Cheese Chemistry, Physics and Microbiology Volume 1
General Aspects
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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:
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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.
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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
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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
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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
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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
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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
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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
4 Cheese: An Overview
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.
Cheese: An Overview 5
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
6 Cheese: An Overview
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
Cheese: An Overview 7
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
8 Cheese: An Overview
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
Cheese: An Overview 9
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.
10 Cheese: An Overview
• 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.
Cheese: An Overview 11
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
12 Cheese: An Overview
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
14 Cheese: An Overview
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).
Cheese: An Overview 15
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
16 Cheese: An Overview
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
Cheese: An Overview 17
References Alexander, J. (1910). Some colloid – chemical aspects of digestion, with ultramicroscopic observations. J. Am. Chem. Soc. 32, 680–687. Alexander, J. (1912). The rennin coagulation of milk from a colloid – chemical standpoint. Proc. 8th Int. Congress Appl. Chem. pp. 12–14. Anifantakis, E.M. (1991). Greek Cheeses: A Tradition of Centuries. National Dairy Committee of Greece, Athens. Berger, W., Klostermeyer, H., Merkenich, K. and Uhlmann, G. (1989). Die Schmalzkäscherstellung. BK Ladenburg, Ladenburg, Germany. Berridge, N.J. (1942). The second phase of rennet coagulation. Nature 149, 194–195. Beuvier, E., Berthaud, K., Cegarra, S., Dasen, A., Pochet, S., Buchin, S. and Duboz, G. (1997). Ripening and quality of Swiss-type cheese made from raw, pasteurized or microfiltered milk. Int. Dairy J. 7, 311–323. Buch Kristensen, J.M. (1995). Cheese Technology – A Northern European Approach. International Dairy Books, Aarhus, Denmark. Burkhalter, G. (1981). Catalogue of Cheeses. Document 141. International Dairy Federation, Brussels. Cantin, C. (1976). Guide Pratique des Fromages. Solar Editeur, Paris. Cheke, V. (1959). The Story of Cheesemaking in Britain. Routledge & Kegan Paul, London. Christian, G. (1984). World Guide to Cheese (English translation by S. Harris). Arnoldo Mondadori Editore S.p.A., Milan. Davies, F.L. and Law, B.A., eds (1984). Advances in the Microbiology and Biochemistry of Cheese and Fermented Milk. Elsevier, London. Davis, J.G. (1965). Cheese, Vol. 1, Basic Technology: Vol. 2: Bibliography. Churchill Livingstone, London. Davis, J.G. (1967). Cheese, Vol. III, Manufacturing Methods: Vol. IV: Bibliography. Churchill Livingstone, London, 1976, Paddington Press Ltd., London. De Koning, P.J. (1979). Rennet and its substitutes. Document 126. International Dairy Federation, Brussels. pp. 11–15. Delfour, A., Jolles, J., Alais, C. and Jolles, P. (1965). Caseino-glycopeptides: characterization of a methionine residue and of the N-terminal sequence. Biochem. Biophys. Res. Commun. 19, 452–455. Eck, A., ed. (1984). Le Fromage. Lavoisier, Paris. Eck, A. and Gilles, J.-C. (2000). Cheesemaking from Science to Quality Assurance. Technique and Documentation, Paris. Eekhof-Stork, N. (1976). World Atlas of Cheese. Paddington Press Ltd, London. Ernstrom, C.A. and Wong, N.P. (1974). Milk clotting enzymes and cheese chemistry, in, Fundamentals of Dairy Chemistry, 2nd edn, Webb, B.H., Johnson A.H. and Alford, J.A., eds, AVI Publishing Co. Inc., Westport, CT. pp. 662–771. Fox, P.F., ed. (1982). Developments in Dairy Chemistry – 1 – Proteins. Applied Science Publishers, London. Fox, P.F., ed. (1983). Developments in Dairy Chemistry – 2 – Lipids. Applied Science Publishers, London. Fox, P.F., ed. (1985). Developments in Dairy Chemistry – 3 – Lactose and Minor Constituents. Elsevier, London.
Fox, P.F., ed. (1987). Cheese: Chemistry, Physics and Microbiology, Vols 1 and 2. Elsevier, London. Fox, P.F., ed. (1992). Advanced Dairy Chemistry, Vol. 1, Proteins. Elsevier, London. Fox, P.F., ed. (1993). Cheese: Chemistry, Physics and Microbiology, Vols 1 and 2, 2nd ed. Chapman & Hall, London. Fox, P.F., ed. (1995). Advanced Dairy Chemistry, Vol. 2, Lipids. Chapman & Hall, London. Fox, P.F., ed. (1997). Advanced Dairy Chemistry, Vol. 3, Lactose, Water, Salts and Vitamins. Chapman & Hall, London. Fox, P.F. and McSweeney, P.L.H. (1997). Rennets: their role in milk coagulation and cheese ripening, in, Microbiology and Biochemistry of Cheese and Fermented Milk, 2nd edn, Law, B.A., ed., Blackie Academic and Professional, Glasgow. pp. 1–49. Fox, P.F. and McSweeney, P.L.H. (1998). Dairy Chemistry and Biochemistry. Blackie Academic and Professional, London. Fox, P.F. and McSweeney, P.L.H., eds (2003). Advanced Dairy Chemistry, Vol. 1, Proteins. Kluwer Academic/Plenum Publishers, New York. Fox, P.F., Guinee, T.P., Cogan, T.M. and McSweeney, P.L.H. (2000). Fundamentals of Cheese Science. Aspen Publishers, Gaithersburg, MD. Fraser, S. (1960). The Cheeses of Old England. Abelard-Schuman, London. Gonzalez, M.A. and del Cerro, C.G. (1988). Quesos de España. Espasa-Calpe, S.A., Madrid. Green, M.L. (1977). Milk coagulants. J. Dairy Res. 44, 159–188. Harbutt, J. (1999). A Cook’s Guide to Cheese. Anness Publishing Ltd, London. Harbutt, J. (2002). The World Encyclopedia of Cheese. Anness Publishing Ltd, London. Hoffmann, F. (1761). A Treatise on the Virtues and Uses of Whey. L. Davis and C. Reymers, London. pp. 1–34. Jenkins, S. (1996). Cheese Primer. Workman Publishing, New York. Kammerlehner, J. (2003). Käsetechnologie. Verlag Reisinger Kuenstlerpresse W Bode, Frising. Kosikowski, F.V. (1977). Cheese and Fermented Milk Foods. Edwards Bros, Inc., Ann Arbor, MI. Kosikowski, F.V. and Mistry, V.V. (1997). Cheese and Fermented Milk Foods, Vols 1 and 2, 3rd edn, Kosikowski, F.V., ed., LLC, Westport, CT. Kosikowski, F.V. and Mocquot, G. (1958). Advances in Cheese Technology. FAO Studies 38, FAO, Rome. Law, B.A., ed. (1997). Microbiology and Biochemistry of Cheese and Fermented Milk, 2nd edn, Blackie Academic and Professional, London. Law, B.A., ed. (1999). The Technology of Cheesemaking. Sheffield Academic Press, Sheffield. Layton, J.A. (1973). The Cheese Handbook. Dover Publications Inc., New York. Linderstrøm-Lang, K. (1929). Studies on casein. III. On the fraction of casein. Comptes-rendus des Travaux du Laboratoire Carlsberg 17(9), 1–116. Lynch, C.M., Muir, D.D., Banks, J.M., McSweeney, P.L.H. and Fox, P.F. (1999). Influence of adjunct cultures of Lactobacillus paracasei ssp. paracasei or Lactobacillus plantarum on Cheddar cheese ripening. J. Dairy Sci. 82, 1618–1628.
18 Cheese: An Overview
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
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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, a N, b N, c N, d N, qN and rN in the N-terminal domain and aC, bC, cC, dC, a C, b C, c C, d C, 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, h N, hC and h C occur in topological intra- and interdomain two-fold symmetry in that they all occur after the d strands. The fifth helix occurs between the c N and d N strands and the sixth occurs at a large insertion in the C-terminal domain.
22 Rennets: General and Molecular Aspects
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).
Rennets: General and Molecular Aspects 23
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
24 Rennets: General and Molecular Aspects
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
Rennets: General and Molecular Aspects 25
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
4CMS 4PEP 2APP 2APR 4APE YPA
40
AEFDGILGMAYPSLASE---YSIPVFDNMMNRHLVAQDLFSVYMDRNG----QESMLTLG APFDGILGLAYPSISAS---GATPVFDNLWDQGLVSQDLFSVYLSSNG---DSGSVVLLG TNNDGLLGLAFSSINTVQPQSQTTFFDTVKSS-L-AQPLFAVALKHQ-----QPGVYDFG -PNDGLLGLGFDTITTVR--GVKTPMDNLISQGLISRPIFGVYLGKAKN--GGGGEYIFG STIDGLLGLAFSTLNTVSPTSQQTFFDNAKA-S-LDSPVFTADLGY-----HAPGTYNFG GKFDGILGLGYDTISVD---KVVPPFYNAIQQDLLDEKRFAFYLGDTSKDTENGGEATFG
170
4CMS 4PEP 2APP 2APR 4APE YPA
30
FDPRKSSTFQNL-GKPLSIHYGT-GSMQGILGYDTVTVSNIVDIQQTVGLSTQEPGDVFTY FNPQDSSTYQST-SGELSITYGT-GSMTGILGYDTVQVGGISDTNQIFGLSETEPGSFLYY YNPSA--TGKELSGYTWSISYGDGSSASGNVFTDSVTVGGVTAHGQAVQAAQQISAQFQQD YDPNQSSTYQAD-GRTWSISYGDGSSASGILAKDNVNLGGLLIKGQTIELAKREAASFASG YTPSKSTSTKLLSGATWSISYGDGSSSSSDVYTDTVSVGGLTVTGQAVESAKKVSSSFTED YDHEASSSYKAN-GTEFAIQYGTG-SLEGYISQDTLSIGDLTIPKQDFAEATSEPGLTFAF Strand d´ 120
4CMS 4PEP 2APP 2APR 4APE YPA
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
26 Rennets: General and Molecular Aspects
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
Rennets: General and Molecular Aspects 27
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
28 Rennets: General and Molecular Aspects
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).
Rennets: General and Molecular Aspects 29
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
30 Rennets: General and Molecular Aspects
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
Rennets: General and Molecular Aspects 31
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
32 Rennets: General and Molecular Aspects
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
Rennets: General and Molecular Aspects 33
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
34 Rennets: General and Molecular Aspects
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
Rennets: General and Molecular Aspects 35
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
36 Rennets: General and Molecular Aspects
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
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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
50 Rennet-induced Coagulation of Milk
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
Rennet-induced Coagulation of Milk 51
(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
52 Rennet-induced Coagulation of Milk
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).
Rennet-induced Coagulation of Milk 53
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
54 Rennet-induced Coagulation of Milk
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
Rennet-induced Coagulation of Milk 55
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.
56 Rennet-induced Coagulation of Milk
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.
Rennet-induced Coagulation of Milk 57
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
58 Rennet-induced Coagulation of Milk
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.
Rennet-induced Coagulation of Milk 59
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,
60 Rennet-induced Coagulation of Milk
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)
Rennet-induced Coagulation of Milk 61
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
62 Rennet-induced Coagulation of Milk
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
Rennet-induced Coagulation of Milk 63
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
Rennet-induced Coagulation of Milk 65
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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Van Hooydonk, A.C.M. and Walstra, P. (1987). Interpretation of the kinetics of the renneting reaction in milk. Neth. Milk Dairy J. 41, 19–47. Van Hooydonk, A.C.M., Boerrigter, I.J. and Hagedoorn, H.G. (1986). pH-induced physico-chemical changes of casein micelles in milk and their effect on renneting. 1. Effect of pH on renneting of milk. Neth. Milk Dairy J. 40, 297–313. Van Hooydonk, A.C.M., de Koster, P.C. and Boerrigter, I.J. (1987). The renneting properties of heated milk. Neth. Milk Dairy J. 41, 3–18. Van Vliet, T. and Walstra, P. (1985). Note on the shear modulus of rennet-induced milk gels. Neth. Milk Dairy J. 39, 115–118. Van Vliet, T., Van Dijk, H.J.M., Zoon, P. and Walstra, P. (1991). Relation between syneresis and rheological properties of particle gels, Colloid Polym. Sci. 269, 620–627. Verissimo, P.C., Esteves, C.L.C., Faro, C.J.F. and Pires, E.M.V. (1995). The vegetable rennet of Cyanara cardunculus L. contains two proteinases with chymosin and pepsin-like specificities. Biotechnol. Lett. 17, 621–626. Verwey, E.J.W. and Overbeek, J.Th.G. (1948). Theory of Stability of Lyophobic Colloids, Elsevier, Amsterdam. Visser, S., Van Roijen, P.J. and Slangen, C.J. (1980). Peptide substrates for chymosin (rennin). Isolation and substrate behaviour of two tryptic fragments of bovine -casein. Eur. J. Biochem. 108, 415–421. Vreeman, H.J. (1979). The association of bovine SH--casein at pH 7.0. J. Dairy Res. 46, 271–276. Vreeman, H.J., Both, P., Brinkhuis, J.A. and Van der Spek, C. (1977). Purification and some physicochemical properties of bovine -casein. Biochim. Biophys. Acta 491, 93–103. Vreeman, H.J., Visser, S., Slangen, C.J. and Van Riel, J.A.M. (1986). Characterization of bovine -casein fractions and the kinetics of chymosin-induced macropeptide release from carbohydrate-free and carbohydrate-containing fractions determined by high performance gel permeation chromatography. Biochem. J. 240, 87–97. Walstra, P. (1979). The voluminosity of casein micelles and some of its implication. J. Dairy Res. 46, 317–323. Walstra, P. and Van Vliet, T. (1986). The physical chemistry of curd making. Neth. Milk Dairy J. 40, 241–259. Walstra, P., Bloomfield, V.A., Wei, G.J. and Jenness, R. (1981). Effect of chymosin action on the hydrodynamic
diameter of casein micelles. Biochim. Biophys. Acta 669, 258–259. Waungana, A., Singh, H. and Bennett, R.J. (1996). Influence of denaturation and aggregation of beta-lactoglobulin on rennet coagulation properties of skim milk and ultrafiltered milk. Food Res. Int. 29, 715–721. Whitney, R.McL. (1988). Proteins of milk, in, Fundamentals of Dairy Chemistry, 3rd edn, N.P. Wong, ed., Avi Books, Van Norstrand Reinhold, New York. pp. 81–169. Wigley, R.C. (1996). Cheese and whey, in, Industrial Enzymology, 2nd edn, T. Godfrey and S. West, eds, MacMillan Press, London. pp. 133–154. Winter, H.H. and Chambon, F. (1986). Analysis of crosslinked polymer at the gel-point. J. Rheology 30, 367–382. Worning, P., Bauer, R., Øgendal, L. and Lomholt, S. (1998). A novel approach to turbidimetry of dense systems. An investigation of the enzymatic gelation of casein micelles. J. Colloid Interf. Sci. 203, 265–277. Zevaco, C. and Ribadeau-Dumas, B. (1984). A study of the carbohydrate binding sites of bovine -casein using high performance liquid chromatography. Milchwissenschaft 39, 206–210. Ziff, R.M. (1980). Kinetics of polymerization. J. Stat. Phys. 23, 241–263. Ziff, R.M. and Stell, G. (1982). Kinetics of polymer gelation. J. Chem. Phys. 73, 3492–3499. Zoon, P., Van Vliet, T. and Walstra, P. (1988a). Rheological properties of rennet-induced skim milk gels. 1. Introduction. Neth. Milk Dairy J. 42, 249–269. Zoon, P., Van Vliet, T. and Walstra, P. (1988b). Rheological properties of rennet-induced skim milk gels. 2. Effect of temperature. Neth. Milk Dairy J. 42, 271–294. Zoon, P., Van Vliet, T. and Walstra, P. (1988c). Rheological properties of rennet-induced skim milk gels. 3. Effect of calcium and phosphate. Neth. Milk Dairy J. 42, 295–312. Zoon, P., Van Vliet, T. and Walstra, P. (1989a). Rheological properties of rennet-induced skim milk gels. 4. The effect of pH and NaCl. Neth. Milk Dairy J. 43, 17–34. Zoon, P., Van Vliet, T. and Walstra, P. (1989b). Rheological properties of rennet-induced skim milk gels. 5. Behaviour at large deformation. Neth. Milk Dairy J. 43, 35–42.
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
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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
74 The Syneresis of Rennet-coagulated Curd
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 a 1/(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. a2 2/(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
The Syneresis of Rennet-coagulated Curd 75
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
76 The Syneresis of Rennet-coagulated Curd
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 G 2 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).
The Syneresis of Rennet-coagulated Curd 77
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
78 The Syneresis of Rennet-coagulated Curd
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
The Syneresis of Rennet-coagulated Curd 79
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
80 The Syneresis of Rennet-coagulated Curd
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).
The Syneresis of Rennet-coagulated Curd 81
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.
82 The Syneresis of Rennet-coagulated Curd
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
The Syneresis of Rennet-coagulated Curd 83
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).
84 The Syneresis of Rennet-coagulated Curd
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).
The Syneresis of Rennet-coagulated Curd 85
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).
86 The Syneresis of Rennet-coagulated Curd
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).
The Syneresis of Rennet-coagulated Curd 87
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.
88 The Syneresis of Rennet-coagulated Curd
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
The Syneresis of Rennet-coagulated Curd 89
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)).
90 The Syneresis of Rennet-coagulated Curd
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
The Syneresis of Rennet-coagulated Curd 91
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
92 The Syneresis of Rennet-coagulated Curd
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
The Syneresis of Rennet-coagulated Curd 93
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)
94 The Syneresis of Rennet-coagulated Curd
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).
The Syneresis of Rennet-coagulated Curd 95
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,
96 The Syneresis of Rennet-coagulated Curd
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 Syneresis of Rennet-coagulated Curd 97
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).
98 The Syneresis of Rennet-coagulated Curd
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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Unger Grundelius, A., Lodaite, K., Östergren, K., Paulsson, M. and Dejmek, P. (2000). Syneresis of submerged single curd grains and curd rheology. Int. Dairy J. 10, 489–496. Vaikus, V., Lubinskas, V. and Mitskevichus, B. (1970). The effect of homogenization on the properties of the acidand rennet-gels of milk. Proc. 18th Int. Dairy Congress, Sydney, Vol. 1E. p. 320. van de Grootevheen, J.G. and Geurts, T.J. (1977). Het Instellen van het Vochtgehalte van Verse Ongezouten Wrongel, Wageningen (unpublished). van den Bijgaart, H.J.C.M. (1988). Syneresis of RennetInduced Milk Gels as Influenced by Cheese-making Parameters. Doctoral Thesis, Wageningen Agricultural University, Wageningen. van der Waarden, M. (1947). Onderzoek naar de factoren die invloed hebben op de wei- uittreding uit gestremde melk, Alg. Ned. Zuivelbond FNZ, The Hague (unpubIished). van Dijk, H.J.M. (1982). Syneresis of Curd. Doctoral Thesis, Wageningen Agricultural University, Wageningen. 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 Dijk, H.J.M., Walstra, P. and Geurts, T.J. (1979). Preliminary note on syneresis pressure in rennet curd. Neth. Milk Dairy J. 33, 60–61. van Dijk, H.J.M., Walstra, P. and Schenk, J. (1984). Theoretical and experimental-study of one-dimensional syneresis of a protein gel. Chem. Eng. J. 28, B43–B50. van Hooydonk, A.C.M. and van den Berg, G. (1988). Control and determination of the curd-setting during cheesemaking. Bulletin 225, International Dairy Federation, Brussels, Vol. 225. pp. 2–10. van Hooydonk, A.C.M. and Walstra, P. (1987). Interpretation of the kinetics of the renneting reaction in milk. Neth. Milk Dairy J. 41, 19–47. van Hooydonk, A.C.M., Boerrigter, I.J. and Hagedoorn, H.G. (1986). pH-induced physicochemical changes of casein micelles in milk and their effect on renneting. 2. Effect of pH on renneting of milk. Neth. Milk Dairy J. 40, 297–313. 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 properties of particle gels. Colloid Polym. Sci. 269, 620–627. Vas, K. (1931). The specific weight, the water content and the role of heating (scalding) in Emmentaler cheesemaking. Milchw. Forsch. 11, 519–529. Vasbinder, A.J., van Mill, P.J.J.M., Bot, A. and de Kruif, K.G. (2001). Acid induced gelation of heat treated milk studied by diffusing wave spectroscopy. Colloid. Surf. B 21, 245–251. Vasbinder, A.J., Alting, A.C., Visschers, R.W. and de Kruif, C.G. (2003). Texture of acid milk gels: formation of disulfide cross-links during acidification. Int. Dairy J. 13, 29–38. Walstra, P. (1990). On the stability of casein micelles. J. Dairy Sci. 73, 1965–1979. Walstra, P. (2000). Fractal particle gels in foods, in, Supramolecular and Colloidal Structures in Biomaterials
and Biosubstrates, Lal, M., Lillford, P.J., Naik, V.M. and Prakash, V., eds, Imperial College Press and The Royal Society, London. pp. 157–173. Walstra, P. (2003). Studying colloids: past, present and future, in, Food Colloids: Biopolymers and Materials, Dickinson, E. and van Vliet, T., eds, Royal Society of Chemistry, Cambridge. pp. 391–400. Walstra, P. and Jenness, R. (1984). Dairy Chemistry and Physics. John Wiley & Sons, New York. Walstra, P. and van Vliet, T. (1986). The physical chemistry of cheesemaking. Neth. Milk Dairy J. 40, 241–259. Walstra, P., van Dijk, H.J.M. and Geurts, T.J. (1985). Syneresis of curd. 1. General-considerations and literaturereview. Neth. Milk Dairy J. 39, 209–246. Walstra, P., van Vliet, T. and Bremer, L.G.B. (1990). On the fractal nature of particle gels, in, Food Polymers, Gels and Colloids, Dickinson, E., ed., Royal Society of Chemistry, London. pp. 369–382. Weber, F. (1984). Drainage of curd, in, Le Fromage, Eck, A., ed., Lavoisier, Paris. pp. 22–36. Whitehead, H.R. (1948). Control of the moisture content and body-firmness of Cheddar cheese. J. Dairy Res. 15, 387–397. Whitehead, H.R. and Harkness, W.L. (1954). The influence of variation in cheesemaking procedure on the expulsion of moisture from Cheddar cheese curd. Aust. J. Dairy Technol. 9, 103–107. Whittle, M. and Dickinson, E. (1998). Large deformation rheological behaviour of a model particle gel. Faraday Trans. 94, 2453–2462. Worning, P., Bauer, R., Ögendahl, L. and Lomholt, S.B. (1998). A novel approach to turbidimetry of dense systems: an investigation of the enzymatic gelation of casein micelles. J. Colloid Interf. Sci. 203, 265–277. Wurster, K. (1934). Whey release in renneting. Milchw. Forsch. 16, 200–214. Zoon, P., van Vliet, T. and Walstra, P. (1988a). Rheological properties of rennet-induced skim milk gels. 1. Introduction. Neth. Milk Dairy J. 42, 249–269. Zoon, P., van Vliet, T. and Walstra, P. (1988b). Rheological properties of rennet-induced skim milk gels. 2. The effect of temperature. Neth. Milk Dairy J. 42, 271–294. Zoon, P., van Vliet, T. and Walstra, P. (1988c). Rheological properties of rennet-induced skim milk gels. 3. The effect of calcium and phosphate. Neth. Milk Dairy J. 42, 295–312. Zoon, P., van Vliet, T. and Walstra, P. (1989a). Rheological properties of rennet-induced skim milk gels. 4. The effect of pH and NaCl. Neth. Milk Dairy J. 43, 17–34. Zoon, P., van Vliet, T. and Walstra, P. (1989b). Rheological properties of rennet-induced skim milk gels. 5. Behaviour at large deformation. Neth. Milk Dairy J. 43, 35–52. Zoon, P., Roefs, S.P.F.M., de Cindio, B. and van Vliet, T. (1990). Rheological properties of skim milk gels at various temperatures – interrelation between the dynamic moduli and the relaxation modulus. Rheol. Acta 29, 223–230.
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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
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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
108 Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels
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
Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels 109
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)
110 Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels
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
Storage and loss modulus, Pa
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).
Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels 111
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
Storage and loss modulus, Pa
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.
112 Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels
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
Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels 113
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,
114 Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels
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
Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels 115
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
116 Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels
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
Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels 117
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
118 Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels
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
Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels 119
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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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
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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
126 Starter Cultures: General Aspects
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
128 Starter Cultures: General Aspects
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
Starter Cultures: General Aspects 129
(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
130 Starter Cultures: General Aspects
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
Starter Cultures: General Aspects 131
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)).
132 Starter Cultures: General Aspects
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
Starter Cultures: General Aspects 133
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
134 Starter Cultures: General Aspects
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
Starter Cultures: General Aspects 135
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).
136 Starter Cultures: General Aspects
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
Starter Cultures: General Aspects 137
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
138 Starter Cultures: General Aspects
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
Starter Cultures: General Aspects 139
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
140 Starter Cultures: General Aspects
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
Starter Cultures: General Aspects 141
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
142 Starter Cultures: General Aspects
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
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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
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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
152 Starter Cultures: Genetics
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
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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).
154 Starter Cultures: Genetics
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
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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
156 Starter Cultures: Genetics
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
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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
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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
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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
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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;
166 Starter Cultures: Bacteriophage
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).
Starter Cultures: Bacteriophage 167
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.
168 Starter Cultures: Bacteriophage
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.
Starter Cultures: Bacteriophage 169
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,
170 Starter Cultures: Bacteriophage
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
Starter Cultures: Bacteriophage 171
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
172 Starter Cultures: Bacteriophage
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.
Starter Cultures: Bacteriophage 173
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
174 Starter Cultures: Bacteriophage
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
Starter Cultures: Bacteriophage 175
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
176 Starter Cultures: Bacteriophage
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
Starter Cultures: Bacteriophage 177
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
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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
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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
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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
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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
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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.
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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
194 Secondary and Adjunct Cultures
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
Secondary and Adjunct Cultures 195
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
Phenotypic from Seiler (1986)
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
Phenotypic from Seiler (1986)
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
Secondary and Adjunct Cultures 197
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; Vald`es-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) (Vald`es-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
198 Secondary and Adjunct Cultures
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
Secondary and Adjunct Cultures 199
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
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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
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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
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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
Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 1: General Aspects ISBN: 0-1226-3652-X Set ISBN: 0-1226-3651-1
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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).
210 Salt in Cheese: Physical, Chemical and Biological Aspects
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).
Salt in Cheese: Physical, Chemical and Biological Aspects 211
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
212 Salt in Cheese: Physical, Chemical and Biological Aspects
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
Salt in Cheese: Physical, Chemical and Biological Aspects 213
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
214 Salt in Cheese: Physical, Chemical and Biological Aspects
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
Salt in Cheese: Physical, Chemical and Biological Aspects 215
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).
216 Salt in Cheese: Physical, Chemical and Biological Aspects
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
Salt in Cheese: Physical, Chemical and Biological Aspects 217
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;
218 Salt in Cheese: Physical, Chemical and Biological Aspects
• 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
Salt in Cheese: Physical, Chemical and Biological Aspects 219
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,
220 Salt in Cheese: Physical, Chemical and Biological Aspects
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
Salt in Cheese: Physical, Chemical and Biological Aspects 221
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,
222 Salt in Cheese: Physical, Chemical and Biological Aspects
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
Salt in Cheese: Physical, Chemical and Biological Aspects 223
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
224 Salt in Cheese: Physical, Chemical and Biological Aspects
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)
Salt in Cheese: Physical, Chemical and Biological Aspects 225
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).
226 Salt in Cheese: Physical, Chemical and Biological Aspects
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
Salt in Cheese: Physical, Chemical and Biological Aspects 227
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.
228 Salt in Cheese: Physical, Chemical and Biological Aspects
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
Salt in Cheese: Physical, Chemical and Biological Aspects 229
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
232 Salt in Cheese: Physical, Chemical and Biological Aspects
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).
Salt in Cheese: Physical, Chemical and Biological Aspects 233
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
234 Salt in Cheese: Physical, Chemical and Biological Aspects
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).
Salt in Cheese: Physical, Chemical and Biological Aspects 235
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).
236 Salt in Cheese: Physical, Chemical and Biological Aspects
(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
Salt in Cheese: Physical, Chemical and Biological Aspects 237
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
238 Salt in Cheese: Physical, Chemical and Biological Aspects
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
Salt in Cheese: Physical, Chemical and Biological Aspects 239
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
240 Salt in Cheese: Physical, Chemical and Biological Aspects
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.
Salt in Cheese: Physical, Chemical and Biological Aspects 241
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).
242 Salt in Cheese: Physical, Chemical and Biological Aspects
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).
Salt in Cheese: Physical, Chemical and Biological Aspects 243
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
244 Salt in Cheese: Physical, Chemical and Biological Aspects
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
Salt in Cheese: Physical, Chemical and Biological Aspects 245
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
246 Salt in Cheese: Physical, Chemical and Biological Aspects
‘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.
Salt in Cheese: Physical, Chemical and Biological Aspects 247
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
248 Salt in Cheese: Physical, Chemical and Biological Aspects
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).
Salt in Cheese: Physical, Chemical and Biological Aspects 249
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 re
