in different cheese varieties Cheese

Age

SN/TN %

References

Mozzarella

25 days

4–5

Quarg Gouda

4 weeks 6 weeks 24 weeks 1 month

Somers et al. (2002) O’Reilly et al. (2002) Guinee et al. (1998) Mara and Kelly (1998) Messens et al. (1999) Exterkate and Alting (1995) Michalski et al. (2003) Sousa and McSweeney (2001) Khidr (1995)

Swiss Feta

16 weeks 2–6 months

⬃12 12–13 23–25 Surface 15–17 Core 9–12 Surface ⬃20 Core ⬃12 16–17 17–20

Mahon Cheddar

2 months 4–6 months

19–20 20–25

Tilsit

28 weeks

Parmesan Gorgonzola Danablu

24 months – –

Surface 24–25 Core 22–23 31–35 43–46 50–53

Camembert

1 month

cheese, s1-CN (f24–199) is produced slowly and -caseins more rapidly, indicating weak chymosin activity and fairly high plasmin activity (Kindstedt, 1993). Plasmin and Lactobacillus proteinases are responsible for extensive proteolysis in Parmigiano-Reggiano cheese that is ripened for a long period (⬃24 months) at an elevated temperature (⬃18–20 °C) (Battistotti and Corradini, 1993). The high cooking temperature used during the manufacture of Parmigiano-Reggiano cheese denatures most of the chymosin.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Figure 12 Urea-polyacrylamide gel electrophoretograms waterinsoluble fraction of a selection of cheese varieties. Lane 1 Na caseinate, lane 2 Cheddar, lane 3 extra-mature Cheddar, lane 4 Cheshire, lane 5 Red Leicester, lane 6 Double Gloucester, lane 7 Emmental, lane 8 Leerdammer, lane 9 Jarlsberg, lane 10 Vorarlberger Bergkase, lane 11 Edam, lane 12 Gouda, lane 13 Norvegia, lane 14 Parmesan, lane 15 Parmesan (from McGoldrick, 1996).

Cooney et al. (2000) Michaelidou et al. (2003) Sarantinopoulos et al. (2002) Katsiari et al. (2000) Moatsou et al. (2002) Taborda et al. (2003) Shakeel-Ur-Rehman and Fox (2002) Barrett et al. (1999) Shakeel-Ur-Rehman et al. (1998) Lane et al. (1997) Churchill et al. (2003) Careri et al. (1996) Zarmpoutis et al. (1997)

Several peptides from Cheddar, Parmigiano-Reggiano, Blue, Swiss and Feta cheeses have been isolated and characterized. Of these varieties, the peptide profile of Cheddar cheese is the best characterized and is summarized in Figs 13 and 14. All the principal water-insoluble peptides in Cheddar cheese are produced either from s1-casein by chymosin or from -casein by plasmin (McSweeney et al., 1994a). After cheese manufacture, residual chymosin acts on Phe239Phe24 of s1-casein to produce the large C-terminal peptide s1-CN (f24–199) and a small peptide s1-CN (f1–23) (Fox and McSweeney, 1997). s1-CN (f24–199) is hydrolysed by chymosin at Leu1019Lys102 (Fig. 15) and more slowly at Phe329Gly33, Leu1099Glu110, Phe289Pro29 and Leu409Ser41; s1-CN (f24–199) is also hydrolysed slowly by plasmin at Lys1039Tyr104 and Lys1059Val106 of s1-CN (f24–199). The large C-terminal peptides s1CN (f24–199), s1-CN (f33–199), s1-CN (f102–199), s1-CN (f110–199), s1-CN (f99–199), s1-CN (f104–199) and s1-CN (f106–199) have been identified in the water-insoluble fraction of Cheddar cheese (McSweeney et al., 1994a; Mooney et al., 1998). The bond Trp1649Tyr165, which is hydrolysed rapidly in solution by chymosin (McSweeney et al., 1993b), does not appear to be hydrolysed in cheese, perhaps due to intermolecular interactions. Peptide s1-CN (f1–23) is hydrolysed at the bonds Gln99Gly10, Gln139Glu14, Glu149Val15 and Leu169Asn17 by lactocepin (Fox and


Figure 13 Principal water-insoluble peptides derived from s1-casein (A) and -casein (B) isolated from Cheddar cheese by McSweeney et al. (1994a) and Mooney et al. (1998) (from Sousa et al., 2001).

McSweeney, 1996). The peptides s1-CN (f1–9), s1-CN (f1–13) and s1-CN (f1–14) accumulate and dominate the RP-HPLC chromatogram of water-soluble fraction of Cheddar cheese (Fig. 15). Although the bond Leu1929Tyr193 of -casein in solution is very susceptible to chymosin, it is hydrolysed very slowly in cheese, probably due to the effect of ionic strength which promotes hydrophobic interactions between susceptible regions of -casein molecules (Fox and McSweeney, 1997). The cleavage of Leu1929Tyr193 in cheese is undesirable, as -CN (f193–209) is very hydrophobic and bitter (Visser et al., 1983). Plasmin preferentially hydrolyses -casein at Lys289Lys29, Lys1059His106 and Lys1079Glu108, producing 1-, 2-, 3-caseins and PPs (5, 8 fast and 8 slow); -caseins are present in the water-insoluble fraction of Cheddar (McSweeney et al., 1994a; Lane and Fox, 1999; McGoldrick and Fox, 1999) and many other cheeses. No large peptides originating from s2-casein have been identified in Cheddar cheese (Mooney et al., 1998) and only four small peptides have been identified in the water-soluble fractions (Singh et al., 1995, 1997). Water-soluble peptides are characteristic of particular cheese varieties and are related to the specificity of the starter and non-starter proteinases and peptidases (Fox

and McSweeney, 1996). In terms of number, most of the peptides in the water-soluble fraction of Cheddar cheeses originate from N-terminal half of -casein (particularly from residues 53 to 91) and a small number from the N-terminal half of s1-casein. However, many of these peptides are present at low levels in cheese, and the water-soluble fraction of Cheddar cheese is dominated by a relatively small number of peptides, originating from s1-casein. Peptides in the water-soluble fraction of Cheddar cheese do not contain intact plasmin or chymosin cleavage sites and hence probably arise, not directly from the caseins, but rather from larger precursor peptides (produced by chymosin or plasmin) by the action of lactocepin or other microbial enzymes. Little work has been done on pH 4.6-soluble peptides in blue-mould cheese, in which extensive proteolysis occurs. González de Llano et al. (1991) studied the production and identification of phosphotungstic acid (PTA)-soluble peptides in Gamonedo blue cheese. Low molecular mass peptides from the PTA-soluble fraction were isolated and their amino acid composition was determined. The isolated peptides contained 7–10 amino acids and the major amino acids were Ser, Glu, Gly/Thr, Ile and Leu (González de Llano et al. 1991).


Figure 14 Water-soluble peptides derived from s1-casein (A), s2-casein (B) and -casein (C) isolated from Cheddar cheese by Singh et al. (1994, 1995, 1997), Breen et al. (1995) and Fernandez et al. (1998). The principal chymosin, plasmin and lactocepin cleavage sites are indicated (from Sousa et al., 2001).


Chymosin 1

1

23

199 Chymosin 199

24

Lc-CEP 102

199

f1-9, f-1-13 Further hydrolysis products

Figure 15 Schematic representation of the early proteolysis of s1-casein during the ripening of many cheeses and the location of peptides produced on a urea-polyacrylamide gel electrophoretogram and a reverse-phase HPLC elution profile.

Low molecular weight peptides formed in Parmigiano-Reggiano cheese during ripening were isolated and identified by Addeo et al. (1992, 1994, 1995) using fast atom bombardment–mass spectrometry. Oligopeptides originating from regions 1–20 and 6–28 of -casein, five phosphopeptides originating from the region 64–84 of s1-casein, three phosphopeptides from the region 1–21/24 of s2-casein and one peptide from C-terminal part of s2-casein were identified. Several water-soluble peptides in Feta were identified by Michaelidou et al. (1998), including s1-CN (f1–14), (f4–14), (f24–30), (f24–32), (f40–49), (f91–98), (f102–109), -CN (f164–180), (f191–205) and -CN (f96–105). Gagnaire et al. (2001) identified 91 peptides in aqueous phase of Emmental cheese, 52 of which originated from s1-casein, 29 from -casein, 9 from s2-casein and 1 from -casein. Significant concentrations of amino acids, the final products of proteolysis, occur in all cheeses that have been investigated. Levels of free amino acids in a number of cheese varieties are given in Table 3. Relative to the level of water-soluble N, Cheddar contains low concentrations of amino acids; the principal amino acids are Glu, Leu, Arg, Lys, Phe and Ser. ParmigianoReggiano contains a very high concentration of amino acids which contribute to the characteristic flavour of this cheese (Resmini et al., 1988). Many amino acids have characteristic flavours (see McSweeney et al., 1997); although none has a cheese-like flavour, it is believed that they contribute to the savory taste of mature cheese. However, the principal role of amino acids in flavour development is as precursors of volatile flavour compounds produced by the range of catabolic reactions

as discussed in ‘Catabolism of Amino Acids in Cheese During Ripening’, Volume 1.

Methods for Monitoring Proteolysis in Cheese A range of analytical techniques have been developed to study proteolysis in cheese and have been reviewed (IDF, 1991, 1999; McSweeney and Fox, 1993, 1997; Fox et al., 1995a; Wallace and Fox, 1998). These methods for assessment of proteolysis in cheese can be classified in two categories, namely non-specific and specific methods. Non-specific methods, which give information about the extent of proteolysis and the activity of proteolytic agents, include determination of the nitrogen soluble in, or extractable by, various solvents or buffers (see Christensen et al., 1991; McSweeney and Fox, 1997; Ardö, 1999) or the measurement of reactive groups (e.g., NH2-groups) (McSweeney and Fox, 1997; Wallace and Fox, 1998). Soluble nitrogen is usually determined by the macro-Kjeldahl method, which is a highly repeatable, but time-consuming and potentially dangerous technique (Wallace and Fox, 1998). A number of techniques for quantifying proteolysis are based on cleavage of the peptide bond, which results in the formation of a new amino group which can react with several chromogenic (e.g., 2,4,6-trinitrobenzenesulphonic acid or ninhydrin) or fluorogenic (e.g., o-phthadialdehyde or fluorescamine) reagents (McSweeney and Fox, 1997; Wallace and Fox, 1998). Non-specific methods give a general idea of proteolysis, but give no information about the specific peptides produced or degraded during ripening. Specific

420 Proteolysis in Cheese during Ripening Table 3 Concentration of amino acids (mg kg 1 cheese) in different cheese varieties (based on Fox and Wallace, 1997)

Amino acid

Cheddar

Edam

Emmental

Cys Asp Thr Ser Glu Pro Gly Ala Val Met Ile Leu Tyr Phe His Lys Arg

44.7 1532.8 – 416.3 3144.1 336.2 306.86 356.2 1096.4 434.8 – 2774.1 464.1 1472.6 – 1127.2 1096.4

– 20.5 144.5 71.1 351.7 153.8 34.6 68.8 167.4 60.3 48.1 426.6 89.2 291.9 49.7 245.5 130.5

– 166.5 688.5 548.9 2680.5 2535.2 430.8 568.2 1561.5 502.7 1051.1 1794.9 285.9 1279.1 868.2 2219.8 19.2

techniques (i.e., electrophoresis and chromatography) have been used extensively to resolve, isolate and identify the peptides that are produced during cheese ripening (Fox et al., 1995a; McSweeney and Fox, 1997; Otte et al., 1999; Singh et al., 1999). In addition, these techniques are used to determine peptide profiles of cheese extracts; data obtained using these techniques are often analysed using multivariate statistical techniques (e.g., Pripp et al., 1998, 1999, 2000a,b; Molina et al., 1999; Shakeel-Ur-Rehman et al., 1999). Urea-PAGE is a powerful tool for monitoring proteolysis during the early stages of cheese maturation and for comparing casein hydrolysis patterns in cheeses manufactured from the milk of different species (Marcos et al., 1979; Sousa and Malcata, 1997). Urea-PAGE is widely used to monitor proteolysis as it resolves proteins based on a combination of charge and mass while sodium dodecylsulphate (SDS)-PAGE, which is used more widely in biochemistry, is less suitable for studying proteolysis in cheese because this technique resolves proteins based on size and the caseins have similar molecular masses. Peptides separated by SDSor urea-PAGE can be isolated by excision of the bands or by electroblotting (McSweeney et al., 1994a; Singh et al., 1995; Sousa and Malcata, 1998a,b) and the N-terminal sequence of isolated peptides determined. Isolation of peptides by electroblotting has been used widely to study proteolysis in cheese (e.g., Singh et al., 1995, 1997; Gouldsworthy et al., 1996; Ferranti et al., 1997; Broadbent et al., 1998). Capillary electrophoresis (CE) is reported to be an excellent technique for resolving the caseins (including different genetic variants), peptides derived therfrom

ParmigianoReggiano – 3241.9 4033.3 4459.5 14489.0 – 2115.6 2260.2 6011.9 2351.5 5205.2 7290.4 2054.7 4314.9 – 10091.0 791.4

Gorgonzola

Danablu

1380 1020 530 1570 3940 2320 390 1140 2220 780 1300 2910 850 1590 800 3050 280

1160 300 190 1020 1730 530 160 340 610 500 300 1530 520 680 610 1540 510

and whey proteins (Otte et al., 1997). Peptide profiles obtained by CE supplement the information obtained by reversed-phase high performance liquid chromatography (RP-HPLC) (Otte et al., 1997; Molina et al., 1998). Capillary electrophoresis has been used to study: • proteolysis in Cheddar (Strickland et al., 1996), Mozzarella, Feta and Danbo (Otte et al., 1997), Tilsit (Bockelmann et al., 1998), Roncal (Irigoyen et al., 2000), Danbo cheeses (Sørensen and Benfeldt, 2001) and Serpa, a raw ewes’ milk cheese (Roseiro et al., 2003); • the effect of the amount of rennet on proteolysis and texture in Feta cheese made from ultrafiltered milk (Wium et al., 1998); • the effect of different strains of Penicillium roqueforti on the ripening of blue-veined cheese (Larsen et al., 1998); • the effect of added proteinases and level of starter cultures on the formation of biogenic amines in Manchego cheese made from raw milk (FernándezGarcía et al., 1999). A number of chromatographic techniques, such as ion-exchange chromatography, SEC and RP-HPLC have been used to fractionate milk proteins or to fractionate cheese extracts for the purification of peptides, or less commonly, as analytical techniques to generate peptide profiles (see McSweeney and Fox, 1997). Ion-exchange and SEC are suitable for the fractionation of large caseinderived peptides. High performance ion exchange chromatography and HP-SEC have the advantage of speed and reproducibility. RP-HPLC is a very good method for resolving water-soluble peptides and has been used to characterize and compare the degree of proteolysis in


cheeses of various ages and quality, and to study the effect of various cheesemaking parameters on proteolysis (Singh et al., 1999). Reversed phase-high performance liquid chromatography has been used extensively to characterize peptides in casein hydrolysates (e.g., Le Bars and Gripon, 1989, 1993; McSweeney and Fox, 1993) as well as in studies on proteolysis of the caseins in cheese during ripening (e.g., González de Llano et al., 1991; Addeo et al., 1992, 1994; McSweeney et al., 1994a; Lynch et al., 1997; Sousa and Malcata, 1998a; McGoldrick and Fox, 1999; Shakeel-Ur-Rehman et al., 2000; Katsiari et al., 2001; Trujillo et al., 2002; Poveda et al., 2003). Numerous peptides from cheese have been purified by a combination of chromatographic procedures and subsequently identified, usually by Edman degradation and mass spectrometry, leading to a better knowledge of the proteolytic pathways in cheese during ripening (Singh et al., 1999; Gagnaire et al., 2001). Free amino acids in cheese have been analysed using amino acid analysers based on ion-exchange chromatography, with post-column ninhydrin derivatization and photometric detection at 570 nm and 440 nm for primary and secondary amino acids, respectively. This method is relatively simple, accurate and quantitative and requires little sample preparation (Bütikofer and Ardö, 1999). Alternatively, fluorescent amino acid derivatives (e.g., dansyl, OPA or N(9-fluorenylmethoxycarbonyl)) can be prepared and separated and quantified by RP-HPLC; amino acids can also be quantified by gas chromatography but this method is rarely used (McSweeney and Fox, 1997). In addition to the above methods, new techniques have been evaluated for indirect measurement of proteolysis. During ripening, the texture of cheese undergoes major changes due to proteolysis, and in recent years, the use of new techniques such as fluorescence spectroscopy and ultrasound have been investigated to monitor changes in protein structure in different types of cheese during maturation. Fluorescence spectroscopy has the advantages of high sensitivity and rapidity for the characterization of molecular interactions and reactions. Tryptophan can be used as an intrinsic probe for monitoring changes in protein structure during cheese ripening as all the major proteins of bovine milk contain at least one tryptophan residue, which has a characteristic excitation in the region 280–295 nm and broad emission spectra (Hebert et al., 2000). Fourier transform infrared spectroscopy (FTIR) has been used to measure: • the levels of protein, fat and moisture in different cheeses (McQueen et al., 1995); • differences between soft cheese varieties (Herbert et al., 2000) and different Emmental-type cheeses (Picque et al., 2002);

• changes in protein structure during cheese ripening (Mazerolles et al., 2001), protein/protein and protein/fat interactions and their relation to the texture of soft cheeses (Dufour et al., 2001); • cheese melting and its correlation with rheological properties (Karoui et al., 2003); • the stability of processed cheese (Christensen et al., 2003b). Mazerolles et al. (2001) investigated changes in the amide I and amide II regions of the FTIR spectra and in the tryptophan fluorescence spectra of 16 experimental semi-hard cheeses, varying in moisture, protein, fat and degree of maturity. The data obtained from mid-infrared and fluorescence spectral data were analysed by PCA, and correlations between spectral data and chemical composition as well as correlations between mid-infrared and fluorescence spectral data were found by canonical correlation analysis (CCA) methods. PCA and CCA of data helped to discriminate between samples at different stages of ripening. Application of low intensity ultrasonics in the food industry has increased during the last decade because it is non-destructive, rapid and cost-effective (McClements, 1997). Ultrasonics have been used in the dairy industry to monitor milk coagulation during cheesemaking (Gunasekaran and Ay, 1996; O’Donnell et al., 1996), to determine the maturity of Mahon, a Spanish semi-soft cheese variety (Benedito et al., 2000a), to determine structural defects in Parmesan cheese (Orlandini and Annibaldi, 1983) and to determine the physical properties of Cheddar cheese (Cho et al., 2001). The use of ultrasonic devices to monitor the maturity of Cheddar cheese non-destructively has been reported. Benedito et al. (2000b) ripened blocks of Cheddar cheeses at 5 or 12 °C; the ultrasonic velocity increased during maturation and decreased with increasing ripening temperature. Cho et al. (2001) measured ultrasound velocity and relative attenuation in Cheddar cheese using a noncontact piezoelectric ultrasound system and correlated results with the physical properties of Cheddar cheese (such as failure strain, failure stress, Young’s modulus and toughness) using multi-linear neutral network analysis. Besides the above applications, ultrasonics can also be used to detect cracks in cheese and to assess eye distribution and size in Emmental cheese (Benedito et al., 2002). Research done so far has been limited to a few varieties of cheeses; research is required for other cheese varieties with different physical properties and ripening behaviour (e.g., mould-ripened cheeses, smearripened cheeses or cheese with eyes) for a clearer understanding of the interaction of ultrasound and cheese and subsequent application of ultrasound as a tool for monitoring ripening.


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Catabolism of Amino Acids in Cheese during Ripening Á.C. Curtin and P.L.H. McSweeney, Department of Food and Nutritional Sciences, University College, Cork, Ireland

Introduction Catabolism of amino acids plays a major role in flavour development in cheese during ripening (McSweeney and Sousa, 2000; Yvon and Rijnen, 2001; Smit et al., 2002; ‘Sensory Character of Cheese and its Evaluation’ and ‘Cheese Flavour: Instrumental Techniques’, Volume 1). In particular, the catabolism of sulphur-containing amino acids (principally methionine), aromatic amino acids and branched-chain amino acids to flavour, or perhaps off-flavour, compounds has received considerable attention. The major aroma compounds produced from these amino acids are listed in Table 1. Recently, many authors have studied amino acid catabolism by cheese-related bacteria, including lactococci, non-starter lactic acid bacteria (NSLAB) and smear bacteria. However, it is important to remember that results obtained under laboratory conditions may not be representative of actual activity under cheeseripening conditions, as cheese constantly undergoes changes which may not be replicated in laboratory experiments. The pathways for amino acid catabolism remain to be characterised fully although much work has been done recently on LAB and Brevibacterium linens (see Yvon and Rijnen, 2001). There appear to be two major pathways by which amino acids are catabolised (Yvon and Rijnen, 2001; Fig. 1). The first series of reactions is initiated by the action of an aminotransferase which transfers the amino group from amino acid A to an -keto acid B (usually -ketoglutaric acid) and results in the production of an -keto acid corresponding to amino acid A and a new amino acid corresponding to -keto acid B (usually glutamic acid). -Keto acids produced by the transamination of aromatic amino acids, branched-chain amino acids and methionine may be degraded further to other compounds by enzyme-catalysed reactions or by chemical reactions. The second major series of reactions by which amino acids are catabolised is initiated by the action of amino acid lyases which cleave the side chains of amino acids. These pathways are particularly important for the catabolism of aromatic amino acids and methionine. Other pathways by which amino acids may

be catabolised include the production of amines by decarboxylases and the production of NH3 by deaminases. There are also specific pathways for the metabolism of threonine, aspartic acid, glutamic acid and arginine. Because of their importance to cheese flavour, the catabolism of methionine, branched-chain and aromatic amino acids will be discussed in separate sections below.

Transamination of Amino Acids Aminotransferases (EC 2.6.1.x) catalyse the transfer of the amino group from an -amino acid to an -keto acid (Hemme et al., 1982). The enzymes have broad substrate specificity and can catalyse reversible transamination reactions (Weimer et al., 1999). Transamination is the first step in the degradation of amino acids by lactococci. In LAB, catabolism of aromatic amino acids, branched-chain amino acids and methionine is initiated by transamination since degradation occurs only in the presence of an -keto acid which acts as an amino group acceptor (see Yvon and Rijnen, 2001). In the context of cheese-related microorganisms, aminotransferases of LAB and B. linens have been researched most intensively; however, enzymes of Propionibacterium freudenreichii have also been studied (Thierry et al., 2002). In a study on the enzyme activities of a range of bacteria present on the surface of smear cheese (brevibacteria, corynebacteria, staphylococci and brachybacteria), Curtin et al. (2002) found methionine aminotransferase activity in only a strain of Staphylococcus equorum. Aminotransferases are pyridoxal-5-phosphate (PLP)dependent enzymes (Hemme et al., 1982; Weimer et al., 1999; McSweeney and Sousa, 2000). The transamination reaction occurs in two steps. The first step involves transfer of the amino group of the amino acid to PLP to yield an -keto acid and an enzyme-bound pyridoxamine-5-phosphate. In the second step, the amino group is transferred from pyridoxamine5-phosphate to an -keto acid to produce an amino acid and to regenerate PLP. The amine acceptor is



436 Catabolism of Amino Acids in Cheese during Ripening

Table 1 Name and chemical nature of the major aroma compounds derived from methionine, branched-chain amino acids and aromatic amino acids (adapted from Yvon and Rijnen, 2001) Amino acid

Aldehydes

Alcohols

Carboxylic acids

Leucine

3-Methylbutanal/ isovaleraldehyde 2-Methylbutanal 2-Methylpropanal/ isobutyraldehyde Phenylacetaldehyde, benzaldehyde OH-phenylacetaldehyde, OH-benzaldehyde Indole-3-acetaldehyde, 3-Methylthiopropanal/methional

3-Methylbutanol

Phenylethanol

3-Methylbutanoic acid/ isovaleric acid 2-Methylbutanoic acid 2-Methylpropanoic acid/ isobutyric acid Phenylacetic acid

OH-phenylethanol

OH-phenylacetic acid

p-Cresol, phenol

Tryptophol 3-Methylthiopropanol

Indole-3-acetic acid 3-Methylthiopropionic acid

Skatole, indole Methanethiol

Isoleucine Valine Phenylalanine Tyrosine Tryptophan Methionine

2-Methylbutanol 2-Methylpropanol

TRANSAMINATION

usually -ketoglutaric acid. Tamman et al. (2000) observed that nine lactobacilli isolated from a 3-yearold Cheddar were able to transaminate amino acids only if exogenous -ketoglutarate was supplied. It

Thiols/Misc.

appears that the activity of transaminases may also rely on the presence of other compounds. For example, Amarita et al. (2001) found that the methionine aminotransferase activity of 29 Lactobacillus strains

Aromatic amino acids Branched-chain amino acids Methionine

Aminotransferase

ELIMINATION Met

HA-DH

Trp

n

io

Hydroxyacid

Tyr

α-Keto acid

at

xid

O

Aldehyde Benzaldehyde Alcohol DH (Phe) Hydroxybenzaldehyde Alcohol (Tyr ) Indole-3-acetate (Trp)

α-KADC ? ?

MGL CBL CGL

α-KADH

TPL

TIL

Acyl-CoA Methanethiol

Indole

aldDH

Phenol

Carboxylic acid

Methyl thioester

Ester

Dimethyldisulphide Dimethyltrisulphide Cresol, skatole (Tyr, Trp) Figure 1 Schematic diagram of pathways for amino acid catabolism found in different microorganisms and some chemical reactions (dotted lines) occurring in cheese during ripening (modified from Yvon and Rijen, 2001). AT: aminotransferase, HA-DH: hydroxyacid dehydrogenase, -KADH: -keto acid dehydrogenase, -KADC: -keto acid decarboxylase, aldDH: aldehyde dehydrogenase, alcohol DH: alcohol dehydrogenase, MGL: methionine--lyase, CGL: cystathionine--lyse, CBL: cystationine--lyase, TPL: tyrosinephenol lyase, TIL: tryptophan-indole lyase.

Catabolism of Amino Acids in Cheese during Ripening 437

increased in the presence of glucose in the reaction mixture. Williams et al. (2002) studied the effects of rate and stage of growth, amino acid type and the presence of glucose on aminotransferase activity in strains of NSLAB from Cheddar cheese. Aminotransferase activity of the cell-free extract of two strains of Lb. paracasei was maximal at, or close to, pH 6.0 and 30 °C, but activity was detected under conditions similar to those in cheese during ripening. The effect of NaCl on activity at pH 8 and 30 °C differed with leucine or phenylalanine as substrate. Glutamic acid was formed most efficiently from -ketoglutarate using aromatic, branched-chain or sulphur-containing amino acids as substrate. Martinez-Cuesta et al. (2002) found that aminotransferase activity was increased in cells treated with a bacteriocin, lacticin 3147, to increase the permeability of the cell wall to amino acids. Aminotransferases have been studied in several cheese-related bacteria. An aminotransferase from Lc. lactis subsp. cremoris NCDO763, with a pH optimum of 6.5–8, was found to be PLP-dependent but metal ionindependent (Yvon et al., 1997). The enzyme acted on leucine, methionine and aromatic amino acids. One of the two substrates of the reaction it catalyses (amino acid or keto acid) must have a hydrophobic group attached to the -carbon of the compound. This enzyme was responsible for the biosynthesis of phenylalanine and tyrosine, but it also had a catabolic role when high concentrations of aromatic amino acids were present. Yvon et al. (1997) suggested that the enzyme is involved in the catabolism of leucine, methionine, phenylalanine, tyrosine and tryptophan, and also in the synthesis of the aromatic amino acids. Engels (1997) purified two aminotransferases from Lc. lactis subsp. cremoris B78. The enzymes were active on methionine, leucine, isoleucine, valine and phenylalanine. Both transaminases were dimeric proteins, had a high temperature optimum and an alkaline pH optimum. The enzymes were found to catalyse the conversion of methionine to L-methylthio-2-ketobutyric acid. Two aminotransferases were also isolated from Lc. lactis subsp. lactis S3 by Gao and Steele (1998). The enzymes were PLP-dependent methionine aminotransferases. Both also had activity on aromatic amino acids and leucine. The aminotransferases were active under cheese-ripening conditions and produced compounds that may be precursors of off-flavour compounds in cheese, e.g., p-hydroxyphenylpyruvic acid produced from tyrosine can breakdown chemically to p-cresol. Dias and Weimer (1998a) detected high levels of methionine aminotransferase activity in several strains of lactococci but found only slight activity in two strains of B. linens. This is in contrast to earlier findings of Lee et al. (1985) who observed aromatic aminotransferases

in 23 coryneforms isolated from cheese. These authors reported that the aminotransferase(s) of B. linens were inducible while the enzymes of other coryneform strains tested were constitutive. The aromatic aminotransferase of B. linens 47 was studied by Lee and Desmazeaud (1985). The inducible enzyme was responsible for removal of the amino group of amino acids for their use as sole nitrogen sources by the bacterium; -ketoglutarate was preferred as the amino group acceptor over pyruvate. An aminotransferase from Lc. lactis LM0230 which acts on branched-chain amino acids was cloned and sequenced by Atiles et al. (2000). The enzyme was active on methionine and phenylalanine in addition to the three branched-chain amino acids. Sequence analysis showed high homology with other branched-chain aminotransferases. Transamination by Lb. helveticus was studied by Klein et al. (2001). The bacterium was incubated with a mixture of amino acids (phenylalanine, tyrosine, methionine, leucine, valine, isoleucine) in ratios similar to those in Emmental cheese. The authors concluded that transamination was the first and main step in the conversion of amino acids by Lb. helveticus, with phenylalanine and tyrosine being converted most efficiently. The transamination of branchedchain amino acids by Lb. paracasei has also been studied (Hansen et al., 2001). It is clear that many bacteria found in cheese are capable of amino acid transamination but what role do these reactions play in cheese flavour development? It has been suggested that the rate-limiting step in flavour development is the conversion of free amino acids to aroma compounds. Yvon et al. (1998) added -ketoglutarate to St Paulin-type cheese in an effort to accelerate flavour development. The level of glutamate in cheese increased when -ketoglutarate was added but the level of some amino acids, including leucine, phenylalanine, tyrosine and valine, decreased. The addition of -ketoglutarate increased the degradation of methionine. However, after six weeks of ripening, large amounts of the resulting -keto acids, produced by transamination, remained in the cheese. Thus, these authors suggested that aminotransferase activity was not limiting, but that subsequent steps in the formation of aroma compounds may have been limiting. Banks et al. (2001) supplemented Cheddar cheese with -ketoglutarate to enhance amino acid catabolism during ripening. -Ketoglutarate (20 g kg 1 milled curd) was added as a mixture with the salt. It was observed that the levels of leucine, phenylalanine, valine, threonine, methionine, alanine and isoleucine decreased on addition of -ketoglutarate. Sensory analysis showed that the addition of -ketoglutarate caused statistically significant changes in aroma intensity, creamy character and fruity notes. In fact, the


aroma intensity of the 12-week-old cheese supplemented with -ketoglutarate was equal to that of a 24-week-old control Cheddar. Supplementation of Cheddar cheese with -ketoglutarate caused statistically significant effects on the production of certain volatile flavour compounds. Shakeel-Ur-Rehman and Fox (2002) supplemented Cheddar cheese with -ketoglutarate, pyruvate or PLP and reported a beneficial effect of added -ketoglutarate and pyruvate on flavour. Cheese supplemented with 1 g -ketoglutaric acid per kg of curd was considered to be as mature at 60 days as 90-day-old commercial Cheddar cheese. Addition of -ketoglutaric acid to cheese also promoted syneresis during pressing of the curd but did not affect plasmin or chymosin activity. In an effort to overcome limitations in the concentration of -ketoglutarate, Rijnen et al. (2000) investigated the possibility of using lactic acid bacteria capable of producing -ketoglutarate from glutamate in cheese, which can be converted to -ketoglutarate by glutamate dehydrogenase. The authors cloned the glutamate dehydrogenase gene (gdh) from Peptostreptococcus asaccharolyticus into Lc. lactis, and followed the conversion of amino acids to aroma compounds in the Ch-Easy model. It was observed that the gdh strain produced -ketoglutarate from glutamate under cheese-ripening conditions and this allowed transamination of aromatic amino acids and branched-chain amino acids. The gdh strain could be used as an alternative to the addition of exogenous -ketoglutarate to cheese to increase amino acid catabolism. A number of groups are now working on the genetics of amino acid-catabolising enzymes with a view to developing strategies for controlling the development of cheese flavour. Rijnen et al. (1999a) characterised the gene (araT) encoding the lactococcal aromatic aminotransferase. It was shown that araT is transcribed as a single gene. This enzyme is essential for the catabolism of aromatic amino acids and is involved in the conversion of leucine and methionine. It also plays a role in the biosynthesis of phenylalanine and tyrosine. Subsequently, Rijnen et al. (1999b) inactivated the lactococcal araT gene and studied the possibility of controlling flavour development by directing the degradation of amino acids by starter bacteria in cheese. These authors followed the production of aroma compounds and the degradation of amino acids in St Paulin-type cheese using radiolabelled amino acids and quantified volatile products by GC–MS. -Ketoglutarate was added to half the cheeses to enhance transamination. It was observed that inactivation of the lactococcal araT gene did not significantly affect the levels of volatile compounds produced from branched-chain amino acids or methionine

during ripening, perhaps because lactococci produce more than one aminotransferase with overlapping specificities (Engels, 1997; Gao and Steele, 1998). Cheeses containing added -ketoglutarate were described as ‘more odorous’ than the control. In cheeses supplemented with -ketoglutarate and made with an araTnegative starter, the formation of aroma compounds was lower than in control cheeses. Since many of the aroma compounds produced from aromatic amino acids, such as phenethanol, phenol and indole, contribute to offflavours in cheese, the use of araT-negative starter strains may prevent the development of certain offflavours in cheese. Amino acids react chemically with carbonyl compounds to form azomethines; if the carbonyl compound has an electron-withdrawing group adjacent to the carbonyl group (e.g., a dicarbonyl), then transamination and decarboxylation occur. This process is known as the Strecker degradation, through which aldehydes are formed (Belitz and Grosch, 1987).

O C

O

C

O

+ H2N

Dicarbonyl

CHR

COOH

C

N

C

O

CHR

C O

H

Amino acid

CO2

H C

H2O NH2

+ C

O

C

N

C

OH

CHR

CHR

O Strecker aldehyde

Transamination via the Strecker degradation forms the same volatile aldehydes as formed by enzymecatalysed transamination, although by a different reaction mechanism. Strecker degradation plays an important role in flavour development of many foods since dicarbonyls are produced by the Maillard reaction. There have been reports of Strecker aldehydes in cheese (e.g., Dunn and Lindsay, 1985) but the extent to which this chemical reaction occurs in cheese is unclear since the same aldehydes are produced from a pathway initiated by the action of aminotransferases. The keto acids produced as a result of transamination of methionine, branched-chain amino acids or aromatic amino acids serve as precursors of aroma compounds (Yvon and Rijnen, 2001), which can be formed by enzymatic or chemical reactions. Four main


degradation pathways for -keto acids are used by cheese-related microorganisms (Fig. 1): • -Keto acids may be reduced to the corresponding hydroxyacid by the action of 2-hydroxyacid dehydrogenases, which have been found in LAB (see Yvon and Rijnen, 2001). Although hydroxyacids are not important flavour compounds, their production reduces the levels of -keto acids available for other reactions. • -Keto acids derived from branched-chain amino acids, aromatic amino acids and methionine may also be decarboxylated to the corresponding aldehydes, although this pathway is not important in most LAB. Aldehydes produced by this pathway may be oxidised to the corresponding carboxylic acid by aldehyde dehydrogenases or reduced to alcohols by alcohol dehydrogenases. • -Keto acids produced by transamination may also be oxidatively decarboxylated to carboxylic acids by the action of -keto acid dehydrogenases, generating acyl-CoAs which are hydrolysed, releasing carboxylic acids (Yvon and Rijnen, 2001). However, this pathway does not appear to be common in microorganisms found in cheese. • -Keto acids may degrade chemically. Phenyl pyruvate and hydroxyphenyl pyruvate (produced from Phe and Tyr, respectively) may be converted to benzaldehyde and hydroxybenzaldehyde, respectively. Likewise, indole-3-pyruvate, which is produced from Trp, is quite unstable and degrades to indole acetic acid, indole-3-aldehyde and skatole. Non-enzymatic degradation of -heto--methylthio butyrate, produced from methionine, to methanethiol has also been reported (Gao et al., 1998).

Production of Volatile Sulphur Compounds by Amino Acid Catabolism Volatile sulphur compounds are found in most cheeses and are important components of flavour (Fox and McSweeney, 1996). Since methionine is present in the caseins at a higher concentration than cysteine, sulphur compounds in cheese presumably originate principally from methionine. The pathways for the production of various flavour compounds from methionine are shown in Fig. 2. Weimer et al. (1999) discussed some of the sulphur compounds in cheese and their importance to flavour. They reported that dimethyldisulphide does not contribute to flavour, while dimethyltrisulphide is a flavour compound. The occurrence of these compounds is related to methanethiol content and the low redox potential of the cheese. Dimethylsulphide and dimethyldisul-

phide can be produced from methanethiol but it is unclear how dimethyltrisulphide is produced in cheese (McSweeney and Sousa, 2000). Methional (-methyl mercaptopropionaldehyde), which is considered to be part of Cheddar cheese aroma (Weimer et al., 1999), can be degraded to methanethiol either spontaneously or by decarboxylation. Methanethiol is a volatile compound with a ‘putrid faecal-like’ aroma at high concentrations. However, at low concentrations, it contributes to the characteristic aroma of cheese (Weimer et al., 1999). It is also a precursor of other volatile sulphur compounds which contribute to the garlic aroma of smear-ripened cheese (Hemme et al., 1982). Starter cultures, flavour adjunct bacteria and non-starter bacteria may form methanethiol in cheese from methionine (Fig. 2). Researchers continue to study the production of methanethiol and its role in cheese flavour. Dias and Weimer (1999) investigated the production of volatile sulphur compounds in Cheddar cheese slurries. It was observed that the production of total volatile sulphur compounds was four times higher in slurries acidified by Lc. lactis subsp. cremoris S3 than in those chemically acidified with gluconic acid--lactone. Addition of brevibacteria and methionine--lyase (ML) to the slurries increased the production of volatile sulphur compounds. They concluded that adjunct cultures and enzymes are required to produce volatile sulphur compounds above their flavour threshold. Molimard and Spinnler (1996) also believed that coryneform bacteria, especially B. linens, are key agents in the production of sulphur compounds in surface mould-ripened cheese. The production of sulphur compounds by LAB was investigated by Seefeldt and Weimer (2000). Lactococci are auxotrophic for methionine while lactobacilli are auxotrophic for both cysteine and methionine. In addition, it was observed that lactococci possess greater cystathionine lyase activity than lactobacilli. The cell-free extract of both lactococci and lactobacilli was able to produce volatile sulphur compounds, including methanethiol, dimethyldisulphide and dimethyltrisulphide. Five bacteria from cheese were analysed by Bonnarme et al. (2001a) for enzymes involved in the production of methanethiol. B. linens had the highest demethiolating activity. S. equorum and M. luteus showed demethiolating activity but they produced only trace amounts of volatile sulphur compounds; all bacteria examined formed methanethiol. The ability of 7 Lb. casei and 22 Lb. plantarum strains to produce flavour compounds from methionine was investigated by Amarita et al. (2001). Several enzyme activities were studied: methionine aminotransferase, hydroxyacid dehydrogenase, methionine

440 O H 2N

CH

C

OH

CH2 CH2 O

S O

H2N

CH2 H2N

CH

C

OH H 2N

CH2

CH

C

OH

OH

Other reactions CH2

Cystathionine-β-synthase

SH

Cysteine

C

CH2

Cystathionine-β-lyase

Cystathionine-γ-lyase O

CH

SH

Cystathionine

Homocysteine Homocysteine methyltransferase

O

C

C

O

O

O

Aromatic aminotransferase or transaminase B

OH

CH2

H2N

CH

C

H2N

OH

Amino acid oxidase

N

N

CH2

CH2

S

CH2

CH3

CH3

CH3

OH

N

S

S

C

CH2

CH2

Methionine adenosyltransferase CH2

CH

NH2

O

H

H

OH

OH

H

H

Methionine α-Keto-γ-thiomethylbutyrate

S-Adenosylmethionine Methionine-γ-lyase

α-Keto-γ-thiomethylbutyrate demethiolase H3C

H3 C

SH

Chemical conversion Other products

S

S

CH3

Dimethyldisulphide

Methanethiol H 3C

S

S

S

CH3

Dimethyltrisulphide

Figure 2 Metabolic pathways for the interconversion of methionine and other sulphur compounds (modified from Dias and Weimer, 1998a; Bonnarme et al., 2000).

N


lyase and amino acid decarboxylase. About 25% of the strains were capable of transaminating methionine to -keto--methylthiobutyrate. Dehydrogenase activity was observed to increase in the presence of glucose, the influence of which may have been due to the supply of energy by the proton motive force generated by lactate efflux. No methionine lyase or amino acid decarboxylase activities were detected. Amarita et al. (2002) studied the production of methional in a cheese slurry system by Lc. lactis IFPL730. The authors reported a decrease in the concentration of methional, and a corresponding increase in 3-methylthiopropanol. It was concluded that the conversion of methional to other volatile compounds, including 3-methylthiopropanol, contributes to cheese flavour. Catabolism of methionine by five cheese-ripening bacteria and five yeasts was investigated by Bonnarme et al. (2001b). For both yeasts and bacteria, dimethyldisulphide was the major sulphur compound produced. In addition, the microorganisms produced different amounts of methanethiol. G. candidum produced a low concentration of S-methyl thioacetate, and was the only yeast with the ability to produce S-methyl thioesters. Berger et al. (1999) observed that G. candidum, a fungus found commonly on the surface of smearripened cheese, can produce sulphur-containing flavour compounds; dimethyldisulphide was the main sulphur compound produced by the ten strains studied. The majority of the strains also produced methanethiol and dimethyltrisulphide. Four strains which produced sulphides could generate S-methyl thioesters. The authors concluded that G. candidum may play an important role in development of flavour in smear-ripened cheese. The pathways for the production of flavourful sulphides by G. candidum were subsequently studied by Demarigny et al. (2000). Two pathways were used to produce sulphides from methionine. The first pathway, which operated at high methionine concentrations, was initiated by the action of -demethiolase and resulted in the production of methanethiol, dimethyldisulphide and dimethyltrisulphide. The other pathway operated at low methionine concentrations and resulted in the production of dimethylsulphide. Bonnarme et al. (2001a) found that G. candidum cultures produced three S-methylthioesthers (S-methylthioacetate, S-methylthiopropionate and S-methylthiobutyrate) from methionine. It was also found that G. candidum metabolised L-methionine using different pathways; L-methionine demethiolating activity was constitutive while L-methionine aminotransferase and 4-methylthio-2-ketobutyrate demethiolating activities were not.

Methanethiol can be formed from methionine by a number of enzyme-catalysed reactions. Methanethiolproducing capacity (MTPC) was found to varying degrees in LAB and brevibacteria (Weimer et al., 1997). B. linens BL2 had the highest MTPC. Cell-free extracts had no measurable MTPC under cheeselike conditions. Dias and Weimer (1998a) examined the conversion of methionine to thiols by lactococci, lactobacilli and brevibacteria. Again, MTPC was not detected under cheese-ripening conditions (pH 5.2, 5% NaCl), using methionine as a substrate, but had activity on cystathionine was found under such conditions. The authors found that lactococci and lactobacilli catabolise methionine using cystathionine - and -lyases while Brevibacterium spp. use an ML. According to the authors, the cystathionine lyases from lysed lactococci make an insignificant contribution to the production of volatile sulphur compounds from methionine in cheese during ripening. Amarita et al. (2001) did not detect methionine lyase activity in 29 strains of Lactobacillus. No cystathionine lyase or methionine lyase activity was detected in the strains of Lactococcus investigated by Gao et al. (1998). Methionine aminotransferase activity was found to be responsible for initiating methionine catabolism. The production of methanethiol from methionine by B. linens CNRZ918 was studied by Ferchichi et al. (1985), who found greatest methanethiol production at pH 8 in the case of both the rod and coccus forms of this microorganism. MTPC decreased as the proportion of coccoid forms increased. Amarita et al. (2002) used a model system to study the ability of Lc. lactis to produce methional and other compounds from methionine. Slurries containing resting cells and the intracellular fraction from Lc. lactis IFPL730 showed the highest production of methional at the outset of incubation, with a concomitant increase in the production of 3-methylthiopropanol. Sensory analysis of slurries indicated the characteristic aroma of methional (cooked potato-like) in samples containing -keto--methylthiobutyrate and the intracellular fraction from Lc. lactis IFPL730. On extended incubation, the intensity of methional aroma decreased but samples developed a cheese-like aroma. S-methylthioesters are important flavour compounds in surface-ripened cheese. Their specific flavour depends on their chain length and configuration (Weimer et al., 1999). B. linens GC71 is capable of esterifying acetic, propionic and methyl branched-chain acids with methanethiol to produce thioesters (Lamberet et al., 1997a). Lamberet et al. (1997b) compared the S-methyl thioester-synthesising ability of some cheese-related


bacteria, including coryneforms, Micrococcaceae, Lactococcus and Leuconostoc. The strains of B. linens tested had a tendency to produce branched-chain thioesters, which was not observed in other coryneforms tested. Lactococci were shown to have poor ability to synthesise these compounds. S-methyl thioacetate was produced by G. candidum, Saccharomyces cerevisiae, Debaryomyces hansenii, Kluyveromyces lactis and Yarrowia lipolytica (Arfi et al., 2002). These authors also reported that L-methionine aminotransferase, L-methionine and 4-methylthio2-oxobutyrate decarboxylase activities were present in all the yeasts studied. Lyases involved in the catabolism of methionine

Methionine--lyase or methioninase (EC 4.4.1.11) is a PLP-dependent enzyme which catalyses the conversion of methionine to -ketobutyrate, methanethiol and ammonia (Soda et al., 1983; Tanaka et al., 1985). The enzyme plays an important role in the bacterial metabolism of methionine.

O H2N

CH

C

CH2

O

OH O

C

CH2

CH2

S

CH3

C

OH

+ H3C

SH + NH3

CH3 Methionine

α-Ketobutyrate

Methanethiol Ammonia

Methionine--lyase have been isolated from several bacteria, including Pseudomonas ovalis (Tanaka et al., 1976), Aeromonas sp., Ps. putida (Nakayama et al., 1984), B. linens CNRZ918 (Ferchichi et al., 1985), B. linens NCDO739 (Collin and Law, 1989), Trichomonas vaginalis (Lockwood and Coombs, 1991) and B. linens BL2 (Dias and Weimer, 1998b). The optimum pH for activity is generally 7.5–8. The enzyme is PLP-dependent and usually composed of four identical subunits, apart from the enzyme of Ps. ovalis which is composed of two non-identical subunits (Tanaka et al., 1976). The ML of Ps. ovalis can also degrade various - and -substituted amino acids in addition to L-methionine, and can perform both , and , elimination reactions. The enzymes from Ps. ovalis and Aeromonas spp. can act on derivatives of L-methionine and L-cysteine in addition to L-methionine (Tanaka et al., 1976; Nakayama et al., 1984). The ML of Ps. putida ICR3460 performed ,and -replacement reactions of L-homocysteine and its

S-substituted derivatives (Nakayama et al., 1984). The enzyme from Trichomonas vaginalis catabolised homocysteine, ethionine and methionine by ,-elimination (Lockwood and Coombs, 1991). The ML of B. linens BL2 is active under cheeseripening conditions (Dias and Weimer, 1998b). Unlike the release of intracellular proteolytic and lipolytic enzymes, the release of which has been shown to be necessary for their activity in cheese, the influence of amino acid catabolic enzymes on ripening may be linked to the ability of the cells to resist lysis and remain metabolically active during ripening. However, the role of enzyme release by cell lysis in the production of flavour compounds by amino acid catabolism is unclear and requires more study. The reactions catalysed by cystathionine--lyase (CL) and cystathionine--lyase (CL) are shown in Fig. 3. CL (EC 4.4.1.8) catalyses the conversion of cystathionine to homocysteine, pyruvate and ammonia while the products of CL are cysteine, ammonia and -ketobutyrate (Weimer et al., 1999). Aubel et al. (2002) studied the genetics of the CL of Lb. delbrueckii subsp. bulgaricus NCDO1489 and concluded that its physiological role is probably in the biosynthesis of methionine. Dwivedi et al. (1982) isolated a CL from E. coli which had a pH optimum of 9–10 and contained 1 mol of PLP per enzyme subunit. The gene coding for cystathionine lyase (metC) of E. coli was cloned by Laber et al. (1996) who also constructed a strain of E. coli which over-produced this enzyme. The CL of Lc. lactis subsp. cremoris B78 is reported to be a tetramer of identical ⬃40 kDa subunits (Alting et al., 1995). The enzyme catalyses the ,-elimination reaction but is able to catalyse the ,-reaction also. It was active at the pH and salt concentration of normal Gouda cheese. Unlike the ML of B. linens BL2, lysis of cells was required for full activity. Fernandez et al. (2000) cloned and characterised the metC gene encoding CL from Lc. lactis strains B78 and MG1363. The proteins encoded by the genes were similar and had high homology to other PLP-dependent enzymes. Enzyme activities were determined in strains which overproduced CL activity or in which this activity was deleted. Results showed that the product of the gene metC is essential for the degradation of cystathionine but that at least one other lyase contributes to methionine degradation by an , elimination. Dobric et al. (2000) reported the nucleotide sequence of the gene for the C/L of Lc. lactis subsp. cremoris MG1363. The enzyme was unique as it could perform either ,- and ,elimination reactions on the same substrate. Yamagata et al. (1993) studied the CYS3 gene encoding the CL of Saccharomyces cerevisae. No detectable


O H2N Cystathionine-γ-lyase O H2N

CH

OH

C

CH

C

O OH

O

C

C

CH2

CH2

SH

CH3

Cysteine

α-Keto butryate

CH2

NH3

OH

Ammonia

CH2 γ S CH2 H2N

CH

O

O

β H2N C

OH

CH

C

OH

H3C

C

C

OH

NH3

Cystathionine-β-lyase

O

Cystathionine

CH2

O

CH2 SH Homocysteine

Pyruvate

Ammonia

Figure 3 The reactions catalysed by cystathionine -lyase and cystathionine -lyase.

homology was found between the CYS3 of S. cerevisae and the cysE gene of E. coli. H2S and methanethiol were the only volatile sulphur compounds found during the degradation of L-cysteine and L-methionine by CL of Lc. lactis subsp. cremoris SK11 (Bruinenberg et al., 1997). Lb. fermentum DT41 was isolated from the starter for traditional Parmesan cheese. A PLP-dependent CL was isolated from this strain by Smacchi and Gobbetti (1998). It was composed of four identical ⬃35 kDa subunits and was optimally active at 37 °C and pH 8. The enzyme was reported to retain activity under cheese-ripening conditions. A 160 kDa homotetrameric CL was purified from Lb. reuteri DSM20016 by de Angelis et al. (2002). The enzyme was optimally active at pH 8 and 35 °C and catalysed the conversion of a range of amino acids, including methionine. This organism, together with other lactobacilli, was used as an adjunct in the manufacture of Canestrato Pugilesetype cheese and cheeses containing an adjunct composed of Lb. fermentum DT41, and Lb. reuteri DSM 20016 had the highest levels of methanethiol, dimethyl sulphide, dimethyl disulphide and dimethyl trisulphide.

Catabolism of Aromatic Amino Acids Tryptophan

Pathways for the catabolism of tryptophan are shown in Fig. 4. Gummalla and Broadbent (1999) studied tryptophan catabolism by lactobacilli used as adjunct

cheese cultures to investigate the contribution of lactobacilli to off-flavour development. The main mechanism for tryptophan catabolism by Lb. casei involved its conversion to indole lactic acid (ILA) by a series of transamination and dehydrogenation reactions with indole-3-pyruvic acid as the sole intermediate. The results suggest that non-starter and adjunct lactobacilli may have important roles in secondary reactions involving indole-3-pyruvic acid and other aromatic metabolites. Tryptophan catabolism by B. linens BL2, under both optimum (pH 6.5, 25 °C with agitation) and cheeselike (pH 5.2, 15 °C, 4% NaCl) conditions, was studied by Ummadi and Weimer (2001). At optimum temperature and pH, B. linens BL2 could degrade tryptophan by various routes simultaneously but under cheeselike conditions, the bacterium did not catabolise it. Cells grown under either conditions did not show tryptophanase, tryptophan decarboxylase or tryptophan 2-monooxygenase activities. Gao et al. (1997) investigated the catabolism of aromatic amino acids in lactococci. They reported that the first step in the catabolism of tryptophan by eight lactococcal strains is due to aminotransferase activity. Aminotransferase activity on L-tyrosine and L-phenylalanine was also found. Tyrosine

Tyrosine is a precursor of several compounds in cheese – tyramine formed by decarboxylation, p-cresol and phenol formed by an atypical Strecker degradation and p-hydroxyphenyl pyruvate formed by aminotransferase


NH3

H N

CO2 H N

Deaminase

CH2

Decarboxylase CH2

CH2 HO

Indole-3-propionate

C

CH

C

HO

NH2

CH2

Tryptamine CH2 NH2

O

O

H N

Tryptophan α-Keto acid Aminotransferase Amino acid H N

CH2

H N HO

C

C

O

O

Indole-3-pyruvate

CH2 HO

C

CH OH

O

H N

Indole-3-lactate

CH2 C

HO

O

Indole-3-acetate

H N

CH3

Skatole (3-methyl-1H-indole) Figure 4 Pathways for the catabolism of tryptophan.

activity (McSweeney and Sousa, 2000), as shown in Fig. 5. Tyramine, a biogenic amine, can cause monamine intoxication (see ‘Toxins in Cheese’, Volume 1). Phenylalanine

Phenylmethanol, phenylethanol, phenylpropane, methylphenyl hydroxyacetate, phenylacetaldehyde,

phenylpyruvate and phenylethyl acetate are flavour compounds derived from phenylalanine and have been found in cheese or model systems (Adda et al., 1982; Dunn and Lindsay, 1985; Jollivet et al., 1992). Enzymes involved in the breakdown of pheylalanine are aminotransferases, L-amino acid oxidases, L-pheylalanine ammonia lyases, L-aromatic amino acid decarboxylases and phenylalanine dehydrogenases. Phenylpyruvate is


CO2

NH3

OH

OH

OH

Decarboxylase

Deaminase CH2

CH2

CH2 C

H2N

CH2

CH

C

OH

OH

CH2

O

NH2

Tyrosine

O

Tyramine

p -Hydroxy phenylpropionate α-Keto acid Aminotransferase Amino acid OH

CH2 O

C

C

OH

O

p -Hydroxy phenylpyruvate OH

CH2 HO

CH C

OH

OH

O

OH

p -Hydroxy phenyl lactate OH

C CH2 C

H

O OH

p-Hydroxy benzaldehyde

H3 C

O

p -Hydroxy phenyl acetate

p-Cresol

Figure 5 Pathways for the catabolism of tyrosine.

formed by the action of an aminotransferase and it can be degraded to phenethanol and benzaldehyde (Fig. 6). During the first week of Camembert ripening, actively growing yeast cells produced phenethyl alcohol from L-phenylalanine (Lee and Richard, 1984). Several compounds with a phenyl group were also identified when the ability of B. linens to produce volatile compounds in liquid cultures was examined (Jollivet et al., 1992). The compounds included phenylmethanol, phenylethanol, phenylpropane and methylphenylhydroxyacetate. The formation of benzaldehyde from phenylalanine by Lb. plantarum is initiated by an aminotransferase

(Nierop Groot and de Bont, 1998). Phenylpyruvic acid was an intermediate in the reaction and its conversion to benzaldehyde was catalysed by Mn (II) which was present at high levels in these cells (Nierop Groot and de Bont, 1999) (Fig. 7). The low pH, low oxygen concentration and low temperature of cheese during ripening would not favour this chemical reaction. However, the authors pointed out that the reaction may still make a significant contribution because of the long ripening time of certain cheeses. Klein et al. (2001) also attributed the production of benzaldehyde from indole pyruvate, which was formed by transamination of phenylalanine by Lb. helveticus, to a chemical reaction.


CO2

NH3

Decarboxylase

Deaminase CH2

CH2

CH2 C

H2 N

CH

OH

CH2 C

OH

CH2

O

NH2

Phenylalanine

O

Phenylethylamine

Phenylpropionate α-Keto acid Aminotransferase Amino acid

CH2 O

C

C

OH

O

Phenylpyruvate

CH2 HO

CH

C

OH

O

Phenyl lactate C

CH2

H

O

CH2

CH2

Benzaldehyde C O

OH

OH

Phenylethanol

Phenylacetate

Figure 6 Pathways for the catabolism of phenylalanine.

An inducible aromatic amino acid aminotransferase was isolated from B. linens 47 by Lee and Desmazeaud (1985), who detected several metabolites of aromatic amino acid catabolism, including phenylpyruvate, phenylacetate, 4-hydroxyphenyl acetate, indolepyruvate and indoleacetate, in the growth medium using HPLC. Hayashi et al. (1993) characterised an aromatic amino acid aminotransferase from E. coli. The haloenzyme contained 1 mol PLP per mol and could catalyse the transamination of a variety of neutral amino acids, in addition to aromatic amino acids. Two aromatic amino acid aminotransferases have also been purified from Lc. lactis subsp. lactis S3 and characterised (Gao and Steele, 1998). Both were active

under cheese-ripening conditions. The authors observed that p-hydroxyphenylacetic acid (pHPA) could be produced from tyrosine; pHPA can degrade chemically to p-cresol. The enzymes could also produce indole-3-acetic acid from tryptophan which could be converted to skatole. It was concluded that further research is required to confirm the role of aromatic amino acids in the development of off-flavours in cheese. An aminotransferase from Lc. lactis NCDO763 catalysed the transamination of L-amino acids only (Yvon et al., 1997). Using -ketoglutarate as an amino group acceptor, the resting cells of this organism produced phenylpyruvate, phenylacetate and phenylactate from phenylalanine. The authors suggested


O H2N

CH

C

CH2

O

O OH

Aminotransferase

O

C

C

HO

OH

C

C

OH

CH

CH2

Metal ion/alkaline pH

Phenylalanine

Enol tautomer

Phenylpyruvate

Chemically O2

COOH CHO

CH2

Benzaldehyde

COOH HC

OH

Phenylacetic acid Mandelic acid

COOH C

O

Phenylglyoxylic acid

Figure 7 Proposed mechanism for benzaldehyde production by both enzymatic and chemical steps (modified from Nierop Groot and De Bont, 1998).

that the aminotransferase could have an important role in flavour development during ripening as it transaminates amino acids (leucine, phenylalanine, tyrosine, tryptophan, methionine) which are precursors of aroma compounds. Gummalla and Broadbent (2001) studied the catabolism of tyrosine and phenylalanine by Lactobacillus adjuncts. It was observed that in the two Lb. casei and two Lb. helveticus strains examined, the specific activity of phenylalanine aminotransferase was higher than that of tyrosine aminotransferase. The strains were incubated under conditions similar to those in cheese during ripening to assess catabolism. No decarboxylase activity was detected during 20 days of incubation but aminotransferase and dehydro-

genase activities were observed. Micellar electokinetic capillary chromatography was used to show that the four lactobacilli catabolised tyrosine to produce p-hydroxyphenyl lactic acid and p-hydroxyphenyl pyruvic acid under conditions similar to those in ripening cheese. Phenyl lactic acid, phenyl acetic acid and benzoic acid were produced from phenylalanine under conditions similar to those in cheese.

Catabolism of Branched-Chain Amino Acids Branched-chain amino acids are degraded by aminotransferases to -keto acids (Fig. 8). The aminotransferase of Lc. lactis subsp. cremoris NCDO 763 was most


O

O H2N

CH

C

CH2 CH

CH3

CH3

Leucine

O H2 N

CH

C

CH

CH3

OH

CH2 CH3

Isoleucine

O H2N

CH

C

CH

CH3

O

OH

OH

CH3

Valine

A M I N O T R A N S F E R A S E

C

C

OH

CH2 CH

CH3

CH3

α-Ketoisocaproate

O O

C

C

CH

CH3

OH

CH2 CH3

α-Keto-β-methylvalerate

O O

C

C

CH

CH3

OH

CH3

α-Keto-isovalerate

Figure 8 Transamination of the branched-chain amino acids to their corresponding -keto acids.

active on leucine, although it was also, but less, active on the aromatic amino acids (Yvon et al., 1997), while that of Lc. lactis subsp. cremoris B78 catalysed the transamination of valine, isoleucine and leucine (Engels, 1997). A branched-chain aminotransferase from Lc. lactis subsp. cremoris NCDO 763 was characterised by Yvon et al. (2000). The enzyme catalysed the transamination of the three branched-chain amino acids and was active under cheese-ripening conditions, although it had pH and temperature optima of 7.5 and 35–40 °C, respectively. Since the enzyme has a role in the degradation of isoleucine and valine, as well as in the transamination of leucine and methionine and was active under conditions similar to those found in cheese during ripening, the authors concluded that the enzyme was involved in flavour development. The branched-chain aminotransferase of Lc. lactis LM0230 has been cloned and sequenced (Atiles et al., 2000). The enzyme has broad specificity, being active on isoleucine, leucine, valine, methionine and phenylalanine.

Lb. paracasei subsp. paracasei LC01 produces at least one aminotransferase, capable of transaminating branched-chain amino acids, which was most active on isoleucine and leucine (Hansen et al., 2001). Responsesurface methodology showed that leucine concentration had a negligible effect on aminotransferase activity, while too high a concentration of -ketoglutarate could inhibit the enzyme. Ayad et al. (2001a) studied the effects of combining selected lactococci on flavour formation in milk. A chocolate-like flavour was produced by a combination of Lc. lactis subsp. cremoris NIZO131157 and Lc. lactis subsp. cremoris SK110. The authors speculated that this flavour was due to branched-chain aldehydes produced from branched-chain amino acids. Subsequently, Ayad et al. (2001b) studied the flavour-generating ability of wild lactococci isolated from artinsanal and non-dairy sources (fermented raw goats’, sheep’s and cows’ milk, as well as from soil, grass, silage and the udder) in milk and in a cheese model. The authors believed that these wild


strains may be able to produce more flavour compounds in cheese than the industrial strains currently used in cheesemaking. The majority of wild strains produced different flavours from industrial strains. Methylated alcohols and methylated aldehydes, probably produced from branched-chain amino acids, were the main volatile compounds formed. It was concluded that wild strains could be used for the development of new cheeses or to alter the flavour of existing types of cheese. However, since the non-dairy wild strains had no proteolytic activity, they would be unable to grow in and acidify cheese milk and would have to be combined with industrial starters. The catabolism of leucine by propionic acid bacteria was investigated by Thierry et al. (2002). P. freudenreichii catabolised leucine to -ketoisocaproic acid, but only if -ketoglutarate was present. The bacterium also converted -ketoisocaproic acid to isovaleric acid via oxidative decarboxylation by -ketoacid dehydrogenase activity yielding an acyl-CoA derivative which was then converted to the acid. The authors noted that the catabolism of branched-chain amino acids by P. freudenreichii was different to the catabolism of branched-chain amino acids by lactococci.

Deaminases There are two types of deamination involving redox reactions (Hemme et al., 1982), differing according to the nature of hydrogen acceptor: • Dehydrogenases (EC 1.4.1) which utilise NAD as the co-enzyme. The general reaction catalysed by these enzymes is: L-amino

acid H2O NAD : -keto acid NH4 NADH

These reactions can produce compounds such as pyruvic acid and -ketoglutaric acid from alanine and glutamic acid, respectively. • Oxidases which use oxygen as hydrogen acceptor. L-amino acid oxidases (EC 1.4.3.2) produce -keto acids according to the following reaction: L-amino

acid O2 : -keto acid NH3 H2O2

L-amine

oxidases (EC 1.4.3.6) form aldehydes according to the reaction: Amine O2 : aldehyde NH3 H2O2

Ammonia, a product of these deamination reactions, is an important constituent of the flavour of cheeses such as Camembert, Gruyère and Comté and

contributes to an increase in pH during ripening (McSweeney and Sousa, 2000). Microorganisms from the smear surface have deaminating ability, e.g., G. candidum (see Fox and Wallace, 1997), while B. linens produces large quantities of ammonia from serine, glutamine, asparagine and threonine. However, most strains of coryneform bacteria from smear cheese were found to have low deaminating activity except on serine, glutamine and asparagine (Hemme et al., 1982). Williams et al. (2001) studied the deaminating ability of LAB isolated from mature Cheddar. Deaminase activity was not widespread in the isolates but this may have been due to the insensitivity or lack of specificity of the assay method used.

Decarboxylases Decarboxylation is the conversion of an amino acid to the corresponding amine with the removal of CO2. Decarboxylases generally have an acid pH optimum (⬃pH 5.5) and usually require PLP as a coenzyme (Hemme et al., 1982). Amines generally have strong and often unpleasant aromas, as evident in certain smearripened cheese types (Fox and McSweeney, 1996). In addition, many amines (e.g., tyramine, histamine, tryptamine, putrescine, cadaverine and phenylethylamine) cause adverse physiological effects (‘biogenic amines’; see ‘Toxins in Cheese’, Volume 1). The relative concentration of amines in cheese depends on the type of cheese and its microflora (McSweeney and Sousa, 2000). The relative concentration of some amines does not compare with that of the parent amino acid, which may be due to differences in the rates of conversion of amino acids (Adda et al., 1982). Most amines in cheese can be formed by decarboxylation, as is the case with the production of tyramine from tyrosine and histamine from histidine. However, the formation of secondary and tertiary amines cannot be explained readily (Fox and McSweeney, 1996). Joosten (1988) studied factors that affect the concentrations of biogenic amines formed in cheese. It was observed that in Gouda cheese, a higher pH, combined with a storage temperature of 21 °C, caused an increase in concentration of histamine, as did low saltin-moisture. Starter type and pasteurisation of milk did not appear to affect the formation of histamine. The role of non-starter bacteria in the formation of biogenic amines in cheese was examined by Joosten and Northolt (1987) who investigated the decarboxylase activity of bacteria including lactobacilli, enterococci, enterobacteriaccae and pediococci. Some strains of lactobacilli could form biogenic amines in cheese. Since the number of enterococcal cells required to produce significant amounts of tyramine is rarely reached


in cheese, these bacteria are not important for amine formation in Dutch cheese, although this may not be true for certain artisanal cheeses for which enterococci are a major part of the starter. The authors concluded that non-starter lactobacilli were the most important agents in Dutch cheese for the formation of biogenic amines. This is in agreement with the findings of Broome et al. (1990) who reported that the concentrations of tyramine and histamine in cheeses inoculated with lactobacilli were twice as high as in control cheeses, indicating that decarboxylases of lactobacilli have a role in their production. Novella-Rodríguez et al. (2002a) studied the effect of defined-strain starters on the production of amines in goats’ milk cheese during ripening. The main amines found were tyramine (94.59 mg kg 1), putrescine and tryptamine. The effect of high hydrostatic pressure on the production of amines in goats’ milk cheese was studied by Novella-Rodríguez et al. (2002b) who found maximum production of amines when the cheeses were treated at 50 MPa for 72 h; rates of production were lower when cheeses received higher pressure treatments (400 MPa for 5 min or 400 MPa for 5 min followed by 50 MPa for 72 h) and in the untreated control cheeses. In addition to being involved in the production of amines, B. linens is able to reduce the amounts of histamine and tyramine in cheese during ripening (Leuschner and Hammes, 1998). During the four weeks of ripening of Münster cheese, B. linens reduced the histamine and tyramine content by 55–70%. Degradation of amines occurs at the surface of the cheese but the concentration of amines on the surface and interior differed only slightly after inoculation with B. linens LTH456. It was suggested that the concentration gradient was removed by diffusion of amines, leading to a decrease in the concentration of biogenic amines in the interior of the cheese. Lactobacilli used as cheese starter adjuncts were incubated by Gummalla and Broadbent (1999) in a defined medium containing L-tryptophan under carbohydrate starvation (CS), or under near-cheese ripening conditions (a chemically defined medium containing 4% salt, at pH 5.2). The specific activity of the tryptophan decarboxylases from Lb. casei strains was lower than those of the corresponding enzymes from Lb. helveticus strains. Generally, activity in either strain did not vary significantly with time or incubation conditions. Twenty-two Lb. plantarum strains and seven strains of Lb. casei had no decarboxylase activity on methionine (Amarita et al., 2001). The combined effects of temperature, pH and salt on the growth of E. faecalis EF37, its proteolytic activity and its ability to produce biogenic amines were studied by

Gardini et al. (2001) who observed that 2-phenylethylamine accounted for more than half of the total content of biogenic amines. The production of biogenic amines was found to be independent of the incubation temperature and in general, was very low at the higher NaCl concentration and was increased by lower pH. Roig-Sagues et al. (2002) studied the ability of 694 strains of bacteria isolated from Spanish artisanal cheeses to produce histamine and tyramine. Tyramineforming activity (mainly by enterococci and some other LAB) was found more frequently than histamine-forming activity, which was formed mainly by enterobacteria, but also by small numbers of other LAB. Most of the tyramine-forming strains of LAB were isolated from cheeses containing the highest levels of tyramine. However, histamine-forming LAB were generally isolated from samples with a low level of histamine. The amount of tyramine found in the samples was significantly higher than that of histamine. The distribution of aromatic L-amino acid decarboxylases in 326 bacteria (four species of E. coli, Erwinia herbicola, Serratia plymuthicum, two species of Proteus, Alcaligenes faecalis, Bacillus natto, Achrombacter hartlebii, 11 species of Micrococcus, one Staphylococcus, three Sarcina spp., Brevibacterium ammoniagenes, Bacterium cadaveris and three Pseudomonas spp.) was studied by Nakazawa et al. (1977). Micrococcaceae were observed to have the highest decarboxylase activity on L-tryptophan, S-hydroxy-L-tryptophan and L-phenylalanine. The amino acid decarboxylase of M. percitreus was reported by this author to be involved in synthesis of aromatic amines such as dopamine and tyramine. A histidine decarboxylase, which did not require PLP as a coenzyme, has been purified from Lactobacillus 30a (Chang and Snell, 1968). The substrates of the enzyme were found to have a heterocyclic nitrogen atom at the same position relative to its alanyl side chain which may be important in the formation of the enzyme–substrate complex. Jetten and Sinskey (1995) studied a decarboxylase isolated from a strain of Corynebacterium glutamicum with activity on oxaloacetate. The enzyme, which catalysed the decarboxylation of oxaloacetate only, a key intermediate in carbon metabolism, had optimum activity between pH 7.0 and 7.5. A glutamate decarboxylase was isolated from Lb. brevis IFO 12005 by Ueno et al. (1997) and was found to be a dimer. Temperature and pH optima were 30 °C and 4.2, respectively. The enzyme could not decarboxylate any other amino acid assayed. Lucas and Lonvaud-Funel (2002) purified the tyrosine decarboxylase of Lb. brevis IOEB 9809. The enzyme had features typical of pyridoxal phosphate-dependent amino acid decarboxylases although this enzyme was


not related by sequence homology to any known tyrosine decarboxylase.

Catabolism of Other Amino Acids Goux et al. (1995) investigated aspartate catabolism in an effort to understand ammonia generation by E. coli. It was reported that arginine may be an intermediate in aspartate catabolism, and may also be an intermediate for ammonia production from aspartate during nitrogen-limited growth. Hayashi et al. (1993) compared an aspartate aminotransferase with the aromatic amino acid aminotransferase of E. coli. Both enzymes were composed of two identical ⬃43.5 kDa subunits, and contained one molecule of PLP per subunit. An aspartate aminotransferase isolated from Lc. lactis LM0230 was cloned and characterised by Dudley and Steele (2001). It was determined using homologous recombination that a mutation in the Asp biosynthetic pathway prevented this strain from growing in milk. According to Kaneoke et al. (1993), at least seven L-arginine degradation pathways are known, and in some species, more than one of these pathways can be operational. These authors studied the arginine oxygenase pathway in two coryneforms, Arthrobacter globiformis IFO 12137 (ATCC 8010) and B. helvolum IFO 12073. This pathway involves four enzymes and produces succinate from L-arginine (Fig. 9). This pathway in the coryneforms studied is not identical to other pathways reported, e.g., the pathways of Pseudomonas aeruginosa and Streptococcus faecalis. E. coli utilises the ammonia-producing succinyl transferase pathway for arginine catabolism, and to a lesser degree, the arginine decarboxylase pathway (Schneider et al., 1998).

3 Arginine

Agmatine 5

1

8

4 Citrulline

N-carbamoylputrescine

2

8

6 Ornithine

Putrescine 7

Figure 9 Pathways of arginine metabolism in bacteria (Arena and Manca de Nadra, 2001). (1) Arginine deiminase, (2) Catabolic ornithine transcarbamylase, (3) Arginine decarboxylase, (4) Agmatine deiminase, (5) Agmatinase, (6) N-carbamoylputrescine hydrolase, (7) Ornithine decarboxylase, (8) Anabolic system.

Lactic acid bacteria isolated from wine can catabolise arginine by at least two pathways (Arena et al., 1999). The arginine deiminase pathway produces orthinine, CO2 and NH3 via three enzymatic reactions. The enzymes involved in this pathway are arginine deiminase (EC 3.5.3.6), catabolic ornithine transcarbamoylase (EC 2.1.3.3) and carbamate kinase (EC 2.7.2.2) (Champomier Verges et al., 1999). Alternatively, the arginase–urease pathway leads to the production of urea. Arginine deiminase, ornithine transcarbamoylase and carbamate kinase from the sourdough microorganism, Lb. sanfranciscencis CB1, were isolated by de Angelis et al. (2003). The enzymes had acidic pH optima and were optimally active at 30–37 °C. Interestingly, arginine has been proposed as a possible growth substrate for the secondary microflora of Swiss cheese (Laht et al., 2002); calculations showed that ATP available from the metabolism of arginine to ornithine was theoretically sufficient to support the growth of non-starter bacteria to populations of 108 cfu g 1. The catabolism of threonine, asparagine, arginine and glutamate in cheese has attracted some study. Aminotransferase or dehydrogenase activities catabolise glutamate to produce -ketoglutarate, while -aminobutyrate is formed from glutamate by the action of a decarboxylase. Threonine is converted to acetaldehyde and glycine (McSweeney and Sousa, 2000). The specific pathways for the catabolism of other amino acids (e.g., glycine, alanine and serine) by cheese-related microorganisms have attracted little attention.

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Sensory Character of Cheese and its Evaluation C.M. Delahunty, Department of Food and Nutritional Sciences, University College Cork, Ireland M.A. Drake, Department of Food Science, Southeast Dairy Foods Research Center, North Carolina State University, Raleigh, North Carolina, USA

Introduction A remarkable variety of cheeses are made in all parts of the world where milk is produced. Cheeses are consumed for their highly regarded nutritional value, and enjoyed for their complex and varied eating quality. The sensory characteristics of cheeses, which determine their eating quality, are properties that are perceived by the human senses, predominantly during consumption. These properties can be described as appearance characteristics, flavour characteristics and texture characteristics. However, cheeses are complex foods, produced using milk from different animals, by many different techniques, and are presented in a variety of sizes, shapes, packages or coatings. Some cheeses are produced in small quantities, such as farmhouse types, sold in local markets and consumed by a relatively small number of people. Others are produced in large quantities in very large automated facilities, may find their way to markets in many different countries and are consumed by very many people. Some cheeses are ripened or matured for years before they are consumed; others are consumed young or unripened. Cheeses may have moulds of different types growing on their surface, may be pierced to allow blue moulds grow within the cheese, or include ingredients such as herbs and/or spices. This considerable diversity in cheesemaking practice, and the number of stages that any single cheese undergoes during its production, results in a wide variety of cheeses each of which has complex sensory characteristics. Sensory evaluation of cheese is absolutely necessary to determine the relative merits of cheesemaking procedures and the influence of measured composition on specific sensory characteristics of cheese. Sensory evaluation is also needed to determine the influence of sensory characteristics on the eating quality of cheese and its consumer acceptability. However, the complexity of cheese presents a considerable challenge for its sensory evaluation. This chapter will focus on human perception of sensory characteristics, on the advantages and disadvantages of sensory evaluation methods, on the intensity

and quality of the sensory characteristics of cheeses, and on the relationships between cheesemaking, cheese composition, cheese sensory characteristics and consumer acceptability of cheese.

A Definition of Sensory Character Sensory characteristics of cheeses are human responses to perceptions of stimuli that are experienced with the cheeses, and can generally be described using terms defined within the categories of appearance, flavour and texture. Sensory characteristics result from interactions of the human sensory modalities of vision, touch, olfaction, gustation and mouthfeel with stimuli induced by rheological, structural and chemical components of the cheese. Sensory characteristics are perceived by consumers when they observe, manipulate, smell and take cheese into the mouth for consumption, and are subsequently expressed as a behavioural response using actions or descriptive terms. A majority of sensory characteristics are complex and are stimulated by the association of many different properties of the cheese, with different sensory modalities acting together. It is this complexity, or component balance, that hinders attempts to adequately represent cheese sensory character using instrumental or chemical analyses. In addition, and unfortunately from the sensory scientists’ point of view, consumers differ from one another. Sensory perception, and particularly its communication, differs between individuals as a result of physiological, psychological, social and cultural differences.

Sensory Characteristics and Cheese Preferences Cheese quality has been defined for many years by manufacturers as cheese produced reliably and economically (Muir et al., 1995a). In the past, limited choices were available to consumers and as a result of this



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limited experience, the consumer’s palate was less discerning. Today, cheese markets are international, and cheesemakers compete openly for consumers, offering them an ever-widening choice. Cheese consumers are more affluent and many have tasted or regularly consume a diversity of cheese types, leading them to become increasingly discerning. These consumers now define the quality standard for cheeses, which is ultimately determined by eating quality. The eating quality of cheese, or a consumer’s liking for cheese, is an integrated response. The stimuli are the sensory characteristics, perceived before and during consumption. However, the response is influenced by other individual consumer-related factors that include sensory abilities, past experiences with cheese, what is expected from a cheese and when and where it will be consumed. Expectations are based on experience, but are created for a specific product by marketing, packaging and familiarity. Finally, liking for, and satisfaction with, a cheese is determined by the context in which it is consumed, and its appropriateness for that context (e.g., would one wish to consume Epoisses for breakfast?). Eating quality determines consumer acceptability and willingness to repeat purchase. Highly regarded eating quality is not that found in a cheese with no defects, but that which offers unique and appealing characteristics consistently. The producer of the cheese with the most acceptable sensory characteristics, if he is aware of this and can ensure that the market presentation of his cheese matches its sensory character, will have an advantage in the market. The concepts of ‘healthy eating’ and ‘conscientious eating’ (e.g., vegetarian and vegan diets) and environmentally friendly eating are now increasingly important to consumers. To meet these consumers’ expectations, cheese producers are challenged to produce new, wholesome products that taste as good as traditional alternatives. This task is proving difficult as dietary guidelines for healthy eating may recommend reducing the intake of ingredients that provide desirable sensory character, such as fat or salt. The production of reduced- and low-fat cheeses to replace traditional types is an example of such consumer-driven product development. However, the majority of new low-fat cheeses do not meet the sensory quality requirements of discerning consumers (Mistry, 2001). This is because fat is not just a provider of desirable sensory character, but it is also important for cheese texture and body, for the development of compounds responsible for flavour and for the release of flavour compounds during consumption. It will be difficult to improve the eating quality of these cheeses unless eating quality is understood better.

Cheesemaking and the Variety of Sensory Character The sensory characteristics of a cheese at the time of its consumption reflect the milk from which it was produced (e.g., a goats’ milk cheese is distinct from a cows’ milk cheese), the processes used in its production and the physical and the chemical changes that occurred during maturation (e.g., proteolysis breaks down proteins to amino acids during cheese maturation, which may subsequently act as substrates for the formation of volatile compounds (see ‘Catabolism of Amino Acids in Cheese during Ripening’, Volume 1)). Milk from the cow, sheep, goat, buffalo or other animals can be used as raw material, and its qualities are determined by breed, diet and stage of lactation. Treatment of milk before cheese production, particularly pasteurisation, can kill micro-organisms and reduce enzyme activity that could otherwise contribute to the development of sensory character during maturation. During cheese production, the coagulant used to form curds, the amount of salt added, the type of starter culture and the use of adjunct cultures will determine sensory characteristics. Finally, the maturation time and the temperature of maturation may be varied. The sensory characteristics of different types of cheese, and the potential variety that may be achieved, are determined by the choices the producer makes at each of the stages in production. Sensory characteristics of many different cheeses are described in the literature and in specialist cheese books. However, the sensory characteristics of relatively few types have been defined, standardised and measured objectively using sensory science methods. Lack of objective knowledge makes it difficult to compare accurately the sensory characteristics of different cheese types, but more importantly, as the cause of sensory characteristics is only partially known, it is difficult to compare accurately cheese appearance, texture and flavour research carried out in different laboratories. Tables 1 and 2 present terms used to describe the appearance, texture and flavour characteristics of cheeses that have been defined and standardised in an objective way. Table 3 presents terms used for descriptive sensory analysis by other researchers, but that have not been defined and standardised adequately. Similar terms are used in many cases even though each descriptive language referenced was developed independently by different research groups. In addition, in many cases, similar terms have been used to describe dominant characteristics of different cheese types. This comparison suggests that even though a remarkable variety of cheese types are produced, that potentially exhibit a wide variety of sensory characteristics, it should

Sensory Character of Cheese and its Evaluation 457

Table 1 Terms used to describe the appearance and texture of cheese using descriptive analysis methods. Terms in this list were developed and defined by trained panels, and in many cases standard materials that help to illustrate the term are provided. Cheeses studied were low-fat, full-fat and smoked Swiss, Cheddar and Gouda (Adhikari et al., 2003), natural and processed cheeses (Drake et al., 1999a; Gwartney et al., 2002), ten different types of cheese (Lawlor and Delahunty, 2000), Cheddar and Camembert (Cooper, 1987) and Mozzarella cheeses (Brown et al., 2003) Term Appearance Chalky Colour/colour intensity

Mottling Mouldy Open/openness Shiny Texture Adhesiveness Chewy Cohesiveness Creamy/creaminess

Crumbly/crumbliness

Crustiness Curdiness Degree of breakdown Dry Firm/firmness

First-bite sticky Fracturability at first bite Grainy

Hardness Mealy Moist Mouth-coating Oily Rate of recovery Residual mouthfeel Residual smoothness of mouth coating

Definition a

Resembling chalk in appearance The colour of Cheddar ranging from pale yellow to orange, the palest yellow representing the start of the scale The colour of cheese ranging from white to orange The evenness of colour shading within the cheese sample, with the most uniform coloured cheese being free from mottling, marbling or any other deficiencies in colour The degree of mouldiness/visible mould growth in the cheese structure The extent to which the interior of the cheese (that is the cut surface) is open, this encompasses cracks, pinholes, irregular-shaped holes and any other openings The extent to which the surface of the cheese is shiny, glossy, moist or sweaty-looking, as opposed to looking matt or dull The degree to which the chewed mass sticks to mouth surfaces, evaluated after five chews Requiring a good deal of mastication, toffee-like texture. Degree of chewing needed to break up the cheese The degree to which the chewed mass holds together, evaluated after five chews The extent to which the texture has broken down to a creamy semi-liquid texture, assessed between tongue and palate during mastication The feeling associated with heavy whipping cream (e.g., 30% fat content) The extent to which the cheese structure breaks up in the mouth, assessed during the first 2–3 chews The feeling in the mouth when the sample falls apart quickly in mouth during mastication The force required to break through the crust of the cheese when taking the first bite, assessed using the front teeth The extent to which a curdy or mealy texture is perceived in the mouth during mastication The amount of breakdown that occurs in the sample as a result of mastication, evaluated after five chews The degree of dryness or moistness sensed in the mouth during mastication Ranging from soft to firm. The extent of resistance offered by the cheese, assessed during the first five chews using the front teeth The force required to squeeze a cube (1.5 1.5 1.5 cm) of cheese flat between the first finger and thumb The amount of force required to take the first bite of cheese, assessed using the front teeth The amount of force required to completely bite through the cheese, assessed using the molars Sticky sensation experienced during the first bite Completely bite through the sample with the molars and evaluate the degree to which the sample fractures The extent to which granular structures are formed as the sample breaks down, perceived in the second half of chewing The feeling of coarse particles in the mouth during mastication The force required to bite the sample (first bite) The feeling in mouth when the sample breaks down in small pieces and it is difficult to gather them for swallowing The perceived moisture content of the cheese. Ranging from dry to moist The extent to which the cheese has a moist or wet texture around the palate during mastication The extent to which the cheese coats the palate and teeth during mastication The degree of coating on the tongue and the palate during mastication Oily, fatty, greasy mouth-feel of any kind Depress sample between thumb and first finger 30%, evaluate the speed or rate at which the sample returns to its original shape The degree of ‘bittiness’ or graininess in the mouth just before swallowing The degree of smoothness felt in the mouth after expectorating the sample

continued


Table 1 continued Term

Definition a

Rubbery/rubberiness

The extent to which the cheese returns to its initial from after biting, assessed during the first 2–3 chews The degree of springiness experienced while biting the sample Of the nature of slime, soft, glutinous or viscous substance, soft, moist and sticky The smoothness of the cheese against the palate as it breaks up during mastication The degree to which the chewed mass surface is smooth, evaluated after five chews Yielding easily to pressure, easily moulded, pliable, easily spreadable Depress sample between thumb and first finger 30%, evaluate the total amount of recovery of the sample The stickiness of the cheese against the palate and around the teeth during mastication Overall sensation of stickiness during mastication The mouthfeel associated with consuming very viscous fluids like heavy whipping cream or honey

Slimy Smooth/smoothness Softness Springiness Sticky/stickiness Viscous

a The precise wording of some definitions has been changed to allow the use of consistent language in this table. However, the meaning of each definition is unchanged.

Table 2 Terms used to describe the flavour of cheese using descriptive analysis methods. Terms in this list were developed, defined and referenced using standard materials by trained panels. Cheeses studied were: Cheddar (Murray and Delahunty, 2000b; Drake et al., 2001), low-fat, full-fat and smoked Swiss, Cheddar and Gouda (Adhikari et al., 2003), aged natural cheese of many types (Heisserer and Chambers, 1993), ewes’ milk cheese (Bárcenas et al., 1999) and cheese flavours (Stampanoni, 1994) Term

Definition a

Standard b, c

Acid/yoghurt, acidic

The taste on the tongue associated with acids (citric, lactic... ) A sour, tangy, sharp, citrus-like taste. The fundamental taste sensations of which lactic and citric acids are typical Flavours indicating age in Cheddar cheese – The combination of aromatics reminiscent of farm animals and barnyards The complex of drying, puckering, shrinking sensations in the oral cavity causing contraction of the body tissues A mouth-drying and harsh sensation Mellow, smooth, clean. In equilibrium, wellarranged or disposed, with no constituent lacking or in excess Aroma associated with freshly cut green peppers The slightly burning, prickling and/or numbness of the tongue and/or mouth surface Fundamental taste sensation of which caffeine or quinine are typical A chemical-like taste – The combination of aromatics associated with the saturated brine used during traditional ewes’ milk cheesemaking Aromatics associated with boiled meat or vegetable stock soup

0.35–0.86 g lactic acid/100 g Ricotta Fermented milk Natural yoghurt Citric acid (0.2% in water)

Age Ammonia Animal, animalic Astringent

Balanced

Bell pepper Biting

Bitter

Blue Brine

Brothy

Aged Cheddar cheese (1 yr or older) Ammonia solution (0.25% in water) 4-Methyl-octanoic acid (2% in PGd) 1-Phenyl-2-thiourea (5000 mg/kg in PG) Alum (0.1% in water) Tea, six bags soaked in watere for 3 h Tannic acid (0.05% in water) Mild Cheddar

Methoxy pyrazines (5 g/kg) Freshly cut bell pepper Horseradish sauce

Caffeine (0.02, 0.06 or 0.08% in water) Tonic water, quinine (0.01% in water) Octan-2-one (1% in PG) Ewes’ milk cheese brine at room temperature

Canned potatoes Low-sodium beef broth cubes Methional (20 mg/kg)



Definition a

Standard b, c

Butter milk Buttery

– Fatty, buttery tasting, of the nature of, or containing butter The aromatics commonly associated with natural, fresh, slightly salted butter Aroma rising from butter at room temperature

Butyric, butyric acid

Sour flavour, similar to baby vomit The aromatics reminiscent of baby vomit; is sour and cheesy

Capric acid Caramel

– The taste and aromatics associated with burnt sugar or syrup; toffee made from sugar that has been melted further – Aroma associated with tom-cat urine The taste and aromatics associated with typical Cheddar Typical aroma and taste of sharp/mature Cheddar cheese – Aromatics associated with cooked milk The combination of sweet, brown flavour notes and aromatics associated with heated milk – Aromas associated with barns and stock trailers, indicative of animal sweat and waste Fatty, creamy tasting, of the nature of, or containing cream

Pasteurised butter milk Unsalted butter Lightly salted butter Pasteurised cooking butter Diacetyl (1% in PG) Diacetyl in vaseline oil (several concentrations) Butyric acid, 2500 mg/kg in vaseline oil SSf. 2 ml SS cotton in 60-ml flask Butyric acid (10 000 mg/kg in PG) Butyric acid (1% in PG) Capric acid (pure) Condensed milk 3-Hydroxy-2-methyl-4-pyrone (2% in PG)

Caseinate Catty Cheddary

Cheese rind Cooked, cooked milk

Cottage cheese Cowy/phenolic

Creamy

Dairy fat Dairy sour Dairy sweet Decaying animal Diacetyl Earthy Fatty Faecal Fermented Fermented fruity / winey

Flavour intensity Free-fatty acid Fresh fish

The oily aromatics reminiscent of milk or dairy fat The sour aromatics associated with dairysoured products The sweet aromatics associated with fresh dairy products The aromatics reminiscent of decaying animal material Aromatics associated with diacetyl – – Aroma associated with complex protein decomposition – The combination of aromatics reminiscent of red wine in general; it is sweet, slightly brown, overripe and somewhat sour The overall intensity of flavour in the sample, from mild to strong Aromatics associated with short chain fatty acids The aromatics associated with fresh fish

Sodium caseinate powder 2-Mercapto-2-methyl-pentan-4-one (20 mg/kg) Processed cheese Mature Cheddar cheese

Cheese rind (Tilsit mild, pasteurised full fat) Skim milk heated to 85 °C for 30 min Evaporated milk UHT milk 3.6% fat, cooked for 10 min Cottage cheese 25% fat p-Cresol (160 mg/kg), bandaids

Mascarpone cheese -Decanolactone (0.1% in PG) UHT Cream 35% fat Whipping cream Unsalted butter Sour cream Vitamin D milk Dimethyl disulfide (bottom notes only; 10 000 mg/kg in PG) Diacetyl (20 mg/kg) Geosmin (0.001% in PG) Palm kernal fat Indole, skatole (20 mg/kg) Fermented milk, 12% fat Burgundy cooking wine

Butyric acid (20 mg/kg) Elodea (an aquatic plant) growing in water continued



Definition a

Standard b, c

Fruity

The taste and aromatic blend of different fruity identities The aromatics associated with different fruits

Goaty

The aromatics reminiscent of wet animal hair; it tends to be pungent, musty and somewhat sour – Aroma associated with blue-vein cheeses Aromatics associated with milkfat

Canned fruit salad (in syrup) trans-2-Hexenal (10 000 mg/kg in PG) Canned fruit cocktail juice Fruit of the forest yoghurt Ethyl butyrate (0.1% in PG) trans-2-Hexenal. 300 mg/kg in vaseline oil SS. 3 ml SS cotton in 60-ml flask Fresh pineapple Ethyl hexanoate (20 mg/kg) Hexanoic acid (5000 mg/kg in PG)

Green-grass Methyl ketone / blue Milkfat / lactone

Milky Mouldy, mouldy/musty

Mushroom

The aromatics commonly associated with ewes’ raw milk The combination of tastes and aromatics generally associated with moulds; they usually are earthy, dirty, stale, musty and slightly sour Aromas associated with moulds and/or freshly turned soil The taste and aromatics associated with raw mushrooms

Musty

Aroma of a damp room or very old book

Nutty

The aromatics reminiscent of several dry fruits such as pecans, walnuts and hazelnuts The non-specific nut-like taste and aromatics characteristic of several different nuts, e.g., peanuts, hazelnuts and pecans The nut-like aromatic associated with different nuts

Overall intensity

Strength of the stimuli perceived by the nose Strength of global stimuli originated by the volatiles released during mastication and perceived on the olfactory receptors via the retronasal route

Oxidised

Aroma associated with oxidised fat The fruity aromatic associated with pineapple

Pineapple

cis-3-Hexenol (1% in PG) 2-Octanone (40 mg/kg) Fresh coconut meat Heavy cream -Dodecalactone (40 mg/kg) Ewes’ milk raw Pasteurised milk, 3.6% fat 2-Ethyl-1-hexanol (10 000 mg/kg in PG) 2-Ethyl-1-hexanol (40 mg/kg) Stilton cheese 2,4,6 Trichloroanisole (1% in PG)

Button mushroom (raw) Brown mushrooms (chopped, raw) 1-Octen-3-ol (0.5% or 1% in PG) 3-Octanol (10 000 mg/kg in PG) 3-Octanol. 5–10 mg/kg in vaseline oil SS. 3 ml SS cotton in 60 ml flask Cola infusion in ethanol (pure) Damp room Very old book Wheat germ 2 g Walnuts 2 g hazelnuts, minced in 60-ml flask (mixed particulates to be sampled) Mixed crushed nuts 2-Acetyl-pyridine (0.01% in PG) Lightly toasted unsalted nuts Unsalted wheat thins Roasted peanut oil extract Roasted peanuts, ground hazelnuts, ground almonds, 1:1:1 1000–73 nut base by Givaudan-Roureg (10% in PG) 4 g cheese aroma/100 ml of pasteurised ewes’ milk 0.5-3.5 g cheese aroma/100 g Quark 91549-24 by Givaudan Roureg 91483-24 by Givaudan Roure 91428-24 by Givaudan Roure 91125-73 by Givaudan Roure 10418-71 by Givaudan Roure 2,4 Decadienal, 20 mg/kg 4-Pentenoic acid (10 000 mg/kg in PG) Canned pineapple chunks



Definition a

Standard b, c

Prickle/bite

Chemical feeling factor of which the sensation of carbonation on the tongue is typical A bland, shallow and artificial taste. Made by melting, blending and frequently emulsifying other cheeses – A physically penetrating sensation in the nasal cavity. Sharp smelling or tasting, irritating Irritative, burnt and/or penetrating sensation in the interior of the mouth

Soda water

Processed

Propionic acid Pungent

Rancid

Rennet Rosy/floral Salty

Sauerkraut Scorched Sharp

Smokey, smoky

Soapy

Sour

Soya sauce

The taste and aroma associated with sour milk and oxidised fats. Having the rank unpleasant aroma or taste characteristic of oils and fats when no longer fresh The aromatics associated with natural lamb rennet Aroma associated with flowers Fundamental taste sensation of which sodium chloride is typical Fundamental taste sensation elicited by salts Fundamental taste sensation produced by aqueous solutions of several products such as sodium chloride The aromatics associated with fermented cabbage Aroma associated with extreme heat treatment of milk proteins The total impression associated with the combination of aromatics that are sour, astringent and pungent Total impression of penetration into the nasal cavity The perception associated with aged and ripened cheeses, from flat to sharp The penetrating, dark brown, acrid aromatic of charred wood Aroma and taste of hickory-smoked ham The penetrating smoky taste and aromatics, similar to charred wood Tainted by exposure to smoke Perception of any kind of smoke odour (hickory, apple, cherry, mesquite or artificial flavouring) A detergent-like taste and smell. Similar to when a food is tainted with a cleansing agent Fundamental taste sensation elicited by acids Fundamental taste sensation of which lactic and citric acids are typical The aromatics that are reminiscent of soy sauce; they are sour, slightly brown and pungent

Cheese strings (a processed cheese snack) Propionic acid (1% in PG) A ratio of 1 part sour cream to 0.68 parts horseradish sauce Danish blue cheese Ammonia (1% in PG) 0.5 g cayenne/100 ml water, boiled in water for 5 min, 1.5 ml of filtration/10 g Quark Cheese stored at 21 °C for 4 days Butyric acid (0.1% in PG)

Natural lamb rennet (33% NaCl) 2-Phenethylamine, 20 mg/kg Sodium chloride (0.25, 0.5, 0.75 or 1% in water) Pecorino Romano sheep cheese, 1200 mg NaCl/100 g Quark

Dimethyl disulfide (top notes only; 10 000 mg/kg in PG) Milk heated to 121 °C for 25 min Propionic acid (100 000 mg/kg in PG) 5000 mg/kg of propionic acid in Vaseline oil SS. 2 ml SS cotton in 60 ml flask

Oil of cade Hickory smoked ham Applewood cheese Guaiacol (0.5% in PG) Guaiacol in vaseline oil (several concentrations) Liquid smoke flavouring. 40 l cotton in 60-ml flask Lauric acid (pure) Mellow processed Cheddar Citric acid (0.08% in water) Lactic acid (0.05 and 0.085% in water) Soya sauce

continued



Definition a

Standard b, c

Spicy/pungent Strength

Valeric acid (1% in PG) English blue Stilton cheese

Yeasty

– The overall intensity of aroma and flavour, the degree of mildness and maturity Aromatics associated with sulphurous compounds The aromatics-associated reminiscent of perspiration-generated foot odour; sour, stale, slightly cheesy and is found in unwashed gym socks and shoes Fundamental taste sensation of which sucrose is typical Fundamental taste sensation elicited by sugars Fundamental taste sensation produced by aqueous solutions of several products such as sucrose or fructose The combination of sweet aromatics produced after food toasting or cooking Chemical feeling factor elicited by certain peptides and nucleotides Aroma described as acidic, fermented and sweaty by the panelists The sweet aromatic that is associated with waxed paper or wax candles Aromatics associated with medium chain fatty acids Aromatics associated with Cheddar cheese whey Aromatics associated with fermenting yeast

Yoghurt

–

Sulfur Sweaty

Sweet

Toasty Umami Vinegary Waxy, waxy/crayon

Whey

Boiled mashed egg. H2S bubbled through water; struck match Isovaleric acid (10 000 mg/kg in PG) Isovaleric acid (0.1% in PG) Isobutyric acid (5% in PG) Cheese stored at 30 °C for 3 h Sucrose (1, 3, 4 or 5% in water) Condensed milk 1.2 g sucrose/100 g Quark

Cooked condensed milk Ciclotene (several concentrations in water) Monosodium glutamate (1% in water) Combination of acetic, butyric and propionic acids Decanoic acid (pure) Capric acid, lauric acid or decanoic acid (100 mg/ml) Fresh Cheddar whey Whey powder Raw yeast dough Yeast in 3% warm sucrose water Yoghurt, 3.2% fat

a The precise wording of some definitions has been changed to allow the use of consistent language in this table. However, the meaning of each definition is unchanged. b Units of measurement are changed to a standard format where possible. c Publications referenced often provided brand names of food standards used. Brand names are not provided in this table as it is recognised that many of these will only be of interest to readers in their country of origin. In addition, as some publications referenced are now more than 10 years old, products may have changed. d Propylene glycol. e Volume of water not given in publication referenced. f Stock solution. g Codes refer to commercially available flavour mixtures that can be provided by Givaudan Roure, Switzerland.

be possible to develop and standardise a terminology that can be used universally, and for all cheese types, eventually leading to a much-improved understanding about the eating quality of cheese.

The Human Senses and the Sensory Properties of Cheese Cheese appearance

Humans are highly visual creatures and allow vision to dominate other sensory modalities. Vision is the perception of shape and texture, size and distance, brightness,

colour and movement. Appearance characteristics of cheese are assessed visually, usually prior to consuming the cheese, or when preparing the cheese for consumption by cutting or spreading. Appearance characteristics include colour, presence of eyes or holes (or openness), mould, rind, and visual texture (Tables 1 and 3). In addition, appearance includes a cheese’s market image (e.g., size, shape, packaging), as most cheese is purchased in this form (Murray and Delahunty, 2000a). Appearance characteristics create sensory expectations, or expectations of how the cheese will ‘taste’, and as vision can dominate other sensory modalities, visual aspects of cheese can often have a strong influence on

Table 3 Studies of cheese sensory character that have used descriptive sensory analysis. Cheese types studied in each case and vocabularies used are listed to allow comparisons References and cheeses studied

Descriptive vocabularies

Adhikari et al., 2003 Low-fat, full-fat and smoked Swiss, Cheddar and Gouda Antoniou et al., 2000 Münster, Emmental, Roquefort, Beaufort, Camembert, Reblochon, Pont l’Eveque, Brie de Meaux, Tomme de Savoie, Valencay, Saint Nectaire, Pyrenees Brebis, Bleu d’Auvergne, Comte Vieux, Fourme de Salers Bárcenas et al., 1999, 2001 Castellano, Idizábal, Manchego, Roncal (ewes’ milk cheeses), Garrotxa (goats’ milk), Tetilla (cows’ milk) Drake et al., 2001, 2002, 2003 Truong et al., 2002 Gwartney et al., 2002 Cheddar, processed cheese

Aroma: Smokey, vinegary, cheddary, buttery, musty, pungent, other (Swiss) Flavour: Smokey, salty, sweet, bitter, acidic, cheddary, sharp, flavour intensity Texture: Grainy, hardness, first-bite sticky, sticky, creamy, mouth-coating, viscous, rubbery, dry, crumbly, mealy Texture: Hardness, elasticity, fracturability, cohesiveness, adhesiveness, chewiness

Heisserer and Chambers, 1993 Asiago, Bel Paese, Blue Cheese, Bond-ost, Brisk, Brie, Butter Cheese, Camembert, Cheddar, Chevre, Colby, Danish Cream Havarti, Edam, Emmentaler, Feta, Fontina, Gorgonzola, Gouda, Gruyere, Jarlsberg, Kreme Kase, Limburger, Manchego, Mozzarella, Monterey Jack, Parmesan, Port du Salut, Provolone, Romano, Roquefort, Sap Sago, Stilton, Swiss Hort and Le Grys, 2001 Cheddar Hough et al., 1996 Reggianito grating cheese

Odour: Overall intensity, sharp, milky, brine, rennet, buttery, toasty, smoky, mushroom Flavour: Overall intensity, fruity, butyric, nutty, buttery, acid/yoghurt, sweet, salty, pungent, rennet, smoky Texture: Surface rugosity, surface moisture, elasticity, firmness, friability, adhesiveness, solubility, moisture, granularity Flavour: Cooked, whey, diacetyl, milkfat/lactone, fruity, sulphur/eggy, sulphur/match, free fatty acid, brothy, nutty, catty, cowy/phenolic, age, yeasty, mouldy/musty, methyl ketone/blue, oxidised, waxy/crayon, faecal, bell pepper, rosy/floral, scorched, bitter, salty, sweet, sour, umami, prickle/bite Texture: Elasticity (evaluated by hand), hardness, cohesiveness, elasticity, adhesiveness between teeth, adhesiveness to teeth, cohesiveness of the mass (mass evaluated after 3–5 chews), adhesiveness of the mass, smoothness of the mass, smoothness of film (evaluated after swallow) Flavour: Buttery, cooked milk, dairy fat, dairy sour, dairy sweet, animalic, butyric acid, decaying animal, fresh fish, fish oil, goaty, sweaty, waxy, fermented/fruity/winey, nutty, pinapple, sauerkraut, smokey, soy sauce, mouldy, mushroom, astringent, biting, pungent, sharp, bitter, salty, sour, sweet

Texture: Creaminess, crumbliness, firmness, hardness, springiness, graininess Visual texture: Grain uniformity, grain size, number round openings, gloss of openings, number cracks, fracturability Manual texture: Crumbly grain, elasticity, fracturability, resistance to cut, resistance to press, ball hardness, ball fracturability, ball cohesiveness Oral texture: Hardness, fracturability, cohesiveness, roughness, water absorption, cohesiveness of mass, adhesiveness to teeth, crystals Aroma: Total intensity, sweet, sour, lipolysis, milky-creamy Flavour: Total intensity, cheese, salty, sweet, bitter, acid, lipolysis, milky-creamy, tongue-tingling, hot, residual intensity continued

463

464

Table 3 continued References and cheeses studied

Descriptive vocabularies

Lawlor et al., 2001, 2002, 2003 Appenzeller, Ambassedeur, Bleu d’Auvergne, Blue Shropshire, Blue Stilton, Cambozola, Cashel Blue, Chaumes, Danish Blue, Dubliner, Emmental, Fontina, Gabriel, Gruyère, Huntsman, Mahón, Old Amsterdam, Raclette, Tête de Moine, Tetilla, Wensleydale Madsen and Ardö, 2001 Danbo cheese

Odour: Pungent, caramel, mushroom, silage, sweaty/sour, fruity, mouldy, Cheddar dairy-sweet, sweet, creamy Flavour: Buttery, caramel, dairy sweet, rancid, mushroom, oily, mouldy, nutty, smoky, soapy, silage, processed, sweet, salty, acidic, bitter, pepper, burnt-aftertaste, strength, balanced Appearance: Colour intensity, crumbly, mottling, mouldy, softness, openness, shiny Texture: Firmness, rubbery, crumbly, smooth, moist, oily, chewy, slimy, grainy, mouth-coating

McEwan et al., 1989 Cheddar Muir and Hunter, 1992a,b,c Banks et al., 1993 Muir and Banks, 1993 Muir et al., 1995a,b,c,d Muir et al., 1996 Muir et al., 1997a,b Cheddar, Farmhouse Cheddar

Texture: Elasticity, firmness, deformability, friability, adhesivity

Odour: Strength, creamy/milky, sour, rindy, manure Flavour: Creamy/milky, strength, sour, manure, salty, acid, smoky, rindy Texture/mouthfeel: Tongue tingling, soft-firm, rubbery, mouth-coating, graininess Odour: Intensity, creamy, sulphur, fruity, nutty, rancid, other Flavour: Cheddar intensity/overall intensity, creamy/milky, sour/acid, sulphur/eggy, fruity/sweet, nutty, rancid, bitter, cowy, unclean/manurial, salty, other Texture: Firmness, rubbery, crumbly, pasty, grainy, mouth-coating

Murray and Delahunty, 2000a,b,c Delahunty and Murray, 1997 Bogue et al., 1999 Fenelon et al., 2000 O’Riordan and Delahunty, 2003b Hannon et al., 2003 Irish farmhouse and Cheddar cheese

Aroma: Pungent, caramel, sweaty/sour, sweet, creamy, fruity Flavour : Pungent, caramel, sweaty, creamy, fruity, buttery, rancid, cheddary, mushroom, mouldy, nutty, smoky, soapy processed, sweet, salty, acidic, bitter, astringent, strength, balanced Appearance: Colour intensity, mottled, uniformity, open, shiny Texture: Firm, rubbery, crumbly, smooth, moist, grainy, mouth-coating

Neilsen and Zannoni , 1998 Hunter and McEwan, 1998 Caerphilly, Cheddar, Comté, Danbo, Edam, Emmental, Fontina, Gouda, Jarlsberg, Parmigiano-Reggiano, Sbrinz, Svenbo

Smell: Strength/intensity, creamy, yoghurt, fruity/citrus fruit/other fruit/nutty, grass, animal/cowshed, caramel, acid/sour, ammonia, hay/grass Aroma/taste: Strength/intensity, creamy/yoghurt, grass, fruity/citrus fruit/other fruit/nutty, animal/cowshed, toasted/caramel, sour, pungent, ammonia, sweet, salty, acid, bitter Texture: Rubbery/elastic, hardness/firmness, crumbly/crumbliness, coating/adhesiveness, dryness, melting/solubility, grainy Odour: pungent, acid, sweet, characteristic, others Taste: pungent, acid, sweet, salty, bitter, characteristic, others Aftertaste: pungent, acid, bitter, others, persistent Texture: elasticity, creaminess, firmness, grainy, others Appearance: paste colour (internal), eyes (internal), shape (external), rind (external)

Ordoˇnez et al., 1998 Idiazábal cheese (ewes’ milk cheese)

Papademas and Robinson, 2001 Halloumi Piggott and Mowat, 1991 Delahunty et al., 1996a,b Jack et al., 1994 Cheddar Roberts and Vickers, 1994 Cheddar

Stampanoni, 1994 Cheese flavours: Cheese general, fresh cheese, soft cheese, hard cheese, goat/sheep cheese Wendin et al., 2000 Cream cheese

Taste and flavour: Salty, bitter, acidity, creamy, milky, minty, intensity Texture: Springy, moist, chewy, crumbly Appearance: Colour, body Appearance: White to orange Flavour: Milky, buttery, cheesy, mouldy, rancid, pungent, sour (aroma), sweet (aroma), salty (taste), sour (taste), bitter (taste), processed, strength, maturity, aftertaste Texture: Dry, hard/soft to firm, coarse, creamy, moist, smooth, sticky, grainy, crumbly, rubbery, chewy, pasty, mouth-coating Aroma: Buttery, fatty, fruity, fermented, mouldy, nutty, sweaty/sour, pungent, rancid, smoky, spoiled dairy, vinegary Flavour: Acid, acid bite, astringent, barny, bitter, buttery, cardboard, chemical, fatty, fruity, metallic, milky, mouldy, peppery, sweaty/sour, rancid, salty, sharp, smoky, soapy, diacetyl (yoghurt), sweet Aftertaste: Acid, bitter, milky, smoky, fishy, mouldy, peppery, soapy, sweaty/sour Texture: Chalk, chewy, creamy, crumbly, firm, grainy, moist, greasy, pasty, squeaky, waxy Flavour: Milky, cooked milk, fatty, buttery, creamy, nutty, butter milk, yoghurt, cottage cheese, caseinate, whey, soapy, fermented, mushroom, earthy, musty, spicy-pungent, blue, ammonia, green-grass, cheese rind, propionic acid, capric acid, butyric acid, fruity, sweaty, animal

Appearance: Yellow colour, granularity, watery, compact Flavour/taste: Sourness, butter, saltiness Texture/mouthfeel: granularity, fat/creamy Texture by hand: Spreadability

465


the perception of other characteristics that, general experience has taught us, are related (even if they may not be physically related). For example, many consumers believe that a coloured cheese is more intensely flavoured than its uncoloured equivalent (Bogue et al., 1999). Cheese texture

Texture can be defined as the attribute of a cheese resulting from a combination of physical properties, including size, shape, number, nature and conformation of the constituent structural elements, that are perceived by a combination of the senses of touch (tactile texture), vision (visual texture) and hearing (auditory texture). For example, the ‘softness’ of a cream cheese can be assessed visually upon cutting the cheese, by proprioceptive sensations when spreading the cheese, and finally by tactile sensations in the mouth during consumption. During mastication and consumption, texture perception occurs in the superficial structures of the mouth, around the roots of the teeth and in the muscles and tendons. Cheese texture characteristics frequently described include firmness, rubberiness, crumbliness, graininess, cohesiveness and adhesiveness (Tables 1 and 3). Cheese flavour

Flavour is most often defined as the integrated perception of olfactory, taste and chemesthesis (or trigeminal) stimuli. Flavour perception begins prior to consumption when a consumer can smell a cheese, but is finally perceived during consumption when compounds that stimulate the olfactory system in the nose, the taste system in the mouth and the trigeminal system in the mouth and nose are released from the cheese and become available to receptors. A large number of flavour characteristics have been described in cheese. Some that have been defined and standardised for application in descriptive sensory evaluations are listed in Table 2. Smell or aroma is usually the first aspect of flavour encountered by a consumer. The stimuli for smell are air-borne compounds of volatile substances that allow them to travel from their source to the olfactory receptors, where perceptions are created that are endowed with distinctive smells. Volatile stimuli are released from cheese into the air, and may be delivered to the nose orthonasally, often consciously, by sniffing (e.g., when one opens a cheese package or removes a trier from the cheese for evaluation). Volatile compounds may also be released into the buccal cavity air during consumption, where they are delivered to the nose retronasally without any conscious effort. Many hundreds of different volatile

compounds, each with a distinctive aroma character, have been identified in cheese, and these provide the largest contribution to the diversity of cheese flavours. Compounds identified in cheeses include fatty acids, methyl, ethyl and higher esters, methyl ketones, aliphatic and aromatic hydrocarbons, short- and longchain alcohols, aromatic alcohols, aldehydes, amines, amides, phenols and sulphur compounds (Maarse and Visscher, 1996). Much of what we commonly refer to as ‘taste’ is incorrectly localised smell detection. The significant contribution of aroma to flavour can be easily demonstrated if one pinches the nose shut whilst eating, effectively blocking air circulation through the nasal passages. Then, a familiar cheese, e.g., Cheddar, will not be recognised, and can easily be confused with one that would otherwise be easily distinguished, e.g., Gruyère. Taste is another aspect of flavour. Tasting occurs in the oral cavity, primarily on the tongue, but also on the soft palate. The primary stimuli for taste are nonvolatile compounds, and these must make contact with the taste receptors. This contact creates perceptions that endow four distinctive taste qualities, referred to as sweet, salty, sour and bitter. A fifth taste, ‘umami’, has been accepted more recently, particularly in Japan and other cultures where it is the most familiar and the most easily perceived. Compounds that contribute directly to cheese taste include lactic acid (sour), sodium chloride (salty), mineral salts of potassium, calcium and magnesium (salty) and free amino acids and peptides of varying types (sweet, bitter, umami) (Warmke et al., 1996; Engel et al., 2000). The last aspect of flavour is chemesthesis. This term is used to describe the sensory system responsible for detecting chemical irritants. Detection is more general than that of taste and smell and occurs primarily in the eyes, nose and mouth. The perception is closely related to the somato-sensory characteristics of pain and temperature change, and provokes a strong behavioural response. The fizz of carbon dioxide (CO2), the cooling sensation of menthol and the burning sensation of chilli are perhaps the best examples of how chemical irritation can provide additional character that is very much desired in a wide range of food products. With regard to cheese, the pungency, the prickle/bite and the sharpness of mature Cheddar are examples of perceived chemical irritation (Table 2). Sensory interactions

Cross-modal sensory interactions also occur, adding complexity to the perception and description of sensory character. Consumers rarely distinguish between stimuli of different sensory modalities (unless trained to do so), and generally describe the integrated sensation as ‘taste’.


However, the factors that cause apparent cross-modal sensory interactions are not always the same and can be difficult to comprehend. A first cause of apparent sensory interactions when perceptible components of a cheese are studied together can be interactions between the components of the cheese prior to introduction to the senses. For example, changing the fat content or salt content of a cheese can influence the physical chemistry of the cheese matrix dramatically, changing the partition coefficients of volatile compounds, and therefore releasing volatiles from the cheese matrix (Delahunty and Piggott, 1995). As a cheese matures, the protein composition changes significantly due to proteolysis, and this may change the binding ability of the cheese for specific volatile compounds (Delahunty and Piggott, 1995). A second cause of sensory interaction is termed a halo effect, and is caused by learning to place greater reliance on one sensory modality over another to make behavioural decisions. This effect was referred to in the context of appearance, as it is most obvious by the dominance, or bias, of the visual sense over the taste or olfactory sense. It can be demonstrated by confusing familiar colour and flavour combinations, or by varying colour intensity beyond expectation (Clydesdale, 1993). With regard to cheese, an influence of added colour on consumers’ perception of flavour has been reported (Bogue et al., 1999). A true cross-modal sensory interaction is one where the function of one sense (e.g., threshold measures, concentration-response functions) is changed by stimulation of another sense. This type of interaction can occur at receptor level, where one component blocks access to the receptor by another (e.g., increasing viscosity may coat the tongue and reduce access of tastants to taste receptors (Lynch et al., 1993)), or where stimulation by both components results in neural convergence as receptor sites are in close proximity and are served by the same nerve (e.g., capsaicin desensitisation reduces perceived taste intensity (Karrer and Bartoshuk, 1995)). The extent of these types of interactions in cheese, and their effect, is not known. Taste–aroma interactions are also observed and appear to be true interactions even though the physiology of the senses of olfaction and taste is independent. In this case, interaction is believed to occur centrally at a cognitive level where stimulus integration takes place (Stevenson et al., 1999). Taste–odour interactions have been observed in many different types of food and are easily demonstrated in model food studies (Noble, 1996). When volatile compounds are introduced to the oral cavity in the absence of taste-active compounds, they are generally perceived to be of low intensity and are described as bland in character. In cheese, it is most likely that the flavour impact of specific volatile compounds will be pronounced (and become familiar) only

when perceived in combination with appropriate tasteactive compounds, such as lactic acid, mineral salts, free amino acids and bitter peptides typically present in cheese (Frank and Byram, 1988). In addition, variations in taste quality and intensity, for example an increase in sourness (i.e., acidity), or an increase in bitterness, will affect how aroma is perceived (although volatile composition may be unchanged) and give the impression that overall flavour has changed considerably. Flavour–texture interactions are also observed widely. The precise nature of many of these interactions is not known, although structural components, such as proteins, can bind volatile compounds; rheology and structure can also influence mass transfer of non-volatile and volatile compounds to the surface of a cheese bolus where they will be released and become available for perception; fat can coat the receptor surface of the tongue, effectively blocking taste transduction (Lynch et al., 1993) and finally, interactions may occur at a cognitive level during perception integration in a way similar to taste–odour interactions (Weel et al., 2002). Texture–flavour interactions can also be influenced by individual consumer physiology, such as mastication behaviour and saliva flow rate and volume.

Sensory Methods Used to Evaluate Cheese Many reported studies on cheesemaking, cheese composition and cheese microbiology had the objective of controlling or improving sensory characteristics such as appearance, flavour and texture. However, it is difficult to compare the success of these studies as the final sensory character was often measured inappropriately. In many studies, judgements of overall sensory quality (i.e., a grade of ‘good’ or ‘bad’), rather than objective measurements of the perceived intensity of specific sensory characteristics, were carried out to determine the influence of cheese composition, counts of micro-organisms, or control of a cheesemaking variable on flavour or texture quality. Although standard procedures may be followed, e.g., International Dairy Federation standards (IDF, 1997), quality judgements are biased by the individual(s) who makes them. In addition, and of greater importance, traditional quality judgements do not allow the application of statistical analyses that would enable relationships between cheese study variables and specific sensory characteristics to be determined. The unaware reader of the literature can very easily confuse measurements of overall sensory quality with descriptions of sensory difference, as it is often reported, for example, that a specific cheesemaking procedure produced cheeses that ‘tasted’ similar, when in fact they were judged to be of similar quality (i.e., had no defects). Cheeses judged to be of similar quality by the same judge may differ


significantly in sensory characteristics (Delahunty and Murray, 1997). The American Society for Testing and Materials (ASTM) Committee E-18 on Sensory Evaluation of Materials and Products has defined sensory evaluation as ‘a scientific discipline used to evoke, measure, analyse and interpret reactions to the characteristics of foods and materials as they are perceived by the senses of sight, taste, touch and hearing’. A key distinction between sensory evaluation and other chemical and instrumental analytical techniques, is that different techniques can be used to evoke, measure and interpret sensory characteristics that have very different objectives and outcomes. Sensory evaluation can be carried out to determine whether cheeses exhibit defects or other undesirable characteristics, whether a difference in overall sensory character can be detected between two or more cheeses, whether specific differences in sensory characteristics can be perceived, to quantify the intensity of one or more sensory characteristics, to quantify the onset, maximum intensity and decline of a sensory characteristic, and to determine whether consumers find the cheeses to be acceptable or not, based on their sensory characteristics. The distinctions in sensory evaluation methodology can be broadly classified as quality scoring, discrimination testing, descriptive testing, time–intensity testing and consumer acceptability testing, respectively. There are some excellent texts that outline sensory tests in detail (Piggott, 1988; Stone and Sidel, 1993; Lawless and Heymann, 1998; Meilgaard et al., 1999). Grading and quality scoring

The manufacture of cheese of consistent quality is extremely difficult due to the number of production factors that ultimately contribute to eating quality (see ‘Factors that Affect the Quality of Cheese’, Volume 1). In addition, cheeses are susceptible to a large number of defects that can originate in milk, transfer to the cheese curd during making and storage, result from microbial contamination or develop during maturation if the composition at manufacture is not controlled. However, to maintain consumer confidence and loyalty towards a cheese, it is very important to control its quality. In addition, as consumers are becoming more brand-conscious, they become less-accepting of variations in sensory characteristics that traditionally would not be considered defects, and expect to find a cheese with near-identical appearance, flavour and texture in the package each time. To test instrumentally for all possible flavours and structural properties that contribute to eating quality would be an extremely laborious task, and may not achieve success. For example, many compounds that contribute to flavour are present

in concentrations below the detection limit of even the most sophisticated instruments. Quality scoring, grading or judging against specified defects on standardised scorecards (Bodyfelt et al., 1988) is the traditional and still most widely used type of formal sensory evaluation in the cheese industry. Cheese grading is carried out to classify the potential of a cheese to develop a satisfactory character during maturation, and to maintain quality at the point of sale. Grading standards generally specify a scoring system, where top grade is awarded a maximum score, and points are deducted when defects are found. For example, the IDF provides standard scorecards for cheese, and specifies a scale that ranges from 5, representing the highest possible quality, to 0, representing the lowest possible quality (IDF, 1997). Each point deducted from the scale is supported by a list of defects that merit the deduction. The defect list that accompanies each score on the scale aims to provide objectivity to the evaluation. The US cheese grading system and the American Dairy Science Association (ADSA) cheese-judging ballot operate in a similar manner (Bodyfelt et al., 1988). Tables 4 and 5 show the United States Department of Agriculture (USDA) standards for grades of Cheddar cheese, effective since 1956, which provide guidelines for the award of four grades – AA, A, B or C. Table 6 shows the ballot used by the ADSA to judge Cheddar cheese quality. McBride and Muir (1999) recently reviewed grading systems used for Cheddar cheese in Australia, United Kingdom, United States, Canada, the IDF and New Zealand. In addition, chapters in recent textbooks by Kosikowski and Mistry (1997) and Robinson and Wilbey (1998) review in detail methods of cheese grading and defects found in cheese. Kosikowski and Mistry (1997) described the sequence of cheese quality judgement. One or more expert evaluators, who have detailed product knowledge built up over many years and maintain a standard in memory of what the ideal product is in terms of sensory characteristics, carry out this evaluation. These experts have the ability to relate their recognition of specific defects to the cause of that defect and to weight the influence of each defect at different levels of severity and how they detract from overall product quality. The overall exterior of a cheese is first judged to determine if it appears deformed or soiled in any way. The rind or surface is judged next as it may be discoloured, cracked or irregular. Internal appearance is judged following cutting, or directly from a cheese trier, as it may have holes, cracks, spots or other opening defects, and colour may be uneven, mottled or dull. Odour, which may be uncharacteristic in many ways, is judged prior to placing a cheese in the mouth,


Table 4 Specifications for Grade AA and Grade A Cheddar cheese (United States Department of Agriculture, Agricultural Marketing Service, Dairy Division) Detailed specifications for US Grade AA Fresh or current

Medium cured

Cured or aged

(a) Flavour : Fine and highly pleasing. May be lacking in flavour development or may possess slight characteristic Cheddar cheese flavour. May possess a very slight feed flavour, but shall be free from any undesirable flavours and odours.

Fine and highly pleasing. Possesses a moderate degree of characteristic Cheddar cheese flavour. May possess a very slight feed flavour but shall be free from any undesirable flavours and odours.

Fine and highly pleasing characteristic Cheddar cheese flavour showing moderate to well-developed degrees of flavour or sharpness. May possess a very slight feed flavour but shall be free from any undesirable flavours and odours.

A plug drawn from the cheese shall be firm, appear smooth, waxy, compact, close, flexible and translucent, but may have a few mechanical openings if not large and connecting. May be slightly or not entirely broken down. May possess not more than one sweet hole per plug but shall be free from other gas holes.

A plug drawn from the cheese shall be firm, appear smooth, waxy, compact, close, and translucent but may have a few mechanical openings if not large and connecting. Should be free from curdiness and possess a cohesive velvet-like texture. May possess not more than one sweet hole per plug but shall be free from other gas holes.

Shall have a uniform, bright attractive appearence; practically free from white lines or seams. May be coloured or uncoloured, but if coloured it should be medium yellow-orange.

Shall have a uniform, bright attractive appearance; practically free from white lines or seams. May show numerous tiny white specks. May be coloured or uncoloured, but if coloured it should be medium yellow-orange.

Bandaged and paraffin dipped. Shall possess a sound, firm rind with a smooth bandage and paraffin coating adhering tightly but may possess very slight mould under bandage and paraffin, and the following other characteristics to a slight degree: Soiled surface and surface mould. The cheese shall be even and uniform in shape. Rindless. Same as for current, except very slight mould under wrapper or covering permitted.

Bandaged and paraffin dipped. Shall possess a sound, firm rind with a smooth bandage and paraffin coating adhering tightly but may possess the following characteristics to a slight degree: Soiled surface and mould under bandage and paraffin; and surface mould to a definite degree. The cheese shall be even and uniform in shape. Rindless. Same as for medium.

(b) Body and texture: A plug drawn from the cheese shall be firm, appear smooth, compact, close and should be slightly translucent, but may have a few small mechanical openings. The texture may be definitely curdy or may be partially broken down if more than 3 weeks old. Shall be free from sweet holes, yeast holes and gas holes of any kind. (c) Colour : Shall have a uniform, bright attractive appearance; practically free from white lines or seams. May be coloured or uncoloured but if coloured it should be a medium yellow-orange.

(d) Finish and appearance: Bandaged and paraffin-dipped. Shall possess a sound, firm rind with a smooth bandage and paraffin coating adhering tightly but may possess soiled surface to a very slight degree. The cheese shall be even and uniform in shape. Rindless. The wrapper or covering shall be practically smooth, properly sealed with adequate overlapping at the seams or by any other satisfactory type of closure. The wrapper or covering shall be neat and adequately and securely envelop the cheese. May be slightly wrinkled but shall be of such character as to protect fully the surface of the cheese and not detract from its initial quality. Shall be free from mould under wrapper or covering and shall not be huffed or lopsided.

continued


Table 4 continued Detailed specifications for US Grade A Fresh or current

Medium cured

Cured or aged

(a) Flavour: Shall possess a pleasing flavour. May be lacking in flavour development or may possess slight characteristic Cheddar cheese flavour. May possess very slight acid, slight feed but shall not possess any undesirable flavours and odours.

Shall possess a pleasing characteristic Cheddar cheese flavour and aroma. May possess a very slight bitter flavour and the following flavours to a slight degree: Feed and acid.

Shall possess a pleasing characteristic Cheddar cheese flavour and aroma with moderate to well-developed degrees of flavour or sharpness. May possess the following flavours to a slight degree: Bitter, feed and acid.

A plug drawn from the cheese shall be reasonably firm, appear reasonably smooth, waxy, fairly close and translucent but may have a few mechanical openings if not large and connecting. May be slightly curdy or not entirely broken down. May possess not more than two sweet holes per plug but shall be free from other gas holes. May possess the following other characteristics to a slight degree: Mealy, short and weak.

A plug drawn from the cheese should be fairly firm, appear smooth, waxy, fairly close and translucent but may have a few mechanical openings. Should be free from curdiness. May possess not more than two sweet holes per plug but shall be free from other gas holes. May possess the following other characteristics to a slight degree: Crumbly, mealy, short, weak and pasty.

Shall have a uniform, bright attractive appearance. May have slight white lines or seams. May be coloured or uncoloured but if coloured, it should be a medium yellow-orange.

Shall have a uniform, bright attractive appearance. May have slight white lines or seams and numerous tiny white it should be a medium specks. May be coloured or uncoloured, but if coloured, it should be a medium yellow-orange.

Bandaged and paraffin dipped. Shall possess a sound, firm rind with the bandage and paraffin coating adhering tightly but may possess very slight mould under bandage and paraffin and the following other characteristics to a slight degree: Soiled surface, surface mould, rough surface, irregular bandaging, lopsided and high edges. Rindless. Same as for current, except very slight mould under wrapper or covering permitted.

Bandaged and paraffin dipped. Shall possess a sound, firm rind with the bandage and paraffin coating adhering tightly but may possess the following characteristics to a slight degree: Soiled surface, rough surface, mould under bandage and paraffin, irregular bandaging, lopsided and high edges; and surface mould to a definite degree. Rindless. Same as for medium.

(b) Body and texture: A plug drawn from the cheese shall be firm, appear smooth, compact, close and should be slightly translucent but may have a few mechanical openings if not large and connecting. May possess not more than two sweet holes per plug but shall be free from other gas holes. May be definitely curdy or partially broken down if more than 3 weeks old. (c) Colour: Shall have a fairly uniform, bright attractive appearance. May have slight white lines or seams or be very slightly wavy. May be coloured or uncoloured but if coloured, it should be a medium yellow-orange. (d) Finish and appearance: Bandaged and paraffin dipped. Shall possess a sound, firm rind with the bandage and paraffin coating adhering tightly, but may possess the following characteristics to a very slight degree: Soiled surface and surface mould; and to a slight degree: Rough surface, irregular bandaging, lopsided and high edges. Rindless. The wrapper or covering shall be practically smooth, properly sealed with adequate overlapping at the seams or by any other satisfactory type of closure. The wrapper or covering shall be neat and adequately and securely envelop the cheese. May be slightly wrinkled but shall be of such character as to fully protect the surface of the cheese and not detract from its initial quality. Shall be free from mould under the wrapper or covering and shall not be huffed but may be slightly lopsided.


Table 5 Specifications for Grade B and Grade C Cheddar cheese (United States Department of Agriculture, Agricultural Marketing Service, Dairy Division) Detailed specifications for US Grade B Fresh or current

Medium cured

(a) Flavour: Should possess a fairly pleasing Should possess a fairly pleasing charactercharacteristic Cheddar cheese flavour, but istic Cheddar cheese flavour and aroma. may possess very slight onion and the May possess very slight onion and the following flavours to a slight degree: Acid, following flavours to a slight degree: Flat, flat, bitter, fruity, utensil, whey-taint, yeasty, yeasty, malty, old milk, weedy, barny and malty, old milk, weedy, barny and lipase; lipase; the following to a definite degree: feed flavour to a definite degree. Feed, acid, bitter, fruity, utensil, and whey-taint.

(b) Body and texture: A plug drawn from the cheese may possess the following characteristics to a slight degree: Coarse, short, mealy, weak, pasty, crumbly, gassy, slitty and corky; the following to a definite degree: Curdy open, and sweet holes.

A plug drawn from the cheese may possess the following characteristics to a slight degree: Curdy, coarse, gassy, slitty, and corky; the following to a definite degree: Open, short, mealy, weak, pasty, crumbly, and sweet holes.

(c) Colour : May possess the following characteristics May possess a very slight bleached to a slight degree: Wavy, acid-cut, mottled, surface; and the following characteristics salt spots, dull or faded; and definitely to a slight degree: Wavy, acid-cut, seamy. May be coloured or uncoloured but mottled, salt spots, dull or faded and if coloured, may be slightly unnatural definitely seamy. May be coloured or uncoloured but if coloured, may be slightly unnatural. (d) Finish and appearance: Bandaged and paraffin dipped. Shall possess a reasonably firm sound rind, but may possess very slight mould under bandage and paraffin. The following characteristics to a slight degree: Soiled surface, surface mould, defective coating, checked rind, huffed, weak rind, and sour rind; and to a definite degree: Rough surface, irregular bandaging, lopsided and high edges.

Rindless. The wrapper or covering shall be fairly smooth and properly sealed with adequate overlapping at the seams or by other satisfactory type of closure. The wrapper or covering shall be fairly neat and adequately and securely envelop the cheese. May be definitely wrinkled but shall be of such character as to protect the surface of the cheese and not detract from its initial quality. Shall be free from mould under wrapper or covering but may be slightly huffed and slightly lopsided.

Bandaged and paraffin dipped. Shall possess a reasonably firm sound rind, but may possess the following characteristics to a slight degree: Surface mould, mould under bandage and paraffin, checked rind, huffed, weak rind, and sour rind; the following to a definite degree: Soiled surface, rough surface, irregular bandaging, lopsided, high edges and defective coating.

Rindless. Same as for current, except slight mould underwrapper or covering permitted.

Cured or aged

Should possess a fairly pleasing characteristic Cheddar cheese flavour and aroma, with moderate to well-developed degrees of flavour or sharpness. May possess very slight onion and the following flavours to a slight degree: Flat, yeasty, malty, old milk, weedy, barny, lipase and sulfide; the following to a definite degree: Feed, acid, bitter, fruity, utensil, and whey-taint. A plug drawn from the cheese may possess the following characteristics to a slight degree: Gassy, slitty, the following to a definite degree: Open, sweet holes, short, mealy, weak, pasty and crumbly. May possess the following characteristics to a slight degree: Wavy, acid-cut, mottled, salt spots, dull or faded; and definitely seamy. May be coloured or uncoloured but if coloured, may be slightly unnatural. Bandaged and paraffin dipped. Shall possess a reasonably firm sound rind, but may possess the following characteristics to a slight degree: Checked rind, huffed, weak rind, and sour rind; the following to a definite degree: Soiled surface, surface mould, mould under bandage and paraffin, rough surface, irregular bandaging, lopsided, high edges and defective coating. Rindless. Same as for medium.

continued


Table 5 continued Detailed specifications for US Grade C Fresh or current

Medium cured

Cured or aged

(a) Flavour : May possess the following flavours to a slight degree: Sour, metallic, onion; and to a definite degree: Acid, flat, bitter, fruity, utensil, whey-taint, yeasty, malty, old milk, weedy, barny, and lipase; feed flavour to a pronounced degree.

May possess the following flavours to a slight degree: Onion and sulfide; and to a definite degree: Flat, sour, metallic, sour, metallic, yeasty, malty, old milk, weedy, barny and lipase; and to a pronounced degree: Feed, acid, bitter, fruity, utensil, and whey-taint.

May possess slight onion and the following flavours to a definite degree: Flat, yeasty, malty, old milk, weedy, barny, lipase and sulfide; and to a pronounced degree: Feed, acid, bitter, fruity, utensil and whey-taint.

(b) Body and texture: A plug drawn from the cheese may possess the following characteristics to a definite degree: Curdy, coarse, corky, crumbly, mealy, short, weak, pasty, gassy, slitty, pinny; and to a pronounced degree: Open and sweet holes. The cheese shall be sufficiently compact to permit the drawing of a plug.

A plug drawn from the cheese may be slightly curdy and may possess the following other characteristics to a definite degree: Coarse, corky, gassy, slitty and pinny; and to a pronounced to a pronounced degree: Open, sweet holes, short, weak, pasty, crumbly and mealy. The cheese shall be sufficiently compact to permit the drawing of a plug.

A plug drawn from the cheese may possess the following characteristics to a definite degree: Gassy, slitty, pinny; and to a pronounced degree: Open, sweet holes, short, weak, pasty, crumbly and mealy. The cheese shall be sufficiently compact to permit the drawing of a plug.

(c) Colour : May have a slight bleached surface and possess the following other characteristics to a definite degree: Wavy, acid-cut, mottled, salt spots, dull or faded; and seamy to a pronounced degree. May be coloured or uncoloured but if coloured, may be definitely unnatural. The colour shall not be particularly unattractive.

May possess the following characteristics to a definite degree: Wavy, acid-cut, mottled, salt spots, bleached surface, dull or faded; and seamy to a pronounced degree. May be coloured or uncoloured but if coloured may be definitely unnatural. The colour shall not be particularly unattractive.

Same as for medium.

Bandaged and paraffin dipped. May possess very slight rind rot and the following other characteristics to a slight degree: Cracks in rind; soft spots and wet rind; and to a definite degree: Surface mould, mould under bandage and paraffin, huffed; and to a pronounced degree: Checked rind, weak rind, sour rind and huffed; and to a pronounced degree: Soiled surface, rough surface, defective coating, irregular bandaging, lopsided and high edges. Rindless. Same as for current, except definite mould under the wrapper or covering permitted.

Bandaged and paraffin dipped. May possess the following characteristics to a slight degree: Rind rot, cracks in rind; and to a definite degree: Checked rind, weak rind, sour rind, wet rind, soft spots and huffed; and to a pronounced degree: Rough surface, soiled surface, surface mould, mould under bandage and paraffin, defective coating, irregular bandaging, lopsided and high edges. Rindless. Same as for medium.

(d) Finish and appearance: Bandaged and paraffin dipped. May possess the following characteristics to a slight degree: Cracks in rind, soft spots and wet rind; and mould under bandage and paraffin; and to a definite degree: Soiled surface, surface mould, defective coating, checked rind, weak rind, sour rind, and huffed; and to a pronounced degree: Rough surface, irregular bandaging, lopsided and high edges. Rindless. The wrapper or covering shall be fairly smooth and properly scaled with adequate overlapping at the seams or by other satisfactory type of closure. The wrapper or covering shall adequately and securely envelop the cheese. May be definitely soiled and wrinkled but shall be of such character as to protect the surface of the cheese and not detract from its initial quality. May have slight mould under the wrapper or covering and may be definitely huffed and lopsided.


Table 6 American Dairy Science Association ballot for judging the quality of Cheddar cheese. A score of 10 is awarded if the judge cannot find fault with the flavour of the cheese. A score of 5 is awarded if a judge cannot find fault with the body and texture of the cheese. When scores of 9 or less, or 5 or less, for flavour or body and texture, respectively, are awarded, the cause for deduction of marks should be indicated


and usually immediately upon opening a packed cheese, cutting a coated cheese or removing a trier plug from a cheese. Flavour judgement is made next, when a sample of cheese is placed in the mouth, chewed and moved around and then expectorated. As for odour, numerous uncharacteristic flavours may be detected in defective cheese, and in addition a cheese that is over-salty or very bitter may be considered defective. Finally, but sometimes simultaneously, body and texture are judged. Defects such as over-hardness, crumbliness, mealy and sticky are judged, most often by working a cheese between the thumb and the fingers. Table 7 presents a list of cheese sensory quality characteristics, which are mostly defects recognised internationally and are described in the IDF standard (IDF, 1997). It should be noted that a characteristic considered to be a defect in one cheese type may be very much desired in another (e.g., the acceptable hardness of Parmigiano-Reggiano would be considered a defect in Cheddar), and therefore judges must take this into account, and evaluate based on their experience of each cheese type individually. In addition, it may be that a characteristic found in the same cheese type produced in two different countries may be considered defective in one country, but acceptable in another. This will be related to the experience of the cheese consumer in each country, which can be very different. However, cheeses produced in automated facilities today are much less likely to suffer from significant defects due to improved hygiene practices at

all stages of milk handling and cheesemaking, beginning on the farm. In addition, control over cheesemaking has improved significantly in recent years. Cheese grading or quality scoring provides a rapid and simple way quickly to assess overall sensory quality, but does not adequately take into account so-called ‘non-quality’ related differences in sensory characteristics that give the cheese of individual producers, or regions of production, a distinctive taste. Traditional ‘quality criteria’ are changing as product ranges expand (e.g., to include low-fat cheeses); variety of cheeses is much greater, and differentiation is increasingly made by purposely developing distinctive sensory characteristics, such as those now given to cheeses by the use of adjunct cultures. Sensory characteristics that are not traditionally considered defects, but which can also differ from one cheese to another, are now also important in determining eating quality for the discerning consumer. What is a negative attribute to one consumer may be a desirable attribute to another consumer. Also, although the characteristics that expert judges seek are those that their market demands, their assessments do not always coincide with those of consumers. It is now well documented that the consumer and the expert opinions of quality often differ. For example, McBride and Hall (1979) found that consumers’ preferences among twelve cheeses, ranging from poor to good quality, were not correlated with their official grade scores. Finally, the current cheese-grading practice does not measure accurately the intensity of a given defect, and

Table 7 Terms used by cheese graders to describe sensory characteristics of cheeses that determine quality with particular emphasis on defects (IDF, 1997; Robinson and Wilbey, 1998) Exterior appearance Rind/surface

Appearance interior: Openings

Appearance interior: Colour

Consistency, body and texture

Flavour, odour and taste

Concave, convex, deformed (bulged), dirty, oblique, soiled, too flat, too high, vaulted (blown) Corroded, cracked, discoloured, dry, fatty, holes, incorrect mould, irregular mould, mould under covering, rotten, rough, smear under covering, smeary, speckled, spots of mould, thick, thin, too little mould, too little smear, too much smear, wet, wrinkled Blown, close, collapsed, cracks, distorted, foreign material, foreign mould, glossy openings, hoop side mould, many holes near the surface, nesty openings, no holes, not typical, pin-holed, spots of putrification, too few, too large, too many, too small, uneven, unevenly mouldy Bleached near the surface, bright, brownish, dirty, discoloured, grey, marbled, mottled, natural, pale/dull, red colour near the surface, speckled, streaky, strong, two-coloured, unevenly coloured, weak, yellow Brittle, chalky, close, coarse, crumbly, curdy, dripping, dry, elastic, firm, flaky, friable, gassy, granular (grainy), greasy, gritty, gummy, hard, harsh, hoop side sift, layered, leathery, long, lumpy, mealy, pasty, runny, rough, short, smeary, smooth, soft, soggy, spongy, springy, squeaky, sticky, stringy, thin (watery), tight, tough, uneven, wet Acid, alcoholic, ammoniacal, aromatic, bitter, bland, burnt, buttery, butyric acid, chemical, clove, cooked, cowy, creamy, ethereal, feedy, fermented, fishy, flat, flowery, foreign flavour, foul, foetid, fruity, garlic, goaty, harsh, malty, metallic, mild, mouldy, musty, musty-flat, nutty, off, oily, oniony, over-ripe, pale, peardrop, putrid, rancid, resinous, rich, ripe, sandy, salty, sharp, soapy, sour, spicy, stale, strong, superfine, sulphide, sweaty, sweet, tangy, tallowy, uncharacteristic, unclean, weedy, yeasty


therefore further statistical analyses that determine the extent to which cheeses differ, and that mathematically relate composition to defect intensity, are not appropriate. It is important to note that there are still industry situations where grading or quality scoring may be appropriate due to a large number of products that must be assessed in a short period of time. However, these sensory tools were not designed to be quantitative or representative of the entire cheese sensory profile and are not ideal tools for research or marketing. Discrimination tests

Sensory discrimination tests differ from quality scoring tests in that they involve direct comparisons of cheeses to determine whether there is either an overall difference between them or whether they differ for a specific and designated characteristic. The most commonly used discrimination tests include the Paired Comparison (ISO, 1983a), Duo-Trio (ISO, 1991), Triangle (ISO, 1983b) and Ranking tests (ISO, 1988). In the Paired Comparison test, two cheeses are presented for comparison with one another and assessors are asked whether they differ; generally, a difference for one specific sensory characteristic is tested. In the Duo-Trio test, assessors are asked which of the two products is the most similar to a third reference product, allowing a common reference to be used again and again. This test has obvious advantages for quality control, although it is not possible to maintain a consistent cheese reference over time. In the Triangle test, assessors are presented with three cheeses and asked to choose which is the most different from the other two. In the Ranking test, four to six cheeses are generally presented for comparison of a single-designated attribute, and the assessor is asked to rank them in order of increasing intensity of that attribute. In best practice, the assessors are forced to make a choice each time for all discrimination tests, thus eliminating response bias. Whether a difference exists or not is determined statistically, based on the number of choices a panel of assessors makes for each cheese in the test, using binomial tables or Chi-squared tests. Therefore, discrimination tests are the most objective and the most sensitive of sensory tests. An additional advantage of these tests is that they do not require well-trained assessors. The only requirement is that all assessors are reasonably sensitive and recognise and understand the designated attribute in a common way. When compared with the traditional quality scoring methods, these discrimination procedures are by far better suited to application to research problems, they follow good sensory evaluation principles and do not encounter problems in scaling and statistical analyses.

For this reason, their principles should now be added to quality scoring methods in an attempt to introduce comparability between the scores of one judge and another. It is also common practice to carry out discrimination tests on cheeses to determine whether a difference exists prior to further testing by more costly methods that aim to describe and quantify differences. Descriptive analyses

A majority of scientists who study cheese are interested in understanding the fundamental reasons why a cheese ‘tastes’ as it does, not just whether the cheese is acceptable, and for this purpose quality control sensory methods are of little value. Descriptive sensory analysis refers to a collection of techniques that seek not only to discriminate between the sensory characteristics of a range of cheeses, but also to determine a quantitative description of all the sensory differences that can be identified. For example, Figs 1a and 1b illustrate quantitative differences in perceived flavour, measured using descriptive analysis, between two hard Swiss cheeses and two Blue cheeses, respectively. All cheeses may be profiled in this way, providing objective and reproducible sensory descriptions of cheeses and providing a basis for determining what characteristics are influenced by changes in cheesemaking practice or composition, and also what characteristics are important for consumer acceptance. The most commonly used descriptive analysis methods for all food types include the Flavour Profile Method (Cairncross and Sjöstrom, 1950), Texture Profile Method (Brandt et al., 1963), Quantitative Descriptive Analysis (QDA)™ (Stone et al., 1974), the Spectrum™ method (Meilgaard et al., 1999), Quantitative Flavour Profiling (Stampanoni, 1993a,b), and Free-Choice Profiling (Langron, 1983; Thompson and MacFie, 1983). A review of descriptive sensory analysis, which details advantages, disadvantages and applications of each of the methods referred to above was published recently by Murray et al. (2001). Each descriptive method has three stages to its implementation. The first involves selecting a panel to conduct the sensory evaluations, the second, establishing terminology or a vocabulary, by which to describe a products’ sensory characteristics and the third, quantifying these sensory aspects. However, for each method, the process is somewhat different. In the cheese industry, as there is a strong tradition of judging that is linked to extensive knowledge of cheese, then it is a wise approach that seeks to build on this knowledge rather than to reinvent the wheel. If the investment in descriptive sensory testing is for the long term, then the Spectrum™ method, or a similar one, is preferable.

476

(a) Balanced Strength

Pungent 50

(b) Silage

45

Astringent

Pungent 80

Caramel odour

40

60

35

Burnt-aftertaste

Fruity

30 25

Pepper

Mushroom

Balanced

Sweaty/sour

Fruity

Strength 40

Dairy-sweet odour

20 15

Bitter

20

Sweet

10

Acidic

Mouldy odour

5 Acidic

Creamy

0

Salty

0

Buttery Salty

Sweet

Cheddar

Caramel flavour

Processed

Dairy-sweet flavour Processed

Silage

Sweet

Rancid Soapy

Mushroom Smoky

Oily Nutty

Appenzeller Gruyere

Mouldy flavour

Oily Blue Shropshire Danish Blue

Figure 1 Comparison of the flavour profiles of two Swiss cheeses, Appenzeller and Gruyère (1a) and two Blue cheeses, Blue Shropshire and Danish Blue (1b) (Lawlor et al., 2002, 2003). Flavour characteristics were measured using descriptive sensory analysis by a trained sensory panel.


Using this method, a group of cheese experts develop and define a descriptive language using a series of universal intensity scales upon which assessors score their perceptions (Drake et al., 2001; Drake and Civille, 2003). The sensory panels that will use the method, often at more than one research site, are then extensively trained. When trained, individual assessors must be able to discriminate between cheeses using each attribute in the descriptive language, repeat their assessments and agree with other panel members on the size and the direction of differences in cheese attributes. The advantages of this descriptive analysis technique are that one panel can be trained readily on several cheese types since one intensity scale is used, different types of cheese can be compared directly and panel scaling is less prone to drift with time (Drake and Civille, 2003). In addition, this approach is objective and allows comparison of results between panels, between laboratories, and from one time to another. For example, if one wishes to study the maturation of a cheese over time, then one must ensure that the differences observed in the results between 3, 6 and 9 months are related specifically to changes that occur in the cheese and not to changes in the performance of the sensory panel. If a cheese type is to be evaluated not very often, or the sensory panel available will not specialise in cheese only, or resources are limited, then the QDA™ approach may be preferable. Using this method, the panel of assessors develop and define the language themselves whilst tasting a wide range of the test cheeses (Murray and Delahunty, 2000b). Assessors must agree with other panel members on the meaning of terms in the descriptive vocabulary and repeat their assessments, but are not required to agree on how to use the attribute scales to rate intensity. When this method is used instead of the Spectrum™ method, it is more difficult to compare the results from one study with those from another in absolute terms. Free-Choice Profiling (FCP) is another useful descriptive analysis method (Williams and Langron, 1984). This method allows the use of untrained assessors, or consumers, to profile the sensory characteristics of cheese. Each assessor may use an individual descriptive vocabulary that they have developed themselves, and which they then readily understand, and data are analysed using Generalised Procrustes Analysis (GPA; Arnold and Williams, 1986). Free Choice Profiling has been used to describe Cheddar cheese (Jack et al., 1993; O’Riordan et al., 1998), Parmigiano-Reggiano (Parolari et al., 1994) and ewes’ milk cheeses (Bárcenas et al., 2003). The advantages of FCP are that accurate discrimination between cheeses in terms of perceived sensory characteristics can be achieved in a very short

time and at a relatively little cost, and that discrimination is based on a large selection of informative words that consumers use and with which they are familiar with. The main disadvantage is that it is difficult to correlate perceived intensity of sensory characteristics obtained in this way, as they are too numerous and imprecise, and there is no consensus vocabulary. To obtain improved accuracy, sensory panels used for descriptive analyses generally comprise of 10–12 assessors instead of a smaller number of experts (with the exception of FCP where 15–20 assessors are needed). These assessors are screened for sensory acuity and relative interest (Stone and Sidel, 1993). A panel or group of individuals is used as factors such as age, saliva flow and onset of fatigue vary between assessors. Assessors also vary in sensitivity to particular stimuli, and it is highly probable that they also vary in their concentration-response functions (Lawless et al., 1994; Williams, 1994). In addition, temporary illness or psychological bias can cause day-to-day changes in sensory ratings. The key point of objective descriptive analysis is that it should be reproducible and independent of consumer preferences. Unlike traditional quality methods that use scorecards, there is no judgment of ‘good’ or ‘bad’ as this is not the purpose of the evaluation. The trained sensory panel operates as an instrument and generates quantitative data analogous to instrumental data. As with any instrument, replication is required. Two guidelines have been published dealing with cheese texture (Lavanchy et al., 1994) and the aroma and flavour of cheese (Bérodier et al., 1997a). These guidelines are very valuable as they define descriptive vocabularies, and then detail a procedure for evaluation of each characteristic, including the use of universal scales that are standardised at a number of points with common food references. In addition, they provide translations of many descriptive characteristics of cheese in Spanish, French, Italian, English and German. However, it is important to note that sensory lexicons or languages are not finite and will continue to evolve with time, usage and application. Time–intensity sensory analyses

The sensory methods discussed above do not account for the dynamics of flavour release from cheeses that occurs during their consumption. Nor do they account adequately for changes to cheese texture, which occur progressively during mastication and breakdown of a cheese in the mouth. When using conventional sensory procedures, particularly descriptive analyses, assessors ‘time-average’ their responses to arrive at a single intensity value. This looses much useful information such as


rate of onset of stimulation, time and duration of maximum intensity, rate of decay of perceived intensity, time of extinction and total duration of the entire process (Lee and Pangborn, 1986). To determine most details about sensory characteristics, changes in sensory character that occur during cheese consumption (which can take up to 30 s for a ‘bite-sized’ piece) can be measured using time–intensity methodology (Lee and Pangborn, 1986), or in the case of texture, using progressive profiling (Jack et al., 1994). Time–intensity methods are useful for the study of new cheese types, such as low-fat cheeses, as the reduction in fat content not only influences sensory character development, but also the breakdown of the cheese in the mouth during consumption and the rate of release of compounds that contribute to flavour. For example, in a study of Cheddar cheese flavour, the time taken to reach maximum intensity for ‘sharpness’, ‘bitterness’ and ‘astringency’ was consistently longer in reduced-fat than in full-fat Cheddar and, more importantly, the rate of flavour release was greater (Shamil et al., 1991/92). Temporal differences in perception indicate an altered flavour balance, caused by reducing the fat content of the cheese, which may be important in consumer acceptability. Delahunty et al. (1996a) showed that a ‘fruity’ note, which might be considered an off-flavour (Aston et al., 1985; Urbach, 1993), became a dominant flavour characteristic sooner during consumption and at a much greater intensity in a low-fat Cheddar-type cheese than in the full-fat equivalent. Delahunty et al. (1996b) also demonstrated that improved relationships between volatile composition and perceived sensory characteristics could be achieved by relating time–intensity sensory data with dynamic volatile compound release data. Jack et al. (1994) found that the texture of Cheddar cheese was perceived to be relatively coarse and crumbly earlier in the chewing sequence, but became increasingly smooth and creamy as chewing progressed. In addition, other more subtle or specific cheese-dependent changes occurred as breakdown in the mouth progressed. It was hypothesised that knowledge of these dynamic changes in texture character is important for understanding consumer acceptability. Consumer acceptability testing

Trained sensory panels should not be asked to express a preference as their expert knowledge will introduce bias. To determine the eating quality of cheese, a naive consumer panel or subjective assessors are used. Ideally, these assessors will be regular consumers of the product type under test or represent the target market for the product. Such consumers bring their subjective experience to this test, for although their preferences

will be based on the sensory characteristics tested, they will be referring to past eating experience. In addition, when one considers that the target market may be children, elderly consumers, consumers in another country or consumers from a culture virtually unknown to the producer, then it becomes clear that the internal expertise in a company or organisation cannot hope to predict acceptability adequately. Consumer acceptability testing makes use of rating scales that measure relative dislikes and likes (e.g., the ninepoint hedonic scale (Peryam and Girardot, 1952)), discrimination tests based on preference (e.g., paired preference, ranked preference) or just right scales that ask a consumer how they feel about the designated sensory characteristic. It is recommended that a minimum of 50–60 targeted consumers be used for consumersensory testing, and a greater number than this if one expects segmentation of preferences (MacFie and Hedderly, 1993). One of the biggest challenges in consumer research is the clarification of consumer language. Consumers may use terms that are ambiguous, have multiple meanings, are associated with ‘good’ or ‘bad’ or are combinations of several terms. Integrated terms, such as ‘creamy’, are often used by consumers to represent a combination of positive attributes. Determining exactly what attribute or attributes ‘creamy’ refers to (flavour or texture or mouthfeel) have been the subject of many studies relating consumer and trained sensory panels (Mela, 1988; Elmore et al., 1999; Bom Frost et al., 2001). Dacremont and Vickers (1994a,b), who used concept matching to clarify consumer perception of Cheddar cheese flavour, found that the concept of Cheddar cheese flavour is a consumer concept and probably varies widely among consumers, as does Cheddar cheese flavour itself. However, the number of consumers questioned was small and further studies with larger consumer groups, and with demographic information, including types (brand, age) of Cheddar cheese normally consumed, would provide additional clarification.

Influence of Cheesemaking Variables on Sensory Character During the past ten years or so, there have been numerous reports of the application of descriptive sensory analysis to determine accurately the influence of cheesemaking variables, e.g., maturation time and temperature, starter culture or use of adjunct cultures, on the sensory characteristics of cheese (Table 3). Studies of Cheddar cheese maturation have found that, overall, the intensity of odour, flavour and aftertaste is determined by the length (Piggott and Mowat,


0.5

6 cheddary

balanced S2 39 mushroom S2

strength S3 PC2 (17%)

1991; Muir and Hunter, 1992a) and the temperature of maturation (Hannon et al., 2003). However, flavours such as milky/buttery and creamy decrease in intensity, while flavours such as sour, bitter, rancid and pungent increase in intensity (Piggott and Mowat, 1991; Muir and Hunter, 1992a; Hannon et al., 2003). Some textural changes, e.g., firmness, are controlled by the cheesemaking procedure and cheese composition, whereas mouth-coating character is related to maturation time (Piggott and Mowat, 1991; Muir and Hunter, 1992a). Hort and Le Grys (2001), who also studied Cheddar, found that springiness decreased, and crumbliness and creaminess increased as maturation progressed. Banks et al. (1993) and Fenelon et al. (2000) used descriptive analysis to determine the sensory properties of low-fat Cheddar cheese, and to compare these with the sensory properties of full-fat Cheddars. Fenelon et al. (2000) found that there were some differences in flavour characteristics related to fat content that were present regardless of the age of cheese. Fullfat cheeses were consistently more buttery, creamy and caramel-like. Adhikari et al. (2003) found that low-fat and full-fat Swiss cheeses, and low-fat Cheddar cheeses were dry and crumbly. Factory and farmhouse Cheddars have also been compared using descriptive sensory analysis (Muir et al., 1997a; Murray and Delahunty, 2000c); farmhouse cheeses were found to have a greater diversity in sensory characteristics. In addition, cheeses produced from pasteurised milk were found to be clearly different from those produced from unpasteurised milk, with the unpasteurised milk cheeses being more diverse in sensory character and more intensely flavoured (Grappin and Beuvier, 1997; Muir et al., 1997a; Murray and Delahunty, 2000c). Numerous studies have used descriptive sensory analysis to address the role of specific adjunct cultures or starter culture enzyme systems in Cheddar cheese flavour (Drake et al., 1996, 1997; Muir et al., 1996; Delahunty and Murray, 1997; Lynch et al., 1999; Banks et al., 2001; Broadbent et al., 2002). Muir et al. (1996) demonstrated that starter culture type and adjunct determined the sensory character of cheese. However, they also found direct and interactive effects of composition. More recently, O’Riordan and Delahunty (2003a,b) found that starter culture type led to consistent differences in sensory characteristics between Cheddar cheeses, but that composition led to significant variation within batches of cheese made using the same starter culture. Delahunty and Murray (1997) also demonstrated differences between Cheddar cheeses based on starter culture type, although these cheeses were awarded the same grade score (Fig. 2). Descriptive sensory analysis has been used to determine the impact of yeast extract and milk standardisation

–0.5 –6

S3 astringent39 S2 S339 S3 39S2 39 39 S339 S1 pungent 39 S3 acidic

39

39

S2

S2

0.5

39

39

S1

S3

39

S1

39

sweet S1

S2 38

6

38

39

S1 S137

processed

39

bitter salty

creamy

S1 rancid 37 mouldy

S1 37

buttery

–6

–0.5

PC1 (43%)

Figure 2 Two-dimensional representation of the result of Principal Components Analysis of descriptive analysis sensory data for Cheddar cheese produced using three different starter cultures (coded S1–S3). Grade scores for flavour, awarded by an expert cheese grader, are also illustrated close to each cheese code (range 37–39).

with milk protein concentrate on reduced-fat Cheddar cheese flavour (Shakeel-ur-Rehman et al., 2003a,b,c) and of smoking parameters on cheese flavour (Shakeelur-Rehman et al., 2003d). There have been many studies of cheese types other than Cheddar, and to discuss them all would be impossible within the scope of this chapter. Of most interest are studies of Comté cheese using a flavour-descriptive vocabulary developed by Bérodier et al. (1997b; published in French). This lexicon has been used to identify naturally existing cheese geo-regions within France (Monnet et al., 2000). In addition, Virgili et al. (1994) used descriptive analysis to study the sensory–chemical relationships in Parmigiano-Reggiano cheese. Descriptive analysis of cheese texture has been conducted recently on a variety of cheeses, on cheeses of different fat contents and on fat replacers (Drake and Swanson, 1996; Drake et al., 1999a; Lobato-Calleros et al., 2001; Madsen and Ardo, 2001; Gwartney et al., 2002). In these studies, descriptive sensory analysis was used to differentiate cheeses and/or the impact of various treatments. A sensory texture language, like a cheese flavour language, is also not necessarily finite. The language will continue to be refined, particularly as additional cheeses are studied or as additional instrumental studies are conducted. The texture languages used by Drake et al.


(1999a) and Gwartney et al. (2002) were merged into one complete language by Brown et al. (2003).

Towards a Universal Cheese-Sensory Language As mentioned previously, some of the key advantages of using descriptive sensory vocabularies with definitions and references are the ability to communicate accurately results between multiple research groups or to reproduce research results at different sites. Hirst et al. (1994) compared the evaluation of cheese between trained British and Norwegian panels using independently developed sensory languages. Cross-cultural differences were attributed to the observed discrepancies in term usage and sample differentiation. More recently, ring trials at seven sites across the European Union were conducted and a core sensory language for evaluation was developed (Hunter and McEwan, 1998; Nielsen and Zannoni, 1998). While similar patterns of differentiation among samples by panels that use different languages are expected (particularly if the vocabularies are comprehensive and the panellists highly trained), standardised language with definitions and references improves communication, cross panel validation and subsequent application of descriptive analysis results to instrumental or consumer data. Further, other sources of variation potentially exist in comparing panel results at different sites within the same country using the same language. Drake et al. (2002) reported on the performance of three descriptive panels trained at different sites by different panel leaders on a previously developed and standardised cheese descriptive language (Drake et al., 2001). Panels were able to communicate accurately attribute differences between cheeses. However, differences were observed between these panels in scale usage and attribute recognition. These differences were attributed to the differences in panel leadership and the duration of panellist training. In a similar study, Martin et al. (2000) compared odour profile results of two panels. Language, scale and method of presentation were standardised. Results obtained from the two panels were similar. However, differences between attribute intensities were reported and were attributed to differences in the experience and/or perception of individual panellists. As with the conclusions of Drake et al. (2002), strong panel–leader interaction was recommended as a means of rectifying these differences, along with regular feedbacks between the two panels. As referred to previously, Tables 1, 2 and 3 present terms used for descriptive sensory analysis by different research groups for a wide variety of cheeses. In many cases similar terms have been used to describe dominant

characteristics of different cheese types, suggesting that it could be possible to develop and standardise a terminology that can be used universally and for all cheese types. The will to achieve this objective is much needed.

Relating Sensory Characteristics to Consumer Preferences Preference mapping is a generic term given to a collection of techniques, which have emerged in recent years to quantify, analyse and interpret consumer preferences for products. A premise can be made that the preferences of a group of consumers of sufficient size (60 or more) will discriminate between comparable products based on intrinsic sensory differences, and that the degree and direction of discrimination will reflect the number and the intensity of sensory differences that can be perceived. Therefore, by simply quantifying and analysing preference, or acceptance for the range, a preference map reflecting sensory differences can be drawn. The preferences of individual consumers can be represented as a map loading, and areas of minimum and maximum preference can be identified. In addition, segmentation techniques, when used in tandem, can illustrate opportunities for a selection of optimised products within the same range (or sensory space). Analysis of consumer preference data in this way is referred to as internal preference mapping (McEwan, 1995; Schlich, 1995). When consumer preference evaluation of a set of cheeses is followed by the application of descriptive analysis to the same set of cheeses, this allows multivariate statistical analysis, e.g., using Partial Least Squares Regression (PLSR; Martens and Martens, 1986), and relation of descriptive properties that describe exactly what attributes are perceived and at what levels with the extent and direction of consumer preferences. This additional analysis facilitates interpretation of the internal preference map, and is referred to as external preference mapping (McEwan, 1995; Schlich, 1995). These techniques provide a powerful research tool for market analysis and new product development. One can extend the preference map by seeking technical extensions, or relationships between preferences, sensory characteristics and physical and chemical properties of products. One can also extend the preference map by seeking behavioural extensions, or by determining characteristics of the consumers and how they have developed their preferences and make their choice decisions. Preference mapping has been conducted with many products, including cheese (McEwan et al., 1989; Lawlor and Delahunty, 2000; Murray and Delahunty, 2000a,c; Bárcenas et al., 2001). Recently, Young et al.


(2003) conducted preference mapping of Cheddar cheeses using consumers at two different locations (Oregon and North Carolina, USA). Seven Cheddar cheeses with distinct descriptive sensory properties were selected. Six distinct consumer clusters were identified, indicating a wide variability in consumer preferences even among one cheese type. Analysis of the consumer concept of ‘aged cheese flavour’ and ‘young cheese flavour’ indicated that consumers could differentiate between young and aged Cheddar cheeses and that these concepts were consistent with descriptive panel language. However, the consumer concept of ‘Cheddar flavour’ varied widely and was not pinpointed to specific descriptive cheese flavour terms. Lawlor and Delahunty (2000) conducted preference mapping with a diverse range of cheese types, and also found wide variability in consumer preferences. Although a Blue Shropshire cheese, described as coloured, mouldy and crumbly, was the least liked overall (162 consumers), it was preferred by two of seven segments of the consumer sample, representing 50% of the total questioned. On the other hand, a Gruyère cheese, described as fruity, sweet and firm, was preferred overall, but was the first choice of only one segment with 10 consumers.

Relating Sensory Perception to Chemical Components and Instrumental Measurements Relating defined sensory flavour and/or texture to specific instrumental tests or chemical compounds is an important and expanding area of research. Cheese flavour chemistry and texture analyses are addressed in detail in ‘Cheese Flavour: Instrumental Techniques’ and ‘Rheology and Texture of Cheese’ of Volume 1, but sensory characteristics of cheeses cannot be addressed without brief attention to this subject. Relating sensory perception to instrumental measurements is important because in certain cases an instrumental test would be more cost-effective and/or convenient than sensory testing. However, more importantly, establishment of key relationships between sensory perception and flavour chemistry or rheology provides the potential to link cheese flavour or texture to the technology of cheese production; this is a key issue in providing a consistent and high-quality product to the discerning consumer. Relating sensory language and chemical volatile compounds represents a challenge for several reasons. The relative concentration of a compound in a cheese is not necessarily a measure of its sensory impact due to different sensory thresholds and the effects of the food matrix on retention and release. The sensitivity and selectivity of the extraction technique must also be taken into account (Delahunty and Piggott, 1995).

Finally, only a small percentage of the volatile components in a food are odour-active (Friedrich and Acree, 1998; see also ‘Cheese Flavour: Instrumental Techniques’, Volume 1). Establishing these relationships is time-consuming and tedious. To use flavour as an example, extensive and relevant instrumental volatile analysis must be conducted, followed by gas chromatography–olfactometry (GC–O) and quantitative analysis to pinpoint volatiles of interest. On the sensory side, descriptive analysis with a defined and anchored language is required. Sensory threshold testing to confirm that key volatile compounds are above detection thresholds must be conducted, followed by descriptive sensory analysis of compounds in model systems across the concentration range found in the cheese to confirm the sensory response (Drake and Civille, 2003). It should also be noted that the perception of the cheese flavour is an integrated response to numerous mixed stimuli, including volatile compounds, nonvolatile compounds and structural properties. The perception of this stimulation is multi-modal, but simultaneous, and therefore very complex. Panelists tasted water-soluble extracts of Comté cheese to identify fractions, which had particular tastes, in an attempt to clarify the effect of peptides and amino acids on flavour (Salles et al., 1995). Preininger et al. (1996) used an unripened cheese matrix to evaluate both volatile and non-volatile flavour components of two Swiss cheese samples. A similar study was conducted on Emmental cheese and reduced-fat Cheddar cheeses (Rychlik et al., 1997; Suriyaphan et al., 1999). Suriyaphan et al. (2001) identified key chemical volatile components of cowy/barny and earth/bell pepper sensory perceived flavours in selected aged British Farmhouse cheeses. In this study, sensory properties were identified by descriptive sensory analysis, aroma volatiles were quantified by gas chromatography–mass spectrometry (GC–MS) and aroma properties described by GC–O. Suspected key volatiles were selected from GC–O data based on aroma properties and flavour dilution values. The selected aroma components were subsequently incorporated into mild (bland) cheese across the concentration range encountered in the Farmhouse cheeses and evaluated by descriptive analysis. Studies such as these provide convincing evidence of the contribution of particular compounds to flavour. Model systems have not as yet provided insights into the role of compound mixtures and the role of compounds at sub-threshold levels. These are complex issues and will require extensive future research. An alternative approach to determining the influence of composition on sensory character is to use multivariate statistical techniques, such as PLS, to determine


relationships between compositional data and quantitative descriptive sensory data. This technique has the advantage of enabling comparison of all mathematically possible combinations of compositional variables with perceived intensity of one or more sensory characteristics, following theoretically the principle of the component balance theory (Mulder, 1952). The validity and value of relationships determined in this way will depend on the amount and type of compositional data collected, and the accuracy of both the compositional and the sensory data. Lawlor et al. (2001, 2002, 2003) determined predictive models using this technique for numerous flavour and texture attributes described in a wide variety of cheese types. Many studies have also been conducted to explore the relationships between sensory properties, compositional measurements and instrumental measurements of cheese texture (Wium et al., 1997; Bachmann et al., 1999; Drake et al., 1999b; Antoniou et al., 2000; Benedito et al., 2000; Truong et al., 2002) and to devise instrumental methods to assess more accurately or predict sensory properties of cheese (Sorensen and Jepsen, 1998; Breuil and Meullenet, 2001; Meullenet and Finney, 2002). Lawlor et al. (2001, 2002, 2003), using PLS, determined relationships between gross composition and perceived texture for a wide variety of cheeses, and found a number of consistent relationships. In particular, it was found that firmness was positively correlated with protein and mineral salt content, and negatively correlated with moisture and pH. Both hand and mouth terms can be used for sensory analysis of cheese texture (Drake et al., 1999c). In general, empirical texture tests and large-strain tests (compression) have been shown to correlate well with sensory bite terms (firmness, elasticity) although the correlation varies with cheese type, instrumental test and specific sensory term and definition. More recently, Brown et al. (2003) demonstrated specific knowledge gaps in relating sensory chewdown terms to rheological tests. Sensory rigidity and resiliency terms were correlated with rheological tests. However, chewdown terms such as ‘degree of breakdown’, ‘cohesiveness’, ‘adhesiveness’, ‘smoothness of mass’ and ‘smoothness of mouth coating’ were not related to instrumental tests. Additional work is needed to investigate the role that fundamental rheological tests can play in differentiating and relating to these important sensory texture parameters in cheese.

Conclusions The sensory characteristics of cheese determine the eating quality of cheese and consumer acceptability. The appearance, flavour and texture of cheese are extremely complex, not simply due to the very wide

diversity of cheese types that are produced, but also the many stages that any cheese goes through during its production and ripening. The complex composition and structure of cheese stimulate each of the human sensory modalities at approximately the same time, resulting in an integrated perception that a consumer responds to during and after cheese consumption. The dairy industry, including cheese production and marketing, has relied on outdated grading and judging methods for quality control and product development for many years. While these methods still have use, objective descriptive analysis techniques are increasingly being applied in cheese quality research in parallel with innovative studies of cheesemaking, cheese composition and consumer acceptability of cheese. Advances in the application of objective sensory science techniques have improved understanding of the relationships between these factors and the sensory attributes of cheese. However, direct comparison of research findings between different laboratories working with the same cheese type, and between studies on different types of cheese, will not be possible until such time as a universal language to describe cheese sensory character is defined and standardised.

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Cheese Flavour: Instrumental Techniques J.-L. Le Quéré, Institut National de la Recherche Agronomique (INRA), Unité Mixte de Recherche sur les Arômes (UMRA), Dijon, France

Introduction The sensory properties of food are important determinants in the choice of foodstuffs by the consumer, and flavour plays a prominent role in this context. Flavour may be defined as the combination of taste and odour, sensations of pain, heat and cold (chemesthesis or trigeminal sensitivity), and tactile sensation. Sensory analysis is clearly the most valid means of measuring flavour characteristics. Applied to cheese flavour, sensory evaluation is a prominent descriptive tool which is used widely in dairy science and industry (Issanchou et al., 1997; see also ‘Sensory Character of Cheese and its Evaluation’, Volume 1). However, determining flavour also means analysing volatile compounds that are sensed in the nose at the olfactory receptors either via the orthonasal (odour) or retronasal (aroma) routes when foods are eaten, non-volatile compounds sensed on the tongue (taste), and compounds perceived as mouthfeel and texture. Instrumental analyses of flavour have been used primarily to analyse volatile components. The main reason for this is the major importance of aroma in the overall flavour of a food, as is easily demonstrated by the difficulties encountered by subjects attempting to identify a particular flavour if the air flow through the nose is prevented, and the fact that volatile components are more amenable to conventional instrumental analysis than non-volatile compounds. Therefore, since the early studies published in the 1960s and the 1970s (Dumont and Adda, 1972, and references cited therein), instrumental methods have concentrated on identification of aroma compounds (Mariaca and Bosset, 1997). Only recently, some significant efforts have been made to develop instrumental procedures to characterise non-volatile components in cheese which are responsible for cheese taste (Salles et al., 1995a; Salles and Le Quéré, 1998; Engel et al., 2000a,b; Le Quéré and Salles, 2001). Instrumental analysis of aroma volatiles has been the subject of important specialised treatises (for the most recent literature on the subject, see Ho and Manley, 1993; Marsili, 1997; Mussinan and Morello, 1998; Stephan et al., 2000; van Ruth, 2001a; Reineccius, 2002, and specifically for instrumental analysis of

volatiles in milk and dairy products see Delahunty and Piggott, 1995; Mariaca and Bosset, 1997). Therefore, the part of this chapter that will be devoted to the analysis of cheese volatiles will focus on particular techniques adapted to the particular characteristics of cheese. Cheese flavour components result from the principal biochemical degradation pathways: glycolysis, lipolysis and proteolysis (see ‘Biochemistry of Cheese Ripening: Introduction and Overview’, ‘Metabolism of Residual Lactose and of Lactate and Citrate’, ‘Lipolysis and Catabolism of Fatty Acids in Cheese’, ‘Proteolysis in Cheese during Ripening’ and ‘Catabolism of Amino Acids in Cheese During Ripening’, Volume 1). The aroma compounds produced are mainly hydrophobic, or lipophilic, and consequently they tend to concentrate in the cheese fat according to their water/fat partition coefficient. Instrumental analysis of cheese volatiles must, therefore, consider, as a first step, an extraction method suitable for separating these volatiles from the cheese fat matrix. However, no single method yields a ‘true’ picture of a food aroma (Reineccius, 2002), and isolation and analysis of aroma remain challenging (Teranishi, 1998). Moreover, not only may the extraction step lead to artefacts, but the total volatile content in most cases is very difficult to relate to the flavour profile determined by a panel in sensory evaluation. Therefore, it appears much more efficient to concentrate efforts on the identification of those compounds that are really relevant to flavour. As no universally suitable extraction method exists, it appears essential to choose a method that yields an extract representative of the sensory properties of the food (Abbott et al., 1993; Etiévant et al., 1994; Etiévant and Langlois, 1998). Once this extraction method has been chosen, the next steps involve various forms of gas chromatography among which gas chromatography–olfactometry (GC–O) plays a prominent role in determining the key volatile compounds that contribute significantly to the flavour of the food (Leland et al., 2001), and gas chromatography–mass spectrometry (GC-MS), which is essential for the identification of those key odorants. Water-soluble extracts (WSE) from cheese have strong flavours (Biede and Hammond, 1979; McGugan



490 Cheese Flavour: Instrumental Techniques

et al., 1979; Aston and Creamer, 1986). Such extracts contain some volatile compounds (Le Quéré et al., 1996; Engels et al., 1997; Le Quéré and Salles, 2001), partly extracted by water according to their water/fat partition coefficient, although flavour compounds are generally more lipophilic than hydrophilic. However, the water extract mainly contains non-volatile compounds. This non-volatile, water-soluble fraction is composed of mineral salts, lactic acid, lactose, amino acids and peptides and has characteristic taste properties (Salles et al., 1995a). Amino acids and small peptides are considered to be mainly responsible for the taste characteristics of water-soluble extracts (McGugan et al., 1979; Aston and Creamer, 1986), their flavour impact being modulated by interaction with calcium and magnesium ions (Biede and Hammond, 1979). Moreover, it has been recognised for a long time that water-soluble, low molecular weight and mainly hydrophobic peptides, which accumulate during ripening as a result of proteolysis, are responsible for bitterness in cheese (Lowrie and Lawrence, 1972; Schalinatus and Behnke, 1975; Furtado, 1984; Lemieux and Simard, 1992). Some fundamental studies on model compounds have characterised the tastes of amino acids and low molecular weight peptides (Salles et al., 1995a and references cited therein); studies conducted on tastes in casein hydrolysates were reviewed by RoudotAlgaron (1996). However, until recently and apart from bitterness, no clear sensory data were obtained on water-soluble extracts from cheese. Although several hundred peptides have been isolated and identified from various types of cheese, only a few small peptides, that are suspected to be responsible for particular tastes, were isolated from the water-soluble fractions of various cheeses and identified (Salles et al., 1995a and references cited therein). However, no direct correlations between these peptides and the organoleptic properties of the fractions have been demonstrated, apart from bitterness. In fact, the watersoluble fraction of cheese generally has a very complex composition, and separation and identification of individual compounds are difficult. Moreover, most analytical techniques require the use of non-food-grade solvents or buffers that make sensory evaluation of sub-fractions difficult or impossible. Part of this chapter will focus on recent advances made to study and identify the taste-active components present in the water-soluble fraction of cheese. A general procedure for the preparation of fractions involves an extraction of grated cheese by water followed by a fractionation scheme, generally adapted from the fractionation protocol used to isolate cheese nitrogen fractions in the study of proteolysis in cheese during ripening (Fox et al., 1994; McSweeney and

Fox, 1997). However, as sub-fractions have to be evaluated sensorially to assess their relative sensory impact and try to link it to their chemical composition, a suitable eluent has to be used in the chromatographic steps. Water (Roudot-Algaron et al., 1993; Salles et al., 1995a; Molina et al., 1999) or water-food-grade ethanol mixtures (Lee and Warthesen, 1996a,b) have been used for this purpose in combination with gel permeation chromatography (GPC) or high-performance liquid chromatography (HPLC). The final identification step generally involves mass spectrometry (MS) and tandem mass spectrometry (MS/MS) of nitrogenous compounds isolated using HPLC, either in a standalone mode or coupled with a mass spectrometer (HPLC–MS) (Roudot-Algaron et al., 1993, 1994b; Sommerer et al., 2001). A specific method for the isolation of small peptides from cheese has been described (Sommerer et al., 1998a). As already outlined for cheese aroma, the relationships between all flavour compounds identified in a food and sensory perception experienced by consumers when eating this food are still not entirely clear. In fact, it is particularly difficult to predict a flavour perception as it is still not known how the various components combine to produce an overall sensory impression. Moreover, interactions between taste and aroma (Noble, 1996) and interactions of trigeminal sensations with taste and aroma (Green, 1996) occur and play an important role in overall flavour perception. However, methods that allow direct analysis of flavour molecules released in the mouth during consumption have been developed in recent years (Taylor and Linforth, 1996; Roberts and Taylor, 2000). Development of instrumental techniques and data obtained recently for volatile and non-volatile flavour compounds in cheese will be presented which may explain the link between flavour perception and cheese composition. Finally, specific instrumental techniques have been developed for the analysis of the complete flavour of cheese. The methods currently used in the quality control of food flavour are still usually based on sensory evaluation by a panel of experts. These panels are able to monitor the quality of a particular food, to detect defects and to compare samples for classification purposes. Nevertheless, obtaining results rapidly at low cost using instruments could be desirable. The so-called ‘electronic noses’ based on gas sensor technology, despite some important drawbacks for some of them (Schaller et al., 2000a), are theoretically able to perform some classification tasks (Schaller et al., 1998), and some applications for the analysis of cheese have been developed (Mariaca and Bosset, 1997; Schaller et al., 1999). However, two other global

Cheese Flavour: Instrumental Techniques 491

analysis methods based on mass spectrometry seem more powerful and reliable for purposes of classification. One of these methods analyses total headspace using a mass spectrometer, without any prior GC separation (Vernat and Berdagué, 1995). This method is often referred to as a mass-based electronic nose. Alternatively, headspace sampling may be replaced by solidphase microextraction (SPME) of food volatiles (Marsili, 1999). Both sampling methods, followed directly by mass spectrometry, have found applications for the rapid characterisation of cheese (Schaller et al., 2000b; Pérès et al., 2001, 2002a). The second method is pyrolysis mass spectrometry (Aries and Gutteridge, 1987), where a small food sample is pyrolysed at up to 500 °C. The resulting volatile fraction, characteristic of the flavour but also of the matrix composition, is analysed by a mass spectrometer. As with the other rapid instrumental methods for classification, a pattern or fingerprint is obtained for each sample, and extensive data treatment, either by conventional multivariate statistics or artificial neural networks, allows the construction of maps useful for classification and quality control purposes (Pérès et al., 2002b).

Characterisation of Aroma (Volatiles) Sample treatment

Volatile aroma compounds in cheese, like in other foodstuffs, are hydrophobic, generally distributed in a heterogeneous manner throughout the matrix and present at low or even traces (10 g/kg) concentrations. Their analysis in cheese requires homogenisation of the sample prior to extraction, where isolation procedures adapted to lipophilic material dispersed in trace amounts in a high-fat food are required. A practice commonly used for cheese is freezing the sample in liquid nitrogen, followed by grating to a fine powder with a blender at low temperature. The rind is generally removed before sample homogenisation. The powder is then used for subsequent steps as such, or after dispersion and homogenisation in water, with possible pH adjustment, if necessary. Extraction methods

As already outlined, all the extraction procedures used to isolate the aroma fraction from the cheese matrix should be adapted to the analysis of trace levels of lipophilic material dissolved in a fatty phase, while minimising losses of highly volatile molecules and preventing modification of compounds or the formation of artefacts. Many techniques have been proposed for the extraction of volatile compounds from cheese, amongst

which, the most traditional and popular methods, based on the volatility of aroma compounds, involve distillation. Although steam distillation methods, and particularly the simultaneous steam distillation/solvent extraction (SDE) technique (Chaintreau, 2001) are still used for dairy products, they have several drawbacks. Highly volatile compounds are recovered poorly, thermally sensitive compounds may disappear and artefacts may appear unless distillation is performed under a reduced pressure, with tight control of temperature. In either approach, at atmospheric pressure or under vacuum, the quality of the aroma extract finally obtained is dependent on the volatility of the aroma compounds and on their solubility in the solvent used. If steam distillation (also called hydrodistillation) is used without simultaneous solvent extraction, it is necessary to add a large quantity of water to the grated cheese to obtain a homogeneous slurry (c. 1 l for 100 g of cheese). The distillate obtained is in fact a dilute aqueous solution of volatile compounds (Dumont and Adda, 1972). A subsequent extraction with large amounts of a suitable solvent, followed by a concentration step, is required. High-vacuum distillation techniques, on the contrary, produce small volumes of concentrated aqueous distillates (cheese moisture content only) that can be extracted with tiny volumes of an organic solvent. A typical experiment involves two steps. In the first step, the frozen grated cheese is transferred to a cone-shaped flask that is connected to a static vacuum (c. 10 Pa) renewed from time to time. Using sub-ambient temperature and rotation of the flask in order to break the continuously dehydrating surface of the sample, the volatiles are condensed with most of the cheese water in traps maintained at the temperature of liquid nitrogen (Dumont and Adda, 1972; Le Quéré and Molimard, 2002). In the second step, the flask containing the dehydrated cheese powder is connected to a molecular distillation apparatus operating under a high vacuum (c. 10 2 Pa). In this step, also called ‘cold-finger molecular distillation’, the remaining water and volatiles are transferred directly to the surface of a cold condenser maintained at the temperature of liquid nitrogen and situated at a very short distance from the surface of the sample (Fig. 1). The condensed ice layer on the surface of the condenser contains less volatile and more lipophilic compounds. This fraction is combined with the aqueous distillate obtained in the first step. Since early workers (see Dumont and Adda, 1972 and references cited therein), high-vacuum distillation techniques have been applied to the extraction of aroma compounds from a large variety of cheeses (see Mariaca and Bosset, 1997 and references cited therein; Moio and Addeo, 1998; Moio et al., 2000). Working at


Figure 1 Apparatus used for cold-finger molecular distillation. A, I, traps for volatiles cooled with liquid nitrogen; E, round-bottom flask containing dehydrated cheese powder, equipped with a ‘cold-finger’ cooled with liquid nitrogen; F, H, J, K, L, high-vacuum stopcocks; M, connection to the high vacuum pumping system; N, guard trap cooled with liquid nitrogen.

ambient or even at sub-ambient temperature, the techniques prevent thermal degradation, but need a substantial amount of sample (c. 50–250 g) and are very time-consuming (a long distillation period up to a few hours). Moreover, the aqueous distillates have to be extracted with a suitable solvent (e.g., dichloromethane or diethyl ether) before performing further analysis. A chemical fractionation by controlling the pH of the aqueous distillate results in separation of the organic extracts into acid, neutral and basic fractions which may be analysed separately (Mariaca and Bosset, 1997; Reineccius, 2002). A nice example on Swiss Gruyère cheese was published 20 years ago (Liardon et al., 1982; Bosset and Liardon, 1984, 1985; Bosset et al., 1993). Specific methods for the analysis of volatile free fatty acids may be found in the literature (see for example Ha and Lindsay, 1990). Cheese volatiles may be extracted directly from samples by a solvent (e.g., diethyl ether). However, further steps are required to separate the aroma from the lipids that are also extracted very efficiently by the solvent. While a simple solvent extraction introduces some bias into an aroma profile, the following steps, which are necessary, may add more bias (Reineccius, 2002). For instance, separation of volatile and nonvolatile compounds that have dissolved in the solvent by distillation under high vacuum has been used by Grosch and co-workers in the study of Swiss (Preininger and Grosch, 1994; Preininger et al., 1994; Rychlik et al., 1997) and Camembert (Kubickova and Grosch, 1997) cheese and more recently by Qian and Reineccius (2002a) in a study on Parmigiano-Reggiano cheese. The risk in this case is that only the most volatile components are selected from an oil-rich phase (Reineccius,

2002). Therefore, direct solvent extraction methods that necessitate a subsequent distillation step under vacuum do not offer a significant advantage compared to other vacuum distillation methods. Dialysis techniques, that are based on molecular size differences and which separate molecules according to their ability to diffuse through a specific membrane at room temperature, may seem a good alternative. Reineccius and co-workers have used this technique (Benkler and Reineccius, 1979, 1980) and compared it to other methods for the isolation of volatiles from Cheddar cheese (Vandeweghe and Reineccius, 1990). More recently, Spinnler and co-workers reinvestigated the technique (Molimard and Spinnler, 1993) and applied it to the extraction of aroma volatiles in the study of the impact of the microflora on the aroma of Camembert-type cheese (Molimard, 1994; Spinnler et al., 1995). In order to eliminate adsorption and artefact formation, these authors used 1% water in the solvent, diethyl ether, to inactivate acidic sites on the perfluorosulphonic acid membrane. They also improved the dialysis yield by recycling the solvent using a distillation device to recycle the solvent attached to the dialysis cell (Fig. 2) in order to maintain a maximum concentration gradient between the two compartments of the cell (Molimard, 1994). Nevertheless, the method is time-consuming (72 h dialysis time), and its efficiency decreases dramatically as the number of carbon atoms of the aroma molecule increases (n 10), modulated by their hydrophobicity (Molimard and Spinnler, 1993). A high-performance size-exclusion chromatographic method has also been described for the purification of aroma compounds from organic extracts of fat-containing food (Lübke et al., 1996). The method was applied

Figure 2 Dialysis cell with solvent recycling device. A, B, cell compartments; C, round-bottom flask containing solvent to distil; D, condenser; E, magnetic stirrers; F, dialysis membrane.


successfully to the clean-up of a dichloromethane extract from goat cheese (Lübke et al., 1996). The main interest in this size-exclusion chromatographic method is the limited number of injections necessary and the reduced final volume of the fractions, which in terms of final useful concentration, appeared significantly quicker and gave rise to less thermally induced artefacts and to reduced losses of the most volatile components than any other distillation method (Lübke et al., 1996). Headspace methods, either static or more often dynamic, also called ‘purge-and-trap’ methods, are popular techniques used to isolate volatiles from cheese. Although direct analysis of the equilibrium headspace would appear to be an ideal method to study aroma compounds, in terms of sensory representativeness and ease of use, static headspace techniques have severe limitations in terms of sensitivity, being restricted to the most volatile and abundant components (Mariaca and Bosset, 1997; Reineccius, 2002). Dynamic headspace, or ‘purge-and-trap’, methods are basically pre-concentration and enrichment techniques. They use stripping of the volatiles from the cheese samples, sometimes dispersed in water, with an inert gas. The volatiles are concentrated in a cold trap or adsorbed onto an inert support (adsorbing polymer, generally of the Tenax® type) and analysed by subsequent thermal desorption or elution by a suitable solvent (Mariaca and Bosset, 1997; van Ruth, 2001a; Reineccius, 2002). Although dynamic headspace methods minimise artefacts developed or introduced during sampling (van Ruth, 2001a), distortion of the aroma profile may result from the trapping of aromas (Reineccius, 2002), especially when polymeric adsorbents are used. However, despite the drawback of relatively poor sensitivity compared to other extraction methods, the main advantages of dynamic headspace techniques are the small amount of sample needed to perform the analysis (c. 20 g) and its speed (Le Quéré and Molimard, 2002). The technique, even though it favours the isolation of the most volatile flavour compounds (Reineccius, 2002), has been applied widely to the analysis of cheese volatiles (see for example Arora et al., 1995; Canac-Arteaga et al., 1999a,b, 2000; Larrayoz et al., 2001; Rychlik and Bosset, 2001a,b). Recent comprehensive reviews on the technique include Wampler (1997) and Pillonel et al. (2002). A comparative study on the advantages of the use of dynamic headspace with cheese samples in the ‘dry’ form or in ‘dispersed suspension’ in water has been published recently (Larrayoz et al., 2001). The ‘dry’ method allowed the extraction of a greater number of compounds and in larger quantities, but a few compounds were extracted better using the ‘suspension’ technique (Larrayoz et al., 2001). Simultaneous distil-

lation extraction (SDE) was also used in this study and compared to dynamic headspace analysis. As expected, the authors concluded that the techniques were complementary; dynamic headspace extracted more highly volatile compounds and SDE was more efficient for phenols, free fatty acids, lactones and heavier aldehydes, ketones, alcohols and esters (Larrayoz et al., 2001). Interference from water in dynamic headspace that could be detrimental to the efficiency of the technique has been discussed in detail by Canac-Arteaga et al. (1999a,b, 2000) and Pillonel et al. (2002). Solid-phase microextraction, first developed for the extraction of volatile organic compounds in water, has been applied recently to the isolation of aroma compounds from food (Harmon, 1997; Pillonel et al., 2002; Reineccius, 2002). Solid-phase microextraction partitions analytes between a liquid or a vapour phase and a thin solid-phase adsorbent, of which there are several choices in terms of polarity and film thickness, coated on inert fibres, generally associated with a syringe which serves as a direct injection device (Harmon, 1997). The method, which is an equilibrium one, can be performed either in the direct extraction mode (immersion of the fibre in the sample matrix, generally in an aqueous solution or suspension) or in a headspace configuration. It can be automated very easily, but the extraction of the solutes depends on polarity, volatility, partition coefficients, sample volume, temperature and the nature of the adsorbentcoating material. Therefore, the technique exhibits a certain degree of selectivity, but with the advantages of sensitivity, ease of use, no solvent and small sample volume (Harmon, 1997; Pillonel et al., 2002; Reineccius, 2002). Solid-phase microextraction, used for the first time for the analyses of cheese volatiles by Chin et al. (1996), has since been used in some significant applications on cheese aroma (Dufour et al., 2001; Pillonel et al., 2002 and references cited therein). Analysing volatiles directly by immersing the fibre in highly complex matrices (as cheese) could damage the fibre, and SPME is, therefore, used almost always in the headspace mode. Comparison of direct SPME and headspace SPME of Camembert volatiles obtained after cryo-trapping of the aqueous phase under vacuum showed only a slight reduction in sensitivity using headspace SPME compared to direct SPME ( Jaillais et al., 1999). The water-soluble extract (WSE) of cheese has been described for a long time as possessing a strong flavour (Biede and Hammond, 1979; Aston and Creamer, 1986; Engels and Visser, 1994; Salles et al., 1995a). Besides non-volatile materials responsible for taste, WSE also contains volatile compounds responsible for its intense aroma. Thus, water-soluble extracts of various


cheeses, obtained by direct extraction with water followed by various centrifugation steps (Le Quéré et al., 1996; Engels et al., 1997; Engel et al., 2002c) or by pressing to obtain an aqueous phase called ‘cheese juice’ (Salvat-Brunaud et al., 1995; Thierry et al., 1999), have been investigated for their volatile components. To be analysed using gas chromatography, WSEs were either extracted with a suitable solvent (Le Quéré et al., 1996), submitted to dynamic headspace analysis (Engels et al., 1997; Thierry et al., 1999) or fractionated using nanofiltration as the final membrane-filtration step (Engel et al., 2002c). Representativeness

As already outlined, because there is no universally applicable method, none of the extraction techniques described above yields an aroma isolate that truly represents either qualitatively or quantitatively the aroma profile of a food (Reineccius, 2002). This fact explains the frequently observed discrepancies between aroma analysis of a food extract and sensory analysis of the food itself. Therefore, the flavour analyst must choose the isolation procedure best suited to address the problem faced: determination of the complete aroma profile, identification of key odorants or off-flavours, monitoring aroma changes with time in foods or prediction of sensory properties (Reineccius, 2002). When the ultimate aim of a particular study is the identification of the compounds that are important for flavour (the key odorants), the most reliable results will be obtained if the odour of the extract resembles closely that of the food itself (Etiévant et al., 1994; Etiévant and Langlois, 1998). Different sensory methods, which necessitate a trained sensory panel, can be used to check the sensory representativeness of the food extract odours (Etiévant et al., 1994). When an estimation of the relative importance of key constituents in a single sample is required, a similarity test is preferred. The panellists are asked to score the similarity of the odour of the extracts obtained by different methods to the odour of the food itself used as reference on an unstructured 10 cm scale. This approach was applied to three French and Swiss hardtype cheeses by Etiévant et al. (1994) and Guichard (1995). It was shown that the distillates obtained at a pressure in the range 10–100 Pa had odours more similar to those of the cheeses than the distillates obtained at a lower pressure (10 mPa). This result means that strongly absorbed and less volatile flavour compounds, extracted only at lower pressure, may not be important for the odour of these cheeses. Similar results were obtained for extracts of Camembert cheese, showing clearly that the second step (molecular distillation operated under a high vacuum) is not necessary to obtain a representative distillate of the cheese odour.

When applied to goat milk cheese, this approach indicated that the best extract was obtained by a direct water extraction of the cheese volatiles (Le Quéré et al., 1996). This result could perhaps be explained by the chemical and hydrophilic nature of the free fatty acids identified as key odorants of goat milk cheese (Le Quéré et al., 1996; Salles and Le Quéré, 1998; Le Quéré and Salles, 2001). A key point in these evaluations of representativeness is the choice of a suitable matrix for testing the olfactory character of the extracts. For cheese, the best results have been obtained when the extracts are added to an emulsion, i.e., a matrix similar to cheese in terms of fat composition (Etiévant et al., 1994). Since, generally, a combination of techniques should be used to obtain a reasonably complete view of an aroma profile (Reineccius, 2002), it is noteworthy that sensory evaluation of headspace or SPME extracts by ‘direct GC-olfactometry’ (i.e., without a chromatographic column) has been demonstrated recently (Lecanu et al., 2002; Rega et al., 2003). Identification of volatile aroma compounds using hyphenated GC techniques

As aroma molecules are essentially volatile, the techniques used to analyse them are usually based on separation using high resolution gas chromatography (HRGC). Substantial progress has been made in this field during the last 20 years and several stationary phases are available which allow almost all separation problems to be overcome. Combined with universal or selective detectors, HRGC is clearly a fundamental technique, essential for all aroma identification studies. A comprehensive review on the use of HRGC for the analysis of milk and dairy products is available (Mariaca and Bosset, 1997). Other interesting comments on qualitative, including multidimensional GC (Wright, 1997), and quantitative aspects may be found in Marsili (1997), van Ruth (2001b) and Reineccius (2002). Among the hyphenated techniques that are coupled to HRGC, the one that uses the human nose as a detector and known as gas chromatography–olfactometry (GC–O, sometimes referred to as ‘GC-sniffing’), has received considerable attention during the past 20 years in aroma research (see for example Blank, 1997; Leland et al., 2001; Reineccius, 2002). The selectivity of this specific detector is based only on the odorous properties of the individual compounds separated by HRGC. As the most abundant volatiles may have little, if any, odour of significance in a food (Mistry et al., 1997), GC-sniffing has been an invaluable tool for identifying target compounds in aroma extracts that are always very complex. The primary aim of this technique is to discriminate the odorous compounds from the many background volatile components. The so-called ‘aromagram’


constructed from the chromatogram obtained by simply smelling a GC effluent (Blank, 1997; Reineccius, 2002) constitutes an interesting interface with sensory analysis, as odour descriptors sensed at the GC sniffing port can be compared to the descriptors generated by a sensory panel. This method is particularly efficient for identifying off-flavours. Selection of key odorants or character-impact compounds in a food is another objective of GC-sniffing. Quantitative approaches (the true GC-olfactometry) based on odour detection thresholds or on odour intensity have been developed and are the subject of specialised treatises (Mistry et al., 1997; Leland et al., 2001; van Ruth, 2001b; Reineccius, 2002). Three different methods have been developed for GC–O: dilution analyses based on determination of detection thresholds, detection frequency methods and intensity measurement methods. Original dilution methods, CHARM (for Combined Hedonic Aroma Measurement) analysis developed by Acree and co-workers (Acree et al., 1984) and Aroma Extract Dilution Analysis (AEDA) developed by Grosch and co-workers (Ullrich and Grosch, 1987) are essentially screening methodologies since the methods, based only on detection threshold determinations, violate certain sensory rules and psychophysical laws (Reineccius, 2002 and references cited therein). They can be used to determine those odorous compounds that are most likely to contribute to the odour of a food. Originally developed by McDaniel et al. (1990), the odour-specific magnitude estimation (OSME) method is basically a crossmodal technique aimed at measuring the perceived odour intensity of eluting volatiles. In OSME and other cross-modality matching methods (Guichard et al., 1995; Etiévant et al., 1999), results are not based on odour detection thresholds, and only one concentration of the extract is evaluated by a panel, unlike dilution methods where several dilutions of the extract are evaluated. Results can be subjected to statistical analysis and more consistent results are obtained when panellists are trained (Callement et al., 2001). The detection frequency methods, originally developed by Roozen and co-workers (Linssen et al., 1993), and referred to as nasal impact frequency (NIF) or surface nasal impact frequency (SNIF) since the work of Chaintreau and co-workers (Pollien et al., 1997), also use a group of assessors who simply have to note when they detect an odour in a single GC run (i.e., also at only one concentration). Those GC peaks being detected as odorous by the greatest number of assessors are considered to be the most important. Not being based on real odour intensities, the method has important drawbacks, especially when all the odorous compounds are present above their sensory threshold for all the assessors (Reineccius, 2002).

There is no perfect GC-sniffing method for finding key odorants in foods. Each of the methods described above has its advantages and weaknesses. Only two studies have compared the methods in terms of performance (Le Guen et al., 2000; van Ruth and O’Connor, 2001). In both cases, the results obtained with the different techniques were found to be very similar and well correlated. Finally, the choice of a GC–O method depends on the objective of the study, on the quality of the panel and on the time scheduled for the analyses (Le Guen et al., 2000). Dilution techniques are clearly time-consuming, intensity methods require a trained panel (Le Guen et al., 2000; Callement et al., 2001) while detection frequency methods are the least demanding but also the least precise (Le Guen et al., 2000). The aim of any GC–O experiment is to determine the relative odour potency of volatiles present in an aroma extract or fraction and to prioritise compounds for identification. This identification step is done mainly through the use of another hyphenated technique that couples HRGC to mass spectrometry (GC–MS). For difficult identifications, GC coupled with Fourier transform infrared spectroscopy (GC/FTIR) provides an interesting complement to GC–MS (Le Quéré, 2000). Mass spectrometry is also used for quantification purposes through the use of a stable isotope dilution assay (Milo and Blank, 1998; Blank et al., 1999 and references cited therein). Such a precise quantitation is required for the determination of odour activity values (OAVs) generally calculated when using AEDA (Grosch, 1994). Odour activity values, calculated as the ratio of concentrations to odour thresholds, despite their limitations in terms of psychophysical validity (Mistry et al., 1997), give a good indication of the respective contributions of key odorants to the aroma of foods. They are the basis of the first attempts at using recombination studies to validate impact odorants sensorially in model cheeses (Grosch, 1994). Aroma-recombination studies are the important last step in sensorially verifying the analytical data obtained by GC–O and for quantification of key odorants of food (Mistry et al., 1997). Either bland unripened cheese (Grosch, 1994; Preininger et al., 1996; Kubickova and Grosch, 1998a) or specially designed odourless model cheese systems (Smit et al., 1995; Salles et al., 1995b) have been used to incorporate potential key odorants. Thus, the importance of methional, 4-hydroxy-2,5-dimethyl-3(2H)-furanone and 2-ethyl-4-hydroxy-5-methyl-3(2H)-furanone, acetic acid and propionic acid was confirmed as key compounds for the aroma of Emmental-type cheese (Preininger et al., 1996). The branched-chain volatile fatty acids, 4-methyloctanoic and 4-ethyloctanoic acids, were confirmed to be essential for the typical goaty note of goat cheese (Le Quéré et al., 1996) and their


retronasal aroma thresholds were determined in a cheese model (Salles and Le Quéré, 1998; Le Quéré and Salles, 2001; Salles et al., 2002). Finally, the odour profile of the aroma model built with a set of 11 potent odorants identified in a GC–O study of an extract from Camembert cheese (Kubickova and Grosch, 1997, 1998b), with four additional volatile compounds identified by headspace–GC–O, has been found to resemble closely the aroma of genuine French Camembert cheese (Kubickova and Grosch, 1998a; Grosch et al., 2001). The GC–O methods that have been developed during the past 20 years, combined with either aroma extracts, headspace or even SPME (Dufour et al., 2001), have facilitated the identification of potent odorants in various cheeses, including Swiss (Preininger and Grosch, 1994; Rychlik et al., 1997; Rychlik and Bosset, 2001a,b), Cheddar (Arora et al., 1995; Christensen and Reineccius, 1995; Dufour et al., 2001), ParmigianoReggiano (Qian and Reineccius, 2002a,b), Blue (Le Quéré et al., 2002; Qian et al., 2002), Mozzarella (Moio et al., 1993), Grana Padano (Moio and Addeo, 1998) and Gorgonzola (Moio et al., 2000) cheeses.

Characterisation of Sapid (Non-Volatile) Flavour Compounds Water-soluble extracts (WSE) of cheese

The water-soluble extract (WSE) of cheese has been reported to possess a strong flavour (Biede and Hammond, 1979; McGugan et al., 1979; Aston and Creamer, 1986). Apart from some water-soluble volatile components responsible for aroma, a WSE of cheese contains mainly non-volatile components that have been considered to be responsible for the taste of cheese (McSweeney, 1997). It has been recognised for a long time that bitterness, which can limit cheese acceptability if too intense, is due to an excessive concentration of low molecular weight and mainly hydrophobic peptides, which accumulate during ripening as a result of proteolysis (Lemieux and Simard, 1992; McSweeney, 1997). Amino acids and small peptides were hypothesised to be mainly responsible for the basic taste of cheese (McGugan et al., 1979; Aston and Creamer, 1986; Engels and Visser, 1994), their flavour impact being supposedly influenced by their interaction with calcium and magnesium ions (Biede and Hammond, 1979). However, the exact role of medium- and smallsize peptides and free amino acids in cheese flavour has not been clearly demonstrated, although it is likely that they contribute to the background flavour of cheese (McSweeney, 1997). In fact, until recently and apart from bitterness, no clear sensory data have been available for

WSEs of cheese and no direct correlations between specific nitrogen-containing compounds and organoleptic properties of fractions have been demonstrated. Among the mineral salts present in the WSE of cheese, the compound responsible for the salty taste is almost always supposed to be NaCl (McSweeney, 1997). The taste of most high molecular weight salts is known to be bitter rather than salty (McSweeney, 1997 and references cited therein). Acid taste is caused by H3O and the principal acid in cheese is lactic acid. However, total lactate concentration does not seem to be a good index of cheese acidity as the pH may increase during ripening caused by the production of ammonia (McSweeney, 1997). Moreover, the perception of acidity in cheese was hypothesised to be influenced by the concentration of NaCl (Stampanoni and Noble, 1991), and no correlation between acid taste and either cheese pH or the amount of lactic acid was found for the flavour of Swiss cheese (Biede and Hammond, 1979), while the acid flavour correlated positively with the levels of triand tetra-peptides and with amino acids (Biede and Hammond, 1979). It has also been hypothesised that short- and medium-chain fatty acids might contribute to the acid taste of cheese (McSweeney, 1997). Although this assumption seems reasonable for short chain acids (e.g., formic, acetic or propionic), their principal contribution to cheese flavour is to its aroma in the unionised form (RCOOH) (Le Quéré et al., 1996; Salles and Le Quéré, 1998; Qian and Reineccius, 2002b). Extraction, separation, identification of sapid compounds in relation to their sensory properties

The study of taste-impact compounds in cheese, or more precisely in its water-soluble fraction, involves the study of soluble low molecular weight material (i.e., small peptides, amino acids, organic acids, minerals, etc.) dispersed in a very complex mixture. As it is necessary to assess the relative sensory impact of potential taste-active compounds, a fractionation scheme suitable for subsequent sensory evaluation is needed, and non-food-grade solvents or buffers must be rejected. Commonly used procedures involve extraction of grated cheese with water, possibly completed by precipitation of caseins and large peptides at pH 4.6, leading to edible fractions with good recovery of nitrogenous compounds (Kuchroo and Fox, 1982). The fractionation scheme that follows is generally adapted from the fractionation protocol used for isolating cheese nitrogen fractions for the study of proteolysis (Fox et al., 1994; McSweeney and Fox, 1997). The following steps (Fig. 3) involve ultrafiltration using membranes with 1, 3 or 10 kDa molecular weight cutoff or precipitation with 70% ethanol (Cliffe et al.,


Figure 3 Possible fractionation schemes used to isolate and evaluate non-volatile compounds from cheese.

1993). The ultrafiltered water-soluble or 70% ethanolsoluble extracts are then subjected to gel filtration chromatography (Fig. 3). Sephadex G10 (Engels and Visser, 1994; Roudot-Algaron et al., 1994a; Engels et al., 1995; Molina et al., 1999), G15 (Roudot-Algaron et al., 1993; Warmke et al., 1996; Kubickova and Grosch, 1998a), G25 (Cliffe et al., 1993; Salles et al., 1995a), or Toyopearl HW-40S (Salles et al., 1995a, 2000; Sommerer et al., 1998a, 2001) media have been used for this purpose, using pure water (generally), 0.01 M NaCl (Engels and Visser, 1994), or aqueous 0.5 M acetic acid (Warmke et al., 1996; Kubickova and Grosch, 1998a) as eluent. The fractions obtained by gel permeation chromatography may be evaluated sensorially (Fig. 3) after freeze-drying and re-dissolution in water, possibly with pH adjustment. Alternatively, liquid chromatographic methods involving Sep Pak C18 cartridges eluted with a stepwise water–ethanol gradient (Engels and Visser, 1994; Engels et al., 1995) or HPLC

using a water/food-grade ethanol gradient (Lee and Warthesen, 1996a,b) have been used instead of gel filtration. This fractionation scheme was developed originally in order to identify small hydrophobic peptides supposedly responsible for taste characteristics such as bitter or umami (Mojarro-Guerra et al., 1991; Cliffe et al., 1993; Roudot-Algaron et al., 1993, 1994a). A dedicated liquid chromatographic purification method has been developed to isolate and identify oligopeptides from the WSE of goat milk cheese (Sommerer et al., 1998a, 2001). Systematic sensory evaluation of the final fractions allows target fractions to be determined that possess interesting tastes, and physicochemical assessment of these key fractions should permit the identification of those compounds that are really relevant to the flavour of cheese (Engels and Visser, 1994; Salles et al., 1995a). Using this approach, some recent studies have been dedicated to the taste of the WSE of various cheeses.


Low molecular weight peptides, with two to four amino residues, were identified in Vacherin Mont d’Or (Mojarro-Guerra et al., 1991). As there was not enough natural material available for sensory evaluation, commercially available analogous synthetic peptides were used in sensory experiments. The dipeptides tested were dissolved in tap water at a rather high concentration (50 mg/100 mL) and were found to be essentially bitter. However, neither quantitative nor threshold data were estimated and the importance of these peptides for the overall taste of the cheese was only an hypothesis (Mojarro-Guerra et al., 1991). In a study on Cheddar cheese, Cliffe et al. (1993) found bitter fractions in material thought to be large hydrophobic peptides while lower molecular weight fractions with savoury notes were thought to be small, more hydrophilic peptides and amino acids. The flavour of the WSE of Comté cheese was the subject of substantial efforts in the early 1990s. A great variety of small peptides was identified in these extracts (Roudot-Algaron et al., 1993, 1994a,b). Some of them were found to be essentially bitter (Roudot-Algaron et al., 1993), -glutamyl dipeptides were found to be sour (Roudot-Algaron et al., 1994a), but all the identified compounds, including non-peptide material (Roudot-Algaron et al., 1993; Salles et al., 1995a), were found at a concentration much lower than their threshold values. Although possible synergistic effects between several molecules found at concentrations below individual threshold values cannot be a priori eliminated, these observations suggest that these components alone could not affect cheese flavour (Salles et al., 1995a). Umami taste was clearly identified in a fraction and easily explained by a substantial amount of monosodium glutamate which was found at a concentration ten times above its threshold value, while the concentrations of the other amino acids were all well below their thresholds (Salles et al., 1995a). Following the same methodology, Grosch and co-workers evaluated the taste compounds of Emmental cheese (Warmke et al., 1996). The contribution of individual free fatty acids, free amino acids, minerals, biogenic amines, lactic and succinic acids, and ammonia was estimated on the basis of taste activity values (TAVs), a concept analogous to the odour activity values (OAVs), and defined as the ratio of concentration to taste threshold. From these results, acetic and propionic acids were confirmed to be important contributors to the taste of Emmental cheese. Glutamic acid was the major taste compound in the fraction containing free amino acids while all the ions investigated might be involved in the taste of Emmental, as were also biogenic amines (tyramine and histamine), ammonia, lactic and succinic acids (Warmke et al., 1996).

However, taste evaluation of mixtures of compounds conducted in tap water suggested that the characteristic taste compounds of Emmental are acetic, propionic, lactic, succinic and glutamic acids, each in the undissociated form and/or as ammonium, sodium, potassium, magnesium and calcium salts, as well as chlorides and phosphates analogues (Warmke et al., 1996). A study conducted on a model based on unripened Mozzarella-type cheese confirmed the importance of acetic, propionic, lactic, succinic and glutamic acids, and sodium, potassium, calcium, magnesium, ammonium, phosphate and chloride ions to the taste of Emmental cheese (Preininger et al., 1996). The same approach applied to Camembert led to the conclusion that the important taste contributors for Camembert are acetic, butyric, 3-methylbutyric, caprylic and succinic acids, monosodium glutamate, ammonia and NaCl (Kubickova and Grosch, 1998a). It was also found that the biogenic amine, cadaverine, and the rare amino acids, ornithine and citrulline, when present, are likely to contribute to the bitter taste of Camembert (Kubickova and Grosch, 1998a). The above results clearly indicated that only low molecular weight compounds found in the WSE contribute significantly to the taste of cheese, while small peptides do not seem to be key flavour compounds, as was previously hypothesised. A study on goat milk cheese led to the same conclusions (Salles and Le Quéré, 1998; Salles et al., 2000; Le Quéré and Salles, 2001). The taste of the various goat milk cheeses investigated was essentially due to mineral salts and lactic acid. Fractions rich in small peptides and free amino acids were found to be essentially tasteless when evaluated either in water (Salles et al., 2000) or in a model cheese (Salles and Le Quéré, 1998; Le Quéré and Salles, 2001). In a comparative study on cheeses made from cows’, ewes’ or goats’ milk, Molina et al. (1999) concluded that, even though differences were found in the intensity and predominance of individual tastes in the fractions of the cheeses made from the milk of the three species, it was difficult to correlate the peptide pattern and the free amino acid content of cheese with the sensory evaluation of the fractions. However, synergistic effects on taste have been demonstrated between peptides, amino acids and mineral salts (Wang et al., 1996) and interactions between tastes in mixtures may exist (Breslin, 1996). Therefore, it appeared interesting to generalise the evaluation of model mixtures of compounds that have been identified and quantified in the WSE of cheese (Warmke et al., 1996; Kubickova and Grosch, 1998a). Moreover, fractionation of the WSE by gel filtration has two main limitations: poor resolution and the necessity of


tedious repetitive steps in order to obtain sufficient peptide material for sensory evaluation. To clarify the putative effect of the small water-soluble peptides on the taste of cheese, it was therefore necessary to develop a new isolation procedure. Nanofiltration using ionisable membranes with a molecular weight cut-off of 500 Da was used by Sommerer et al. (1998b). A nanofiltrate was prepared from the 1-kDa permeate obtained by ultrafiltration of the WSE (Fig. 3). A large proportion of mineral salts and a substantial proportion of amino acids were thus eliminated from the nanofiltration retentate in which the majority of small peptides were concentrated (Sommerer et al., 1998b). This relatively pure and edible peptide-containing fraction could be used in sensory analysis, after incorporation into a bland model cheese system (Salles et al., 1995b), on its own or with the addition of putative synergistic effectors such as mineral salts or amino acids (Sommerer et al., 1998b). Using omission tests (see Engel et al., 2002a,b, and references cited therein for a comprehensive review), it was shown that small peptides have no effect on the taste of goat milk cheese, and no additive or synergistic effects were found between those peptides and salts or amino acids (Sommerer et al., 1998b). This unexpected result has been confirmed after complete physicochemical assessment of the WSE from goats’ milk cheese has allowed the development of a model mixture that was validated sensorially (Engel et al., 2000a). Using omission tests, the relative impact of WSE components on goat cheese taste has been determined (Engel et al., 2000b). Among the main taste characteristics of the WSE from goats’ milk cheese (salty, sour and bitter), saltiness was explained by additive effects of Na, K, Ca2 and Mg2, sourness was due to synergism between NaCl, phosphates and lactic acid, and bitterness resulted entirely from CaCl2 and MgCl2. Amino acids, lactose and peptides had no significant impact on the taste properties of the WSE of goats’ milk cheese (Engel et al., 2000b). The same procedure was applied recently to a specially selected bitter Camembert cheese (Engel et al., 2001a,b,c) and confirmed that the WSE from cheese contained taste-active compounds, the impact of which could be modulated by an effect of the cheese matrix (Engel et al., 2001a). Sourness of Camembert WSE was explained by an enhancing effect of NaCl on the acid taste due to the concentration of H3O, saltiness was due to NaCl whereas bitterness was mainly due to the bitter peptides found in the fraction with a molecular weight in the range 500–1000 Da (Engel et al., 2001b). The intense proteolytic activity of the strain of Penicillium camemberti, specially selected to develop bitterness in this case, has been demonstrated to be responsible for the accumula-

tion of small (MW 1000 Da) bitter peptides during ripening (Engel et al., 2001c).

Dynamic Methods for Flavour Characterisation Even if the ‘best’ extraction and identification methods are used, poor correlations are often found between the overall levels of flavour components (volatile and non-volatile) and sensory perception experienced by a consumer. In other words, it is not enough to know the exact composition of food in terms of flavour compounds to understand perfectly the perception of its flavour. In fact, the perception of flavour is a dynamic process (Piggott, 2000). During the consumption of food, the concentration of aroma compounds at the olfactory epithelium and of sapid compounds at the taste buds varies with time. Flavour components are released progressively from the food matrix during chewing. Kinetics of the release of flavour depends on the nature of the food matrix composition and of individual mastication pattern. Sensory methods, such as time-intensity, have been used to study the dynamicand time-related aspects of flavour perception (Piggott, 2000). Release of volatiles in vivo

Techniques which measure volatiles directly in the mouth or in the nose have been developed to obtain physico-chemical data that reflect the pattern of aroma molecules released from food and that are effectively present at the olfactory receptors during consumption (Linforth and Taylor, 1993; Taylor and Linforth, 1994). Among the various approaches aimed at sampling aroma from the nose (nose-space), the collection of expired air samples on Tenax® traps (Fig. 4) provided the first robust results (Linforth and Taylor, 1993; Taylor and Linforth, 1994). When applied to

Tenax trap

GC

MS

Analysis Pump Sampling

Figure 4 Collection and analysis of expired air by Tenax trapping and GC–MS (reproduced from Roberts and Taylor (2000), with permission from the American Chemical Society).


Cheddar cheese (Delahunty et al., 1994), the ‘buccal headspace’ method demonstrated that, despite a similar composition of volatiles found with conventional headspace analysis, some cheeses, depending on their fat content, released a different balance of volatiles during consumption (Delahunty et al., 1996a). Gas chromatography–olfactometry of buccal headspace showed a number of volatile compounds which have been suspected to contribute primarily and most likely to Cheddar cheese flavour (Delahunty et al., 1996b). It was presumed that the buccal headspace extract was representative of the aroma compounds that a consumer perceives during consumption (O’Riordan and Delahunty, 2001). By overlapping the sampling time periods, release curves can be constructed and temporal changes reflecting relative concentrations of volatiles at a particular moment during consumption can be determined (Linforth et al., 1996). When applied to Cheddar cheese, ‘temporal buccal headspace’ results, obtained on an accumulated ‘time-concentration’ basis (four time periods: 15, 30, 45 and 60 s of cheese consumption), were correlated with sensory time–intensity data (Delahunty et al., 1996c). Time-course data confirmed the results of conventional analysis while providing improved sensory predictions from the instrumental results (Delahunty et al., 1996c). Mastication behaviour using electromyography and saliva production rates of individuals have also been measured during consumption of Cheddar cheese (Delahunty et al., 1998; O’Riordan et al., 1998). Combined to nose-space analysis and sensory evaluation using free choice profiling, these authors demonstrated that although there were differences in chewing styles and saliva production rates, the assessors’ individual nose-space profiles were very similar for all Cheddar cheeses examined (Delahunty et al., 1998). Partial least-squares regression analysis allowed the most important flavour differences between cheeses to be predicted from the volatiles released during consumption (O’Riordan et al., 1998). Recently, atmospheric pressure ionisation–mass spectrometry (API–MS) has been developed to monitor aroma release during chewing (Taylor et al., 2000). Air from the nose (nose-space) is sampled directly into the API-MS source through an interface (Fig. 5), making real time breath-by-breath analysis possible (Linforth et al., 1996; Taylor and Linforth, 1996). Therefore, by combining time–intensity sensory studies with nose-space analysis, it is now possible to relate temporal parameters of aroma release to perception (Linforth et al., 2000). The method, reviewed in detail in specialised treatises (Roberts and Taylor, 2000; Taylor, 2002), has been applied recently to soft French

Nitrogen 10 l/min

Cone

Venturi

Quad

Breath flow

Corona pin

Figure 5 Principle of the API-MS interface used for breath by breath analysis (reproduced from Roberts and Taylor (2000), with permission from the American Chemical Society).

cheeses (Salles et al., 2003). Three French mouldripened soft cheeses (Brie made from pasteurised milk, Camembert made from pasteurised milk and from raw milk) were evaluated by a panel of 15 assessors (Salles et al., 2003). Retronasal aroma profiles made by citation frequency of attributes revealed four main descriptors for the three cheeses. The sulphury note (cabbage/cauliflower/vegetable) was particularly intense for the Camembert cheeses, while the buttery/creamy note was important for the three cheeses studied; the mushroom attribute was less intense in the Camembert cheeses, and ammonia was perceived in all cheeses but was found particularly difficult to score by the panellists (Salles et al., 2003). Therefore, the three main aroma notes (sulphury, buttery and mushroom) were selected for subsequent time–intensity (TI) scoring (15 assessors evaluated each attribute, with three replicates of each cheese). Gas chromatography–olfactometry of the dynamic headspace sampling of the three cheeses allowed odour-active compounds to be identified, amongst which sulphur compounds (methanethiol, dimethylsulphide (DMS), S-methylthioacetate, dimethyldisulphide (DMDS), 2,4-dithiapentane, dimethyltrisulphide, 2,3,5-trithiahexane and dimethyltetrasulphide) could be related to the sulphury attribute scored by the panellists. However, API-MS nose-space experiments allowed the detection of only six compounds of which three contained sulphur ones (DMS, S-methylthioacetate and DMDS). Simultaneous TI scoring of the sulphury note allowed a perfect superposition of the time–intensity curve with the release of the sulphur compounds (Fig. 6). The most significant perception and flavour release parameters allowed the three cheeses to be well discriminated by principal component analysis (PCA), showing a good agreement between perception scored by assessors and consistency in their release of aroma compounds while eating cheeses (Salles et al., 2003). Another PCA analysis showed a positive correlation for the sulphury note between the perception parameters derived from the TI curves and parameters derived from the aroma

Perceived intensity


10 9 8 7 6 5 4 3 2 1 0

T-I Sulphury/cabbage attribute

I (m/z 90.8)

25 20

S-methyl thioacetate m/z 90.8

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I (m/z 93.7)

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I (m/z 62.9)

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Time in min Figure 6 Flavour release from Camembert cheese for one assessor. Simultaneous time–intensity scoring of the sulphury/ cabbage attribute and API-MS analysis of S-methyl thioacetate, dimethyldisulphide (DMDS) and dimethylsulphide (DMS) in the nose-space.

release curves (Salles et al., 2003), as suggested by the characteristic curves presented in Fig. 6 for one panellist within one session. Non-volatiles in vivo

Development of methods to study flavour release has concentrated mainly on the volatile fraction, while only a limited number of studies have been devoted to the release of non-volatile compounds in the mouth. Conductivity measurements have been used to relate the release of salt during chewing to Cheddar cheese texture (Jack et al., 1995), and a similar approach with additional in-mouth measurement of pH has been used with a variety of foodstuffs, including Cheddar cheese (Davidson et al., 1998). However, in these approaches, the sensors available for in vivo measurements only give the best estimate for salt (non-specific to sodium) and acid release. Saliva sampling using cotton buds coupled to a direct liquid mass spectrometry procedure has been described to study the rate of release of sucrose (Davidson et al., 1999). Panellists were instructed to take a swab from a specific location on the tongue at different times during mastication using a cotton bud. The weight of saliva swabbed was measured and sucrose concentration was monitored using liquid-API–MS after extraction by a methanol– water solution (Davidson et al., 1999). A continuous

sampling technique using a motor-driven ribbon placed across the tongue while a panellist chews a food sample has also been described (Davidson et al., 2000). At the end of the eating process, the ribbon was cut into 5 cm lengths after estimation of the saliva weight adsorbed on the ribbon, each piece representing a certain time. Non-volatile components were extracted from the pieces of ribbon with a solvent and their concentration determined by direct liquid phase API- or electrospray (ES)–MS (Davidson et al., 2000). Temporal release of sucrose and glucose from biscuits, of sodium from potato crisps, of sucrose, glucose and fructose, citric and malic acids from fresh orange and finally minerals (sodium, calcium and potassium) from Cheddar cheese was monitored successfully (Davidson et al., 2000). The cotton bud technique has been applied recently to a model processed cheese in which aroma and non-volatiles compounds consistent with literature data had been incorporated (Pionnier et al., 2003). As it was demonstrated that with certain foodstuffs the increased frequency of sampling affected the chewing pattern (Davidson et al., 2000), each panellist produced only one saliva sample per mastication, at a time-consuming cost, however. Using ES–MS in negative ionisation mode, time-course release curves for minerals (sodium, calcium, magnesium and potassium), amino acids (leucine, phenylalanine, glutamic acid), organic acids (citric, lactic, propanoic and butyric) and phosphoric acid have been obtained (Pionnier et al., 2003). As a typical example, Fig. 7, shows release curves from cheese for phenylalanine, glutamic acid, leucine, phosphoric and lactic acids obtained for one assessor. The first conclusion that could be stressed from the analyses of the release curves is that individual physiological parameters (mainly mastication behaviour and salivation rate) are related more closely to the temporal release of taste compounds than to their physico-chemical properties (Pionnier et al., 2003). Model mouth systems

A number of mechanical devices which mimic in more or less detail the processes that occur in the mouth during eating ‘model mouths’ have been developed (Piggott, 2000 and references therein). These are often variants of dynamic headspace analysis, but their aim is to obtain time-resolved samples containing volatiles as similar as possible to those present during actual eating. The various parameters like temperature, air flow, mastication rate and addition of artificial saliva can be varied to study their effects on volatile flavour release. The main advantages of model mouths are the large quantities of food samples that can be handled, overcoming some sensitivity problems encountered


Phenylalanine Glutamic acid Leucine Phosphoric acid Lactic acid

Concentration (g/100 g saliva)

0.6 0.5 End of mastication 0.4 0.3 0.2 0.1 0.0 0

10

20

30

40

50 Time (s)

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Figure 7 Flavour release from a flavoured model cheese for one assessor. In mouth, time-course release of non-volatile compounds (concentration in g/100 g saliva) using the cotton bud technique. Data points measured in electrospray-MS in negative ionisation mode are the mean of three replicates.

when monitoring volatiles at low concentrations (Taylor, 2002), and the suppression of inter-individual variations, always encountered when working with a panel, that can be detrimental to a robust interpretation of the data. Recently, using an imitation cheese preparation, the release of volatile flavour compounds from the Retronasal Aroma Simulator (RAS), originally developed by Roberts and Acree (1995), has been compared with flavour release in vivo using API-MS detection in both cases (Deibler et al., 2001). While delivering higher concentrations of volatiles than from human breath, the RAS gave a good approximation of time-averaged flavour release in the mouth, with volatile compounds present at similar ratios (Deibler et al., 2001). Volatiles in the RAS effluent from Cheddar, Brie and vanilla ice cream were measurable (Deibler et al., 2001). The model-mouth device originally developed by Roozen and co-workers (van Ruth et al., 1994) has been used to investigate the relationships between the gross, non-volatile and volatile compositions and the sensory attributes of eight Swiss-type cheeses (Lawlor et al., 2002). Eight flavour attributes were found to be correlated with subsets of volatiles, amino acids, free fatty acids and gross compositional constituents with, for instance, the nutty flavour of Emmental that was positively correlated with the concentrations of propionic acid, ethyl acetate and 2-pentanone (Lawlor et al., 2002). Flavour release and flavour perception are dynamic processes and must be studied using dynamic methods (Piggott, 2000). Dynamic physico-chemical methods have been developed to study the parameters of flavour release from foods. Parallel increased applications of dynamic sensory methods provide a better understanding of food flavour, with important results obtained for

cheese flavour. However, further work is needed to improve our knowledge of various interactions arising at different levels in the process of food consumption: e.g., interactions between food ingredients (Delahunty and Piggott, 1995; Pionnier et al., 2002; Taylor, 2002), and interactions at the perceptual levels such as tastearoma interactions (Noble, 1996; Given and Paredes, 2002; Hollowood et al., 2002; Taylor, 2002), or trigeminal interferences (Green, 1996; Given and Paredes, 2002), as these play a fundamental role in overall flavour perception.

Global and Fast Assessment of Cheese Flavour The methods currently used to evaluate and control the quality of cheese flavour are still essentially based on sensory evaluation by a panel of experts. These trained panels are able to handle such difficult tasks like quality monitoring through descriptive analysis, off-flavour detection and comparison of samples for classification purposes. It could be interesting for such tasks to substitute humans by instruments that could give quicker answers at a reduced cost. Electronic nose

Evaluation of the complete aroma emitted from food using gas sensors, the so-called ‘electronic noses’, is now theoretically feasible (Hodgins, 1997; Schaller et al., 1998). Electronic noses are composed of arrays of non-specific gas sensors which are based on different physical principles (Hodgins, 1997; Schaller et al., 1998). The most common sensors are semiconducting metal oxides and conducting organic polymers, but


they all give rise to a response with a typical pattern. Therefore, pattern recognition software, using either standard statistics or artificial neural network technology, must be used for data treatment and final presentation of the results (Hodgins, 1997; Schaller et al., 1998). The electronic nose is particularly attractive for quality control applications where conformity/nonconformity answers are expected. Some discriminative studies have been conducted on cheese samples (Schaller et al., 1998 and references cited therein). Using metal oxide semiconductors, it was possible to distinguish between five Swiss cheese varieties (Mariaca and Bosset, 1997). However, some problems occurred with the repeatability of the system that could be possibly related to the product itself, the sampling technique or the moisture content of the air used for sampling, precluding its use in routine tests (Schaller et al., 1998). Samples of Swiss Emmental cheese at different stages of ripening have been evaluated using different technologies over a period of one year (Schaller et al., 1999). The metal oxide semiconductors technology has given the best discriminative results. However, the sensors seemed to be damaged by short-chain fatty acids released from cheese. Conducting organic polymer sensors showed poor sensitivity to volatile components of cheese, the main problem being that these sensors are unstable (Schaller et al., 2000a). The other technologies tested were not sensitive enough to cheese volatile compounds and electronic noses containing these sensors showed poor discriminative power (Schaller et al., 1999). However, recently, the ripening of Danish Blue cheese was monitored by means of an electronic nose which contained 14 conducting polymer (polyaniline) sensors; results were found to be highly correlated to those of sensory analysis and GC–MS analysis of volatile compounds during a 5–12-week ripening period (Trihaas et al., 2003). The close control of the experimental sampling conditions (quality of dry air with a humidity 0.5% and equilibration time at controlled temperature) might explain this success (Schaller et al., 1998). Nevertheless, despite some success in some classification tasks when using perfectly controlled sampling conditions, electronic noses hardly meet the requirements of the food industry in terms of precision, reproducibility, sensitivity and stability. Of particular importance, the sensors are known to deteriorate over time or can be poisoned, therefore changing their response. Even with frequent calibration, the inherent weaknesses of the technique make the general applicability of the databases problematic. Moreover, these instruments cannot be used to identify single odorants or to differentiate samples with subtle differences in distinctive sensory attributes. Therefore, in off-flavour studies,

where identification of the off-flavour compound is a pre-requisite and in quality control assessment, they may be used successfully only after recognising their inherent weaknesses (Reineccius, 2002). Mass spectrometry-based systems

For classification purposes, two other global and fast analytical methods, based on mass spectrometry, have been used for dairy products and seem more powerful and reliable than electronic noses. The first consists of a global analysis of a headspace sample by a mass spectrometer operated in electron ionisation mode, without GC separation (Vernat and Berdagué, 1995). The feasibility of the method was originally demonstrated for rapid classification of four rather different French cheeses (Vernat and Berdagué, 1995). This method is often described as a ‘MS-based electronic nose’ (Schaller et al., 2000b). The mass patterns obtained, considered as fingerprints of the food products analysed, also need data treatment, either by conventional statistics or artificial neural networks. The technique has been used successfully to discriminate four Swiss Emmental cheeses differing in age (Schaller et al., 2000b), and Camembert-type cheeses according to their origin, manufacturing process or ripening stage (Pérès et al., 2002a). Solid-phase microextraction may be used as a preconcentration technique instead of dynamic headspace analysis (Marsili, 1999). Applied to rapid characterisation of cheeses, SPME has been demonstrated to be a very efficient pre-concentration technique (Schaller et al., 2000b). In the task of discriminating Swiss Emmental cheeses ripened for different times, SPME has been found to be superior to dynamic headspace analysis in terms of repeatability, simplicity and compatibility with an autosampler (Schaller et al., 2000b). However, when applied to the characterisation of Camembert-type cheese (Pérès et al., 2001), SPME yielded less satisfactory results than those obtained by dynamic headspace analysis (Pérès et al., 2002a). The better performance of the dynamic headspace method in that case was attributed to the absence of signal drift (ageing of the SPME fibres causes drift, as demonstrated by Pérès et al., 2001) and to automation of the injection of sample into the mass spectrometer. According to the authors, the protocol chosen for the analysis by dynamic headspace-MS was more efficient than SPME in terms of extraction yield, and reduced thermal, mechanical and chemical modification of the samples (Pérès et al., 2002a). Developed in the 1980s for food applications, direct pyrolysis-MS is another method that delivers ‘fingerprints’ which can be used for classification/authentication


purposes (Aries and Gutteridge, 1987). With this method, a tiny sample is pyrolysed rapidly at up to 530 °C and the resulting volatile fraction, characteristic of the flavour but also of the matrix breakdown, is analysed immediately by a mass spectrometer operated in low energy electron ionisation mode. Here again a mass pattern, this time rather complex, is obtained for each sample and several data pre-processing steps are often necessary to select a reduced number of mass fragments that allow satisfactory classification. Curiepoint pyrolysis–mass spectrometry with associated multivariate data analysis techniques is considered as a powerful classification tool in microbiology for the recognition of micro-organisms (Talon et al., 2002 and references cited therein) and food science (Aries and Gutteridge, 1987; Pérès et al., 2002b and references cited therein). However, when applied to the discrimination of five Camembert-type cheeses, it appeared less competitive than SPME–MS or dynamic headspace–MS in terms of sample preparation and analysis time (Pérès et al., 2002b). The main advantage of the method is that it provides a specific fingerprint of the cheese matrix which could be potentially related to textural parameters (Pérès et al., 2002b). Recently, in a similar approach, the proton transfer reaction mass spectra (PTR-MS, another atmospheric pressure ionisation mode MS source) of the static headspace of Mozzarella cheese have been found to display comparable discrimination power to sensory descriptive analysis (Gasperi et al., 2001).

Concluding Remarks Cheese is a biochemically active product that undergoes many changes during ripening. The development of flavour is one of the consequences of these biochemical changes that occur over the entire ripening period. Modern instrumental methods allow for detailed analyses of volatile compounds, and some pertinent complementary sensory information can be obtained by combining gas chromatography with olfactometry. Recent developments have allowed the identification of the role of non-volatile components in the overall flavour of cheese. Nevertheless, the relationship between flavour–aroma and sapid compounds present in a foodstuff and sensory perception of that food by a consumer is not so easy to establish. It is still not well understood how the various flavour-active components combine to produce a particular sensory perception. Recent developments in dynamic instrumental methods that can follow the in vivo sequential release of the flavour molecules are valuable tools that can account for the balance of flavour compounds released, a balance that changes with time. With more complete and

accurate information, combined flavour chemistry and sensory evaluation should help understand the relationship between flavour stimuli and perceived flavour and explain the mechanisms of flavour perception. Authentication of cheese (for instance, varieties with protected designations of origins) is another challenge. Tools developed recently that combine analytical instrumentation for global assessment of flavour with multivariate data analyses have demonstrated their usefulness for classification purposes.

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Rheology and Texture of Cheese D.J. O’Callaghan and T.P. Guinee, Dairy Products Research Centre, Teagasc, Ireland

Introduction – Overview of Cheese Rheology and Texture Rheology of materials, e.g., cheese, may be defined simply as the study of their deformation and flow when subjected to a stress or strain. The rheological properties of cheese are those that determine its response to stress or strain, as applied, for example, during compression, shearing or cutting. In practice, such stresses and strains are applied to cheese during processing (e.g., portioning, slicing, shredding and grating) and consumption (slicing, spreading, masticating and chewing). The rheological properties include intrinsic characteristics such as elasticity, viscosity and viscoelasticity that are related primarily to the composition, structure and the strength of attractions between the structural elements of the cheese. The rheological characteristics of cheese are quantified by rheological quantities that are measured in tests involving the application of stress or strain under defined experimental conditions. The output variables from these tests (e.g., creep, stress relaxation, compression tests), which may include change in dimensions over time, the ratio of stress-to-strain for certain strain levels, stress or strain required to induce fracture, enable the determination of quantities such as shear modulus, fracture stress and firmness. In lay terms, the behaviour of the cheese when subjected to these stresses and strains is referred to by descriptive terms such as hardness, firmness, springiness, crumbliness or adhesiveness. Owing to the variations in manufacturing conditions and composition, different cheese varieties exhibit a wide range of rheological behaviour, ranging from the viscous behaviour of soft cheese to the elastic behaviour of hard cheeses at low strain. The rheological properties of cheese are of considerable importance as they affect: 1. its handling, portioning and packing characteristics; 2. its texture and eating quality, as they determine the effort required to masticate the cheese or alternatively the level of mastication achieved for a given level of chewing. The degree of chewing required may, in turn, influence the flavour/aroma properties and the suitability of the cheese for different consumer groups (e.g., children, aged);

3. the use of cheese as an ingredient, as they influence its behaviour when subjected to different size reduction methods (such as shredding, grating or shearing) and how it interacts and blends with other ingredients in foods in which cheese is an ingredient. 4. its ability to retain a given shape at a given temperature or when stacked; 5. its ability to retain gas and hence to form eyes or cracks or to swell. Hence, the rheological properties of cheese are significant quality attributes of importance to the manufacturer, pre-packer, distributor, retailer, industrial user and consumer. The rheology of cheese is a function of its composition, microstructure (i.e., the structural arrangement of its components), the physico-chemical state of its components, and its macrostructure, which reflects the presence of heterogenities such as curd granule junctions, cracks and fissures. The physicochemical properties include parameters such as the level of fat coalescence, ratio of solid-to-liquid fat, degree of hydrolysis and hydration of the paracasein matrix, and the level of inter-molecular attractions between para-casein molecules. Hence, the rheological characteristics differ markedly with the cheese variety and age. The effect of variety on the rheological properties is readily apparent on comparison of an almost-flowable mature Camembert with a firm, brittle Parmesan or of a crumbly Cheshire cheese or with an elastic springy Swiss-type cheese or String cheese (Table 1). Similarly, the influence of age is clear on comparison of a young (e.g., 1–2 months) rubbery Cheddar with a fully mature pliable Cheddar (Table 1). Cheese rheology and factors that affect it have been reviewed extensively (Sherman, 1969; Eberhard, 1985; Visser, 1991; van Vliet, 1991a; Rao, 1992; Prentice et al., 1993; Ustunol et al., 1995; Beal and Mittal, 2000; Fox et al., 2000; Madsen and Ardö, 2001; Guinee, 2003). In this chapter, the basic rheological characteristics of cheeses in general and the methods for their quantification will be examined. The effects of compositional and biochemical factors on the rheological properties of cheese are discussed in ‘Cheese as an Ingredient’, Volume 2. For detailed information on cheese texture,



512 Rheology and Texture of Cheese

Table 1 Rheological properties of raw cheese and their definitionsa Cheese type displaying property

Rheological property

Definition

Elasticity (rubberiness)

Tendency of cheese to recover its original shape and dimensions upon removal of an applied stress Tendency to recover from large deformation (strain) after removal of deforming stress Tendency of hard cheese to crack, with very limited flow (confined to vicinity of crack); after fracture, the broken surfaces can be fitted to each other Tendency of hard cheese to fracture at a relatively low permanent deformation High resistance to deformation by applied stress

Springiness Elastic fracturability

Brittleness Firmness (hardness) Longness Toughness (chewiness)

The resistance of cheese to fracture until a relatively large deformation is attained A high resistance to breakdown upon mastication

Softness Plastic fracturability

Low resistance to deformation by applied force The tendency of cheese to flow on fracture

Shortness

The tendency to plastic fracture at a small deformation; low resistance to breakdown upon mastication The tendency to resist separation from another material with which it makes contact (e.g., another ingredient or a surface such as a knife blade or palate) The tendency to break down easily into small, irregularly shaped particles (e.g., by rubbing) The tendency to increase in apparent viscosity when subjected to an increasing shear rate (especially upon heating) The tendency to exhibit a decrease in apparent viscosity when subjected to an increasing shear rate

Adhesiveness (stickiness)

Crumbliness Shear thickening

Shear thinning

Swiss-type cheese, low-moisture Mozzarella Swiss-type cheese, low-moisture Mozzarella Parmesan, Romano, Gruyere

Romano, Parmesan Cheddar, Swiss-type cheese, Romano, Parmesan, Gouda Mozzarella, Swiss Mozzarella, String cheese, Halloumi Blue cheese, Brie, Cream cheese Mature Cheddar, Blue cheese, Chaumes, Raclette Camembert, Brie Mature Camembert

Cheshire, Wensyledale, Blue cheese, Stilton, Feta Cream cheese (when heated), ‘creaming’ of processed cheese products Quarg (especially at low temperatures, i.e., 4 °C)

a Definitions modified from Szczesniak (1963a), van Vliet (1991a) and Fox et al. (2000).

the reader is referred to ‘Sensory Character of Cheese and its Evaluation’, Volume 1 and the following reviews: Szczesniak (1963a,b, 1998), Brennan (1988) and Rosenthal (1999).

Terminology of Rheology and Texture General rheological terminology

The general terminology used to describe the rheology of materials has been discussed extensively (Sherman, 1983; Rao and Steffe, 1992; Whorlow, 1992; Collyer and Clegg, 1998; Sharma et al., 1999). The terms most commonly applied to the rheology of cheese are described in Table 1. Deformation and strain

Any rheological measurement involves deforming a sample of material by applying a force, e.g., by compression or by shear (Fig. 1). The displacement in response to the force at the point of application is known as deformation. The term ‘deformation’ used in this sense

does not imply permanent deformation but rather a change in shape (i.e., form) which may be temporary, permanent or partly recoverable. A series of instantaneous measurements of force and associated displacement describe the rheological characteristics of a material under the measurement conditions. The conditions which affect the force–displacement response include temperature, type of deformation (compression, extension, shear or pressure), level of deformation in relation to the elastic limit and fracture point of the material, rate of deformation, previous history of deformation. Strain may be defined as the fractional displacement that occurs under an applied stress. Stress

Stress is defined as the distribution of force over an area of a material. The ‘area’ over which a force is distributed may be a surface (e.g., the surface of a cylindrical sample exposed to a compression plate) or an imaginary section within a material (e.g., an internal fracture plane). The force applied at a surface is distributed throughout the material and is borne by the structural elements, e.g., in

Rheology and Texture of Cheese 513

Table 2 Rheological properties derived from stress/strain curves obtained from large strain deformation of cheese Textural characteristic to which parameter is related

Rheological Property

Abbreviation

Interpretation

Elastic, or compression, modulus Apparent elastic modulus, or Deformability Modulus

E /

Fracture stress

f

Fracture strain

f

Measure of elasticity at low strain Ratio of stress to strain in a viscoelastic region below the fracture point Stress required for fracture and collapse of cheese mass beyond point of recovery. Deformation required to induce fracture

Firmness (maximum stress)

max

Fracture work

Wf

Stress to required to compress cheese sample to a given deformation The energy required to fracture the cheese

the case of cheese, the casein strands of the matrix and the occluded fat globules. The rheological behaviour of the material is effected by the response of the structural elements to the applied stress. The initial response of a cheese sample to an applied stress is determined mainly by the para-casein matrix. At larger deformations, the moisture and fat phases, which are occluded in the matrix, contribute to the rheological response. Shear and normal modes of stress and strain

Two modes of stress can be applied on a surface, namely shear or normal. Shear stress is created when a force is applied parallel to the plane of a surface element, whereas normal stress is created by a force applied perpendicularly to a surface element. Normal stress, , is defined as:

F A

Strength of cheese matrix

brittleness and “shortness” or “longness” of cheese firmness or hardness toughness

where A is the cross-sectional area over which the force (F) is applied (Fig. 1). Normal stress can occur in tension or compression. In a large strain compression situation, two expressions are used for normal stress, differing in respect of the calculation of area. Apparent stress is the applied force divided by the original crosssectional area of the sample, while true (or, more strictly, corrected) stress is the applied force divided by the instantaneous area of the sample, allowing for the fact that the sample spreads as it is compressed. When true stress is plotted against strain, a more distinct peak is observed around the fracture point (Fig. 2). However, the instantaneous area is not easily measured and is often approximated by a calculation based on constant volume (Ak and Gunasekaran, 1992). Shear stress, , is defined as:

F

Elasticity Elasticity

F A

kPa ΔL F A

A

450 400 350

σ

300 250 200

ΔL Lo

150

Lo

(a)

σt

100 50 0 (b)

Figure 1 Deformation of a solid material by the application of a force, F, (a) in a direction normal to the surface (area, A), resulting in a compression deformation, or (b) tangential to the surface resulting in shear deformation. The stress is calculated as F/A; strain is calculated as L/Lo , where Lo is the original length of the sample of material.

0

0.2

0.4

ε

0.6

0.8

Figure 2 Apparent stress, , and true stress, t, plotted against strain, ␧, from force–displacement data obtained in the compression of Cheddar cheese on a texture analyser, Stable Micro Systems, model TA.HDi, showing a more distinct peak near the fracture point in the t plot.


where A is the cross-sectional area over which the shear force (F) is distributed (Fig. 1). Two alternative expressions for normal strain have been used in tensile or compression situations, namely Cauchy strain and Hencky strain. Cauchy strain ( ), also referred to as strain, apparent strain or engineering strain, is defined as the deformation relative to the original sample dimension, i.e.,

L Lo

where Lo is the original height of the sample and L is the displacement under applied stress, (Fig. 1). Hencky strain, sometimes referred to as natural strain or true strain, is defined as the natural logarithm of the ratio between the sample length upon application of force and the original length, i.e.,

t ln

冢 LL 冣, o

where Lo is the original sample length and L is the length under load. Hencky strain is thought to be more relevant than engineering strain in describing fluid (or non-recoverable) behaviour, e.g., squeeze flow patterns of deformation, as occur in large strain compression and in spreading cheese on a cracker. However, for small strains, the Hencky strain approaches the Cauchy strain (Fig. 3). The relationship between Cauchy and Hencky strain can be derived as:

t ln(1 ). Ideally, engineering strain should be used for recoverable (elastic) deformations and true strain for nonrecoverable (viscous) deformations. Obviously, this is

Initial height, Lo = 25 mm Cauchy strain

Hencky strain

0 5 10 15 20 25

0 0.20 0.40 0.60 0.80 1.00

0.00 0.22 0.51 0.92 1.61 8

Displacement, ΔL (mm)

Figure 3 Deformation of a sample of cheese, originally 25 mm high (L o), under axial compression, showing equivalence between displacement (L), Cauchy strain ( ) and Hencky strain ( t ).

impractical, as the transition from elastic to viscous behaviour is gradual. Throughout this text, strain is used in the engineering or Cauchy sense, unless otherwise stated. Shear strain, is defined as:

L Lo

where L is the shear (tangential) displacement on the application of shear stress, . Compression testing is generally used for the rheological evaluation of cheese because of the relatively low tensile strength of most cheeses, e.g., compared to its compression strength. Exceptions include members of the pasta-filata family of cheeses, such as Mozzarella and Haloumi (see ‘Pasta-Filata Cheeses’, Volume 2), which when heated are able to undergo a high degree of stretching when pulled. As discussed in ‘Pasta-Filata Cheeses’ and ‘Cheese as an Ingredient’, Volume 2, this characteristic is associated with the presence of para-casein fibres which are formed during the exposure of the curds to high temperatures (e.g., 58–60 °C) at a low pH (e.g., 5.1–5.4) during manufacture and stretching. In practice, normal and shear stresses occur simultaneously during testing, size reduction at industrial level (e.g., comminution, shredding, grating) and consumption (mastication). In a compression test, a normal force is applied but fracture generally occurs as a consequence of shear stresses built up in the sample. Likewise, in a torsion test, a normal force must be applied to maintain sufficient contact between the sample and the plate delivering the shear stress. In general, the simplest fundamental rheological properties (e.g., Young’s modulus, shear modulus) are defined for one mode of stress in one dimension, and for this reason, rheological measurements often attempt to confine stresses to one mode and one dimension. However, this is possible only in some low deformation situations, since stresses in one dimension tend to produce structural displacements, and hence stresses, in other dimensions and modes. Thus, it is not possible to create large deformations in one dimension in isolation, as for example during compression testing when deformations exceed the linear viscoelastic limit (see ‘Fundamental Measurements: Oscillatory Rheometry for Linear Viscoelastic Measurements in Cheese’). However, cheese generally undergoes relatively large deformation during handling and consumption, and hence it is necessary to describe its rheological characteristics under large deformation conditions, e.g., during compression by the molar teeth (⬃70%). It is difficult to measure shear and compression stresses simultaneously


in all dimensions under large strain deformation, and, consequently, much use is made of empirical or semiempirical methods to describe the rheology of cheese or other foods under large strain deformation conditions to which cheese is subjected in practice. In contrast, low strain linear viscoelastic tests, while giving precise rheological quantities (i.e., storage and loss moduli) and indirect information on structure, tell little about the expected rheological behaviour of the cheese during processing and eating. Bagley and Christianson (1987) suggested a generalised approach to the measurement and interpretation of the rheological properties of foods aimed at dealing with the difficulties in describing behaviour that is highly viscous and highly elastic at the same time. With this approach, constants can be derived which enable the rheological property being measured in a given test, e.g., compression modulus at low strain during compression testing, to be related to a rheological property measured in another test, e.g., shear modulus in a torsion test or shear test. In practice, inhomogeneities and graininess in cheese can confound such comparisons and it is difficult to interpret the significance of such results (Bagley and Christianson, 1987).

Under low , solids, including some cheeses (e.g., a young – medium-aged, low-moisture, part-skim Mozzarella cheese), exhibit a simple linear relationship between and which can be expressed in terms of various moduli. In compression or tensile testing, Young’s Modulus (E) may be defined as: E

where is the normal stress and is the strain on the material. In shear tests, the shear modulus (G) is given by: G

where is the shear stress and is the shear strain. The above elastic moduli are intrinsic rheological characteristics of the material, that are independent of sample dimensions, time and strain rate. However, for most cheeses, the elastic region is small (e.g., 0.006; Guinee et al., 2000a,b) and of little consequence because most strains applied in practice are 0.05. Bulk modulus and compressibility of cheese

The relationship between stress and strain

Stress and strain at a micro level result from an externally applied force at a macro level, the displacement at the point of application being the cumulative effect of a strain at every point along the length of the sample (Fig. 4). The relationship between stress and strain is characteristic of the material but depends on temperature, and for viscoelastic materials, on other factors including the time over which the stress is applied and the pre-test stress–strain history and the rate of strain (see ‘Uniaxial Compression’).

Isotropic stress, or pressure, is sometimes described as a third mode of stress. This usually occurs in fluid materials and is really a normal force applied equally in each of three dimensions. In general, the application of isotropic stress to a material results in a slight reduction in volume, or shrinkage. From the volume reduction, a bulk modulus (K) may be determined and is defined as: K

PV V Sample surface

---- Sample interior -----

Area

Force

Force

Displacement

Stress Modulus

Stress

Strain

L

Strain

Original height X Displacement

(a)

(b)

Figure 4 The application of an external force over a surface area results in stress and strain throughout the sample, as illustrated in (a). The displacement at the surface of application is the cumulative effect of a strain at every point, e.g., X, along the length of the sample. For low strain deformations there is a linear relationship between stress and strain, and a modulus, equal to the ratio between them, may be determined (b).


where P is the applied pressure, V is the initial volume of material and V is the change in volume. There are simple relationships between compressibility, usually expressed in terms of bulk modulus (K), Young’s modulus (E) and Poisson’s ratio () (Whorlow, 1992; Rosenthal, 1999): E 3K(1 2) and E 2G(1 ) Since ⬃0.5 for most cheeses (see Poisson effect in Glossary), these relationships simplify further to:

Rheological concepts applied to cheese Cheese structure

Cheese is essentially a concentrated protein gel, which occludes fat and moisture. Gelation is brought about by either of the following mechanisms (see ‘Rennetinduced Coagulation of Milk’ and ‘Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels’, Volume 1): 1. slow quiescent acidification (e.g., using a starter culture or food-grade acid and/or acidogen), at a temperature of 20–40 °C, to the isoelectric pH of casein, i.e., ⬃4.6; 2. sensitisation of the casein to calcium via the hydrolysis of the principal micelle-stabilising casein, -casein, by added acid proteinases (i.e., rennets); or

E 3G

(b) Force, F velocity, v at depth, y

Figure 5 Two situations where viscous forces are at work: (a) flow between parallel plates which move relative to each other; (b) flow in a pipe.

Strain, γ

However, all of the above relationships apply only in the linear (i.e., elastic) region. The volume reduction up to the point of fracture is about 9% for Cheddar cheese (Calzada and Peleg, 1978). Cheese with a large vacuole volume (e.g., where eyes occupy a significant proportion of the volume, as in Swiss cheese), are more compressible and therefore have lower K values than cheeses without eyes. However, unlike their behaviour under normal or shear stresses, most cheeses are relatively non-compressible under isotropic stress. Consequently, from a practical point of view, their bulk modulus is of little interest, but may be of interest in the calculation of true stress in uniaxial compression (see ‘Uniaxial Compression’).

(a)

ar

pe

Slo

=

e sh

e

rat

Time

Flow is normally the result of shear displacement. Shear forces occur when a liquid flows inside a pipe or when a molten mass (e.g., melted cheese) flows along a surface (Fig. 5). In a fluid, strain is not recoverable and applied stress results in a continuously changing strain. A viscous material behaves as a fluid and responds to shear (stress), in terms of strain, in a time-dependent manner (Fig. 6). For an ideal viscous material, i.e., a Newtonian fluid, the rate of strain is proportional to applied stress (Fig. 7). The relationship between stress () and rate of strain . ( ), or shear rate, is described by the coefficient of viscosity ():

.

Figure 6 Ideal viscous (Newtonian) response to constant . applied stress, i.e., strain () increases at a constant rate ( ).

Shear rate, γ arbitrary units

Viscous deformation

.

10 8 6 4 2 0 0

2

4 6 8 Stress, σ, arbitrary units

10

Figure 7 Relationship between shear stress (s) and shear rate . ( ) for an ideal (Newtonian) liquid.


3. a combination of acid and heat, e.g., heating milk to ⬃90 °C at ⬃pH 5.6. The micro-structure of milk gels and cheeses has been studied extensively (Hall and Creamer, 1972; Kalab and Harwalkar, 1974; Kimber et al., 1974; Kalab, 1977, 1979; de Jong, 1978; Green et al., 1981a,b, 1983; Green, 1990a,b; Kiely et al., 1992, 1993; Mistry and Anderson, 1993; Bryant et al., 1995; Desai and Nolting, 1995; Everett et al., 1995; Guinee et al., 2000a). The physico-chemical properties of the para-casein matrix and occluded components may be deduced from micro-structural observations, compositional analyses and theoretical considerations of the chemistry of the conversion of milk to cheese and partition of components (e.g., milk salts) between the whey and the cheese curd (Walstra and van Vliet, 1986). Natural rennet-curd cheese is essentially a particulate calcium phosphate–para-casein matrix, composed of interconnected and overlapping strands of partially fused para-casein aggregates (in turn formed from fused para-casein micelles). The integrity of the matrix is maintained by various intra- and inter-aggregate hydrophobic and electrostatic attractions. In young cheese, the matrix has an ‘internal’ structure consisting of a relatively loose network of clearly recognisable particles (para-casein micelles and aggregates of paracasein micelles) which are in contact with neighbouring particles over part of their surfaces. Ongoing fusion of para-casein particles during maturation leads to a gradual reduction in the extent of internal matrix structure, as reflected by the disappearance of interparticle boundaries and the formation of a more homogeneous mass (Kimber et al., 1974; de Jong, 1978). The para-casein network is essentially continuous, extending in all directions, although some discontinuities exist in the matrix at the micro- and macro-structural levels. Micro-structural observations made using transmission electron microscopy (TEM) suggest that hydrolysis of para-casein (e.g., by rennet) to watersoluble peptides results in parts of the matrix losing contact with the main para-casein network, an occurrence that leads to discontinuities or ‘breaks’ in the para-casein matrix at the micro-structural level (de Jong, 1978). Hence, it is noteworthy that ageing of Mozzarella for 50 days results in the degradation of ⬃50% s1-casein to s1-CN f 24–199 and an increase in the porosity of the defatted para-casein matrix, as observed using scanning electron microscopy (SEM) (Kiely et al., 1993). Discontinuities at the macro-structural level exist in the form of curd granule junctions or curd chip junctions (in Cheddar and related dry-salted varieties) (Kalab and Harwalkar, 1974; Kalab, 1979; Lowrie et al., 1982; Paquet and Kalab, 1988). Curd

granule junctions in low-moisture Mozzarella are well defined, ⬃3–5 m wide and appear as veins running along the perimeters of neighbouring curd particles (Kalab, 1977). Unlike the interior of the curd particles, the junctions are comprised mainly of casein, being almost devoid of fat. Factors that contribute to the formation of these junctions include leaching of the fat from the surface of the curd particles and dehydration of surface protein, during the cutting, acidification, cooking and pressing stages of cheese manufacture. Chip junctions in Cheddar and related dry-salted varieties are clearly discernible on examination of the cheese by light microscopy and, like curd granule junctions, have a higher casein-to-fat ratio than the interior. The difference in cheese composition at junctions, compared to the interior of the curd particles, probably leads to differences in the molecular attractions between contiguous para-casein layers in the interior and exterior of curd particles, and thus to differences in structure–function relationships. From a rheological viewpoint, the occurrence of structural discontinuities may result in the lack of tensile strength in many cheeses which in practical terms may be reflected as crumbliness, shortness, fracturability, e.g., Feta, Stilton and Cheshire. Discontinuities probably also contribute to poor replication of rheological measurements. The matrix encases fat globules (in varying degrees of coalescence), moisture, dissolved solutes and enzymes within its pores (Kimber et al., 1974; Laloy et al., 1996; Guinee et al., 2000a). Clumping and coalescence of fat globules occur during manufacture due to the combined effects of shear stress on the fat globule membrane and shrinkage of the surrounding paracasein matrix which forces the occluded globules into close contact. Evidence for fat clumping is provided by scanning electron micrographs which show fissures, or irregular-shaped openings, in the para-casein matrix, which remain after removal of fat during sample preparation (Mistry and Anderson, 1993; Bryant et al., 1995; Fig. 8). The frequency of these fissures decreases as the fat content is reduced, e.g., from 33.2 to 8.2%, w/w, fat (Mistry and Anderson, 1993; Guinee et al., 2000b). Major physico-chemical changes occur in the protein and fat phases of cheese during maturation. These include partial hydrolysis of the matrix comprising para-casein, increase in hydration of the para-casein, and coalescence of fat globules, resulting in the formation of fat pools (Fox et al., 1996, 2000; Guinee and Law, 2001; Guinee, 2002). These changes are mediated by the residual rennet, micro-organisms and their enzymes, and changes in mineral equilibrium between the serum and para-casein matrix. The type and level


(a)

(b)

5 μm

1 μm

Figure 8 Scanning electron micrographs of Cheddar cheese, showing the continuous para-casein matrix (arrow heads) permeated by holes and fissures, corresponding to discrete, clumped or coalesced fat globules (solid arrows). Bar, i.e., 5 m in (a) and 1 m in (b) (from Guinee et al. (1998), reproduced with permission from the society of Dairy Technology).

of physico-chemical changes depend on the variety and composition of cheese and ripening conditions. These changes assist in the conversion of fresh ‘green’ curd to a mature cheese and markedly influence its rheological, textural, functional and flavour characteristics (see ‘Biochemistry of Cheese Ripening: Introduction and Overview’, ‘Metabolism of Residual Lactose and of Lactate and Citrate’, ‘Lipolysis and Catabolism of Fatty Acids in Cheese’, ‘Proteolysis in Cheese during Ripening’, ‘Catabolism of Amino Acids in Cheese During Ripening’ and ‘Sensory Character of Cheese and its Evaluation’, Volume 1). Thus, a storage period is generally required before rennet-coagulated cheeses attain the desired rheological and textural attributes (e.g., fracturability, firmness, spreadability, brittleness) associated with the particular variety. Creep and stress relaxation in cheese

The time-dependent rheological behaviour of cheese has been studied (Visser, 1991; Ma et al., 1996; Pereira et al., 2001; Venugopal and Muthukumarappan, 2001). Creep is the time-related change in strain on application of a constant stress to a material such as cheese. Practical examples of creep occur when curd or cheese is compressed gradually under its own weight (e.g., Camembert), is pressed or stacked, e.g., during retailing. Creep (J) may be expressed in terms of strain or compliance, which is the ratio of strain to applied stress. When a constant stress, , applied for a time, t, results in a strain, (t), then the creep compliance is: J(t)

(t)

A creep curve for Cheddar cheese is shown in Fig. 9. Three characteristic regions can be identified. In the elastic region (A–B), is instantaneous and fully reversible; in this region, the creep compliance is elastic (J0). Viscoelastic deformation occurs in region B–C, where the material is partly elastic and partly viscous;

the creep compliance is retarded elastic (JR) and the recovery of the elastic component of on the removal of is delayed. In the viscous region (C–D), increases linearly with time and permanent deformation occurs; the creep compliance is referred to as being Newtonian ( JN). On removal of the stress at point D, the strain recovery curve shows three identifiable regions: an instantaneous elastic recovery (D–E), a delayed recovery (E–F), and an eventual flattening. The vertical distance from the flat portion of the recovery curve to the time axis is the non-recoverable per unit , which is related to the amount of structural damage to the sample during the test. In the elastic region of the creep curve, the strands of the cheese matrix absorb and store the stress energy, which is instantly released on removal of , enabling the cheese to regain its original dimensions. The extent and duration of the elastic region depends on the magnitude of and the structural and compositional characteristics of the cheese. At critical strain, the structure of the cheese is altered via the breaking of bonds between structural elements, which are stressed beyond their elastic limit. Eventually, when the stress-bearing structural casein matrix has fractured, the cheese is said to flow. At short time scales and low , most hard cheese varieties are essentially elastic, whereas after a long time, they flow, albeit very slowly, and do not recover to their original shape on removal of the stress. Failure to appreciate this characteristic can often lead to loss of shape (e.g., manifested by bulging, inclined surfaces) during storage, distribution and retailing, especially if cheeses of different consistencies are laid haphazardly upon each other. A stress relaxation test generally entails the instantaneous application of a constant deformation or strain,

(typically 0.10–0.20), by compression of the cheese sample between two parallel plates of a texture analyser (e.g., TA HDi Texture Analyser, Stable Micro Systems, Godalming, England; Instron Universal Testing Instrument (UTM); Instron Corporation, Massachusetts, USA.). On the application of , increases instantaneously to o (i.e., zero-time value) but decays exponentially with time (t) (Shama and Sherman, 1973). The resultant -time curve is used to determine the stress relaxation time, t, which may be defined as the time required for to decrease to a fraction of o, e.g., t at which o/e, where e is the base of the natural logarithm. In a variation on such a test, Emmons et al. (1980) compressed full-fat (35%) and reduced-fat (17%) Cheddar cheeses, having a common level of moisture-in-non-fat-substance, at a constant speed to a strain of 0.2 and held the strain for 1 min. They


Recovery after removal of stress

Sustained constant stress

0.3

D

Strain, ε

0.2 E

C

0.1

F

B Non-recoverable strain

A

0 0

50

100

150

200

250

300

Time, s Figure 9 Creep–relaxation curve for mature Maasdammer cheese (fat, 29%, w/w, protein, 28%, w/w). A stress of 3700 Pa was applied to a cheese disc (diameter, 40 mm; height, 2.27 mm), placed between the parallel plates of a controlled strain rheometer (TA Carrri-Med csl2500) at 20 °C, and removed after 180 s. The curve is divided into regions indicating elastic, viscoelastic and viscous behaviour.

showed that the initial compression slope (or modulus of deformability), the relaxation slope and the residual force (after 1 min) were much higher for reduced-fat cheese, made from milk with or without homogenisation, than for full-fat cheese. Mechanical models of cheese rheology

From its creep and stress-relaxation behaviour (Fig. 9), it can be inferred that cheese is a viscoelastic material. It exhibits elastic and viscous characteristics, but unlike true elastic or viscous materials, the relationship between stress and strain depends on the magnitude and the duration of the applied stress or strain. On the application of a low stress, that is sufficiently small so as not to induce permanent damage or fracturing (breaking of bonds between the structural elements) of the microstructure, for short times, cheese behaves as an elastic solid. However, a low stress applied over a relatively long time scale results in an increasing strain, a gradual failure of the structure and an eventual flow. Hence, the relationship between (or ) and (or ) is linear only at very low and short time scales. The at which linearity between and is lost is referred to as the critical strain (i.e., at the end of the linear viscoelastic range), which for most solidlike foods, including cheese, is relatively small, e.g., 0.02–0.05 (Walstra and van Vliet, 1982). The modelling of cheese rheology begins with simple relationships such as Hooke’s Law for small displacements in the elastic region. In the region beyond the elastic limit, sometimes referred to as the elastoplastic region (i.e., where recovery following deformation is partial on removal of stress), modelling the rheology of cheese requires more complex models.

Mechanical models have been used to simulate creep and relaxation effects in materials (Rao, 1992; Tanner, 2000). The viscoelastic behaviour of cheese may be simulated by various mechanical models that contain different arrangements of dashpots (representing the fluid element) and springs (representing the elastic element) in series and/or in parallel. A simple model consisting of a spring in parallel with a dashpot is referred to variously as a Kelvin or Voigt element (Whorlow, 1992) or Kelvin-Meyer solid (Tanner, 2000) (Fig. 10). In contrast, a Maxwell element consists of a spring in series with a dashpot, which gives an exponentially decaying response to a suddenly applied constant strain (Fig. 11). Several models have been based on multiple Kelvin bodies in series, or Maxwell bodies in parallel, to simulate creep and stress relaxation, respectively, in viscoelastic solids (Whorlow, 1992); elements with a spectrum of time constants are employed in these models to approximate viscoelastic

σ γ

γ

Time

Figure 10 Kelvin model and its response to constant applied stress.


Applied strain

the force–displacement equations of these models (Whorlow, 1992; Steffe, 1996).

σ

Large strain deformation Definitions and terminology

Time Figure 11 A Maxwell model and its stress relaxation response to a constant applied strain.

behaviour (Fig. 12). Subramanian and Gunasekaran (1997b) showed that a model consisting of eight Maxwell elements could simulate the shear modulus over a wide dynamic range in low amplitude oscillation (0.1–20 Hz). Ma et al. (1996) showed that a six-element Kelvin model could simulate creep compliance in full-fat and reduced-fat Cheddar cheese. The Burgers body, which consists of a combination of Maxwell and Kelvin elements in series (Fig. 13), affords a close approximation to both the creep and stress relaxation behaviour of cheese. The mechanical representation of these models provides an intuitive guide to the nature of viscoelasticity and a simulation of rheological behaviour based on

(a)

(b)

τ1

τ1 Applied

τ2

τ3

strain

τ2 τ3

Large strain measurement implies permanent deformation and measurement of non-linear rheological characteristics which are related to deformation of the microstructure. In contrast to linear viscocelastic deformation where applied strains are generally 0.05, large strain deformation may be defined as that which occurs at strains in the range of ⬃0.1–0.9 during compression, and even at higher strains in the case of shear deformation (e.g., 1). Consideration of the forces that are applied to cheese from manufacture to consumption, indicates a very broad range of deformation. In some situations, the strains are of a relatively low magnitude and do not result in visible damage (e.g., during ripening, transport, retailing), while in others the strain results in fracture (e.g., during portioning) or complete disintegration of the cheese mass (e.g., comminution, as in shredding, grating, grinding, as for example in the preparation of cheese ingredients and in the manufacture of processed cheese products and cheese powders). Hence, in the current context, large strain deformation is arbitrarily subdivided into two regions, i.e., large strain deformation-elastoplastic (LSD-E; e.g., strains ⬃0.1–0.5; Fig. 14), where deformation does not result in fracture and the structure can partially recover, and large strain deformation-fracture (LSD-F; 0.3–0.9), where the cheese mass undergoes fracture or disintegrates and cannot recover. In the following discussion, the LSD-E and LSD-F regions will be treated jointly (Fig. 14). Measurement using texture analyser

Figure 12 (a) Series of three Kelvin elements with a spectrum of time constants, which may be used to simulate creep and (b) A combination of Maxwell elements with a spectrum of time constants, which may be used to simulate relaxation behaviour in a viscoelastic solid.

Large strain deformation testing of cheese usually involves the application of strains (e.g., ⬃ 0.8) that result in fracture, by compression of the cheese sample

Typical linear visco-elastic limit

Applied strain Strain, ε

Typical fracture point

Time

Typical extent of compression to which cheese is subjected in chewing and in compression testing

0.05 0

0.40

0.80 Strain, ΔL/Lo

Figure 13 Burgers four element model, which simulates creep and relaxation behaviour of cheese.

Figure 14 Range of strain in compression tests on cheese.

1.0


between two parallel plates of a texture analyser (Culioli and Sherman, 1976; Dickinson and Goulding, 1980; Creamer and Olson, 1982; Tunick et al., 1991; Guinee et al., 1996; Fenelon and Guinee, 2000; Truong et al., 2002). The cheese sample is placed on a base plate and is compressed at a fixed rate (typically 20 mm/min 1) to a pre-determined level (e.g., 75% of its original height) by the mobile plate (cross-head). However, the rate of compression used in various studies has differed widely, e.g., 5–500 mm/min 1 (Table 3). The force (F) developed during compression is recorded as a function of distance (or displacement); alternatively, the force may be converted to and the displacement to . The resultant versus curves for a range of hard rennet-curd cheeses (Fig. 15) typically show a number of distinct regions and enable the determination of a number of rheological parameters: • A–B; increases proportionally with . The slope of this linear region defines the compression modulus, E (i.e., E = / ), which is of little practical significance in relation to cheese behaviour during processing or consumption, where strains are 0.05. However, in the commercial grading of cheese, E may be an indication of springiness (e.g., where a grader sensorically monitors the resistance to small deformation, as in pressing the thumb into the outside of the cheese block; the force applied during this hand deformation is typically 18 N or ⬃ 40 kPa). • B–C, increases less than proportionally with . The slightly lower slope of the curve in this region compared to that in A–B is probably due to the formation of microcracks that do not spread throughout the sample but which allow some stress to be dissipated; • C–D, the slope of the / curve decreases markedly. The cheese begins to fracture at C, as cracks grow and spread throughout the entire sample at an increasing rate. Eventually, at D the rate of collapse of the stress-bearing para-casein matrix overtakes the build-up of within the matrix through further compression and a peak , denoted as the fracture stress, is reached. The fracture stress, f, and strain,

f, are measures of the stress and strain, respectively, required to cause complete fracture of the sample. Strength, or fracturability, is defined as the stress required to fracture the sample (at D), while toughness, or fracture work, is defined as the area under the curve up to the point of fracture. • D–E, decreases with further compression due to the collapse of the stress-bearing structure. The decrease in may be attributable to: (i) shattering of the samples into pieces that spread over the base plate, resulting in an increased surface area and (ii) the probable loss of contact between some

of the pieces of cheese and the base plate which results in dissipation of stress energy stored within the individual pieces. • E–F, increases as the cross-head begins to compress the fragmented pieces of cheese. The at the end of the compression (point F) is a measure of firmness, as judged in the first bite of mastication (Sherman, 1969; van Vliet, 1991a). The various quantities obtained from the – curve and their interpretation are given in Table 2. The application of a strain to a segment of cheese (e.g., cube or cylinder) and monitoring the resultant by a texture analyser, as above, is a typical method for measuring the large strain deformation behaviour of cheese. However, many variations of both the procedure of stress or strain application, and the levels, are possible. A so-called apparent elastic modulus can be calculated at a strain well below the fracture point, e.g., ⬃ 0.1, as the ratio between and . A preferred term for this parameter is modulus of deformability, as the deformation in question may include some plastic flow (Ak and Gunasekaran, 1995; Johnston, 2000). However, such a parameter needs to be interpreted with caution as some apparent initial deformation may occur before complete contact is made between the compression plate and the sample surface, an occurrence that could lead to erroneous values. Fracture and work of fracture. Rheological behav-

iour over such a range of in the form of shear or compression, can be explored in several ways, such as applying a gradually increasing , a fixed , a defined followed by its removal, a gradually increasing up to a point followed by its reversal. Stress–strain cycles, often referred to as bites (analogous to compression between the molar teeth during mastication), may be repeated at interval(s) or applied in a given sequence (e.g., pre-test compression). Depending on the level of applied strain, cheese exhibits a combination of rheological behaviours, such as non-linear elastic (e.g., region B–C, Fig. 15), sometimes referred to as viscoelastic, or inelastic (e.g., region D–E, Fig. 15), sometimes referred to as plastic behaviour.

Rheological Measurements in Cheese: Sensoric Methods The methods used to assess the rheological characteristics of cheese may be broadly classified as sensoric or instrumental, where instrumental methods can be categorised further as empirical or fundamental. The aim of sensoric methods, which are performed routinely by cheese graders, is to acquire an impression

522

Table 3 Test conditions used with uniaxial compression of cheese

Cheese type

Sample dimensionsa (mm)

Speed (mm/min)

Maximum strain ( )

Instrument b

Tc (°C)

Gouda Cheddar Cheddar, Cheshire, Leicester Cheddar, Mozzarella, processed cheese spread Italico, Montasio, Sbrinz, Grana Padana, Pecorino Cheddar, Cheshire

15 15 20 20 29 30 57 20–40 20 20 20 17 15

25–500 10–100 5–1000 5 5–200 50

0.80 0.50 0.50 0.75 0.80 0.80

UTM UTM UTM UTM UTM UTM

20–21 not stated 0–40 22 20 21

Processed cheese analogue Brie Gouda

17 15 15 19 15 20–30

5–500 33 1–200

0.80 0.80 ⬃0.75

21 5 and 20 20

Cheddar Munster, Carré de l’Est, Camembert, Coulommiers and three speciality soft cheeses Mozzarella Cheese analogs Munster, Emmental, Roquefort, Beaufort, Camembert, Reblochon, Pont l’ Eveque, Brie de Meaux, Tomme de Savoie, Valencay, St. Nectaire, Pyrenees Brebis, Blue d’Auvergne, Comte Vieux, Fourme de Salers Cheddar cheese with varying fat content (6–33%, w/w) Cheddar Cheddar, Swiss, Romano, Havarti Cheddar Cheddar

19 19 (or 27) 16 17

2.5–125 10

0.70

UTM Alwetron TCT5 Overload Dynamics UTM UTM

Culioli and Sherman (1976) Calzada and Peleg (1978) Dickinson and Goulding (1980) Casiraghi et al. (1985) Casiraghi et al. (1985) Green et al. (1985); Green et al. (1986) Marshall (1990) Molander et al. (1990) Luyten et al. (1991a,b)

22 15

Ak and Gunasekaran (1992) Hennequin and Hardy (1993)

67 12 10 10 21 21

5–500 250 10

0.70–0.75 0.80 0.80

TA.XT2 UTM

30–60 20 20

Ak and Gunasekaran (1995) Lobato-Calleros et al. (1997) Antoniou et al. (2000)

30 29 30 29 10 10 10 19 26 18 24

50 50

0.70 0.75

50 20

0.75 0.75

UTM UTM TA.XT2 UTM UTM

8 8 20 20 20

Tilsit Queso Fresco

20 20 20 15

10 100

0.70 0.30

UTM TA.XT2

15 RTd

Fenelon and Guinee (2000) Guinee et al. (2000b) Halmos (2000) Hort and LeGrys (2000) Johnston (2000); Johnston et al. (2002) Weinrichter et al. (2000) Hwang and Gunasekaran (2001)

Gruyere, processed Mozarella Commercial cheeses: Swiss, Colby, Edam, Cheddar, Gouda, Mozzarella, Romano, White American, Yellow American Montasio Cheddar

20 5–20 12.7 12.7 12.7

1250 3.6–14 60

0.90 0.52 0.70

UTM UTM TA.XT2

RT 7

Charalambides et al. (2001) Breuil and Meullenet (2001)

21 10 25 15 15 15 15

12–600 50 24

0.90 0.40 or 0.80 0.80

TA.XT2 UTM TA.XT2

4 22 25

Xiong et al. (2002) Innocente et al. (2002) Truong et al. (2002)

a b c d

, diameter of cylinder. UTM, Universal Testing Machine (Instron); TA.XT2, texture analyser (Stable Micro Systems). T, Temperature. RT, Room temperature.

Reference


kPa 400 Stress, σ

350

F

300 250 200

D

E

150 C

100 50 0 0.0

A

B 0.2

0.4 Strain, ε

0.6

0.8

Figure 15 Large strain deformation test: typical stress–strain curve of a 6-month-old mature Cheddar cheese sample compressed to 25% of its original height; several regions of the curve are identifiable, based on slope variation (see text for details).

of how the texture of the cheese is perceived during consumption. Cheese texture may be defined as a composite sensory attribute resulting from a combination of physical properties that are perceived by the senses of touch (including kinaesthesis and mouthfeel), sight and hearing. The test conditions are arbitrary, frequently involving deformation which results in visual fracture, e.g., as when rubbing cheese between the fingers until it becomes pliable, three (finger) point bending of a cylindrical cheese plug or slice, and mentally gauging the force required to bend or break it. Alternatively, the cheese may be assessed by the application of forces or deformations which cause no visible fracture, e.g., pressing the ball of the thumb into the surface of a whole cheese and mentally assessing the degree of indentation or the force exerted on the fingers. In all cases, a sensory impression is formed and the grader assigns a score, based on one or more criteria, such as test conditions and response. The sensory properties of cheese, including texture, are discussed comprehensively in ‘Sensory Character of Cheese and its Evaluation’, Volume 1.

Rheological Measurements in Cheese: Empirical Instrumental Methods A wide range of instrumental techniques is used for characterising the rheology of cheese (Table 4). Instrumental methods may be arbitrarily classified as empirical or fundamental. In general, the nature of the stresses and strains in empirical methods is less well defined than in fundamental methods. Moreover, unlike fundamental methods, the measurements obtained with some empirical methods are on an arbitrary scale (e.g., the ball compressor). Empirical instrumental measurements

Many textural studies have involved rheological measurements to imitate the sensory evaluation of cheese texture. The aim of empirical tests is to measure a parameter, which experience indicates, or suggests, is related to the textural characteristics of the cheese. Hence, while the test conditions are arbitrary and the stresses and strains involved may not be well defined,

Table 4 Typical rheological testing techniques applied to cheese

Test Oscillatory shear (parallel plate) Uniaxial compression Cone penetration Puncture Bending test Wire cutting test Torsion test a , diameter of cylinder.

Type of instrument used

Typical sample shape and dimensionsa (mm)

Reference

Rheometer

Cylinder: 30 3

Ma et al. (1996)

Texture analyser or UTM Texture analyser or Instron, 30° cone Texture analyser or Instron, 2–5 mm diameter needle Texture analyser Texture analyser Torsion gelometer

Cube: 25 25 25 Cube: 12.7 12.7 12.7

(see Table 3) Breuil and Meullenet (2001)

Cube: 12.7 12.7 12.7

Hennequin and Hardy (1993); Breuil and Meullenet (2001) Rosenthal (1999) Green et al. (1986) Truong and Daubert (2000)

Finger: 25 25 50 Finger: 25 25 50 Capstan: 19 max 27.8


a value is obtained which gives some indication of the textural characteristics of the cheese and differentiates one sample from another. However, they provide only single datum values that are an overall measure of the many different facets of rheological behaviour. In these tests, a sample is compressed or penetrated in one or more bites, thereby simulating the compressive and penetrative actions of the teeth on cheese during mastication. Likewise, the action of a cheese grader who presses the ball of the thumb into the cheese is imitated by the ball-compressor test. Some empirical instrumental tests are discussed briefly below. Imitative tests

Imitative instruments include the bite tenderometer and the denture tenderometer which measure the forces involved in chewing using strain gauges, and typically involve compression to 60% of the original height. In the Volodkevich bite tenderometer, which was designed to simulate the motions of mastication, a pair of tooth-like jaws, or wedges, compress a sample of about 6 mm thickness, imitating the squeezing and biting action of teeth (Szczesniak, 1963b). Later instruments used plungers to penetrate a sample, or parallel plates to compress a sample, e.g., to ⬃20–30% of its original height (Szczesniak, 1963b). Early devices for evaluating the hardness of cheese involved compression by a ball, in an instrument known as the ball-compressor, where deformation resulting from applying a fixed force for a specified time was measured (Szczesniak, 1963b). The action simulated that of a thumb pressing against cheese when making a sensory evaluation of the product. The General Foods Texturometer was designed to simulate the biting of food by the jaws and teeth (Friedman et al., 1963; Bourne, 1978). A food sample (⬃12.6 mm high) was loaded onto a fixed plate and then subjected to a deforming force by a tooth-shaped plunger, which was mounted on a hinge and actuated to simulate the vertical action of a human jaw. The area of the samples is at least that of the plunger base, which is available in sizes from 16 to 50 mm . The instrument compresses samples to a height of 3.2 mm, i.e., 75% compression. When the plunger deforms the sample, strain gauges detect the movement of the plunger and a force–time trace is recorded and is known as a texture profile. The sample is subjected to two successive deformations (referred to as bites). The Texturometer has been superceded by uniaxial compression instruments, such as the Instron UTM, for the purpose of texture profile analysis. One distinction between the Texturometer and other instruments is that the Texturometer simulates the action of the human jaw, whereby the plunger decelerates as it

reaches the end of the compression stroke, and then accelerates upward as it withdraws. The usual practice with other instruments is compression at constant speed. Cutting tests

Cutting tests measure the resistance to the passage of a knife or a wire through a cheese (e.g., Cherry-Burell Curd tension meter). As wire-cutting tests tend to be more fundamental, they are discussed in more detail in ‘Fundamental Measurements: Large Strain Deformation’. Penetration tests

Penetration tests involve measurement of the force required to insert a probe (cone or cylinder) a given distance into cheese, or alternatively the depth of penetration of a probe under a constant load for a given time. As the probe penetrates the sample, the cheese in its path is fractured and forced apart. The progress of the probe is retarded to an extent depending on the hardness of the cheese in its path, the adhesion of the cheese to its surface (which depends on the depth of penetration into the cheese and the thickness of the needle, or angle of the cone, used). Hennequin and Hardy (1993) used a cylindrical probe (5 mm diameter at a speed of 10 mm/min to a depth of 10 mm) to penetrate soft cheeses (e.g., Camembert, Coulommier, Munster) and found that the force at 10 mm penetration gave a high correlation with sensory firmness (r 0.94, n 19). They concluded that the technique is suitable as a rapid method for texture measurement in soft cheese. Breuil and Meullenet (2001) found a significant correlation between measurements obtained using a cone penetrometer (30°), or a 2-mm needle, and textural characteristics of a wide range of commercial cheeses (e.g., Colby, Edam, Cheddar, Mozzarella and Cream cheese) as measured by a sensory panel.

Fundamental Measurements: Oscillatory Rheometry for Linear Viscoelastic Measurements in Cheese Elastic shear (G) and loss modulus (G)

As discussed in ‘General rheological terminology’, there is a range of strain, typically 0.05, over which cheese recovers fully, although not instantaneously, when stress is removed. This behaviour is referred to as viscoelastic. Even at these low strains, ideal elastic (Hookean) or viscous (Newtonian) behaviour, represents two extremes, and cheeses, like most organic materials, show some characteristics of both. Hence, the accurate characterisation of cheese rheology requires the measurement of both elastic and viscous responses. In the elastic region, where there is a linear


relationship between dynamic stress and strain, the behaviour is described as linear viscoelastic. Linear viscoelastic measurements are typically made by applying torsion, using oscillatory rheometry (van Vliet, 1991b). This involves using a precision actuator to apply a low oscillating strain to a sample (which could be a liquid, a ‘soft’ solid, or a rigid solid), and the measurement of the resultant stresses within the sample, using a sensitive transducer. Alternatively, a small stress is applied to the sample and the resultant strain is measured. The former is referred to as controlled strain rheometry, whereas the latter is known as controlled stress rheometry. Several geometries are possible for applying torsion, but the most convenient for a solid material, like cheese, are parallel circular plates. Parallel plates have the advantage that the sample can be easily prepared to fit the plates; moreover, plates with serrated surfaces minimise the risk of slippage, associated with fat liquefaction (Subramanian and Gunasekaran, 1997a; Guinee et al., 1999). With parallel plate geometry, the cheese sample, which is disc-shaped, is gripped between the plates, one of which is fixed, while the other applies a low-amplitude torsional harmonic motion (Fig. 16). At any time t, the angle of rotation, &, of the oscillating plate is defined by:

& a sin t where a is the maximum angle of rotation and is the angular velocity. The shear applied by the plate varies throughout the sample, from zero at the central axis to a maximum at the edge. At a point on the oscillating plate, at a radial distance, r, from the axis, the displacement due to rotation by an angle & is r&, and the shear strain is:

r& w

where w is the thickness of the sample. This displacement results in a strain, (t), at any radius r:

冢

ar sin t o sin t w冣

where o is the amplitude of (t). (In this notation, (t) is used interchangeably with .) In general, the resultant oscillating stress is out of phase with the applied shear by a phase angle . The stress wave can be reconstructed as the sum of two sine waves, one in-phase with, and the other out-of-phase (by 90°) with, the strain wave (Fig. 17). Thus,

' " o' sin t o" cos t where o' and o" indicate the stress components which are in-phase and out-of-phase with the strain , and are related by the phase angle (Fig. 18): tan

o" o'

The stress wave has an amplitude o, defined by:

o

√(o')2 (o")2

Two dynamic moduli, elastic shear modulus (or storage modulus), G, and viscous modulus (or loss modulus), G, may be defined from the relationship between and , where,

G'

o' o (a)

δ (c)

Strain Stress

(b)

Shear

(a)

ωt (b) Radial distance from centre of sample

Figure 16 Schematic showing viscoelastic deformation of cheese during low-amplitude oscillation rheometry: (a) sample before test; (b) sample being subjected to a torsional shear displacement; (c) shear displacement as a function of radial distance from the central axis.

90°

Elastic, or in-phase, component, τo' sin ω t Loss, or out-ofphase, component, τo'' cos ω t

Figure 17 Stress and strain traces in dynamic oscillation, showing phase delay, , between stress and strain (a), and resolution of in-phase and out-of-phase components of stress (b).


(b)

(a) , τo

ess

Str

Loss component, τo″

δ

cos ω t sin (ωt + δ ) sin ω t

Elastic component, τo′ Strain, γo

Oscillatory shear also implies an oscillatory shear rate . (), since .

d . (o sin t) ' cos t o cos t dt .

Figure 18 Trigonometrical representation of phase relationship between dynamic strain and dynamic stress in oscillatory measurement in viscoelastic region, (a), with vector direction indicating phase, as in (b).

and G"

Complex viscosity

' o" o

.

where o is, by definition, the amplitude of . This enables a complex viscosity to be calculated, with viscous and elastic components, by dividing the appropriate component of shear stress by the shear rate. The ‘viscous’ component of viscosity (in-phase with shear rate) is: G" '" . since o o . o

The ‘elastic’ component of viscosity (out-of-phase with shear rate) is:

Therefore,

"

G" tan G' Consideration of a spring-dashpot mechanical model, e.g., a Kelvin element, indicates that the same relationship between stress and strain applies (Fig. 19). However, it is noteworthy that the magnitudes of G and G for the spring and dashpot components of the mechanical model in the viscoelastic region differ markedly from those which characterise large strain deformation. Ma et al. (1996) reported decreases (of ⬃50%) in G and G for full-fat and reduced-fat cheeses on increasing stress by a factor of 10. The tendency of to approach a constant value as the strain was reduced towards zero shows that cheese behaves in a viscoelastic manner rather than an elastic manner at very low strains.

'' G" . o

Thus, complex viscosity equals the complex (shear) modulus divided by the angular frequency, , with the in-phase and out-of-phase components interchanged (Fig. 20). According to the Cox-Merz rule, the complex viscosity determined by dynamic rheometry is virtually identical to the (steady) viscosity when compared at numerically equal values of shear rate and . Dynamic rheometry is often used for this purpose since it is easier to perform than viscometry on viscoelastic materials. Cylindrical geometry may be used in the case of liquids or gels, or cone-and-plate or parallel plate geometry in the case of more solid samples. It is preferable to operate in a shear-rate control mode to avoid the occurrence of

Stress τ Oscillatory strain γο sin ω t

Shear rate, . γo

ss,

e

Spring G'

Dashpot G''

(b)

(a)

Str

τo"

τo

Loss component, τo"

η

δ

Elastic component, τo'

Figure 19 Kelvin element and stress response () to oscillatory strain ( ). The spring represents the elastic, or in-phase, component, G. The dashpot represents the loss, or out-of-phase component, G.

η' = . γο

τ ' η" = .o γo

Figure 20 Trigonometrical representation of phase relationship between shear rate and dynamic stress in oscillatory measurement in viscoelastic region (a), with corresponding components of complex viscosity illustrated in (b).


* Avisc exp

冢 ERT 冣 visc

where Avisc is a pre-exponential factor, Evisc is the activation energy, R is the gas constant and T is absolute temperature. A logarithmic form of this equation gives a linear plot:

log10 *

冢冣 A T

B

where A Evisc/(2.3 R) and B log10 Avisc. Cheddar cheese had a higher activation energy (137 000 J/mol) than Cheshire (84 800 J/mol) at 60 weeks of age, implying a greater sensitivity to temperature for the rheological properties of Cheddar cheese. However, the viscosity–temperature curves for the two cheeses crossed around 25 °C, i.e., Cheddar had a higher viscosity than Cheshire below 25 °C and vice versa above 25 °C (Fig. 21). The sensitivity of viscosity to temperature was found to decrease with age for Cheshire cheese (Fig. 21).

+ Cheshire at 20 weeks 6.5 6.0 log η∗

unpredictably large shear. The strain is kept low if one wishes to measure without structural damage to the sample but may be increased if one wishes to determine a fracture stress or to simulate a practical situation. Ma et al. (1996) found that elastic and loss moduli, G and G, respectively, increase only to a second order with increasing frequency, i.e., by a factor of about 3 for a 1000-fold increase in frequency. This implies a 300-fold decrease in the viscosity components, and , respectively (i.e., multiplying by 3/1000 ⬃ dividing by 300), showing that the complex modulus is much more stable with respect to frequency for cheese than is complex viscosity. Various studies on different cheese varieties have indicated that G and G decrease, while increases, with increasing temperature in the range 4–40 °C, the range normally encountered during consumption and mastication (Taneya et al., 1979; Horne et al., 1994; Guinee et al., 2000b; Guinee, 2002). These changes, mark a transition from a cheese which is largely elastic in character at low temperature ( ⬃ 12–16°), to one which is more viscous in character at the higher temperature ( ⬃ 40°) and may be attributed to fat liquefaction. Tunick et al. (1990) made shear measurements on Cheddar and Cheshire cheeses in the range 20–40 °C and fitted the Arrhenius equation to the complex viscosity in the form:

Cheshire at 60 weeks

5.5

Cheddar at 60 weeks

5.0 4.5 4.0 20

30

40

Temperature, °C

Figure 21 Variation of complex viscosity of Cheddar and Cheshire cheese with temperature in the range 20–40 °C. Cheddar at 60 weeks, ; Cheshire at 20 and 60 weeks, O, , respectively. Data taken from Tunick et al. (1990).

Fundamental Measurements: Large Strain Deformation Large strain deformation measurements on cheese are usually undertaken using uniaxial compression, shear (or torsion), wire-cutting or bending. The methodology and instrumentation used for these measurements, and factors affecting the measurements, are discussed below. Uniaxial compression

The most common types of rheological measurement in cheese involve linear (uniaxial) displacement, e.g., using Instron UTM (Lee et al., 1978; Weaver et al., 1978; Imoto et al., 1979; Creamer and Olson, 1982; Visser, 1991; van Vliet, 1991b; Guinee et al., 1996; Pons and Fiszman, 1996; Fenelon and Guinee, 2000), the Stevens Response Compression Analyser (Stevens Group Ltd, Blackburn, UK) (Brennan, 1984), the TA.XT2 texture analyser or its derivatives from Stable Micro Systems (Truong et al., 2002; Xiong et al., 2002) (Table 3; Fig. 22).

(a)

(b)

(c)

Figure 22 TA. HDI texture analyser (Stable Micro Systems) (a), and sample of Cheddar cheese before (b) and after (c) uniaxial compression to 20% of original height.


Measurements are generally made in compression mode rather than in tensile mode because (a) compression behaviour is relevant to sensory perception arising from chewing and mastication, (b) it is difficult to grip cheese samples to carry out a tensile test and (c) hard cheese has inhomogeneities which occur in random positions and directions with respect to sample dimensions, making it difficult to obtain reproducible results for tensile fracture. A compression measurement involves compression of a rectangular or cylindrical sample between parallel plates. Compression testing is more suited to large strain deformation than to linear viscoelastic deformation (low strain). This is because initial contact between the parallel plates and the sample usually involves some realignment of the sample surface due to imperfections in the surface smoothness, as a result of intrinsic macrostructural characteristics of the cheese (e.g., veins, cracks, openness). Moreover, the difficulty in fine precision cutting can give rise to lack of accuracy in low strain measurements (up to 0.5 mm of deformation). Ideally, the cheese sample may be conditioned by applying one or more low compression deformations (e.g., ⬃ 0.05) prior to testing. A typical force–displacement curve, obtained by compression of a sample of Cheddar cheese at constant velocity to a strain of 0.8 (i.e., final height of the sample is 20% of original height), is shown in Fig. 23. Compared to other rheological methods for evaluating cheese, large-strain deformation compression offers several advantages: the strains applied are in the range of those applied to cheese during size-reduction operations as applied commercially; it is a dynamic method for which the calculated parameters (e.g., f,

f) depend on a range of stress–strain data accumu-

250

lated during the test; sample preparation does not require specialised cutting equipment; all cheeses, apart from soft cheeses with a very high f, e.g., mature Camembert with an almost-runny consistency, can be prepared easily and tested; and the test method is simple and rapid. However, for reproducible results, sample shape and dimensions need to be precise, which can be difficult where cylindrical samples are acquired by pushing a cylindrical borer into a portion. As is common to all large strain deformation methods, a serious limitation is the difficulty in obtaining results for cheeses with eyes. Relationship between shear and normal stresses in uniaxial compression

A proper interpretation of uniaxial compression tests requires that the complexity of internal forces in a sample be understood. A sample undergoing uniaxial compression is distorted in various directions at any one time, e.g., vertically, horizontally and diagonally (Fig. 24). Even though the applied force is uniaxial, a combination of compressive and shear forces is created in different planes within the sample (Fig. 25). A fracture is more likely to be caused by shear rather than compression forces, since cheese is relatively incompressible. Thus, large strain uniaxial compression indirectly measures shear behaviour. Mohr’s circle, a construction widely used in the analysis of the strength of materials, enables the shear and compression stresses (up to the fracture point) on an inclined plane, at any angle, &, to the horizontal to be calculated (Fig. 26). Analysis shows that the maximum normal stress (max F/A) is twice the maximum shear stress (i.e., max F/(2A)).

(a)

(b)

200 Force, N

fracture point

150 100 θ1

θ2

50 0

0

5

10

15

20

Displacement, mm Figure 23 Force–displacement curve obtained from uniaxial compression of 6-month-old Cheddar cheese at 4 °C, on a TA.HDi texture analyser, showing the fracture point. The shaded area represents the fracture work or toughness.

Figure 24 Illustration of strains in relation to the principal axes of strain (vertical, y and horizontal, x ) in a sample undergoing uniaxial compression. Compression along the y-axis results in a simultaneous extension along the x-axis. This coincides with a reduction in the angle of inclination, from &1 to &2 of a diagonal line implying shearing along an inclined plane.


(a) Compression forces

(b) Shear forces

Frictional force

Figure 25 Compression (a) and shear forces (b) within a sample during a uniaxial compression test.

Effect of pre-test strain history

Fracture stress decreases significantly if the sample has been cycled through successive compressions. This was shown for Cheddar, Cheshire and Leicester cheeses at 20 °C by Dickinson and Goulding (1980). The effect was noticeable even when the previous strains were relatively low, e.g., f in Cheddar and Cheshire cheeses fell by ⬃ 30% after 50 cycles of ⬃ 0.1; the reduction in f in Leicester cheese was about 12% under the same conditions. However, pre-test strain history (50 cycles at

⬃ 0.2) produced no significant effect on f. This indicates that a history of recoverable deformation causes some internal structural weaknesses, which reduce the subsequent strength of the cheese, but the flow conditions under which fracture occurs are not affected. Effect of sample-machine interface conditions and sample dimensions

The expression of compression characteristics in the form of stress–strain curves rather than force–displacement F Shear stress τ

F/A σ 2θ

σ

Normal stress

τ

θ

Figure 26 Mohr’s circle, for computing normal and shear stresses on any plane in a sample under uniaxial compression. Each point on the circle represents the normal (, x-axis) and shear (, y-axis) stresses which occur on a plane inclined at an angle &. It can be inferred from the diagram that maximum occurs at & 45° and equals half of the uniaxially applied , i.e., F/A, at & 0°, where F is the applied force and A is the cross-sectional area of the sample.

curves is meant to remove the effect of sample dimensions. At low values, the effect of sample dimensions may be eliminated in this way. However, for large

(0.1), especially f, the distribution of stress and strain within the sample depends on sample dimensions, as the sample may be deformed into an irregular shape, due to fracturing, barreling and squeezing. Squeezing flow is an intrinsic aspect of large strain uniaxial compression of cheese, i.e., as sample height is reduced, the cheese spreads in a lateral direction (Fig. 24). This implies that shearing takes place within the sample and that frictional shear forces occur at the points of contact between the sample and the compression plates. Friction can be reduced by lubrication of the contact surfaces with mineral oil or grease; in contrast, surface friction can be increased by the use of emery paper, or the surfaces can be bonded using glue, both of which eliminate slippage as a result of cheese ‘sweating’. Because lubrication allows lateral movement at the contact surfaces during compression, the sample shows a slight tendency towards an hour-glass shape, as opposed to the relatively large barreling effect. Lubrication can reduce the stresses in squeezing flow by as much as 50% and increase the observed f from ⬃0.45 to 0.55, in the case of Gouda cheese at 20 °C with an aspect ratio (i.e., height/width) of unity and a cross-head speed of 500 mm/min (Culioli and Sherman, 1976). However, the frictional effect increases with cross-head speed. At a low cross-head speed (5 mm/min), lubrication decreased f by ⬃20% in Cheddar cheese where the aspect ratio was 0.35, with the effect becoming more pronounced (of the order of 20–30%) at f (Casiraghi et al., 1985). In contrast, the bonding of the cheese surfaces to the compression plates (e.g., using cyanoacrylate ester adhesive) had relatively little effect on f, f and max. A similar trend was found for Mozzarella and processed cheese spread (Casiraghi et al., 1985). At low compression plate speeds (20 mm/min), friction had only a negligible effect in Gruyere and processed Mozzarella cheese with aspect ratios near unity (Charalambides et al., 2001). However, at aspect ratios 0.5, at a given was slightly higher when the samples were not lubricated. Similar findings were reported by Ak and Gunasekaran (1992), using mineral oil as a lubricant on Cheddar cheese with aspect ratios of 0.65 and 1.0. Hence, accurate – curves can be obtained from unlubricated testing provided the aspect ratio is sufficiently high, e.g., 1. Effect of deformation rate

Due to viscous effects, at a given (e.g., 0.1) and f depend on rate of deformation and increase by ⬃40–50% per 10-fold increase in compression plate speed in cheeses at 4–20 °C (Luyten et al., 1991a; Ak


and Gunasekaran, 1992; Pons and Fiszman, 1996; Xiong et al., 2002). f is not significantly affected by deformation rate. Influence of shape

Cylindrical and rectangular-shaped samples have been used in uniaxial compression (Table 3). For both shapes, only slight differences have been reported in the – characteristics up to the fracture point (⬃40% compression). However, at f, compression of cubic samples resulted in significantly greater forces (and ) than cylindrical samples (Culioli and Sherman, 1976). For a given shape (e.g., cylindrical) and aspect ratio (e.g., unity), doubling the absolute dimensions had little effect on at a given deformation (Culioli and Sherman, 1976). Large strain shear measurements

A rheometer or a viscometer may be used also, in addition to a texture analyser, to apply large strain shear to cheese. In the rheometer, the parallel plate geometry already described (see ‘Fundamental Measurements: Oscillatory Rheometry for Linear Viscoelastic Measurements in Cheese’) for linear viscoelastic deformation, becomes applicable for large shear strain (i.e., torsion testing, where a shear is applied in rotational mode). A large shear strain may be applied also with a rheometer using samples of cheese with capstan geometry (Truong and Daubert, 2000, 2001; Truong et al., 2002). The effect of cutting the cheese into a capstan shape is that the greatest shear stress occurs at the cross-section of minimum radius (Fig. 27). Bowland and Foegeding (1999) used this technique to determine the fracture properties of model processed cheese. Torsion shear tests, as above, have been applied recently to cheese and offer few if any distinct advantages above large strain deformation in (a)

(b)

compression/extension mode. However, for highly deformable cheeses, e.g., fresh low-moisture part-skim Mozzarella and young reduced-fat cheeses, e.g., Cheddar, which generally do not undergo elastic fracture (i.e., where the sample breaks into distinct pieces), but rather plastic fracture, on compressing to strains of 0.7–0.8 (Fenelon and Guinee, 2000; Guinee et al., 2002), torsion shear may be useful in determining fracture stress and fracture strain. The latter parameters may be important in some cheeses, e.g., in the formation of cheese strings containing two different-coloured cheeses in a twisted helical (rope-like) configuration. However, preparation of capstan-shaped samples requires specialised milling equipment and is time-consuming. Vane rheometry has been used for large strain shear deformation tests in processed and natural cheeses, including Cheddar and Mozzarella. In this method a probe, typically with four blades or vanes, is inserted into a sample and rotated slowly at a constant rate (e.g., 10 cycles per minute), while the torque is measured (Fig. 28). The technique produces a shear stress versus strain characteristic with a distinct peak at the point of failure, i.e., fracture. Fracture stress is shear rate-dependent in both cases, being smaller by a factor of 2–3 with the vane technique than with the torsion technique. Using vane rheometry and torsion, it was possible to separate different cheese types on fracture stress/fracture strain ‘texture maps’, which were similar in both cases (Truong and Daubert, 2001). The vane method, which is rapid and does not require sample preparation, as in cutting, has been found to give trends comparable to those obtained using capstan geometry for processed cheese, Cheddar and Mozzarella (Truong and Daubert, 2001). However, disadvantages of vane rheometry may include the difficulty of inserting the vane without cracking the cheese mass (e.g., hard cheeses such as Romano or Parmesan, with a low fracture strain and soft cheeses with a low fracture strain, such as Feta), and variability of results for cheeses, such as Blue or Gouda, with macrostructural heterogeneities, such as cracks, veins, small openings and openness. Bending tests

2r 2R

2R

Figure 27 Illustration of sample shapes used in torsion tests, using parallel plate geometry (a), and capstan geometry (b). The capstan shape is obtained by milling a cylindrical sample using a purpose-built milling machine.

Hard cheeses can be subjected to bending tests which may involve three- or four-point loading (Whorlow, 1992; Rosenthal, 1999); a schematic for three-point bending of a ‘finger-shaped’ cheese sample is shown in Fig. 29. Such tests produce compression and tension on alternate sides of a neutral axis, with the maximum tensile strain occurring on the bottom surface of the sample and the maximum compressive strain on the top surface (Fig. 29). A force–displacement curve obtained from three-point bending of a cheese sample, e.g., Cheddar cheese (Fig. 30), enables the


(a)

(b)

Figure 28 Vane rheometer probe before (a) and during (b) shear test on process cheese. Photos courtesy of Truong and Daubert (2000) Gel Consultants Inc. (See Colour plate 11.)

(a)

estimation of fracture stress (f). With the assumption that the sample deforms into an arc shape on bending, the tensile strain ( ) at any point on the bottom surface may be estimated for any displacement (y) as:

H

W

4Hy L2 4y2

L (b)

compression

E

neutral axis C

E1

D

tension

Figure 29 Schematic of a bending test with three-point loading: (a) Geometry with cheese sample in place prior to testing, resting on the two support beams, C and D; (b) cheese sample being deformed by mobile beam E during testing.

where y is the displacement at any point of contact along the axis where the sample makes contact with the moving beam, H is the sample height and L is the span between the supporting beams (Fig. 29). Assuming that is proportional to , the tensile or compressive stress (M) at any point on either surface of the sample can be approximated, subject to assumptions about linearity, as:

M ⬇

3FL 2WH2

50

Force, N

40

f

30 20 10 0

0

5 10 Displacement, y, mm

15

Figure 30 Force–displacement curve for three-point bending of a 180-day-old Cheddar cheese sample (25 25 50 mm), ripened at 4 °C. The sample was deformed at a rate of 20 mm/min (by the mobile beam) on a TA.HDi texture analyser, using a three-point bend rig (model HDP/3PB) with 40 mm span between the supporting beams. A distinct fracture point (f) at point E1 (Fig. 29) on the bottom surface of the sample coincides with the maximum extension.

where F is the force (obtained from the force-displacement curve) and W is the width of the sample (Fig. 29). Even though the – behaviour departs from linearity before the point of fracture, the formula can be used to approximate the fracture stress (f). Since fracture is more likely to occur in tension than in compression, fracture behaviour in tensile mode can be compared more easily using bending tests than by using tensile tests, which are difficult to carry out because of the difficulty of grabbing a sample without deforming it. The fracture strain ( f) obtained during threepoint bending may give a better estimation of the cutting behaviour of cheese than f obtained from compression testing (see ‘Uniaxial Compression’), as cutting involves a combination of tensile and shear strains. In compression testing, fracture is due to shear displacement.


Wire-cutting

Fracture energy during cutting is quantified by measuring the force required to push wires of different diameters at constant velocity through a cheese mass (Green et al., 1986; Marshall, 1990). Luyten et al. (1991b) investigated the fracture properties of Gouda cheese using wire-cutting. A typical force–time curve shows an initial rise in force, which reaches a maximum as the wire penetrates the sample surface. Once the surface has been broken, the force rapidly drops to a constant level, Fc, as the wire ‘ploughs’ through the sample. Fc increases somewhat with cutting speed (⬃ doubling for a 20-fold increase in speed) and with wire diameter (by 3–4 fold for 300 m compared with 25 m diameter). Since fracture develops around a crack, a specific fracture energy ( J/m2), Rf, can be defined as the energy needed per unit area (of crack) to cause a fracture to spread. While it is not possible to determine specific fracture energy precisely, because of the inherent heterogeneity in cheese structure (see ‘Cheese structure’), its order of magnitude can be determined by measuring cutting force using wires with a series of diameters and extrapolating to a diameter of zero. The specific fracture energy is calculated as: Rf

Fc0 d

where Fc0 is the cutting force, extrapolated to cutting with a wire of zero diameter, and d is the sample width, i.e., the length of wire in contact with the cheese (Luyten et al., 1991b). The fracture energy obtained with the wire-cutting method may give a more accurate prediction of the behaviour of cheese during cutting (e.g., portioning, slicing) than that obtained using large-strain shear or compression deformation tests.

Measurement of Time-Dependent Rheological Characteristics As has been stated (see ‘Terminology of Rheology and Texture’ and ‘Fundamental Measurements: Large Strain Deformation’), the rheological behaviour of viscoelastic materials, like cheese, is generally influenced not only by instantaneous stress or strain, as in the case of ‘ideal’ materials, but also by rheological history of the material, i.e., the stresses and strains which have already been experienced. This is verified by natural occurrences, such as a gradual deformation of a pane of glass under its own weight in a window of an old building. Indeed the compression of rock or ice under gravitational force results in flow, albeit very slow, analogous to a creep experi-

ment. Similarly, hard cheeses can eventually bulge, especially if stacked. Such time-dependent behaviour may be measured in creep and stress-relaxation tests, and may be carried out by means of compression, tension or torsion in the viscoelastic region (e.g.,

0.1; cf. Fig. 9).

Effect of Sample Temperature on Large Strain Deformation Characteristics in Cheese Early research showed that increasing the temperature of Gouda cheese in the range 10–20 °C reduced the value of f, f and max, as measured by compression to 80% using the Instron UTM (Culioli and Sherman, 1976). While f in Cheshire and Leicester cheeses decreased exponentially as the temperature was increased from 0 to 40 °C, the effect on fracture strain depended on the type of cheese; fracture strain for Cheshire cheese increased by ⬃2 over the range of temperature, while fracture strain for Leicester cheese was not affected by temperature (Dickinson and Goulding, 1980). Molander et al. (1990) reported a similar trend for f and max in 4-week-old Brie between 5 and 20 °C; however, in contrast to the results of Culioli and Sherman (1976), f increased slightly on raising the temperature. The discrepancy between the latter studies in relation to strain may be attributable to differences in the degree of fat coalescence, proteolysis and therefore fat separation and slippage. On heating cheese to a temperature (30–60 °C) greater than those (e.g., 4–25 °C) normally associated with retailing, domestic refrigeration and consumption, compression results in squeezing flow behaviour (Ak and Gunasekaran, 1995), i.e., stress increases with strain as the cheese is squeezed between the plates and no fracture point is observed. The deformability modulus (initial slope of the stress–strain curve) showed an Arrhenius type of characteristic, decreasing exponentially with temperature from 18 kPa at 30 °C to 3 kPa at 60 °C. Such a trend is expected, as milkfat is essentially fully liquid at 30 °C (Norris et al., 1973). Indeed, heating cheese to 60 °C in the absence of an applied stress generally results in flow of the part-molten cheese mass to an extent dependent on cheese type and heating time.

Techniques for Measurement of Viscosity In some situations, cheese products may occur in ‘liquid’ form, either in the course of processing or in their usage. Typical examples are processed cheese, cheese dips and cheese sauces. The viscosity of these products may be measured by a number of instruments, e.g., the


Bostwick consistometer, which has been used to give an empirical measurement of viscosity of a soft processed cheese spread (Rosenthal, 1999). In the latter instrument, a sample of the material being tested is placed in a cell and released by opening a simple guillotine slide gate, allowing the product to flow horizontally across a scale marked in centimeters. The length of flow in a given time period (usually 30 s), known as the Bostwick number, is taken as a measure of viscosity. Alternatively, viscosity can be measured under defined shear or low amplitude stress or strain in a rheometer, using different geometries such as concentric cylinders, a cone and plate, or parallel plates. Online measurements of viscosity of cheese products may be important, e.g., as an early measure of indicating the susceptibility of a formulation to ‘creaming’ (see ‘Pasteurized Processed Cheese and Substitute/Imitation Cheese Products’, Volume 2). A range of commercial on-line viscometers are available for measuring viscosity in a continuous flow situation.

Terminology Applied to Cheese Texture Cheese texture may be defined as a composite sensory attribute resulting from a combination of physical properties that are perceived by the senses of touch (including kinaesthesis and mouth-feel), sight and hearing (Brennan, 1988). Thus, cheese texture is directly measurable only by sensory analysis. Sensory analysis requires definition and classification of textural attributes or descriptors. Descriptors applied to cheese texture have been grouped into mechanical, geometrical and other characteristics (Fig. 31). The mechanical characteristics are sensed as forces on the teeth, tongue and the mouth

Primary characteristics Initial perception on palate

The Texture Profile Analysis (TPA) method, involving instrumental measurement using double bite compression, was developed to imitate the compressing action

• analytical characteristics • particle size, shape and size distribution • oil content; size, shape and size distribution of oil particles

Secondary characteristics

Residual masticatory impression

Relationships between cheese texture and rheology

• visual appearance • sampling and slicing characteristics • spreading, creaming characteristics, pourability

Initial perception (before placing in mouth)

Mastication (high shearing stress)

generally during eating, and by hearing in the case of fracture, whereas geometrical characteristics are mostly sensed visually but may also be partly sensed by touch. The other characteristics are ‘mouth-feel’ qualities, described subjectively by terms such as hard, soft, firm, springy, crumbly, adhesive, moist or dry. These terms are thought to have significance in relation to consumer appeal and satisfaction (Szczesniak, 1963a). The mechanical characteristics, in turn, have been divided into five primary parameters and three secondary parameters (Table 5, Fig. 31). The secondary parameters are considered to be composed of various intensities of hardness and cohesiveness. The geometrical parameters are divided into two classes, i.e., those related to particle size and hardness, and those related to particle shape and orientation. Experience shows that panelists found hardness relatively easy to sense but that adhesiveness was much more difficult to judge (Halmos, 2000). Sensory texture terms, as distinct from rheological terms, have linguistic boundaries, i.e., they are susceptible to different interpretation in different languages (Lawless et al., 1997; Bourne, 2002). Some texturerelated characteristics can be measured by machines and these are not bound by language. These characteristics include hardness, cohesiveness, adhesiveness, elasticity, viscosity, brittleness, chewiness and gumminess, definitions for which are given in Table 5. The measurements give objective quantifiable data, provided the measurement conditions are well defined.

Tertiary characteristics

• elasticity, cohesion • viscosity • adhesion (to palate) • • • • •

hard, soft brittle, plastic, crisp, rubbery, spongy smooth, coarse, powdery, lumpy, pasty creamy, watery, soggy sticky, tacky

• greasy, gummy, stringy • melt down properties on palate

Figure 31 Classification of food texture into primary, secondary or tertiary characteristics, based on Sherman (1969).


Table 5 Classification of the mechanical characteristics of cheese into primary and secondary parametersa Primary parameters

Secondary parameters

Hardness – the force necessary to attain a given deformation Cohesiveness – strength of internal bonds making up the body of the product Elasticity – the rate at which a deformed material returns to its original form after the deforming force is removed Viscosity – rate of flow per unit force Adhesiveness – the work necessary to overcome the attractive forces between the surface of a food and surface of other materials with which it comes in contact, e.g., the teeth, palate and tongue

Brittleness – the force at which the material fractures Chewiness – the energy required to masticate a solid food, e.g., some cheeses such as Mozzarella, to a state ready for swallowing Guminess – the energy required to disintegrate a semi-solid food, e.g., some cheeses such as ripe Camembert, to a state ready for swallowing

a Modified from Szczesniak (1963a), Bourne (1978).

Texture profile analysis (TPA)

A system of rheological parameters (e.g., firmness, elasticity) related to texture and known as TPA was developed (Fig. 32; Table 6; Friedman et al., 1963). The rheological measurements were originally carried out using the General Foods Texturometer (see ‘Imitative tests’), using double-bite compression (Bourne, 1978). Texture profile analysis parameters were later calculated from measurements using uniaxial doublebite compression at constant speed, using texture analysers including the Instron UTM (Breene, 1975; Bourne, 1978; Lee et al., 1978) and the texture analyser (TA series from Stable Micro Systems) (Halmos, 1997; Meullenet and Gross, 1999). Use of TPA to evaluate cheese texture

Szczesniak (1963b) found a curvilinear relationship between TPA hardness and an organoleptic rating of hardness. Casiraghi et al. (1989), working with five different Italian cheese varieties, including Grana Padano and Italico, showed that sensory hardness was highly correlated with instrumental hardness. 1st compression stroke 2nd compression stroke Stress, σ

of molar teeth while masticating food in the mouth (Szczesniak, 1963a; Peleg, 1976; Bourne, 1978). Classification of the mechanical attributes of cheese texture, as described above, was designed with the aim of integrating sensory data for foods evaluated by trained panels, with texture-profile data obtained on the same foods using compression testing. For this purpose, objective rheological parameters, some of which correspond in name to the sensory-determined parameters, were defined (Table 5) and are known as TPA parameters (see ‘Texture profile analysis’). While this classification system has been modified, the textural descriptors and their interpretation as devised by this classification scheme (Table 5) are still widely used in textural evaluation of food (Brennan, 1988; Drake et al., 1999). Sherman (1969) proposed an alternative classification of food texture (Fig. 31). The characteristics contributing to the texture of cheese, and other foods, during eating have been classified as primary, secondary (e.g., adhesiveness) or tertiary (e.g., firmness) (Fig. 31). The primary characteristics, from which all others are derived, include the food’s composition, its micro- and macro-structure, and its molecular properties. The secondary and tertiary categories of textural properties include many characteristics which are directly related to the rheological properties as it is subjected to various stresses and strains during eating, e.g., hardness, brittleness and adhesiveness (Sherman, 1969). According to this classification, the secondary characteristics are associated with initial perception in the mouth, i.e., upon contact with tongue, palate and teeth prior to mastication. Sherman (1969) claimed that the main characteristics sensed at this stage are elasticity (E), viscosity () and adhesion to the palate, where elasticity is understood as the tendency to recover its shape after removal of the stress. Two of those characteristics, namely elasticity and viscosity, can together be represented by the Burgers mechanical model (see ‘Cheese texture’).

H1 A1 0

A2 A3

Time Figure 32 Typical stress trend during a double-bite compression test, from which TPA parameters are calculated (see Table 5).


Table 6 Texture profile analysis (TPA) parameters and physical definitionsa Terminology b

Physical definition (TPA term)

Units

Fracturability Firmness Springiness (or elasticity) Cohesiveness Gumminess Chewiness Adhesiveness

Stress (or sometimes, force) to fracture point, H1 (Fig. 32) Stress (or sometimes, force) at a given deformation Percentage of deformation which is recovered between the first and second bites

Pa, kPa Pa or kPa –

Area of second bite over area of the first bite (A2/A1) in Fig. 32 Hardness Cohesiveness Hardness Cohesiveness Springiness Work necessary to pull the plunger (or compression plate) away from the sample (Area 3 in Fig. 32)

– Pa, kPa Pa, kPa J/m3

a Sources: Bourne (1978), van Vliet (1991a), Szczesniak (1963a), Yang and Taranto (1982). b Fracturability was originally known as brittleness (Bourne, 1978), and firmness as hardness (Szczesniak, 1963a).

Green et al. (1985) found significant correlations between five sensory attributes (firmness, springiness, crumbliness, graininess and stickiness) and instrumental parameters (f and f ). Hennequin and Hardy (1993) reported that TPA-hardness, i.e., force at 70% compression, also had a high correlation with sensory hardness (r 0.78, n 19, P 0.001) for four soft cheeses. Halmos (2000) compared sensory and instrumental measurements of hardness, cohesiveness and adhesiveness of six cheeses with a wide range of texture (including Havarti, Swiss and Romano). The sensory measurements increased with the corresponding instrumental readings, apart from one parameter for Romano cheese, for which the cohesiveness as measured instrumentally was ranked higher than the corresponding sensory measurement. The significant correlations, which were characteristic of the overall study, confirm the value of objective measurements in support of sensory measurements. However, the deviation in the trend for the Romano cheese highlights the complexity of textural (i.e., tactile sensory) characteristics as compared with instrumental measurements. Antoniou et al. (2000) performed sensory and TPA analyses on 15 French cheeses (Munster, Emmental, Roquefort, Beaufort, Camembert, Reblochon, Pont l’Eveque, Brie de Meaus, Tomme de Savoie, Valencay, St Nectaire, Pyrenees Brebis, Blue d’Auvergne, Comte Vieux and Fourme de Salers). The cheeses fell into three compositional groups on the basis of moisture (means 34, 45 and 51%, w/w). This grouping carried through to the results of mechanical and sensory analysis. The mechanical (TPA) terms which were most significant in differentiating the groups were: force at 10% deformation, relaxation force (after holding sample for ⬃12 s at 10% compression), force at 80% deformation (hardness), fracture force and adhesiveness. The most significant sensory terms were: hardness, fracturability and chewiness. Some of the mechanical parameters were highly correlated with

each other (e.g., force at 10% deformation, fracture force and hardness). Likewise, some of the sensory parameters were inter-correlated, e.g., hardness with adhesiveness. In agreement with previous studies (Green et al., 1985; Casiraghi et al., 1989; Hennequin and Hardy, 1993; Halmos, 2000), sensory parameters were highly correlated with mechanical parameters, e.g., mechanical hardness with sensory hardness. It is noteworthy that the 10% compression measurements (a level of deformation that is mostly recoverable) predicted cheese texture (i.e., as judged in sensory terms) better than the 80% compression tests. Despite the significant correlations between some sensory textural parameters and rheological measurements, instrumental analysis of texture, e.g., using texture analysers, is not considered a complete substitute for sensory evaluation (see Halmos, 2000), because of several factors: complexity of mastication, differences between individuals in the perception of texture, effect of time of day upon perception of texture, and others. While instrumental methods alone cannot be relied upon to determine consumer acceptance, their value resides in their ability consistently to enable small changes in physical characteristics, which contribute to texture, to be quantified. Use of instrumental shear deformation to evaluate cheese texture

Three techniques for large strain shear deformation testing have been described (see ‘Large strain shear measurements’). Truong et al. (2002) compared instrumental textural measurements on Cheddar cheese, as obtained using vane rheometry (shear), uniaxial compression (single bite) or TPA (double bite), with the corresponding sensory texture measurements. Instrumental texture maps of ten commercial Cheddar cheeses, generated by the vane method and by compression testing, clearly separated the cheeses and showed similar distribution patterns. Highly significant


correlations were found between vane parameters and TPA parameters (i.e., by uniaxial compression), and between TPA parameters and sensory texture parameters (by mouth). Correlations between vane parameters and sensory parameters were significant, but not as highly significant as between sensory and TPA parameters. The higher correlation between sensory texture and TPA texture could be due to the fact that TPA parameters were developed in conjunction with compression (i.e., General Foods Texturometer), while no corresponding texture-related parameters have been developed for torsional techniques, such as the vane method.

Conclusions The rheological properties of cheese have a large influence on its texture and behaviour during size reduction, and hence, its suitability as an ingredient (see ‘Cheese as an Ingredient’, Volume 2). Many factors influence the rheological properties, including manufacturing procedure, variety, composition and biochemical changes during ripening. The latter parameters have a major influence on the degree of hydration, or aggregation, of the para-casein matrix, which is the major structural element controlling deformation on the application of a stress. Many methods are available for measuring the rheological properties of cheese; some measure within the linear viscoelastic range to yield precise rheological quantities. In contrast, rheological measurements made under large strain or stress yield quantities which are more empirical in nature, but which are typically related to the stresses and strains experienced during consumption and size reduction.

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Glossary Cauchy strain. See Engineering strain. Compliance. Symbol J, is the ratio of strain to stress. In the elastic region, J 1/G, where G is shear (or storage) modulus. Cox-Merz rule. This rule states that the (steady) viscosity versus shear rate curve is virtually identical to the viscosity versus frequency curve, determined by dynamic oscillation. Creep. The response to a constant applied (normal or shear) stress. Creep can be expressed in terms of strain or compliance. Creep compliance. The ratio of strain, (t), resulting from an applied constant stress, c, to the stress, i.e., (t)/c. Creep modulus. The inverse of creep compliance, i.e., c/(t). Deformability modulus. Slope of the stress–strain curve in an approximately linear region, typically up to a strain of ⬇0.10. Elastic material behaviour. An elastic deformation is one where the material recovers fully upon removal of applied stress without time dependency, i.e., recovery is instantaneous and complete upon removal of stress. Elastoplastic material behaviour. When the stress in the material exceeds a certain limit, irreversible deformation results with negligible time dependency, i.e., partial recovery is instantaneous upon removal of stress; also known as elastoplastic deformation. Engineering strain. Deformation relative to original sample dimension, i.e., L/Lo, is called engineering strain, or Cauchy strain, or strain. Engineering stress. The ratio between applied force, F, and original sample area, Ao, is known as engineering stress or stress. Fracture work. See Toughness. Kelvin element. Also known as a Voigt element or a Kelvin-Meyer solid. This is a mechanical model consisting of a spring in parallel with a dashpot. A number of such elements in series, with a spectrum of time constants, can be used to simulate creep compliance. Kinematic viscosity. This is the ratio between dynamic viscosity and density. Units: m2/s 104 stokes. Linear behaviour. If one measured parameter varies in proportion to another, e.g., stress in proportion to a range of applied strain, their behaviour is described as linear and a modulus may be defined as the ratio between the parameters, e.g., Young’s modulus. Linear viscoelastic deformation. Cheese and other organic materials exhibit a combination of elastic and viscous behaviour at low strains, i.e., they recover their shape upon removal of applied stress, but not instantly. The elastic and viscous effects can be determined using


low-amplitude oscillatory rheometry. At low amplitudes of oscillation there is a constant relationship between the elastic and viscous components of complex modulus. Consequently, displacements of this type are referred to as linear viscoelastic deformation. Loss modulus. The ratio between the out-of-phase component of shear stress and shear strain (G /) in a dynamic oscillatory measurement; also referred to as viscous modulus. Maxwell element. This is a mechanical model consisting of a spring in series with a dashpot. A combination of such elements in parallel, with a spectrum of time constants, may be used to simulate relaxation behaviour in a viscoelastic material. Modulus of deformability. See Deformability modulus. Poisson effect. When a sample is compressed it bulges in the lateral direction, i.e., the cross-section increases with compression; this is the Poisson effect. The ratio between lateral strain and longitudinal strain is known as Poisson’s ratio. Poisson’s ratio equals 0.5 in the absence of a volume change, and is less than 0.5 for a compressible material. Shear modulus. The ratio between the in-phase components of shear stress and shear strain (G /) in a dynamic oscillatory measurement; also referred to as storage, elastic, or in-phase, modulus. Storage modulus. See Shear modulus. Strength. The maximum stress a material withstands before it breaks (i.e., fractures) or flows (i.e., becomes plastic). Stress. See Engineering stress. Stress relaxation modulus. The stress that is required to maintain a constant deformation is observed, as a function of time (i.e., in a stress relaxation test). The ratio of shear stress to strain is known as stress relaxation modulus, or relaxation modulus. The relaxation modulus depends on the applied strain if the strain exceeds the limit of linear viscoelasticity. Thus, G(t, ) (t)/. Stress relaxation test. This test involves an initial application of (a normal or shear) strain at a constant rate up to a pre-determined level of strain and then measuring

the decay of stress as a function of time while holding the sample at constant strain; also known as a step strain transient test. Toughness. The work required to fracture; this is measured as the area under a force–deformation curve up to the point of fracture (Fig. 23). True strain. The accumulated strain during the applied loading, ln (L/Lo), where ln is the natural logarithm, L is the sample length under load, and Lo is the original sample length, is known as the true strain, Hencky strain or natural strain. This is applicable where the strain is large and sample cross-section changes appreciably under the load. True strain is not used very much in cheese rheology. True strain can be related to engineering strain, , using, ln (1 ). True stress. The ratio between applied force, F, and actual area of cross-section, A, is termed true stress. Thus, true F/A, where A is the actual area, taking the Poisson effect into account. Uniaxial compressive strength. The apparent stress at fracture, i.e., Fo/Ao, where Fo is the compression force at fracture and Ao is the initial cross-sectional area of the sample. Viscoelastic material behaviour. Where rheological behaviour can be resolved into elastic and viscous components, e.g., as represented by a Maxwell model. Viscoplastic material behaviour. In contrast to elastic behaviour, this is a time-dependent and irreversible deformation that occurs when a certain stress level has been exceeded, i.e., strain does not respond instantaneously to applied stress, but instead strain keeps on growing while the stress is applied and does not return to zero upon removal of stress; also referred to as viscous material behaviour. Viscosity or dynamic viscosity. Coefficient of dynamic viscosity, , is the ratio between shear stress and shear rate. /" where is shear stress and " is shear rate. Units: Pa.s or N.s/m2 10 poise. Viscous modulus. See Loss modulus. Young’s modulus. The ratio between normal stress and engineering strain (E / ).

Growth and Survival of Microbial Pathogens in Cheese C.W. Donnelly, Department of Nutrition and Food Science The University of Vermont, Burlington, USA

Introduction Cheesemaking evolved centuries ago as a means of preserving raw milk via fermentation. Selection of the beneficial natural flora in milk, such as lactobacilli, streptococci and lactococci, or direct addition of these as starter cultures, preserves products and in many instances allows competition with bacterial pathogens. However, cheeses can become contaminated with pathogens as a result of their presence in the raw milk used for cheesemaking and subsequent survival during the cheesemaking process. Alternatively, bacterial pathogens can contaminate cheese via post-processing contamination if sanitation and other measures in the processing plant are not sufficient to prevent re-contamination (Linnan et al., 1988; Johnson et al., 1990a). The characteristics of the specific cheese variety will dictate the potential for growth and survival of microbial pathogens, with ripened soft cheeses presenting a higher risk for growth and survival of pathogens than aged hard cheeses where a combination of factors, including pH, salt content and aw, interact to render cheeses microbiologically safe. Although cheeses have been linked with documented outbreaks of food-borne illness, epidemiological evidence collected from around the world confirms that this occurs infrequently ( Johnson et al., 1990a; Altekruse et al., 1998; De Buyser et al., 2001). This chapter will provide an overview of factors which affect growth and survival of microbial pathogens in cheese.

Factors that Influence the Safety of Cheese The pathogens, Salmonella enterica, listeria monocytogenes, Staphylococcus aureus and enteropathogenic E. coli (ETEC) pose the greatest risk to the safety of cheese (Johnson et al., 1990a; De Buyser et al., 2001; Leuschner and Boughtflower, 2002). If active lactic acid starter cultures are used, Staph. aureus is considered to be a low-risk pathogen (Johnson et al., 1990a). However, in traditional cheeses where active starter cultures are not used, Staph. aureus may pose a significant risk for toxin production in cheese if numbers are sufficiently high. The factors that contribute to

the safety of cheese with respect to pathogenic bacteria include milk quality, starter culture or native lactic acid bacterial growth during cheesemaking, pH, salt, control of aging conditions and chemical changes that occur in cheese during aging (Johnson et al., 1990c). Other technologies (e.g., use of starter cultures that produce substances inhibitory to pathogens) may provide opportunities to add additional barriers to the growth of bacterial pathogens. It is particularly important for the producers of raw milk cheeses to have a documented and systematic approach to ensure product safety. Pathogens in raw milk

S. enterica, L. monocytogenes, Staph. aureus and ETEC are associated with raw milk. E. coli 0157:H7 can readily contaminate raw milk on the farm with contamination levels of 4.2–10% and 2% reported in the US and Canada, respectively (D’Aoust, 1989; Padhye and Doyle, 1991). Over 70 cases of E. coli infection, characterized by bloody diarrhea, haemolytic uremic syndrome (HUS) and kidney failure, have been traced to the consumption of raw milk (Martin et al., 1986; Borczyk et al., 1987; Bleem, 1994) with a few additional cases in England linked to yoghurt (Morgan et al., 1993). E. coli 0157:H7 was first characterized in 1982 during epidemiological investigations of two outbreaks which occurred in North America. Cattle are thought to be the principal reservoir for this important human pathogen, and in investigations where food has been identified as the vehicle of transmission, ground beef is the product most frequently linked to human illness. Shere et al. (1998), in a longitudinal study of E. coli dissemination on four Wisconsin dairy farms, identified contaminated animal drinking water as the most probable vehicle for infection of animals and a potential intervention point for on-farm control of dissemination of this pathogen. Since shedding of this pathogen by cattle is intermittent, re-inoculation from an environmental source rather than colonization of the pathogen is the more likely explanation than intermittent shedding.



542 Growth and Survival of Microbial Pathogens in Cheese S. enterica serovars Enteritidis, Typhimurium and Dublin have been associated with food-borne disease outbreaks involving raw milk and milk products (Maguire et al., 1992; Cody et al., 1999; Villar et al., 1999; De Valk et al., 2000). A 1987 FDA survey revealed the presence of salmonella in 32 of 678 (4.7%) samples of raw milk obtained from bulk-tank trucks in Wisconsin, Michigan and Illinois, with 10 of 16 (62.5%) collection sites also testing positive (McManus and Lanier, 1987). Salmonella spp. were isolated from 26 of 292 (8.9%) of farm bulk tank samples collected in eastern Tennessee and southwest Virginia (Rohrbach et al., 1992). Wells et al. (2001) examined recovery of salmonella from faecal samples obtained from dairy cows in 91 herds from 19 US states. Salmonella spp. were recovered from 5.4% of the samples. Recovery levels from cows on farms with less than 100 animals were much lower (0.6%) than those from farms with over 100 cows, where recovery levels were 8.8%. The incidence of Salmonella spp. in milk samples would be expected to occur at a much lower frequency than in faecal samples. Most farmstead cheesemakers maintain small dairy herds, where the lower incidence data would apply. S. enterica serotype Typhimurium definitive type (DT) 104 emerged in the UK as an important source of human infection in the late 1980s (Threlfall et al., 1996). Subsequent outbreaks of human illness traced to dairy sources were reported in Vermont (Friedman et al., 1998), Nebraska, California (Cody et al., 1999) and Washington State (Villar et al., 1999). This organism is notable because it is resistant to multiple antibiotics. Two outbreaks of S. enterica subsp. enterica serotype Typhimurium DT104 infection were recently linked to the consumption of Mexican-style soft cheese manufactured from raw milk (Cody et al., 1999; Villar et al., 1999). Aceto et al. (2000) conducted a survey to assess the herd prevalence of S. enterica subsp. enterica serotype typhimurium DT 104 in Pennsylvania dairy herds. Of 51 farms surveyed, 11 were positive for salmonella species and 4 for S. typhimurium, 2 of which were DT-104 positive. S. enterica serovar Dublin is present in dairy cattle and was identified as the most invasive of the salmonella bacteria for humans in studies conducted in Denmark (Lester et al., 1995). Beckers et al. (1987) and Lovett et al. (1987) estimate that extremely low levels of L. monocytogenes (0.5–1.0 ml) exist in commercial bulk-tank raw milk. Listeria is inactivated by pasteurization, and contamination of processed dairy products is therefore most likely a function of post-pasteurization contamination from the dairy plant environment. In fact, numerous surveys document the presence of listeria within the dairy plant environment, including floors in coolers, freezers, processing rooms, particularly entrances, cases

and case washers, floor mats and foot baths and the beds of paper fillers (Charlton et al., 1990; Klausner and Donnelly, 1991). Pritchard et al. (1994), in a study of dairy processing facilities, found that processing plants near a farm had a significantly higher incidence of listeria contamination than those without an on-site dairy farm. Arimi et al. (1997) demonstrated the link between on-farm sources of listeria contamination (dairy cattle, raw milk and silage) and subsequent contamination of dairy-processing environments. These investigators subjected listeria strains collected from farms and dairy processing environments over a 10-year period to strain-specific ribotyping using the automated Riboprinter™ microbial characterization system. A total of 388 listeria isolates from 20 different dairy processing facilities were examined along with 44 silage, 14 raw-milk bulk tank and 29 dairy cattle isolates. These 475 isolates included 93 L. monocytogenes, 362 L. innocua, 11 L. welshimeri, 6 L. seeligeri, 2 L. grayii and 1 L. ivanovii strains. Thirty-seven different listeria ribotypes (RTs) comprising 16 L. monocytogenes (including five known clinical RTs responsible for food-borne listeriosis), 12 L. innocua, 5 L. welshimeri, 2 L. seeligeri, 1 L. ivanovii and 1 L. grayii were identified. Greatest diversity was seen among the isolates from dairy-processing facilities with 14 of 16 (87.5%) L. monocytogenes RTs (including 5 clinical RTs), and 19 out of 21 (90.5%) non-L. monocytogenes RTs detected. Sixty-five of the ninety-three L. monocytogenes isolates belonged to the group of the five clinical RTs, which included one RT unique to dairy-processing environments, two RTs common to dairy-processing environments and silage, and one RT common to dairyprocessing environments, silage, raw milk and dairy cattle with the last RT appearing in dairy-processing environments, silage, raw-milk bulk tanks and dairy cattle. The finding of eight L. monocytogenes and twelve non-L. monocytogenes RTs common to both dairyprocessing and farm environments clearly implicates the farm as a natural reservoir for listeria RTs capable of entering dairy-processing facilities. These findings, which support the link between on-farm sources of listeria contamination (dairy cattle, raw milk and silage) and subsequent contamination of dairy-processing environments, stress the importance of farm-based Hazard Analysis and Critical Control Points (HACCP) programmes for controlling listeria. This work also showed that two important clinical L. monocytogenes ribotypes which were previously identified as RT 19092 and RT 19161 and epidemiologically linked to listeriosis cases involving pasteurized milk and turkey frankfurters were recovered from dairy-processing facilities A and B for 12 and 3 months, respectively, with L. innocua RT 19094 also present in these same two facilities for at least five years.

Growth and Survival of Microbial Pathogens in Cheese 543

Abou-Eleinin et al. (2000) analysed 450 goats’ milk samples obtained from the bulk tanks of 39 goat farms for listeria spp. over a 1-year period. Modified versions of the USDA-FSIS (McClain and Lee,1989) and FDA (Lovett et al., 1987) protocols were used for recovery of listeria. Overall, 35 (7.8%) samples yielded listeria, with L. monocytogenes identified in 17 of the 35 (3.8%) listeria-positive samples and L. innocua in 26 (5.8%) of samples. Eight milk samples contained both L. monocytogenes and L. innocua. Milk samples from 18 of the 39 (46.2%) farms were positive for listeria at least once during the year-long study. Five different listeria RTs were identified from 34 selected L. monocytogenes isolates, 2 of which were deemed to be of clinical importance. Isolation rates of listeria were markedly higher during the winter (14.3%) and spring (10.4%), compared to autumn (5.3%) and summer (0.9%). Similar trends have been previously reported for cows’ milk (Rea et al., 1992; Ryser, 1999). Milk quality

Raw-milk quality is important in producing all cheeses, but particularly for those made from raw milk. Low bacterial counts and low somatic cell counts are the key indicators of milk quality, and as their numbers increase, there is a higher risk for contamination of milk and cheese with pathogens. Monitoring and controlling bacteria and somatic cell counts in milk should be components of a HACCP programme to ensure product safety. As rapid, cost-effective methods become available for detection of bacterial pathogens in raw milk, the use of specific pathogen testing could become part of a HACCP programme. In general, when rawmilk bacteria and somatic cell counts are high, there will be other negative impacts on cheese quality that may reduce consumer acceptability and cheese yield. In most artisanal cheesemaking, the time from milking to cheesemaking is very short and in some cases the milk is made into cheese immediately on the farm without cooling. Minimizing the time from milk collection to the initiation of cheesemaking reduces the opportunity for the growth of undesirable bacteria in raw milk. Conversely, when milk is cooled and held in transport, the opportunity for pathogen growth, particularly growth of psychrotrophic pathogens, is increased. The European Community Directives 92/46 and 92/47 (Anonymous, 1992) contain regulations for the hygienic production and placing on the market of raw milk, heat-treated milk and milk-based products. These regulations establish hygienic standards for raw-milk collection and transport that focus on issues such as temperature, sanitation and microbiological standards, enabling the production of raw milk of the highest

possible quality. Raw cows’ milk must meet quality standards, e.g., a standard plate count at 30 °C of 100 000 cfu/ml and somatic cell counts of #400 000 per ml of milk. To meet these and other established standards, countries employ HACCP principles in the production of fluid dairy products. This involves identification of sites to be monitored and evaluated to ensure that products are produced under the correct conditions, as well as the development of critical limits established by valid and verifiable parameters. In the case of fluid milk products, many processors have identified length of shelf-life as a critical limit. Shelf-life is influenced by a number of factors, including cleaning and sanitizing of pipelines and milking equipment, condition of raw milk used to produce product and storage temperature. Pasteurization will eliminate some of the indigenous microflora in the raw milk, including pathogenic bacteria; however, thermoduric organisms survive pasteurization. Post-pasteurization contamination of milk is problematic if the processing/packaging environment is not maintained. Moreover, many contaminants, including listeria, are able to form biofilms which protect them from cleaning and sanitizing agents. Some regulations, such as those of the EU, have established microbiological limits at the sell-by-date for products such as cheeses. With respect to regulations which govern the use of raw milk for cheesemaking, limits have been established for Staph. aureus in raw milk. Finished cheeses must meet specific hygienic standards, in which case the presence of Staph. aureus and E. coli indicates poor hygiene. Heat treatment of milk

Milk contains heat-labile compounds (e.g., lactoferrin, lysozyme and lactoperoxidase) that are inhibitory to the growth of some pathogens. Recent work by Pitt et al. (2000) has demonstrated that the growth of Staph. aureus, S. enteritidis and L. monocytogenes was slower in raw milk held at 37 °C for 72 h than in pasteurized milk held under the same conditions. During the first 16 h of incubation, the number of organisms increased in both raw and pasteurized milks, but after 16 h, the number of recoverable viable pathogenic organisms in the raw milk began to decrease; an overall decrease of 2–5 logs was found. The inhibitory effect of raw milk on the survival of the above three pathogens in milk, reported by Pitt et al. (2000), is probably of great importance for cheesemaking from raw milk, and additional research needs to be undertaken. The lactoperoxidase system (LPS) is a naturally occurring inhibitory system in raw milk and comprises three components, lactoperoxidase, thiocyanate and hydrogen peroxide. All three components are required to exhibit maximum antimicrobial effects.

544 Growth and Survival of Microbial Pathogens in Cheese Gram-negative psychrotrophs, such as pseudomonads, are extremely sensitive to this system. Approximately 0.5–1.0 g/ml lactoperoxidase is needed for effective inhibition, and bovine milk typically contains 30 g/ml (Björck, 1978). Quantities of thiocyanate and hydrogen peroxide in milk are variable. For a full inhibitory effect, about 10 g/ml of hydrogen peroxide is required, and bovine milk normally contains 1–2 g/ml hydrogen peroxide. The thiocyanate levels in milk range from 0.02 to 0.25 mM but 8–25 mM is needed for optimum activity. Pitt et al. (2000) hypothesized that the inhibitory effect of raw milk in their study was due to activation of the lactoperoxidase system by hydrogen peroxide-producing lactic acid bacteria naturally present in the raw milk, which grew at 37 °C. The authors postulated that the reduction in growth of these organisms in the raw milk could result from inhibitory products produced by activation of the LPS. However, it is highly unlikely that pasteurization inactivates lactoperoxidase in milk, and so there must be an alternative explanation for the much greater inhibition of Staph. aureus, S. enteritidis and L. monocytogenes by the raw milk. Some cheeses are made from milk that has been given a sub-pasteurization heat treatment at the farm, but are technically classified as raw-milk cheeses. This process can be beneficial when milk has to be transported and stored at refrigeration temperature at a cheesemaking facility and when there will be a time delay before cheese manufacture. These near-pasteurization thermal treatments are often called thermization and they help to reduce the gowth of psychrotrophic bacteria that cause quality defects in cheese. However, the thermization process may partially inactivate some indigenous antimicrobial milk components that were mentioned previously. Comprehensive studies conducted by the US Food and Drug Administration (FDA) and the US Department of Agriculture (USDA; Bunning et al., 1986, 1988) and by Health and Welfare Canada (Farber et al., 1992) have shown that listeria are unable to survive normal pasteurization conditions. Knabel et al. (1990) found that growing L. monocytogenes at 43 °C prior to heat-inactivation caused an increase in thermotolerance, but a study conducted by Farber et al. (1992) demonstrated that even under worst-case scenario conditions, which included cultivation of L. monocytogenes populations at 43 °C prior to inactivation, pasteurization would render a 4.5–6.2-D process. Therefore, while populations of L. monocytogenes have been shown to survive minimum pasteurization (71.1 °C/16 s) in various laboratory studies, survival under actual conditions of commercial milk pasteurization and processing is unlikely. Studies which define the impact of commercial heat treatment of raw milk,

either naturally or artificially contaminated with bacterial pathogens, are still relatively scarce. However, L. monocytogenes is generally regarded as being more heat resistant than salmonella or E. coli 0157:H7 (D’Aoust et al., 1987; Line et al., 1991). Using raw milk inoculated to contain various pathogens at a level of 105 cfu/ml, D’Aoust et al. (1987) concluded that salmonella were inactivated in milk after heating to 64.5 °C (148.2 °F) or above for 16.2 sec, except for S. senftenberg which survived until the treatment exceeded 67.5 °C (153.5 °F) for 16.2 sec. Heating at 63 °C (145.4 °F) for 16.2 sec reduced populations of S. senftenberg by 3 orders of magnitude. Thermal inactivation of E. coli 0157:H7 was complete at temperatures 64.5 °C (148.2 °F) for 16.2 sec (Line et al., 1991) which is similar to that required for most salmonella except S. senftenberg. Much of the aged raw milk cheese produced in the US is subjected to some form of heat treatment, generally thermization. This treatment generally consists of heat treatment at 55 °C for a period ranging from 2 to 16 sec. The specific impact of this heat treatment combined with the interactive effects of salt and pH during subsequent ripening on pathogens such as listeria, salmonella and E. coli has not been well explored. Extrinsic and intrinsic parameters in cheese which dictate microbial growth

Growth of microbial pathogens in cheese is dictated by extrinsic and intrinsic parameters. The important intrinsic parameters include moisture content, pH and acidity, nutrient content, redox potential, presence of antimicrobial compounds, either those occurring naturally or those which are added as food preservatives, e.g., NO3, and the presence of competitive microflora (ICMSF, 1986). All of these factors dictate the potential for bacterial pathogens to grow, persist or decline in cheeses. Extrinsic parameters include factors such as type of packaging/packaging atmosphere, time and temperature of storage and holding conditions, processing steps, product history and traditional use. The interaction of these factors dictates the potential for microbial growth in cheese. Depending on the cheese variety, intrinsic parameters such as pH may serve to enhance or inhibit the growth of bacterial pathogens. Ryser and Marth (1987a) studied the behaviour of L. monocytogenes in Camembert cheese. The high moisture content and the neutral pH of this surface-ripened cheese facilitate growth and survival of pathogens such as listeria. Growth of listeria in Camembert cheese was found to parallel the increase in cheese pH during ripening and reached a final population of 106–108 per g. This contrasts with Blue cheese, where listeria failed to grow and decreased in number during


56 days of storage (Papageorgiou and Marth, 1989). These authors suggested that Penicillium roqueforti may produce bacteriocins against L. monocytogenes. In hard cheese varieties like Colby and Cheddar, L. monocytogenes populations decline during aging, with survival strongly influenced by the moisture content and the pH (Ryser and Marth, 1987b; Yousef and Marth, 1990). Cheeses such as Camembert and Feta have nearly identical composition in terms of moisture content, water activity, % salt-in-water and ripening temperature. However, fully ripened Camembert has a pH of 7.5 versus Feta which has a pH of 4.4 that prevents the growth of listeria. Cheeses made from raw milk

In the US and other parts of the world, the manufacture of cheese from raw milk is a topic which is being revisited from the perspective of microbiological safety. Pasteurization of milk prior to cheesemaking is but one step that may reduce the risk of the presence of pathogenic bacteria in cheese. Current US regulations which govern the use of raw, heat-treated and pasteurized milk for cheesemaking were promulgated in 1949 (Anonymous, 1950; 21 CFR Part 133). One of the two options can be selected by cheesemakers to assure the safety of cheese – pasteurize milk destined for cheesemaking or hold cheese at a temperature of not less than 1.7 °C (35 °F) for a minimum of 60 days. Recent research has shown that S. typhimurium, E. coli 0157:H7 and L. monocytogenes can survive well beyond the mandatory 60-day holding period in Cheddar cheese prepared from pasteurized milk (Reitsma and Henning, 1996; Ryser, 1998). In a referral to the National Advisory Committee on Microbiological Criteria for Foods in April 1997, the FDA asked if a revision of policy requiring a minimum 60-day aging period for raw-milk hard cheeses was necessary. The FDA, in its communication, noted that such a duration may be insufficient to provide an adequate level of public health protection. The FDA cited numerous studies and outbreak investigations documenting the presence of listeria, salmonella and E. coli 0157:H7 in raw milk. Of particular concern was the report by Reitsma and Henning (1996) detailing the survival of E. coli 0157:H7 in aged Cheddar cheese. The FDA did note, however, that there was ‘limited epidemiological evidence that food-borne illness results from consumption of raw-milk hard cheeses that have been aged for 60 days’, citing work by Fabian (1947), D’Aoust et al. (1985) and Johnson et al. (1990b) in support of this claim. Groups outside of the US have recently expressed concern about the safety of raw-milk cheeses. The Institute of Food Science and Technology (IFST, 2000) in the UK issued a position statement drawing attention to the

potential public health hazards posed by pathogenic bacteria in cheeses made from raw milk. The IFST indicates that these hazards apply particularly to soft and semi-soft cheeses (IFST, 2000). Codex Alimentarious is presently recommending a ‘combination of control measures’ (including pasteurization) to achieve the appropriate level of public health protection (Groves, 1998). In a comprehensive review of all outbreaks of human illness associated with the consumption of aged rawmilk cheese, in the majority of instances, confounding parameters other than use of raw milk contributed to pathogens being present in the product at the time of consumption (Donnelly, 2001). Further, in challenge studies which examine the fate of pathogens in aged cheese, confounding factors can also explain the appearance of pathogens following 60 days of aging. Such confounding parameters in actual outbreaks or challenge studies involve the use of pasteurized versus raw milk in cheesemaking trials, inadequate development of acidity during cheesemaking, a low salt level, contamination by ill employees during manufacture, temperature abuse of milk designed for cheesemaking and environmental contamination during cheesemaking.

Previous Reviews on the Safety of Raw Milk Cheeses Two comprehensive reviews have been published regarding outbreaks of human illness linked to consumption of cheese. Johnson et al. (1990b) conducted a comprehensive review of the epidemiological literature during the 40-year period, 1948–1988. These authors identified only six outbreaks of illness transmitted by cheese produced in the US during this period. Post-pasteurization contamination was the most frequent causative factor in these outbreaks. Improper pasteurization equipment and/or procedures were implicated in only one outbreak each in the US and Canada, and use of raw milk was a factor in one outbreak in each of these countries. No outbreaks were linked to hard Italian cheese varieties such as Parmesan, Romano and Provolone. In rare instances, Swiss and Cheddar cheeses were linked to food-poisoning outbreaks. Factors other than pasteurization cited by Johnson et al. (1990b) as contributors to cheese safety include milk quality and management, lactic starter management, pH, salt, controlled aging conditions and natural inhibitory substances in the raw milk. These authors proposed three actions to improve the safety of raw milk cheeses: (1) Establish a guideline for minimum heat-treatment of milk for cheesemaking, e.g., 64.4 °C (148 °F) for 16 sec or equivalent with adequate process control, (2) Evaluate current safety

546 Growth and Survival of Microbial Pathogens in Cheese technology and practices used for cheese manufacture and (3) Evaluate technologies not currently used in cheese manufacture for safety potential (Johnson et al., 1990c). Altekruse et al. (1998) reviewed all cheese-associated outbreaks reported to the Centers for Disease Control and Prevention (CDC) during the period 1973–1992. These authors noted the infrequency of large, cheeseassociated outbreaks reported during this period and suggested that improvement of cheesemaking methods and process control have resulted in cheese being a safer product. There were 32 cheese-associated outbreaks, 11 of which could be attributed to contamination at the farm, during manufacturing or during processing. Of the 11 outbreaks attributed to contamination prior to distribution, 5 were associated with the consumption of Mexican-style soft cheese versus only one outbreak linked to Cheddar cheese. It is notable that no outbreaks reported to the CDC during 1973–1992 were associated with raw milk cheese that was aged for a minimum of 60 days. The authors indicated that salmonella, E. coli 0157:H7 and L. monocytogenes may survive the aging process. However, the literature reference for survival of listeria points to Camembert cheese (Ryser and Marth, 1987a), and the authors failed to note the rapid decline of listeria populations in aged Cheddar cheese as documented by Ryser and Marth (1987b). Altekruse et al. (1998) suggest that aging alone may not be a sufficient pathogen control step to eliminate salmonella, listeria and E. coli 0157:H7 from cheese. Outbreaks involving Cheddar cheese

In 1976, seven lots of Cheddar cheese manufactured from pasteurized milk were contaminated with S. heidelberg and were responsible for 339 confirmed cases of illness and an additional 28 000–36 000 cases of illness (Fontaine et al., 1980). The cheese involved was aged for less than 60 days, and improper pasteurization was cited as the cause of the outbreak. Follow-up with the first few patients led epidemiologists to suspect cheese eaten in Mexican-style restaurants as the vehicle of infection. Seven lots of Cheddar cheese produced from pasteurized milk by a Kansas manufacturer and purchased from a single Denver distributor were identified as the potential sources of contamination. The epidemic began in July in two widely separated Colorado cities, Denver and Pueblo. Levels of S. heidelberg in these cheeses were estimated to be 0.36–1.8 per 100 g. The pH of contaminated cheese was 5.6, which may have been a factor in this outbreak. Poor manufacturing practices coupled with inadequate control programmes at the cheese plant were cited as causative factors in this outbreak. The Kansas State Health Department had

recorded 25 instances of non-compliance with good manufacturing practices by that particular food-processing plant. The Kansas Board of Agriculture required that raw milk contain 3 000 000 organisms/ml. Routine microbial analysis of the grade B or surplus grade A milk used at the plant revealed that counts greatly exceeded this standard. In the production of cheese, raw milk was stored for 1–3 days in an insulated but unrefrigerated holding tank prior to pasteurization at 71.6 °C for 15 sec. The milk was filtered after pasteurization, which is a violation of FDA guidelines for pasteurization. Salmonella outbreaks in Ontario, Canada, during the period 1980–1982 occurred in raw-milk Cheddar cheese. S. muenster was identified in the cheese and traced to a single farm where one cow was shedding the organism (Wood et al., 1984). Subsequent trials using milk from this infected cow were conducted to determine potential for survival during commercial preparation of raw milk cheese. Curd tested positive in 11 of 181 vats. During curing, one lot was negative after 30 days but one lot was positive after 125 days. It would be unlikely for this scenario to be repeated as cheese is rarely manufactured from milk from a single cow. Milk is co-mingled, and the dilution effect with milk from other animals and other farms reduces the level of pathogens, if present. A large Canadian outbreak of salmonellosis linked to the consumption of Cheddar cheese was reported in four Canadian Atlantic provinces (Newfoundland, New Brunswick, Prince Edward Island and Nova Scotia) between January and July 1984. This outbreak proved to be the largest single epidemic of salmonellosis ever to occur in Canada, ultimately involving more than 2700 cases of illness (Bezanson et al., 1985; Johnson et al., 1990b). Production of the cheese, which was manufactured from either pasteurized (73.8 °C (165 °F) for 16 sec) or heat-treated (66.7 °C (152 °F) for 16 sec) milk, was traced to a single plant on Prince Edward Island. Testing of the raw milk supply identified two cows in separate herds, one which shed S. typhimurium and one which shed S. heidelberg. D’Aoust et al. (1985) reported on the distribution and survival of S. typhimurium phage type 10 isolated from Cheddar cheese in this outbreak. Levels of salmonella ranged from 0.36 to 9.3 per 100 g. The pH of the cheese ranged from 4.97 to 5.40, consistent with normal Cheddar, which has a pH range of 5.0–5.5. S. typhimurium phage type 10 was found to survive in Cheddar cheese for up to 8 months at 4 °C. The data provided by D’Aoust et al. (1985) is very interesting. The authors compare salmonella recovery as a function of whether mild Cheddar cheese was manufactured from heat-treated (16 s at 66.7 °C; not pasteurized) or pasteurized (16 sec at 73.8 °C) milk.


Tested samples of mild Cheddar manufactured from heattreated milk were found to contain 0.36–9.3 salmonella/100 g. However, four lots of mild Cheddar manufactured from pasteurized milk were also found to contain 0.36–4.3 salmonella/100 g. Certain lots of cheese contained Staph. aureus at high levels (105 per g), which may indicate poor starter activity (Johnson et al., 1990b) or contamination through handling. It is difficult to understand how D’Aoust et al. (1985) could support their concluding statement in this article ‘Although pasteurization of milk used in cheesemaking increases the safety of the finished product, use of heat-treated (unpasteurized) milk in the manufacture of medium and old Cheddar cheese and survival of salmonella during prolonged periods of refrigerated storage raises legitimate doubts of the safety of current manufacturing practices.’ In the data presented, pasteurization did not result in the unequivocal safety of mild Cheddar cheese. An evaluation of the pasteurization process, described by Johnson et al. (1990b), indicated that the employee in charge of the process manually overrode the electronic controls, which shut down the pasteurizer while milk continued to flow through the unit and into the vat. The pasteurizer was shut down after filling three vats and later restarted to fill the next three vat series. The first and the third vats of each three vat sequence tested positive for salmonella, except for the first vat of the day and the middle vat of each three vat series which consistently tested negative. This pattern only occurred when raw milk which included milk from the cow shedding S. typhimurium was used. Bezanson et al. (1985) subsequently subjected outbreak strains to molecular analysis by biotyping, antibiotic resistance patterns, plasmid restriction and endonuclease analyses and revealed that two genetically distinct organisms were the aetiologic agents in this outbreak. These studies revealed the existence of a double infection, indicating that the incriminated cheese likely had two sources of contamination. S. typhimurium phage type 10 subgroup I strains were identified among cultures from raw milk and cattle associated with the incriminated dairy. S. typhimurium phage type 10 subgroup I and II strains were recovered from individuals employed at the dairy along with their family members. S. typhimurium subgroup I and II strains were present in cheese curd samples obtained from the plant as well as from a consumer pack obtained from a distributor. Cheese plant workers from whom both subgroup I and II strains were cultured were involved in the production and/or packaging of Cheddar cheese, raising questions about the possibility of contamination of the cheese by ill workers. Salmonella were confirmed in a cheese-trim bucket. Plant inspections revealed that employees used their bare hands to transfer cheese to a forming machine, and an employee tested

positive for S. typhimurium. It is likely that this incriminated cheese was also responsible for an outbreak of illness reported at the same time in Ontario linked to S. typhimurium phage type 10 biotype 4 (D’Aoust et al., 1985). Hedberg et al. (1992) reported on a multi-state outbreak of S. javiana and S. oranienburg linked to the consumption of contaminated Mozzarella cheese and shredded cheese products. Cases were more likely to have consumed cheese manufactured at a single cheese plant or cheese shredded at processing plants that also shredded cheese from the single plant, than matched controls. The outbreak strains were isolated from 2 of 68 unopened 16-oz blocks of Mozzarella cheese. Inspections revealed deficiencies in plant sanitation and cleaning, and equipment was not routinely cleaned and sanitized between shredding different types of cheese from different manufacturers. However, no deficiencies in pasteurization were identified. Cheese-manufacturing equipment was found to be susceptible to environmental contamination and contamination by aerosols. Investigators believed that the contaminated Mozzarella cheese sent to four processing plants for shredding, crosscontaminated other cheese products at those plants. It is most likely that the cheese was contaminated from environmental sources or from infected production workers. Four outbreaks occurring in the late 1990s were reported in the UK, although detailed epidemiologic data on these outbreaks is lacking. An outbreak of E. coli 0157:H7 (phage type 8, Verotoxin gene 2) infection involving 22 cases was reported in Scotland in 1994. This outbreak was associated with the consumption of raw-milk cheese (Anonymous, 1997a). A December 1996 outbreak of salmonella gold-coast which occurred in England and Wales was linked to the consumption of a brand of mild, coloured, Cheddar cheese produced in August and September 1996 in Somerset, England. Phosphatase tests and examination of recording chart records from the pasteurizer indicated that pasteurization had failed at the plant on several occasions (Anonymous, 1997b). An outbreak of infection caused by E. coli 0157:H7 (phage type 21/28 VT2) was reported in 1999 in north-east England (Anonymous, 1999a,b). The vehicle of infection was Cotherstone cheese, a rawmilk cheese, manufactured in small quantities and distributed to specialty cheese shops in England. Samples from the dairy herd, slurry and environmental samples from the cheese manufacturing facilities were negative for E. coli 0157:H7. In March of 1999, a large outbreak of infection was reported in England and Wales due to consumption of contaminated milk from a single dairy. An outbreak of E. coli 0157:H7 infection was reported which was linked to the consumption of fresh cheese

548 Growth and Survival of Microbial Pathogens in Cheese curd, which was held for 60 days, from a dairy plant in Wisconsin (Durch et al., 2000). Nineteen of 55 laboratory-confirmed patients had purchased cheese curds from an unrefrigerated display at the cheese plant. To be legal, cheese curds must be manufactured from pasteurized milk. Vats of raw-milk Cheddar cheese were inadvertently used to make fresh curds, which were incorrectly labelled as ‘pasteurized’ Cheddar cheese curd. A comprehensive risk assessment would consider, among other factors, the degree to which the consuming population is exposed to risks associated with the consumption of aged raw-milk cheeses. Cheddar cheese is produced worldwide and is therefore considered an important variety of hard cheese. The USDA, National Agricultural Statistics Service, reports that Cheddar cheese was the most popular variety of cheese produced and consumed in the US in 1999, with a production level of 2.8 billion pounds (1.2 million tonnes) or 35.4% of the total cheese produced (Anonymous, 1999c). Given that a large amount of this cheese is produced from raw or heat-treated milk, the high degree of exposure (consumption) of this product coupled with the low incidence of disease outbreaks attests to the safety of aged cheese made from raw and heat-treated milk. Table 1 summarizes outbreaks involving Cheddar cheese which have occurred since 1976. Listed in this table are confounding parameters which contributed to the presence of pathogens in the finished product and the subsequent onset of human illness.

Challenge Studies Reitsma and Henning (1996) examined the survival of E. coli 0157:H7 during the manufacture and ripening of Cheddar cheese. E. coli 0157:H7 was inoculated at two levels into pasteurized milk, 1 103 cfu/ml and 1 cfu/ml. The organism showed a sharp decrease in numbers over the 158-day testing period. Treatment 1 (1000 cfu/ml) showed a 2-log CFU/g reduction after 60 days of ripening; however, E. coli 0157:H7 was still present even after 158 days of ripening when viable cells were detected in four of five replicates. Treatment 2 (1 cfu/g) showed a reduction to 1 cfu/g in 60 days, with no viable E. coli 0157:H7 detected at 158 days. As the authors state, ‘the results of this study cannot predict the behaviour of heat-injured cells which could result from the pasteurization of naturally contaminating E. coli.’ Further, the low salt-in-moisture content (SM) and absence of natural inhibitors present in raw milk create an artificially protective environment for E. coli 0157:H7 in pasteurized milk. The SM determines the water activity, which, in turn, dictates the potential for growth of a micro-organism in the cheese environment. The SM in that study ranged from 2.75 to

3.76% with a mean of 3.25%, whereas in normal Cheddar, the SM ranges from 4 to 6%. The low SM could have affected the results in the study of Reitsma and Henning (1996) and the authors recommend further research with Cheddar containing a higher SM to determine if similar results would be obtained with an SM more commonly encountered in Cheddar cheese. NaCl is an important inhibitor of microbial growth in cheese. The major roles of NaCl in Cheddar cheese are to check lactic acid fermentation after an optimum peak has been attained, reduce moisture through syneresis of the curd, suppress the growth of spoilage micro-organisms and create physical changes in cheese proteins which influence cheese texture, protein solubility and protein conformation (Fox et al., 2000; ‘Salt in Cheese: Physical, Chemical and Biological Aspects’, Volume 1). While there are no state or federal standards for the amount of salt added to Cheddar cheese, variations in salt content from 0.8 to 2% are common. The minimum aw (adjusted with NaCl) for the growth of E. coli is 0.950 (Fennema, 1985). Further, most raw milk receives some form of heat treatment, albeit subpasteurization. The combination of heat, salt and natural inhibitors could provide barriers to the survival of E. coli 0157:H7. The experimental design used by Reitsma and Henning (1996) failed to consider these potential safeguards. It is plausible that the use of pasteurized milk for cheesemaking provides E. coli 0157:H7 with a more protective environment than raw milk, thus heat treatment could create more of a problem to food safety. The authors state ‘The low number of outbreaks seem to indicate that pathogens in cheese are not a major problem.’ The authors further state ‘treatment 1 (1000 cfu/ml) would not likely be encountered in industry because of co-mingling of milk from several or many farms, thus creating a dilution effect.’ Concern is expressed about the authors’ concluding statement ‘The current requirement for ripening of Cheddar cheese will not assure consumers of a safe product if the cheese is made from raw milk and a pathogen such as E. coli 0157:H7 is present in the cheese at the beginning of ripening.’ This statement is contradicted by the authors’ own data which show that E. coli 0157:H7 present at 60 cfu/g in curd after salting was reduced to 1 cfu/g after 60 days, even in the artificially low SM of cheeses in the study. A subsequent study by Zhang and Henning (1999) described mathematically the decline of E. coli populations during cheese ripening. The authors inoculated pasteurized whole milk with E. coli biotype 1 at populations of 100–1000/ml. The authors used a complete factorial design to investigate the effects of high- and low-level environmental factors such as moisture (34–40%), pH (5.1–5.6), temperature (4–13 °C) and salt


Table 1 Data from outbreak investigations involving aged raw milk cheese and confounding parameters which contribute to the presence of pathogens Number of cases

Cheese type

Confounding parameter

339 confirmed; 28 000– 36 000 suspected

Cheddar made from pasteurized milk

1. Raw milk did not meet standards 2. Raw milk stored 1–3 days in holding tank – no refrigeration 3. Milk filtered after pasteurization 4. Cheese pH, 5.6 5. 25 instances of noncompliance with GMP Milk traced to single farm; lack of co-mingling 1. Employee manually shut down pasteurizer 2. Group II type shed by workers 1. Deficiencies in cleaning and sanitation 2. Equipment not routinely cleaned and sanitized between shredding of different cheese types from different makers 3. Cheese equipment susceptible to contamination from environment/aerosols 4. Cheese contaminated by infected workers 5. No deficiencies in pasteurization Incorrectly labelled as a pasteurized product

Date

Location

Isolate

1976

Colorado

Salmonella heidelberg

1980–1982

Ontario

Salmonella muenster

1984

4 Canadian Atlantic Provinces and Ontario

Salmonella typhimurium phage type 10, group I and II

2700 confirmed cases

Cheddar made from pasteurized and/or heattreated milk

1989

Multistate (Minnesota, Wisconsin, Michigan, New York)

Salmonella javiana and Salmonella oranienburg

164

Shredded cheese

1999

Raw-milk Cheddar

E. coli 0157:H7

concentration (0.8–1.7%) on survival of E. coli. Temperature and pH were found to have the most significant impact on survival, and there was no significant interaction among the four parameters studied. Salt concentration within the ranges used in this study (0.8–1.7%) was found to have no impact on survival of E. coli. Teo and Schlesser (2000) examined the survival of three groups of bacteria in raw-milk Cheddar cheese during cheesemaking and ripening; naturally occurring

Fresh cheese curd held for 60 days

Reference Fontaine et al., 1980

Wood et al., 1984 Bezanson et al.,1985; D’Aoust et al., 1985

Hedberg et al., 1992

Durch et al., 2000

coliforms, a streptomycin-resistant strain of E. coli K12 (ATCC 35695) and E. coli 0157:H7. Populations of naturally occurring coliforms present at levels of ⬃105 cfu/ml experienced a 1-log reduction after 60 days of aging at 7 °C, and a further 3–4-log reduction after 180 days (Teo and Schlesser, 2000). In contrast, E. coli K12 populations exhibited a less than 1-log reduction during 60 days of aging, and only a 1–2-log reduction by 90 days. Similar results were recorded with a five-strain

550 Growth and Survival of Microbial Pathogens in Cheese cocktail of E. coli 0157:H7 where populations declined by 1 log following 60 days at 7 °C, and by 1–2-logs following 90 days at the same temperature. A number of questions are raised by the data presented by Teo and Schlesser (2000). The coliform levels used were extremely high, and, in practice, such levels would raise concerns about raw milk quality. The FDA has set standards for ETEC and E. coli in cheese at levels of 103 and 104 cells/g, respectively (Anonymous, 1998). Thus the cheese produced by Teo and colleagues did not comply with these standards, and further, exceeded these standards by 103–104/ml as shown in their Figure 6 where initial populations of E. coli 0157:H7 exist at approximately 5 107/ml. This study has a biased objective, to ‘confirm prior work that suggests 60-day aging inadequate to protect public health.’ Figure 2 presented in the paper documents a decline in populations over time, thus if a reasonable starting population of 1–10 E. coli 0157:H7 were used, no viable cells should be present following aging. Studies by Ryser and Marth (1987a,b,c) examined the fate of L. monocytogenes during the manufacture of Cheddar, Camembert and Brick cheeses. Rapid growth to populations of 5 107 cfu/ml is observed in Camembert cheese, in which the pH normally increases during ripening, thereby creating a favourable growth environment for listeria (Ryser and Marth, 1987a). In contrast, listeria populations show a marked decline in viable population levels during the ripening of Cheddar cheese. However, population levels do not decline to undetectable levels. Current US regulations call for cheese made from raw or sub-pasteurized milk to be ripened at 1.7 °C (35 °F) for at least 60 days prior to sale. Ryser and Marth (1987b) have shown that aging alone will not ensure the production of listeria-free Cheddar cheese. This stated, it is clear, that the greatest threat posed to the safety of cheese is due to post-process environmental contamination with listeria. While outbreaks of illness have resulted from the presence of L. monocytogenes in softripened and Hispanic-style cheeses (Linnan et al., 1988), no outbreaks of listeriosis have been reported as a result of survival of listeria in cheese aged for a minimum of 60 days. Genigeorgis et al. (1991) evaluated the ability of 24 types of market cheeses to support the growth of L. monocytogenes. Cheeses able to support growth included soft Hispanic-type cheeses, Ricotta, Teleme, Brie, Camembert and Cottage cheeses (pH range, 4.9–7.7). Cheeses which did not support growth, and which resulted in the gradual death of L. monocytogenes, included Cotija, Cream, Blue, Monterey Jack, Swiss, Cheddar, Colby, String, Provolone, Münster, Feta and Kasseri (pH range, 4.3–5.6). A correlation was observed between the growth of listeria in cheeses having a pH of greater than 5.5, and in cheeses which were manufactured without a starter culture.

Approximately 80% of the cheeses made in Switzerland are manufactured from raw milk. However, the term ‘raw milk cheese’ as applied to Swiss cheese is a misnomer because Swiss cheese receives an extensive heat treatment during manufacture. Bachman and Spahr (1995) assessed the safety of Swiss hard and semi-hard cheeses made from raw milk. These authors inoculated Aeromonas hydrophila, Campylobacter jejuni, E. coli, L. monocytogenes, Pseudomonas aeruginosa, Salmonella spp., Staph. aureus and Yersinia enterocolitica into raw milk at levels ranging between 104 and 106 cfu/ml for the manufacture of hard (Swiss-type) and semi-hard (Tilsit-type) cheese. In the hard cheese, no pathogens were detected beyond 1 day. This was attributed to the curd-cooking temperature of 53 °C (127.4 °F) for 45 min and 42 °C (107.6 °F) for 15 min for Swiss hard and semi-hard cheeses. Further, the rapid decrease of the redox potential of Swiss cheese is likely to impart additional inhibitory effects. Pathogens were found to survive longer in the semi-hard than in the hard cheese. After 90 days of aging at 11–13 °C, when ripening was complete, all pathogens except L. monocytogenes were below detectable limits. Growth of L. monocytogenes was not observed in the interior of the cheese, but they grew well on the cheese surface. Thus, manufacturing parameters used in the production of semihard cheese are bacteriostatic, not bacteriocidal, for L. monocytogenes. Based upon these studies, the Swiss dairy industry has adopted a listeria-monitoring programme for cheese and other dairy products. The synergistic effects of active antimicrobial enzyme systems in raw milk coupled with antagonistic effects of starter cultures, fast acidification, inhibitory effects of lactic acid and high curd-cooking temperatures render a microbiologically safe hard cheese when produced under good manufacturing practices. Spahr and Schafroth (2001), in studies which examined the fate of Mycobacterium avium subsp. paratuberculosis, recorded pH values associated with Swiss hard and semi-hard cheese manufacture. After 24 h, cheeses manufactured under these curd-cooking conditions reached a pH value of 5.3 in hard cheese and 5.2 in semi-hard cheese, and these pH conditions remain for 10 days for hard cheese and 25 days for semi-hard cheese. Further, the rapid decrease of the redox potential of Swiss cheese likely imparts additional inhibitory effects. The synergistic effects of active antimicrobial enzyme systems in raw milk coupled with antagonistic effects of starter cultures, fast acidification, inhibitory effects of lactic acid and high curd-cooking temperatures render a microbiologically safe hard cheese when produced under good manufacturing practices. Pellegrino and Resmini (2001) examined the safety of the Italian hard cheeses, Grana Padano and Parmigiano Reggiano. The authors noted several parameters


associated with these cheeses which contribute to their microbiological safety; (1) cooking of cheese curd to a temperature between 53 and 56 °C for 15–20 min, with a total holding time of up to 85 min at these temperatures, (2) moulding of the cheese, whereby it is held at temperatures of 52 °C (126 °F) and 56 °C (133 °F) for at least 10 h at pH 5.0, (3) brine-salting of the cheese which lowers the aw to 0.9 and (4) extended ripening for periods of 9 months (Grana Padano) to 12 months (Parmigiano Reggiano) which promotes a further decrease in the aw to levels inhibitory for growth of bacterial pathogens. Resmini and Pellegrino (1996) demonstrated that the high-temperature–low-pH conditions occurring within Grana cheeses, which they describe as self-pasteurization, result in the inactivation of alkaline phosphatase, except within the outermost 3–4-cm layer. However, in this outer layer the SM ranges between 8 and 24% in the ripened cheese and the aw is close to 0.8. Staph. aureus, which is more tolerant of low aw, cannot survive below an aw of 0.86 and can produce toxins only above an aw of 0.90 (Sperber, 1983). Pecorari et al. (2001) examined the fate of pathogens during the production and ripening of Parmigiano Reggiano cheese. E. coli, S. typhimurium, Staph. aureus and L. monocytogenes were inoculated into raw milk at levels ranging between 104 and 106 cfu/ml. None of the inoculated pathogens were detected 24 h after cheesemaking, confirming that the cheesemaking conditions of Grana cheeses do not support pathogen growth or survival. These results are consistent with those obtained by Yousef and Marth (1990) who reported a rapid decline of L. monocytogenes from an initial level of ⬃104 g of Parmesan cheese to undetectable levels within 14–112 days of ripening. These authors attributed the decline of L. monocytogenes viability in Parmesan cheese to the following parameters – addition of lipase (for US Parmesan) for flavour development, heat treatment of the curd and reduction in moisture content (aw) during ripening. Battistotti (1995), in an analysis of more than 100 samples of mature Italian Grana cheeses, failed to detect salmonella, Staph. aureus, L. monocytogenes, coliforms or enterococci, further confirming the microbiological safety of hard Italian cheeses. The results of the aforementioned challenge studies are summarized in Table 2. Most studies, which show the survival of pathogens, have been based on the use of pasteurized milk rather than raw milk in the experimental design. The growth rate of listeria (and presumably other pathogens) in milk is a function of the degree and extent of heat treatment. The fastest rate of growth is observed in UHT milk, followed in turn by HTST, heat-treated and raw milks (Northolt et al., 1988; Rajikowski et al., 1994). Therefore, challenge studies, which assess the survival of pathogens when

inoculated into pasteurized milk, may overestimate survival during 60 days of aging. The UK Institute of Food Science and Technology (IFST) has stated that the total health risk to the consumer is less from cheese made from pasteurized milk than from cheese of similar composition made from unpasteurized milk (IFST, 2000). Alternative hypotheses could be offered, including consideration that the use of raw milk provides protective effects from pathogens in milk and that post-pasteurization environmental contamination poses a far greater threat to the safety of cheese. As a result, the use of pasteurized milk in cheesemaking may provide an environment, which provides for optimal growth of pathogens whereas, in raw milk, the normal flora and natural inhibitors provide a margin of control over pathogen growth. In fact, a study conducted by Rudolf and Sherer (2001) showed a higher incidence of L. monocytogenes in cheeses made from pasteurized milk (8%) than in cheese made from raw milk (4.8%). Phage typing of isolates revealed persistent listeria contamination within dairy plant environments for periods of weeks to several months and documented cross-contamination within the plant environment as a significant factor associated with the contamination of cheese. The recommendation for mandatory pasteurization may ultimately lead to use of milk of inferior quality for cheesemaking. Pathogens harboured in this inferior quality milk can be transported to a processing facility and become established as environmental pathogens. A wiser strategy may involve routine testing of incoming lots of raw milk and working with producers when infected animals are identified to allow treatment and confinement of animals to control infectious disease. There is no evidence in the literature to support the view that cheese made from raw milk where pathogens are not present is a dangerous food. Thus, raw milk screening coupled with the use of good manufacturing practices to control environmental contamination during cheesemaking may be the most effective control strategy to improve the safety of aged cheese. The US FDA has recently stated ‘a review of the literature relating to the potential for growth of pathogens in hard cheeses that are aged for at least 60 days shows that such growth is not likely to occur because of the combined effect of decreased pH, decreased water activity, and possibly other factors inherent to these cheeses’ (Anonymous, 1999d). Although survival during aging is possible, the FDA cited a considerable body of evidence which showed that certain cheeses do not support the growth of pathogens during the aging process and subsequent storage. Both facultative and obligate heterofermentative lactobacilli have been isolated from Cheddar cheese, such as Lactobacillus casei, Lb. paracasei, Lb. plantarum, Lb.

552 Growth and Survival of Microbial Pathogens in Cheese Table 2 Results of selected challenge studies which examine the fate of pathogens in raw milk cheeses and parameters which promote survival/decline of pathogens

Reference

Cheese type/pathogen

Milk inoculation levels

Reitsma and Henning, 1996

Cheddar/E. coli 0157:H7

1 cfu/ml and 1000 cfu/ml

Ryser and Marth, 1987b

Cheddar/ L. monocytogenes

5 102 cfu/ml

Teo et al., 2000

Cheddar/E. coli 0157:H7

105/ml

Bachman and Spahr, 1995

Swiss hard/ semihard Aeromonas, Campylobacter, E. coli, L. monocytogenes, P. aeruginosa, Salmonella, Staphylococcus, Yersinia Italian Grana

104–106 cfu/ml

Parmesan/ L. monocytogenes

104–105 cfu/ml

Pellegrino and Resmini, 2001

Yousef and Marth, 1990

60 days of aging

Factors promoting survival/decline

No survival at Cheese 1 cfu/ml; Survival at manufactured 1000 cfu/ml from pasteurized milk; low salt levels Survival during Decline in aging at 6 or 13 °C populations after 35 days of storage 1-log decrease E. coli populations in cheese exceeded FDA standards No detection of Cook at 53 °C; pathogens redox potential beyond 1 day

Undetectable

Curd cooked at 53–56 °C, brine-salting, extended ripening to lower aw Addition of lipase, heat treatment of curd, reduction of aw

SMP, Skim Milk Powder.

casei subsp. pseudoplantarum, Lb. curvatus, Lb. brevis, Lb. rhamnosus and unclassified strains (Broome et al., 1990; Jordan and Cogan, 1993; McSweeney et al., 1993; Fitzsimons et al., 1999; Fox et al., 2000; Tammam et al., 2000). These are usually termed the non-starter lactic acid bacteria (NSLAB; see ‘The Microbiology of Cheese Ripening’, Volume 1). Lactobacilli are usually present at low numbers (50/g) in cheese immediately after manufacture, but grow at a temperature-dependent rate during ripening and eventually become the dominant viable micro-organisms in cheese, reaching a population of 107 cfu/g in 10–60 days. The NSLAB population decreases with storage and usually approach 5 106 cfu/g after one year (Prentice and Brown, 1983). Conditions, which dictate the rate and extent of growth of NSLAB in cheese, include pH, moisture content, salt concentration and ripening temperature (Martley and Crow, 1993). In commercially produced cheese, NSLAB may originate from raw milk, postpasteurization environmental contamination and/or ingredients. These organisms may well offer a protective effect against the growth of pathogens and this role

should be studied as this may be a positive contribution of raw milk to the safety of raw-milk Cheddar cheese, consistent with competitive exclusion theories which have helped to advance the safety of products such as poultry. A lower total bacterial load in raw milk entering the pasteurizer results in a lower total bacterial count in pasteurized milk, but after 10–16 h, an increase in the number of NSLAB still occurs.

Growth and Survival of Bacterial Pathogens in Soft and Semi-Soft Cheeses Legitimate concerns can be raised regarding the safety of soft and semi-soft cheeses manufactured from raw milk, as well as high-moisture, low-salt aged cheeses. An outbreak of food-borne listeriosis linked to cheese was reported by Bille et al. (1992). This outbreak occurred in Vaud, Switzerland, and was linked to the consumption of Vacherin Mont D’Or cheese. A total of 122 cases during the period 1983–1987 were reported. The normal endemic rate of listeriosis in Switzerland is 5–10 cases/million persons. During the outbreak


period, the rate of listeriosis rose to 50 cases/million persons. Sixteen cases were reported in 1983, 24 in 1984, 13 in 1985, 28 in 1986 and 41 in 1987. A mortality rate of 28% was associated with these cases. Of the clinical isolates available from the epidemic period, 111 of 120 (93%) were serotype 4b of two unique phage types, and 85% of these strains matched the epidemic phage types isolated from Vacherin Mont D’Or cheese. L. monocytogenes in Mexican-style cheese has been responsible for two major outbreaks of food-borne disease in the US. Mexican-style cheeses comprise a range of cheese products which include Queso Blanco, Quesco Fresco, Panela Ranchero, Queso de Hoja and soft Hispanic cheese (Bolton and Frank, 1999). These cheeses do not have a standard of identity, and most are coagulated and using rennet, may have added organic acids (citric, acetic and lactic); usually a lactic starter culture is not used (Bolton and Frank, 1999). The first documented link between cheese consumption and an outbreak of listeriosis was reported in California in 1985. Jalisco brand Mexican-style cheese was implicated as the vehicle of infection (Linnan et al., 1988). A total of 142 cases involving 93 pregnant women or their offspring and 49 non-pregnant, immuno-compromised adults were documented in Los Angeles County, CA. Forty-eight deaths were recorded, giving a mortality rate of 33.8%. The majority of afflicted individuals (62%) were pregnant Hispanic women. Although an additional 160 cases occurred in other parts of California, for logistical reasons, the study reported by Linnan et al. (1988) was limited to Los Angeles County. In this outbreak, the cheese was most likely manufactured from a combination of raw and pasteurized milks, and the cheese plant that manufactured the incriminated cheese was found to harbour listeria as an environmental contaminant. The epidemic strain in this outbreak was a serotype 4b, and this serotype was recovered from unopened packages of Queso Fresco and Cotija Mexican-style cheese. An outbreak of listeriosis associated with homemade, Mexican-style, fresh, soft cheese occurred in North Carolina between October 2000 and January 2001 (Boggs et al., 2001). The outbreak involved 12 cases, consisting of 10 pregnant women, 1 post-partum female and a 70- year-old immuno-compromised male. The 11 women, upon hospital admission, reported symptoms of fever, chills, headache, abdominal cramps and vomiting. The cheese implicated in the outbreak was purchased from door-to-door vendors. L. monocytogenes isolates obtained from nine patients, three cheese samples from two stores, one cheese sample from a patient’s home and one raw milk sample from a dairy all had indistinguishable PFGE patterns, indicating a common link. It is important to note that the manufacturing con-

ditions in this outbreak would not be those encountered in a licensed, inspected commercial cheese-processing facility. Microbiological surveys of raw milk conducted in the US have shown the presence of L. monocytogenes in 1.6–7% of samples. This incidence is similar to that in Canadian (1.3–5.4%) and Western-European (2.5–6.0%) raw milks. In the recently released Health and Human Services (HHS) and USDA listeria Risk Assessment and listeria Action Plan, USDA and FDA advise pregnant women, older adults and people with weakened immune systems that ‘Cheeses that may be eaten include hard cheeses, semi-soft cheeses such as Mozzarella, pasteurized processed cheeses such as slices and spreads, Cream cheese and Cottage cheese.’ However, persons in these risk groups are advised ‘do not drink raw (unpasteurized) milk or eat foods that contain unpasteurized milk.’ This advice may be ambiguous with respect to aged rawmilk cheeses (Anonymous, 2001).

Stress Adaptation of Pathogens and Impact upon Cheese Safety Over the past several years, microbiologists who study stress adaptation in bacterial pathogens are aware of genetic mechanisms which allow a number of Grampositive and -negative bacteria to adapt to hostile environments. Rpos is a sigma factor which is thought to allow induction of specific stress-related components in tolerant isolates. The rpos-regulated proteins enhance acid tolerance and cross-protect E. coli 0157:H7 against subsequent heat and salt challenges. The acid-tolerance response (ATR) gene encodes for the ability to withstand lethal pH conditions following adaptation to sublethal pH in L. monocytogenes, S. typhimurium, E. coli and A. hydrophila. These mechanisms play a role in predicting the fate of pathogens in acidic foods. Acid adaptation increases the general resistance, including acid tolerance, of L. monocytogenes, S. typhimurium and E. coli, so that they survive better in both acidic and fermented foods than unadapted cultures. These findings have important implications for the safety of hard cheeses that are aged for at least 60 days where the combined effects of pH, salt and decreased aw dictate potential for pathogen survival. Leyer and Johnson (1992) inoculated the surfaces of commercially produced cheeses with adapted and nonadapted S. typhimurium at an initial level of 104/ml. Acid-adapted salmonella survived in Cheddar cheese through 74 days of storage at 5 °C under aerobic storage compared with non-adapted salmonella, which were not detected after 14 days. In Swiss cheese, end products such as propionate and acetate produced by propionic acid bacteria were found to inhibit salmonella.

554 Growth and Survival of Microbial Pathogens in Cheese Dineen et al. (1998) examined the persistence of E. coli 0157:H7 in fermented dairy products (yoghurt). The authors concluded that post-processing contamination of fermented dairy products with E. coli 0157:H7 represents the greatest potential health hazard to humans. Those strains of E. coli 0157:H7 which possessed the rpos system appeared to contribute most effectively to bacterial survival under moderately lethal conditions, but did not appear to play much of a role in survival under sub-lethal conditions. The authors offered the following recommendations: (i) coliforms, including E. coli 0157:H7, may be present in raw milk, (ii) coliforms are destroyed by pasteurization, (iii) the primary objective of a comprehensive sanitation programme should be to prevent recontamination of pasteurized products and (iv) the presence of active starter cultures may help minimize the presence of bacterial pathogens in finished products. L. monocytogenes is able to withstand low pH following sub-lethal exposure to acidic conditions (O’Driscoll et al., 1997). According to Chawla et al. (1996) and Chen et al. (1997), temperature and acidity had a significant effect on the fate of acid-injured L. monocytogenes, with complete repair occurring at pH 6.6. At pH values 5.6 (which are typically found in Cheddar cheese), refrigeration temperatures were bacteriostatic, whereas higher temperatures were bacteriocidal. These findings are consistent with those reported by Ryser and Marth (1987b) where L. monocytogenes was inactivated faster in Cheddar cheese ripened at 13 °C versus 6 °C. Since repair of sub-lethal injury requires optimal conditions, decreased survival of sub-lethally injured bacteria in Cheddar cheese would be expected due to low pH and high salt conditions. Mathew and Ryser (2002) assessed the ability of sublethally heat-injured L. monocytogenes cells to compete with a commercial mesophilic lactic acid starter culture during fermentation of UHT milk. L. monocytogenes strains were heat-injured by two treatments (low heatinjured (LHI) and high heat-injured (HHI)) to yield greater than 99% injury. The UHT milk was inoculated to contain 104–106 LHI, HHI or untreated L. monocytogenes together with 0, 0.5 or 2% of a commercial Lactococcus lactis subsp. lactis/Lc. lactis subsp. cremoris starter. While listeria populations grew to levels of approximately 109 cfu/ml after 8 h of fermentation, after 24 h, 93% of the non-injured control population became injured. In starter-free controls, 80% of both HHI and LHI cells were repaired within 10 h of incubation. These findings document the suppression of listeria growth by the starter culture which causes microbial injury, resulting in cells which are unable to grow or express pathogenicity. The potential of listeria and other pathogens to become inactivated and/or sub-lethally

injured during cheesemaking should be investigated. The combined effects of acid production by starter cultures, salt and mild heat alone or in combination all have the potential to injure bacterial pathogens such as L. monocytogenes, E. coli and salmonella. These interactive effects could provide an explanation for the remarkable safety record of aged raw-milk cheese.

Improvement in Cheese Safety Utilization of more sensitive methods for the detection of pathogens existing at low levels in Cheddar and aged raw-milk cheeses could do much to assure cheese safety. Baylis et al. (2000) compared the Oxoid Ltd SPRINT salmonella system (Oxoid, Ltd) against the ISO 6579:1993, Qualicon BAX PCR (Wilmington, DE, USA), bioMerieux VIDAS (Hazelwood, MO, USA) and Tecra Unique methods (Willoughby, NSW, Australia). The SPRINT system was developed for the rapid detection of low levels of injured salmonella in foods. This system utilizes an enrichment broth that contains a specifically developed peptone that allows consistent and rapid recovery of injured salmonella cells, coupled with a Recovery Supplement which contains an Oxyrase® Enzyme System that assists recovery through reduction in oxidative stress of the medium. After 5 h of incubation, selective agents are added to the medium. When tested with ice cream and skimmed milk powder containing low levels of heat-injured S. typhimurium, the SPRINT method was superior (61% confirmed positive samples) to the ISO (37% positive), BAX (36% positive), VIDAS (30% positive) and Tecra (25% positive) methods. Similar improvements have been advanced by Pritchard and Donnelly (1999) for recovery of injured listeria in dairy products, where continuing work on enrichment of dairy environmental samples at the University of Vermont (UVM) and listeria Repair Broth (LRB) has shown that combining these two primary enrichment media into a single tube of Fraser broth for secondary enrichment yields a significantly higher (p0.05) percentage of listeria-positive samples than when either LRB or UVM is used alone. Altekruse et al. (1998) stated that ‘Because of inherent problems of statistical sampling of foods for microbial pathogens (ICMSF, 1986), end-point testing may not assure the safety of cheese. These problems are amplified when organisms are present in small numbers below the sensitivity of the test or when there is intermittent contamination and the tested specimens do not contain pathogens.’ Raw ingredient testing, i.e., screening of the raw milk supply, may overcome these shortcomings. In recent years, cheese and cheese products have been recalled due to the presence of pathogenic bacteria


such as salmonella, L. monocytogenes and E. coli. In some instances, cheeses, both domestic and imported, have been linked to outbreaks of human illness. In November 1998, the FDA issued the Domestic and Imported Cheese and Cheese Products Food Compliance Program (Anonymous, 1998). The objectives of this programme are for the FDA to conduct inspections of domestic cheese firms, to examine samples of imported and domestic cheese for microbiological contamination, the presence of phosphatase, and filth and to take appropriate regulatory action when violations are encountered. Target pathogens for analysis include L. monocytogenes, salmonella, E. coli (ETEC), enterohemorrhagic E. coli 0157:H7 and Staph. aureus. Under this initiative, direct reference seizure or detention of cheese based on the presence of L. monocytogenes is authorized. It should be noted that ETEC analysis is performed only if E. coli is present at 104 cfu/g. A review of the FDA’s Product Recalls, Alerts and Warnings Archive (http://www.fda.gov/oc/po/firmrecalls/archive.html) for the calendar years 1999, 2000 and 2001 revealed several recalls due to the presence of L. monocytogenes in cheese, one recall involving E. coli contamination of Blue and Gorgonzola cheeses, and one recall involving salmonella contamination of Mexican White cheese. The strain of E. coli identified was not 0157:H7. Listeriacontamination appears to be a function of post-process contamination. In no instance during this period were aged cheeses made from raw milk the subject of a recall.

Future Research and Conclusions A number of gaps in the scientific literature have been identified as a result of this review. Future research is suggested in a number of areas: 1. Fully explore the impact of pasteurization of milk on the microbial ecology of cheeses aged for more than 60 days. Does pasteurization increase the susceptibility of cheese to the growth of pathogens introduced via post-processing contamination? 2. Evaluate the potential for survival of S. typhimurium DT 104 intentionally added at low levels (10 and 100 cfu/ml) to raw milk destined for aged raw-milk cheesemaking. 3. Evaluate improved microbiological methods for rawmilk screening and aged raw-milk cheese analysis. 4. Understand the contributions of microbial injury to the interactive effects of salt, pH and mild heat in the suppression of growth of listeria, E. coli and salmonella. Do acid-adapted cultures of these microbial species show enhanced ability to persist in aged raw-milk cheese by withstanding salt, aw

and mild heat conditions encountered during aged raw-milk cheesemaking? 5. Develop microbiological criteria for raw milk destined for aged cheesemaking by setting tolerance limits for coliforms, E. coli, ETEC, enterohemorrhagic E. coli 0157:H7, salmonella, L. monocytogenes and Staph. aureus. Explore the impact of utilization of raw milk with stringent microbiological standards on the safety of raw-milk cheese. 6. Explore the development of risk reduction procedures and practices at both the primary production level (milk screening) and the cheese production level to improve the safety of aged raw-milk cheese. Aged cheeses made from raw milk are microbiologically safe when manufactured under conditions that use milk-screening procedures, GMPs and HACCP. The raw and heat-treated aged cheese issue is not unlike the listeria and potential for survival during pasteurization issue, which confronted the dairy industry in the 1980s. There was a body of scientific evidence which indicated that listeria was able to survive the pasteurization process. Through careful research and analysis over a period of years, it was established that pasteurization, in fact, offered adequate public health protection and that the greatest risk from listeria posed to dairy products was the threat of post-processing contamination. Careful investigation of the safety of aged, raw-milk cheeses may indicate that raw milk provides protective effects from pathogens in milk and that environmental contamination poses a far greater threat to the safety of cheese. This issue deserves the benefit of full study, careful evaluation of published research information and new research to fully assess all potential risks and benefits.

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Pritchard, T.J. and Donnelly, C.W. (1999). Combined secondary enrichment of primary enrichment broth increases Listeria detection. J. Food Prot. 62, 532–535. Pritchard, T.J., Beliveau, C.M., Flanders, K.J. and Donnelly, C.W. (1994). Increased incidence of Listeria species in dairy processing plants having adjacent farm facilities. J. Food Prot. 57, 770–775. Rajikowski, K.T., Calderone, S.M. and Jones, E. (1994). Effect of polyphosphate and sodium chloride on the growth of Listeria monocytogenes and Staphylococcus aureus in ultra-high temperature milk. J. Dairy Sci. 77, 1503–1508. Rea, M.C., Cogan, T.M. and Tobin, S. (1992). Incidence of pathogenic bacteria in raw milk in Ireland. J. Appl. Bacteriol. 73, 331–336. Reitsma, C.J. and Henning, D.R. (1996). Survival of enterohemorrhagic Escherichia coli 0157:H7 during the manufacture and curing of Cheddar cheese. J. Food Prot. 59, 460–464. Resmini, P. and Pellegrino, L. (1996). Evaluation and interpretation of alkaline phosphatase activity in hard cheeses made with raw milk. Food Australia 48, 452–454. Rohrbach, B.W., Draughon, F.A., Davidson, M.P. and Oliver, S.P. (1992). Prevalence of Listeria monocytogenes, Campylobacter jejuni, Yersinia enterocolitica and Salmonella in bulk tank milk: risk factors and risk of human exposure. J. Food Prot. 55, 93–97. Rudolf, M. and Sherer, S. (2001). High incidence of Listeria monocytogenes in European red smear cheese. Int. J. Food Microbiol. 63, 91–98. Ryser, E.T. (1998). Public health concerns, in, Applied Dairy Microbiology, Marth, E.H. and Steele, J.L., eds, Marcel Dekker, Inc., New York. Ryser, E.T. (1999). Incidence and behavior of Listeria monocytogenes in unfermented dairy products, in, Listeria, Listeriosis and Food Safety, 2nd edn, Ryser, E.T. and Marth, E.H., eds, Marcel Dekker, Inc., New York. pp. 331–404. Ryser, E.T. and Marth, E.H. (1987a). Fate of Listeria monocytogenes during manufacture and ripening of Camembert cheese. J. Food Prot. 50, 372–378. Ryser, E.T. and Marth, E.H. (1987b). Behavior of Listeria monocytogenes during the manufacture and ripening of Cheddar cheese. J. Food Prot. 50, 7–13. Ryser, E.T. and Marth, E.H. (1987c). Behavior of Listeria monocytogenes during manufacture and ripening of brick cheese. J. Dairy Sci. 72, 838–853. Shere, J.A., Bartlett, K.J. and Kaspar, C.W. (1998). Longitudinal study of Escherichia coli 0157:H7 dissemination on four dairy farms in Wisconsin. Appl. Environ. Microbiol. 64, 1390–1399. Spahr, U. and Schafroth, K. (2001). Fate of Mycobacterium avium subsp. paratuberculosis in Swiss hard and semihard cheese manufactured from raw milk. Appl. Environ. Microbiol. 67, 4199–4205. Sperber, W.H. (1983). Influence of water activity on foodborne bacteria. J. Food Prot. 46, 142–150. Tammam, J.D., Williams, A.G., Noble, J. and Lloyd, D. (2000). Amino acid fermentation in non-starter Lactobacillus spp.


isolated from Cheddar cheese. Lett. Appl. Microbiol. 30, 370–374. Teo, A. and Schlesser, J. (2000). Survival of naturally occurring Escherichia coli and a streptomycin-resistant strain of E. coli K12 (ATCC 35695) during the aging period of hard cheeses made from raw milk. J. Dairy Sci. 83 (Suppl. 1), 109 (abstr.). Threlfall, E.J., Frost, A.J., Ward, L.R. and Rowe, B. (1996). Increasing spectrum of resistance in multiresistant Salmonella typhimurium. Lancet 347, 1053–1054. Villar, R.G., Macek, M.D., Simons, S., Hayes, P.S., Goldoft, M.J., Lewis, J.H., Rowan, L.L., Hursh, D., Patnode, M. and Mead, P.S. (1999). Investigation of multidrug-resistant Salmonella serotype Typhimurium DT104 infections linked to raw milk cheese in Washington State. JAMA 281, 1811–1816.

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Toxins in Cheese N.M. O’Brien and T.P. O’Connor, Department of Food and Nutritional Sciences, University College, Cork, Ireland J. O’Callaghan and A.D.W. Dobson, Department of Microbiology, University College, Cork, Ireland

Biogenic Amines and Mycotoxins In this chapter, we will discuss the formation of toxic compounds, such as biogenic amines and mycotoxins, in cheese. Both these classes of compounds have been reported to be present in cheese and are produced as a result of the activity of micro-organisms, both fungi and bacteria, either in the raw materials used in cheese manufacture or during the production and storage/ripening process. The factors that affect the formation of biogenic amines and mycotoxins will be discussed, together with their occurrence and potential toxic effects.

Biogenic Amines Biogenic amines are non-volatile, low molecular mass aliphatic, alicyclic or heterocyclic organic bases which cause physiological effects (Davidek and Davidek, 1995). Typically, they originate in foods from the decarboxylation of specific amino acids. Decarboxylation can occur due to indigenous decarboxylases in foods or to decarboxylases produced by microorganisms in the food. Biogenic amines are found in a variety of foodstuffs, most commonly fish of the families Scombridae and Scombereoscidae, but also in cheese (Maga, 1978; Smith, 1981; Chang et al., 1985; McCabe, 1986; Joosten, 1988; Lopez-Glaria et al., 2001; Innocente and D’Agostin, 2002). In cheese, biogenic amines are produced by decarboxylation of amino acids during ripening (see ‘Catabolism of Amino Acids in Cheese during Ripening’, Volume 1). Levels produced vary as a function of ripening period and microflora (Renner, 1987; Leuschner et al., 1998). High levels of biogenic amines are most likely to be detected in cheeses heavily contaminated with spoilage micro-organisms ( Joosten, 1987). The principal biogenic amines detected in cheese are histamine, tyramine, tryptamine, putrescine, cadaverine and phenylethylamine (El Sayed, 1996; Roig Sagues et al., 1998; Vale and Gloria, 1998; Novella-Rodriguez et al., 2000; Finoli et al., 2001a). Roig Sagues et al. (1998) reviewed the literature on the concentrations of histamine and

tyramine and other biogenic amines reported in cheese (Table 1). The ingestion of biogenic amine-containing foods may cause adverse toxic reactions (Stratton et al., 1991). Some of the biogenic amines have vasoactive properties (e.g., histamine, tyramine, phenylethylamine, tryptamine) while others act primarily by inhibiting histamine-detoxifying enzymes, e.g., the putrefactive amines, putrescine and cadaverine (Hui and Taylor, 1985). Histamine

Histamine has been reported to exert a wide range of effects in the body (Taylor et al., 1984). It stimulates both the sensory and the motor nerves, modulates gastric secretion and stimulates both vascular and extravascular smooth muscle. Histamine toxicity can result in a wide variety of symptoms such as rash, urticaria, inflammation, nausea, vomiting, diarrhoea, abdominal cramping, hypotension, tingling sensations, flushing, palpitations and headache (Taylor, 1986; Bartholomew et al., 1987). In general, toxic symptoms are relatively mild and many patients may not need medical attention. Thus, the exact prevalence of histamine toxicity worldwide is unclear. The prevalence of cheese-related toxicity is also unclear although, as discussed below, several incidences have been reported in the literature. Taylor (1986) comprehensively reviewed the toxicological and clinical aspects of histamine toxicity. He noted that the most common effects of histamine are on the cardiovascular system, causing dilation of peripheral blood vessels, capillaries and arteries with resultant hypotension, headache and flushing. Abdominal cramping, vomiting and diarrhoea may be related to histamine effects on H1 receptors. Urticaria may also be related to the interaction of histamine with H1 receptors, resulting in sensory and motor neuron stimulation. However, for most individuals, ingestion of even large concentrations of biogenic amines, such as histamine, does not elicit symptoms of toxicity since they are rapidly converted to aldehydes by monoamine



562 Toxins in Cheese

Table 1 Concentration (mg/kg) of biogenic amines in different cheeses* Cheese

Histamine

Tyramine

Tryptamine

Emmental Blue cheese Camembert Dutch (Edam and Gouda) Cheddar Parmesan

69–650 3–910 nd–480 nd–450

0–917 40–1100 10–210 0.1–670

nd–1100 nd–60 nd–200

nd–2120 nd–293

nd–1530 85–280

nd–300

Phenylethylamine

Putrescine

Cadaverine

0.1 10

0.5 44

16 42

0.1

7–20

17–48

nd–300

* Adapted from Roig Sagues et al. (1998). nd, not detected.

oxidase (MAO) and diamine oxidase (DAO) and then to carboxylic acids by oxidative deamination (Edwards and Sandine, 1981). These enzymes, present in the gastrointestinal tract, may prevent/reduce the absorption of unmetabolised histamine into the bloodstream (Taylor and Lieber, 1979; Lyons et al., 1983; Hui and Taylor, 1985). However, if the activities of MAO and DAO are impaired due to a genetic defect or the presence of potentiators such as food-borne putrefactive amines (e.g., putrescine, cadaverine) or pharmacologic agents (e.g., isoniazid), adverse reactions may occur on ingestion of biogenic amines (Rice et al., 1976; Diamond et al., 1987; Joosten, 1988). Putrescine and cadaverine have been reported to inhibit two histamine-detoxifying enzymes, DAO and histamine N-methyltransferase (HMT) (Hui and Taylor, 1985). Taylor and Sumner (1986) noted that many bacteria, especially Enterobacteriaceae, are capable of producing putrescine and cadaverine as they possess ornithine decarboxylase and lysine decarboxylase. Stratton et al. (1991) noted that putrefactive amine potentiators of histamine toxicity are usually formed by bacteria other than those responsible for histamine production since only relatively few bacteria possess histidine decarboxylase. Tyramine, tryptamine and phenylethylamine can also act as potentiators. Tyramine inhibits MAO, tryptamine inhibits DAO and phenylethylamine inhibits both DAO and HMT. Joosten (1988) reported that tyramine is the only inhibitor, of MAO and DAO, present in significant quantities in cheese. The anti-tuberculosis drug, isoniazid, inhibits histamine-metabolising enzymes and has been reported to result in histamine poisoning in conjunction with cheese consumption (Smith and Durack, 1978; Lejonc et al., 1979; Uragoda and Lodha, 1979). Other drugs administered as antidepressants, antihistamines or antimalarials can sometimes inhibit histamine-metabolising enzymes (Stratton et al., 1991).

Histamine is a normal constituent of the body; it is formed from histidine by a pyridoxal phosphatedependent decarboxylase and modulates several important bodily functions (Douglas, 1980). The concentration of histamine in the blood is strictly regulated. Orally administered histamine causes poisoning only when regulatory mechanisms fail to counteract the ingested dose, i.e., caused by consumption of a very high dose or inhibition of histamine-metabolising enzymes. Oral ingestion of up to 1 mmol (⬃100 mg) of histamine does not elicit toxic symptoms in normal individuals (Motil and Scrimshaw, 1979). However, vasodilation and increased heart rate result following intravenous administration of 0.07 mol histamine, demonstrate the important detoxifying role of intestinal histaminemetabolising enzymes. Factors influencing formation of histamine and other biogenic amines

The presence of histamine-producing bacteria in foods such as cheese is a key factor in histamine formation. Enterobacteriaceae is the main family implicated in histamine production. However, Clostridium, Lactobacillus and some strains of Klebsiella, Morganella and Hafnia have also been reported to possess histidine decarboxylase, and hence are potential histamine producers (Sakabe, 1973; Taylor et al., 1978, 1979; Taylor, 1986; Marino et al., 2000). Low concentrations of free histidine are present in milk. However, proteolysis during cheese ripening can liberate large amounts of histidine (Hinz et al., 1956). Histamine formation can be controlled primarily by good hygienic practices and by low storage temperatures. Joosten (1988) reported that lactobacilli play a significant role in histamine formation in Gouda cheese; he reported that ripening temperature, pH and salt concentration influence the ability of Lactobacillus to produce histamine in cheese. Ripening for one year at 21 °C resulted in 6.8 mmol histamine/kg cheese compared to 2.2 mmol/kg cheese after 1 year at 9 °C;

Toxins in Cheese 563

6.5 mmol histamine/kg cheese was detected after 2 weeks of ripening when the pH was 5.39 whereas only 3.4 mmol/kg was detected when the pH was 5.19. A high salt concentration in the Gouda (salt-in-moisture, 4.8%) resulted in 3.5 mmol histamine/kg cheese while a salt-in-moisture of 2.6% resulted in 2.1 mmol histamine/kg cheese. Chambers and Staruszkiewicz (1978) reported that higher levels of biogenic amines are formed in cheese made from pasteurised milk than in raw milk cheese. It appears that bacteria responsible for biogenic amine formation are present in milk prior to processing rather than as post-processing contaminants. Thus, adherence to high standards of cleanliness during milk production can play a role in reducing the formation of biogenic amines in cheese. Storage temperature also appears to play a role in histamine formation in cheese. Elevated storage temperature increases the potential for histamine formation in cheese, particularly if significant numbers of bacteria with decarboxylase activity are present (Sumner et al., 1985). As noted earlier, increasing the storage temperature for Gouda cheese from 9 to 21 °C results in higher histamine levels (Joosten, 1988). Enhancing proteolysis during cheese ripening by addition of proteolytic enzymes has been reported to increase the concentration of biogenic amines in cheese (Leuschner et al., 1998; Fernandez-Garcia et al., 2000). Biogenic amines in cheese

Only a few cases of histamine poisoning due to cheese consumption have been reported in the literature. Gouda containing 85 mg histamine/100 g cheese was implicated in an outbreak in Holland (Doeglas et al., 1967). Salt-tolerant lactobacilli, which contaminated the rennet, were considered the most likely factor responsible for the high levels of histamine (Stadhouders and Veringa, 1967). Cheese-related histamine poisoning has also been reported in the United States. In 1978, 38 people exhibited symptoms of toxicity following consumption of Swiss cheese containing more than 9 mmol/kg of histamine (Chambers and Staruszkiewicz, 1978), and in 1980, 6 people aboard a naval ship were poisoned by Swiss cheese containing 16.8 mmol/kg of histamine (Taylor et al., 1982). An individual in Canada being treated with isoniazid exhibited toxicity after consuming Cheddar containing 40 mg histamine/100 g (Kahana and Todd, 1981). Similar reactions to histamine-containing cheese by individuals taking isoniazid have been reported by Uragoda and Lodha (1979) and Taylor (1986).

Swiss cheese was implicated in an outbreak of histamine poisoning reported by Sumner et al. (1985). A strain of Lactobacillus buchneri, which possessed histidine decarboxylase activity, was isolated from the cheese. However, other strains of Lb. buchneri did not possess this enzyme activity and were incapable of producing histamine. Recsei and Snell (1982) reported that a strain known as Lactobacillus 30a, which closely resembled Lactobacillus delbrueckii, is capable of producing large amounts of histamine. Sumner et al. (1985) reported that the ability to produce histamine appears to be limited to a few strains of lactobacilli, making them difficult to characterise. Other organisms such as Enterococcus faecium, Streptococcus mitis, Lb. delbrueckii subsp. bulgaricus, Lb. plantarum, Lb. casei, Lb. acidophilus and Lb. arabinose have been shown to possess histidine decarboxylase activity (Stratton et al., 1991). Joosten and Northolt (1989) isolated five histamineproducing strains similar to Lb. buchneri from Gouda cheese. Tham (1988) reported that enterococci are probably irrelevant in cheese-related histamine toxicity. However, Gardin et al. (2001) reported that Enterococcus faecalis produced 2-phenylethylamine and also substantial amounts of tyramine in skim milk. They noted that the main biological feature influencing the formation of biogenic amines was the extent of growth of micro-organisms, such as Ec. faecalis, characterised by decarboxylase activity. In traditional and artisanal cheeses produced from raw milk, enterococci often reach levels of 107 cfu/g. Gardin et al. (2001) cautioned that it is important that the presence of biogenic amines due to the activity of these micro-organisms is maintained within safe levels, without affecting the positive effects of enterococci on the final organoleptic characteristics of the cheese. In addition to histamine, tyramine in cheese has also been reported to induce adverse reactions, such as headache and hypertension, in patients taking MAO inhibitors (Blackwell, 1963; Smith and Durack, 1978; Lejonc et al., 1979). Tyramine is found at levels ranging from non-detectable to 70 mg/100 g in cheese (Voigt et al., 1974). These workers detected tyramine in 81 of 85 samples of Cheddar cheese tested. Ingles et al. (1985) reported high levels of tyramine (625 g/g) as well as histamine (490 g/g) in Danish Blue cheese. Voigt and Eitenmiller (1978) concluded that many organisms may be responsible for generating biogenic amines in cheese but that most are adventitious rather than part of the starter culture population. The build-up of amines is influenced by the availability of substrate, pH, salt concentration and temperature ( Joosten and van Boekel, 1988).


Mycotoxins Mycotoxins are a group of secondary metabolites produced by various filamentous fungi which can cause a toxic response, termed a mycotoxicosis, when ingested at low concentrations by higher vertebrates and other animals (Fig. 1). The biosynthetic pathways for many of these mycotoxins have been extensively characterised, particularly the aflatoxin biosynthetic pathway (Fig. 2). Ingestion of mycotoxins can lead to the deterioration of liver or kidney function. Some mycotoxins are neurotoxins, while others produce effects ranging from skin sensitivity or necrosis to extreme immunodeficiency. This, coupled with the fact that aflatoxin B1 (AFB1) is regarded as the most potent liver carcinogen known for a wide variety of animal species, makes contamination of the human food chain, including dairy produce, with mycotoxins a significant problem in global food safety. The presence of mycotoxins in cheese is fundamentally due to three main reasons: (1) the presence of aflatoxin M1 (AFM1) in fresh or reconstituted milk (Blanco et al., 1998) used in cheese production, as a consequence of feed contaminated with AFB1 eaten by dairy cattle (Lund et al., 1995), which is often termed indirect contamination, (2) synthesis of mycotoxins by fungi such as Penicillium and Aspergillus species, which grow on cheese, termed direct contamination and (3) the production of mycotoxins by fungi which are used in the manufacture of mould-ripened cheeses. Indirect contamination

It is now well-established that the intake by dairy cows of feedstuffs contaminated with either aflatoxin B1 (AFB1) or aflatoxin B2 (AFB2) results in the excretion of the monohydroxylated AFM1 and AFM2 (Fig. 1) derivatives in their milk within a few hours (Allcroft and Carnaghan, 1963). It has been calculated that if cows ingest AFB1 in their diet at a level of 300 ng/g feed, they will produce milk containing 1–3 ng/ml AFM1 24 h later (Smith et al., 1994). According to two other studies (Veldman et al., 1992; Chopra et al., 1999), normal carry-over is about 0.4–0.6% and a daily intake of AFB1 70 g by cows results in greater than the regulatory limit (0.05 g/l of AFM1) in milk accepted in most countries. The amount of AFM1 formed depends on the individual cow, with the excretion of AFM1 in the milk decreasing markedly about 1 day after the feeding of AFB1 had ceased, although small amounts are found for a further 2–3 days. The conversion ratio of AFB1 to AFM1 varies from 1:100 to 1:300. There is some evidence to suggest seasonal variations in the level of AFM1 in milk, with higher levels being observed in Albanian farm milk in winter than

in summer (Panariti, 2001), while in a Greek study, no seasonal effects were observed on AFM1 levels in milk (Markaki and Melissari, 1997). While AFM1 is much less toxic, less mutagenic and less carcinogenic than AFB1, it is nonetheless classified as a possible human carcinogen (Group 2B) and as such its presence in milk-derived products, such as cheese, must be a cause for concern. In addition, it is important to note that the consumption of AFM1contaminated infant formula and other milk products by infants is to be avoided and very low limits have been set (0.01–0.05 g/kg) for infant foods, owing to the relatively high consumption level of these products by infants, their low body weight and the possibly greater susceptibility of younger children to aflatoxins (Aksit et al., 1997). The indirect contamination of milk with other mycotoxins, such as sterigmatocystin, T-2 toxin (van Egmond and Paulsch, 1986), fumonisins (Maragos and Richard, 1994) or cyclopiazonic acid (CPA; Dorner et al., 1994), has been reported. However, it is widely believed that these toxins do not represent a significant public health risk (Prelusky et al., 1990; Charmley et al., 1993), even though CPA can potentially be carried over into processed milk products (Prasongsidh et al., 1997). There is some evidence that ochratoxin A (OTA) can be present in cows’ milk. A Swedish study showed OTA in 14% of 36 cows’ milk samples at a level ranging from 10 to 40 ng/ml (Breitholtz-Emanuelsson et al., 1993). The results of quantitative surveys of the level of AFM1 in milk and milk products carried out in the late 1960s and 1970s in a number of countries were summarised by Smith et al. (1994). When compared with the results of surveys undertaken in the 1980s, it appears that the incidence of AFM1-contaminated milk in general decreased but this trend did not occur in all countries surveyed. This lowering of the incidence of AFM1 contamination may be as a result of the effect of legislation implemented in many countries on the contamination of feedstuffs with aflatoxins. In a recent review by Pittet (1998), including data from the Czech Republic, Slovakia, Czechoslovakia, France, Greece, Germany, Iran, Japan, Switzerland, Syria, the USA and the Netherlands, the incidence of AFM1 in cheese appears to be very varied. In another study in the south of Spain in which 35 samples of local cheese were analysed, AFM1 was detected in 16 samples (44.7%) at a concentration between 20 and 200 g/g cheese (Barrios et al., 1996). In a survey in the Bursa Province in Turkey, the level of AFM1 in 7 of 57 samples of full-fat white cheese analysed exceeded 250 ng/kg (Oruc and Sonal, 2001). In other studies, the level of AFM1 was low, with only 4 of 204 samples of pasteurised milk, powdered


CH2

COOH

O

OH

O

O NH

N

O

CH3

N

N H

H H Cl

O

H

HN N

Ochratoxin A

Roquefortine C O

O

O

O

O O

O

OCH3

O

O

O

OCH3

Aflatoxin B2

Aflatoxin B1

O

O

O

O

O

O

O

OCH3

OCH3

O

O

O

O

O

Aflatoxin G2

Aflatoxin G1

CH3 O OH

O

H

N H CH3

O O

CH3

Me

O

OH

O

NH acid

OH

O

O

O

O

COOH O

O

CH3 CH3COO

O

H

O

OH

CH3

CH3 Citrinin Figure 1 Structure of some toxins produced by fungi.

Patulin

PR Toxin

CH3

CH3

CHO


(a)

Acetate + Malonyl-CoA

(b)

HO O HO O

Hexanoyl-X HO

OH

(c)

O Norsolorinic acid OH HO O HO OH

OH O OH

HO

O HO

OH

O HO

O

HO

OHMe

OH O

OH

Averantin HO O

O

OH

HO

Me

O

O

O

O HO

O

HO

OH

1′-Hydroxyversicolorane

OH O

Me

(e)

OH

O O

HO

O

O Versiconal acetate

O

Averufin

OH

(d)

O HO

OH O

O 5-Hydroxyaverantin

O Averufanin

OH O

HO

OH

O

O

HO

OH

(f1) (f2)

O

O Versicolorin A

O

HO

OH

OH

O OH

O

OH OH

O

Versicolorin B

Versiconal OH

O O

O

O OH

O O

O Demethylsterigmatocystin

(i)

O

O

O OH

(h)

Dihydromethlysterigmatocystin OH

O

OH O OMe

O

OH

O OMe

O

O

Dihydrosterigmatocystin

Sterigmatocystin

(j) (j) O

O

OMe

O OMe O O O-methylsterigmatocystin

O

O

OMe O OMe

Dihydro-O-methylsterigmatocystin O

O

O

O O

O

O

O OMe

Aflatoxin B1/G1

O

O

OMe

Aflatoxin B2/G2

Figure 2 Aflatoxin biosynthetic pathway. Enzymes involved: (a) fatty acid synthase, (b) polyketide synthase, (c) norsolorinic acid reductase, (d) versiconal hemiacetal reductase, (e) esterase, (f1) versicolorin B synthase, (f2) versiconal cyclase, (g) desaturase, (h) O-methyltransferase (MT-II), (i) O-methyl-transferase, (j) O-methyltransferase (MT-I) (compiled from Trail et al., 1995; Bennet et al., 1997; Minto and Townsend, 1997).


milk and cheese analysed in Campinas, Brazil, being positive (De Sylos et al., 1996) and only 2 of 50 cheese samples tested in Argentina being positive, with levels of 0.33 and 0.20 g/l (Lopez et al., 1998). Fate of AFM1 in cheese during manufacture and ripening

Initially, it was believed that the processing of milk reduced the level of AFM1 present. However, later it became clear that the AFM1 content of milk is not reduced by heat treatments such as pasteurisation or sterilisation (Yuosef and Marth, 1989). The fate of AFM1 in milk during cheese manufacture is affected by the principal manufacturing steps. Contradictory data have been reported for the recovery of AFM1 after cheese production. Some early studies showed variable losses of AFM1 during cheese manufacture, e.g., Purchase et al. (1972) reported that Cottage cheese made by acid coagulation of naturally contaminated milk contained no AFM1, which was present in the whey. However, a number of other studies have indicated that AFM1 in milk partitions between the curds and the whey in both acid-coagulated and rennetcoagulated cheeses. A number of studies have shown that AFM1 is stable during cheesemaking, and that 40–57% of total AFM1 is found in the curd (Stubblefield and Shannon, 1974; Stubblefield et al., 1980). Considering the partition coefficient of AFM1 in water, it would be expected that most of the toxin should partition into the whey. However, a greater than expected proportion of the toxin ends up in the curd, possibly due to the fact that AFM1 binds to casein (Brackett and Marth, 1982). Thus, the presence of AFM1 in cheese may be due to the fact that, on the one hand, this toxin binds to casein and, on the other hand, that a part of the whey remains in the curd. An examination of different types of cheese showed high stability of AFM1 during maturation and storage (Applebaum and Marth, 1982; Yuosef and Marth, 1989) and that while fluctuations in the level can occur during cheese maturation and storage, it appears that little if any of the AFM1 is lost during the cheesemaking process. Therefore, the presence of AFM1 in cheese and indeed in other casein-containing products is to be expected if contaminated milk is used as the starting material. The best way to control the presence of AFM1 in milk and cheese is to restrict its presence in the feed. With this in mind, the European Union has established an acceptable limit for AFB1 in animal feed of 10 g/kg (Moss, 1998). Production of toxic metabolites in cheese

The moulds, Penicillium camemberti and P. roqueforti, have long been used in the manufacture of mould-

ripened cheeses which are eaten throughout the world. P. roqueforti is an essential component of the microflora of a number of cheeses such as Roquefort (France), Stilton (UK), Tulum (Turkey), Gorgonzola (Italy), Blauschimmelkase (Switzerland) and Danish Blue (Denmark). P. camemberti produces cyclopiazonic acid while P. roqueforti produces at least three toxins, PR toxin, roquefortine and patulin (Fig. 1), and some strains can also produce mycophenolic acid, penicillic acid, cyclopiazonic acid, penitrem A, isofumigaclavine A and B, festuclavine and chaetoglobosin A. Cyclopiazonic acid (CPA) is produced by all strains of P. camemberti, and screening of P. camemberti isolates has failed to identify an isolate incapable of producing toxin (Leistner and Eckardt, 1979; Fig. 1). Cyclopiazonic acid has been reported in samples of commercial Blue cheese, at a level ranging from 0.05 to 1.5 g/g (Le Bars, 1979). It appears to be found predominantly in the rind of the cheese but has also been reported to migrate to the core in Taleggio-type cheese (Finoli et al., 1999). Cyclopiazonic acid is produced by P. camemberti in cheese usually after 5 days at 25 °C, but not during normal storage at refrigeration temperatures (Still et al., 1978). The low level of CPA found in cheese (Le Bars, 1979), coupled with the relative instability of the toxin (Noroozian et al., 1998) and its low toxicity make consumption of these cheeses safe for the consumer. Finoli et al. (2001b) reported the presence of roqueforine C in cheese ranging from 0.05 to 1.47 mg/kg, but PR toxin was not found. In any case, PR toxin has been reported to be unstable in Blue cheese (Scott and Kanhere, 1979). Roquefortine C has also been reported to be present in Valdeon, a naturally ripened Blue cheese from Spain (Lopez-Diaz et al., 1996), while mycophenolic acid has been reported in Manchego cheese (Lopez-Diaz et al., 1996). Thus, while there are reports of mycotoxin contamination of mould-ripened cheese, the low levels present, coupled with the fact that large quantities are seldom eaten, suggest that they are not a hazard to human health. In any case, there is no evidence to date of human toxicity resulting from the consumption of mould-ripened cheeses. Direct contamination of cheese with mycotoxins

Cheese is very susceptible to mould growth and is normally kept under refrigeration conditions; many retail packs are either vacuum-packed or flushed with an inert gas. Therefore, spoilage generally results from psychrotolerant moulds that can grow at low oxygen tensions. Mould growth during ripening and storage often necessitates trimming. Moulds have been reported to cause spoilage of vacuum-packaged Cheddar cheese during maturation (Hocking and Faedo, 1992). This


defect occurs sporadically in Cheddar blocks which are matured for up to 9–12 months at 8–12 °C and is caused by the growth of fungi in folds and wrinkles of the plastic film in which the Cheddar is packaged. In one study, 195 fungi were isolated from vacuumpackaged Cheddar, about 27% of which were Penicillium species, with P. commune and P. glabrum being dominant (Hocking and Faedo, 1992). Given that P. commune can produce CPA, the presence of this fungus must be a concern. Indeed, the potential for mycotoxin production by mycotoxigenic fungi which contaminate cheese is a constant concern for both the manufacturer and the consumer. The most important spoilage organisms in hard, semi-hard and semi-soft cheeses from several countries, made without added preservatives, are P. commune and P. nalgiovense (Lund et al., 1995). Less-important species include P. verrucosum, P. solitum, P. roqueforti, P. discolor, P. crustosum, P. palitans and Aspergillus versicolor (Filtenborg et al., 1996; Kure et al., 2001). Cheese has been reported to contain mycotoxins that are teratogenic (OTA, AFB1), nephrotoxic (OTA, citrinin), neurotoxic (penitrem A, CPA) or carcinogenic (AFB1, AFG1, OTA, sterigmatocystin; Filtenborg et al., 1996; Creppy, 2002). Others, including patulin, penicillic acid and PR toxin have also been reported but are known not to persist in cheese (Stott and Bullerman, 1976). A number of other secondary metabolites produced by different Penicillium species have also been reported to be present in cheese. These include novel metabolites such as cyclopeptin, viridicatol, rugulovasine A, meleagrin, chaetoglobosin A, compactin, viridic acid, PC-2, verrucolone, diportinic acid, anacine, verrucine A and sclerotigenin, which had not previously been reported from cheese (Larsen et al., 2002). A known metabolite of Penicillium associated with blue-veined cheese is mycophenolic acid, which exhibits antibiotic properties. Since mycotoxin-producing moulds are obligate aerobes, the appropriate packaging of cheese is important. For example, growth of potential mycotoxigenic moulds in cheese can be prevented by modified atmosphere packaging (Taniwaki et al., 2001). In addition, it appears that the production of roquefortine C and CPA by P. roqueforti and P. commune can be prevented or at least reduced considerably in cheese if adequate modified atmosphere packaging is used; atmospheres of 20–40% CO2 and 1% O2 reduce CPA production to very low levels (Taniwaki et al., 2001). Work has also been undertaken on the incidence of mycotoxins in cheese contaminated with Aspergillus spp. The ability of toxigenic aspergilli to produce aflatoxin during growth on Cheddar cheese was first demonstrated by Lie and Marth (1967), who demonstrated that aflatoxin can penetrate into cheese to a

depth of up to 4 cm from the surface. Aflatoxin has also been shown to be produced in Manchego-type cheese, at a level up to 130 g/g cheese, following ripening at 15 °C for 60 days (Blanco et al., 1988). Aflatoxin was also detected in both the outer 10-mm layer and in the second 10-mm layer following incubation at 28 °C for 30 days. Sterigmatocystin was found in Ras cheese inoculated with A. versicolor; toxin was detected after 45 days of ripening and reached a maximum after 90 days (Abde Alla et al., 1996). The presence of mould growth on the surface of the cheese does not automatically imply that mycotoxins are present in cheese, as the minimum water activity (aw) for growth and toxin production can be quite different in mycotoxigenic fungi (Moss, 1991; Sweeney and Dobson, 1998). Therefore, even if mould growth does occur on cheese, the level of mycotoxin contamination is likely to be low, based on research findings to date. In any case, it is recommended that if cheese is visually contaminated with mould growth, the contaminated portion of the cheese be removed to a depth of a least 2.5 cm. As previously stated, there is no direct evidence of human toxicity resulting from the consumption of cheese contaminated with mycotoxins. However, this might simply reflect a lack of suitable human-related assay techniques rather than the actual absence of toxins.

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Taylor, S.L., Kiefe, T.J., Windham, E.S. and Howell, J.F. (1982). Outbreak of histamine poisoning associated with consumption of Swiss cheese. J. Food Protect. 45, 455–457. Taylor, S.L., Hui, J.Y. and Lyons, D.E. (1984). Toxicology of scombroid poisoning, in, Seafood Toxins, Pagelis, E.P., ed., ACS Symposium Series 262, Washington, DC. pp. 417–430. Tham, W. (1988). Histamine formation by enterococci isolated from home-made goat cheeses. Int. J. Food Microbiol. 7, 103–108. Trail, F., Mahanti, N. and Linz, J.E. (1995). Molecular biology of aflatoxin biosynthesis. Microbiology 141, 755–765. Uragoda, C.G. and Lodha, S.C. (1979). Histamine intoxication in a tuberculosis patient after ingestion of cheese. Tubercule 60, 56–61. Vale, S. and Gloria, M.B.A. (1998). Biogenic amines in Brazilian cheeses. Food Chem. 63, 343–348. van Egmond, H.P. and Paulsch, W.E. (1986). Mycotoxins in milk and milk products. Neth. Milk Dairy J. 40, 175–188. Veldman, V., Meijst, J.A.C., Borggreve, J., Heeres-van der Tol, J.J. (1992). Carry-over of aflatoxin from cows food to milk. Anim. Prod. 55, 163–168. Voigt, M.N. and Eitenmiller, R.R. (1978). Role of histidine and tyrosine decarboxylases and mono- and diamine oxidases in amine build-up in cheese. J. Food Prot. 41, 182–186. Voigt, M.N., Eitenmiller, R.R., Koehler, P.E. and Handy, M.K. (1974). Tyramine, histamine and tryptamine content of cheese. J. Milk Food Technol. 37, 377–388. Yuosef, A.E. and Marth, E.H. (1989). Stability and degradation of aflatoxin M1, in, Mycotoxins in Dairy Products, van Egmond, H.P., ed., Elsevier, London. pp. 127–161.


Nutritional Aspects of Cheese N.M. O’Brien and T.P. O’Connor, Department of Food and Nutritional Sciences, University College, Cork, Ireland

Introduction Milk and dairy products are important components of our food supply. On average, these foods contribute 4% of total energy intake worldwide and approximately 10% of total energy intake in Europe, North America and Australia (FAO Food Balance Sheets, 1995–1999). Cheese is a nutritious, versatile dairy food. A wide variety of cheese types are available to meet specific consumer requirements and allow convenience of use. Per caput consumption of cheese in the European Union has been reported to be 15.2 kg/year. Greece has the highest per caput consumption of 23.5 kg/year (Burrell, 1996). Cheese contains a high concentration of essential nutrients relative to its energy level. Its precise nutrient content is influenced by the type of milk used (species, stage of lactation, full-fat, low-fat, skim), the manner of manufacture and, to a lesser extent, the degree of ripening. As outlined in detail elsewhere in this book, the water-insoluble nutrients of milk (coagulated casein, colloidal minerals, fat, fat-soluble vitamins) are retained in the cheese curd whereas the water-soluble milk constituents (whey proteins, lactose, water-soluble vitamins and minerals) partition into the whey. However, loss of water-soluble B vitamins in the whey may be compensated to a certain extent by microbial synthesis during ripening. Milk and dairy products, including cheese, contain components which may increase the risk of certain chronic diseases but reduce the risk of others (Norat and Riboli, 2003). Cholesterol and saturated fat are potential risk factors for atherosclerosis. A recent paper (Moss and Freed, 2003) has suggested that non-fat constituents of milk, particularly the calcium– magnesium ratio, lactose and milk fat globule membrane antigens, have specific coronary atherogenic effects. However, other components may reduce risks, e.g., conjugated linoleic acid (CLA) which may have antioxidant and anticancer properties, calcium which may protect against hypertension and osteoporosis, and folic acid, vitamin B6 and vitamin B12 which may exert beneficial effects on plasma homocysteine levels (an independent risk factor for atherosclerosis). The

epidemiological evidence for an association between dairy products, including cheese, and colorectal cancer has been reviewed by Norat and Riboli (2003); no significant association between cheese consumption and colorectal cancer was noted. Epidemiological studies which attempt to investigate the effect of a specific food item (e.g., cheese) on disease risk are fraught with difficulty in interpretation as it is more likely that it is the overall dietary profile, made up of a balance of a wide variety of different foods, that may influence risk of chronic disease.

Protein Cheese contains a high content of biologically valuable protein. As shown in Table 1 (Holland et al., 1989), the protein content of cheese ranges from approximately 4–40%, depending upon the variety. The protein content of different cheese varieties tends to vary inversely with the fat content. During traditional cheese manufacture, most of the whey proteins pass into the whey. Whey proteins represent only 2–3% of the total protein in cheese, the remainder being casein, which is slightly deficient in sulphur amino acids. Thus, the biological value of cheese protein is slightly less than that of total milk protein. If the essential amino acid index of total milk protein is given a value of 100, then the corresponding value of the proteins in cheese varieties ranges from 91 to 97 (Renner, 1987). Cheese protein is almost 100% digestible, as the ripening phase of cheese manufacture involves a progressive breakdown of casein to water-soluble peptides and free amino acids. Hence, a significant degree of breakdown of cheese protein has occurred before it is consumed and subjected to the effects of gastrointestinal proteolytic activity. Milk proteins are a key source of a range of bioactive peptides (BP) which can exert hormone-like regulatory effects in the human body (Meisel, 1998; Gobbetti et al., 2002; Pihlanto-Leppala, 2002; Fitzgerald and Meisel, 2003). These peptides may be released from their parent protein by proteolysis in products such as cheese. The production of BP is influenced by



574 Table 1 Composition of selected cheeses, per 100 g (Holland et al., 1989) Energy Cheese type

Water (g)

Protein (g)

Fat (g)

Carbohydrate (g)

Cholesterol (mg)

kcal

kJ

Brie Caerphilly Camembert Cheddar (normal) Cheddar (reduced-fat) Cheshire Cottage cheese Cream cheese Danish blue Edam Emmental Feta Fromage frais Gouda Gruyere Mozzarella Parmesan Processed cheese* Ricotta Roquefort Stilton

48.6 41.8 50.7 36.0 47.1 40.6 79.1 45.5 45.3 43.8 35.7 56.5 77.9 40.1 35.0 49.8 18.4 45.7 72.1 41.3 38.6

19.3 23.2 20.9 25.5 31.5 24.0 13.8 3.1 20.1 26.0 28.7 15.6 6.8 24.0 27.2 25.1 39.4 20.8 9.4 19.7 22.7

26.9 31.3 23.7 34.4 15.0 31.4 3.9 47.4 29.6 25.4 29.7 20.2 7.1 31.0 33.3 21.0 32.7 27.0 11.0 32.9 35.5

Tr 0.1 Tr 0.1 Tr 0.1 2.1 Tr Tr Tr Tr 1.5 5.7 Tr Tr Tr Tr 0.9 2.0 Tr 0.1

100 90 75 100 43 90 13 95 75 80 90 70 25 100 100 65 100 85 50 90 105

319 375 297 412 261 379 98 439 347 333 382 250 113 375 409 289 452 330 144 375 411

1323 1554 1232 1708 1091 1571 413 1807 1437 1382 1587 1037 469 1555 1695 1204 1880 1367 599 1552 1701

Tr, trace. * Variety not specified.

Nutritional Aspects of Cheese 575

the starter culture and ripening conditions. An important class of BP are peptides that inhibit the activity of angiotensin I-converting enzyme (ACE), inhibition of which mainly gives rise to antihypertensive effects but may also modulate immuno-defense and nervous system activity (Meisel, 1993). Angiotensin I-converting enzyme-inhibitory peptides have been reported in several ripened cheeses (Stepaniak et al., 1995; Meisel et al., 1997; Smacchi and Gobbetti, 1998). It appears the BP liberated by starter proteolytic enzymes during cheese ripening can be degraded further to inactive fragments, as the ripening progresses. For example, an antihypertensive peptide derived from s1-casein was observed in 6-month-old Parmesan cheese but was not detected in 15-month-old cheese (Meisel et al., 1997). Anticancer effects have been reported for peptides derived from a slurry of cheese made using Lc. lactis subsp. lactis as a starter culture (Kim et al., 1995). Bioactive peptides have potential as ingredients in functional foods and pharmaceuticals.

Carbohydrate Most of the lactose, the principal carbohydrate in milk, is lost in whey during cheese manufacture and hence most cheeses contain only trace amounts of carbohydrate (Table 1). Furthermore, the residual lactose in cheese curd is usually fermented to lactic acid by the starter bacteria. Thus, cheeses can be consumed without ill-effects by lactose-intolerant individuals who are deficient in the intestinal enzyme, -galactosidase.

Fat and Cholesterol The fat content of cheese varies considerably, depending on the milk used and the method of manufacture (Table 1). Fat affects firmness, adhesiveness, mouthfeel and flavour of cheese (see ‘Rheology and Texture of Cheese’, Volume 1). In some varieties of cheese, free fatty acids and their catabolites are important flavour constituents (see ‘Lipolysis and Catabolism of Fatty Acids in Cheese’, Volume 1). From a nutritional point of view, the digestibility of the fat in different varieties of cheese is in the range 88–94% (Renner, 1987). Most cheeses are potentially significant dietary sources of fat. For example, a 50-g serving of Cheddar provides 17 g fat (Table 1) which is a significant amount when compared with typical intakes of fat in affluent Western societies. A typical Western diet providing 2000 kcal with 40% energy derived from fat contains approximately 88 g fat. Cheese fat generally contains ⬃66% saturated, 30% monounsaturated and 4% polyunsaturated fatty acids. Thus, cheese represents a significant dietary source of

both total fat and saturated fatty acids. Of the many saturated fatty acids in milk, only C12:0, C14:0 and C16:0 have the property of raising blood cholesterol and palmitic acid, C16:0, is relatively ineffective (Hayes et al., 1991). Many sets of dietary guidelines issued by expert panels worldwide have recommended reductions in the intake of both total and saturated fat in Western societies. In large measure, these recommendations are based on evidence that increased intakes of some saturated fatty acids elevate both total and low-density lipoprotein cholesterol in blood, which is associated with an increased risk of coronary heart disease. While some nutritionists dispute this hypothesis, the overwhelming body of medical opinion worldwide supports the concept of dietary guidelines. Market forces and consumers have responded to these guidelines and the market for food products low in fat, cholesterol and sodium has expanded significantly. The cheese industry has responded by developing ‘light’ cheese products with a reduced fat content (Olson and Johnson, 1990). The cholesterol content of cheese is a function of its fat content (Table 1) and ranges from approximately 10–100 mg/100 g, depending on the variety. Despite considerable consumer confusion and the widespread prevalence of misinformation, dietary cholesterol has much less influence on blood cholesterol level than dietary saturated fat (Keys, 1984). Thus, the cholesterol content of cheese is of lesser importance than its saturated fat content. The majority of individuals show little or no response in blood cholesterol level to increased dietary cholesterol intake in the range 250–800 mg/day. However, a minority (approximately 20%) of adults do exhibit an increased level of blood cholesterol in response to increased dietary intake (McNamara, 1987). In recent years, there has been widespread interest in the potential role of oxidation products of cholesterol on the aetiology of atherosclerosis (Brown and Jessup, 1999; Leonarduzzi et al., 2002). However, cholesterol oxides are formed to a negligible extent in cheese under normal conditions of manufacture, ripening and storage (Stanton and Devery, 2002). Conjugated linoleic acid (CLA) is a potentially beneficial component of milk products, including cheese (MacDonald, 2000). Conjugated linoleic acid is a mixture of positional and geometric isomers of linoleic acid (C18:2) that contain conjugated unsaturated double bonds. The principal isomer is cis-9, trans-11-octadecadienoic acid which accounts for more than 82% of total CLA in dairy products (Chin et al., 1992). Conjugated linoleic acid has been reported to have antioxidant and anticarcinogenic

576 Nutritional Aspects of Cheese

properties in vitro and in animal models (Ha et al., 1987, 1990; Ip et al., 1991). However, these suggested anticarcinogenic properties of CLA could not be confirmed in a recently published epidemiological study on humans (see Voorrips et al., 2002). These authors noted that cheese contributed approximately 21% of total CLA intake in their study group. Conjugated linoleic acid may also be anticholesterolaemic and antiathrogenic (Lee et al., 1994; Mougios et al., 2001). On average, the concentration of CLA in milk and dairy products ranges from 0.2 to 1.6 g/100 g fat (Lin et al., 1995; Fritsche and Steinhart, 1998). Fritsche and Steinhart (1998) have estimated that the intake of CLA in Germany is 0.35 g/d for women and 0.43 g/d for men. Zlatanos et al. (2002) reported that Greek Feta and hard cheeses contain 1.9 (average of 0.8) g CLA/100 g fat. These authors reported higher levels of CLA in Greek cheese derived from sheep’s and goats’ milk than the level of CLA reported by others (Lin et al., 1995; Jiang et al., 1997; Ma et al., 1999) in cheese derived from cows’ milk.

Vitamins The concentration of fat-soluble vitamins in cheese is influenced by the same factors that affect its fat content. Most of the fat-soluble vitamins in milk are retained in the cheese fat. The concentration of water-soluble vitamins in cheese is generally lower than in milk due to losses in the whey. The loss of some of the B vitamins is offset, to a certain extent, by microbial synthesis during cheese ripening. In particular, propionic acid bacteria synthesize significant levels of vitamin B12 in hard cheeses such as Emmental (Renner, 1987). In general, most cheeses are good sources of vitamin A, riboflavin, vitamin B12 and, to a lesser extent, folate. The vitamin content of a range of cheeses is shown in Table 2 (Holland et al., 1989). Cheese contains negligible levels of vitamin C.

Minerals Cheese is an important dietary source of several minerals, in particular calcium, phosphorus and magnesium (Table 3). A 100-g serving of hard cheese provides approximately 800 mg calcium. However, acid-coagulated cheeses, e.g., Cottage, contain considerably less calcium than rennet-coagulated varieties (Renner, 1987). Bioavailability of the calcium from cheese is equivalent to that from milk. Recker et al. (1988) reported that 22.9, 26.7 and 25.4% of total calcium was absorbed from cream cheese, whole milk and yoghurt, respectively.

While the aetiology of osteoporosis is very complex, it appears that adequate calcium intake during childhood and in the teenage years is important in assuring the development of high-peak bone mass. Maximizing bone mass early in life is considered to be a major preventative factor against the development of osteoporosis in later years (Heaney, 1991). Cheese has a potential role in supplying extra, highly bioavailable, calcium. Dairy products, including cheese, contribute little dietary iron (Table 3). Iron deficiency is commonly observed in both developing and developed countries. Hence, there has been interest in fortifying dairy products with iron to enhance their nutritional value. Cheddar and processed cheese have been successfully fortified with iron (Zhang and Mahoney, 1989a,b, 1990, 1991). As discussed elsewhere in this book, NaCl serves several important functions in natural and processed cheeses (see ‘Salt in Cheese: Physical, Chemical and Biological Aspects’, Volume 1). A wide range of sodium levels are found in cheese due to different amounts of salt added during cheesemaking (Table 3). In general, the salt content of natural cheeses tends to be lower than that of many processed cheeses. There is considerable evidence that high sodium intake contributes to hypertension in a susceptible minority (20%) of individuals who are genetically saltsensitive. Unfortunately, there is no simple diagnostic test to identify salt-sensitive individuals. Hence, dietary guidelines for the general public usually recommend that salt intake be restricted. However, it is important to note that even in countries with a high consumption, cheese contributes only about 5–8% of total sodium intake (Renner, 1987).

Cheese and Dental Caries The aetiology of dental caries involves metabolism of sugars by oral micro-organisms to acids which gradually dissolve tooth enamel. However, it is now recognized that a number of dietary factors and nutrient interactions can modify the expression of dental caries (Herod, 1991; Kashket and DePaola, 2002). The cariogenic potential of food is influenced by its composition, texture, solubility, retentiveness and ability to stimulate saliva flow (Morrissey et al., 1984). Dental caries has been acknowledged as a ‘silent epidemic’ that represents a serious threat to children and adults (Surgeon General, 2000). A considerable body of research has been published on the cariostatic effects of cheese (see reviews by O’Brien and O’Connor, 1993; Kashket and DePaola, 2002). Early work (Shaw et al., 1959; Dreizen et al.,

Table 2 Vitamin content of selected cheeses, per 100 g (Holland et al., 1989) Cheese type

Retinol (g)

Carotene (g)

Vitamin D (g)

Vitamin E (mg)

Thiamine (mg)

Riboflavin (mg)

Niacin (mg)

Vitamin B6 (mg)

Vitamin B12 (g)

Folate (g)

Pantothenate (mg)

Biotin (g)

Brie Caerphilly Camembert Cheddar (normal) Cheddar (reducedfat) Cheshire Cottage cheese Cream cheese Danish blue Edam Emmental Feta Fromage frais Gouda Gruyere Mozzarella Parmesan Processed cheese* Ricotta Roquefort Stilton

285 315 230 325

210 210 315 225

0.20 0.24 0.18 0.26

0.84 0.78 0.65 0.53

0.04 0.03 0.05 0.03

0.43 0.47 0.52 0.40

0.43 0.11 0.96 0.07

0.15 0.11 0.22 0.10

1.2 1.1 1.1 1.1

58 50 102 33

0.35 0.29 0.36 0.36

5.6 3.5 7.6 3.0

165

100

0.11

0.39

0.03

0.53

0.09

0.13

1.3

56

0.51

3.8

350 44 385 280 175 320 220 100 245 325 240 345 270

220 10 220 250 150 140 33 Tr 145 225 170 210 95

0.24 0.03 0.27 0.23 0.19 N 0.50 0.05 0.24 0.25 0.16 0.25 0.21

0.70 0.08 1.0 0.76 0.48 0.44 0.37 0.02 0.53 0.58 0.33 0.70 0.55

0.03 0.03 0.03 0.03 0.03 0.05 0.04 0.04 0.03 0.03 0.03 0.03 0.03

0.48 0.26 0.13 0.41 0.35 0.35 0.21 0.40 0.30 0.39 0.31 0.44 0.28

0.11 0.13 0.06 0.48 0.07 0.10 0.19 0.13 0.05 0.04 0.08 0.12 0.10

0.09 0.08 0.04 0.12 0.0 0.09 0.07 0.10 0.08 0.11 0.09 0.13 0.08

0.9 0.7 0.3 1.0 2.1 2.0 1.1 1.4 1.7 1.6 2.1 1.9 0.9

40 27 11 50 40 20 23 15 43 12 19 12 18

0.31 0.40 0.27 0.53 0.38 0.40 0.36 N 0.32 0.35 0.25 0.43 0.31

4.0 3.0 1.6 2.7 1.8 3.0 2.4 N 1.4 1.5 2.2 3.3 2.3

185 295 355

92 10 185

N N 0.27

0.03 0.55 0.61

0.02 0.04 0.03

0.19 0.65 0.43

0.09 0.57 0.49

0.03 0.09 0.16

0.3 0.4 1.0

N 45 77

N 0.50 0.71

N 2.3 3.6

N, the nutrient is present in significant quantities but there is not reliable information on the amount; Tr, trace. * Variety not specified.

577


Table 3 Mineral content of selected cheeses, mg/100 g (Holland et al., 1989) Cheese type Brie Caerphilly Camembert Cheddar (normal) Cheddar (reduced-fat) Cheshire Cottage cheese Cream cheese Danish blue Edam Emmental Feta Fromage frais Gouda Gruyere Mozzarella Parmesan Processed cheese* Ricotta Roquefort Stilton

Na 700 480 650 670 670 550 380 300 1260 1020 450 1440 31 910 670 610 1090 1320 100 1670 930

K

Ca

Mg

P

Fe

Zn

100 91 100 77 110 87 89 160 89 97 89 95 110 91 99 75 110 130 110 91 130

540 550 350 720 840 560 73 98 500 770 970 360 89 740 950 590 1200 600 240 530 320

27 20 21 25 39 19 9 10 27 39 35 20 8 38 37 27 45 22 13 33 20

390 400 310 490 620 400 160 100 370 530 590 280 110 490 610 420 810 800 170 400 310

0.8 0.7 0.2 0.3 0.2 0.3 0.1 0.1 0.2 0.4 0.3 0.2 0.1 0.1 0.3 0.3 1.1 0.5 0.4 0.4 0.3

2.2 3.3 2.7 2.3 2.8 3.3 0.6 0.5 2.0 2.2 4.4 0.9 0.3 1.8 2.3 1.4 5.3 3.2 1.3 1.6 2.5

* Variety not specified.

1961) showed that the incorporation of dairy products in the diet greatly reduced the development of dental caries in rats. Reynolds and Johnson (1981) confirmed these findings. Later work (Jenkins and Ferguson, 1966; Weiss and Bibby, 1966) indicated that if enamel is treated with milk in vitro and subsequently washed, the solubility of the enamel is greatly reduced. This effect was attributed to the high levels of calcium and phosphate in milk ( Jenkins and Ferguson, 1966) or to casein adsorption onto enamel surfaces (Weiss and Bibby, 1966). Reynolds and del Rio (1984) reported that both casein and whey proteins significantly reduced the extent of caries, with the former exerting the greater effect. Further evidence for the protective effect of casein was provided in a study on rats fed with caseinenriched chocolate (Reynolds and Black, 1987). Calcium and phosphate appear to become available under the acidic conditions of the plaque and reduce demineralization of enamel (Reynolds, 1997; Reynolds et al., 1999). Concentrates containing various levels of whey protein, calcium and phosphate but negligible amounts of casein, significantly reduced the incidence of dental caries in rats (Harper et al., 1987). Thus, there is evidence that milk proteins, calcium and phosphate all exert an anticariogenic effect. Guggenheim et al. (1999) reported that micellar casein inhibits oral colonization by the cariogenic Streptococcus sobrinus and dental caries in rats. Vacca-Smith et al. (1994) demonstrated that -casein can reduce the adherence

of the cariogenic Sc. mutans to hydroxyapatite (the mineral of enamel). Rugg-Gunn et al. (1975) provided the first evidence that the consumption of cheese had an anticariogenic effect in humans. They showed that the consumption of Cheddar cheese after sweetened coffee or a sausage roll increased plaque pH, possibly due to increased salivary output. Similar effects were reported by Imfeld et al. (1978) who used a more sophisticated continuous wire telemetry procedure to monitor variations in plaque pH. The effect of eating patterns on dental caries in rats was investigated by Edgar et al. (1982). Rats fed additional meals of cheese while on a high-sucrose diet, developed fewer smooth surface carious lesions and exhibited increased salivary output (which buffers acid formed in plaque) and a reduction in the number of Sc. mutans. Harper et al. (1983) suggested that the cariostatic effect of cheese in rats is due to its calcium phosphate and/or casein; the fat or lactose appeared to exert little influence. Further work by Rosen et al. (1984) on the effect of cheese, with or without sucrose, on dental caries and the recovery of inoculated Sc. mutans in rats indicated beneficial cariostatic effects of cheese consumption but little effect on Sc. mutans numbers. These data suggest that the cariostatic effects of cheese may not be directly related to effects on Sc. mutans. Work on the protective effects of four cheese varieties in an in vitro demineralization system suggested that most, but not all, of the protective


effects of cheese could be explained by mass action effects of soluble ions, particularly calcium and phosphate (Jenkins and Harper, 1983). The effect of Cheddar cheese on experimental caries in humans was investigated by Silva et al. (1986) using an ‘intraoral cariogenicity test’ (ICT). Demineralization and consequent reduction in the hardness of enamel monitored in this test is assumed to be typical of the early stage of the development of caries. Enamel slabs were polished and their initial micro-hardness determined using a Knoop Indenter. The slabs were then wrapped in Dacron and fastened on a prosthetic applicance made specifically for each subject to replace a missing lower first permanent molar. The subjects chewed 5 g of cheese immediately after rinsing their mouths with 10% (w/v) sucrose. Chewing cheese immediately after sucrose rinses resulted in a 71% reduction in demineralization of the enamel slabs, raised plaque pH but caused no significant change in the microflora of plaque compared with controls. Silva et al. (1987) investigated the effects of the water-soluble components of cheese on human caries using the ICT procedure and an experimental protocol which avoided salivary stimulus caused by chewing cheese. An average reduction of 55.7% in the cariogenicity of sucrose was reported, indicating the presence of one or more water-soluble anticariogenic components in cheese. Further evidence that cheese may inhibit dental caries in the absence of saliva was provided by Krobicka et al. (1987); rats that had their saliva-secreting glands surgically removed developed fewer and less-severe lesions when fed with cheese in addition to a cariogenic diet when compared to appropriate controls. Trials on human subjects have confirmed that the consumption of hard cheese leads to significant rehardening of softened enamel surfaces (Jenkins and Hargreaves, 1989; Gedalia et al., 1991). Jensen and Wefel (1990) showed that processed cheese was both antiacidogenic and enamel-protective in human subjects fed with processed cheese four times a day for one month. Saliva flow is greatly reduced in individuals who receive head and neck irradiation for malignancies. These individuals are at high risk of developing dental caries. Sela et al. (1994) reported that hard cheese consumption by these individuals was effective in controlling caries. Moynihan et al. (1999) noted that the concentration of calcium in plaque was significantly higher in human subjects fed with cheese-containing meals than in control subjects fed with meals without cheese. The beneficial effects of cheese were observed even when it was incorporated into other foods, e.g., pasta with cheese sauce. Epidemiological studies (Pappas et al., 1995a,b) indi-

cate that high intake of cheese is negatively associated with root caries in elderly populations, many of whom are at high risk for such lesions. While more research is needed to define the precise mechanism(s) involved in the cariostatic effects of cheese, there is ample evidence to support the consumption of cheese as an anticaries measure (Herod, 1991; Kashket and DePaola, 2002). The most plausible mechanisms for the protective effect of cheese appear to be related to the mineralization potential of the casein–calcium phosphate of cheese, to the stimulation of saliva flow induced by its texture and/or flavour, the buffering effects of cheese proteins on acid formation in dental plaque and the inhibition of cariogenic bacteria.

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Ha, Y.L., Storkson, J. and Pariza, M.W. (1990). Inhibition of benzo[a]pyrene-induced mouse forestomach neoplasia by conjugated dienoic derivatives of linoleic acid. Cancer Res. 50, 1097–1101. Harper, D.S., Osborn, J.C. and Clayton, R. (1983). Cariostatic potential of four cheeses evaluated in a programmedfed rat model. J. Dent. Res. 62, 283 (abstr.). Harper, D.S., Osborn, J.C., Clayton, R. and Hefferren, J.J. (1987). Modification of food cariogenicity in rats by mineral-rich concentrates from milk. J. Dent. Res. 66, 42–45. Hayes, K.C., Pronczuk, A., Lindsey, S. and Diersen-Schade, D. (1991). Dietary saturated fatty acids differ in their impact on plasma cholesterol and lipoproteins in non-human primates. Am. J. Clin. Nutr. 53, 491–498. Heaney, R.P. (1991). Evaluation of Publically Available Scientific Evidence Regarding Nutrient–Disease Relationships. 3. Calcium and Osteoporosis. Life Sciences Research Office, Federation of American Societies for Experimental Biology, Rockville Pike, MD. Herod, E.L. (1991). The effect of cheese on dental caries. Aust. Dental J. 36, 120–125. Holland, B., Unwin, I.D. and Buss, D.H. (1989). Milk Products and Eggs: The Fourth Supplement to McCance and Widdowson’s The Composition of Foods, 4th edn, Royal Society of Chemistry/Ministry of Agriculture, Fisheries and Food, Cambridge, UK. Imfeld, T.H., Hirsch, R.S. and Muhlmann, H.R. (1978). Telemetric recordings of interdental plaque pH during different meal patterns. Br. Dent. J. 139, 351–356. Ip, C., Chin, S.F., Scimeca, J.A. and Pariza, M.W. (1991). Mammary cancer prevention by conjugated dienoic derivatives of linoleic acid. Cancer Res. 51, 6118–6124. Jenkins, G.N. and Ferguson, D.B. (1966). Milk and dental caries. Br. Dent. J. 120, 472–477. Jenkins, G.N. and Hargreaves, J.A. (1989). Effect of eating cheese on Ca and P concentrations of whole mouth saliva and plaque. Caries Res. 23, 159–164. Jenkins, G.N. and Harper, D.S. (1983). Protective effect of different cheeses in an in vitro demineralization system. J. Dent. Res. 62, 284 (abstr.). Jensen, M.E. and Wefel, J.S. (1990). Effects of processed cheese on human plaque pH and demineralization and remineralization. Am. J. Dent. 3, 217–223. Jiang, J., Bjorck, L. and Fonden, R. (1997). Conjugated linoleic acid in Swedish dairy products with special reference to manufacture of hard cheeses. Int. Dairy J. 7, 863–867. Kashket, S. and DePaola, D.P. (2002). Cheese consumption and the development and progression of dental caries. Nutr. Rev. 60, 97–103. Keys, A. (1984). Serum cholesterol response to dietary cholesterol. Am. J. Clin. Nutr. 40, 351–359. Kim, H.D., Lee, J.H., Shin, Z.I., Man, H.S. and Woo, H.J. (1995). Anticancer effects of hydrophobic peptides derived from a cheese slurry. Food Biotechnol. 4, 268–272. Krobicka, A., Bowen, W.H., Pearson, S. and Young, D.A. (1987). The effects of cheese snacks on caries in desalivated rats. J. Dent. Res. 66, 1116–1119.

Lee, K.N., Kritschevsky, D. and Pariza, M.W. (1994). Conjugated linoleic acid and atherosclerosis in rabbits. Atherosclerosis 118, 19–25. Leonarduzzi, G., Sottero, B. and Poli, G. (2002). Oxidized products of cholesterol: dietary and metabolic origin and proatherosclerotic effects (review). J. Nutr. Biochem. 13, 700–710. Lin, H., Boylston, T.D., Chang, M.L., Luedecke, L.O. and Shultz, T.D. (1995). Survey of the conjugated linoleic acid contents of dairy products. J. Dairy Sci. 78, 2358–2365. Ma, D.W.L., Wierzbicki, A.A., Field, C.J. and Clandinin, M.T. (1999). Conjugated linoleic acid in Canadian dairy and beef products. J. Agric. Food Chem. 47, 1956–1960. MacDonald, H.B. (2000). Conjugated linoleic acid and disease prevention: a review of current knowledge. J. Am. Coll. Nutr. 19, 111S–118S. McNamara, D.J. (1987). Effects of fat-modified diets on cholesterol and lipoprotein metabolism. Ann. Rev. Nutr. 7, 273–290. Meisel, H. (1993). Casokinins as inhibitors of angiotensinconverting-enzyme, in, New Perspectives in Infant Nutrition, Sawatzki, G. and Renner, B., eds., Thieme, Stuttgart. pp. 153–159. Meisel, H. (1998). Overview on milk protein-derived peptides. Int. Dairy J. 8, 363–373. Meisel, H., Goepfert, A. and Gunther, S. (1997). ACE inhibitory activities in milk products. Milchwissenschaft 52, 307–311. Morrissey, R.B., Burkholder, B.D. and Tarka, S.M. (1984). The cariogenic potential of several snack foods. J. Am. Dent. Assoc. 109, 589–591. Moss, M. and Freed, D. (2003). The cow and the coronary: epidemiology, biochemistry and immunology. Int. J. Cardiol. 87, 203–216. Mougios, V., Matsakas, A., Petridou, A., Ring, S., Sagredos, A., Melissopoulou, A., Tsigilis, N. and Nicolaidis, M. (2001). Effect of supplementation with conjugated linoleic acid on human serum lipids and body fat. J. Nutr. Biochem. 12, 585–594. Moynihan, P.J., Ferrier, S. and Jenkins, G.N. (1999). Cariostatic potential of cheese: cooked cheese-containing meals increase plaque calcium concentration. Br. Dent. J. 187, 664–667. Norat, T. and Riboli, E. (2003). Dairy products and colorectal cancer: a review of possible mechanism and epidemiological evidence. Eur. J. Clin. Nutr. 57, 1–17. O’Brien, N.M. and O’Connor, T.P. (1993). Milk, cheese and dental caries. J. Soc. Dairy Technol. 46, 46–49. Olson, N.F. and Johnson, M.E. (1990). Light cheese products: characteristics and economics. Food Technol. 44 (10), 93–96. Pappas, A.S., Joshi, A. and Palmer, C.A. (1995a). Relationship of diet to root caries. Am. J. Clin. Nutr. 61, 423S–429S. Pappas, A.S., Joshi, A. and Belanger, A.J. (1995b). Dietary models for root caries. Am. J. Clin. Nutr. 61, 417S–422S. Pihlanto-Leppala, A. (2002). Bioactive peptides, in, Encyclopedia of Dairy Sciences, Roginski, H., Fuquay, J.W. and Fox, P.F., eds., Academic Press, London. pp. 1960–1967.


Recker, R.R., Bammi, A., Barger-Lux, M.J. and Heaney, R.P. (1988). Calcium absorbability from milk products, an imitation milk and calcium carbonate. Am. J. Clin. Nutr. 47, 93–95. Renner, E. (1987). Nutritional aspects of cheese, in, Cheese: Chemistry, Physics and Microbiology, Vol. 1, General Aspects, Fox, P.F., ed., Elsevier Applied Science, London. pp. 345–363. Reynolds, E.C. (1997). Remineralization of enamel subsurface lesions by casein phosphopeptide-stabilized calcium phosphate solutions. J. Dent. Res. 76, 1587–1595. Reynolds, E.C. and Black, C.L. (1987). Reduction of chocolate’s cariogenicity by supplementation with sodium caseinate. Caries Res. 21, 445–451. Reynolds, E.C. and del Rio, A. (1984). Effect of casein and whey protein solutions on caries experience and feeding patterns of the rat. Arch. Oral Biol. 29, 927–933. Reynolds, E.C. and Johnson, I.H. (1981). Effect of milk on caries incidence and bacterial composition of dental plaque in the rat. Arch. Oral Biol. 26, 445–451. Reynolds, E.C., Black, C.L. and Cai, F. (1999). Advances in enamel remineralization: casein phosphopeptide–amorphous calcium phosphate. J. Clin. Dent. 10, 86–88. Rosen, S., Min, D.B., Harper, D.S., Harper, W.J., Beck, E.X. and Beck, F.M. (1984). Effect of cheese, with or without sucrose, on dental caries and recovery of Streptococcus mutans in rats. J. Dent. Res. 63, 894–896. Rugg-Gunn, A.J., Edgar, W.M., Geddes, D.A.M. and Jenkins, G.N. (1975). The effect of different meal patterns upon plaque pH in human subjects. Br. Dent. J. 139, 351–356. Sela, M., Gedalia, I. and Shah, L. (1994). Enamel rehardening with cheese in irradiated patients. Am. J. Dent. 7, 134–136. Shaw, J.H., Ensfield, B.J. and Wollman, D.H. (1959). Studies on the relation of dairy products to dental caries in caries-susceptible rats. J. Nutr. 67, 253–273. Silva, M.D. de A., Jenkins, G.N., Burgess, R.C. and Sandham, H.J. (1986). Effect of cheese on experimental caries in human subjects. Caries Res. 20, 263–269. Silva, M.F. de A., Burgess, R.C., Sandham, H.J. and Jenkins, G.N. (1987). Effects of water-soluble components of cheese on experimental caries in humans. J. Dent. Res. 66, 38–41. Smacchi, E. and Gobbetti, M. (1998). Peptides from several Italian cheeses inhibitory to proteolytic enzymes of lactic

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Factors that Affect the Quality of Cheese P.F. Fox, Department of Food Science, Food Technology and Nutrition, University College, Ireland T.M. Cogan, Dairy Products Research Centre, Teagasc, Fermoy, Ireland

Introduction As discussed in ‘Cheese: An Overview’, Volume 1 and ‘Diversity of Cheese Varieties: an Overview’, Volume 2, the manufacture of cheese exploits either of two properties of the casein system: precipitation/coagulation at the isoelectric pH (4.6), which is exploited in the production of fresh, acid-coagulated cheeses (⬃25% of total cheese production), or by limited proteolysis using rennets which specifically hydrolyse the micellestabilizing protein, -casein, following which the rennetaltered micelles coagulate in the presence of Ca2 at a temperature 20 °C, usually 30–35 °C (⬃75% of cheese production). Most acid-coagulated cheeses are consumed fresh (unripened) whereas the vast majority of rennet-coagulated cheeses are ripened for a period ranging from ⬃3 weeks to 2 years. Although there are recognizable differences between the unripened curds for different cheeses, mainly with respect to moisture content and texture, the characteristic differences between the 1000 or so varieties of cheese develop during ripening. The quality of acid-coagulated cheeses is subject to some variation but the fact that they are consumed fresh and that no modifications are required after manufacture, makes them relatively easy to produce with consistent quality (see ‘Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels’, Volume 1 and ‘Acid- and Acid-Rennet-Curd Cheeses: Part A Quark, Cream Cheese and Related Varieties’, Volume 2). In contrast, the characteristic quality of rennet-coagulated cheeses develops mainly during ripening and frequently depends on the growth of a secondary microflora, which are not readily reproducible. During ripening, a complex array of microbiological, biochemical and chemical reactions occur and therefore there are many opportunities for problems to develop. In this chapter, the quality aspects of rennet-coagulated cheeses will be considered. Some of the principal areas of cheese science through which cheese quality can be improved through research will be discussed. Cheese is the quintessential convenience food – it can be consumed as it is without preparation, can be

used as a sandwich filler, grated or diced and used as a condiment or as a component of several cooked dishes. At least 50% of cheese is used, at home or in the factory, as an ingredient or component of other foods. The principal applications of cheese as an ingredient are discussed in ‘Cheese as an Ingredient’, Volume 2. The use of cheese as a food ingredient is increasing and for many of these applications, the principal criterion of quality depends on the functionality of the cheese, which depends mainly on the physico-chemical properties of the proteins. When used as an ingredient in food applications, cheese is expected to perform one or more functions and there is considerable commercial interest in producing cheese products with functional properties tailor-made for particular applications. Cheese may be used as an ingredient in several forms: • natural cheese: sliced, diced or grated; • processed cheese products; • dried cheese products: – dried, grated natural cheese (a traditional product); – cheese powders. • enzyme-modified cheese products, which may be produced from mild cheese or fresh cheese curd or from blends of enzyme-treated casein and fats and are used mainly as high-intensity cheese flavours. There are several aspects to the quality of cheese; some are applicable to all cheese products and applications, others are significant for specific cheeses. Probably the most important aspects of cheese are: • • • • • •

safety from a public health viewpoint nutritional flavour texture functionality appearance, e.g., – Cheese must conform to the expected characteristics of the variety. There are obvious differences between the principal families of cheese but it may not be so easy to differentiate between cheeses claimed to



584 Factors that Affect the Quality of Cheese

belong to similar varieties, e.g., Gouda and Edam, Cheddar and Gloucester, Brie and Camembert. The borderlines are blurred and within each variety, a certain degree of variability is tolerated and acceptable. – Reproducibility/reliability – consumers expect that a product will be reproducible and consistent between batches and over time with respect to flavour, texture, appearance and functionality, especially for the principal, ‘mass-produced’ varieties; some variability is tolerated, perhaps even expected, in artisanal cheeses.

Milk Selection Pre-treatment Standardization

Cheesemilk Addition of: starter culture (acidification) colour (optional) CaCl2 (optional) Coagulation [rennet or acid (produced in situ or pre-formed) or heat/acid]

Production of Rennet-Coagulated Cheese The production of rennet-coagulated cheese can be divided into two phases: • Conversion of milk to curd • Conversion of curd to cheese The key operations are summarized in Fig. 1. The production of good-quality cheese depends on a good milk supply from the chemical and microbiological viewpoints. Raw milk is a rather variable commodity and is subjected to a range of processes aimed at modifying, standardizing and optimizing the cheesemaking properties of milk. Given a good milk supply, the first objective is to produce curd of the desired chemical composition with the desired microflora. Unless these criteria are met, the curd will not evolve into cheese with the characteristic flavour, texture and functionality during ripening. The ripening of cheese, and hence its quality, is due to the activity of micro-organisms and enzymes from four or five sources: • • • • •

Milk Rennet Primary starter Secondary cultures Adventitious (non-starter) bacteria

It is reasonable to expect that it should be possible to produce premium-quality cheese consistently by controlling these agents; however, in spite of considerable research and quality control efforts, it is not yet possible to do so. A very wide and diverse range of factors interact to affect the composition of cheese curd and hence the quality of the final cheese; an attempt to summarize these is shown in Fig. 2, which follows the same sequence as Fig. 1. Some of these factors/agents can be manipulated easily and precisely while others are more difficult, or perhaps impossible, to control. Indeed, the precise influence of many of the factors included

Coagulum (gel) Cut coagulum Stir Heat Acidification (rennet-coagulated cheeses) Separation of curds from whey

Curds Acidification Special operations (e.g., cheddaring, stretching) Salting (some varieties) Moulding Pressing (some varieties)

Fresh cheese Salting (most varieties) Ripening (most rennet-coagulated cheeses)

Mature cheese Figure 1 General protocol for cheese manufacture (from Fox et al., 2000).

in Fig. 2 on cheese ripening and quality are not known precisely and many of the factors are interactive. It is possible to apply the principles of Hazard Analysis Critical Control Point (HACCP) analysis to cheese production and it is hoped that this article may stimulate efforts to apply HACCP principles to cheesemaking. The principal items in Fig. 1 and 2 will be discussed individually in the following sections.

Milk Supply It is well recognized that the quality of the milk supply has a major impact on the quality and consistency of the resultant cheese. Three aspects of quality must be considered: microbiological, enzymatic and chemical.

Species Breed Stage of lactation Plane of nutrition Animal health

Composition Somatic cell count

Microbiological quality RAW MILK

On-farm

hygiene

Transport

temperature

In-factory

time

Public health and safety natural creaming centrifugal milk powder UF

Thermization Standardization Pasteurization

Selection criteria Acid production Phage sensitivity Ripening properties Rate of lysis

CHEESE MILK

Colour CaCl2 GDL Starter culture Secondary/adjunct culture Rennet Type Amount COAGULUM

Gel strength subjective/objective assessment - cheese yield - curd composition

Cut

- curd syneresis - curd composition - acidification - retention of coagulant

- Cook - syneresis - curd composition - curd structure - retention of coagulant - solubilization of CCP

Type

- cheese yield - curd composition

CURDS/WHEY

Cheese quality

Composition - casein - fat - calcium - pH - enzymes

- Agitate Acidification -

curd syneresis curd composiiton

- Drain Whey CURDS - Acidification - Dehydration - Texturization? - Salting? - Moulding - Pressing? UNRIPENED/FRESH CHEESE

Rennet Milk enzymes Starter enzymes Secondary culture Adventitious microflora

- Proteolysis - Lipolysis - Glycolysis - Secondary changes

- Salting - Special secondary cultures - Coating/packaging Moisture pH RIPENING NaCl - Composition Fat - Temperature - Humidity - Time

MATURE CHEESE

Flavour

Texture Functional properties Figure 2 Interaction of compositional and technological factors that affect the quality of cheese.

585


Microbiological

Three aspects of the microbiology of cheese milk are of interest/concern: • Public health • Off-flavours and spoilage • Desirable bacteria Public health aspects

As a product of animal origin, milk may become contaminated with pathogenic micro-organisms, and this, clearly, is of great concern. Previously, the principal pathogens of concern in milk were Mycobacterium bovis and Brucella abortus, but in developed dairying countries today, these pathogens have been largely or entirely eliminated from the dairy herd. Today, a wide range of pathogens are of concern, mainly Listeria monocytogenes, enterotoxigenic strains of E. coli, e.g., E. coli O157 H7, Shigella, Erwinia, Campylobacter, Staphlyococcus, Salmonella spp. and M. paratuberculosis. Many of these micro-organisms do not grow in milk which simply acts as a vector. In cheese, these pathogens die off under the rather hostile conditions in well-made cheese which has a relatively low pH (5.3), a relatively high salt content (5–10% salt-inmoisture; S/M) and perhaps bacteriocins. For this reason, Public Health Authorities in many counties require that cheese made from raw milk be aged for 60 days, although this practice may not be fully effective. Alternatively, cheese must be made from pasteurized milk or the cheese itself must be pasteurized, as in processed cheese. Cheese, the pH of which does not decrease at the desired rate or to the desired extent during manufacture (e.g., due to bacteriophage infection or contamination with antibiotics) or if the pH increases substantially during ripening, e.g., surface mould- or smear-ripened cheese, are most at risk. High-moisture, fast-ripening cheeses are at a greater risk of harbouring pathogens than low-moisture, slowripening varieties. A considerable amount of cheese is made from raw milk, especially in France, Germany and southern European countries, but there is increasing pressure in northern Europe and North America to produce all cheese from pasteurized milk. There have been very few reported outbreaks of food poisoning from the consumption of raw milk hard, long-ripened cheese varieties. In all outbreaks for which adequate data are available, mitigating circumstances, e.g., lack of a starter culture or poor starter activity, have also been involved. Clearly, raw milk cheese is safe if adequate precautions are taken. However, it should be remembered that most raw milk cheeses are high-cooked, hard

or extra-hard varieties – many of these cheeses, e.g., Parmigiano Reggiano, Grana Padano, Swiss Emmental and Gruyère de Comté, are cooked at ⬃55 °C; the cooked curds are transferred while hot to moulds with a capacity of 20–60 kg curd and consequently, the curds cool slowly – most of these ‘raw milk’ cheeses are in fact pasteurized, as indicated by a negative alkaline phosphatase test. High-moisture, raw-milk cheeses are of more concern but most of these have a low initial pH (4.6) and appear to be safe. It is probably significant that raw-milk cheeses are made on a small/very small scale from very fresh milk from healthy cows. For further discussion on pathogens in cheese, see ‘Growth and Survival of Microbial Pathogens in Cheese’, Volume 1. It is unlikely that it will be possible to produce raw milk guaranteed free of pathogenic bacteria. However, milk with very low numbers of pathogens can be produced from healthy animals and any pathogens that do enter milk can be: • killed (pasteurization or novel alternatives); • removed (bactofugation or microfiltration (MF); see ‘Application of Membrane Separation Technology to Cheese Production’, Volume 1); • prevented from growing or killed, e.g., by use of low pH, selected bacteriocin-producing starters. To date, efforts to remove pathogens from cheese milk have concentrated on adequate pasteurization. There is ongoing research on alternative methods and it is likely that work in this area will continue and probably expand. Off-flavours and spoilage

A second beneficial effect of pasteurization is the killing of spoilage micro-organisms, e.g., coliforms, pseudomonads and yeasts. In countries with a developed dairy industry, the quality of the milk supply has improved markedly during the past 30 years – total bacterial counts (TBC) are now usually 20 000 cfu/ml ex-farm. The TBC probably increases during transport to and storage at the factory, but growth can be minimized by thermization (65 °C 15 s) of the milk on reception at the factory, which is a standard practice in some countries. The presence of Clostridium tyrobutyricum poses special problems. Although many cheeses are made from raw milk, quantitatively, most cheese is made from milk pasteurized at or close to 72 °C 15 s. If produced from goodquality raw milk and if subsequently handled under hygienic conditions, pasteurized milk should have a very low TBC (a few hundred cfu/ml) and therefore

Factors that Affect the Quality of Cheese 587

represents a very uniform raw material from a microbiological viewpoint. Some alternatives to pasteurization are likely to become industrially significant. Desirable indigenous bacteria

Some of the adventitious micro-organisms in raw milk, especially the non-starter lactic acid bacteria (NSLAB), probably contribute positively to cheese flavour – it is generally accepted that the flavour of raw-milk cheese is more intense, although more variable, than that of pasteurized milk cheese. Although the reason(s) for the differences in flavour between raw and pasteurized milk cheese has not been explained to everybody’s satisfaction and is still under investigation, there is broad support for the view that adventitious NSLAB are mainly responsible. These indigenous LAB are killed by pasteurization; attempts are being made to replace them through the use of adjunct cultures (see also ‘Secondary and Adjunct Cultures’, ‘The Microbiology of Cheese Ripening’ and ‘Raw Mild Cheeses’, Volume 1). While raw milk may be acceptable for cheesemaking in small-scale, manually operated factories, the cheesemaking quality of even good-quality raw milk is too variable to be used successfully in very large, highly automated factories such as those involved in the manufacture of Cheddar, Gouda or Mozzarella (Pizza) cheese. In these cases, it is highly probable that pasteurized milk will continue to be used, with selected adjunct cultures added to simulate the superior quality of raw-milk cheese. Alternatives to pasteurization

There are a number of alternatives to pasteurization for the decontamination of cheese milk: • Thermization – heat treatment at a sub-pasteurization temperature, e.g., 65 °C 15 s; thermization is intended to reduce the microflora of raw milk and extend the period for which it may be held at the factory without a risk of spoilage. Although thermization does not meet the requirements for pasteurization from the public health viewpoint, it is fairly widely used for cheese milk and in combination with other hurdles, e.g., cooking of the cheese curd, low pH, high S/M, is probably adequate to render good-quality milk free of pathogens and foodpoisoning bacteria; • Microfiltration; • Bactofugation, which may be used as a general, quite efficient method for the removal of bacteria and spores from milk but which is not widely used;

• The LPO-H2O2-SCN% system, which is not used commercially for cheese milk; • Addition of H2O2, which is not used in developed dairying countries; • Pre-ripening milk with bacteriocin-producing cultures. Microfiltration is a very efficient method for the removal of bacteria from milk – 99.9% of the bacteria can be removed, i.e., it is more efficient than pasteurization. Microfiltration has the added advantage over pasteurization that no heat damage is caused to the whey proteins. In addition to killing bacteria, MF removes somatic cells, which are significant sources of enzymes and are generally believed to have a negative effect on cheese quality. However, the quality of Cheddar (McSweeney et al., 1993) and Gruyère de Comté (Beuvier et al., 1997) cheese made from pasteurized or MF milk was similar and different from that of raw-milk cheese, suggesting that NSLAB, rather than other factors, the distinguishing factors. At present, MF is not used commercially for the general removal of bacteria from cheese milk and it is not acceptable as an alternative to pasteurization from a public health viewpoint because it cannot guarantee milk free from pathogens. However, MF is provisionally accepted in France as a suitable alternative to HTST pasteurization for the decontamination of beverage milk (see ‘Application of Membrane Separation Technology to Cheese Production’, Volume 1). If no problems are encountered, it is likely that MF will become an acceptable alternative to pasteurization of cheese milk. A serious microbiological problem in many/most cheeses arises from the growth of Clostridium tyrobutyricum in the cheese during ripening which catabolizes lactic acid to butyric acid and H2, with the production of off-flavours and late gas blowing. Cheddar-type cheese is an exception owing to the rapid decrease in pH and the rapid increase in S/M to an inhibitory level. The principal sources of Cl. tyrobutyricum are soil and silage; the feeding of silage to cows, the milk of which is to be used for cheesemaking, is prohibited in Switzerland and parts of France. However, in most countries, the outgrowth of Cl. tyrobutyricum is prevented by the use of NaNO3 or lysozyme or the spores are removed by bactofugation or MF. Indigenous enzymes

Milk contains about 60 indigenous enzymes (see Fox et al., 2003), the significance of which for cheese quality has not yet been researched adequately. Several indigenous enzymes have the potential to affect cheese quality, especially lipoprotein lipase (LPL), proteinase(s), acid


and alkaline phosphatase, xanthine oxidase (XO) and perhaps sulphydryl oxidase (SO), lactoperoxidase and -glutamyl transpeptidase. Some of these enzymes are active in milk prior to cheesemaking and adversely affect the yield and/or quality of cheese. Many of the indigenous milk enzymes survive HTST pasteurization (72 °C 15 s) and at least some, e.g., plasmin, acid phosphatase and XO, are active during cheese ripening. Lipoprotein lipase has the potential to cause significant lipolysis in milk and the resulting fatty acids are concentrated in the cheese curd where they may cause hydrolytic rancidity, especially in mild-flavoured cheese. Normally, LPL has low activity in milk where it is separated from its triglygeride substrates by the milk fat globule membrane (MFGM). However, the MFGM is quite susceptible to damage due to rough handling of milk, leading to activation of the LPL, and rancidity. Plasmin, the principal indigenous proteinase in milk, hydrolyses s1-, s2- and, especially, -casein, producing - and -caseins, some of the proteose peptones (PP) and other peptides. Plasmin activity reduces cheese yield because the PPs are not incorporated into the cheese curd and is reported to damage the quality of the rennet-induced coagulum. Plasmin activity increases with advancing lactation, age of cow and mastitis and its action may result in a weak coagulum with poor syneresis properties – the consequences are reduced yield of cheese and a high moisture content. The formation of -caseins in cheese during ripening clearly indicates that plasmin is active in cheese – it is mainly responsible for the hydrolysis of -casein in lowcooked cheeses and for total primary proteolysis in highcooked varieties in which the rennet is extensively or totally inactivated (see ‘Proteolysis in Cheese during Ripening’, Volume 1,). Studies on the effect of the plasmin inhibitor, 6-amino hexanoic acid, have shown that plasmin makes a significant, but not essential, contribution to proteolysis in Cheddar cheese; addition of exogenous plasmin accelerates proteolysis. A study of the effect of plasmin inhibitors on the ripening of high-cooked cheeses should be interesting. Milk contains at least four times as much plasminogen as plasmin. The indigenous plasminogen may be activated by added plasminogen activators (there are some indigenous plasminogen activators in milk), which accelerate proteolysis in cheese (Barrett et al., 1999). Dephosphorylation by acid phosphatase may be responsible for some of the variability in the level of phosphorylation exhibited by caseins but incomplete phosphorylation may also be responsible. The significance of the variability in the level of phosphorylation in cheese quality is unknown but dephosphorylation of casein-derived peptides in cheese may be significant. It is claimed that alkaline phosphatase is active in Grana

Padano cheese during ripening and is responsible for the dephosphorylation of casein phosphopeptides, which is significant for proteolysis. Alkaline phosphatase is inactivated by HTST pasteurization. Acid phosphatase survives pasteurization and since it is concentrated in the MFGM, it is concentrated in cheese curd. Many of the small water-soluble peptides produced by primary proteolysis are phosphopeptides and are partially dephosphorylated during ripening, either by milk acid phosphatase or by bacterial phosphatases. Since phosphopeptides are resistant to the action of proteinases and peptidases, dephosphorylation by phosphatase action is an important pre-requisite for secondary proteolysis in cheese. However, objective studies on the significance of phosphatase activity in cheese ripening and quality have not been reported. Xanthine oxidase reduces nitrate to nitrite which is needed for anti-clostridial activity. Eventually, all nitrate and nitrite are decomposed to N2, probably by XO. Degradation of nitrate is important since it may react with amino acids to form carcinogenic nitrosamines. Sulphydryl oxidase oxidizes sulphydryl groups to disulphides: 2RSH : R9S9S9R Several small sulphydryl compounds, e.g., H2S, methanethiol, dimethyl sulphide and dimethyl disulphide, are important for cheese flavour. Sulphydryl oxidase oxidizes and protects the sulphydryl groups of proteins and this may affect the redox potential (Eh) of cheese and the stability of thiol compounds and hence the quality and stability of cheese. Somatic cells are an important source of enzymes, especially proteinases, in milk. Somatic cell count (SCC) is negatively correlated with cheese yield and quality; an SCC 300 000 per ml is recommended. As discussed above, the somatic cells in milk can be removed by MF, which should, therefore, improve cheese quality and reduce variability. Although precise information is lacking, it is not likely that indigenous enzymes in milk are a major cause of variability in cheese quality; some of these enzymes contribute to cheese ripening and may contribute to the superior quality of raw milk cheese, a possibility that warrants investigation. Chemical composition

The chemical composition of milk, especially the concentrations of casein, fat, calcium and pH, has a major influence on several aspects of cheese manufacture, especially rennet coagulability, gel strength, curd syneresis, and hence cheese composition and cheese yield. When


seasonal milk production is practised, as in New Zealand, Ireland and Australia, milk composition varies widely but there is some variability even with random calving patterns mainly due to nutritional factors. The constituents of milk are influenced by several factors, including species, breed, individuality, nutritional status, health and stage of lactation of the producing animal. Owing to major compositional abnormalities, milk from cows in the very early or late stages of lactation and those suffering from mastitis should not be used for cheesemaking. Somatic cell (leucocyte) count is a useful index of quality. Some genetic polymorphs of the milk proteins improve cheese yield and quality and there is an increasing interest in breeding for these. The milk should be free of chemical taints and free fatty acids, which cause offflavours in the cheese, and antibiotics which inhibit bacterial cultures. There is considerable information on the effects of protein content, [Ca] and pH on the various renneting parameters of milk in model systems and quite an amount of information on their effects in cheesemaking experiments. However, there is less information on the effects on the simultaneous change in two or more of these factors, especially in actual cheesemaking experiments. Studies on the interactive effects of these and other compositional factors on the cheesemaking properties of milk and on the quality of the resulting cheese are warranted. It is possible to reduce, but not eliminate, the variability in the principal milk constituents by standardizing the concentrations of fat and casein, not just the ratio (protein content can be standardized by adding UF retentate), the pH (using gluconic acid--lactone) and calcium content (by adding CaCl2). Standardization of milk composition Fat and casein

Different cheese varieties have a defined fat-in-dry matter (FDM) content, in effect, a certain fat-to-protein ratio, and this situation has legal status in the ‘Standards of Identity’ for many varieties. While the moisture content of cheese, and hence the levels of fat and protein, is determined mainly by the manufacturing protocol (including size of curd particles, pH, cook temperature, agitation, pressing), the fat to protein ratio is determined mainly by the fat to casein ratio in the cheese milk. Depending on the ratio required, it can be modified by: • removing some fat by gravity creaming, as practised in the manufacture of Parmigiano Reggiano, or by centrifugation; • adding skim milk;

• adding cream; • adding milk powder or ultrafiltration retentate; such additions also increase the total solids content of the milk and hence cheese yield. Calcium

Calcium plays a critical role in the coagulation of milk and in the subsequent processing of the coagulum; it is common practice to add CaCl2 (e.g., 0.01%) to cheese milk, i.e., 40 mg Ca/l milk. This is small in comparison with the indigenous concentration of Ca in milk, 1200 mg/l. Addition of 40 mg/l Ca to milk increases the concentrations of soluble, colloidal and ionized Ca and reduces the pH of milk, all of which have positive effects on the various renneting parameters (see ‘Rennet-induced Coagulation of Milk’ and ‘The Syneresis of Rennet-coagulated Curd’, Volume 1). pH

The pH of milk is a critical factor in cheesemaking (see ‘Rennet-induced Coagulation of Milk’ and ‘The Syneresis of Rennet-coagulated Curd’, Volume 1). The addition of 1–2% starter culture to milk reduces the pH of the milk immediately by about 0.1 unit. Starter concentrates (direct-to-vat starters; DVS), which are now used widely, especially for small and medium factories, have no immediate, direct acidifying effect. Previously, it was standard practice to add the starter to the cheese milk 30–60 min before rennet addition. During this period, the starter began to grow and produce acid, a process, referred to as ‘ripening’, which served a number of functions: • It allowed the starter bacteria to enter the exponential phase of growth and hence to be highly active during cheesemaking; this is not necessary with modern high-quality starters. • The lower pH favoured rennet action and gel formation. However, the practice increases the risk of bacteriophage infection of the starter; phage become distributed throughout liquid milk but after it has coagulated, the phage cannot move through the coagulum and hence can infect only those cells in the immediate vicinity of an infected cell. This practice has been discontinued for most cheese varieties. The pH of milk on reception at the dairy is higher today than it was previously, owing to improved hygiene during milking and the widespread use of refrigeration at the farm and factory. In the absence of acid production by contaminating bacteria, the pH of milk increases slightly during storage due to the loss of CO2 to the atmosphere. The natural pH of milk is ⬃6.7 but varies somewhat (e.g., it increases in late lactation and during mastitic infection).


To off-set these variations in pH and to reduce it as an alternative to ripening, the pre-acidification of milk by 0.1–0.2 pH units is recommended, either through the use of the acidogen, gluconic acid--lactone, or by limited growth of a lactic acid starter, followed by pasteurization (referred to as pre-maturation). Pre-acidification improves the uniformity of rennet-coagulated milk gels, which is reflected in the production of cheese of more uniform quality. Pre-acidification through the growth of a starter culture, which is fairly widespread in France, would appear to pre-dispose the system to the growth of phage, which are not killed by pasteurization, and undesirable bacteria. O

O

C

C

OH

HC

OH

HC HO

C HC

OH H

O

OH

HC CH2OH Gluconic acid-δ-lactone

H2O

HO

C

H

HC

OH

HC

OH

CH2OH Gluconic acid

In addition to variations in gross composition, there are numerous minor differences and variations which are not easily removed or standardized. Some of the more significant of these are due to inter-species differences. Although the vast majority of cheese, worldwide, is produced from cows’ milk, sheep’s and goats’ milk are very significant for cheese in southern Europe and in the Middle East – many world-famous cheeses are made from sheep’s milk, e.g., Manchego, Feta, Roquefort and the Italian Pecorino varieties. Sheep’s milk is used mainly for the production of cheese and yoghurt. Goats’ milk or mixtures of sheep’s and goats’ milk are also used widely for cheese production (see ‘Cheeses Made from Ewes’ and Goats’ Milk’, Volume 2). Buffalo milk is used for the production of cheese in southern Italy (Mozzarella di Buffala) and especially in Egypt. Bovine, ovine, caprine and buffalo milk differ from each other in many respects: concentrations of fat, protein, many inorganic salts, enzymes, fatty acid profile, types and proportions of caseins. These differences cannot be changed and are reflected in the quality of the cheese produced from these milks. The most obvious difference arises from differences in fatty acids – ovine and caprine milk-fat have considerably higher concentrations of hexanoic, octanoic and decanoic acids and branched, medium-chain fatty acids than bovine milkfat and these are readily apparent as differences in the

flavour of the cheese. Ovine and caprine caseins are considerably different from the bovine caseins. It is likely that there are differences in the peptide and amino acid profiles of cheese produced from bovine, ovine, caprine or buffalo milk and that these affect the flavour of the cheeses. A notable example of this is that the rennet from the thistle, Cynara cardunculus, produces very satisfactory cheese from sheep’s milk, e.g., Sera de Estrala (see ‘Cheeses Made from Ewes’ and Goats’ Milk’, Volume 2), but this rennet produces very bitter cheese from cows’ milk. Cows transfer high levels of carotenoids from their feed to their milk or meat whereas sheep, goats and buffalo do not. Consequently cheese, butter and other dairy products produced from cows’ milk are much more yellow than those made from milk of the other species and may be unattractive to certain consumers. The yellow colour can be destroyed by bleaching (H2O2 or benzoyl peroxide) or masked (by chlorophyll or TiO2). The milk of all species contains the same range of enzymes but at different levels; the significance of these differences is not known. Several sapid compounds are transferred from the animal’s feed to its milk and affect the flavour of cheese made therefrom. There is a widely held view that the milk of cows fed on unimproved pasture yields better and more distinctive cheese than that from cows fed a more homogeneous diet. Further work in this area is warranted.

Coagulant (rennet) The key and characteristic step in the manufacture of rennet-coagulated cheeses is the coagulation of milk through the limited proteolytic action of certain proteinases, called rennets. Several proteinases can coagulate milk but only a few are suitable for cheese production. Traditionally, rennets were extracts of the gastric tissue of calves, kids or lambs, in which the principal enzyme is chymosin. Owing to increased production of cheese, concomitant with a reduced supply of calfs’ stomachs, the supply of calf rennet has been inadequate for many years. This led to a search for ‘rennet substitutes’, four of which are commercially successful: bovine pepsin and proteinases from the fungi, R. meihei, R. pusillus and C. parasitica (porcine pepsin was used previously to a limited extent). All successful rennet substitutes are aspartyl (acid) proteinases. The gene for calf chymosin has been cloned in several micro-organisms and the product (referred to as fermentation-produced chymosin; FPC) is now widely used for cheesemaking in many, but not all, countries. The extract of the thistle, Cynara cardunculus,


is used in the manufacture of certain cheeses in Portugal and Spain. The active enzyme is cardosin, which is an acid proteinase (which are rare in plants). Thistle rennet is unsuitable for cheesemaking in general. The mechanism by which chymosin coagulates milk is well established at the molecular level (see Fox and McSweeney, 1997; Fox et al., 2000; Hyslop, 2003; ‘Rennetinduced Coagulation of Milk’, Volume 1). Chymosin specifically hydrolyses -casein, the protein responsible for the stability of the casein micelles, at Phe1059Met106, releasing the hydrophilic C-terminal peptide (referred to as the glyco- or caseino-macropeptide) and destabilizing the micelles. All commercial rennet substitutes hydrolyse the Phe1059Met106 bond except C. parasitica proteinase. which hydrolyses Ser1049Phe105. The rennetaltered micelles coagulate in the presence of Ca2 at a temperature 20 °C (in cheesemaking, at 30–35 °C). It has been proposed (Andreeva et al., 1992; Gustchina et al., 1996) that chymosin normally exists in an inactive conformation but is activated when the substrate binds in the active site cleft of the enzyme. It has been suggested that the sequence —H.P.H.P.H— (residues 98–102 of -casein) is responsible for this conformational change. This sequence occurs in the -casein of cow, goat, sheep and buffalo but not in the -casein of the mare, camel, pig, rat or human, in which the corresponding sequence is ..HPRPH.., ..RPRPR.., ..RPRPH.., ..HPINP.. and ..RPNLH.., respectively (Iametti et al., 2001; Martin et al., 2003). Therefore, one would expect that calf chymosin would not coagulate the milk of the mare, camel, pig, human or rat. There have been few studies on the coagulation of non-bovine milk by calf chymosin. The commercial use of calf rennet in cheesemaking from sheep, goat or buffalo milk indicates that calf chymosin can hydrolyse the -casein in these milks, as expected from the above hypothesis. Calf chymosin can also coagulate porcine milk (Fox, 1975b); in fact, porcine milk is coagulated by calf rennet at 4 °C whereas bovine milk is not, due to the nature of the non-enzymatic secondary phase. Some investigators have reported that camel milk is not coagulated by calf rennet but Farah (1993) reported that it is coagulated slowly to a weak gel. The status of mares’ milk with respect to -casein remained unclear until very recently. Ochirkhuyag et al. (2000) reported that equine milk does not contain -casein and that the micelle-stabilizing function is played by -casein; however, Malacarne et al. (2002) reported that it contains a low level (7%) of -casein which has been isolated and sequenced (Egito et al., 2001, 2002; Iametti et al., 2001). Equine -casein is hydrolysed at Phe979Ile98 (Egito et al., 2001) (which correspond to Phe1059Met106 of bovine -casein). However, equine milk does not appear (Fox, unpublished) to

be coagulated by calf chymosin or Fromase (R. miehei proteinase) at pH 6.6 (normal pH ⬃7.0 or higher). Studies on the coagulability of equine milk by different rennets and under different conditions appears to be warranted. We are not aware of studies on the coagulation of human or rat milk by calf chymosin. Studies on the action of chymosins from various species on the caseins from a range of species would be interesting. For a proteinase to be successful as a rennet substitute, two characteristics are important: • Specific hydrolysis of -casein at or close to Phe1059Met106; if other bonds in any of the caseins are hydrolysed, the resulting peptides may be lost in the whey, causing a reduction in cheese yield. • Its general proteolytic specificity during cheese ripening must be low and similar to that of chymosin (see below). It is generally accepted that calf chymosin produces the best quality cheese. An adequate supply of chymosin from genetically engineered micro-organisms is now available (although its use is not permitted in all countries) and therefore rennet quality should not be a cause of variability in cheese quality. In the presence of Ca2, the rennet-altered micelles in bovine milk coagulate to form a gel at a temperature 20 °C; this is referred to as the secondary phase of rennet coagulation. Renneted bovine milk does not coagulate ⬃18 °C, above which coagulation has a Q10 of 16. The very high temperature dependence of the secondary phase of coagulation has not been explained fully. Presumably, hydrophobic interactions are involved; perhaps the temperature-dependent dissociation of -casein from the casein micelles is a contributory factor. The temperature dependence of the coagulation of rennet-altered micelles is reduced by reducing the pH and increasing the [Ca2] or casein concentration, e.g., by UF. As mentioned above, porcine milk is coagulated by rennet at 4 °C. The reason(s) for the difference between bovine and porcine milk in this regard has not been explained. In spite of many studies on the mechanism of coagulation of rennet-altered casein micelles and kinetics thereof, a generally applicable model of the phenomenon has not been developed. The subject is comprehensively reviewed in ‘Rennet-induced Coagulation of Milk’, Volume 1. Further research on various aspects of the secondary phase of rennet coagulation of, and the effect of low temperatures on, bovine milk and that of other species appear warranted. Some of the added rennet is retained in cheese curd. The amount retained varies with rennet type, cook temperature and pH at draining; these variables


should be standardized if cheese of consistent quality is to be produced. The proportion of rennet retained in the curd is proportional to its moisture content, reflecting the presence of rennet mainly in the aqueous phase of cheese. However, everything else being equal, more chymosin than other rennets is retained in the curd, suggesting that chymosin is adsorbed more strongly on the caseins. The amount of chymosin and pepsin retained in low-cooked cheeses increases strongly as the pH of the curds-whey is reduced. In the case of chymosin, this is due to increased adsorption, for unknown reasons, onto the casein; for pepsin, which is very pH-sensitive (irreversibly denatured at pH 7), greater stability at a lower pH is also a major factor. Surprisingly, pH has no effect on the retention of fungal rennets, a lower proportion of which is retained in the curd than chymosin (see Fox and McSweeney, 1997). Obviously, cook temperature has a major effect on the level of residual rennet in the curd – chymosin and bovine pepsin are extensively or totally denatured in high-cooked cheese, e.g., Parmigiano Reggiano or Emmental; porcine pepsin is extensively denatured even in low-cooked cheeses due to its sensitivity to pH 6.5. Low-cooked, low-pH, highmoisture cheese, e.g., Camembert, retains ⬃30% of the added chymosin activity; Cheddar retains ⬃6% and Emmental ⬃0%. Everything else being equal, increased retention of the coagulant in cheese curd results in greater initial hydrolysis of s1-casein; however, the significance of this variable on the flavour and texture of cheese has not been studied thoroughly. It has been suggested that the activity of chymosin in cheese curd is the limiting factor in cheese ripening; however, excessive rennet activity leads to bitterness. Proteolysis in cheese during ripening is discussed later; there have been relatively few studies on the significance of chymosin activity to cheese quality, an aspect which appears to warrant further research. Considering the importance of proteolysis in the ripening and quality of cheese and the significance of the coagulant thereto, studies on various factors that affect the retention of the coagulant in cheese curd appear warranted, e.g., • the adsorption of chymosin on casein micelles and the apparent lack of adsorption of fungal proteinases; • stability of various rennets under various conditions of temperature, pH and other factors.

Starter The second key reaction in cheesemaking is acidification – the pH of all rennet-coagulated cheeses should decrease to a value in the range 4.6–5.2 within a few

days of manufacture, or in some varieties, at the end of curd manufacture (5–6 h). Acidification at the appropriate rate and time is an essential and characteristic feature of cheesemaking – it is, in fact, a sine qua non. Among the important consequences of acidification are: • activity of the coagulant; • survival and retention of coagulant in the curd; • firmness of the coagulum, which affects the loss of fat and protein in the whey on cutting and hence reduces the yield of cheese; • syneresis of the curds and hence the composition of the cheese; • solubilization of colloidal calcium phosphate (CCP), which has a major effect on the texture, meltability and stretchability of the cheese; • inhibition of the growth of undesirable bacteria, most importantly pathogenic and food poisoning bacteria; • the activity of various enzymes in the cheese during ripening and consequently the rate of ripening and the quality of the cheese. Originally, acidification was due to the production of lactic acid from lactose by adventitious LAB. Acidification of some cheese varieties still depends on the activity of the adventitious microflora but most cheeses now are acidified using selected LAB added to the cheesemilk as a culture (starter). The idea of using starter cultures was introduced in ⬃1870 in Denmark. The cultures used today in cheesemaking can be divided into two groups: 1. Mesophilic – with an optimum growth temperature of ⬃28 °C; 2. Thermophilic – which grow optimally at ⬃42 °C. Mesophilic cultures are used for cheese curds which are cooked at a temperature 40 °C while cheeses in which thermophilic cultures are used are cooked at 50–55 °C. Mesophilic cultures contain strains of Lactococcus lactis subsp. lactis and/or Lc. lactis subsp. cremoris. Starters used for some cheeses, e.g., Gouda, Edam, Danbo, also include citrate-utilising strains of Lc. lactis subsp. lactis and/or Leuconostoc subsp., the principal function of which is the production of CO2 and certain flavour compounds. Thermophilic cultures contain species of thermophilic lactobacilli, e.g., Lb. helveticus and Lb. delbreuckii subsp. bulgaricus or Lb. delbreuckii subsp. lactis, alone or with Streptococcus thermophilus. It is possible to simulate the acid-producing function of the starter LAB by using acid or acidogen (usually GDL). Fresh acid-curd cheese (e.g., Cottage,


Quarg, Cream) of satisfactory quality may be produced by direct acidification and some is produced commercially. Some rennet-coagulated cheeses are also produced by direct acidification, usually when the flavour is very mild or masked by other components or is less important than physico-chemical functional properties; examples include Feta-type and Mozzarella-type cheeses. Chemically acidified Mozzarella has better, more consistent and stable functionality than the biologically acidified product. However, most ripened rennet-coagulated, chemically acidified cheese does not develop a flavour typical of the variety. Possible explanations for this situation are: • the high redox potential (Eh) of chemically acidified cheese (c. 150 mV compared with c. 450 mV for biologically acidified cheese); • a high concentration of lactose which may result in a high count of NSLAB. Both mesophilic and thermophilic cultures may by mixed-strain or defined-strain. Mixed-strain cultures contain an unknown number of strains of the same or different species. Several such systems were in use 20–50 years ago – the cultures were selected based on cheesemaking characteristics and various propagation procedures were used. These cultures were capable of producing very good quality cheese but they were susceptible to phage infection because many of the strains in the culture were sensitive to the same phage. The maintenance and propogation of mixedstrain cultures is laborious and not very reproducible. With the objective of improving resistance to phage, work commenced in New Zealand in the 1930s on the development of defined-strain cultures. The principal criterion for selecting strains for these cultures was phage unrelatedness, i.e., phage which infects one strain does not affect other strains in the culture; while a phage infection may kill off one strain, the other strains grow normally. Other essential criteria are acid-producing ability and strain compatibility; the ability to produce good quality cheese was assessed from experience and undesirable strains were removed from the culture. Originally, blends of 5–6 strains were usually used but blends of 2–3 strains are more common today. The definedstrain approach was introduced initially for Cheddar cheese and is now very widely used in New Zealand, Australia, Ireland and the USA. Although a different approach was used in the Netherlands to select definedstrain cultures for Gouda cheese, the essential outcome was similar. Defined-strain thermophilic cultures are now used also but less widely than mesophilic cultures. The use of defined-strain, phage-unrelated cultures has greatly reduced the risk of phage infection but to

reduce the risk even further, various techniques have been introduced to improve starter activity and ensure cheese quality. For discussions on starter technology, see Cogan and Hill (1993), Cogan and Accolas (1996) and ‘Starter Cultures: General Aspects’, Volume 1. There have been very considerable advances in the genetics of lactococci during the last 10 years and the complete sequence of the genome is known (see ‘Starter Cultures: General Aspects’ and ‘Starter Cultures: Genetics’, Volume 1). The genes for many of the important cheesemaking characteristics of lactococci, e.g., lactose metabolism, proteolysis and phage resistance, are carried on plasmids and hence are easily manipulated. Many genetically engineered strains of Lactococcus have been constructed but are not used in practice. However, lactococci can be genetically modified by natural mating (conjugation) and such genetically modified strains are being used commercially. The principal limitation with engineering superior starters is the lack of knowledge on the key enzymes in cheese ripening. The availability of the complete chromosome sequence of Lc. lactis (see ‘Starter Cultures: General Aspects’ and ‘Starter Cultures: Genetics’, Volume 1) opens up new avenues for research on cheese starter cultures. For example, five potential or rudimentary prophages were identified, suggesting that starter cultures are the ultimate source of phage. Examination of the chromosome for putitive proteinases, peptidases and lipases, especially those with unique activities, should be useful. Since 1999, the chromosome of 20 other LAB and other cheese-related bacteria have been or are being sequenced. These include other strains of Lactococcus, Lb. delbruckii, Lb. helveticus, Sc. thermophilus and B. linens. Comparative genomics of these different bacteria should be useful in delineating the differences that occur between them. However, the gene sequences are of little value unless the protein products (enzymes) are produced. Identifying how to turn on those genes which encode enzymes with potential cheese ripening properties, but which are normally not expressed, could be rewarding in studies on flavour development in cheese. Defined-strain cultures give very reproducible results in terms of acid production and overall cheese quality. However, the flavour of the cheese is considered to be rather bland, probably because of a lack of microbial diversity, both in the starter and also in the modern milk supply, which, if pasteurized, is essentially sterile. Attempts to overcome the lack of flavour will be discussed later. Traditionally, cheese starters were produced at the cheese factory from mother-cultures obtained from a culture supplier; this is still the usual practice in larger factories. However, the proper maintenance of starters is


technically demanding and expensive. Consequently, starter concentrates, referred to as direct-to-vat starter (DVS) or direct vat inoculum (DVI), produced by culture suppliers have become quite widespread among small to medium cheese factories or as a back-up starter system for larger manufacturers. Another starter system warrants mention, i.e., artisanal or natural cultures. These cultures are produced in-house by the cheesemaker, who incubates some warm whey under conditions that select bacteria with desirable cheesemaking characteristics. Today, such cultures are usually used for high-cooked cheeses – hot whey, perhaps at ⬃55 °C, is transferred to insulated containers in which it cools slowly; these conditions are selective for thermophilic bacteria and by the time the whey has cooled sufficiently to allow mesophilic bacteria to grow, the pH has become inhibitory. These cultures are very complex and their composition is not known, certainly at the strain level, and probably not at the species level. Although the primary function of the starter culture is to produce acid at the appropriate rate and time, the starter bacteria or their enzymes also play an essential role during cheese ripening – a typical and desirable flavour does not develop in starter-free cheese and many flavour defects, e.g., bitterness and fruitiness, are related to characteristics of the starter. The microbiology and biochemistry of cheese ripening will be discussed in a later section. In view of the significance of the starter LAB, both for the acidification and ripening of cheese curd, it is not surprising that the starter LAB have been the subject of extensive research since the very early days of microbiology and consequently, LAB are now very well characterized at the cellular, molecular and genetic Cultures: genetics levels (see ‘Starter Cultures: General Aspects’, ‘Starter Cultures: Genetics’ and ‘Starter Cultures: Bacteriophage’, Volume 1). Research on these very industrially important bacteria will very probably continue for the immediate future. Among the areas likely to attract attention are: • Characterization of Lactobacillus and Streptococcus, which at present are less well characterized than Lactococcus; • Further work on the protection of LAB against phage; • Selection and improvement of starter strains, through genetic engineering techniques with respect to acid production and especially ripening attributes; • Selection of LAB strains with probiotic properties for cheese production; • The existing trend towards DVI starters will probably continue; success will depend on economic factors;

• It is likely that the lack of diversity caused by the use of such highly defined starter systems will be offset by the use of adjunct starters, mainly Lactobacillus spp., but possibly also Streptococcus spp., and perhaps Enterococcus, for some varieties.

Post-Coagulation Operations A rennet-coagulated milk gel is quite stable if left undisturbed but if cut or broken, it synereses strongly, thereby creating the possibility of removing water and concentrating fat and protein. When the coagulum has reached the desired degree of firmness, usually 30–60 min after the addition of rennet, the gel is ready for further processing. The firmness of the gel at cutting should be optimized so as to reduce the loss of fat and protein from the curd particles into the whey. If the coagulum is too soft, extensive shattering will occur with high losses of fat and protein in the whey. If the coagulum is too firm, the coagulum may be difficult to cut using the usual equipment; it may run before the cutting knives and shattering may occur. Excessively firm curd is particularly problematic when using UF retentate. A uniform gel firmness at cutting also results in curd particles of more uniform size, leading to a cheese curd of more uniform composition and ultimately in cheese of more uniform quality. Traditionally, the point at which the gel was considered to be ready for cutting was determined subjectively by the cheese-maker but several devices are now available which permit objective assessment of gel firmness (see Fox et al., 2000; ‘General Aspects of Cheese Technology’, Volume 2). Some of these devices can be used to activate the cutting knives. The size of the curd particles affects the extent of syneresis – the coagulum for low-moisture cheeses is cut into small pieces while the gel for high-moisture varieties is cut into large pieces or is not cut at all but is scooped directly into moulds. Fat globules are lost from the cut surfaces; hence, finely cutting the coagulum increases the fat loss. If it were possible to achieve the same degree of syneresis by other means, it may be possible to increase cheese yield by using a larger cut. Contrary to what one might first think, the fat lost from the cut surfaces is mainly in large globules because large globules are more exposed on the cut surface on cutting. Would a very low degree of homogenization improve fat retention? The curd can be sedimented from a rennet-induced milk gel by a low gravitational force. Would it be technically feasible to coagulate milk by some form of centrifuge and to prepare curd of the desired moisture content by centrifuging the coagulum at an appropriate centrifugal force? It might be possible to eliminate


cooking of the curds and all variability associated with syneresis of the curds. Once the gel has been cut, the pieces begin to synerese and expel whey. In addition to the size of the curd pieces, the rate and extent of syneresis are affected by several factors through which the cheesemaker can control the composition of cheese. The syneresis of rennet-induced milk gels is discussed in ‘The Syneresis of Rennet-coagulated Curd’, Volume 1. As discussed below, the composition of cheese has a major influence on the microbiology and biochemistry of cheese ripening and ultimately on the quality of the cheese. Syneresis is promoted by: • • • •

increasing the temperature (cooking); reducing the pH; vigorous stirring during cooking; removing some or most of the whey and continuing to stir.

The relative importance of these factors depends on the variety – syneresis of some varieties depends mainly on cook temperature, e.g., Emmental, Parmigiano Reggiano and Cheddar, while others depend mainly on pH, e.g., Camembert. The cook temperature ranges from ⬃30 °C (no cooking) to 55 °C; the extent of syneresis is related directly to the cook temperature. For reproducible cheese composition and quality, it is critical to control the rate and extent of syneresis. The cook temperature, pH, curd particle size and the extent of agitation are characteristic of the variety and were probably introduced/applied emperically long ago by artisanal cheesemakers. It seems reasonable to suggest that the manufacture of many varieties could be improved by changing the combination of the above parameters; research in this area appears to be warranted. The composition of the curd affects syneresis and this is a major reason for standardizing the composition of the cheese milk. The most important compositional factors are: • Fat, which has a negative effect, i.e., a high fat content causes poor syneresis, because fat is essentially an inert filler in the gel. • Protein, increasing the protein content, up to a certain value, promotes syneresis, but if the protein content is too high, the gel is too firm and synereses poorly. • Calcium, like other aspects of renneting, the extent of syneresis is directly affected by the concentration of calcium. • pH, syneresis is promoted by decreasing pH. The syneresis of rennet-induced milk gels has not yet been fully described at the molecular level. While

the effects of processing parameters (cutting, cooking, stirring, etc.) on syneresis are fairly well described, the effects of compositional factors, such as amount and type of casein, genetic polymorphs, casein micelle structure and size, effect of plasmin and other proteinases, fat globule size and integrity, milk salts, pre-treatments of cheese milk (e.g., heat treatment, high pressure treatment), have not been studied in detail. The deficiency is explained partly by a lack of good and appropriate analytical methods for the measurement of syneresis (see ‘The Syneresis of Rennetcoagulated Curd’, Volume 1). When the moisture content (as assessed subjectively by the cheesemaker based on evaluation of the texture of the curd) and the pH of the curd have reached desired values, the curds are separated from the whey and subjected to one of several treatments designed to regulate the composition, and in some cases the texture, of the curd. These include: • washing of the curd to reduce its lactose content (and thereby control its final pH) and perhaps to increase its moisture content, which occurs on washing with cold water, e.g., Monterey Jack, washedcurd Cheddar, low-fat cheeses; • replacing some of the whey by warm water, e.g., Gouda; this practice was probably used initially as a means of cooking the curds on farms lacking jacketed cheese vats and the ability to generate steam, but it has become a standard method for reducing the lactose content and acidity of the curd for some varieties; • cheddaring the curd, e.g., Cheddar, Mozzarella (Pizza cheese), which affects the texture of the curd but its main effect is probably to allow the pH of the curds to fall and the CCP to dissolve, thereby affecting texture; • kneading and stretching, as used for pasta-filata varieties, mainly to give the cheese a fibrous texture which affects the meltability and stretchability, i.e., the functionality, of the cheese; • moulding, applied to all varieties to give a characteristic shape and size, which are not simply cosmetic but are significant for the ripening of the cheese, e.g., if smear-ripened cheeses are too large, the surface will become over-ripe while the core is still immature; on the other hand, Swiss-type cheeses must be large so as to retain a substantial portion of the CO2 necessary for eye development; some cheeses have a characteristic open texture, with many mechanical openings, e.g., Samsoe, Havarti and blue varieties; in the latter, the openings are necessary for good mould development throughout the cheese; • pressing, applied to semi-hard and hard cheeses with the objective of removing some moisture but mainly


to consolidate the cheese mass which is important for texture and the retention of gas for eye development; • salting, which is discussed further below. In large-scale factories, all the above operations are mechanized and/or automated which improves the consistency of the product if executed properly; however, it is not possible for the cheesemaker to make ad hoc adjustments if the process is not progressing as planned. Very large cheese vats (e.g., 30 000 l capacity) are used in large modern factories producing Cheddar, Gouda or Mozzarella, and possibly other varieties. The use of such large vats eliminates certain causes of variation in cheese but introduces others. About 30 min is required to empty these large vats and separate the curds from the whey; as a result, the curds at the start of draining differ from those at the end in several respects, e.g., moisture content (due to extra syneresis), pH and calcium content. These differences are probably reflected in the quality of the cheese but definitive studies have not been reported.

Salting Salting, one of the classical methods for food preservation, operates by reducing the water activity, aw, of the product. Most, probably all, cheeses are salted by one of four methods: • mixing dry salt with milled or chipped curd, e.g., for Cheddar-type cheese; • brine salting of the moulded/pressed cheese; NaCl diffuses into the cheese in response to the difference in osmotic pressure between the brine and the aqueous phase of the cheese; • surface application of dry salt to the surface of pressed cheese, e.g., Blue cheeses; • salting of cheese milk – for a few varieties, e.g., Domiati, a substantial amount of salt is added to the milk before renneting, traditionally, to control the microflora of the milk. There is a substantial literature on the technology, physics and significance of salting, which has been reviewed by Guinee and Fox (1993), Fox et al. (2000) and in ‘Salt in Cheese: Physical, Chemical and Biological Aspects’, Volume 1. The principal effects of salt in cheese are: • A major inhibitory and selective effect on the microflora. • A significant effect on the activity of many enzymes. • Through its effects on the microflora and enzymes, salt has a major indirect effect on the ripening, flavour and quality of cheese.

• A direct effect on flavour. • An excessive dietary intake of NaCl has several undesirable effects, e.g., hypertension and osteoporosis. Although cheese makes a relatively small contribution to dietary NaCl intake, there is an economic incentive to reduce the NaCl content of cheese, which may adversely affect its quality, or partially replace it by KCl. The significance of salt, and especially the uniformity of salt concentration, on the quality of cheese is well recognized. The physics of salt diffusion in cheese, the effect on various micro-organisms and the technology of salting are well known. Thus, it should be possible to achieve a very reproducible level of salt in cheese. However, this is not always achieved in practice and variations in salt concentration are probably a significant but avoidable cause of variations in cheese quality.

Use of UF in Cheese Production Ultrafiltration (UF) has many applications in cheese technology, as discussed in ‘Application of Membrane Separation Technology to Cheese Production’, Volume 1. Some problems with cheese quality persist but the potential for UF in cheese technology is great and research in its application will continue.

Ripening Fresh rennet-coagulated cheese curd is suitable for consumption and a little is consumed, e.g., Burgos cheese, but most is ripened (matured) for a period ranging from ⬃3 weeks (e.g., Mozzarella/Pizza cheese) to 2 or more years (e.g., Parmigiano-Reggiano, extramature Cheddar). During ripening, the characteristic flavour, texture, appearance and functionality develop along lines pre-determined by the microbiology and composition of the curd, as established during the manufacturing stage. However, the cheesemaker can influence the rate and, to some extent, the pattern of ripening by controlling the temperature and, for some varieties, the humidity of the environment. Many cheeses develop a characteristic microflora (bacteria, yeasts, moulds) during ripening and this microflora has a major effect on the sensory qualities of the cheese. Traditionally, this secondary microflora was adventitious, acquired from the milk and/or environment, and the growth of certain, desirable, contaminating micro-organisms was promoted by selecting certain environmental conditions such as pH, temperature, humidity, oxygen concentration, salt concentration and moisture level. However, the adventitious microflora was likely to be variable, leading to inconsistencies in


cheese quality. In modern cheese technology, the adventitious microflora is replaced by selected secondary cultures, although adventitious micro-organisms may still grow, and even dominate in some cases. The principal secondary micro-organisms are: • Mesophilic lactobacilli, probably in all varieties but especially in internal bacterially ripened varieties, e.g., Cheddar and Gouda. Traditionally, these NSLAB were adventitious, probably variable and uncontrolled; today, it is becoming increasingly common to inoculate cheese milk with selected NSLAB (see below and ‘Secondary and Adjunct Cultures’ and ‘The Microbiology of Cheese Ripening’, Volume 1). • Propionic acid bacteria, characteristic of Swiss-type cheese (see ‘Metabolism of Residual Lactose and of Lactate and Citrate’, Volume 1 and ‘Cheese With Propionic Acid Fermentation’, Volume 2). • Penicillium camemberti and P. roqueforti in surfacemould and blue-mould varieties, respectively. The inoculation of some mould-ripened cheeses with mould spores is adventitious but increasingly, the cheeses are inoculated with selected strains. The metabolic activity of the mould dominates the ripening, and hence the quality, of these cheeses (see ‘Surface Mould-ripened Cheeses’ and ‘Blue Cheese’, Volume 2). Therefore, ensuring the optimum growth of the mould is paramount. • Coryneform bacteria, e.g., Brevibacterium, Arthrobacter and Corynebacterium spp. are the characteristic microflora of surface smear-ripened cheeses and are responsible for their characteristic appearance, aroma and taste. Traditionally, surface smear-ripened cheeses acquired their secondary microflora from the environment and from older cheeses via smearing. However, for hygienic and consistency reasons, it is becoming increasingly common to inoculate the surface of the cheeses with selected coryneform bacteria (see ‘Secondary and Adjunct Cultures’, Volume 1 and ‘Bacterial Surface-ripened Cheeses’, Volume 2). Several species of yeast, e.g., Debaryomycos hansenii and Yarrowia lipolytica, have been isolated from cheese. These yeasts are adventitious contaminants on many varieties; since they are aerobic and acid-tolerant, they grow mainly on the surface of all cheeses but their growth on many varieties is prevented through packaging or rind formation. The growth of yeasts is essential on surface smear-ripened cheeses because they catabolize lactic acid, increase the pH of the curd and enable the corynebacteria, which cannot grow at pH 5.8, to grow. Apart from their significance in the deacidification of smear-ripened cheese, their precise contribution to ripening has not been quantified. However, since yeasts are metabolically active, it is likely that their

contribution is considerable. With the objective of improving the consistency of cheeses in which they are a significant part of the microflora, the inoculation of such cheeses with selected strains of yeasts is becoming increasingly common. Geotricum candidum is part of the adventitious surface microflora of many cheeses. A further variable through which the cheesemaker can influence the pattern of ripening and the quality of the final cheese is by preventing the loss of moisture from the cheese surface by appropriate packaging (rindless cheese) or by controlling its loss to form a rind. Scientific work on the significance of cheese packaging on cheese quality is lacking. The main focus has been on the prevention of mould growth on the surface and the loss of cheese yield. Undoubtedly, the changing composition of cheese (through evaporation of moisture) and the loss of gases and probably other volatile compounds affect cheese microflora and enzyme activity and consequently cheese quality. Although the technological advantages accruing from the packaging of cheese are great and perhaps cannot be off-set by other factors, a scientific comparison of various aspects of rindless and rinded cheese, e.g., Cheddar, may be interesting. Cheese ripening involves a very complex set of biological, biochemical and chemical reactions which can be classified into four groups: • Glycolysis and the catabolism of lactic and citric acids. • Lipolysis and the catabolism and modification of fatty acids. • Proteolysis and catabolism of amino acids. • Interactions between the products of the previous reactions. These reactions are catalysed by living micro-organisms or enzymes derived from four or five sources: • • • • •

Milk Coagulant Primary starter Secondary starter (for most varieties) Adventitious microflora

The biochemistry of cheese ripening has been studied quite intensely in recent years and an extensive literature has accumulated, which has been the subject of several reviews, including Fox and McSweeney (1997), Fox et al. (1996a, 2000), Fox and Wallace (1997), and ‘Biochemistry of Cheese Ripening: Introduction and Overview’, ‘Metabolism of Residual Lactose and of Lactate and Citrate’, ‘Lipolysis and Catabolism of Fatty Acids in Cheese’, ‘Proteolysis in Cheese during Ripening’, ‘Catabolism of Amino Acids in Cheese During Ripening’, Volume 1. The principal features of glycolysis, lipolysis and proteolysis are summarized in Figs 3, 4 and 5, respectively. The principal


Lactose Sc. thermophilus

Glu. + Gal Lc. lactis Lc. cremoris

Lactobacillus Sc. thermophilus D,L-Lactate

Swiss L-Lactate

Propionibacterium Mould and Surface smear

Propionate, acetate, CO2

Cheddar Dutch

CO2, H2O NSLAB D,L-Lactate

Pediococcus (lactobacilli)

Acetate

Figure 3 Summary of lactose metabolism in cheese.

products of the various reactions have been characterized and include numerous large, medium and small peptides, amino acids, amines, ammonia, fatty acids and partial glycerides, other organic acids, aldehydes and ketones, thiol compounds, CO2, H2, hydrocarbons, pyrazines and furanones. Several hundred sapid and aromatic compounds have been isolated from several cheese varieties and identified (see ‘Sensory Character of Cheese and its Evaluation’ and ‘Instrumental Techniques’, Volume 1). The spectrum of these compounds is generally similar between the varieties that have been studied in detail but the varieties differ with respect to their concentration and proportions. There are four aspects to the quality of cheese; the relative importance of these depends on the variety and application of the cheese: • • • •

Appearance Flavour (aroma and taste) Texture Functionality The appearance of cheese includes such features as:

• Depth and uniformity of colour • Presence or absence of mechanical opening or eyes due to gas production • Presence or absence of mould

Triglycerides δ-Keto acid δ-Hydroxyacids

n-Fatty acids CH3SH

Alcohols Lactones

Methyl ketones

Thioesters Esters

Figure 4 Summary of lipolysis in cheese.

Usually, the appearance of cheese is the only attribute by which the purchaser can assess the quality of cheese and hence is of the utmost importance. Today, it is unlikely that cheese produced by large manufacturers and sold through reputable outlets will be defective in appearance although the appearance of artisanal cheese may vary. For cheese consumed as a table cheese, flavour is probably its most important quality attribute although flavour and texture are strongly interactive. The flavour of cheese is due to a subtle balance between several hundred compounds. It has been the subject of research since the beginning of the twentieth century, especially since the development of gas chromatography (GC) in the 1950s and the interfacing


Casein

Rennet

Ca-para-casein

Rennet, Plasmin

Large peptides Lactococcal CEP Oligopeptidases Aminopeptidases Small peptides Aminopeptidases Dipeptidases

Deaminases

Carbonyls Acids

Alcohols

in as es

C

Tr an sa m

Esters

Amino acids

CO2

Decarboxylase

Amines α-Keto acid

C and C Lyases

S

Various products, including sulphur compounds

Amino acid α-Keto acid

Figure 5 Summary of proteolysis and amino acid catabolism in cheese.

of GC with mass spectrometry (MS). Considerable progress has been made on the characterization of cheese flavour by instrumental methods (see ‘Instrumental Techniques’, Volume 1). However, commercially, cheese quality is usually assessed by subjective sensory evaluation (see ‘Sensory Character of Cheese and its Evaluation’, Volume 1). It is still not possible to describe completely the flavour of cheese, especially more highly flavoured varieties. More research is needed in this area and it is very likely that it will continue. An objective method for grading cheese would be very useful. Analysis of cheese volatiles by GC–MS is probably the best approach at present but the present instruments are not capable of handling large numbers of samples and are too expensive. The electronic nose appears promising but considerably more work is required. The texture of cheese is important, both directly and indirectly. It is important directly because such important functional attributes as sliceability, grateability, crumbliness and eye development are, in fact, related to texture. To the consumer, texture is an indirect index of cheese flavour and general quality. In spite of its undeniable importance, the texture of cheese has received much less research attention than cheese flavour although as described in ‘Rheology and Texture of Cheese’, Volume 1, the texture of cheese can be described quite accurately by certain rheological parameters. The influence of various compositional parameters and the changes that occur during ripening have been described in rheological terms. It should be possible to develop some of the present rheometers based on the principle of compression, penetration or cutting to take whole cheese

samples and hence could be used in a factory environment – it would appear that research in this area is warranted. Further work on the molecular basis of cheese texture and rheological properties is also required. All cheeses are expected to exhibit certain physicochemical or functional properties when cold, e.g., sliceability or crumbliness or grateability or when heated, e.g., meltability or stretchability. Most cheese (⬃70% in the USA) is used as an ingredient in other foods, either domestically or in a factory context. The flexibility and ease of use of cheese as a food component or ingredient are among its main attributes and are discussed in detail in ‘Cheese as an Ingredient’, Volume 2. Being able to provide the user, especially the industrial user, of cheese with a product with the correct functionality is a challenge to the cheese manufacturer. Considerable progress has been made but further work is required. The quality of cheese is determined in the first instance by the composition of the curd both directly and indirectly by its influence on the various ripening agents. The significance of principal ripening agents is described in the following sections.

Indigenous Enzymes Milk contains 60 indigenous enzymes (see Fox et al., 2003). Some of these enzymes, including LPL, acid phosphatase, alkaline phosphatase and XO, are located on the fat globule membrane, some, including plasmin, are adsorbed on the casein micelles while others are in the serum (whey) phase. Since the fat globules and casein micelles are concentrated in the cheese


curd, cheese is enriched with many enzymes. Several enzymes in milk are quite heat stable and survive HTST pasteurization. The most significant of the indigenous enzymes as far as cheese ripening is concerned are: • Plasmin, which fully survives HTST pasteurization (in fact, its activity is increased due to inactivation of inhibitors of plasmin and plasminogen activators) and makes a significant contribution to primary proteolysis, especially in high-cooked cheeses in which the coagulant is extensively or totally inactivated; it is mainly responsible for the hydrolysis of -casein in most cheese varieties. Although the level of plasmin activity in milk is variable, it is unlikely that this variability causes significant variations in cheese quality although it may affect cheese yield and composition (through retarded syneresis) and functionality which is strongly influenced by the integrity of the casein network. • Cathepsin D, an acid lysozomal proteinase with a specificity similar to chymosin, occurs mainly in the serum phase of milk and therefore most of it is lost in the whey; furthermore, it is relatively heat labile. Therefore, there is probably little cathepsin D activity in cheese and, in any case, it is probably overshadowed by the much greater activity of chymosin. • Lipoprotein lipase, which is extensively or totally inactivated by HTST pasteurization, has little or no impact on the quality of cheese made from pasteurized milk although it probably contributes to lipolysis in raw-milk cheese; it is associated with the casein micelles and is incorporated into cheese curd. • Acid phosphatase is probably active in cheese but its significance has not been established. Many of the small peptides produced from casein by chymosin or plasmin are phosphopeptides and are partially dephosphorylated during ripening, indicating the action of an acid phosphatase. Work is needed to establish the contributions and significance of the indigenous and bacterial acid phosphatases in cheeses. One of the important nutritional features of cheese is its anticariogenic property due to the Ca-binding properties of the caseins and casein phosphopeptides (see ‘Nutritional Aspects of Cheese’, Volume 1). In this regard, the activity of acid phosphatase may be negative but dephosphorylation is necessary to enable the further degradation of phosphopeptides which may be important for flavour and texture development. • Xanthine oxidase reduces nitrate to nitrite, which is the active agent against clostridia and coliforms in cheese and contributes to the eventual disappearance of NO3 and NO2 : this is important from a toxicological viewpoint since nitrate may lead to the formation of nitrosamines.

It is possible that other indigenous milk enzymes are active in cheese during ripening and affect its quality; their activity may contribute to the differences in flavour between raw and pasteurized milk cheese but definitive studies have not been reported. Research on the significance of indigenous milk enzymes in cheese quality is warranted.

Coagulant The coagulant is mainly responsible for primary proteolysis in low-cooked cheese and for the desirable textural changes during the early stages of ripening. The peptides normally produced by rennet are too large to affect flavour but they serve as substrates for microbial proteinases and peptidases which produce small peptides and amino acids which contribute to background flavour. Amino acids serve as substrates for various catabolic reactions, the products of which (amines, NH3, acids, carbonyls, sulphur compounds) are major sapid compounds in cheese (see ‘Proteolysis in Cheese during Ripening’ and ‘Catabolism of Amino Acids in Cheese During Ripening’, Volume 1). However, excessive rennet action or incorrect specificity may lead to bitterness. The C-terminal region of -casein is very hydrophobic and peptides released from this region, which contains the primary chymosin cleavage sites on -casein, are very bitter. The hydrolysis of -casein by chymosin is inhibited by NaCl and it is not normally hydrolysed in Cheddar but is hydrolysed to some extent in surface-salted cheese, in which the concentration of NaCl is sub-inhibitory for a considerable period. Chymosin is generally regarded as the best rennet; since there is now an unlimited supply of fermentationproduced chymosin, there is no excuse for rennetrelated problems in cheese. The natural function of chymosin is to coagulate milk in the stomach of the neonate, delay its discharge into the small intestine and thereby increase the efficiency of digestion. Chymosin is the most efficient milk coagulant known but it was not intended for cheesemaking although it is the best for this task also. However, it is likely that through genetic engineering, chymosin could be modified to improve its cheese-ripening properties, i.e., to increase its action on certain peptide bonds to yield desirable peptides or reduce it on other bonds to avoid defects such as bitterness. Some mutant chymosins have been produced (see ‘Rennets: General and Molecular Aspects’, Volume 1) but we are not aware of their use in cheesemaking trials. C. parasitica proteinase is much more active on -casein in cheese than chymosin, pepsins or Rhizomucor proteinases but it does not cause bitterness. Probably, it


preferentially hydrolyses in the N-terminal region of -casein, which is hydrophilic. Characterization of the specificity of C. parasitica proteinase on caseins in cheese and in model systems is warranted. Perhaps a combination of chymosin and C. parasitica proteinase might produce cheese with interesting characteristics. The texture and functionality of cheese are affected strongly by even a low level of proteolysis, e.g., the stretchability of biologically acidified Mozzarella deteriorates after ⬃2 weeks at 4 °C due to proteolysis. Thus, it is important to control the level and activity of rennet in cheese. As discussed earlier, the amount of rennet retained in cheese curd is affected by the moisture content of the cheese, the pH and temperature of cooking; activity during ripening is affected by pH, moisture, S/M and temperature.

Starter In addition to its essential role in the production of acid in the manufacture of cheese curd, the starter LAB also play a key role in cheese ripening. Experiments with chemically acidified Cheddar and Gouda cheese have shown that the starter is essential for normal flavour development. Even inoculation of chemically acidified cheese with NSLAB, which reached 108 cfu/g, did not produce good-flavoured cheese (Lynch et al., 1997). The starter LAB reduce the redox potential (Eh) of cheese curd to about 250 mV and this may be of major significance in flavour development. There is very little information on the development and significance of Eh in cheese, possibly because it is very difficult to measure the Eh of cheese accurately; research in this area would appear to be highly desirable. The precise route and mechanism for flavour generation by the starter have not been elucidated fully but considerable progress has been made (see Fox and Wallace, 1997; McSweeney and Sousa, 2000; Yvon and Rijen, 2001; ‘Biochemistry of Cheese Ripening: Introduction and Overview’, ‘Metabolism of Residual Lactose and of Lactate and Citrate’, ‘Lipolysis and Catabolism of Fatty Acids in Cheese’, ‘Proteolysis in Cheese during Ripening’ and ‘Catabolism of Amino Acids in Cheese During Ripening’, Volume 1, and Volume 2, on the principal families of cheese). The growth of the starter ceases at the end of the curd manufacturing stage and the cells die at a rate characteristic of the strain. Therefore, it seems reasonable to conclude that starter enzymes rather than viable cells are involved in ripening and that differences in the enzyme profile of starter strains affect cheese quality. Modern defined-strain starters produce acid very reproducibly and, if properly selected and managed, show good resistance to phage. Lactococcus strains

have been selected mainly on the basis of acid-producing ability, phage resistance and compatibility. Based on pilot-scale studies and commercial experience, strains that produce unsatisfactory, especially bitter, cheese have been identified and excluded from commercial usage. However, there are substantial and recognizable differences in flavour quality and intensity between cheeses made using different defined-strain cultures, which presumably reflect differences in the enzyme profile of the component strains; systematic studies on strains are lacking. This probably reflects the lack of information on precisely what attributes of a starter are desirable from a flavour-generating viewpoint. Studies on genetically engineered strains that superproduce proteinase and/or the general aminopeptidase, PepN, showed that, although proteolysis was accelerated, cheese quality was not improved. Lactococcus strains lacking one or more peptidases in various combinations are available; mutants lacking any one or two peptidases can grow in milk but strains lacking three or more peptidases cannot. Published studies on the use of these peptidase-deficient mutants in cheese are lacking. Since all lactococcal enzymes, except the cell wallassociated proteinase, are intracellular and since the cells do not grow in cheese, the cells must lyse before these enzymes can participate in ripening; therefore, the rate of lysis of Lactococcus strains is being studied with the objective of selecting strains with improved cheesemaking properties. A bacteriocin-producing strain of Lactococcus has been isolated which accelerates lysis of the starter and consequently accelerates cheese ripening (Morgan et al., 1995). The enzymes of the glycolytic and proteolytic systems of Lactococcus are very well studied at the molecular, biochemical and genetic levels (see ‘Biochemistry of Cheese Ripening: Introduction and Overview’, ‘Metabolism of Residual Lactose and of Lactate and Citrate’, ‘Lipolysis and Catabolism of Fatty Acids in Cheese’, ‘Proteolysis in Cheese during Ripening’ and ‘Catabolism of Amino Acids in Cheese During Ripening’, Volume 1). Although less thoroughly studied, the phosphatase and lipase/esterase of a few strains have been isolated and characterized. During the past few years, there has been increasing awareness of the significance of the amino acid-catabolizing enzymes of the starter and a number of reports on lactococcal deaminases, transaminases, decarboxylases and lyases have been published (see McSweeney and Sousa, 2000; Yvon and Rijnen 2001; ‘Catabolism of Amino Acids in Cheese During Ripening’, Volume 1). It is very likely that work on the lipolytic and amino acid-catabolizing enzymes of Lactococcus and their significance in cheese ripening and quality will be intensified in the immediate future.


The enzymes of Lactobacillus and Streptococcus strains have been studied less thoroughly than those of Lactococcus but the systems of the three genera appear to be generally similar. Thermophilic lactobacilli lyse very rapidly and are more proteolytic than lactococci. Although considerable information is available on the individual enzymes of Lactococcus and to lesser extent of Lactobacillus, especially on the glycolytic and proteolytic systems, there have been few comparative studies on the different enzyme activities in starter strains. There have been even fewer studies on the relationship between different starter enzyme profiles and cheese quality. It would appear to be highly desirable that studies should be undertaken to relate cheese quality to the enzyme profile of natural starter strains or genetically engineered cultures. The availability of starter strains deficient in or over-producing one or more enzymes will facilitate such studies. It is very likely that the desirable cheesemaking properties of starters are due to a balance between certain, perhaps secondary, enzymatic activities, which have not yet been identified.

Non-Starter Lactic Acid Bacteria In addition to starter bacteria, cheese curd contains adventitious bacteria acquired from the milk and environment. When raw milk was used widely, it was probably the principal source of bacteria in cheese curd, especially since it was heavily contaminated during milking and was not cooled; counts 106 cfu/ml were common and 90% of the bacteria in milk are retained in the cheese curd. However, in the modern dairy industry, the microbial quality of the raw milk is very high and the milk is usually pasteurized; typically, the milk entering the cheese vat contains 1000 cfu/ml. In large factories, the cheese is made in enclosed vats, with very little contamination from the environment. In all ripened cheeses, a NSLAB flora, which varies within and between cheeses made in the same plant, develops (see ‘The Microbiology of Cheese Ripening’, Volume 1). Cheese is quite a hostile environment due to: • • • • •

a low pH a relatively high S/M anaerobic conditions lack of a fermentable carbohydrate the presence of bacteriocins and other inhibitory substances produced by the starter.

Hence, relatively few species of bacteria can grow, or even survive, in the centre of a well-made cheese. Recent studies have shown that the non-starter

microflora of Cheddar cheese is dominated by mesophilic lactobacilli, especially Lb. casei, Lb. paracasei, Lb. plantarum and Lb. curvatus. In cheese produced from good quality pasteurized milk in a modern plant these NSLAB typically grow from a few hundred per gram at the end of manufacture to 107–108/g within 2–3 months. Thus, while the population of starter LAB declines, the number of NSLAB increases and dominates the viable microflora of long-ripened cheese after 2–3 months (see ‘The Microbiology of Cheese Ripening’, Volume 1). Although less well studied that Cheddar, the NSLAB in Gouda, Emmental and Grana-type cheeses are also predominantly mesophilic lactobacilli and this is probably the normal situation in long-ripened cheeses. The significance of NSLAB for Cheddar and Dutch cheese quality is controversial. Many researchers consider their contribution to be negative (in the Netherlands, a maximum of 2 106 NSLAB/g is specified for Gouda). Although there are several studies on cheese with a controlled microflora, the ripening and quality of NSLAB-free cheese do not appear to have been compared with ‘control’ cheeses containing ‘wild’ NSLAB. Several comparative studies on cheese made under aseptic or non-aseptic conditions using Lactococcus starter alone or with selected Lactobacillus adjuncts indicate that inoculation of cheesemilk with selected strains of Lactobacillus improves cheese flavour and possibly accelerates ripening. However, a typical but mild flavour develops in Cheddar, Gouda and Emmental free of NSLAB, i.e., NSLAB do not appear to be essential for cheese ripening although they do affect the ripening pattern and cheese quality. Since the numbers and strains of NSLAB in cheese are uncontrolled, it is likely that they contribute to variability in cheese quality. Therefore, it appears worthwhile to determine what factors affect their growth. The number of NSLAB in Cheddar is strongly influenced by the rate at which the curd is cooled and subsequently ripened. Rapid cooling of the curd after moulding and pressing is the most effective way of retarding the growth of NSLAB, although they will grow eventually to ⬃107 cfu/g in cheese ripened at 4 °C. The growth of NSLAB can be prevented by ripening at ⬃1 °C but all ripening reactions are retarded to an unacceptably slow rate. The growth of NSLAB does not appear to be influenced by the composition of cheese (moisture, salt or pH) within the ranges normally found in commercial cheese. Non-starter lactic acid bacteria grow mainly after the lactose in the cheese curd has been metabolized by residual starter activity. Although the growth substrates in cheese for Lactobacillus are not known, they


can derive energy from the sugars of glycoproteins of the MFGM (Diggin, 1999). It is likely that available, suitable substrates are limiting (NSLAB normally plateau at ⬃107–108 cfu/g) and hence it might be possible to out-compete wild NSLAB by adding selected strains of Lactobacillus to cheese milk, thereby offering better control of the ripening process. Non-starter lactic acid bacteria may also be controlled by including a broad spectrum bacteriocin-producing strain in the starter culture (Fenlon et al., 1999).

Lactobacillus Species as Adjunct Cultures Cheddar and Cheddar-type cheeses do not have an intentional secondary microflora but there has been considerable interest in recent years in the use of an adjunct secondary culture (usually mesophilic lactobacilli) for the following reasons: • to intensify cheese flavour which is considered to have become too mild owing to the improved microbial quality of the cheese milk, pasteurization of the milk, the use of enclosed vats and other equipment (which reduce contamination from the environment) and the use of defined-strain starters, i.e., the cheese microflora have become too narrow; • to accelerate cheese ripening; the ripening of cheese, especially low-moisture, highly flavoured varieties, is a slow, and consequently an expensive, process. Various approaches to accelerate ripening have been assessed, including the use of mesophilic lactobacilli (see Fox et al., 1996b); • to give identifiable flavour characteristics to cheese produced by a particular manufacturer or sold by a particular retailer; • to improve the flavour of reduced-fat cheese, which generally lacks flavour; • inoculation of cheese with mesophilic lactobacilli which suppresses the growth of adventitious lacto-

bacilli (NSLAB). Since the NSLAB are uncontrolled (they are the only really uncontrolled component of cheese), they probably contribute at least to some extent to variability. In this case, the adjunct lactobacilli need not contribute to the biochemistry of ripening, just suppress the growth of adventitious NSLAB. A considerable amount of research on the significance of mesophilic lactobacilli in cheese has been reported during the past 10 years and the results seem promising. Further research in this area is warranted. Thermophilic Lactobacillus spp. are more effective as adjuncts than mesophilic lactobacilli (Tobin, 1999), probably because they die rapidly in cheese, lyse and release intracellular enzymes. Both mesophilic and thermophilic lactobacilli and Sc. thermophilus are being used commercially as adjunct cultures for Cheddar cheese, and possibly for other varieties. Sc. thermophilus is used mainly to improve the phage resistance of the culture (since it is resistant to lactococcal phage) and to permit the use of a higher cook temperature, facilitating better control over cheese composition and hence ripening and quality.

Cheese Composition The quality of cheese is influenced by its composition, especially moisture content, NaCl concentration (preferably expressed as % S/M), pH, moisture-in-non-fat substances (MNFS; essentially the ratio of protein to moisture) and % fat-in-dry matter. At least five studies (O’Connor, 1971; Gilles and Lawrence, 1973; Fox, 1975a; Pearce and Gilles, 1979; Lelievre and Gilles, 1982) have attempted to relate the quality of Cheddar cheese to its composition. These authors agree that moisture content, % S/M and pH are the key determinants of cheese quality but they disagree as to the relative importance of these parameters (see Fig. 6).

FDM 52–55%

pH 4.85–5.20

pH 4.95–5.10 FDM 52–55%

Salt > 1.4%

pH 4.95–5.15

Premium quality

Premium quality

Premium quality

MNFS 52–56% MNFS 50–57%

S/M 4.0–6.0% S/M 4.0–6.0%

Gilles and Lawrence (1973) Composition of cheeses was determined at 14 days and related to quality of mature Cheddar cheese.

Moisture < 38%

pH < 5.4

Fox (1975a) Relationship between the quality and composition of 10-week-old Cheddar cheese.

MNFS 52–54%

S/M 4.2–5.2%

Pearce and Gilles (1979) Composition of cheeses was determined at 14 days and related to quality of Cheddar cheese.

Figure 6 Relationships between composition (determined at various stages during ripening) and the quality of mature Cheddar cheese (moisture-in-non-fat substances (MNFS); fat-in-dry-matter (FDM), and salt-in-moisture (S/M)).


O’Connor (1971) found that the flavour, texture and total score of Cheddar were significantly correlated with % NaCl and particularly with pH; moisture content had less effect on cheese quality. Salt content and pH were strongly correlated with each other, as were salt and moisture. Based on the results of a study on experimental and commercial cheeses in New Zealand, Gilles and Lawrence (1973) proposed a grading (selection) scheme which has since been applied commercially in New Zealand to young (14 day) Cheddar cheese. The standards prescribed for Premium grade cheese were: pH: 4.95–5.10; % S/M: 4.0–6.0%; MNFS: 52–56%; FDM: 52–55%. The corresponding values for First Grade cheeses were: 4.85–5.20%, 2.5–6%, 50–57% and 50–56%; young cheeses with a composition outside these ranges were considered unlikely to yield goodquality mature cheese. Quite wide ranges of FDM are acceptable; Lawrence and Gilles (1980) suggested that since relatively little lipolysis occurs in Cheddar cheese, fat content plays a minor role in determining cheese quality but if FDM is below about 48%, the cheese is noticeably more firm and less attractive in flavour. Pearce and Gilles (1979) reported that the grade of young (14-day-old) cheeses produced at the New Zealand Dairy Research Institute was most highly correlated with moisture content; the optimum compositional ranges were: MNFS: 52–54%; S/M: 4.2–5.2%; pH: 4.95–5.15. Fox (1975a,b) reported a weak correlation between grade and moisture, salt and pH for Irish Cheddar cheeses but a high percentage of cheeses with compositional extremes was downgraded, especially those with low salt (1.4%), high moisture (38%) or high pH (5.4). Salt concentration seemed to exercise the strongest influence on cheese quality and the lowest percentage of down-graded cheeses can be expected in the salt range 1.6–1.8% (S/M: 4.0–4.9%); apart from the upper extremes, pH and moisture had little influence on quality in the sample studied. High salt levels tend to cause a curdy texture, probably due to insufficient proteolysis; a pasty body, often accompanied by off-flavours, is associated with low salt and high moisture levels. In the same study, the composition of extra-mature Cheddar cheeses was found to vary less and the mean moisture content was 1% lower than that of regular cheeses. A very extensive study of the relationship between the composition and quality of nearly 10 000 cheeses produced at five commercial New Zealand factories was reported by Lelievre and Gilles (1982). As in previous studies, considerable compositional variation was evident but was less for some factories than others. While the precise relationship between quality

and composition varied between plants, certain generalizations emerged: • within the compositional range suggested by Gilles and Lawrence (1973) for ‘premium’ quality cheese, composition does not have a decisive influence on grade, which decreases outside this range; • composition alone does not provide an exclusive basis for grading; • MNFS was again found to be the principal factor affecting quality; • within the recommended compositional bands, grades declined marginally as MNFS increased from 51 to 55% and increased slightly as S/M decreased from 6 to 4; pH had no consistent effect within the range 4.9–5.2 and FDM had no influence in the range 50–57%. • there were specific intra-plant relationships between grade and composition; therefore, each plant should determine the optimum compositional parameters pertinent to it. The results of the foregoing investigations indicate that high values for moisture and pH and a low salt content lead to flavour and textural defects. The desired ranges suggested by Gilles and Lawrence (1973) appear to be reasonable, at least for New Zealand conditions, but within the prescribed zones, composition is not a good predictor of Cheddar cheese quality. Presumably, several other factors, e.g., starter, NSLAB, activity of indigenous milk enzymes, relatively small variations in cheese composition and probably other unknown factors, influence cheese quality but become dominant only under conditions where the principal determinants, moisture, salt and pH, are within appropriate limits. Although the role of calcium concentration in cheese quality has received occasional mention, its significance has been largely overlooked. Lawrence and Gilles (1980) pointed out that the concentration of calcium in cheese curd determines the cheese matrix and, together with pH, indicates whether proper procedures were used to manufacture a specific cheese variety. As the pH decreases during cheese manufacture, CCP dissolves and is removed in the whey. The whey removed after cooking comprises 90–95% of the total whey expressed during cheesemaking and under normal conditions contains ⬃85% of the calcium and ⬃90% of the phosphorus lost from the cheese curd. Thus, the calcium content of cheese reflects the pH of the curd at whey drainage; there are strong correlations between the calcium content of cheese and the pH at 1 or 14 days and the amount of starter used (see Lawrence et al., 1984). Since the pH of cheese increases during ripening, the pH of mature


cheese may be a poor index of the pH of the young cheese. Therefore, calcium concentration is probably a better record of the history of a cheese with respect to the rate of acidification than the final pH. Reduction in calcium phosphate concentration by excessively rapid acid development also reduces the buffering capacity of cheese and hence the pH of the curd will fall to a lower value for any particular level of acid production. No recent work on the level and significance of calcium in Cheddar cheese appears to be available. The calcium content of cheese has a major effect on its meltability and stretchability, e.g., pasta-filata cheese does not stretch well, or not at all, until the pH falls below ⬃5.4. Biologically acidified Mozzarella has poor stretchability and meltability immediately after manufacture but these properties improve during the early stages of ripening and are optimal after about 2–3 weeks; functionality deteriorates on continued ripening due to proteolysis. In contrast, directly acidified cheese is functional immediately after manufacture. The difference in behaviour is due to the lower calcium concentration in the directly acidified cheese owing to the faster decline in pH to ⬃5.6. Under such conditions, much of the CCP dissolves and is removed in the whey at drainage; the concentration of calcium per unit of protein, which is very important for cheese functionality, in biologically and chemically acidified Mozzarella cheese was 27.7 and 21.8 mg/g, respectively (Guinee et al., 2002). There is little published information on the relationships between composition and quality for other cheese varieties. However, it is very likely that similar factors affect the quality of all cheeses more or less to the same extent.

Ripening Temperature Ripening temperature has a major influence on the rate of ripening and quality of cheese. Traditionally, cheese was ripened in caves or cellars at a relatively constant temperature. This practice is still widespread for some varieties but artificially refrigerated rooms are now used by large-scale manufacture. The ripening temperature is fairly characteristic of the variety, e.g., Cheddar, 6–8 °C; Gouda, 12–14 °C; ParmigianoReggiano, 18–20 °C; Emmental, 6 °C for ⬃2 weeks, then at 22 °C for 4–6 weeks to allow the propionic acid bacteria to grow rapidly and produce adequate CO2 for good eye development, then at ⬃4 °C for several months to complete ripening; Camembert, 14 °C for ⬃2 weeks to induce the growth of P. camemberti, then at 4 °C for 2–4 weeks.

Ripening can be accelerated by increasing the ripening temperature but all reactions, desirable and undesirable, are accelerated and an unbalanced flavour or off-flavour may develop. Ripening at an elevated temperature is normally considered with the objective of accelerating ripening (see Fox et al., 1996b). Cheese flavour can probably be modified by manipulating temperature; however, this is rarely practised except for Swiss-type cheeses. The rate at which the curd is cooled after moulding has a major effect on the growth of starter LAB and NSLAB. The curds for most cheeses are moulded immediately after cooking and acidification occurs mainly in the moulds. Hence, the rate at which the curd cools in the moulds has a major effect on starter growth and rate of acid development, and is strongly affected by the size of the cheese and ambient temperature. The effect of cooling on starter growth is particularly noticeable for high-cooked cheeses, e.g., Swiss and Grana types. The thermophilic starters used for these cheeses do not grow at the cook temperature but begin to grow as the curd cools in the moulds. For consistency, it is important to control the ambient temperature. For Cheddar-type cheeses, acidification is almost complete at moulding. Traditionally, the moulded cheeses were pressed overnight at ambient temperature and the cheeses cooled close to ambient during this period, although ambient temperature probably varied significantly with season. In modern practice, the cheeses exit the Wincanton tower at ⬃36 °C and are packaged and stacked on pallets (5 10 cheeses ⬃1 tonne) and transferred to ripening rooms. The cheeses at the centre of the pallet do not decrease to ambient (store) temperature for about 4 weeks and this causes considerable variation in the number and probably the type of NSLAB, and hence in the quality of the cheese. Many factories now cool the packaged cheese in a cooling tunnel overnight before stacking on pallets. If the cheese is cooled to 1 °C and ripened at this temperature, the cheese will be free of NSLAB but the rate of ripening will be very slow. The humidity of the environment must be controlled, at 85–90% RH, for the ripening of many varieties, mainly those with a surface microflora, which will not grow if the cheese develops a rind. Traditionally, rind development was encouraged on internal bacterially ripened cheeses by reducing the RH slowly. The rind serves to protect the cheese against undesirable surface growth and the loss of moisture (weight). Today, many varieties, e.g., Cheddar and Gouda, are coated or wrapped in plastic, i.e., rindless cheese, to prevent weight loss and to protect the surface of the cheese against undesirable bacterial growth.


Conclusions Through increased knowledge of the chemistry, biochemistry and microbiology of cheese, it should be possible to produce cheese of a very high quality consistently, although this is not always achieved owing to failure to control one or more of the key parameters that affect cheese composition and ripening. Milk is a variable raw material and although it is possible to eliminate major variations in the principal milk constituents, some variations persist. Variability in milk composition can also be compensated by manipulating some process parameters in the cheesemaking process. Most large factories operate on a strict time schedule and hence subtle manipulation of the process on an individual vat basis may not be possible. Therefore, strict control of milk composition and starter activity is critical. From a microbiological viewpoint, the milk supply to modern cheese factories is of very high quality and after pasteurization contains only a few hundred bacteria per ml. In modern factories where enclosed vats and other equipment is used, the level of contamination from the environment is very low; cheese curd containing 103 NSLAB/g at 1 day is normal. However, these adventitious NSLAB grow to c. 107–108 cfu/g and dominate the microflora of cheese after about 3 months. Since the adventitious NSLAB grow slowly in cheese, they are most significant in long-ripened cheese. Although the significance of the adventitious NSLAB in long-ripened cheese is unclear, it would appear to be desirable to control them, either by eliminating them or standardizing their number and type. In industrial-scale manufacture of cheese, it is not possible to eliminate NSLAB. It is possible to prevent their growth by ripening at ⬃1 °C but the overall ripening process is also reduced to an unacceptable rate. Outcompeting indigenous NSLAB by an adjunct Lactobacillus culture, which does not have to contribute to ripening, is a possibility but this approach has not been investigated. Although it is now possible to avoid major defects in cheese produced using modern technology, further research on the biochemistry of cheese ripening is required to enable the process of cheese manufacture and ripening to be refined to an extent that will allow the consistent production of premium quality cheese. The key to successful cheesemaking is a good reliable starter, both from the viewpoint of reproducible acid production and subsequent ripening. If properly managed, modern starters are generally satisfactory and their performance is being improved progressively.

The use of starter adjuncts, usually mesophilic lactobacilli, for some varieties, especially Cheddar, is increasing, with the objective of intensifying and modifying flavour, accelerating ripening and perhaps controlling adventitious NSLAB and thus standardizing quality. Basically, cheesemaking is a relatively simple process, consisting of two phases: conversion of milk to cheese curd and transformation of the curd to mature cheese; both phases involve a number of steps. The key steps in curd manufacture are: acidification, coagulation, syneresis/dehydration and salting. With the knowledge currently available on the mechanism of these processes and the scale and quality of the cheesemaking equipment, it should be possible to produce cheese curd of consistently premium quality from chemical and microbiological viewpoints. Unfortunately, this is not the case in practice. Undoubtedly, variability in the composition and microflora of the milk contribute to the variability of cheese curd but there is variability in curd produced during the course of a single day from a single large batch of bulked milk using the same rennet and starter. One factor likely to be responsible for this variability is the time-lag in performing certain cheesemaking operations, e.g., it requires ⬃30 min to separate the curds and whey in the very large (30 000 l) vats now used for Cheddar, Gouda or Mozzarella. This time-lag continues during later operations, e.g., cheddaring, milling, salting and pressing. The solution to this problem is the development of a continuous curd production system, such as the ALPMA system, but this is not used for hard cheeses. Work in this area appears warranted. The objective of cheesemaking is to consistently manufacture cheese with the desired, characteristic flavour, texture and functionality in the highest yield possible, as cheaply and as quickly possible. The closest we have come to achieving that objective is the production of enzyme-modified cheeses, which do not resemble closely the flavour, texture or functionality of any natural cheese but are used successfully to replace natural cheese in some applications (see ‘Sensory Character of Cheese and its Evaluation’, Volume 2). With improved knowledge of the biochemistry of cheese ripening, it may be possible to produce some of the milder, less complex cheese using the EMC approach – research in this area is warranted. Cheese ripening is a very complex biological, biochemical and chemical process which is determined and directed by the composition and microflora of the cheese curd; if these are reproducible and consistent, it should be possible to produce cheese of excellent quality consistently.


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Fox, P.F., Olivecrona, T., Vilaro, S., Bengtsson-Olivecrona, G., Kelly, A.L., McSweeney, P.L.H., Shakeel-Ur-Rehman, Fleming, C.M., Stepaniak, L., Gobbetti, M., Corsetti, A., Pruitt, K.N. and Farkye, N.Y. (2003). Indigenous enzymes in milk, in, Advanced Dairy Chemistry, Vol. 1, Proteins, 3rd edn, P.F. Fox and P.L.H. McSweeney, eds., Kluwer Academic/Plenum Publishers, New York. pp. 465–601. Gilles, J. and Lawrence, R.C. (1973). The assessment of cheese quality by compositional analysis. NZ J. Dairy Sci. Technol. 8, 148–151. Guinee, T.P. and Fox, P.F. (1993). Salt in cheese; physical, chemical and biological aspects, in, Cheese: Chemistry, Physics and Microbiology, Vol. 1, General Aspects, 2nd edn, P. Fox, ed., Chapman & Hall, London. pp. 257–302. Guinee, T.P, Auty, M.A.E., Feeney, E.P. and Fox, P.F. (2002). Effect of pH and calcium concentration on some textural and functional properties of Mozzarella cheese. J. Dairy Sci. 85, 1665–1669. Gustchina, E., Rumsh, L., Ginodinan, L., Majer, P. and Andreeva, N. (1996). Post X-ray crystallographic studies on chymosin: the existence of two structural forms and the regulation of activity by the interaction with the histidine–proline cluster of -casein. FEBS Lett. 379, 60–62. Hyslop, D.B. (2003). Enzymatic coagulation of milk, in, Advanced Dairy Chemistry – Vol. 1 – Proteins, 3rd edn, P.F. Fox and P.L.H. McSweeney, eds., Kluwer Academic/Plenum Publishers, New York. pp. 839–878. Iametti, S., Tedeshi, G., Oungre, E. and Bonomi, F. (2001). Primary structure of -casein isolated from mares’ milk. J. Dairy Res. 68, 53–61. Lawrence, R.C. and Gilles, J. (1980). The assessment of the potential quality of young Cheddar cheese. NZ J. Dairy Sci. Technol. 15, 1–12. Lawrence, R.C., Heap, H.A. and Gilles, J. (1984). A controlled approach to cheese technology. J. Dairy Sci. 67, 1632–1645. Lelievre, J. and Gilles, J. (1982). The relationship between the grade (product value) and composition of young commercial Cheddar cheese. NZ J. Dairy Sci. Technol. 49, 1098–1101. Lynch, C.M., McSweeney, P.L.H., Fox, P.F., Cogan, T.M. and Drinan, F.D. (1997). Contribution of starter lactococci and non-starter lactobacilli to proteolysis in Cheddar cheese with a controlled microflora. Lait 77, 441–459. Malacarne, M., Martuzzi, F., Summer, A. and Mariani, P. (2002). Protein and fat composition of mare’s milk: some nutritional remarks with reference to human and cows’ milk. Int. Dairy J. 12, 869–877. Martin, P., Ferranti, P., Leroux, C. and Addeo, F. (2003). Non-bovine caseins: quantitation variability and molecular diversity, in, Advanced Dairy Chemistry – Vol. 1 – Proteins, 3rd edn, P.F. Fox and P.L.H. McSweeney, eds., Kluwer Academic/Plenum Publishers, New York. pp. 277–317. McSweeney, P.L.H. and Sousa, M.J. (2000). Biochemical pathways for the production of flavour compounds in cheese during ripening. Lait 80, 293–324.


McSweeney, P.L.H., Fox, P.F., Lucey, J.A., Jordan, K.N. and Cogan, T.M. (1993). Contribution of the indigenous microflora to the maturation of Cheddar cheese. Int. Dairy J. 3, 613–634. Morgan, S.M., Ross, R.P. and Hill, C. (1995). Bacteriolytic activity due to the presence of novel plasmid-encoded lactococcins, A, B and M. Appl. Environ. Microbiol. 61, 2995–3001. Ochirkhuyag, B., Chobert, J.-M., Dalgalarrondo, M. and Haertlé, T. (2000). Characterization of mare caseins. Identification of s1- and s2-caseins. Lait 80, 223–235.

O’Connor, C.B. (1971). Composition and quality of some commercial Cheddar cheese. Ir. Agric. Creamery Rev. 26 (10), 5–6. Pearce, K.N. and Gilles, J. (1979). Composition and grade of Cheddar cheese manufactured over three seasons. NZ J. Dairy Sci. Technol. 14, 63–71. Tobin, J. (1999). Effects of Adjunct Cultures and Starter Blends on the Quality of Cheddar Cheese. PhD Thesis, National University of Ireland, Cork. Yvon, M. and Rijnen, L. (2001). Cheese flavour formation by amino acid catabolism. Int. Dairy J. 11, 185–201.

Index

Abondance, 199 Acid-coagulated milk gels, 6, 77–8, 105 casein micelles in, 105–106 effect of compositional/processing parameters on textural properties: fat content/homogenization, 118 heat treatment, 116–17 inoculation/gelation temperature, 115–16 pH/calcium content, 118–19 rennet addition, 117 solids non-fat (SNF) content, 117–18 mechanisms: physico-chemical aspects, 106–109 theoretical models, 106 physical properties: appearance, 113–14 microstructure, 112 permeability, 112–13 rheological, 109–11 texture/sensory, 111–12 whey separation/syneresis, 114–15 study by electron microscopy (EM)/ confocal scanning laser microscopy (CSL), 112 syneresis, 114–15 see also Coagulation; Rennets Adjunct cultures see Secondary/adjunct cultures Amino acids, catabolism of see Catabolism of amino acids Appenzell, 199 Aroma, compounds: extraction methods, 491 dialysis, 492 headspace, 493 high-performance size-exclusion chromatography, 492–3 high-vacuum distillation, 491–2 solid-phase microextraction, 493 solvent, 492 steam distillation, 491 water-soluble extract (WSE), 493–4 identification using hyphenated GC techniques, 494–6 representativeness, 494 sample treatment, 491 see also Sensory characteristics of cheese; Flavour Arzua, 323 Aspartic proteinases see Chymosin/aspartic proteinases Autolysis, 126, 289 Avenato, 323 Bacteriocins, 136, 153 Bacteriophage, 154, 163–70, 287, 351, 375 classification, 165 DNA homology, 167 host range, 165 morphology, 165 serology, 165–6 structural protein profiles, 166–7

control of, 164 engineered phage resistance systems, 179 antisense mRNA, 180 bacteriophage-triggered defence, 181–2 current status/future perspectives, 182 gene replacement/insertional mutagenesis, 180–1 phage-encoded resistance (Per), 179–80 recombinant superinfection exclusion/ immunity, 181 epidemiology, 167 Lactobacillus phage, 170–1 Sc. thermophilus phage, 170 genome organisation/evolution, 171–6 lysogenic life cycle, 174 maintenance of lysogeny, 175–6 site-specific recombination, 174–5 superinfection exclusion, 176 lytic life cycle: bacteriophage lysis, 174 DNA packaging, 173 DNA replication, 172–3 lysogenic/lytic switch, 172 phage adsorption/DNA injection, 171–2 structural proteins, 173–4 multiplication, 163 natural resistance systems in lactic acid bacteria, 176–7 abortive infection, 178–9 adsorption inhibition, 177 injection blocking, 177 restriction/modification, 177–8 proteolytic enzymes, 131 raw milk cheeses, 321 resistance systems: engineered phage, 179–82 natural bacteriophage, 176–9 starter cultures, 129–38 Bavarian Blue, 193 Beaufort, 197 Bel Paese, 193, 195 Biogenic amines, 201, 561–3 Bleu d’Auvergne, 193 Blue cheese, 123 contamination, 550 fatty acids, 378 mesophilic starters, 149 ultrafiltration, 269 use of salt, 211–12, 219 Brevibacterium linens, 149, 192 Brie: flavour, 502 lipolytic agents, 376 moulds, 193 rheology/texture, 535 Brine-salted cheese see Salt Buffalo milk cheese, 5

610 Index

Cabrales, 193, 306 Camembert, 123 contamination, 550 coryneform bacteria, 195, 197 fatty acids, 377 flavour, 492, 498, 499, 500 lipolytic agents, 376 Listeria growth, 544 mesophilic starters, 149 microbial growth, 544–5 moulds, 193 raw milk, 321 rheology/texture, 528, 535 starter cultures, 126 ultrafiltration (UF), 271 use of salt, 219, 240 yeast flora, 306 Canestrato, 5, 323 Casein, 7, 48–50, 55, 60, 71–2, 105–106, 351, 413, 415, 417, 589, 591 activity of cell envelope proteinase, 132 chemistry, 48 micelle assembly, 49 micelle properties, 49–50, 105–106 micelle stability, 50 self-assembly, 49 structures, 48–9 chymosin activity on, 52, 392–3 effect of NaCl, 212–13, 214, 220–3 enrichment of cheese milk by MF, 277–8 gel formation/properties of, 71–4, 78–81, 91, 94, 106–109, 111, 115, 116 hydration in cheese, 220–5 hydrolysis by cathepsin D, 396 mares’ milk, 591 quality of cheese, 591–2 raw milk cheese, 324, 336, 339 use of capillary electrophoresis for analysis of casein, 420 Catabolism of amino acids, 152–3, 201, 302–303, 350–2, 435, 451 aromatic: phenylalanine, 444–7 tryptophan, 443 tyrosine, 443–4 branched-chain: deaminases, 449 decarboxylases, 449–51 production of volatile sulphur compounds: lyases involved in methionine catabolism, 442–3 transamination, 435–9 Catabolism of fatty acids, 376–80 Catabolism of lactate, 348–9 Cathepsins, 396, 399 Cauchy strain, 539 Cheddar, 5, 10, 12, 14 contamination, 545, 546–9 dry-salted varieties, 245–6 fatty acids, 377 flavour, 335, 337, 492, 502, 605 lipolytic agents, 374, 375 lysis, 136 mesophilic starters, 149 microbiology, 289, 290, 296, 297, 301 moulds, 193 propionic acid bacteria, 200 quality, 595, 596, 604, 605 raw milk, 323, 327, 330

reduced sodium, 226–7 rheology/texture, 516, 517, 518–19, 520, 527, 529, 530, 535 ripening, 348 sensory characteristics, 478–9 starter cultures, 126, 128 use of salt, 208–209, 211, 216–19, 240 Cheddar curd: salt uptake: curd depth during holding, 238 degree of mixing, 238 level of salting, 237–8 mellowing period, 238 method, 237 moisture content of curd, 238–9 other factors, 239 temperature, 238 Cheese: composition, 7–8, 603–605 fat-in-dry matter (FDM), 7, 589 moisture in non-fat substances (MNFS), 7 pH, 8 history of, 1–5, 261, 605 Cheshire, 5, 208, 527 Chymosin/aspartic proteinases, 19–33, 351, 392, 600–601 active site, 23–7 catalytic mechanisms, 27–9 inhibitors, 33 natural sources, 19–20 physical properties/stability, 20–1 structure, 21–4 substrate-binding pockets/specificity, 30–3 zymogen activation, 29–30 crystals, 23 fermentation-produced chymosin, 51–2 milk clotting mechanism, 33–4 mutant chymosins, 32–3 physical properties/stability: enzyme solubility, 20–1 enzyme stability, 20 molecular weight/isoelectric point, 20 recombinant calf chymosin: eukaryotic expression, 35–6 prokaryotic expression, 34–5 ripening process, 351, 356 stability (solubility), 20 structure: active site, 24–7 catalytic mechanisms, 27–9 gene sequences, 21 inhibitors, 33 primary, 21 secondary, 21, 23 substrate-binding pockets/specificity, 30–3 tertiary, 23–4 zymogen activation, 29–30 substrate specificity, 32 yield, 34–5 see also Pepsin Citrate metabolism, 130–1, 151–2, 367–8 Clostridia, 7, 153, 202, 327, 328, 365–6, 562, 600 Clotting see Acid-coagulated milk gels; Coagulation; Rennets Coagulation, 10–11, 47 Cynara cardunculus, enzymes from, 52 enzymes, 47, 391, 392–3 influence of NaCl, 212–13

Index 611

physico-chemical mechanisms involved in gel formation from unheated milk, 106–109 post-coagulation operations, 11–12, 594–6 theoretical models, 106 visual, 11 see also Acid-coagulated milk gels; Rennets Comté cheese, 127 Conjugated linoleic acid, 575–6 Contamination see Pathogens; Safety; Toxins Cooking, 224–5 health/safety, 551 Coryneform bacteria, 195, 197, 597 antimicrobial activities, 198 form/use of adjunct culture, 198 lipolysis, 198 proteolysis, peptidolysis, amino acid catabolism, 198 selection as adjuncts, 197–8 useful properties in selecting surface bacteria, 197 Cottage cheese, 2, 9 acid milk gels, 105, 112, 116 addition of rennet, 117 contamination, 550 heat treatment, 116 reduced sodium, 227 ultrafiltration, 268 Cream cheese, 2 acid milk gels, 105, 112 compliance, 539 modulus, 539 whey removal, 114, 115 Cultures see Secondary/adjunct cultures; Starter cultures Curd, 12, 14 behaviour during processing: axial drainage, 95 compaction of curd column, 94–6 curd fusion, 93–4, 97 syneresis under pressure, 93 water content of cheese, 96–8 cooking temperature, 11 manufacturing process, 11–12 salt absorption/diffusion: direct mixing with milled curd, 229, 232 dry surface-salting of moulded pressed curd, 232 factors affecting uptake in Cheddar, 237–9 initial moisture content, 236–7 pH of curd/brine, 237 salt-in-moisture level/pre-salting, 208–11, 235 temperature of curd/brine, 235 syneresis during curdmaking: effects of grain size, 86–7 methods of estimation, 85–8 rate equations, 85–6 stirring, 87–8 washing, 90 Cynara cardunculus proteinase, 3, 52 Danablu, Danish Blue, 193, 194, 306 Danbo: mould, 193 ultrafiltration, 268 use of salt, 220 yeast flora, 306 Debaryomyces hansenii, 191, 192, 196 Deformability modulus, 539 Dental caries, 576, 578–9 Domiati, 10, 219 Dry-salted cheese see Salt

Dutch cheese, 128 lipolytic agents, 374 ripening, 348 see also Gouda; Edam Eastern European cheeses, 299 Edam: fatty acids, 378 mesophilic starters, 149 starter cultures, 126 ultrafiltration, 268 Elastic material behaviour, 539 Elastoplastic material behaviour, 539 Emmental: fatty acids, 378 flavour, 502 propionic acid bacteria, 200 starter cultures, 123, 126, 127 use of salt, 215 whey cultures, 127 see also Swiss cheese Enterococci, 290, 295 Enzymes, 19, 133, 288, 378, 448 coagulation, 47, 50–2, 391, 392–3 indigenous, 587–8, 600 microbial, 214–15 proteolytic, 19, 413–15 ripening process, 352–7 secondary starter microorganisms, 195, 413–15 sources: lyosomes, 19 micro-organisms, 19–20 plants, 19 stomach, 19 tissues, 19 see also Chymosin/aspartic proteinases; Lactic acid bacteria; Catabolism of amino acids; Catabolism of fatty acids; Lipolysis; Peptidases; Proteolysis; Ripening of cheese Ewe’s milk cheese, 199, 340 Exopolysaccharides, 137, 158 Fatty acids, 349, 373–80 Feta, 5, 268–9 contamination, 550 microbial growth, 545 microbiology, 290 ultrafiltration (UF), 271 use of salt, 219 Flavour, 3–4, 129, 289, 306, 332, 347, 466, 489–91 acid milk, 112 contribution of lipolysis/catabolism of FFA, 379–80 dynamic methods for characterisation: model mouth systems, 501–502 release of non-volatiles in vivo, 501 release of volatiles in vivo, 499–501 fatty acids, 349 global/fast assessment, 502 electronic nose, 502–503 mass spectrometry-based systems, 503–504 peptides, 352 quality, 586–7, 599, 603 raw milk, 335–8 sapid (non-volatile) compounds: extraction, separation, identification in relation to sensory properties, 496–9 water-soluble extracts (WSE), 496 smell/aroma, 466

612 Index

Flavour – contd. taste, 466 see also Sensory characteristics of cheese; Aroma, compounds Free-Choice Profiling, 477 Fungi, 36 Gammelost, 306 Gaziantep, 219–20 Gel formation see Acid-coagulated milk gels; Coagulation; Rennets; Syneresis of rennet-coagulated curd Genetic engineering, 155, 601 Geotrichum candidum, 191 Goat cheese, 1, 5, 14, 193, 325, 498 Gorgonzola, 193 Gouda, 14 fatty acids, 378 lysis, 136 mesophilic starters, 149 propionic acid bacteria, 199 quality, 595, 596 starter cultures, 126 use of salt, 223, 237 Grana, 8, 126 microbiology, 289 whey cultures, 127 Gruyère: catabolism of fatty acids, 378 Coryneform bacteria in, 197 flavour, 492 rheology/texture, 529 starter cultures, 126, 127 whey cultures, 127 HACCP see Hazard Analysis and Critical Control Points Havarti, 535 Hazard Analysis and Critical Control Points, 542, 543, 584 see also Safety Health see Nutrition; Pathogens; Safety; Toxins Heterofermentative lactobacilli, 200 form/use of adjunct cultures, 202 species found in cheeses, 200–201, 291 useful properties to select as adjuncts: antagonistic activities, 201–202 formation of biogenic amines, 201 lipolytic activities, 201 probiotic properties, 202 proteolysis/amino acid catabolism, 201 Histamine, 561–3 Hygiene, 321–3 Illness see Pathogens; Safety; Toxins Italian cheeses, 298–9, 338 Jarlsberg, 5 Kelvin element, 539 Kinematic viscosity, 539 Kluyveromyces, 195 Lactate: catabolism, 348–9 changes during ripening: metabolism by Clostridium tyrobutyricum, 365–6 metabolism by Propionibacterium, 366–7

oxidation, 364 oxidative metabolism in surface mould-ripened varieties, 364 racemization, 362–4 see also Lactose metabolism Lactic acid bacteria, 399–400 aminopeptidases, 411–12 carboxypeptidases, 403, 411 di- and tri-peptidases, 403 endopeptidases, 403 Lactobacillus delbrueckii, 290 Lactobacillus helveticus, 123 Lactococcus lactis, 123 peptidases, 400–403 proline-specific peptidases, 412–13 proteinases, 400 see also Starter cultures, Non-starter lactic acid bacteria, Bacteriophage Lactobacillus spp, 123, 290 see also Non-starter lactic acid bacteria Lactococcus lactis, 123 chromosome, 149–50 genetic manipulation, 155 genetics of industrially important traits: bacteriocins, 153–4 bacteriophage, 154–5 lactose/citrate metabolism, 151–2 proteolysis/amino acid catabolism, 152–3 Lactose metabolism, 130, 151–2, 361–2 Leuconostoc, 155, 290 Limburger, 195 Linear viscoelastic deformation, 539 Lipolysis, 198, 199–200, 201, 303, 349 agents, 373–6 contribution of FFA to flavour, 379–80 measurement of, 380, 384–5 patterns of, 380 raw milk cheese, 325, 327 Liquid pre-cheese (LPC), 269–73 Listeria monocytognes, 542–4, 550, 551, 553–5 Livarot, 195 Loss modulus, 540 Low-concentrated retentates, 267–8 Lysis, 126, 289 see also Autolysis Lysogeny, 174 Maasdam, 5, 199, 378 Mahon, 220 Manchego, 5, 335 Membrane processing, 261 applications of: liquid pre-cheeses, 269–75 medium/intermediate concentrated retentates, 268–9 microfiltration, 276–8 milk protein concentrates, 278–9 on-farm concentration, 275–6 properties of UF retentates, 265–7 protein-standardized milk, 267–8 reverse osmosis, 275 UF in cheesemaking, 267–75 APV-sirocurd process: definitions, 262–5 design/configuration, 261 hollow fibre, 262–3 microfiltration (MF), 262 nanofiltration (NF), 262 plate and frame, 263

Index 613

reverse osmosis (RO), 262 spiral-wound, 263–4 tubular, 262 ultrafiltration (UF), 262 vibrating membrane system, 264–5 Mesophilic starters, 149–52, 597 Leuconostoc, 155–6 plasmids, 150–1 Mexican-style cheese, 553 Microbial pathogens see Pathogens Micrococcus, 197, 304 Microfiltration: applications: casein enrichment of cheese milk, 277–8 microbial epuration of raw milk, 276–7 modifications, 278 selective fractionation of globular fat, 278 Milk: antiobiotics in, 7 casein chemistry, 48–50 chemical composition, 7, 588–9 clotting mechanism, 33–4, 47 composition, 91 fat, 47 gel formation, 106–109 heat treatment, 88 homogenization, 88 indigenous enzymes, 587–8 indigenous proteinases: others, 396–9 plasmin, 213–14, 393–6, 600 microbiology: desirable indigenous bacteria, 587 off-flavours/spoilage, 586–7 public health aspects, 586 pasteurized, 8, 355 alternatives to pasteurization, 587 protein, 47–8, 573, 575 protein-standardization, 267–8 quality, 584 rennet-induced coagulation, 50–65 safety: heat treatment, 543–4 pathogens, 541–3 quality, 543 standardization: calcium, 589 fat/casein, 589 pH, 589–90 syneresis of renneted-milk gel, 80–1 toxins in, 564 various additions to, 88–9 Milk gels see Acid-coagulated milk gels; Rennet coagulation of milk Milk protein concentrates, 278–9 Modulus of deformability, 540 Morbier, 195, 199, 330 Moulds, 304–306, 395, 597 contamination with mycotoxins, 567–8 form/use of adjunct culture, 194–5 species found in cheese, 193–4 useful properties in selecting as adjuncts: appearance on/in cheese, 194 de-acidification activity, 194 interactions with other microorganisms, 194 lipolytic activity, 194 production of aroma, 194 production of mycotoxins, 194

proteolytic activity, 194 Mozzarella, 9, 12 contamination, 553 flavour, 498 quality, 595, 596 raw milk, 321 rheology/texture, 529, 530 starter cultures, 123, 126 ultrafiltration (UF), 268, 272–3 use of salt, 222, 240 Münster, 195 Mycotoxins, 564, 567–8 Nitrogen metabolism in lactic acid bacteria, 131–2 amino acid degradation, 133–4 role of proteinase, 132 transport systems/peptidases, 132–3 see also Proteolysis; Peptidases; Lactic acid bacteria Non-starter lactic acid bacteria (NSLAB), 7, 289, 291, 353 biochemical activities: amino acid catabolism, 302–303 citrate utilisation, 302 lipolysis, 303 proteolysis, 302 enterococci, 290, 295 growth/survival: environmental conditions, 296 interactions, 297 nutrient availability, 296–7 non-starter lactobacilli, 289–90 pediococci, 290 population dynamics: Cheddar, 297 Greek/eastern European cheeses, 299 Italian cheese varieties, 298–9 Portuguese cheese varieties, 298 Spanish artisanal cheeses, 297–8 Swiss cheeses, 297 quality, 602–603 ripening process, 356–7 significance: adjunct as probiotics, 301–302 influence on quality, 299, 301 use of other adjunct cultures, 301 source of, 295–6 see also Lactobacillus NSLAB see Non-starter lactic acid bacteria Nutrition, 573 carbohydrate, 575 cheese and dental caries, 576, 578–9 fat/cholestrol, 575–6 minerals, 576 protein, 573, 575 vitamins, 576 Parmigiano-Reggiano, Parmesan: contamination, 545, 550 fatty acids, 377–8 flavour, 492 raw milk, 322 sensory characteristics, 474 starter cultures, 123 whey cultures, 127 Pathogens, 7, 541 challenge studies, 549–52 Escherichia coli, 541, 544, 545, 548, 549–50, 551, 555 growth/survival in soft/semi-soft cheeses, 552–3 reviews on safety of raw milk, 545–6

614 Index

Pathogens – contd. safety of cheese, 541 extrinsic/intrinsic parameters affecting microbial growth, 544–5 heat treatment of milk, 543–4 milk quality, 543 raw milk, 541–3, 545 Salmonella, 542, 544, 546–8, 551, 553, 555 Salmonella enterica, 542, 546, 548, 553 stress adaptation and impact on safety, 553–4 Pecorino, 5, 124, 126, 127 Pediococcus, 290, 292 Penicillium camemberti, 193 Penicillium roqueforti, 193 Pepsin, 29, 31, 354, 393 Peptidases, 132–3, 400, 403, 417 aminopeptidases, 411–12 carboxypeptidases, 403, 411 di- and tri-peptidases, 403 endopeptidases, 403 proline-specific, 412–13 Phage see Bacteriophage Pichia spp., 196 outbreaks involving Cheddar, 546–9 Poisson effect, 540 Pont l’Eveque, 535 Port Salut, 193 Portuguese cheese, 298, 306 Processed cheese, 227 Prochymosin, 20, 30, 34–6 Propionibacterium, 303, 449, 597 see also Emmental; Swiss cheese Propionic acid bacteria, 198, 303, 449, 597 as adjunct cultures, 200 characteristics of species found in cheeses, 198–9 useful properties for selecting as adjuncts: lactate metabolism, 199 lipolysis, 199–200 probiotic properties, 200 proteolytic activities/amino acid catabolism, 199 Proteolysis, 152–3, 156, 302, 350–2 amino acids, 350–2 lactocepins, 400 monitoring: amino acid analysis, 421 capillary electrophoresis (CE), 420 chromatographic techniques, 420–1 fluorescent spectroscopy, 421 Fourier transform infrared spectroscopy (FTIR), 421 tryptophan, 421 ultrasonics, 421 urea-PAGE, 420 patterns, 415–19 primary, 323–4 raw milk cheese, 323–5 ripening, 391 coagulant, 391, 392–3 exogenous proteinases/peptidases, 391–2 indigenous proteinases, 391, 393–9 non-starter lactic acid bacteria, 200, 391 secondary starter, 191, 391, 413–15 starter lactic acid bacteria, 391, 399–413 secondary, 198, 199, 201, 324 water-soluble peptides, 416–19 Provolone, 545

Quality of cheese, factors affecting: cheese composition, 603–605 coagulant (rennet), 590–2, 600–601 cultures, 592–4, 601–602 indigenous enzymes, 600 Lactobacillus species as adjunct cultures, 603 milk supply: alternatives to pasteurization, 584, 587 chemical composition, 588–9 indigenous enzymes, 587–8 microbiology, 586–7 standardization of composition, 589–90 non-starter lactic acid bacteria, 602–603 packaging, 597 post-coagulation operations, 594–6 production parameters, 584 ripening, 596–9, 605–606 salting, 216–20, 596 starter, 592–4, 601–602 use of ultrafiltration (UF), 596 Quarg, 2, 5 acid milk gels, 105, 112 addition of rennet, 117 ultrafiltration, 270 whey removal, 115 see also Acid-coagulated milk gels Queso Blanco, 5 Raclette, 323, 328, 335, 338 Raw milk cheese, 8, 319–20, 545–6 biochemical aspects: lipolysis, 325, 327 proteolysis, 323–5 volatile compounds, 327–35 safety aspects: diversity of microorganisms, 320–1 hygiene, 321–3 numbers of microorganisms, 320 sensory aspects: flavour/odour, 335–8 texture, 338–40 Reconstituted skim milk, 64, 139, 208 Reduced-sodium cheese, 207, 225–6 Cheddar, 226–7 Cottage cheese, 227 other cheeses, 227 Rennet coagulation of milk, 2, 3, 6, 8, 10–11, 19, 584 adhesive sphere models/viscosity, 55–6 development of rheological properties, 56–7 effect of acidification, 117 fractal models/rearrangements, 62–3 heat treatment, 64–5 high pressure treatment, 64 kinetic models, 53–5 measurement of clotting time/curd-cutting time, 53 mechanisms of milk-clotting, 33–4 milk processing/gel formation, 63–5 modelling gel-firming kinetics, 60–2 post-coagulation operations, 594–6 preparation, 52 primary enzymatic phase, 11, 50–2 production, 584 quality, 590–2, 600–601 rennet, 354–5 ripening process, 354–5 secondary non-enzymatic phase, 11 substitutes, 10 theoretical basis of viscoelasticity, 57–60

Index 615

UF retentates, 266–7 see also Acid-coagulated milk gels; Coagulation; Chymosin/aspartic proteinases Rennets see Chymosin/aspartic proteinases; Pepsin Rheology, 511–33 cheese structure, 516–18 compliance, 539 creep/stress relaxation, 518–19 development of properties during rennet coagulation, 56–7 effect of NaCl, 223–4 empirical instrumental methods of measurement, 523 exopolysaccharides, use of, 158 gel formation, 75–7, 109–10, 113–14, 117–18 large strain deformation: bending tests, 530–1 definition/terminology, 520 effect of sample temperature, 532 fracture/work to fracture, 521 measurement using texture analyser, 520–1 shear measurements, 530 uniaxial compression, 527–30 wire-cutting, 532 mechanical models, 519–20 oscillatory rheometry for linear viscoelastic measurements: complex viscosity, 526–7 elastic shear modulus, 524–6 loss modulus, 524–6 overview, 511–12 sensoric methods, 521–3 terminology: bulk modulus/compressibility, 515–16 deformation and strain, 512 relationship between stress/strain, 515 shear/normal modes of stress/strain, 513–15 stress, 512–13 viscous deformation, 516 time-dependent measurement, 532 viscosity measurement, 532–3 see also Texture Rhizomucorprotease, 20 Ripening of cheese, 12, 14, 347, 375, 395 acceleration, 357 agents: cathepsin D, 355–6 NSLAB, 356–7 other indigenous enzymes, 356 plasmin, 355 rennet, 354–5 starter enzymes, 356 biochemical activities of NSLAB, 302–303 catabolism of amino acids, 350–2, 435 aromatic, 443–7 branched-chain, 447–9 deaminases, 449 decarboxylases, 449–51 other, 451 transamination, 435–9 volatile sulphur compounds, 439–43 glycolysis of residual lactose/catabolism of lactate, 347–9 lipolysis/metabolism of fatty acids, 349 proteolysis, 350–2, 391–2 quality, 596–9, 605–606 see also Proteolysis, Lipolysis; Catabolism of lactate; Catabolism of amino acids; Catabolism of fatty acids

Romano, 535, 545 Roquefort, 5 lipolytic agents, 376 mould, 193, 194 rheology/texture, 535 yeast flora, 306 Saccharomyces cerevisiae, 196 Safety, 541 Cheddar, 546–9 improvements, 554–5 microbial growth, 544–5 milk: heat treatment, 543–4 quality, 543 raw, 541–3, 545–9 soft/semi-soft cheeses, 552–3 stress adaptation and impact of pathogens, 553–4 St Nectaire, 193–4, 306 St Paulin, 136, 273, 323 Salt, 10, 207–208, 348, 576, 596 absorption/diffusion: brine- and surface dry-salted cheeses, 244–5 brine concentration/concentration gradient in brine-salted cheese, 232–3 brine-salted cheese, 228–9 Cheddar/dry-salted varieties, 245–6 cheese geometry, 233–4, 244 concentration gradient in dry-salted cheeses, 239–40 concentration of calcium in brine, 240 direct mixing of salt/milled curd, 229, 232 dry surface-salting of moulded pressed cheese curd, 232 fat content of cheese, 243–4 initial moisture content of curd, 236–7 initial salt-in-moisture level of curd/pre-salting, 235 mechanisms, 228–32 method of brine-salting, 233 methods of salting, 228 moisture content of cheese, 98, 241–3 pH of curd/brine, 237 salting time, 234–5 temperature of brine/cheese, 240 temperature of curd/brine, 235 uptake in Cheddar curd, 237–9 casein hydration/physical properties of cheese: cooking properties, 224–5 microstructure, 223 model systems, 220–3 rheology, 223–4 control of microbial growth, 208–12 effect on cheese composition: lactose content/pH, 249 moisture level, 247–9 salt content, 249 enzyme activity: coagulant, 212–13 microbial enzymes, 214–15 milk proteinase, 213–14 gel formation, 83–4 quality, 596 reduced-sodium cheese: Cheddar, 226–7 Cottage cheese, 227 other cheeses, 227

616 Index

Salt – contd. ripening/quality: Blue cheese, 219 Camembert, 219 Cheddar, 216–19 other cheeses, 219–20 salt/moisture equilibria in brine-salted cheese after salting, 244–7 salt/moisture equilibrium in Cheddar cheese, 246–7 water activity (aw), 215–16 Sbrinz, 127 Secondary/adjunct cultures: coryneform bacteria and staphylococci, 195, 197–8 effect on quality, 603 heterofermentative lactobacilli, 200–202 moulds, 193–5 non-starter lactic acid bacteria, 7, 289, 291, 353 propionic acid bacteria, 198–200 yeasts, 191–3 see also Starter cultures Semi-hard cheese, 273 Sensory characteristics of cheese, 455, 463–5 cheese preferences, 455–6 consumer preferences, 14, 480–1 definition, 455 evaluation methods, 467–8 consumer acceptability testing, 478 descriptive analysis, 475–7 discrimination tests, 475 grading/quality scoring, 468–75 time-intensity analyses, 477–8 human senses/sensory properties: cheese appearance, 462, 466 cross-modal interactions, 466–7 flavour, 466 texture, 466 universal language, 480 variety, 456–62 see also Aroma, compounds; Flavour Shear modulus, 540 Sheep milk cheese, 1, 5, 14 Soft cheese, 270–3, 552–3 Spanish artisanal cheeses, 297–8, 306 Staphylococcus, 195, 197, 304 antimicrobial activities, 198 form/use of adjunct culture, 198 lipolysis, 198 proteolysis, peptidolysis, amino acid catabolism, 198 selecting surface bacteria as adjuncts: effect on colour of cheese surface, 197–8 growth, 197 Starter cultures, 123, 149, 287, 288–9, 348 defined-strain, 164–5 genomics, 129–30, 158–9 mesophilic starter genetics: bacteriocins, 153–4 bacteriophage, 154–5 chromosome, 149–50 industrially important traits, 151–5 lactose/citrate metabolism, 151–2 Leuconostoc, 155–6 manipulation, 155 plasmids, 150–1 proteolysis/amino acid catabolism, 152–3 metabolism: autolysis, 136 bacteriocins, 136–7 citrate, 130–1

exopolysaccharide production, 137–8 growth, 134–5 lipases/esterases, 134 metabolic engineering, 135–6 nitrogen, 131–4 stress responses, 137 sugar, 130 mixed-strain mesophilic, 9–10, 164–5 pH control, 139–40 phage infection, 127, 139 preparation: preservation/distribution, 140–2 propagation, 138–40 time/temperature combination, 140 quality, 592–4 taxonomy, 123–4 types, 126, 128, 164 new sources, 129 primary, 123, 124, 126, 191 secondary, 123, 191, 195 see also Bacteriophage Stilton, 193, 194, 208 Streptococcus thermophilus, 123, 157 Stress: relaxation modulus, 540 relaxation test, 540 Swiss cheese, 128 contamination, 545, 552 flavour, 335, 338, 492 microbiology, 289, 296, 297 raw milk, 323, 327, 328 Syneresis of rennet-coagulated curd, 71, 114 during curdmaking, 84–5 effects of other process variables: acidity, 90 coagulation, 89 heat treatment of milk, 88 high-pressure treatment, 91 homogenization of milk, 88 temperature, 89–90 ultrafiltration, 90–1 various additions to milk, 88–9 washing of curd, 90 mechanisms, 78–9 methods for estimating: effects of curd grain size, 86–7 modelling process, 81–4 rate equations, 85–6 stirring, 87 renneted milk, 80–1 review of, 91–2 unified approaches to gel formation/syneresis: acid gels, 77–8 behaviour during processing, 92–3 compaction of curd column, 94–5 curd fusion, 93–4 effect of milk composition, 91 gel formation, 73–5 renneting, 72–3 rheological characteristics, 75–7 under pressure, 93 water content of cheese, 95–8 Texture: terminology, 533 evaluation: instrumental shear deformation, 535–6 texture profile analysis (TPA), 534 see also Rheology

Index 617

Thermophilic starters, 56, 126 Lactobacillus spp.: genetic manipulation, 157 important traits, 156–7 Streptococcus thermophilus: genetic manipulation, 158 important traits, 157–8 Tilsit, 306 Tomme, 193, 199 Torulospora delbrueckii, 196 Toughness, 540 Toxins: biogenic amines: formation, 562–3 histamine, 561–2 in cheese, 563 mycotoxins: direct contamination of cheese, 567–8 fate in cheese during manufacture/ripening, 567 indirect contamination, 564, 567 production of toxic metabolites in cheese, 567

protein-standardized milk, 267–8 see also Membrane processing Uniaxal compression, 527–8 compressive strength, 540 effect of deformation rate, 529–30 effect of pre-test strain history, 529 effect of sample-machine interface conditions/sample dimensions, 529 influence of shape, 530 relationship between shear/normal stresses, 528

Ultrafiltration (UF), 8–9, 90–1, 265–75, 596 cheese quality: functionality, 274–5 proteolysis/ripening characteristics, 274 texture, 273–4 liquid pre-cheeses: fresh unripened cheeses, 269–70 other applications, 273 semi-hard cheese, 273 soft cheese, 270–3 medium/intermediate concentrated retentates: APV-sirocurd process, 268 general considerations, 269 other cheeses, 269 structured Feta-like cheese, 268–9 properties of UF retentates: buffering capacity, 265–6 rennet coagulation, 266–7 rheological behaviour, 266

Water-soluble extract (WSE), 493–4, 496 Whey, 105 heat treatment, 9 incubation, 9 preparation, 127 separation/syneresis, 114–15

Viscoplastic material behaviour, 540 Viscosity/dynamic viscosity, 540 Volatile compounds, 327–8 alcohols, 331–2 carbonyl compounds, 330–1 esters, 332 lactones, hydrocarbons, 334–5 sulphur compounds, 332–4 volatile fatty acids (VFA), 328–30

Yeast, 36, 191, 306–307, 597 forms/use as adjunct culture, 193 interactions with other microorganisms, 192–3 species found in cheeses, 191–2 useful properties in selecting adjuncts: effect on appearance of cheese surface, 192 lipolytic activity, 192 production of aroma, 192 proteolytic activity, 192 utilisation of residual sugars/lactate de-acidification activity, 192 Young’s modulus, 540


Plate 1 An arrangement of multi-channel geometry ceramic membranes (courtesy of GEA filtration, Hudson, WI, USA). (See page 263.)

Plate 2 Plate and frame UF system (courtesy of GEA filtration, Hudson, WI, USA). (See page 263.)

Plate 3 Spiral-wound UF membranes (courtesy of GEA filtration, Hudson, WI, USA). (See page 264.)

Plate 4 Vibrating membrane system (courtesy of Pall Corporation, Portsmouth, UK). (See page 264.)

Plate 5 Commercial UF system for the production of fresh cheese from pH 4.6 milk (courtesy of TIA, Bollene, France). (See page 270.)

Plate 6 A selection of cheeses made by UF using the liquid pre-cheese concept. (See page 272.)

Plate 7 A UF plant for producing Feta cheese by the MMV process (courtesy of TIA, Bollene, France). (See page 272.)

Plate 8 Farm with an UF facility in Dexter, New Mexico, USA (courtesy of North American Milk Products, LLC, St Louis, Missouri, USA). (See page 276.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Plate 9 Urea-polyacrylamide gel electrophoretograms water-insoluble fraction of a selection of cheese varieties. Lane 1 Na caseinate, lane 2 Cheddar, lane 3 extra-mature Cheddar, lane 4 Cheshire, lane 5 Red Leicester, lane 6 Double Gloucester, lane 7 Emmental, lane 8 Leerdammer, lane 9 Jarlsberg, lane 10 Vorarlberger Bergkase, lane 11 Edam, lane 12 Gouda, lane 13 Norvegia, lane 14 Parmesan, lane 15 Parmesan (from McGoldrick, 1996). (See page 416.)

1 1

Chymosin

199 Chymosin

23

199

24

Lc-CEP

102

199

f1-9, f-1-13 Further hydrolysis products

Plate 10 Schematic representation of the early proteolysis of s1-casein during the ripening of many cheeses and the location of peptides produced on a urea-polyacrylamide gel electrophoretogram and a reverse-phase HPLC elution profile. (See page 419.)

(a)

(b)

Plate 11 Vane rheometer probe before (a) and during (b) shear test on process cheese. Photos courtesy of Truong and Daubert (2000) Gel Consultants Inc. (See page 531.)

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