Cheese: Chemistry, Physics & Microbiology

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.E Olson Department of Food Science, University of Wisconsin, Madison

List of Contributors

Dr J.M. Banks

Ms Y.E Collins

CHARIS Food Research Hannah Research Institute Ayr KA6 5HL Scotland

Dairy Products Research Centre Teagasc, Moorepark Fermoy Cork Ireland

Dr T. Beresford

Dairy Products Research Centre Teagasc, Moorepark Fermoy Cork Ireland Dr E. Beuvier

Station de Recherches en Technologie et Analyses Laitieres Institut National de La Recherche Agronomique F-39801 Poligny Cedex France Dr S. Buchin

Station de Recherches en Technologie et Analyses Laitieres 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.-E 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

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 A.C. Curtin

Department of Food and Nutritional Sciences University College Cork Ireland Professor E 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. D o b s o n 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

Dr A.A.A. Magboul

Department of Food Science North Carolina State University Campus Box 7624 Raleigh NC 27695-7624 USA

DAL Food Industries Industrial Area No. 1/15 Block 4F Khartoum North, PO Box 708 Sudan

Dr G.E Fitzgerald

National Food Biotechnology Centre Departments of Microbiology and Food & Nutritional Sciences University College Cork Ireland Professor P.E 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

Professor J.-L. Maubois Laboratoire de Recherches kaiti~res 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

Dr E Irlinger

Institut National de La Recherche Agronomique 78850 Thiverval-Grignon Cedex France Dr J.-L. Le Qu~r~

Institut National de la Recherche Agronomique Unit6 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

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

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

Professor P. Walstra

Dipartimenta Biologia Universit/~ della Basilicata Campus di Macchia Romana 85100 Potenza Italy

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

Department of Life Sciences University of Limerick Castletroy Limerick Ireland

Dr T.P. O'Connor

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

Dr M.G. W i l k i n s o n

Professor A. Williams

CHARIS Food Research Hannah Research Institute Ayr KA6 5HL Scotland

xi

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 NaC1. 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 - 1 0 7 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 ultrafihration 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, theology 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. RE Fox

Preface to the Second Edition

The first edition of this book was very well received by the various groups (lecturers, students, researchers and industrialists) interested in the scientific and technological aspects of cheese. The initial printing was sold out faster than anticipated and created an opportunity to revise and extend the book. The second edition retains all 21 subjects from the first edition, generally revised by the same authors and in some cases expanded considerably. In addition, l0 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.

RE Fox

Preface to the Third Edition

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

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

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

Milk is also a rich source of nutrients for bacteria which contaminate the milk, some species of which utilize milk sugar, lactose, as a source of energy, producing lactic acid. Bacterial growth and acid production would have occurred during storage or during attempts to dry milk in the prevailing warm, dry climate to produce a more stable p r o d u c t - 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 p o i n t s - --~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 e v e n t s - 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 9 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 furore 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 quantifies 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

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-

3

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

'Some degree of heat' I F (Stand not far from the fire) Coagulum

Drain whey quickly when milk coagulated using wicker baskets or moulds. Aid whey drainage using weights

j WHEY

Curds

~'

I Place cheese in a cool shady place Surface application of dry salt

l

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' I Cheese can be 'exported beyond the sea'

I

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 pressure) 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 ContrOl~e). 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

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 c e n t u r y - 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 Schwangenk&se Grana Taleggio

897 1000 1070 1174 1178 1200 1282

Cheddar Parmesan Gouda Gloucester Stilton Camembert St Paulin

1500 1579 1697 1783 1785 1791 1816

5

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 O v e r v i e w

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 uhrafiltration and nutritional aspects, which apply, more or less, to most cheese varieties, are considered

Milk

Manufacture (5-24 h)

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:

Cheese

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

Milk

Acidification

= Coagulum

Cut/break

(biological chemical) or

Cold-pack cheese ~

~ Curds and whey

1 Cook Separate Wash Curds

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:

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

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).

7

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 Clostridiurn 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 CI. 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 ~ 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~ 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 Ca 2+ 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 CaC12. 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-g-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 K-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 ~ 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 etal., 1999; 'Secondary and Adjunct Cultures' and 'The Microbiology of Cheese Ripening', Volume 1). Thermization (--65 ~ • 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

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 ~ • 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-g-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 ~ a starter consisting of Lactococcus lactis subsp. lactis and/or Lc. lactis subsp, cremoris is normally used while cultures of Streptococcus therrnophilus 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

9

used for varieties that are 'cooked' to higher temperatures, e.g., Swiss and hard Italian varieties. Probably the earliest form of starters were 'slopback' c u l t u r e s - 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.: 9 Coagulant activity during coagulation. 9 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. 9 Strength of the coagulum, which influences cheese yield.

10

Cheese: An Overview

9 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. 9 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). 9 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.

'Salt in Cheese: Physical, Chemical and Biological Aspects', Volume 1. In a few special cases, e.g., Domiati, a high level of NaC1 (10-12%) is added to the cheesemilk, traditionally to control the growth of the indigenous microflora. This concentration of NaC1 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).

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 NaC1 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 NaC1 in cheese will be discussed in

The majority of cheeses are produced by enzymatic (rennet) coagulation. With a few exceptions (e.g., Serra da Estrela (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.

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: 9 limited proteolysis by selected proteinases; 9 acidification to pH --~4.6; 9 acidification to about pH 5.2 in combination with heating to - 9 0 ~

Cheese: An Overview

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 ~ while the secondary phase has a Q10 of --~16 and occurs very slowly or not all at temperatures '

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lie

13

14 Cheese: An Overview During ripening, an extremely complex set of biochemical changes occur through the catalytic action of the following agents: 9 coagulant; 9 indigenous milk enzymes, especially plasmin and lipoprotein lipase, which are particularly important in cheese made from raw milk; 9 starter bacteria and their enzymes; 9 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 (]3-oxidation of fatty acids, esterification, formation of thioesters) and the catabolism of lactic acid to CO2 and H20 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.

C h e e s e Production and C o n s u m p t i o n World cheese production was ---16.5 • 100 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 - 2 8 % 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 O v e r v i e w

Table 2

15

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

9375O 102029 1020712 123000 3662 6750 210 3500 12400 21564 57900 1540 282 2058 153861 5250 1260 7716 31976 7O 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 Taji kistan 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 C6te d'lvoire 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

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 Schmalzkascherstellung. BK Ladenburg, Ladenburg, Germany. Berridge, NJ. (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. Burkhaher, 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). Amoldo Mondadori Editore S.p.A., Milan. Davies, EL. 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. andJolles, 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.E, ed. (1982). Developments in Dairy Chemistry- 1 Proteins. Applied Science Publishers, London. Fox, P.E, ed. (1983). Developments in Dairy Chemistry- 2 Lipids. Applied Science Publishers, London. Fox, P.E, ed. (1985). Developments in Dairy Chemistry- 3 Lactose and Minor Constituents. Elsevier, London.

17

Fox, P.E, ed. (1987). Cheese: Chemistry, Physics and Microbiology, Vols 1 and 2. Elsevier, London. Fox, P.E, ed. (1992). Advanced Dairy Chemistry, Vol. 1, Proteins. Elsevier, London. Fox, P.E, ed. (1993). Cheese: Chemistry, Physics and Microbiology, Vols 1 and 2, 2nd ed. Chapman & Hall, London. Fox, RE, ed. (1995). Advanced Dairy Chemistry, Vol. 2, Lipids. Chapman & Hall, London. Fox, P.E, ed. (1997). Advanced Dairy Chemistry, Vol. 3, Lactose, Water, Salts and Vitamins. Chapman & Hall, London. Fox, P.E 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.E and McSweeney, P.L.H. (1998). Dairy Chemistry and Biochemistry. Blackie Academic and Professional, London. Fox, P.E and McSweeney, P.L.H., eds (2003). Advanced Dairy Chemistry, Vol. 1, Proteins. Kluwer Academic/Plenum Publishers, New York. Fox, P.E, Guirlee, 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 Espafia. 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, E (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). Kdsetechnologie. Verlag Reisinger Kuenstlerpresse W Bode, Frising. Kosikowski, EV. (1977). Cheese and Fermented Milk Foods. Edwards Bros, Inc., Ann Arbor, MI. Kosikowski, EV. and Mistry, V.V. (1997). Cheese and Fermented Milk Foods, Vols 1 and 2, 3rd edn, Kosikowski, EV., ed., LLC, Westport, CT. Kosikowski, EV. 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. Linderstr~m-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.E (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 Ober 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.E, 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.E, Cogan, T.M., Drinan, ED. 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. IrishJ. Agric. Food Res. 33, 183-192. Meyer, A. (1973). Processed Cheese Manufacture. Food Trade Press, London. Montandon, J. (1981). Kdse 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, RE (1993). Some nonEuropean cheese varieties, in, Cheese: Chemistry, Physics and Microbiology, Vol. 1, 2nd edn, Fox, RE, 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 Faction 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 Faction de la pressure. Le Lait I0 (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, RL.H. and Fox, RE (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.E (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, RE (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.E (2000c). Influence of ripening temperature on the volatiles profile and flavour of Cheddar cheese made from raw or pasteurized milk. Int. DairyJ. 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). Forrnaggi 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.E and von Hippel, P.H. (1956). K-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, VL. 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 Universityof 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, or-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 (Fohmann, 1970), piglet (Fohmann 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 (Andr4n and Bj6rck, 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), ,5. cerevisiae proteinase A (N67 and N263), rhizomucor protease (N173) and human renin (N07). Specific receptors for phosphorylation have been found in porcine pepsin at $68 (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 (Fohmann, 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 ~ than at room temperature (Fohmann, 1959b). Kawaguchi etal. (1987) reported the rapid loss of the activity of chymosin when the temperature is increased from 45 to 55 ~ Photo-oxidation of histidine, as well as modification of the e-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/1 urea at 37 ~ 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 (Fohmann, 1992). Prochymosin is more stable than chymosin at neutral pH (Fohmann, 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 etal., 1986; Bailey and Siika-aho, 1988). Pepsin shows greater general stability than chymosin; for example, after incubation in 6 mol/1 urea at 37 ~ 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 etal., 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 proteolyric 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 E-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 (Fohmann, 1959b). Non-crystallized chymosin is soluble in solution containing 1 molA NaC1 and at pH 5.5. In a solution of >2 molA NaC1, chymosin appears to be insoluble. Crystallized chymosin shows higher solubility at 25 ~

Rennets: General and Molecular Aspects

than at 2 ~ (Fohmann, 1970); however, amorphous precipitates of chymosin are more stable at 2 ~ than at 25 ~ 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 etal., 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.

21

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 Games and Sielecki, 1986; Fohmann, 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 [3-hydroxy amino acid residues (Fohmann et al., 1977, 1979; Harris et al., 1982; Moir etal., 1982; Hidaka etal., 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 [3-sheets with a few small or-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, co, de, 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 [3-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 [3-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

PPI

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

PI0

P20

CTT

CTG

GAG

GAC

TTC

CTG

CAG

AAA

CAG

CAG

TAT

GGC

ATC

AGC

AGC

AAG

TAC

TCC

GGC

TTC

L

L

E

N

F

L

E

K

E

E

Y

G

I

S

S

K

Y

S

G

F

P30

G

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

i0

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

9O

i00

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

ii0

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

TAC

150

160

GCC

ATC

AAC

CCG

TCC

TAC

TAC

ACA

GGG

TCC

CTG

CAC

TGG

GTG

CCC

GTG

ACA

GTG

CAG

CAG

A

I

N

P

S

Y

Y

T

G

S

L

H

W

V

P

V

T

V

Q

Q

170

180

Y 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

V

G

L

A

K

A

I

320

Figure 1

* GGG

TGA

TCACATCGcTGACCA

...........

323

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

Rennets: General and Molecular Aspects

Secretion signal peptides Fungi A. A. R. R. R. R.

awamori 1 oryzae 2 miehei 3 pusillus 4 niveus 5 chinensis 6

Mammals Bovine chymosin 7 Lamb chymosin 8 Porcine pepsin 9 Human pepsin 1~ Rat pepsin1 Rat cathepsin D12

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

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

Yeast S. cerevisiae (YPA) 13 MFSLKALLPLALLLVSANQVAA*K S. cerevisiae (BAR1) TM M S A I N H L C L Y L I L A S F A I I N T I T A * L C. tropicalis15 M A T I F L F T K N V F I A . LA. FA. L

Plant Barley AP R16

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 etal. (1986); (5) Chen etaL (1991); (6) Horiuchi etaL (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 etaL (1989); (12) Birch and Loh (1990); (13) Ammerer etaL (1986); (14) MacKay etaL (1988); (15) Togni etal. (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 etal., 1989; Miller etal., 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 A resolution (Gilliland et al., 1990) and at 2.2 ft, resolution (Newman et al., 1991). Crystallographic studies at 2.0 A resolution have also been performed on a site-specific mutant of chymosin, in which Vtll 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.

23

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 A (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-A 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 etal., 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, 2 0 6 . . . 210 and 2 4 9 . . . 282. In addition, ion-pairs are found between R59... D57, R157... E308, R157. 9 9 1326 (COO-), R307... Dll and R315... D138 (Gilliland et al., 1990; Newman et al., 1991). Chymosin also contains a single cis-proline, P23, o n the w-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 etal., 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, Asp--Thr--Gly, 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 APR 2 A. miehei APR 3 M. pusillus APR 4 R. niveus APR 5 R. chinensis APR 6

APAPRTRKGFTINQIARPANKTRTINLPGMYARS

.......

LA-KFGGTVPQSVKEA-A*SK

LPTGPSHSPHARRGFTINQITRQTARVGPKTASFPAIYSRALA-KYGGTVPAKLKSAVA*GH RPVSKQSESKDKLLALPLTSVSRKFSQTKFGQQQ RPVSKQSDADDKLLALPLTSVNRKYSQTKHGQQ

.......

LAEKLAG

.....

LKPFSE*AA

........

AAEKLGG

.....

IK-A-F*AE

PNGKKINIPLAKNN

....

SY-KPSA--KNALNKA

......

LA-KYNRRKVGSGGITTE*AS

PGEKKISIPLAKNP

....

NY-KPSA--KNAIQKA

......

IA-KYNKHKINTSTGGIV*AG

Mammals AEITRIPLYKGKSLRKAL-KEHGLLE-DFLQKQQYG-ISSKYS ....... GF*GE Bovine prochymosin 7 AEITRIPLYKGKPLRKAL-KERGLLE-DFLQKQQYG-ISSEYS ....... GF*GE Lamb prochymosin 8 SIHRVPLKK GKSLRKQL-KDHGLLE-DFLKKHPYN-PASKYHPV ...... L*TA Chicken pepsinogen 9 LVKVPLVRKKSLRQNLIKD-GKLK-DFLKTHKHN-PASKYFPE---AAAL*IG Porcine pepsinogen10 IMYKVPLIRKKSLRRTL-SERGLLK-DFLKKHNLN-PARKYFPQWE-APTL*VD Human pepsinogenl 1 Human progastricsin12 AVVKVPLKKFKSIRETM-KEKGLLG-EFLRTHKYD-PASKYRFGD ..... L*SV TFSLPTRTATFERIPLKKMPSVREIL-EERG--V-DMIRLSAEWGVFTK Mouse prorenin 13 TFGLPTDTTTFKRIFLKRMPSIRESL-KERG--V-DMARLGPEWSQPMK Human prorenin14 SALVRIPLHKFTSIRRTM-SEVGGSVEDLIAK .... GPVSKYSQAV-PAVTE*GP Human procathepsin D 15 SALIRIPLRKFTSIRRTM-TEVGGSVGDLI .... LKGPITKYSMQSSPRTKE*PV Rat procathepsin D 16 Yeast KVHKAKIYKHELSDEMKEVTFEQHLAHLGQKYLTQFEKANPEVVFSREHPFFTE*GG S. cerevisiae APR 17 LAFALFAQGLTIPD ..... GIEKRTDKVVSLDFTVIRKPFNATAHR---LIQKR*SD C. tropicalis APR 18 Plant EGLVRIALKKRP-IDRNSRVATGLSGGEEQP---LLSG ...... AN---PLR*SE Barley APR 19

-R*PS -R*LT

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 etaL (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 F]]9 and L32 (Gilliland et al., 1990). In other aspartic proteinases, Y77 hydrogenbonds to W39. In pepsin, the location of the hydroxyl group of Yrr 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 VlllF 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 S 1 and $3 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 5. cerevisiae proteinase A agrees overall with other uninhibited aspartic proteinases, although the conformation of Y75 occupying the $1 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 ( n H i s m P r o - - H i s m P r o m H i s m ) of K-casein, thereby explaining the catalytic specificity of chymosin towards K-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 (t~-structures

Rennets" General and Molecular Aspects

I0 4CMS

20

30

40

50

GEVASVPLTNY-LDSQYFGKIYLGTPPNEFTVLFDTGSSDFWVPSIYCKSNAC-KNHQR

4PEP

IGDEPLENY-LDTEYFGTIGIGTPAQDFTVIFDTGSSNLWVPSVYCSSLAC-TNHNL

2APP

AASGVATNTPTANDIEEYIPVTIG--GTTLNLNFDTGSSDLWVFSTELP-ASQQSGHSV

2APR

AGVGTVPMTDYGNDIEYYGQVTIGTPGKKFNLDFDTGSSDLWIASTLCT--NCGSGQTK

4APE

STYSATTTPIDSLDDAYITPVQIGTPAQTLNLDFDTGSSDLWVFSSETTASE-VDGQTI

YPE

GGH-DVPLTNYLNA-QYYTDITLGTPPQNFKVILDTGSSNLWVPSNECGSLAC-FLHSK

Strand

a"

Strand

60 4CMS

70

b ~

Strand

80

c"

90

i00

ii0

FDPRKSSTFQNL-GKPLSIHYGT-GSMQGILGYDTVTVSNIVDIQQTVGLSTQEPGDVFTY

4PEP

FNPQDSSTYQST-SGELSITYGT-GSMTGILGYDTVQVGGISDTNQIFGLSETEPGSFLYY

2APP

YNPSA--TGKELSGYTWSISYGDGSSASGNVFTDSVTVGGVTAHGQAVQAAQQISAQFQQD

2APR

YDPNQSSTYQAD-GRTWSISYGDGSSASGILAKDNVNLGGLLIKGQTIELAKREAASFASG

4APE

YTPSKSTSTKLLSGATWSISYGDGSSSSSDVYTDTVSVGGLTVTGQAVESAKKVSSSFTED

YPE

YDHEASSSYKAN-GTEFAIQYGTG-SLEGYISQDTLSIGDLTIPKQDFAEATSEPGLTFAF Strand

d"

Strand

120

i30

4CMS

AEFDGI

4PEP

AP FDG I LGLAY

2APP

TNNDGLLGLAFS

2APR

- PNDGLLGLGFDT

LGMAYPSLAS

S INTVQPQSQTTFFDTVKS I TTVR-

ST I DGLLGLAFSTLNTVS GKFDGI

LGLGYDTI Strand

Strand

FSVYMDRNG

.... QESMLTLG

QDL F SVYLS SNG- - -DS GSWLLG ..... QPGVYD

I SQGL I SRP I FGVYLGKAKN-

FDNAKA-

S - LDS PVFTADLGY

-GGGGEY

a

Strand

180

..... HAPGTYNFG

b

Strand

190

200

c

Strand 210

AIDPSYYTGSLHWVPVTV-

QQYWQFTVDSVTI

G I DS SYYTGS

LNWVPVSV-

EGYWQ

I T LDS I TMDGET

2APP

F I DS S KYTGS

LTYTGVDNS

QGFWS

FNVDS YTAGS

Q - SG - DG - FSG IADTGTTLLLDD

2APR

GYDSTKFKGS

LTTVP

I TVDRATVGT

S TVA- S S - FDGI LDTGTTLL

I DNS RGWWG

SGVVVACEGGCQAI

d 220

4CMS

4APE

F IDTTAYTGGITYTAVSTLQHFWEWTSTGYAVGSGTFKSTS

YPA

G I D E S KF KGD I TWL PVRRKa"

230

FG I FG

SVD- - - KWPPFYNAIQQDLLDEKRFAFYLGDTSKDTENGGEATFG

4PEP

Strand

r

160

S - L -AQ PL FAVALKHQ

-GVKT PMDNL PTSQQTF

q 150

E - - -YS I PVFDNMMNRHLVAQDL

4APE

170

140

P S I SAS - - - GAT PVFDNLWDQGLVS

YPA

4CMS

25

AYWEVKF

Strand

VDTGT

S LLTGPTSA SVV

I L PNN I

- IDGIADTGTTLLYLPATV

E G I GLGD E YAE L E S -HGAA I DTGT S L I T L P S GL

b"

240

LDTGTSKLVGPSSD

IACS GGCQAI

Strand

250

260

270

c" 280

ILNIQQAI-GATQNQ-YGEFDIDCDNLSYMPTVVFEINGKMYPLTPSAYTSQD---QGFC

4PEP

IANIQADI-GASENS-DGEMVISCSSIDSLPDIVFTIDGVQYPLSPSAYILQD---DDSC

2APP

VSQYYSQVSGAQQDSNAGGYVFDCST--NLPDFSVSISGYTATVPGSLINYGPSGDGSTC

2APR

AASVARAY-GASDNS-DGTYTISCDT-SAFKPLVFSINGASFQVSPDSLVFEEF--QGQC

4APE

VSAYWAQVSGAKSSSS-VGYVFPCSAT--LPSFTFGVGSARIVIPGDYIDFGPISTGSSC

YPA

AEMINAEI-GAKKGW-TGQYTLDCNTRDNLPDLIFNFNGYNFTIGPYDYTLEV---SGSC Strand

d" 290

Strand

q

300

Strand

r

310

320

4CMS

TSGFQSENHS

.... QKWILGDVFIREYYSVFDRANNLVGLAKAI

4PEP

TSGFEGMDVPTSSGELWILGDVFIRQYYTVFDRANNKVGLAPVA

2APP

LGGIQSNSGI

.... GFSIFGDIFLKSQYV~FDSDGPQLGFAPQA

2APR

IAGFGYG-NW

.... GFAIIGDTFLKNNYVVFNQGVPEVQIAPVAE

4APE

FGGIQSSAGIG

.... INIFGDVALKAAFWFNGATTPTLGFASK

YPA

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 etaL, 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 t w o 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 F3i, respectively, and to the peptide N atoms of T216 and T33, respectively. The carboxyl

26

Rennets: General and Molecular Aspects

~N

t

b~

d/v

eN,

t ~45t "

'

/ h;.

hN

hr: i

{

.....m:,,L ,_

"~SL .J ~_~,, .... !~~"~

c

[~

"

,

.~

Fag.,

i~

h.,v2

{. jlS.~

.

!/

~_

t

qtv

hN / ' g u rrr

/

-

'~ - i l l ~

t t

dc

_2

l-|e-

cc

t

bc

.

t

at.

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 $35 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

"'M,.

Nr

chymosin

rcine pepsin

I

~A aa.

,I

Cryphonectrl

Rhizomucor protease

mouse renin human renin

yeast proteinase A

HIV proteinase

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 etal., 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 Tz16-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 etal., 1977, 1982; James and

28

Rennets: General and Molecular Aspects

i 9 ""

ii

Figure 7 A plot of the Ce~ 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 (Polgar, 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 CmN bond but not to the hydroxyl C n O 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 $35 (Suguna et al., 1987) or residues G76, D77 or Y75 on the flap (Blundell et al., 1987; Pearl, 1987). Yeerapandian 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,

HE 31 ..~~,~.

"',

:3~~

c:J 217

218

Figure 8 The 'fireman's grip' at the active site of chymosin. Hydrogen bonds (broken lines) involved are T216N . . . m33oy1 (2.8 A), T330 "/1 . . . K2140 (2.7 ,~,), T 3 3 N . . . m216Oy1 (2.9 ,~,) and T2160"/1 . . . F310 (2.8 ,~). Other hydrogen bonds contributing to the stability of 032 and D215 are also shown (adapted from Newman et al., 1991).

Rennets: General and Molecular Aspects

H~ /l:r

/~..0,..

N

R

H'""

~'O ~

29

-

H. H

R/~O""~\H ~

ASP 21 $

aS,,' 2~s

\ M.... "'~"O

"'"O

\o I ASP 32

ASP 32 (a)

(b)

reactants

o,,olvent protonal~on.

tetrahedral intermediate I nitrogen ,twerson

/

C-N bonr mta1~n

fission, p r o t o n a h o f /

4./Fr'

H~

Asp 215 prolonation.

o\

H H

ASP 21 $ 0

..... ON

fission.

H....~. H

prolonalion

"-.

/ ASP 215

0

ASP 32

ASP 32 (d) products

o"

(c)

tetrahedral internted/ate il

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 [3-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 [3-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 Fohmann, 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 L44p--I1 bond is not susceptible to proteolytic cleavage but becomes susceptible at higher pH. At low pH, the cleavage sites differ among the aspartic proteinases- F27p--Lx8p for calf prochymosin, human progastricsin and chicken pepsinogen, M16p--ElFp for porcine pepsinogen B and L26p--I27P for procathepsin D (Barkholt and Foltmann, 1975; Barkhoh et al., 1979; Truk et al., 1985; Fohmann, 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 F42p--G1 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 N38p--T39p and V45p--A1, respectively (p = prochymosin). Moore et al. (1995) have studied the crystal and the molecular structures of human progastricsin at 1.62 A 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 F27p--L28P--Q29p--K30p--Q31P to F27P--P28p--R29P--Q30P--Q31P 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 (S37e--V38p) 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 ~ activation is completed in two or three days (Rand and Ernstrom, 1964), while at pH 2, "-20 ~ and an ionic strength of 0.1, activation is completed in 5-10min (Fohmann, 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). oLB-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 cxB-crystallin, the yield of activity is increased (Chitpinityol et al., 1998b). Substrate-binding pockets and specificity

A high concentration of NaC1 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

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 etal., 1987; Cooper etal., 1987; Foundling et al., 1987; James and Sielecki, 1987; Suguna et al., 1987). In chymosin, the subsites S1 and $1' are shallow pockets within the active site cleft. The S1 subsite (for binding of F105) has greater specificity than SI', and is blocked by YF5 (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 SI' in which an additional charged residue, E290, is near to the K-casein M106 side chain. The $2 pocket has low specificity and allows the peptide side-chains to adopt a range of conformations whereas at subsites $1 and $3, 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 K-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 Yr5 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 $3 (El3) and $2 (T222, E287, L289, 1300) 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 SI' subsite is less specific (Bang-Jensen et al., 1964; Fohmann, 1964; Guillou e t a l . , 1991; Nedjar etal., 1991).

31

The fungal and yeast proteinases have an S1 subsite with a deeper pocket and broader specificity. Therefore, the $1 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 E and at PI' 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 (Fohmann, 1960) which may be the result of the enhanced binding affinity of K-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 $35, 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 pKal value (Ido et al., 1991). In contrast, mutation of $35A in porcine pepsin lowered pKal 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 K-casein cleavage site (adapted from Gilliland et aL, 1990; Newman et aL, 1991) Subsite

K-Casein residues

Chymosin residues

S4 S3 S2 $1 $1' $2' $3'

His102 Leul03 Serl04 Phel05 Met106 Alal07 Ile108

Ser219, Lys220, Gin288 Ser12, Gin13, Tyr75, Phe117, Gly217, Thr218, 8er219 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

1 F

Chymosin 1 PeP sin2 Rhizopuspep sin3 Penicillopep sin3 Endothiapep sin4 Proteinase A s Cathepsin E 6 R. miehei APR 7 R. pusillus APR 8

i0 V

N

Q

H

L

C

G

S

H

20 L

1"

V

E

1" 1" 1" 1" 1" 1"

1]" 1" 1]" 1" 1"

1"

1]" 1" 1~ It

A

L

Y

L

V

C

G

30 E

R

1]" 1" 1" 1" It 1]' 1" 1" 1" 11" 1]" 1" 1" 1]" 1]" 1" 1" 11" 1]" 1]" 1" 1]" 1" 1]' 1]" 1]' 1]" 1" 1]" 1]" 1"

G

F

F

Y

T

P

KA

1" 1" 1" 1]"

1~ 1~ 1~ 1~ 1~

1" 1]" it "1" 1]" 1" 1]" 1]" 1]' 1]" 1]" 1]" 1)" 1~ It 1` It

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: (1]') Main cleavage site and (1') 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 $3 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 ( C O 0 - ) in P2 of the substrate is suggested to cause the Table 2

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 (mHmG) (mutant PC + 2) to the C-terminus of the protein, on the activity of the enzyme on a synthetic hexapeptide, L m S ~ F ( N O 2 ) ~ N I ~ A ~ L m O M e , 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

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 o~-,13-Caseins

3.4 3.5 4.5

Milk-clotting activity Synthetic peptides K-Casein H--P--H--P--H--L--SmF--M--A--Imp--p--K--K Oxidized B-chain of insulin Trypsinogen Haemoglobin Haemoglobin K-Casein Milk clotting Hammerten casein z--Phe--Leu--Ala--Ala 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

References

proteinase A A. niger proteinase A

Rennets: General and Molecular Aspects similar to the native enzyme. The optimum pH of T r r D 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 TrrD mutant was increased relative to the wild-type enzyme, from approximately 45 ~ for the wild type and PC + 2 mutant, to 55 ~ for the TrrD 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/1 and 3.2 • 10-8mol/1, respectively (Powell etal., 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) N H ~ L ~ S - - S t a ~ A ~ I m P ~ P ~ K ~ K (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 molA at pH 5.6) but not by its own pro-segment (Strop et al., 1990).

M e c h a n i s m of Milk Clotting In milk, the primary soluble proteins are the whey proteins, ot-lactalbumin and [3-1actoglobulin. The insoluble proteins are found in large colloidal particles, called casein micelles. K-Casein is a calcium-insensitive protein which forms a protective layer around the calciumsensitive caseins (ORS1-, Ors2- , ~ - and y-), 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 K-casein at the F105~M106 bond which results in the release of a hydrophilic glycopeptide (residues 106-169) that passes into the whey, and para-K-casein that remains in the micelles, para-K-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 ors1-, ors2- and [3-caseins and ot-lactalbumin monomer, by chymosin has been reported with a much slower rate

33

of proteolysis (Caries and Dumas, 1985; Miranda et al., 1989). The proteolytic action of microbial proteases on K-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 (F105~M106), but C. parasitica proteinase cleaves the $104~F105 bond (Dr~nse and Foltmann, 1989). Chymosin causes limited hydrolysis of K-casein, with the formation of only macropeptide and para-K-casein, while fungal proteinases cause extensive non-specific hydrolysis of both K-casein and para-K-casein (Shammet etal., 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 K-casein ( H - - P m H - - P m H m L - - S - - F - - M m A m I m ProP--K) 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-K-casein and other caseins occurs under the influence of Ca 2+ and eventually results in gel formation (Bringe and Kinsella, 1986a; Merin et al., 1989). The formation of a clot is Ca 2+ 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 etal., 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 ~ or reducing the pH from 6.6 permits flocculation at a lower percentage of K-casein hydrolysis (Dalgleish, 1982). However, the induction of gel formation at 35 ~ requires approximately 65% hydrolysis of K-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 Ca 2+ 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 etal., 1972; Martin etal., 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 etal., 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 etal., 1984), tac promoter (McCaman etal., 1985; Strop etal., 1990), APR promoter (Caulcott etal., 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 tzm (Marston et al., 1984; McCaman et al., 1985; Strop et al., 1990; Kapralek 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; Kapralek 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; Kapr~ilek 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/1 urea or 6 mol/1 guanidine HC1) 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; Kapr~ilek 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 tool/1 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/I urea buffer, pH 8. The urea mixture was incubated at 25 ~ 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 A 8 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 (Kapralek 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/1 (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 lxg/ml of cell-free culture fluid (Kapralek 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; Golf 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). Kluyverornyces 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. awarnori has been used to express prochymosin cDNA under different expression cassettes (Ward, 1989; Ward et al., 1990). The level of active extracellular chymosin was 250mg/1 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/1 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 or-amylase promoter (Boel et al.,

1987) whereas in the same expression system, a more than 3 g/1 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/1 (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/1 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), Kluyverornyces 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).

References

Abad-Zapatero, C., Rydel, TJ. and Erickson, J. (1990). Revised 2.3 /~ structure of porcine pepsin: evidence for a flexible subdomain. Proteins: Struct. Funct. Genet. 8, 62-81. Abdel Malak, C.A. (1992). Calf chymosin as a catalyst of peptide synthesis. Biochem. J. 288, 941-943. Aikawa, J., Yamashita, T., Nishiyama, M., Horinouchi, S. and Beppu, T. (1990). Effects of glycosylation on the secretion and enzyme activity of mucor rennin, an aspartic proteinase

Rennets: General and Molecular Aspects

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tional contribution of Thr218, Lys220 and Asp304 in chymosin. Protein Eng. 4, 69-71. Takahashi, K. (1995). Proteinase A from Aspergillus niger. Meth. Enzymol. 248, 146-155. Tanaka, T. and Yada, R.Y. (1996). Expression of soluble cloned porcine pepsinogen A in Escherichia coli. Biochem. J. 315, 443-446. Tanaka, T. and Yada, R.Y. (2001). N-Terminal portion acts as an initiator of the inactivation of pepsin at neutral pH. Protein Eng. 14, 669-674. Tang, J., Sepulveda, P., Marciniszyn, J., Chen, K.C.S., Huang, W.-Y., Tao, N., Liu, D. and Lanier, J.P. (1973). Amino acid sequence of porcine pepsin. Proc. Natl. Acad. Sci. USA 70, 3437-3439. Tang, J., James, M.N.G., Hsu, I.N., Jenkins, J.A. and Blundell, T.L. (1978). Structural evidence for gene duplication in the evolution of the acid proteases. Nature 271, 618-621. Tang, J., Lin, L., Co, E., Hartsuck, J.A. and Lin, X. (1992). Understanding HIV-proteinase: Can it be translated into effective therapy against AIDS? Scand. J. Clin. Lab. Invest. 52 ($210), 127-135. Tichy, P.J., Kapr~ilek, E and Jecmen, P. (1993). Improved procedure for a high-yield recovery of enzymatically active recombinant calf chymosin from Escherichia coli inclusion bodies. Protein Expr. Purific. 4, 59-63. Togni, G., Sanglard, D., Falchetto, R. and Monod, M. (1991). Isolation and nucleotide sequence of the extracellular acid protease gene (ACP) from the yeast Candida tropicalis. FEBS Lett. 286, 181-185. Toh, H., Ono, M., Saigo, K. and Miyata, T. (1985). Retroviral protease-like sequence in yeast transposon Tyl. Nature 315,691. Tonouchi, N., Shoun, H., Uozumi, T. and Beppu, T. (1986). Cloning and sequencing of the gene for mucor rennin, an aspartate protease for Mucor pusillus. Nucl. Acids Res. 14, 7557-7568. Tropea, J.E., Nashed, N.T., Louis, J.M., Sayer, J.M. and Jerina, D.M. (1992). Effect of salt on the kinetic parameters of retroviral and mammalian aspartic proteases. Bioorg. Chem. 20, 67-76. Turk, V., Lah, T., Puizdar, V., Babnik, J., Kotnik, M. and Kregar, I. (1985). Cathepsins D and E: molecular characteristics and mechanism of activation, in, Aspartic Proteinases and Their Inhibitors, Kostka, V., ed., Walter de Gruyter and Co., Berlin. pp. 283-297. Uchiyama, H., Uozumi, T., Beppu, T. and Arima, K. (1980). Purification of prorennin mRNA and its translation in vitro. Agric. Biol. Chem. 44, 1373-1381. Umezawa, H., Aoyagi, T., Morishima, H., Matsuzaki, M., Hamada, M. and Takeuchi, T. (1970). Pepstatin, a new peptide inhibitor produced by actinomycetes. J. Antibiotics 23,259-262. van den Berg, J.A., van der Laken, K.J., van Ooyen, A.J.J., Renniers, T.C.H.M., Rietveld, K., Schaap, A., Brake, A.J., Bishop, R.J., Schultz, K., Moyer, D., Richman, M. and Schuster, J.R. (1990). Kluyveromyces as a host for heterologous gene expression: expression and secretion of prochymosin. Bio/Technology 8, 135-139.

van Hooydonk, A.C.M., Olieman, C. and Hagedoorn, H.G. (1984). Kinetics of the chymosin-catalysed proteolysis of K-casein in milk. Neth. Milk Dairy J. 38, 207-222. Veerapandian, B., Cooper, J.B., Sali, A. and Blundell, T.L. (1990). X-ray analyses of aspartic proteinases. III. Threedimensional structure of endothiapepsin complexed with a transition-state isostere inhibitor of renin at 1.6 A resolution. J. Mol. Biol. 216, 1017-1029. Visser, S., van Rooijen, P.J., Schattenkerk, C. and Herling, K.E.T. (1976). Peptide substrates for chymosin (rennin): kinetic studies with peptides of different chain length including parts of the sequence 101-112 of bovine K-casein. Biochim. Biophys. Acta 438, 265-272. Visser, S., van Rooijen, P.J. and Slangen, C.J. (1980). Peptide substrates for chymosin (rennin): isolation and substrate behaviour of two tryptic fragments of bovine K-casein. Eur. ]. Biochem. 108,415-421. Visser, S., Slangen, C.J., and van Rooijen, P.J. (1987). Peptide substrates for chymosin (rennin): interaction sites in K-casein related sequences located outside the (103-108) haxapeptide region that fits into the enzyme's active site cleft. Biochem. J. 244,553-558. Visser, S., Vanalebeek, G.J., Rollema, H.S. and Friedenthal, M.K. (1988). Spectrophotometric method for the determination of chymosin and pepsin in calf and adult bovine rennets. Neth. Milk Dairy J. 42,221-232. Ward, M. (1989). Production of calf chymosin by Aspergillus awamori, in, Genetics and Molecular Biology of Industrial Microorganisms, Hershberger, C.L., Queener, S.W. and Hegeman, G., eds, American Society for Microbiology, Washington. pp. 288-294. Ward, M. and Kodama, K.H. (1991). Introduction to fungal proteinases and expression in fungal systems, in, Structure and Function of the Aspartic Proteinases, Dunn, B.M., ed., Plenum Press, New York. pp. 149-159. Ward, M., Wilson, L.J., Komada, K.H., Rey, M.W. and Berka, R.M. (1990). Improved production of chymosin in Aspergillus by expression as a glucoamylase-chymosin fusion. Bio/Technology 8,435-440. William, D.C., Whitaker, J.R. and Caldwell, P.V. (1972). Hydrolysis of peptides bonds of the oxidized B-chain of insulin by Endothia parasitica protease. Arch. Biochem. Biophys. 149, 52-61. Wlodawer, A., Miller, M., Jaskolski, M., Sathyanarayana, B.K., Baldwin, E., Weber, I.T., Selk, L.M., Clawson, L., Schneider, J. and Kent, S.B.H. (1989). Conserved folding in retroviral proteases: crystal structure of a synthetic HIV-I protease. Science 245,616-621. Wong, R.N.S. and Tang, J. (1986). Cloning and sequence: analysis of cDNA for human progastricsin. Federation Proc. 45, 105. Yada, R.Y. and Nakai, S. (1986). Use of principal component analysis to study the relationship between physical/chemical properties and milk-clotting to proteolytic activity ratio of some aspartyl proteinases. J. Agric. Food Chem. 34, 675-679. Yamada, T. and Ogrydziak, D.M. (1983). Extracellular acid proteases produced by Saccharomycopsis lipolytica. J. Bacteriol. 154, 23-31.

Rennets: General and Molecular Aspects

Yamauchi, T., Nagahama, M., Hori, H. and Murakami, K. (1988). Functional characterization of Asp-137 mutant of human renin expressed in COS cells. FEBS Lett. 230, 205-208. Ye, X.Y., Yoshida, S. and Ng, T.B. (2000). Isolation of lactoperoxidase, lactoferrin, alpha-lactalbumin, beta-lactalbumin B and beta-lactoglobulin A from bovine rennet whey using ion exchange chromatography. Int. J. Biochem. Cell Biol. 32, 1143-1150.

45

Yonezawa, M., Suzuki, J., Nishiyama, M., Horinouchi, S. and Beppu, T. (1993). Role of the amino-terminal amino acid sequences determinating the in vitro refolding process of prochymosin polypeptide. J. Biotechnol. 28, 85-97. Yu, J., Iwasaki, S., Tamura, G. and Arima, K. (1968). Physical properties and amino acid composition of mucorrennin crystal isolated from Mucor pusillus var. Lindt. Agric. Biol. Chem. 32,1051.

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 Lomhoh 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 K-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 K-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 c o n -

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 P r o p e r t i e s

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

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

Copyright 9 2004 Elsevier Ltd All rights reserved

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 K-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 OLsl-, OLsz-, [3- and K-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 K-casein is found glycosylated with several threonine, and occasionally serine, residues in the hydrophilic C-terminal end of the K-casein molecule carrying relatively short sugar chains (Zevaco and Ribadeau-Dumas, 1984; Vreeman etal., 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 K-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, mSer--X--A, 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 Ors1-, C~sz-and [3-caseins ensures that most of the phosphorylated residues are found in clusters in these molecules, one in [3-casein, two in OLsl-casein and three in oLsz-casein (Swaisgood, 1992). K-Casein is unique amongst the caseins in the absence of phosphoseryl clusters along its sequence. Most K-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 Ors1- and [3-caseins. Most gaps are found with O~sz-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, Otsz-casein is the most calcium sensitive, precipitating at Ca 2+ concentrations less than 2 mM (Aoki et al., 1985), while OLsl-casein precipitates in the range 3-8 mM (Parker and Dalgleish, 1981; Aoki et al., 1985; Farrell et al., 1988) and [3-casein precipitates in the range 8-15 mM Ca 2+ at 37 ~ but remains in solution at 1 ~ (Parker and Dalgleish, 1981; Farrell etal., 1988). K-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 [3- and K-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 [3-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 C~sl-casein suggest that it can be sub-divided into three blocks, a hydrophobic

Rennet-induced Coagulation of Milk

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 %2casein, 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. K-Casein is seen to be a mirror image of [3-casein, with a hydrophilic C-terminus, the caseinomacropeptide cleaved off by chymosin, and a hydrophobic N-terminal block preceding the Phel05--Metl06 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, OLsl-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). K-Casein also self-associates in solution, interacting via its hydrophobic C-terminal, exhibiting a monomer ~ 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 Otsl- and [~-caseins, it has been demonstrated that the size of the otsl-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 [3-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.

49

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 ~Xsl-, OLs2- or [3-casein. Again, more than two casein molecules could be involved with any one calcium phosphate nanocluster. If the casein molecule is [3-casein, further extension of this chain is through a hydrophobic linkage. Both routes permit branching and hence lead to a three-dimensional network structure. K-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 K-casein limits the micelle size. In consequence, the micelle acquires an external coat of K-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 - 1 0 8 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 H20 g-1 protein, depending on the method of measurement. The structure is not rigidly fixed but dynamic. Cooling the milk from the 37 ~ of the udder to storage at refrigeration temperatures brings about solubilization of a significant fraction of [3-casein, some K-casein and much lower levels of %1- and Ots2-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 ~ 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 K-casein, inverse relationship of micellar size and K-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 - 2 0 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 K-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 K-casein, splitting it at the Phel05mMetl06 bond and thus shave off the hairy layer, so that the subsequently exposed micelle cores begin to aggregate, once sufficient of their K-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, K-CNf 106-169, or the formation of para-K-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 K-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 K-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 K-casein, termed the para-K-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 K-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 (,)

'0' 0 , , 0 . ,00 0 0 , / , , 0 w'O, 0,0"o

0

0

(B) Partially renneted micelles

(C) Aggregating micelles in small clusters

~d)& co oo o~,8 ~

~

c~'co

0 o

c~~ ~ @0

co @ CK) (D) Percolating clusters

co 0

0

0

(~

)O O0 C~

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 Phel05~Metl06 of K-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 K-casein is limited with only formation of the caseinomacrpeptide and para-K-casein, while in the case of fungal proteinases, extensive non-specific hydrolysis of both K-casein and para-K-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 PhemMet 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 Phe--Met 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 His98--Lys111 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 K-casein (see Fox and McSweeney, 1997). The Phe and Met residues in the chymosin-sensitive bond of K-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 PhemMet bond in a similar fashion to chymosin but the C. parasitica proteinase cleaves the Serl04--Phe]05 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 Phel05--Metl06 bond of K-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

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; Lomhoh 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

53

and Walstra, 1987; Zoon et al., 1988a,b,c, 1989a,b; Van Vliet et al., 1991; Home, 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 Fohmann, 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 Fohmann (1959) to give the familiar equation:

RCT =

k [El

+ A

(1)

where k and A are constants and [E] is the enzyme concentration, R C T 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 K-casein is very high. Dalgleish (1979) suggested 60-80% of the K-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 K-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 Ca 2+ 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-K-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 K-casein. Beginning with the model calculations of Payens (1976, 1977, 1989), Payens etal. (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 K-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 K-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. Iff = 1, only dimers are possible, iff = 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 K-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 etal., 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

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 K-casein is proteolyzed. They write the relative viscosity of skim milk as: f i r = 1 + 2.54}+

5.9 +

1.9 ) T

(4)

B2 = 4

B2

(5)

vHs

s=-hkTln([P]~-[P]~[P])

(3)

(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 "rra3, 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, 4}, as the K-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 (Home and Davidson, 1993) micellar suspensions. In the adhesive sphere model, however, the loss of the K-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

,0, o"

4}2

55

.I

I1

~A4

where 4} is the micellar volume fraction and r a stickiness parameter related through the second virial coefficient (B2) to the depth (s) of an attractive square well potential created as the K-casein hairs are proteolyzed. The relevant equations are:

Fi~lure 2 A schematic defining the terminology of the adhesive spnere model. The micelle of diameter (o-) has a hairy layer of thickness (A) equivalent to the width of the square well potential. The depth of this attractive potential (s) 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.

Thus, when 8>45 ~, the viscous component dominates, whereas when 8 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:

O=O~

-

k,-kf kf ) e x p ( - k l t * ) ] + ( k l - kf

(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 K-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, K-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 (Co) was set much less than the micellar concentration (C). The calculations require that the ratio C/Co 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 =

(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 (Linet 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 =

,

(20)

the volume fraction of the cluster is given by:

~bcluster- Nc

(21)

The average volume fraction therefore decreases as the cluster grows. When it reduces to the volume fraction of particles in the system, ~b0, 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 exampies. 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, ~b0. 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

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: c~

where

oz= 2s + 8 + 1

(22)

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

Fusion

63

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 o~, as the gel aged. This necessitated assuming that the fractal dimension, Dr, 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 oz 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.

Particle shift

Chain rearrangement

Syneresis 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 [3-1actoglobulin and possible interaction of [3lactoglobulin with micellar K-casein (Trujillo etal., 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 etal., 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 ~ reduced micelle size, at 20 ~ resulted in no change and at 40 ~ 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 3 0 m i n 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 ~ or >--300 MPa at 50 ~ 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 ~ has been associated with the interaction of high-pressure-denatured /3-1actoglobulin with glycosylated K-casein, which would hinder the action of chymosin on K-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 ]3-1actoglobulin to bind to the surface of micelles via interaction with K-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 K-casein), while increasing [3-1actoglobulin denaturation reduced the rate of aggregation. Coagulation time was not related to the degree of K-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 [3-1actoglobulin 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 K-casein-[3-1actoglobulin cross-linking

Rennet-induced Coagulation of Milk reduces the susceptibility of K-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 [3-Lg-K-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 K-casein to paraK-casein, aggregation would occur mostly between micelles not fully covered with 6-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 CaC12 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

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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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Mercier, J.C. (1981). Phosphorylation of caseins: present evidence for an amino acid triplet code posttranslationally recognized by specific kinases. Biochimie 63, 1-17. Morrissey, P.A. (1969). The rennet hysteresis of heated milk. J. Dairy Res. 36,333-341. Needs, E.C., Stenning, R.A., Gill, A.L., Ferragut, V. and Rich, G.T. (2000). High pressure treatment of milk: effects on casein micelle structure and on enzymic coagulation. J. Dairy Res. 67, 31-42. Niki, R., Kim, G.Y., Kimura, T., Takahashi, K., Kohyama, K. and Nishinari, K. (1994). Physical properties of gels and microstructure of rennet gels from casein micelles of different sizes. Milchwissenschaft 49,325-329. O'Callaghan, D.J., O'Donnell, C.P. and Payne, EA. (2002). Review of systems for monitoring curd setting during cheesemaking. Int. J. Dairy Technol. 55, 65-74. Olson, N.E (1977). Factors affecting cheese yields. Dairy Ind. Int. 42, 14-15, 19. O'Reilly, C.E., Kelly, A., Murphy, P.M. and Beresford, T.P. (2001). High pressure treatment: applications in cheese manufacture and ripening. Trends Food Sci. Technol. 12, 51-59. Parker, T.G. and Dalgleish, D.G. (1981). Binding of calcium ions to bovine [3-casein. J. Dairy Res. 48, 71-76. Payens, T.A.J. (1976). On the enzyme-triggered clotting of casein: a preliminary account. Neth. Milk Dairy J. 30, 55-59. Payens, T.A.J. (1977). On enzymatic clotting processes. II. The colloidal instability of chymosin-treated casein micelles. Biophys. Chem. 6, 263-270. Payens, T.A.J. (1979). Casein micelles: the colloid-chemical approach. J. Dairy Res. 46, 291-306. Payens, T.A.J. (1989). The enzyme-triggered coagulation of casein micelles. Adv. Colloid Interf. Sci. 30, 31-69. Payens, T.A.J. and Brinkhuis, J. (1986). Mean-field kinetics of the enzyme-triggered gelation of casein micelles. Colloids Surf. 20, 31-69. Payens, T.A.J., Brinkhuis, J.A. and Van Markwijk, B.W. (1969). Self-association in non-ideal systems. Combined light-scattering and sedimentation measurements in [3-casein solutions. Biochim. Biophys. Acta 175, 434-437. Payens, T.A.J., Wiersma, A.K. and Brinkhuis, J. (1977). On enzymatic clotting processes. 1. Kinetics of enzymetriggered coagulation reactions. Biophys. Chem. 6, 253-261. Pearse, K.N. (1976). Moving boundary electrophoresis of native and rennet-treated casein micelles. J. Dairy Res. 43, 27-36. Raymond, M.N., Garnier, J., Bricas, E., Cilianu, S., Blasnic, M., Chain, A. and Lefrancier, P. (1972). Studies on the specificity of chymosin (rennin). I. Kinetic parameters of the hydrolysis of synthetic oligopeptide substrates. Biochimie 54, 145-154. Roefs, S.P.EM. (1985). Structure of Acid Casein Gels. PhD Thesis, Wageningen Agricultural University, Wageningen, The Netherlands. Ross Murphy, S.B. (1995). Rheological characterization of gels. J. Texture Studies 26, 391-400. Schmidt, D.G. (1970). The association of C~sl-casein at pH 6.6. Biochim. Biophys. Acta 207, 130-138.

69

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70

Rennet-induced Coagulation of Milk

Van Hooydonk, A.C.M. and Walstra, P. (1987). Interpretation of the kinetics of the renneting reaction in milk. Neth. Milk DairyJ. 41, 19-47. Van Hooydonk, A.C.M., Boerrigter, IO. 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, RC. and Boerrigter, IJ. (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 DairyJ. 39, 115-118. Van Vliet, T., Van Dijk, HJ.M., Zoon, P. and Walstra, R (1991). Relation between syneresis and rheological properties of particle gels, Colloid Polym. Sci. 269,620-627. Verissimo, RC., Esteves, C.L.C., Faro, CJ.E 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, EJ.W. and Overbeek, J.Th.G. (1948). Theory of Stability of Lyophobic Colloids, Elsevier, Amsterdam. Visser, S., Van Roijen, pJ. and Slangen, CJ. (1980). Peptide substrates for chymosin (rennin). Isolation and substrate behaviour of two tryptic fragments of bovine K-casein. Eur. J. Biochem. 108,415-421. Vreeman, H.J. (1979). The association of bovine SH-K-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 K-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 K-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.E 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, E (1986). Analysis of crosslinked polymer at the gel-point. J. Rheology 30, 367-382. Worning, P., Bauer, R., Ogendal, L. and Lomhoh, 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 K-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, E, Van Vliet, T. and Walstra, E (1989a). Rheological properties of rennet-induced skim milk gels. 4. The effect of pH and NaCI. Neth. Milk Dairy J. 43, 17-34. Zoon, E, 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: 9 regulation of the water content of the cheese implies controlling syneresis; 9 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; 9 rate of syneresis in relation to other changes (e.g., acidification, proteolysis, inactivation of rennet enzymes) affects cheese composition and properties; 9 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; 9 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; 9 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.E 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 lxm. 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 K-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

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

Copyright 9 2004 Elsevier Ltd All rights reserved

72 The Syneresis of Rennet-coagulated Curd the surface appears to be covered by K-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 [3-casein, goes into solution and additional 'hairs' of partly protruding [3-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 p H 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 K-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 K-casein molecules to para-K-casein and soluble caseino-

% 4O1 / 0_ -((mY)

300C

r

lo-

~

,, ~

/ 001

O-

I i

soo

',

20 oc

tan~5 0 . 4 - ~ 0oC [

4.5

T

5.5 pH

1

I

6.5

Figure 1 The proportion of calcium (Ca) and inorganic phosphate (Pin) and the electro-kineticpotential(~) of casein micelles, as wellas the dynamic shear modulus (G', frequency 1 s-l) and the loss tangent (tan 6, 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 K-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 K-casein has been hydrolysed (see also Fig. 2). The reactivity is roughly an inverse exponential function of the concentration of unhydrolysed K-casein molecules on the

The Syneresis of Rennet-coagulated Curd

100

f

0 0

j

I

20

I

40

Time (min)

Figure 2 Approximate example of the changes that occur in milk after adding rennet. Degree of hydrolysis of K-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 virtually zero, at least at physiological pH and 30 ~ If the pH is lowered, the enzymic reaction becomes much faster and, moreover, flocculation starts at a lower proportion of hydrolysed K-casein molecules (van Hooydonk et al., 1986). It appears that at low pH, the chymosin becomes adsorbed onto the micelles and this causes the hydrolysis of the K-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) micelle on which to act. At such a bare spot, the micelle is reactive. This implies that at a lower pH, flocculation starts at a stage where less K-casein has been hydrolysed. The reactivity of fully renneted micelles, i.e., those that are fully converted into paracasein micelles, depends little on pH, increases with Ca 2+ concentration, decreases with increasing ionic strength (NaC1) and increases markedly with temperature, especially from 15 to 30 ~ (Dalgleish, 1983). Above 50 ~ 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 [3-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

73

somewhat thread-like; these grow to form large tenuous flocs, until they start to touch and form a continuous network (Mulder etal., 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 I~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 -

(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:

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

qbfl~ = Na

(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, qb (for para-casein micelles at 30 ~ 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 cJSI/(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 etal., 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 Ixm 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:

(5) which relates the superficial velocity, v, of a liquid of viscosity, r/, flowing through the gel due to a pressure gradient Vp. The permeability of a 'fractal' gel is, under some assumptions, given by: B = const, a2qD2/(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

~

~

4~.~ .,

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 ~ The bars indicate 10 i*m (from Bremer, 1992).

(in agreement with experiments), which implies that the permeability of the gel depends strongly on the initial q5 and thus on casein concentration. A similar strong dependence on q~ 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 Ixm) and sufficient in number (about 2.1016 m -3) to accommodate the fat

globules (average diameter- about 3.4 lxm; number of globules larger than 1 I x 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, a). 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'7o) can be seen as a viscosity. We further have G 2 = G '2 + G ''2. 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 o0; the timescale of the deformation is about o)-1. Values of G are shown in Fig. 1. The moduli were observed to depend generally on a~ and to increase steeply with as, 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 8 = 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 ~ tan ~ = 0.45 - 0.6 at ~o = 10 -3 s -I, 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 l/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 p h e n o m e n o n 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., dT/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 o n l y - 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

I

I

I

I

I

77

I

2

/

238 Pa

~

Pa

0

0

i

i

100

200

I

-1--

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 ~ 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 co = 10 -3 s -1, the storage modulus at 20 ~ is about 2.4 times that at 30 ~ 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 etal., 1989a; Roefs etal., 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 ~ of about 3.4 ml g-1 (leading to 45 = 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~ 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~-Iactone at 30 ~ Approximate results at 30 ~ (from various sources)

Acid gel Property

Rennet gel

Type 1 Type2

pH G' at ~o= 0.01 rad s-1 (Pa) Tan ~ at ~o = 0.01 rad s-1 Fracture stress a (Pa) Fracture straina ( - ) Permeability B (l~m2) Fractal dimensionality b dB/dt (nm 2 s -1) Initial syneresis rate c

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, equations (8-10) predict that ( V o - V) is proportional to t, n o t tl/2; more generally, different authors find different relations. The only conclusion can be that, under constant conditions, the rate of syneresis ( - d V / d t ) decreases as syneresis proceeds. This need not always be true for the relative rate of syneresis ( - d In V/dt), although this quantity will also eventually approach zero. Effects of curd gram 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) o~

10o0o

0 od

V-

Vinf- [(Vo- Vinf)exp(-Kt)]

(10)

0 t~

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 etal. (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,

~-g

1000

O

o'~ Z~ Ill

E 100

i

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 etal., 2000 and Lodaite et al., 2000).

T h e S y n e r e s i s of R e n n e t - c o a g u l a t e d

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 tirring

c-

o~

40

9

'

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 ~ as a function of time after cutting, with or without stirring (from Lawrence, 1959).

Curd

87

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; Thome 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 . 0 4 % 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

100

,

effect of heating, presumably because [3-1actoglobulin now reacted primarily with K-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).

I /I S

(D (-

50

#

o~

,v..L

0

50

100

10 4

External pressure (Pa)

Figure 18 The amount of whey expelled from curd (as % of the original milk volume) after 2 h at 30 ~ 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), Thom6 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 FlClckiger, 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 [3-1actoglobulin. Heat treatment of synthetic milk free of serum proteins hardly affected syneresis. Addition of K-casein to milk diminished the detrimental

Homogenization of the milk 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 CaC12 is frequently added to enhance coagulation. Most authors report that small additions (e.g., up to 10 mM) of CaC12 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 etal., 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 CaC12 is due to its lowering the pH; if the pH was kept constant, addition of CaC12 caused syneresis to decrease, while MgC12 caused a marked increase (van der Waarden, 1947; Stoll, 1966; Kovalenko and Bocharova, 1973). Presumably, one has to consider two p o i n t s - increasing of the calcium ion activity (enhancing syneresis) and of the colloidal calcium phosphate (diminishing syneresis). Presumably, Mg 2+

The Syneresis of Rennet-coagulated Curd

act much the same as Ca 2+, 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 Ca 2+ activity. Addition of phosphate, citrate, oxalate or EDTA (van der Waarden, 1947; Stoll, 1966) at constant pH, all reduce syneresis; these additions considerably reduce Ca 2+ 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., NaC1) 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 Ca 2+ 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 A1C13 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 In VIdT) decreases with increasing temperature, but otherwise the results are fairly different. At 25 ~ reported values of Q10 vary from about 2.5 to 15, at 45 ~ from about 1.1 to 1.5.

100

Coagulation 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 Andren, 1990). Renneting with proteolytic enzymes from Rhizornucor rniehei 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 E1-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

89

.

.

.

.

.

.

.

.

.

.

. IO j O

fO j

o/

80

60

iiii

(D e-

9

o~ 40

20

0

,

|

20

n

u

u

,

40

i

u

|

,

|

60

Temp. (~

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 9 Sammis etaL (1910), V Wurster (1934), 9 Koestler and Petermann (1936), 9 Gyr (1944), 9 Lawrence (1959), A 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 ~ (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 etal., 1983), a small detrimental effect on syneresis (Nilsen, 1982) or a considerable e f f e c t holding for 20 h at 5 ~ 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

.

.

.

.

.

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).

O

80

60 (D c-

[]

40

XX

20X X X

0

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; O Gyr (1944); 9 Cheeseman (1962); A Stoll (1966); 9 Berridge (1970); 9 Patel etal. (1972); V Marshall (1982); [] Pearse etal. (1984); + Weber (1984). See text for the results of x Emmons et al. (1959)).

Washing of the curd 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.

Ultraffitration Uhrafihration 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

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 - W i n f ) 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 etal. (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 K-casein is still unhydrolysed at the moment of cutting, whereas this may be about 12% for a milk concentrated two-fold by uhrafihration (van Hooydonk and van den Berg, 1988); this proportion is higher for a more concentrated milk. 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 [3-1actoglobulin 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,

91

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 Uhrafihration). 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; Thome et al., 1958; Kammerlehner, 1974; Grandison e t a l . , 1984a,b), but addition of some CaC12 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 (Thome 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 etal. (1986a) made milk with synthetic micelles of variable casein composition. The proportions of [3- and K-caseins clearly affected clotting time, but syneresis far less. Dephosphorylation of [3-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 [3-1actoglobulin 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: 9 the geometrical constraints (dimension of the curd grain); 9 pressure applied to the curd (grains), where the relative effect is greatest in the low pressure range; 9 pH; 9 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

Table 2

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 ~ 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

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

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, it, as: / x - 0.5

(

v)

1 - dln--~eH

(11)

where V is grain volume and eH is the relative deformation expressed as the Hencky strain. He found, as an average over the first 15 min, /.t = 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/x agrees with the deformability (i.e., the rheological properties) of the

6

J

E"

=

0.30 k 00Pa

0

0

500 Time (s)

'

~--~

1000

0

' t=900s

] ]

I

I

2

4

I

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).

93

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:

( i t - iinf) = e x p ( - K p e sqrt(t)) ( i o - iinf)

(12)

iinf = 0.1 and where the rate constant K = 4 • 10-5 pa-1 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: with

p~ = 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 ~ the fracture stress obtained was about 60% higher than at 32 ~ whereas at 36 ~ 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 R e n n e t - c o a g u l a t e d

Curd

Compaction

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:

e

20

i , i n

i

15 i m i i e e lip

10 e lad

~"

I

I

u = 0.1 mm min -1 I I I

I

I

I

I

I

Tw

|

|

|

|

|

0.0

0.1

0.2

0.3

0.4

0.5

500

13_ mat) m

m .-> mat)

400 300

m ~D Q.

E

200

O

o

100 min-1

1500 1200 900

600 300

Pe = Pc + Pl + Pw

21

0.6

Relative deformation

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).

(14)

A part of the pressure is lost by Diction 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 1). 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 p]. Also, the pressure loss at the wall, Pw, markedly increases with total pressure, being, for instance, proportional to pe 2.5. 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

in-1 0

of a c u r d c o l u m n

,---, a. m

m

9 0-2 kPa, 500 s 9 1 kPa, 0-1500 s

1.I

.i--,

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 ~

~- 0 . 5 ! 0

0

9

tt

9

,

,

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 o-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

I

1

3O E

.9.o

450 2100

0.1

-

530

i

2-

E E v "13 1 -

i

04

E 10-1~

10-12 0

0.5 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

95

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 etal. (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, P1 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 ~ 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) c h e e s e - 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 arid Naudts (1970), on Herve cheese; results obtained in the Wageningen laboratory on renneted milk uhrafihration 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 NaC1 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. 9 Moulding the curd, followed by further drainage under its own weight; this is applied only for fairly soft cheese. 9 Moulding and pressing the curd; this is the common method for semi-hard and several hard cheeses. 9 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. 9 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 I3 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 m 2, 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 m 2 or less, and even a layer of a few mm then makes a substantial barrier.

T h e S y n e r e s i s of R e n n e t - c o a g u l a t e d Curd

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.

60] 50

I

I

I

I

I

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

- ....... i

4O

0

I

5 Time (h)

0

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).

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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,

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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 Wfillerstorf, 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 modem 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).

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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).

The Syneresis of Rennet-coagulated Curd

References Akkerman, J.C. (1992). Drainage of Curd. Doctoral Thesis, Wageningen Agricultural University, Wageningen. Akkerman, J.C., Lewis, R.O. and Walstra, P. (1993). Fusion of curd grains. Neth. Milk Dairy J. 47, 137-144. Akkerman, J.C., Fox, EH.J. and Walstra, P. (1994). Drainage of curd: expression of single curd grains. Neth. Milk Dairy j. 48, ~-17. Akkerman J.C., de Gee, M. and Schenck, J. (1995). On upscaling a curd particle model to batch processing scale. J. Food Process. Eng. 18, 219-231. Akkerman, J.C., Buijsse, C.A.P, Schenk, J. and Walstra, P. (1996). Drainage of curd: role of drainage equipment in relation to curd properties. Neth. Milk Dairy J. 50,371-406. Andersson, H. and Andren, A. (1990). Influence of chromatographically pure bovine chymosin and pepsin-A on cheese curd syneresis. J. Dairy Res. 57, 119-124. Araki, T. and Tanaka, H. (2001). Three-dimensional numeric simulations of viscoelastic phase separation: morphological characteristics. Macromolecules 34, 1953-1963. Beeby, R. (1959). A method for following the syneresis of the rennet coagulum in milk. Aust. J. Dairy Technol. 14, 77-87. Beltman, H. (1975). Verdikken en Geleren: Een Fysisch Chemisch Onderzoek Naar de Invloed van Polymeren op de Reologie van Waterige Systemen als Model Voor Levensmiddelen. Doctoral Thesis, Wageningen Agricultural University, Wageningen. Berridge, N.J. (1970). The influence of various treatments on the drainage of continuously made curd. J. Dairy Res. 37, 417-429. Bijsterbosch, B.H., Bos, M.T.A., Dickinson, E., van Opheusden, J.H.J. and Walstra, P. (1995). Brownian dynamics simulation of particle gel formation: from argon to yoghurt. Faraday Discuss. 101, 51-54. Biot, M.A. (1941). General theory for three-dimensional consolidation.J. Appl. Phys. 12, 155. Birkkjaer, H.E., Sbrensen, BJ., Jorgensen, J. and Sigersted, B. (1961). The effect of cheesemaking techniques on cheese quality. Beretn. Statens ForsOgsmejeri, 128. Bos, M.T.A. and van Opheusden, J.H.J. (1996). Brownian dynamics simulation of gelation and aging in interacting colloidal systems. Phys. Rev. E 53, 5044-5050. Bremer, L.G.B. (1992). Fractal Aggregation in Relation to Formation and Properties of Particle Gels. Doctoral Thesis, Wageningen Agricultural University, Wageningen. Bremer, L.G.B. and van Vliet, T. (1991). The modulus of particle networks with stretched strands. Rheol. Acta 30, 98-101. Bremer, L.G.B., van Vliet, T. and Walstra, P. (1989). Theoretical and experimental study of the fractal nature of the structure of casein gels. J. Chem. Soc., Faraday Trans. 1 85, 3359-3372. Bremer, L.G.B., Bijsterbosch, B.H., Schrijvers, R., van Vliet, T. and Walstra, P. (1990). On the fractal nature of the structure of acid casein gels. Colloid. Surf. 51,159-170. Bushell, G.C., Yan, YD., Woodfield, D., Raper, J. and Amal, R. (2002). On techniques for the measurement of the mass fractal dimension of aggregates. Adv. Colloid Interf. Sci. 95, 1-50.

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Calvo, M.M. and Balcones, E. (2000). Some factors influencing the syneresis of bovine, ovine, and caprine milks. J. Dairy Sci. 83, 1733-1739. Caron, A., Pouliot, Y. and StGelais, D. (2001). Whey syneresis differs from curd made with uhrafiltered or microfiltered milk retentate powders. Milchwissenschaft 56, 387-391. Casiraghi, E.M., Peri, C. and Piazza, L. (1987). Effect of calcium equilibria on the rate of syneresis and on the firmness of curds obtained from milk UF retentates. Milchwissenschaft 42,232-235. Cheeseman, G.C. (1962). Syneresis of milk and caseinate solutions. Proc. 16th Int. Dairy Congress, Copenhagen, Vol. B. pp. 465-472. Cheeseman, G.C. and Chapman, H.R. (1966). Adapting cheesemaking to a continuous process. Dairy Ind. 31, 99-102. Cheeseman, G.C. and Mabbitt, L.A. (1968). Studies on change in properties of fat globule membrane during concentrating of milk. J. Dairy Res. 35, 135. Creamer, L.K. (1985). Water absorption by renneted casein micelles. Milchwissenschaft 40, 589-591. Dalgleish, D.G. (1983). Coagulation of renneted bovine casein m i c e l l e s - dependence on temperature, calciumion concentration and ionic strength. J. Dairy Res. 50, 331-340. Dalgleish, D.G. (1998). Casein micelles as colloids: surface structures and stabilities. J. Dairy Sci. 81, 3013-3018. Daviau, C., Famelart, M.H., Pierre, A., Goudedranche, H. and Maubois, J.L. (2000a). Rennet coagulation of skim milk and curd drainage: effect of pH, casein concentration, ionic strength and heat treatment. Lait 80, 397-415. Daviau, C., Pierre, A., Famelart, M.H., Goudedranche, H., Jacob, D., Garnier, M. and Maubois, J.L. (2000b). Characterisation of whey drainage kinetics during soft cheese manufacture in relation with the physicochemical and technological factors, pH at renneting, casein concentration and ionic strength of milk. Lait 80, 417-432. Daviau, C., Pierre, A., Famelart, M.H., Goudedranche, H., Jacob, D., Gamier, M. and Maubois, J.L. (2000c). Residual amount of water in a draining curd of Camembert cheese and physicochemical characteristics of the drained curd as modified by the pH at renneting, the casein concentration and the ionic strength of milk. Lait 80, 555-571. Daviau, C., Pierre, A., Famelart, M.H., Gouderanche, H., Jacob, D., Gamier, M. and Maubois, J.L. (2000d). Whey drainage during soft cheese manufacture and properties of drained curd as modified by casein concentration, whey protein to casein ratio, and pasteurisation of milk. Lait 80, 573-587. de Kruif, C.G., Jeurnink, T.J.M. and Zoon, P. (1992). The viscosity of milk during the initial stages of renneting. Neth. Milk Dairy J. 46, 123-137. de Kruif, C.G., Hoffmann, M.A.M., van Marle, M.E., van Mil, P.J.J.M., Roefs, S.P.EM., Verheul, M. and Zoon, N. (1995). Gelation of proteins from milk. Faraday Discuss. 101, 185-200. Delbeke, R. and Naudts, M. (1970). The preparation of Hevre cheese form raw milk. Revue Agric. Brux. 23,333-355.

100 The Syneresis of Rennet-coagulated Curd Dickinson, E. (2000). Structure and theology of simulated gels formed from aggregated colloidal particles. J. Colloid Interf. Sci. 225, 2-15. Dimov, N.D. and Mineva, R (1962). Some factors influencing the syneresis of fresh curd and the loss of dry matter in whey of cow's, buffalo's and sheeps' milk. Proc. 16th Int. Dairy Congress, Copenhagen, Vol. B. pp. 817-825. Dinsmore, A.D. and Weitz, D.A. (2002). Direct imaging of three-dimensional structure and topology of colloidal gels.J. Phys. Condens. Matter 14, 7581-7597. Emmons, D.B., Price, W.V. and Swanson, A.M. (1959). Tests to measure syneresis and firmness of Cottage cheese coagulum, and their application in the curd-making process. J. Dairy Sci. 42,866-869. Emmons, D.B., Lister, E.B., Beckett, D.C. and Jenkins, K.J. (1980). Quality of protein in milk replacers for young calves. 5. Effect of method of dispersing fat on curd formation and whey syneresis. J. Dairy Sci. 63, 417-425. Famelart, M.H., Le Graet, Y. and Raulot, K. (1999). Casein micelle dispersions into water, NaC1 and CaC12: physicochemical characteristics of micelles and rennet coagulation. Int. Dairy J. 9,293-297. Family, E and Landau, D.R eds. (1984). Kinetics of Aggregation and Gelation. North-Holland, Amsterdam. Feagan, J.T., Erwin, L.J. and Dixon, B.D. (1965). Methods of moisture expulsion in relation to fat losses in Cheddar cheese. Aust. J. Dairy Technol. 20,214-220. Geurts, T.J. (1978). Some factors which affect moisture content of cheese before salting. Neth. Milk Dairy J. 32, 112-124. Geurts, T.J., Walstra, R and Mulder, H. (1974). Water binding to milk protein, with particular reference to cheese. Neth. Milk Dairy J. 28, 46-72. Ghosh, B.C., Steffl, A., Hinrichs, J. and Kessler, H.G. (1994). Rennetability of whole milk homogenized before or after pasteurization. Milchwissenschaft 49,363-367. Gillies, A.J. (1959). Inhibitory factors in cheese milk. Proc. 15th Int. Dairy Congress, London, Vol. 2. pp. 523-529. Gouda, A. and E1-Shabrawy, S.A. (1987). Microstructure and some properties of curd made with calf rennet, Rennilase and Suparen rennets. Chem. Mikrob. Technol. Lebensm. 10, 129-133. Grandison, A.S., Ford, G.D., Owen, A.J. and Millard, D. (1984a). Chemical-composition and coagulating properties of renneted Friesian milk during the transition from winter rations to spring grazing. J. Dairy Res. 51, 69-78. Grandison, A.S., Ford, G.D., Millard, D. and Owen, A.J. (1984b). Chemical-composition and coagulating properties of renneted milks from cows during early lactation. J. Dairy Res. 51,407-416. Green, M.L., Hobbs, D.G., Morant, S.V and Hill, V.A. (1978). Intermicellar relationships in rennet-treated separated milk. II. Process of gel assembly. J. Dairy Res. 45,413-422. Green, M.L., Marshall, R.J. and Glover, EA. (1983). Influence of homogenization of concentrated milks on the structure and properties of rennet curds. J. Dairy Res. 50, 341-348. Gyr, A. (1944). Rate of contraction of rennet curd. Ann. Paediatricia 163,314-327.

Heerink, GJ. and Geurts, TJ. (1981). Comprimeerpaarheid van een wrongelbed de wei. Wageningen (unpublished). Henstra, S. and Schmidt, D.G. (1970). A study of the rennet coagulation of cow's milk by the micro-capsule method, Proc. 7th Int. Congress Electron Microscopy, Grenoble, Vol. 1. pp. 389-390. Holt, S.A. and White J.W. (1999). The molecular structure of the surface of commercial cow's milk. Phys. Chem. Chem. Phys. 1, 5139-5145. Horne, D.S. (1998). Casein interactions: casting light on the black boxes, the structure in dairy products. Int. Dairy J. 8, 171-177. Huber, E, Fertsch, B., Schreiber, R. and Hinrichs, J. (2001). Dynamic model system to study the kinetics of thermally-induced syneresis of cheese curd grains. Milchwissenschaft 56, 549-552. Humbert, G., Driou, A., Guerin, J. and Alais, C. (1980). Effects of high-pressure homogenization on the properties of milk and its enzymic clotting characteristics. Lait 60, 574-594. Johnston, D.E. and Murphy, RJ. (1984). Effects of fat content on properties of rennet-induced milk gels. Milchwissenschafi 39,585-587. Johnston, D.E., Murphy, RJ. and Whittaker, M.R. (1983). Effects of thermization and cold-storage on some properties of rennet-induced milk gels. J. Dairy Res. 50, 231-236. Kalab, M. and Harwalkar, V.R. (1973). Milk gel structure. 1. Application of scanning electron-microscopy to milk and other food gels. J. Dairy Sci. 56,835-842. Kammerlehner, J. (1974). Study of different effects on rennet coagulation time and syneresis of rennet curd and of cheese as well as on drying of the latter in the ripening room. Deutsche Molkerei-Ztg. 95, 306-310, 342-350, 377-379. Kaytanli, M., Erdem, Y.K. and Tamer, I.M. (1994). Factors affecting whey drainage rate of renneted skim milk gels a kinetic approach. Milchwissenschafi 49, 197-200. Kiermeier, E and Keis, K. (1964). The behaviour of milk in cheesemaking from cows affected by secretory distubances. Milchwissenschaft 19, 79-82. Kiermeier, E and yon Wullerstorf, B. (1963). Laboratory plant for cheese experiments. Milchwissenschaft 18, 75-77. Kiermeier, E, Rennet, E. and Djafarian, M. (1967). Effect of high leucocyte count on chemical and bacteriological properties of milk. VII. Effect on cheese-making properties of milk. Z. Lebensm. Unters. Forsch. 132,352-358. Kirchmeier, O. (1972). Vorl~ufiges Synarese - Gesetz fur Labgallerten. Milchwissenschaft 27, 99-102. Knoop, A.M. and Peters, K.H. (1975a). The curd structures obtained by rennet and acid coagulation of milk. Kieler Milchw. Forsch. Ber. 27, 227-248. Knoop, A.M. and Peters, K.H. (1975b). Structural changes of rennet coagulation during ageing. Kieler Milchw. Forsch. Ber. 27,315-329. Koestler, G. and Petermann, R. (1936). The conditions for whey release by renneted milk. Landw. Jahrb. Schweiz. 50, 103-120. Kovalenko, M.S. and Bocharova, S.G. (1973). Effect of change in structural and mechanical properties of milk coagulum on syneresis. Dairy Sci. Abstr. 35,372.

The Syneresis of Rennet-coagulated Curd

Lawrence, AJ. (1959). Syneresis of rennet curd. Part 2. Effect of stirring and the volume of whey. Aust. J. Dairy Technol. 14, 169-172. Lawrence, AJ. and Hill, R.D. (1974). A method for measuring the syneresis of cheese-curd. Proc. 19th Int. Dairy Congress, New Delhi, Vol. 1E. pp. 204-205. Lehner, D., Worning, P., Fritz, G., Ogendahl, L., Bauer, R. and Glatter, O. (1999). Characterization of enzymatically induced aggregation of casein micelles in natural concentration by in situ static light scattering and ultralow shear viscosimetry. J. Colloid Inter_[. Sci. 213,445-456. Lelievre, J. (1977). Rigidity modulus as a factor influencing the syneresis of renneted milk gels. J. Dairy Res. 44, 611-614. Lelievre, J. and Creamer, L.K. (1978). NMR-study of formation and syneresis of renneted milk gels. Milchwissenschaft 33, 73-76. Lelievre, J., Kelso, E.A. and Stewart, D.B. (1978). Effects of Pseudomonas species on the syneresis of renneted milk gels. Proc. 20th Int. Dairy Congress, Paris, Vol. E. pp. 760-762. Lodaite, K. (2002). Physical Properties of Rennet-Induced Skim Milk Gels. Doctoral Thesis, Lund University, Lund. Lodaite, K., Ostergren, K., Paulsson, M. and Dejmek, P. (2000). One-dimensional syneresis of rennet induced gels. Int. Dairy J. 10,829-834. Lodaite, K., Ostergren, K., Santos, O., Archambauh, E.-L., Paulsson, M. and Dejmek, P. (2002). Fusion of skim milk cheese curd grains: development of a method to measure the fracture stress of the bonds between fused curd grains. Int. Dairy J. 12, 455-461. Lucey, J. (2001). The relationship between rheological parameters and whey separation in milk gels. Food Hydrocolloids 15,603-608. Lucey, J.A., van Vliet, T., Grolle, K., Geurts, T. and Walstra, P. (1997a). Properties of acid casein gels made by acidification with glucono-8-1actone. 1. Rheological properties. Int. Dairy J. 7, 381-388. Lucey, J.A., van Vliet, T., Grolle, K., Geurts, T. and Walstra R (1997b). Properties of acid casein gels made by acidification with glucono-8-1actone. 2. Syneresis, permeability and microstructural properties. Int. Dairy J. 7, 389-397. Lucey, J.A., Tamehana, M., Singh, H. and Munro, P.A. (1998). A comparison of the formation, rheological properties and microstructure of acid skim milk gels made with a bacterial culture or glucono-delta-lactone. Food Res. Int. 31,147-155. Lucey, J.A., Tamehana, M., Singh, H. and Munro, P.A. (2000). Rheological properties of milk gels formed by a combination of rennet and glucono-delta-lactone. J. Dairy Res. 67,415-427. Lucey, J.A., Tamehana, M., Singh, H. and Munro, P.A. (2001). Effect of heat treatment on the physical properties of milk gels made with both rennet and acid. Int. Dairy J. 11,559-565. Luyten, H. (1988). The Rheological and Fracture Properties of Gouda Cheese. Doctoral Thesis, Wageningen Agricultural University, Wageningen. Marshall, RJ. (1982). An improved method for measurement of the syneresis of curd formed by rennet action on milk. J. Dairy Res. 49,329-336.

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McLean, D.M. and Schaar, J. (1989). Effects of [3-1actoglobulin and K-casein genetic-variants and concentrations on syneresis of gels from renneted heated milk. J. Dairy Res. 56,297-301. Meakin, P. (1988). Fractal aggregates. Adv. Colloid Interf. Sci. 28, 249-331. Mellema, M., van Opheusden, J.H.J. and van Vliet, T. (1999). Relating colloidal particle interactions to gel structure using Brownian dynamics simulations and the Fuchs stability ratio.J. Chem. Phys. 111, 6129-6135. Mellema, M., Heesakkers, J.W.M., van Opheusden, J.H.J. and van Vliet, T. (2000). Structure and scaling behavior of aging rennet-induced casein gels examined by confocal microscopy and permeametry. Langmuir 16, 6847-6854. Mellema, M., van Opheusden, J.H.J. and van Vliet, T. (2002a). Categorization of rheological scaling models for particle gels applied to casein gels. J. Rheol. 46, 11-29. Mellema, M., Walstra, P., van Opheusden, J.H.J. and van Vliet, T. (2002b). Effects of structural rearrangements on the rheology of rennet-induced casein particle gels. Adv. Colloid Interf. Sci. 98, 25-50. Mulder, H. and Walstra, E (1974). The Milk Fat Globule. Emulsion Science as Applied to Milk Products and Comparable Foods. Pudoc, Wageningen. Mulder, H., de Graaf, J.J. and Walstra, P. (1966). Microscopical observations on the structure of curd and cheese. Proc. 17th Int. Dairy Congress, Munich, Vol. D. pp. 413-420. Needs, E.C., Stenning, R.A., Gill, A.L., Ferragut, V. and Rich, G.T. (2000). High-pressure treatment of milk: effects on casein micelle structure and on enzymic coagulation. J. Dairy Res. 67, 31-42. Nilsen, K.O. (1982). Effect of cooling and pasteurization on renneting properties of milk and on the rigidity and syneresis properties of the gel. II. Syneresis properties of the gel. Meieriposten 71,123-127, 162-163. O'Connell, J.E., Kelly, A.L., Auty, M.A.E., Fox, RE and de Kruif, K.G. (2001). Ethanol-dependent heat-induced dissociation of casein micelles. J. Agric. Food Chem. 49, 4420-4423. Olson, N.E and Price, W.V. (1970). Effect of curd deformation during cheddaring on characteristics of Cheddar cheese. J. Dairy Sci. 53, 1676-1702. Pastorino, A.J., Hansen, C.L. and McMahon, D.J. (2003). Effect of salt on structure-function relationships of cheese. J. Dairy Sci. 86, 60-69. Patel, M.C., Lund, D.B. and Olson, N.E (1972). Factors affecting syneresis of renneted milk gels. J. Dairy Sci. 55, 913-918. Paulson, B.M., McMahon, D.J. and Oberg, C.J. (1998). Influence of sodium chloride on appearance, functionality, and protein arrangements in nonfat Mozzarella cheese. J. Dairy Sci. 81, 2053-2064. Pearse, M.J. and Mackinlay, A.G. (1989). Biochemical aspects of syneresis - a review. J. Dairy Sci. 72, 1401-1407. Pearse, M.J., Mackinlay, A.G., Hall, R.J. and Linklater, P.M. (1984). A microassay for the syneresis of cheese curd. J. Dairy Res. 51,131-139.

102 The Syneresis of Rennet-coagulated Curd Pearse, MJ., Linklater, P.M., Hall, RJ. and Mackinlay, A.G. (1985). Effect of heat-induced interaction between [3-1actoglobulin and K-casein on syneresis. J. Dairy Res. 52, 159-165. Pearse, MJ., Linklater, P.M., Hall, RJ. and Mackinlay, A.G. (1986a). Effect of casein micelle composition and casein dephosphorylation on coagulation and syneresis. J. Dairy Res. 53,381-390. Pearse, MJ., Linklater, P.M., Hall, RJ. and Mackinlay, A.G. (1986b). Extensive degradation of casein by plasmin does not impede subsequent curd formation and syneresis. J. Dairy Res. 53,477-480. Peri, C., Lucisano, M. and Donati, B. (1985). Studies on coagulation of milk ultrafiltration retentates. 2. Kinetics of whey syneresis. Milchwissenschaft 40,650-652. Poon, W.C.K. and Haw, M.D. (1997). Mesoscopic structure formation in colloidal aggregation and gelation. Adv. Colloid Interf. Sci. 73, 71-126. Prasad, V., Trappe, V, Dinsmore, A.D., Segre, P.N., Cipelletti, L. and Weitz, D.A. (2003). Universal features of the fluid to solid transition for attractive colloidal particles. Faraday Discuss. 123, 1-12. Raynal, K. and Remeuf, E (2000). Effect of storage at 4 ~ on the physicochemical and renneting properties of milk: a comparison of caprine, ovine and bovine milks. J. Dairy Res. 67, 199-207. Renault, C., Gastaldi, T.E., Lagaude, A., Cuq, J.L. and de la Fuente, B.T. (1997). Mechanisms of syneresis in rennet curd without mechanical treatment. J. Food Sci. 62,907-910. Roberts, P.A. and Knackstedt, M.A. (1996). Transport and elastic properties of fractal media. Physica A 233,848-858. Roefs, S.P.EM. and van Vliet, T. (1990). Structure of acid casein gels. 2. Dynamic measurements and type of interaction forces. Colloid. Surf. 50, 161-175. Roefs, S.P.EM., Walstra, P., Dalgleish, D.G. and Horne, D.S. (1985). Preliminary note on the change in casein micelles caused by acidification. Neth. Milk Dai~y J. 39, 119-122. Roefs, S.P.EM., de Groot-Mostert, A.E.A. and van Vliet, T. (1990a). Structure of acid casein gels. 1. Formation and model of gel network. Colloid. Surf. 50, 141. Roefs, S.P.EM., van Vliet, T., van den Bijgaart, HJ.C.M., de Groot-Mostert, A.E.A. and Walstra, P. (1990b). Structure of casein gels made by combined acidification and rennet action. Neth. Milk DairyJ. 44, 159-188. Rzepiela, A.A., van Opheusden, J.HJ. and van Vliet, T. (2001). Brownian dynamics simulation of aggregation kinetics of hard spheres with flexible bonds. J. Colloid Inter[. Sci. 244, 43-50. Rzepiela, A.A., van Opheusden, J.HJ. and van Vliet, T. (2002). Large shear deformation of particle gels studied by Brownian dynamics simulations. Comput. Phys. Commun 147,303-306. Sammis, J.L., Suzuki, S.K. and Laabs, EW. (1910). Factors controlling the moisture content of cheese curds. Bur. Animal Industry, USDA, Bulletin 122. pp. 1-61. Schorsch, C., Carrie, H. and Norton, I.T. (2000). Cross-linking casein micelles by a microbial transglutaminase: influence of cross-links in acid-induced gelation. Int. Dairy J. 10,529-539.

Schwartzberg, H.G., Huang, B., Abularach, V. and Zaman, S. (1985). Force requirements for water and juice expression from cellular foods. Lat. Am. J. Chem. Eng. Appl. Chem. I5,141-176. Scott Blair, G.W. and Coppen, EM.V (1940). An objective measure of the consistency of curd at the pitching point. J. Dairy Res. 11, 187-195. Siegenthaler, B. and Fl~ickiger, B. (1964). A simple method for the determination of differences of whey expulsion from curd. Schweiz. Milchztg. 90, 766-768 (Wissensch. Beilage 96). Stoll, W.E (1966). 5yneresis of Rennet-formed Milk Gels. Doctoral Thesis, University of Minnesota, St Paul (Diss. Abstr. 27B(3), 851). Storry, J.B., Grandison, A.S., Millard, D., Owen, A.J. and Ford, G.D. (1983). Chemical composition and coagulating properties of renneted milks from different breeds and species of ruminant. J. Dairy Res. 50, 215-229. Straatsma, J. and Heijnekamp, A. (1988). Kwantitatieve Invloed van Diverse Factoren op het Kaasbereidingsproces. NIZO-Meddeling M21. Straatsma, J., de Vries, E., Heijnekamp, A. and Kloosterman, L. (1984). Control of moisture content during cheesemaking. Voedingsmiddelentechnologie 17, 26-29. Tanaka, H. (1999). Viscoelastic model of phase separation in colloidal suspensions and emulsions. Phys. Rev. E 59, 6842-6852. Tanaka, H. (2000). Viscoelastic phase separation. J. Phys. Condens. Matter 12, R207-R264. Tanaka, T. and Fillmore, D.J. (1979). Kinetics of swelling of gels.J. Chem. Phys. 70, 1214-1218. Tanaka, H., Koyama, T. and Araki, T. (2003). Network formation in viscoelastic phase separation. J. Phys. Condens. Matter 15, $387-$393. Tarodo de la Fuente, B. and Alais, C. (1975). Physicochemical studies on enzymatic coagulation of milk. Chimia 29, 379-383. Tellier, C., Mariette, E, Guillement, J.P. and Marchal, E (1993). Evolution of water proton nuclear magneticrelaxation during milk coagulation and s y n e r e s i s structural implications. J. Agric. Food Chem. 41, 2259-2266. Teo, C.T., Munro, RA., Singh, H. and Hudson, R.C. (1996). Effects of pH and temperature on the water-holding capacity of casein curds and whey protein gels. J. Dairy Res. 63, 83. Thom~, K.E. and Liljegren, G. (1959). Determining whey expulsion from curd on laboratory scale as a method of controlling the cheesemaking ability of milk. Proc. 15th Int. Dairy Congress, London, Vol. 3. pp. 1922-1928. Thomr K.B., Axelsson, I. and Liljegren, G. (1958). Method for the estimation of whey separation in rennet coagulation. Milk Dairy Res. Rep. Alnarp, 53. Tranchant, C.C., Dalgleish, D.G. and Hill, A.R. (2001). Different coagulation behaviour of bacteriologically acidified and renneted milk: the importance of fine-tuning acid production and rennet action. Int. Dairy J. 11,483-494. Unger Grundelius, A. (2004). The Permeability of RennetInduced Curd. Licenciate Thesis. Lund University, Lund.

The Syneresis of Rennet-coagulated Curd Unger Grundelius, A., Lodaite, K., Ostergren, 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 DairyJ. 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

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and Biosubstrates, Lal, M., Lillford, RJ., 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, E (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., Ogendahl, 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 NaC1. 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.EM., de Cindio, B. and van Vliet, T. (1990). Rheological properties of skim milk gels at various t e m p e r a t u r e s - interrelation between the dynamic moduli and the relaxation modulus. Rheol. Acta 29, 223-230.

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

micelles. The stability of casein micelles of milk is attributed to their net negative charge and steric repulFresh acid-coagulated cheese varieties include Cream sion by the flexible macropeptide region of K-casein cheese, Cottage cheese and Quarg and other cheeses (the so-called 'hairs'). Different types of interaction are where the coagulation of milk occurs by acid rather responsible for micelle integrity, including Ca-induced than by rennet, as in most other cheese varieties interactions between protein molecules, electrostatic, (e.g., Cheddar). Fresh acid cheeses differ from ferhydrophobic and hydrogen bonding. These intermented milk products in having a significant amount actions are probably also involved in the formation of the moisture (whey) removed after coagulation. and structural properties of acid casein gels. Whey removal methods, such as centrifugal separVarious models for the structure of casein micelles ation and ultrafiltration (UF), are used for Quarg and have been proposed, and it has been the source of conCream cheese whereas cutting of the coagulum into troversy over the years. The latest model by Home granules and a high cook temperature are used for (1998) envisages a polymerization scheme (dual-binding Cottage cheese. Cultures of mesophilic lactic acid bacmodel) for the assembly of casein micelles. Cross-linking teria (i.e., usually Lactococcus spp. and Leuconostoc of the molecules proceeds via two routes, hydrophobic spp.) and sometimes probiotic species are used as interactions between groups on different molecules cultures for most fresh acid curd cheeses. A common forming one pathway, with more than two molecules factor in all of these acid cheese products is that the possibly joining at such junctions, and a second pathinitial step involves the formation of an acid-induced way where chain extension is through a CCP nanoclusgel, which is then further processed. The formation ter acting as a neutralizing bridge between two and physical properties of acidified milk gels have phosphoseryl clusters on separate molecules of Ors1-, been reviewed recently (Lucey and Singh, 1997, Ors2- or [3-casein. Both routes permit branching and 2003; Horne, 1999; Lucey, 2002a). There has been hence lead to a three-dimensional network. However, considerable research on acid milk gels made with K-CN can link only to hydrophobic residues on another thermophilic cultures for the production of yogurt CN molecule. Because it has no phosphoseryl cluster to (e.g., Tamime and Robinson, 1999). The manufacture permit further extension, the polymer chain ends there. and technologies involved in the production of fresh As a consequence, the K-CN acquires an external suracid cheeses have also been reviewed (Guinee et al., face position where it acts as a steric stabilizer. 1993; Puhan etal., 1994; Kosikowski and Mistry, As the pH of milk is reduced, CCP dissolves and 1997; Fox et al., 2000; Lucey, 2002b). This chapter the caseins are liberated into the serum phase (Dalgleish focuses primarily on the formation of these acid-milk and Law, 1988). The extent of liberation of caseins gels and their physical, rheological and microstrucdepends on the temperature at acidification (Dalgleish tural properties. and Law, 1988), which has little effect on the solubilization of CCP. Apparently, little change in the average Casein micelles hydrodynamic diameter of casein micelles occurs durCaseins constitute approximately 80% of the protein ing acidification of (unheated) milk to pH ---5.0 in bovine milk, with four main types (Ors1-, Ors2-, (Roefs et al., 1985; de Kruif, 1997), although the [3- and K-caseins (CN)) in combination with appre- internal structure of the casein micelles is altered due ciable quantities of micellar or colloidal calcium phos- to the loss of CCP (Walstra, 1993). Aggregation of phate (CCP) in the form of aggregates called casein casein occurs as the isoelectric point (pH --4.6) is Introduction

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

Copyright 9 2004 Elsevier Ltd All rights reserved

106

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

0.25 o~" 0.20 ~J tO -..~

= 0.15

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0.05

0.00 1

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|

,

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2

3

4

5

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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 e t a l . , 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 n-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

-10

-8 v

-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 [3-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 ~ which are commonly used for the formation of acid milk gels, no preferential dissociation of [3-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 K-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 - 2 0 ~ and at 30 ~ there is virtually no liberation of caseins (Dalgleish and Law, 1988). K-Casein dissociates to a greater extent than the other caseins during acidification at low temperatures (Dalgleish and Law, 1988); since the K-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--4.7) the texture will be soft and the cheese will lack flavour. At a very low pH (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 flesh acid products by cultures is generally performed by either of two methods: slow, 12-16 h at 20-23 ~ (long set) or 4-6 h at 30-32 ~ (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 ~ acid casein gels made at 40 ~ 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 ~ 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 ~ 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 ~ (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 ~ 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 ~ At very high temperatures, the gelation pH may increase. At very low temperatures (e.g., 4 ~ no coagulation of casein occurs even at pH 4.6. Heat treatment of milk at a temperature _>78 ~ 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, 13-1actoglobulin, 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 (- 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 ~ 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 (, ~ 1.=7 6 .=

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37 ~ but generally 48-52 ~ 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~ (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 a r o m a - p r o d u c i n g 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

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 ~ and is incubated overnight at a controlled temperature (45 ~ or in large containers in which the temperature decreases to 37-40 ~ to a final pH as low as 3.3 (Limsowtin etal., 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. ferrnentum, Sc. therrnophilus) 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 etal., 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 etal., 1996; Parente etal., 1997), hard cheese varieties in Argentina (Reinheimer et al., 1996), and Comte 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, Pr~.sure a la 'recruite') which are used for the manufacture of Swiss-type cheeses (Emmental, Sbrinz, Gruyere; 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. therrnophilus, but also lactococci and enterococci) often dominate cultures incubated at a relatively low temperature ( < 4 2 ~ 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 ~ for 10-15 min) followed by incubation at a high temperature (37-45 ~ until the desired titrat-

127

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 ~ and 42 ~ 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) (Gl~ttli, 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 (Gl~ttli, 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 isdefined, 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

(Limsowtin etal., 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, crernoris 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 etal., 1995; Cogan etal., 1997; Wouters et al., 2002). Any potential new starter

129

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 ~ (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 etal., 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 ApH 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. mesenteroicles 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

Transporta

Pathway b

Cleavage enzyme c

Lactococcus lactis Leuconostoc spp.

PEP-PTS Permease

GLY PK

pl3gal 13gal

Sc. thermophilus Lb. delbrueckii Lb. helveticus

Permease Permease Permease

GLY GLY GLY

13gal 13gal 13gal

a b c d

PEP PTS, phosphotransferase system. GLY, glycolysis; PK, phosphoketolase. 13gal, phospho-13-galactosidase; 13gal, 13-galactosidase. These species metabolize only the glucose moiety of lactose.

Products (mol/mol lactose)

4 Lactate 2 Lactate + ethanol + 2 002 2 Lactate d 2 Lactate d 4 Lactate

Isomer of lactate L D L D DL

Starter Cultures: General Aspects

(Magni et al., I999) that the induction of the citrate metabolic pathway under acidic conditions makes the cells more resistant to the inhibitory effects of lactate.

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;

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

IN peptidase

amino acids " ~ transport systems

fPro-specific peptidases-'~

9 XDAP (PepXs) / 9 proline iminopeptidase I Pepl) / ~ prolidase (PepQm) / ~.. prolinase (PepPm) J (1) v ~ -o ~. ~c~ EL

casein

PrtP

aa

4' amino acid catabolism arginine deiminase pathway aldolases arninotransferases decarboxylases dehydrogenases

/'qeneral peptidases 9 aminopeptidases (PepN m, PepCc) 9 dipeptidases (PepVm) 9 tripeptidases (PepT m) 9 endopeptidases

biosynthetic path protein synthesis

Glu-specific peptidases 9aminopeptidases (PepA

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 (CEE 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

PrtH~'~ILB

H

PrtB

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).

125-130 of PrtP, corresponding to the substrate binding cleft, result in a variety of specificities towards degradation of Ors1-, 13- and K-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 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

PrtP protease domain I N ~ +1~s

+12

C

+6 +18

+15

N -8

+3 H

+3

substrate

J

Figure 3 Three-dimensional model of the o>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 etal., 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 ot-ketoacids which are potent aroma compounds, e.g., c~-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 ot-ketoglutaric acid as a substrate, and the concentration of this ketoacid is limiting in cheese. In fact, addition of ot-ketoglutaric acid (oLKG) to cheese has been shown to greatly enhance flavour formation. Alternatively, ot-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 5c. 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 5c. 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 5c. 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). N CDO 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

in Lc. lactis C2 (Wang et al., 2000). Lc. lactis NCDO 2118 was able to grow in a CDM containing otKG but no glutamate, but the lag phase depended on the concentration of otKG added (Lapujade et al., 1998). No glutamate dehydrogenase activity, the enzyme which is used in many bacteria to produce glutamate directly from otKG, was detected but transaminase activities, with several amino acids as amino group donors and otKG as acceptor, were detected. Indeed, addition of otKG 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 etal. (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 H202. Under these conditions, various NADH oxidases and peroxidases are produced to reduce the toxic H202 (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-

135

tis during aerobic growth if heme is also present. Lc. lactis MG1363 grown in M17 (glucose) in the presence of 10 tzg haemin/ml produced increased biomass and retained almost 100% viability over 70 days at 4 ~ (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 H202 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 etal., 2002; Hugenholtz et al., 2002). Diacetyl is produced chemically from ot-acetolactate (AL) which is produced from pyruvate which, in turn, is produced from citrate, ot-Acetolactate is highly unstable and breaks down to diacetyl when 02 is present and to acetoin when 02 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 (Hugenhohz 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 SKl l, 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 etal., 1987; Chapot-Chartier etal., 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 ~ 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 90 ~ 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 Ca 2+ 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 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.

165

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 d e f i n e d - 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; Br(issow 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; Prevots et al., 1989; Benbadis et al., 1990; Fayard et al., 1993; Le Marrec et al., 1997; Stanleyet 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

Table 1

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; Br(issow 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; Prevots 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 'R (Mata et al., 1986; Lahbib-Mansais etal., 1988; Sechaud etal., 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

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, skl, blL41, blL66, 4~US3 P335, 4~LC3, rlt, Tuc2009, TP901-1, 4~31,4~50, Q30, Q33, ul36 P107 1483 P087 1358 BK5-T a 949 c2, blL67, 4~vML3, ~197, P001 P034 KSY1

Podovi ridae

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

Starter Cultures: Bacteriophage

167

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 blL67 being 22 195 bp, compared to 22 163 bp for c2. c2 and blL67 share about 80% of the overall nucleotide sequence identity (Lubbers etal., 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 etal., 1995; Lubbers etal., 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

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).

Prolate-headed phage The two sequenced Lc. lactis prolate-headed phage, blL67 (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)

Late

Early .......----q ori

cos

~

L ~

Replication ~

COS

Lysis ~---[MorphogenesisH PackagingH

MorphogenesiHs Lysis

(B) 936 (sk 1 )

Early

Late

Middle

ori

cos

acka0in0 H "or0ho0enesisH ',sis I--

I Replicati~e (c) P335 (TP901-1)

Lysogenic Lytic early sttP

II genetic switch

Lytic middle 1. .. ac/cos

IntegrationF~-~:!:~":', q Replication

Lytic late ..-~1

Morphogenesis H Lysis F

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 bacteriophagelife 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 blL67 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, b i l l 7 0 and ski are mem-

Table 2

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, skl (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

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 22 28 31 33 38 36 40 36 35 41 36 14 14 15

Prolate, lytic Prolate, lytic S.la, 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. delbrueckfi) 4,gle (Lb. plantarum) 4,adh (Lb. gassed)

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 4,01205 4,7201 DT1 Sfi19 Sfi21 Sfil 1

pac cos cos cos cos pac

43 35 34 37 40 39

S.I, S.I, S.I, S.I, S.I, S.I,

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 blL67 c2 skl biLl70 rl-t Tuc2009 ul36 BK5-T TP901-1 blL285 b blL286 b blL309 b blL310 b blL311 b blL312 b

Cos/pac site

195 163 451 754 350 347 798 003 667 538 834 949 957 510 179

075 466 820 392 739 807

temperate lytic lytic lytic temperate lytic

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

Starter Cultures: Bacteriophage

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 (~skl (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 (40RFs) 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 biLl70 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 biLl70 genome to that of skl 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 biLl70, 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 ski (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 skl (Parreira et al., 1996; Chandry et al., 1997).

169

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 r l t (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 r l t 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 lyric 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 u136, 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, d~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 4)01205 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

DNA replication and synthesis of phage proteins

DNA packaging and morphogenesis

Cell lysis and release of progeny phage

;t;2 4'

/~ DNA injection blocking

Adsorption interference

/~

/~

systems

Late

infection

4.

s

Insertional mutagenesis

'1'. Recombinant superinfection exclusion

Antisense

4'

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), qbadh (Ahermann etal., 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 my4 (Auvray et al., 1997)) can be discerned, indicative of a direct evolutionary relationship between these two Lb. delbrueckii phages (Mikkohen etal., 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 - 4 8 % 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 (Prevots 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. Brassow 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 lyric 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 etal., 1993; Monteville etal., 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 lyric 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

@

-e>

~*--

~ ~

g#

Attachmentto host cell and injectionof DNA

O

..

DNA replication and synthesis of phage proteins

Integration of DNA into the host chromosome

".~'

, ,o'7

AL

Cell division

r

Induction

DNA packaging and hage morphogenesis

event

e.o o ~~ Cell lysis and release of progeny phage Integrated phage DNA replicates along with the host chromosome

Lysogenic life cycle Figure 4

i

i

9

9

Lytic life cycle

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

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 k 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 otis and the exploitation of the interactions between phage/hostencoded replication proteins and their cognate phage otis 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 etal., 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 ori2oo9 sequence is located within the gene coding for the putative replisome organiser protein (rep2oo9), and its encoded protein specifically interacts with the ori2oo9 DNA (McGrath et al., 1999). In a further study, genes highly homologous to rep2oo9, that contained sequences identical to ori2oo9, were identified in two other lactococcal phages, Q30 and Q33 (McGrath et al., 2001), while a third repzoo9 homologue was identified in another closely related phage, u136 (Bouchard and Moineau, 2000). The ability of plasmids harbouring ori sequences to confer a phage-resistance phenotype was also used to identify otis in the 5c. 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 otis, each of which is capable of independently mediating a per phenotype (Stanley et al., 2000). Phages infecting 5c. thermo-

173

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 otis of 7201 (Stanley et al., 2OOO). Another method used to identify phage otis 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 hellcase 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 etal., 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 ATE 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; Garcia 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 TPg01-1 (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 5c. therrnophilus 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 01205, whereas all cos-containing phages tested exhibited homology to the gene specifying one of the structural components of the cos-containing phage ~b7201.

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 ~bc2 (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 15 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 17 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, 15, were suggested to be involved in forming covalently linked muhimers, 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-k-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 (Garcia 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

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 Blasi, 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 [3-turn linker region and a hydrophilic N-terminus (Young and Bl~isi, 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 (Bl~tsi and Young, 1996). Three distinct holin groups have been described (Young and Blasi, 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

175

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 rlt, 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 qbO1205, 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; Brussow 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 )t (see above).

Table 3 Identified genes encoding restriction/modification systems in Lc. lactis and Sc. thermophilus R/M system or identified subunits Lc. lactis Lla14031 Lldl

Unnamed (HsdR, HsdM, HsdS) L0308 (HsdR) L0309 (HsdM) L0310 (HsdS) Unnamed (HsdS) Unnamed (HsdS) Unnamed (HsdS) Unnamed (HsdR, HsdM, HsdS) Llal LlaDII LlaCI LiaBI LlaKR21 ScrFI LlaDCHI LlaFI Sc. thermophilus

Unnamed (HsdS) Unnamed (HsdR, HsdM, HsdS) Unnamed (HsdS) Unnamed (HsdR, HsdM, HsdS) Sth3681

Type

Location

Reference

I I I

plL2614 pND861 pAH82 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)

pCIS3 plL7 piLl03 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)

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)

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 rlt, all of which possess integrases belonging to the type I-Int family of site-specific recombinases (Van de Guchte et al., 1994b; Boyce etal., 1995; van Sinderen etal., 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 X, 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 (Garc/a 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 etal. (1997) characterised the lysogeny module of the Sc. thermophilus phage ~bSfi21. 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

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,

177

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 qbc2. Following infection with ~bc2, 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 ~bc2 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 sie2oo9 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

178

Starter Cultures: Bacteriophage

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 5crFIBM and 5crFIAM independently confer protection against 5crFI 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 etal., 1998b). Schouler etal. (1998a) were the first to describe a type I system in Lactococcus. This system, named Llal4031, was found to be encoded on a native plasmid harboured by Lc. lactis II1403. 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. therrnophilus, 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. therrnophilus 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, ~bS0, 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 ~bS0 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

Starter

Table

4

Cultures:

Bacteriophage

179

Lactococcal abi genes for which DNA sequence data are available

Abi

Location

%G + C

Phage species affected

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

pTR2030; pCI829 Unspecified; pCI642 pTN20 pBF61 piLl05 pNP40 pNP40, pAJ2074 pCI750 Chromosome pND852 pND859 pSRQ800 pND861 Chromosome pPF144 plL2614 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

Mechanism

Reference

Early P335 Late 936 Late 936 Late 936 Late c2 Late 936 Early 936 Latee36/c2/EarlyP335

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) Prevots et al. (1996) Suet aL (1997) Deng et al. (1997) Emond et aL (1997) Deng et aL (1999) Prevots et aL (1998) Prevots and Ritzenthaler (1998) Schouler et al. (1998a) Emond et aL (1998) Twomey et aL (2000) Bouchard et aL (2002) Dai et al. (2001)

-

Late 936 Latee36/EarlyP335 Late c2 -

Late 936/c2 Earlyc2 Late 936/P335 -

* 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. therrnophilus (Larbi etal., 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 ~ and that intracellular phage DNA replication was affected as for phages infecting Lactococcus. However, at 37 or 42 ~ 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 ~b50 genomic DNA fragment on a plasmid vector in trans conferred a phage-resistance phenotype on the lactococcal host against ~bS0, 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 perSO fragment was in fact the origin of replication of ~bS0 and that the resistance phenotype conferred was due to the titration of

180

Starter Cultures: Bacteriophage

essential phage DNA replication factors by the plasmidborne oris. The putative ori for the P335-type lactococcal bacteriophage, Tuc2009 (designated ori2oo9), has also been used in the construction of a per system. The ori2oo9 sequence is located within a gene coding for a putative replisome organiser protein (rep2oo9), 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 otis 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 Suare7, 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 (b7-9 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 d~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 etal., 1992; Kim etal., 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 d~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 ~b31 origin of replication (ori31). Following r 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 ~b31 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 methyhransferase 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 u136, 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

Starter Cultures: Bacteriophage

181

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 Still, 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 WM 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.

off203, which when supplied in trans on a multicopy vector provided resistance to Sc. thermophilus Still against 12 out of 25 phages tested. The off203 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 off203 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 (hA2 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 [3-recombinase gene. The resulting strain was completely immune to d~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 LlalR + restriction cassette so that infection of a cell harbouring this plasmid with (b31, causes the lethal gene product of LlalR + 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, d~31 mutants considerably lesssensitive to the +31p-LlalR + 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 qb3lp (ORF2). Furthermore, the ability of these mutant phages to induce the native qb31p promoter was demonstrated to be reduced, compared to that of the parent +31. Pairing the +31p-LlalR + system with other abortive infection systems, Per31 and AbiA, resulted in a reduction in numbers of qb31

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 etal. (1997) described a gene from the lysogeny module of the Sc. thermophilus phage Sfi21,

Bacteriophage-triggered defence

182

Starter Cultures: Bacteriophage

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 etal., 1992; Nauta etal., 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 etal., 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, ThivervaI-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, E 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 NaC1, which may also contain some of the surface micro flora (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'Eveque, 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.

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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192 Secondary and Adjunct Cultures Table 1 Main yeast species encountered in/on the surface of cheese

Perfect form

Imperfect form

Galactomyces geotrichum Debaryomyces hansenfi 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 vinL Candida zeylanoides. From Nunez et al., 1981; Baroiller and Schmidt, 1990; Nahabieh and Schmidt, 1990; B&rtschi et aL, 1994; Eliskases-Lechner and Ginzinger, 1995b.

Useful properties in selecting yeast adjuncts Effect on a p p e a r a n c e o f c h e e s e s u r f a c e

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 etal., 1993). It is generally recognised that Yarrowia lipolytica, Saccharomyces cerevisiae and K. marxianus subsp. marxianus are more proteolytic than D. hansenii (Vannini etal., 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 etal., 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

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 NaC1 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 m1-1 of cheese milk or 106-107 cfu m1-1 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-

193

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 m l - 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. cyclopiurn; 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 caseifulvurn 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 domesticurn (Cylindrocarpon sp.) and Verticillium lecanii. C. sulfureurn 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 ITFE a cheese technical centre in France. Useful properties for which to select moulds as adjuncts

Appearance of mould on~in cheeses 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. 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. Proteotytic 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 OLsl-, ~ - and K-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 [3-oxidation of free fatty acids, produced by lipolysis (see 'Lipolysis and Catab-

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. Production of aroma 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. 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. carnemberti 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 u s e 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

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, NaC1 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 Verticilliurn 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 Gruyere, Beaufort and Comte (Piton-Malleret and Gorrieri, 1992). Generally, counts

195

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 Gruyere 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; Kampfer 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

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1%, w/w, KC1 (see 'Reduced Sodium Cheese' for discussion on lowsodium cheese). The third major feature of the use of NaC1 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 NaC1 resides in the Na moiety since KC1 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, NaC1 is probably sufficient to overcome the insipid taste (Schroeder et al., 1988). In this chapter, we will concentrate on the significance of NaC1 in cheese ripening rather than on its dietary and direct flavour effects. NaC1 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

Copyright 9 2004 Elsevier Ltd All rights reserved

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) Gruyere 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: 9 control of microbial growth and activity; 9 control of the various enzyme activities in cheese; 9 syneresis of the curd and thus in a reduction in cheese moisture, which also influences the above; 9 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 NaC1 for this purpose in cheese is in the manufacture of Domiati cheese from milk to which 12-15%, w/w, NaC1 is added to inhibit bacterial growth and thus maintain milk quality (Naguib et al., 1979; Su~muth, 1998; 'Cheese Varieties Ripened in Brine', Volume 2). For all other major varieties, NaC1 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 NaC1 to the regulation of cheese pH, which in turn influences cheese ripening and texture. The pH of cheese may be regulated by: 9 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; 9 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; 9 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 NaC1 > 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 NaC1 but was strongly inhibited by >-2.5%, w/w, NaC1. However, even at 5%, w/w, NaC1, acid was produced by all starters to

Salt in Cheese: Physical, Chemical and Biological Aspects

a level - 4 5 - 5 5 % 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, NaC1 at --38 ~ 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, NaC1 brine but did not fall below 5.9 at 5%, w/w, NaC1 brine; the pH of curd held in 4%, w/w, NaC1 brine was similar to that of curd held in water (0%, w/w NaC1 brine) (Fig. 1). The pH decreased to 5.65, 5.53, 5.62 and 5.90 at 0, 2, 4 and 5%, w/w, NaC1 brine, respectively (Fig. 1). These results suggest that starter activity is stimulated by 2%, w/w, NaC1. Overall, the experiments of Irvine and Price (1961) suggest that the growth of lactococci in Cheddar curd is generally not inhibited by -casein is the principal substrate for proteolysis with little degradation of [3-casein (see 'Coagulant'); proteolysis of [3-casein is more extensive at low salt levels (Phelan et al., 1973; Kelly etal., 1996). However, Thomas and Pearce (1981) noted that while the normal products of [3-casein degradation ([3-CNf1-192, [3-CNf1-189 and [3-CNf1-165 produced by rennets, and y-caseins by milk proteinase) were not apparent in their studies, the concentration of unhydrolysed [3-casein decreased, suggesting that proteolysis of [3-casein in low-sah cheese may be due to bacterial proteinases. Kelly et al. (1996) noted that cleavage of Leu192-193 in [3-casein and LeUl01-LySl02 in Otsl-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, NaC1 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

217

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 etal., 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 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: 9 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-sah 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 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 [3-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 CXsl-casein. Capillary gel electrophoresis showed that the hydrolysis of [3-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 etal., 1999); the opposite trend was noted for Otsl-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 [3-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 NaC1 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; Ruegg 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 (Sanchez et al., 2001). This trend is similar to that noted by Najera et al. (1994) for Idiazabal cheeses but disagrees with that reported by Thakur etal. (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 NaCI on C a s e i n H y d r a t i o n and the Physical P r o p e r t i e s of C h e e s e 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 etal., 1985; Pearse and Mackinlay, 1989; Abou-E1-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, NaC1 is added to the milk (Abou-E1-Nour, 1998), the effects of NaC1 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 CaC12 (M.M. Hewedi, personal communication). Owing to the importance of casein hydration on cheese quality, the effects of NaC1 on hydration are discussed below.

Effect of NaCI on casein hydration in model systems and in cheese The significance of casein hydration, as affected by NaC1, on the physical properties of cheese has been demonstrated using dilute model systems. Creamer (1985) studied the effect of NaC1 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, NaC1 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

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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 (9 or 5%, w/w, NaCI (O) and the pH adjusted to different values using 20%, w/v, lactic acid. The dispersions were held at 15 ~ for 22 h and then centrifuged at 3000 g for 30 min at 15 ~ 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 NaC1 may be attributed to the binding of Na + by the casein (G~I and Banky, 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 NaC1 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 (R~iegg 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) CaC12 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 - 1 2 % , w/w, at 1.4%, w/w, Ca. These findings indicate that the addition of calcium increases caseincasein 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 - 1 2 500 g at 25 ~ 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, NaC1; 2.8%, w/w, salt-in-moisture) decreased from - 1 6 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 f r o m - 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, NaC1) 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 NaC1 rather than to differences in proteolysis of the casein. Thus, electrophoretic analysis of the sera showed that the levels of intact Otsl-, Ors2-, 13- and paraK-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, NaC1 (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, NaC1 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, NaC1 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, NaC1) 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, NaC1) and plasticizing in hot dilute brine (i.e., 5%, w/w, NaC1) 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, NaC1) 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 NaC1 (---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: 9 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); 9 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, NaC1 and 0.5%, w/w, CaC12), cheese looses water during brining, resulting in a net weight

Salt in Cheese: Physical, Chemical and Biological Aspects

loss, as discussed in 'Moisture level'. In contrast, salting in freshly prepared dilute brine (e.g., _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 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 NaC1 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, NaC1 prior to stretching the curd in hot water containing 0, 5 or 10%, w/w, NaC1 (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%, wlw, 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

225

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 etal., 1988; Morris and Dillon, 1992; Narhinen etal., 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 NaC1 on Enzyme Activity in Cheese', 'Influence of NaC1 on the Water Activity (aw) of Cheese', 'Overall Influence of NaC1 on Cheese Ripening and Quality' and 'Effect of Salt on Cheese Composition'). The sodium level (%, w/w) in cheese ranges from - 0 . 2 6 , 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: 9 reducing the level of added salt per se (Kosikowski, 1983; Wyatt, 1983; Lindsay et al., 1985); 9 partial or complete substitution of NaC1 by other salts such as KC1, MgCI2 or CaC12 (Fitzgerald and Buckley, 1985; Demott etal., 1986; Lefier etal., 1987; Anonymous, 1993; Iwanczak etal., 1996; Katsiari et al., 1997; Salem and Abeid, 1997; Abou-E1Nour, 1998); 9 a reduced salt level in combination with flavourenhancing substances such as autolysed yeast extract (Karahadian and Lindsay, 1984; Demott et al., 1986); 9 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); 9 alterations of cheesemaking procedure, e.g., washing curd at a low temperature ( - 2 0 ~ to remove lactose) and heating moulded curd to a core temperature curd of 85 ~ (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, NaC1 was also acceptable. Fitzgerald and Buckley (1985) studied the influence of KC1, MgC12, CaC12 and 1:1 mixtures of these salts with NaC1 on the quality of Cheddar cheese salted to give an ionic strength similar to the control (i.e., 1.44%, w/w, NaC1 in the cheese) ripened at 4 ~ over a 4-month period. The use of KC1, MgC12 or CaC12 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 MgC12 and CaC12 but not in the case of KC1, 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 NaC1/MgC12 blend which gave softer cheese than the control. Both flavour and texture scores for the CaC12/NaC1- and MgC12/NaCl-salted cheeses were significantly lower than the controls. A KC1/NaC1 (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 NaC1/KC1 (1:1) mixtures were observed for Cheddar (Lindsay etal., 1982), and Camembert, Gouda and Camping type cheeses (Iwanczak et al., 1996). Moreover, both of these studies reported that reduction of salt (i.e., NaC1 or 1:1 NaCI/KC1 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 NaCI/KC1 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 NaC1, KC1 or mixtures of NaC1/KC1 (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 NaC1 (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 ~ 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, NaC1) 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 NaC1) 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

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., 200, respectively. However, reduction of salt in dry matter (SDM) in Gouda cheese by - 2 0 % 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

...
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

(a)

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012 0

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9

Figure 13 Influence of cheese shape on salt uptake (A,m) and moisture loss (A,F1) by Romano-type cheese during salting in 19.5%, w/w, NaCI brine at 23 ~ Rectangular cheeses (A,A), cylindrical cheeses (re,l-l) (redrawn from Guinee and Fox, 1986c).

Salt in Cheese: Physical, Chemical and Biological Aspects

relationship for the quantity of salt absorbed through a flat surface as a function of brining time: Mt = 2(C - Co) (D*t/zr) 1/2 Vw

(1)

where Mt = quantity of salt absorbed over time, g NaC1/ cm 2, C - salt content of brine, g NaC1/ml, Co - o r i ginal salt content of the cheese, g/ml, t = duration of the salting period, days, D * - pseudo-diffusion coefficient, cm2/day, V w - 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 cm 2 planar surface (Mp) were in close agreement with the experimental values (Mr) 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 ~ on salt uptake by Cheddar curd cubes (1 cm 3) in brines (20 or 25%, w/w, NaC1) at the same temperature as the curd. Salt uptake was lowest at 32 ~ similar at 27 and 38 ~ and highest at 43 ~ The reduction in salt content on changing the brining temperature from either 27 or 38 ~ to 32 ~ was -6.5%. The low salt uptake at 32 ~ 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 > 3 2 ~ exuded fat was liquid and dispersed in the brine. Increasing brine temperature increases the mobility of NaC1 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 Kaletung, 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 etal., 1974b). The increase in D* for Romano and Gouda cheeses in the range 5-25 ~ in 19.2% NaC1 brine was similar at - 0 . 0 0 8 3 cmX/day (Guinee, 1985). Geurts etal. (1974b) reported that D* for Gouda cheeses salted in - 2 0 % NaC1 increased by - 4 0 - 5 0 % on increasing the temperature from 12.5 to 25 ~ compared to an increase in D in pure water o f - - 2 0 % over the same temperature range.

235

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-sahing 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 . 2 5 (control, unsalted curd) to - 1 4 % , 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 ~ for 1 day, covered with wax on three sides and the unwaxed side placed in contact with 19.2%, w/w, NaC1 for 3 days. On completion of brine-salting, the SAVI levels decreased with distance from the cheese-brine interface until it approached that of the pre-sahed 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, NaC1 at 18 ~ (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-sahing 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 m m thick) layer of cheeses pre-sahed to >6%, w/w, S/M exceeded the NaC1 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 A s p e c t s

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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 (r-l), 6.0 (B), 8.4 (A), 9.5 (A), 10.5 (9 or 12.5 (O) %, w/w, NaCI before brine-salting at 15 ~ in 19.2%, w/w, NaCI 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 NaCI 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 NaC1 diffuse, an increase in the relative pore width of the protein matrix and hence a reduced frictional effect on the inward-diffusing Na + and C1-. 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

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 NaC1 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 NaC1 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 NaC1 on casein hydration in model systems and in cheese'). While little information is available on

237

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 etal. (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" P h y s i c a l , C h e m i c a l a n d B i o l o g i c a l A s p e c t s

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Salting level, g/100 g Figure 15 Relationship between salting level of curd and the concentration of salt (O) and salt-in-moisture ( O ) i n 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 ~ 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 - 1 1 % on raising the curd temperature from 24 to 41 ~ and the percentage of fat lost increased markedly, e.g., by - 0 . 6 kg fat/tonne curd per 1 ~ rise in temperature in the normal working temperature range of 29-35 ~ 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

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). 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 NaC1 in cheese during salting. The first such study was made by Georgakis (1973), who related the diffusion of NaC1 in Greek Feta to cheese surface area, Manufacturing conditions

1

Curd acidity at salting

Moisture ~ content of curd

1

Salt uptake., !

L Salt-in-moist re

Curd particle size at milling I Salting rate

Extent of mixing Method saltingof f -Drg salting -Brine salting

Figure 16

Principal factors that affect the uptake of salt by

Cheddar curd (from Fox, P.E, ed., Cheese: Chemistry, Physics and Microbiology, 2nd edn, Chapman & Hall, p. 281).

239

Manufacturing conditions

J

Cheese dimensions at salting

Curd acidity at salting

Shape

Surface area to volume

Size

ratio

Moisture content of curd Salt uptake

Brining conditions (e.g. temperature, % NaCI)

.~ Salt-in-moisture

Figure 17

Principal factors that affect the uptake of salt by

brine-salted cheeses (from Fox, P.E, 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 NaC1 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, NaC1; 0.2% Ca; 10-20 ~ 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 NaC1 in the moisture phase of a dry-salted, 12-week-old Cheddar ( 5 0 % FDM, 37.9% H20) (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 NaC1 diffusion in Gouda cheese has been studied by Geurts et al. (1974b), Guinee (1985), and Turhan and Kaletung (1992). Concentration gradient 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., i999; 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, NaC1 (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~ i.e., from ---0.205 to 0.17 cm2/day (Guinee, 1985). Similarly, increasing the brine concentration from 15 to 20%, w/w, NaC1 reduced the moisture diffusivity in Turkish white cheese, with the reduction at 20 ~ (---17%) being more pronounced than that at 4 or 12.5 ~ (---8%) (Turhan and Gunasekaran, 1999). While the apparent D* decreases on increasing NaC1 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~rine 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 SAVIequilibrium 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 NaC1 and H20 in cheese (Geurts etal., 1974b; Turhan and Kaletunq, 1992); an increase of---0.008 cm2/dayPC was found for Gouda-type cheeses for brine temperature in the range 5-25 ~ (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 CaCI2 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 s@ rind, especially where the NaC1 concentration in the brine is relatively low, e.g., 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% NaC1 brine), the value of p varied more, i.e., from 3.75 at the rind to 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 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 NaC1 on casein hydration in model systems and in cheese' and 'Concentration of calcium in the brine'), especially in weak brines (., 2~

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The Microbiology of C h e e s e Ripening

isolated m o s t frequently are Ec. faecalis, Ec. faecium and Ec. durans.

Enterococci are a major component of the bacterial population of cheeses produced in Italy (Senini et al., 1997; Suzzi etal., 2000; Andrighetto etal., 2001), France (Bouton et al., 1998), Spain, Portugal (Freitas and Malcata, 2000), Greece (Papageorgiou et al., 1998; Nikolaou et al., 2002), Turkey, The Balkans (Bintsis and Papademas, 2002; Hayaloglu et al., 2002) and Egypt (Hemati et al., 1998). Their numbers at the end of ripening range from 105 to 107 cfu g-a, although numbers vary with cheese type (Fig. 2A). The species

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Ripening period (days) Figure 2 Changes in the populations of (A) enterococci, (B) non-starter lactobacilli and (C) Leuconostoc spp. during ripening of Afuega'l Pitu (9 Cuesta etal., 1996), Armada (x; Tornadijo etaL, 1995), Cabrales (A; Nunez, 1978), Canestrato Pugliese ( t ; Albenzio et aL, 2001), Cheddar (x; Dasen et aL, 2003), Fossa Pit (~; Avellini et aL, 1999), La Serena (A; Fernandez del Pozo et aL, 1988), Penamellera (rq; Estepar et aL, 1999), Serra da Estrela (m; Dahl et aL, 2000), Swiss-type (O; Beuvier et aL, 1997 and Demarigny et aL, 1996) and Tenerife caprine (+; Z~.rate et aL, 1997) cheeses.

296

The Microbiology of Cheese Ripening

The diversity of the non-starter population is greater in cheeses made from raw than from pasteurised milk, and Berthier et al. (2001) were able to demonstrate, using molecular techniques, that most of the nonstarter lactobacilli in Comte originated from the milk. The presence of high numbers of enterococci in artisanal cheeses is usually associated with poor hygienic practices (Franz et al., 1999), although bovine faeces was not considered to be the source of enterococci in the farm-house raw milk Cheddar type cheese studied by Gelsomino et al. (2002). Although some lactobacilli are inactivated by pasteurisation (Turner et al., 1986), other strains may survive the heat treatment and proliferate in the cheese during ripening (McSweeney et al., 1994; Jordan and Cogan, 1999). Enterococci are also likely to survive pasteurisation. The production of natural milk cultures from pasteurised milk that is incubated at 42-44 ~ for 12-15 h inevitably promotes the selection of heattolerant LAB, including enterococci (Giraffa et al., 1997). It has been suggested (Martley and Crow, 1993) that milk is not the principal source of NSLAB in cheeses made from pasteurised milk and that the manufacturing equipment was the more probable source. Serological typing methods confirmed that air-borne lactobacilli in the plant during cheesemaking are recovered from the cheese (Naylor and Sharpe, 1958b). Non-starter lactic acid bacteria have been isolated from the floor and drains in the dairy environment and from the surfaces of equipment used in cheese manufacture and vacuum packaging (Somers et al., 2001). Lactobacilli are able to form and persist in biofilms on cheesemaking equipment and could be re-isolated from batches of cheese produced after the plant had been cleaned, implying that they survive cleaning and sanitising treatments (Somers et al., 2001). The source of enterococci in cheese milk has been identified as the milking equipment (Gelsomino et al., 2002). The proposition that a factory-specific flora could impart distinctive flavour characteristics to the cheese produced in a given plant (Chapman and Sharpe, 1981) implies that contamination during manufacture is a critical component of the process. However, studies of the non-starter Lactobacillus populations of cheeses made in a single plant over an extended time period failed to detect the recurrence of specific strains (Fitzsimons et al., 2001; Williams et al., 2002a), indicating that, if the contamination was from within the plant, the source was either intermittent or at a low level. Factors affecting growth and survival of NSLAB Environmental conditions Non-starter lactic acid bacteria, in particular nonstarter lactobacilli and enterococci, are not adversely affected by environmental conditions in the cheese

curd and are able to proliferate during maturation. Non-starter lactobacilli have a generation time of approximately 8.5 days in cheese ripened at 6 ~ (Jordan and Cogan, 1993) and viable cells can be recovered from cheese stored at 10 ~ for 3 years. The growth rate and final population density of non-starter lactobacilli and enterococci are not affected significantly over the pH range, salt and moisture levels that normally occur in the curd during Cheddar cheese manufacture (Lane et al., 1997). Their growth rate is temperature-dependent but ripening temperature had little influence on the final numbers of lactobacilli in the cheese. Rapid block cooling and ripening at low temperatures reduces their growth rates (Folkertsma et al., 1996). In cheese ripened at 1 ~ the non-starter lactobacillus population was 3 log cycles lower than in a cheese ripened at 8 ~ (Shakeel-Ur-Rehman et al.,

2000). Nutrient availability Non-starter lactic acid bacteria require an energy source for growth. The level of residual lactose in fresh curd is usually low but nevertheless some is likely to be present when the non-starter Lactobacillus population is becoming established in the cheese. However, the subsequent increase in the non-starter Lactobacillus population is likely to occur after the lactose has been utilised, indicating that it is not the sole energy source (Turner and Thomas, 1980). Waldron (1997) showed that growth of mesophilic lactobacilli was independent of the lactose content of the cheese. Mesophilic lactobacilli possess glycoside hydrolases (Williams and Banks, 1997) and can utilise sugars derived from the glycomacropeptide of casein and the glycoproteins of the milk-fat globule membrane (Fox et al., 1998; Diggin et al., 1999; Williams et al., 2000). In addition, starter culture autolysis during ripening releases sugars, e.g., ribose (Thomas, 1987; Rapposch et al., 1999). However, studies by Lane et al. (1997) on the growth rate of non-starter lactobacilli in cheeses made with fast- and slow-lysing starter cultures indicated that cell lysate was not a major source of growth substrates. Citrate is present in small amounts (--~8 mmol kg -1) in unripened Cheddar cheese but is not used as an energy source by non-starter lactobacilli (Palles et al., 1998; Williams et al., 2000); high numbers of lactobacilli also develop in cheese in which there has been no significant citrate utilisation (Jordan and Cogan, 1993). Lipids and bacterial catabolites are not effective substrates but peptides and amino acids are catabolised by lactobacilli, provided that a keto acid acceptor is present to facilitate the aminotransferase involved (Tammam et al., 2000; Williams et al., 2000,

The Microbiology of Cheese Ripening 297 2001). Proteolytic products formed during ripening stimulate the growth of Lb. casei (Nath and Ledford, 1972), whilst Laht et al. (2002) concluded that arginine was one of the main energy sources for non-starter lactobacilli in Swiss-type cheese. The ATP available from arginine metabolism is theoretically sufficient to support growth to 108 cfu g-1 Interactions The microbial flora of cheese are complex and it is inevitable that interactions between members of the population will occur. The complex nature of cheese ecosystems complicates the interpretation of these interactions; however, Martley and Crow (1993) were able to demonstrate interactions between NSLAB during ripening. It has also been reported that Lb. casei, Lb. rharnnosus and Lb. plantarum inhibit PAB and enterococci in cheese as a result of competition for limiting nutrients (Jimeno et al., 1995; Lynch et al., 1996). In addition to the inter-species competition for nutrients, some of the metabolic products formed by NSLAB, e.g., lactate, acetate and formate, may interfere with the growth of other species (Lindgren and Dobrogosz, 1990; Vandenbergh, 1993; Ouwehand, 1998). In addition, many NSLAB are able to produce bacteriocins with broad or narrow spectra of activity (Stiles, 1994; Ouwehand, 1998; Franz et al., 1999). The inclusion of bacteriocin-producing starter LAB offers a means of suppressing the growth of undesirable bacteria. This approach has been used to control the growth of both food-borne pathogens and NSLAB in cheese (Ryan etal., 1996; Buyong etal., 1998; Benkerroum et al., 2000; Benech et al., 2002), and as a means of manipulating the ripening process (Fenelon et al., 1999; Oumer et al., 2001; Garde et al., 2002). Non-starter lactic acid bacteria may undergo autolysis during ripening. However, Kiernan et al. (2000) were unable to find evidence for the autolysis of mesophilic lactobacilli during the ripening of Cheddar cheese. The ability of Leuconostoc spp. to autolyse is strain-dependent (Cibik and Chapot-Chartier, 2000).

Population dynamics Although the population size remains relatively stable from 3 months to the end of maturation (Fig. 2), the population is not static but is in a dynamic state as the balance of the species and the strains change. Cheddar cheese The initial non-starter Lactobacillus level in commercially produced Cheddar curd is - 10 a cfu g- 1 which increases to --~107 cfu g-1 within 3 months of manufacture and remains at this level throughout the remainder of maturation (Peterson and Marshall,

1990; Fox et al., I998). However, the heterogeneity of the population of non-starter lactobacilli in Cheddar cheese decreases during ripening. While several species are detected in young cheeses, they are replaced, as the cheese ages, by strains of Lb. paracasei which dominate throughout the remainder of the ripening period (Fitzsimons et al., 2001; Williams et al., 2002a). Similar dynamics was observed in New Zealand Cheddar except that the dominant species were Lb. paracasei and Lb. rhamnosus (Crow et al., 2001). Population shifts at strain level also occur during the ripening of Cheddar cheese. Although some strains may persist throughout ripening while others may recur in the latter stages of ripening, having been undetected in the intervening period, the overall trend is for the number of strains present to decline during maturation. Most mature cheeses are dominated by no more than six strains (Crow et al., 2001; Fitzsimons et al., 2001; Williams et al., 2002a; Dasen et al., 2003). Swiss-type cheeses The number of non-starter lactobacilli and enterococci are higher in Swiss-type cheese made from raw milk than in cheese made from pasteurised milk but the diversity of non-starter lactobacilli declines during ripening (Beuvier etal., 1997). The population of young cheese was comprised of Lb. paracasei, Lb. plantarum and Lb. brevis but as the cheese matured Lb. paracasei dominated (Demarigny et al., 1996). Similar results were obtained in Emmental cheese made from thermised milk (Thierry et al., 1998). The non-starter Lactobacillus population in Comt~ cheese was dominated by Lb. paracasei, although Lb. plantarum and Lb. rhamnosus were also present (Bouton et al., 1998; Grappin et al., 1999). Enterococci remained at low levels throughout ripening (Fig. 2A). Berthier et al. (2001), using PCR methods, detected fewer strains in the mature cheese than at other stages throughout ripening. Spanish artisanal cheeses There is very little information available on changes in the species profiles in Spanish cheeses during ripening. Most data are restricted to counts of the dominant genera at various times during maturation, and significant variation occurs between the different groups of bacteria in different cheeses (Fig. 2A-C). This variation may be due to the artisinal nature of many of these cheeses. Lb. plantarum and Lb. brevis dominated, and the population remained constant at "~ 108 cfu g-1 throughout the 60-day ripening of Afuega'l Pitu cheese but the numbers of leuconostoc and enterococci decreased during ripening (Cuesta et al., 1996). In San Simon cheese, enterococcal numbers were maximal

298

The Microbiology of Cheese Ripening

during the first week of ripening, and stayed constant throughout the remainder of the 6-week maturation period (Fontan et al., 2001). The numbers of leuconostoc, lactobacilli and enterococci varied little during the ripening of Penamellera cheese (Estepar et al., 1999), but leuconostocs were one of the major groups at the drying room stage in the maturation of Cabrales cheese (Nunez, 1978). Lb. plantarum was dominant in the cheese's interior during the cave-ripening stage. Non-starter lactobacilli and leuconostocs varied little during the ripening of La Serena and ovine milk cheeses, coagulated with Cyanara extracts (Fernandez del Pozo et al., 1988; Vioque et al., 2000), and the enterococcal population declined; in contrast, the proportion of enterococci in Manchego cheese increased throughout ripening (Ordol~ez et al., 1978). Variations in the individual populations during the ripening of caprine milk cheeses were cheesespecific. The development of the LAB population of Valdeon, a hand-made blue cheese, exhibited an initial dominance of lactococci and enterococci. However, from the drying stage, lactobacilli and leuconostoc replaced the lactococci, and the mature cheese was dominated by enterococci (Lopez-Diaz et al., 2000). The population of I-week old Armada cheese was dominated by lactococci whereas Lb. casei and Lb. plantarum were the most abundant during the latter stages of ripening (Tornadijo et al., 1995). The numbers of enterococci, leuconostocs and lactobacilli declined during ripening in summer-made cheese, but the latter two microbial groups remained at ---108 cfu g-1 throughout the 16-week period of ripening in autumn-made cheese. Leuconostoc and enterococcal numbers remained at ---107 cfu g-~ during the 60-day ripening period of Tenerife cheese whereas the numbers of lactobacilli increased from 105 cfu g-1 after 2 days to >107 cfu g-1 at the end of ripening. Lb. plantarum was dominant in the young cheese whereas in the mature cheese Lb. paracasei was predominant among the lactobacilli (Z~irate et al., 1997). Portuguese cheese varieties

The microbiology of Appelation d'Origine Protegee (AOP) Portuguese cheeses was reviewed by Freitas and Malcata (2000). The more dominant LAB in 9-day-old Picante da Beira Baixa cheese were Leuc. mesenteroides (19%), Lb. plantarum (15%), Lb. paracasei (15%) and Ec. faecalis (8%). Leuconostoc spp. were not detectable after 40 days, with Lb. plantarum and Lb. paracasei persisting throughout ripening (Freitas et al., 1996). Lb. brevis and Lb. fermentum were also detected in the mature cheese. The geographical location and season of manufacture influence the micro-

bial population of Serra da Estrela (Tavaria and Malcata, 2000). The numbers of LAB are maximal after a ripening period of 7 days, and, in this population, Leuc. lactis and enterococci are the most abundant (Macedo et al., 1995; Dahl et al., 2000). The proportion of Leuc. rnesenteroides and Lb. plantarum in the population tended to increase throughout ripening, whilst that of Ec. faecium, Ec. faecalis and Lb. pentosus declined (Tavaria and Malcata, 1998). Italian cheese varieties

Ricotta forte is produced by ripening cottage ricotta cheese for 12 months, during which time the curds are mixed regularly to prevent mould growth. At the end of ripening, the dominant lactobacilli are Lb. paracasei, Lb. acetotolerans, Lb. alimentarius and Lb. brevis, and of these only Lb. paracasei was detected early in ripening. Lb. kefiri, Lb. gasseri, Lb. hilgardii, Lb. plantarum, Lb. paraplantarum and Lb. zeae were detected transiently (Baruzzi et al., 2000). Major shifts in species profile also occurred during the ripening of the pasta-filata cheese, Caciocavallo Pugliese. The two dominant species at the end of the 60-day ripening period were Lb. parabuchneri and Lb. paracasei, while Lb. fermenturn was the dominant species in the young cheese (Gobbetti et al., 2002). The proportion of Ec. faecalis and Ec. durans in the population decreased from --6% to 0.1% during ripening whilst that of Pd. pentosaceus increased. The Lactobacillus community involved in traditional Mozzarella production has been investigated (Morea et al., 1998). Lb. fermentum, which was dominant in the natural whey starter, was not detected during manufacture or ripening. Following heat treatment associated with the stretching process the complexity of the population decreased from 11 to 5 strains of Lb. casei, Lb. fermentum, Lb. plantarum and Weissella hellenica. Only Lb. plantarum was detected during ripening. The most numerous non-starter lactobacilli in Scamorza Ahamurana cheese ripened for 6 days were Lb. fermentum and Lb. paracasei (Baruzzi et al., 2002). Lb. paracasei was not detected in the whey or curd whereas Lb. fermentum was present at all stages in the manufacturing process. Lb. gasseri and W. viridescens were also detected in the whey but not in the ripening curd whereas Ec. durans was only detected in the mature cheese. Dramatic changes in the diversity of the microbial communities of the Sicilian artisanal pastafilata-type cheese, Ragusano, during the manufacturing process were revealed by classical and cultureindependent PCR and density gradient gel electrophoresis techniques (Randazzo et al., 2002). Mesophilic LAB, including Leuconostoc spp. and Lc. lactis, dominated the raw milk population but disappeared during cooking

The Microbiology of Cheese Ripening

and fermentation of the curd. However, Lb. delbrueckii and Lb. fermentum grew during ripening, and enterococci were also present in reasonable numbers as the microbial population stabilised in 15- and 30-day-old cheeses. Marked shifts in the species profiles have also been observed in cheeses ripened for longer periods of time. Non-starter lactobacilli and enterococci increased during the 60-day ripening period of Pecorino Sardo ovine milk cheese, though significant differences occurred between batches (Mannu etal., 2002). Lb. casei numbers were constant throughout the ripening of one batch of cheese, appeared after 60 days in a second batch whereas, in a third batch, although they were present in high numbers during ripening, they only became dominant when the starter LAB population declined. The numbers of non-starter lactobacilli in traditional farm-house Fiore Sardo cheese increased from 105 cfu g- 1 in i-day-old cheese to 108 cfu g- 1 after ripening for 30 days; the population then decreased slowly, and by 7 months the level was reduced to 104 cfu g-1 (Mannu et al., 2000). Lb. plantarum decreased dramatically during maturation whereas Lb. paracasei, when present, dominated the cheese microflora. Lb. paracasei was also dominant in Montasio cheese, and although it was not detectable immediately after manufacture its population increased to 107 cfu g-1 during the first month of ripening and remained at that level up to 120 days (Lombardi et al., 1995). Enterococcal numbers decreased by one to two orders of magnitude during the 9-week ripening of Canestrato Pugliese cheese (Albenzio et al., 2001), whilst the non-starter Lactobacillus population increased to 28 days and remained at that level for the remainder of maturation (Fig. 2A,B). The population in cheese made from raw milk (108 cfu g - l ) was 3 log cycles higher than that in cheeses made from thermised or pasteurised milk. A similar population developed during the 60-day maturation period of Fossa (pit) cheese; the numbers of nonstarter lactobacilli then declined by two orders of magnitude during the 3-month aging process (Avellini et al. , 1999). Prolonged ripening times are a feature of Parmigiano Reggiano cheese. During ripening the number of non-starter lactobacilli decreased from 108cfu g-1 after 5 months to approximately 104 cfu g-1 at 24 months (Coppola et al., 1997). Lb. paracasei/Lb, casei and Lb. rhamnosus, which persisted throughout the 24-month maturation, dominated the population, and Pd. acidilactici was present for 22 months. Enterococci disappeared during ripening of Parmesan for 14 months (Thompson and Marth, 1986).

299

Greek and eastern European c h e e s e s

Kefalotyri is a hard, salted cheese, which traditionally is produced without starters. Lactobacilli and enterococci are present throughout the 120-day ripening period. Lactobacilli predominate for 30 days when enterococci became increasingly dominant (LitopoulouTzanetaki, 1990). The proportion of Lb. plantarum, Ec. faecium, Ec. durans and Pediococcus spp. recovered increased during ripening whereas the proportion of Lb. casei remained at 16-20% throughout the period. Lb. brevis, Lb. buchneri and Leuconostoc spp. decreased during ripening and were absent in 120-day-old cheese. Ripening-related changes in the LAB population of two other Greek ovine milk soft cheeses, Feta and Teleme, have also been studied (Tzanetakis and LitopoulouTzanetaki, 1992). Lb. plantarum was the dominant isolate recovered from both cheeses. Phenotypic characterisation of isolates indicated that different strains dominate at different stages of ripening in Feta cheese (Xanthopoulos et al., 2000). The proportion of enterococci and pediococci in Feta after ripening for 90 days was lower than that in the original curd. After 180 days of ripening, the proportion of enterococci in Teleme cheese had increased from the levels in the curd whereas that of the Leuconostoc spp. present was similar at both stages after a transient increase between 30 and 60 days. In Turkish white cheeses, species of Enterococcus, Lactobacillus, Leuconostoc and Pediococcus dominate during ripening (Bintsis and Papademas, 2002; Hayaloglu et al., 2002). Enterococci numbers remained constant during the ripening of Turkish Kashar cheese, with Ec. faecium and Ec. durans being the most frequently isolated, while Lb. casei, Lb. plantarum and Lb. rhamnosus were the dominant non-starter lactobacilli in the mature cheese (Aran, 1998). Significance of NSLAB in cheese manufacture Influence of non-starter lactobacilli on cheese quafity

Non-starter lactobacilli can impact on cheese quality in both beneficial and detrimental ways; however, an increasing number of studies have shown that selected adjunct strains of Lactobacillus spp. positively influence cheese quality (Fox et al., 1998; Table 2). Cheddar cheese produced under controlled bacteriological conditions in aseptic vats can develop full mature flavour in the absence of non-starter lactobacilli, although non-starter lactobacilli are believed to add desirable flavour notes and reduce harshness and bitterness associated with some starter cultures (McSweeney etal., 1994; Shakeel-Ur-Rehman et al., 2000). The presence of non-starter lactobacilli in commercial cheese is associated with the development of

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329

330

Raw Milk Cheeses

Ortigosa et al. (2001) in ovine Roncal cheese (comparison of R, P and P + Lb. casei). In Cheddar cheese, the variations in VFAs originating from amino acid catabolism were not the same as those of other VFAs. In a study by Shakeel-Ur-Rehman et al. (2000c), 2- and 3-methyl butanoic acids were found at higher levels in P cheeses than in R cheeses, and in another of their studies (Shakeel-Ur-Rehman et al., 2000a), 2-methyl propanoic and 3-methyl butanoic acids were found at higher levels in cheeses ripened at low temperature (1 ~ compared to 8 ~ These compounds correlated negatively with the number of NSLAB in the cheeses, so the authors hypothesised that NSLAB have a minor role in the formation of these compounds or that NSLAB broke them down further.

Carbonyl compounds Tables 6 and 7 summarise the differences between ketones and aldehydes found in R, P or MF cheeses. Diacetyl (2,3-butanedione) and acetoin (3-hydroxy2-butanone) are products of the metabolism of citrate by Lc. lactis spp. lactis bv. diacetylactis, Leuconostoc spp. or some Lactobacillus species (McSweeney and Sousa, 2000). They are important aroma compounds in numerous varieties of cheese (Curioni and Bosset, 2002). Nevertheless, they tend to decrease during ripening. In 6-month-old Cheddar, diacetyl is present in quantities too low to participate in flavour (Urbach, 1997). In cheese, diacetyl can be converted into acetoin, then 2,3butanediol and 2-butanone and finally 2-butanol (Urbach, 1993). It is notable that 2-butanone and 2-butanol, in contrast to diacetyl and acetoin, generally increase during ripening, like other methyl ketones and secondary alcohols (Bosset and Liardon, 1985; Barlow etal., 1989; Urbach, 1993; Carbone|l etal., 2002). Therefore, it is likely that the transformation of diacetyl into the more reduced compounds progresses throughout ripening due to the enzymatic activities of microorganisms, so the raw milk microflora can be expected to influence these compounds. Indeed, diacetyl and acetoin were found in lower quantities in R cheeses than in P or MF cheeses in semi-hard (Buchin et al., 1998; Shakeel-Ur-Rehman et al., 2000a,c; Ortigosa et al., 2001) or Swiss-type cheeses (Buchin et al., unpublished). The contrasting result was observed in only one study on Morbier-type cheese (Buchin et al., unpublished). The quantities of 2-butanone in ripened cheese were affected differently by the presence of the raw milk microflora. Ortigosa et al. (2001) found less 2-butanone in Roncal cheese made from raw ewes' milk than in cheese made from P milk, but the opposite was true for the Morbiertype cheeses studied by Buchin etal. (1998). These contradictory results could be expected because the amount of 2-butanone in a ripened cheese arises from

the balance between its formation and its reduction to 2-butanol. It is difficult to establish a correlation between the presence of microbial populations and the level of 2-butanone at a given time of ripening, because of the continuous production and degradation of this compound. To follow the evolution of microbial growth and the level of 2-butanone throughout the ripening period may provide valuable information. In contrast, as expected, 2-butanol was more abundant in R cheeses than in P cheeses in all studies (Buchin et al., 1998; Shakeel-Ur-Rehman et al., 2000c; Ortigosa et al., 2001). The other carbonyl compounds found in cheeses are methyl ketones and aldehydes. Their characteristics have been summarised in a recent review by Curioni and Bosset (2002). Methyl ketones originate from the B-oxidation of fatty acids by microorganisms. Their aromatic impact is of primary importance in blue and surface mould-ripened cheeses, but they are also likely to have an influence on the flavour of other varieties, e.g., 2-heptanone in Emmental, Gruyere and Grana Padano. This ketone is typical of blue cheese flavour, whereas the others have fruity, floral or musty notes. Straight-chain aldehydes originate from the oxidation of unsaturated fatty acids. They are characterised by green, fatty odours. The branched-chain aldehydes with four or five carbon atoms originate from the catabolism of the amino acids, valine (2-methyl propanal), leucine (2-methyl butanal) and isoleucine (3-methyl butanal). In cheese, they have green or malty notes. As previously noted, the variations of these compounds in the presence of the raw milk microflora differed according to the study. In the studies of ShakeelUr-Rehman etal. (2000c) and Buchin etal. (1998) on Cheddar and Morbier-type cheese, respectively, R cheeses contained less of the different methyl ketones and aldehydes than P cheeses. This is in contrast with the results of the studies of Shakeel-Ur-Rehman et al. (2000a) comparing R and P Cheddar, and Buchin et al. (2002) comparing R and MF Morbier-type cheeses. The results of Buchin et al. (2002) showed less ketones but more aldehydes in R Swiss-type cheeses compared to MF ones. The explanation for these contrasting results may be the same as that for the products of pyruvate metabolism. Methyl ketones and aldehydes are intermediate products in the degradation of fatty acids or amino acids. Due to the enzymatic activities of microorganisms in cheese, methyl ketones are progressively reduced to 2-alkanols, aldehydes are oxidised to acids or reduced to n-alkanols. Therefore, their levels depend on the balance between production and degradation, which is linked to the degree of maturity of the cheese. The maturity of the cheeses for each of the studies may be

Raw Milk Cheeses

Table 6

Ketones in raw (R), pasteurised (P) and microfiltered (MF) milk cheeses

Cheese

Emmental

Cheddar

Cheddar

Propanone 3-Hydroxy-2butanone (acetoin) 2,3-Butanedione (diacetyl) 2-Butanone

R < MF R < MF

RP

n.d.

n.d.

n.d.

R>MF ShakeeI-UrRehman et aL (2000a)

Buchin et aL (1998)

Buchin et aL (unpublished)

Ortigosa et al. (2001)

n.d.: no difference. var: variable. 120 d: 120 days of ripening. 240 d: 240 days of ripening.

different, which could explain the different results from one study to another, even using the same technology. Alcohols

Table 8 summarises the differences between alcohols obtained in R, P or MF cheeses. The observation of the levels of alcohols in the cheeses confirms the previous hypothesis. The presence of the native microflora in milk has a major influence on

the production of alcohols in cheeses. All studies agreed that R cheeses contained greater amounts of the different alcohols, compared to P or MF cheeses (Buchin et al., 1998; Shakeel-Ur-Rehman et al., 2000a,c; Ortigosa et al., 2001; Buchin et al., 2002). This was valid for n-alkanols as well as 2-alkanols. Because alcohols result from the reduction of previously formed compounds, it can be concluded that the raw milk microflora induced faster cheese ripening.

332

Raw Milk Cheeses

Table 7

Aldehydes in raw (R), pasteurised (P) and microfiltered (MF) milk cheeses

Cheese

Emmental

Acetaldehyde Propanal 2-Methyl propanal Butanal 3-Methyl butanal 2-Methyl butanal 2-Methyl butenal Pentanal Hexanal Heptanal 2,4-Heptadienal Octanal Nonanal Decanal Undecanal Dodecanal Tetradecanal Hexadecanal Furancarboxaldehyde Benzaldehyde Phenylacetaldehyde

R > MF

n.d.

R > MF

R>MF

Reference

Buchin et aL (unpublished)

a b c d

Cheddar

Cheddar

R > MF R > MF n.d. n.d.

n.d. R > MF R > MF

n.d.

Semi-hard

n.d. R

R P>R

n.d. n.d.

R P (120 d)d

n.d. n.d.

R>MF n,d, R > MF R > MF

R < P (240 d) R > P (240 d)

n.d. Buchin et al. (1998)

Buchin et al. (unpublished)

Ortigosa et al. (2001)

n.d.: no difference. var: variable. 240 d: 240 days of ripening. 120 d: 120 days of ripening.

Esters Esters are formed by the condensation of an acid and an alcohol. In cheese, this reaction may be spontaneous, or may be mediated by microbial esterases. According to Urbach (1995), esters are not likely to be formed by the starter culture, although Yvon and Rijnen (2001) reported that esterification reactions can be mediated by various LAB, including lactococci, lactobacilli, Sc. thermophilus, leuconostocs and pediococci. This ability is highly strain-dependent. Esters generally have fruity odours and may also influence cheese flavour. They are particularly numerous in hard cheeses such as Swiss Emmental and Parmesan, in which they play an important role in flavour (Urbach, 1997). In contrast, their presence in Cheddar cheese is limited; in fact, fruity flavour in this cheese is a defect. Table 9 summarises the differences between esters obtained in R, P or MF cheeses. In all studies (Buchin et al., 1998, 2002; Shakeel-UrRehman et al., 2000a,c; Ortigosa et al., 2001), whatever the cheese variety, the presence of the raw milk microflora was linked to a greater formation of esters. As expected, ethyl esters were the most important, in rela-

tion to the levels of ethanol, and they were more diversified in Swiss-type cheese (Buchin et al., unpublished).

Sulphur compounds Sulphur compounds contribute to cheese flavour. They are numerous in mould- or smear-surface cheeses, and provide typical cabbage or garlic flavours (Urbach, 1997; Yvon and Rijnen, 2001). Hydrogen sulphide, methional, methanethiol, dimethyldisulphide and dimethyhrisulphide are related to Cheddar flavour (McSweeney and Sousa, 2000); methional, methanethiol and dimethyltrisulphide are key flavour compounds in Emmental cheese (Rychlik and Bosset, 2001), 3-methyhhio-1propanol is present in premium quality Cheddar cheese, ethyl 3-methyhhiopropanoate in Parmesan cheese, while methanethiol is related to unpleasant odours in Grana cheese (Urbach, 1997). The sulphur compounds in cheese derive from the sulphur amino acids. Several mechanisms are involved in their formation. In the reducing environment of cheese, purely chemical decomposition of methionine or cysteine could occur to produce compounds such as methanethiol or H2S. A negative redox potential is a necessary condition for the production of volatile

Raw Milk Cheeses

Table 8

Alcohols in raw (R), pasteurised (P) or microfiltered (MF) milk cheeses

Cheese

Emmental

Ethanol 1-Propanol 2-Methyl propanol 1-Butanol 3-Methyl butanol 1-Pentanol 1-Hexanol 1-Heptanol 1-Octanol 1-Nonanol 1-Decanol 2-Ethyl 1-decanol 2-Propanol 2-Propen-l-ol 2-Butanol 3-Methyl 2-butanol 2-Pentanol 2-Hexanol 2-Heptanol 2-Octanol 2-Nonanol 2-Decanol 3-Methyl 3-buten-l-ol 3-Methyl 2-buten-l-ol 3-Penten-2-ol 2-Methyl 3-pentanol 2,3-Butanediol 1,3-Butanediol Furan methanol Phenol Phenethyl alcohol

R > MF R > MF

Reference

a b c d

333

R > MF R > MF

R > MF n.d. R > MF R > MF R > MF

Cheddar

Cheddar

PR10 d > P PR10 > P PR10 > P R< P PR10 > P n.d. PR10 > P PR10 > P PR 10 > P

R > R > n.d c R < n.d. R< R > n.d.

P P P

Semi-hard

Semi-hard

Roncal

R>P R>P

n.d. R>MF

R>P n.d. n.d. R>P

R>MF R > MF

R > R > R > n.d. R >

n.d.

n.d.

n.d. n.d. R>MF

R > P (240 d) R > P (120 d)

n.d.

R > P (120 d)

PR10 > P R > P

R>P R>P R>P

P R>MF P P P

R > MF

n.d.

R > MF

n.d. n.d. n.d. n.d. PR10 > P

n.d.

R< P

n.d.

ShakeeI-UrRehman et aL (2000c)

ShakeeI-UrRehman et al. (2000a)

n.d. Buchin et aL (unpublished)

P (240 d)

P P

n.d. R>P

R> P PR10 > R> P PR10 > PR10 > PR10 >

P (240 d) a P (120 d) b P (240 d)

n.d. n.d. n.d.

n.d. Buchin et al. (1998)

Buchin et al. (unpublished)

Ortigosa et al. (2001)

240 d 240 days of ripening. 120 d" 120 days of ripening. n.d.: no difference. PR10: mix of 90% pasteurised milk with 10% of raw milk.

sulphydryl compounds in cheese, but enzymatic reactions may also be involved (Urbach, 1997). On the one hand, native milk enzymes may produce disulphide linkages as precursors of sulphydryl groups, and heating of milk stops the production of H2S and reduces the production of methanethiol by inactivating these enzymes (Urbach, 1995). On the other hand, the surface microorganisms of smear cheeses are high producers of sulphur compounds, like methanethiol or methylthioesters. Lactic acid bacteria may also contribute to the production of sulphur compounds. Cheese lactobacilli can produce H2S and Lc. lactis has the ability to cleave methionine

and produce methanethiol (Yvon and Rijnen, 2001), and starters may contribute by providing a reducing environment. The further formation of dimethyldisulphide or dimethyltrisulphide and of most of the methyl thioesters from methanethiol is due to chemical rather than biological reactions. Table 10 summarises the differences in sulphur compounds between R, P or MF cheeses. As expected, comparisons of R and P cheeses showed higher levels of sulphur compounds in the R cheeses (Buchin et al., 1998; Shakeel-Ur-Rehman et al., 2000c; Ortigosa et al., 2001), except in the study by

334

Raw Milk Cheeses

Table 9

Esters in raw (R), pasteurised (P) and microfiltered (MF) milk cheeses Semi-hard

Semi-hard

Emmental

Methyl acetate Methyl propanoate Methyl butanoate Methyl hexanoate Methyl octanoate Ethyl methanoate Ethyl acetate Ethyl propanoate Ethyl butanoate Ethyl hexanoate Ethyl heptanoate Ethyl octanoate Ethyl decanoate Ethyl dodecanoate Ethyl tetradecanoate Propyl acetate Propyl propanoate Propyl butanoate Butyl acetate Butyl propanoate Pentyl acetate 1-Methyl-propyl acetate 2-Methyl-propyl propanoate 2-Methyl-propyl butanoate 3-Methyl-butyl acetate 2-Methyl-butyl butanoate 3-Methyl-butyl butanoate

R > MF n.d. a n.d. n.d.

R>MF R>MF n.d. n.d. n.d.

R R R R

R>MF R>MF n.d. R>MF

Reference

Buchin et aL (unpublished)

a b c d

> > > >

MF MF MF MF

R > MF

Cheddar

Cheddar

Cheese

R>P

R>P

R>P PR10 d > P PR10 > P R>P

R>P R>P R>P R>P

n.d. n.d. n.d. R > MF n.d.

R>P n.d.

Roncal

n.d. R > P (120 d) b

R > P (240 d) c n.d.

R>MF n.d.

n.d.

R>MF R>MF R>MF n.d. n.d. R>MF

R > MF R > MF

R>MF

R > MF R > MF

R>MF n.d.

n.d.

n.d. ShakeeI-UrRehman et al. (2000c)

ShakeeI-UrRehman et al. (2000a)

Buchin et al. (1998)

Buchin et al. (unpublished)

Ortigosa et al. (2001)

n.d.: no difference. 120 d: 120 days of ripening. 240 d" 240 days of ripening. PR10: mix of 90% pasteurised milk with 10% of raw milk.

Shakeel-Ur-Rehman etal. (2000a), where dimethyldisulphide and dimethyhrisulphide were absent from R cheeses. In comparisons of R and MF cheeses, Buchin et al. (2002) found no differences in Morbier-type cheeses, and only a higher level of dimethyldisulphide in raw milk Swiss-type cheeses. This would indicate that the inactivation of native enzymes by heating the milk may be a major event in the diminution of sulphur compound formation, compared to the elimination of the native flora. It is noteworthy that in all these studies, the diversity of the sulphur compounds reported was very poor.

Lactones, hydrocarbons Table 11 summarises the differences in lactones between R and P cheeses.

Lactones are the result of spontaneous cyclisation of the hydroxy-acids naturally present in milk fat. In the studies by Shakeel-Ur-Rehman et al. (2000a,c), heattreatment of the milk influenced the levels of some lactones, but the results were inconsistent. Their occurrence in cheese may also be linked to feeding (Urbach, 1997). Whether aliphatic or aromatic, the levels of hydrocarbons in cheeses do not seem to be influenced by the presence of the native microflora in milk. In conclusion, the presence of the native microflora in R cheeses is of primary importance for the formation of most volatile compounds. Nevertheless, considering the present state of knowledge, it is difficult to establish precisely the role of this microflora. This role can be direct, in transforming the milk constituents into volatile

Raw Milk Cheeses

Table 10

335

Sulphur compounds in raw (R), pasteurised (P) and microfiltered (MF) milk cheeses

Cheese

Emmental

Cheddar

Cheddar

Carbon sulphide Carbon disulphide Dimethyl sulphide Dimethyl disulphide Dimethyl trisulphide Methional Methane sulfonylbis

n.d. a

n.d.

n.d. R > MF

n.d. n.d.

Reference

Buchin et al. (unpublished)

n.d. n.d. R>P

R< P R

P

Semi-hard

R> P R>P

Semi-hard

Roncal

n.d. R > P (240 d) b n.d.

n.d.

ShakeeI-UrRehman et al. (2000c)

ShakeeI-UrRehman et al. (2000a)

Buchin et aL (1998)

Buchin et al. (unpublished)

Ortigosa et al. (2001)

a n.d no difference. b 240 d: 240 days of ripening.

compounds, or indirect, by modifying the composition of the cheese, with the production of precursors of volatile compounds or of molecules that influence chemical reactions or the activity of other microorganisms. In particular, the activities of the indigenous populations can interfere with those of the starter bacteria. Within the complexity of the native milk microflora, it is difficult presently to establish the role of each population, at the species and at the strain level. It is likely that many of the metabolic pathways producing volatile compounds are strain-dependant (Yvon and Rijnen, 2001), which would make their elucidation all the more difficult. The development of molecular techniques for the discrimination of microbial populations at the strain level could be very beneficial to such studies. This situation underlines the importance of maintaining a high diversity of strains in the milk, to retain the diversity of the molecules produced.

Table 11

Lactones in raw (R) and pasteurised (P) milk cheeses

Cheese

Cheddar

Cheddar

-,/-Octanolacton e y-Decanolactone -,/-Dodecanolactone y-Hexadecanolactone y- Decan olacton e y-Dodecanolactone y-Dodecenolactone

PR 10b< P> R n.d. n.d. n.d. R< P R< P

n.d. a R> P R> P n.d. n.d. R> P R> P

Reference

ShakeeI-UrRehman et aL (2000c)

ShakeeI-UrRehman et aL (2000a)

a n.d.: no difference. b PR10: mix of 90% pasteurised milk with 10% of raw milk.

Sensory Aspects In order to avoid any ambiguity, due to the different use of the same terms by different authors, we have chosen to define sensory perceptions as follows: odour is perceived by the nose, with no introduction of the food into the mouth, while flavour is the perception of the food during mastication, either retronasaly or by the tongue (five basic tastes: sweet, acid, bitter, salty, umami). Flavour/odour

Table 12 summarises the differences between the flavour and odour attributes reported for R, P or MF cheeses. Raw milk cheeses ripen faster than cheeses made from milk, the microflora of which has been removed. As a consequence, R cheeses tend to develop a stronger odour/flavour at the same age than those made from P or MF milk (Johnson et al., 1990b; Lau et al., 1991). This has been observed in all types of cheese studied: Cheddar (McSweeney et al., 1993; Shakeel-Ur-Rehman et al., 2000a,b), Manchego (Gaya et al., 1990; FernandezGarcia et al., 2002; Gomez-Ruiz et al., 2002), Raclette (Gallmann and Puhan, 1982), other hard and semi-hard cheeses (Lau et al., 1991; Van den Berg and Exterkate, 1993; Buchin etal., 1998; Skie and ArdO, 2000), Bergk~se (Ginzinger et al., 1999a), Swiss-type cheeses (Bouton and Grappin, 1995; Beuvier etal., 1997; Demarigny et al., 1997) and soft goats' milk cheese (Morgan et al., 2001). In all cases, this phenomenon seems to be directly linked to the activity of the indigenous microflora of the milk. In Cheddar, it has been attributed, in part, to the presence of NSLAB (composed mainly of lactobacilli, but also of pediococci and micrococci) in the raw milk, which are the major part of the natural microflora of this variety of cheese

336

Raw Milk Cheeses

Table 12

Characteristic flavour and odour attributes of raw (R), pasteurised (P) and microfiltered (MF) milk cheeses

Cheese variety

Raw milk

Bergk&se

Odour: intense Flavour: intense Flavour: intense, typical, acid, pungent Flavour: intense, pungent, salty Odour: intense, creamy/milky, fruity/sweet, acid/sharp, pungent Flavour: intense, sour/ acid, sulphur/eggy, bitter, rancid, unclean Only cheeses ripened at 8 ~ (vs 1 ~ Odour: intense, acid Flavour: intense, sour Odour: intense, fruity/ sweet, pungent Flavour: sour/acid Flavour: intense, of acid milk, of rind, Flavour: of fresh milk, fruity, of garlic, spicy, animal, chemical, rancid, bitter, pungent Flavour: intense, animal, spicy, sour

Swiss-type Swiss-type Cheddar

Cheddar

Cheddar

Semi-hard cheese, Morbier-type

Semi-hard round-eyed cheese Roncal

Idiazabal

Idiazabal

Odour: intense (120 d), animal (240 d) Flavour: characteristic, pungent (240 d), animal Aftertaste :intense Odour: characteristic, pungent, sour Flavour: characteristic, pungent, salty Aftertaste: characteristic, pungent Flavour: characteristic, creamy, pungent, acid

Pasteurised milk

Microfiltered milk

Flavour: bitter Flavour: bitter Flavour: acid, bitter, salty Odour: musty

Reference

Ginzinger et al. (1999a) Bouton and Grappin (1995) Beuvier et aL (1997) ShakeeI-UrRehman et aL (2000a)

ShakeeI-UrRehman et aL (2000b) ShakeeI-UrRehman et aL (2000c) Buchin et aL (1998)

Skie and Ard5 (2000)

Odour: animal (120 d)

Ortigosa et aL (2001)

Flavour: torrefied (240 d) Odour: sweet

Mendia et aL (1999)

Flavour: sweet, bitter, sour Aftertaste: bitter, sour Flavour: sweet

Ordonez et aL (1999)

Odour: sweet

(McSweeney et al., 1993). In Swiss-type cheeses (Beuvier et al., 1997), flavour intensity was correlated with counts of FHL, propionibacteria and enterococci, which occur naturally in the raw milk. In pasteurised milk cheeses, denaturation of enzymes and whey proteins by the heat treatment may also be involved; the aggregation of whey proteins on the surface of the caseins micelles also prevents proteolysis of the caseins. This difference in maturity is enhanced by the temperature of ripening and depends on the age of the cheese (Klantschitsch et al., 2000; Shakeel-Ur-Rehman et al., 2000b).

Besides the intensity of flavour, differences in the flavour profile of cheese can be observed. The flavour of the ripened cheese is richer and more complex when the indigenous microflora is present in the milk to be processed. Some observations are constant from one study to another, whereas others vary. In almost all studies comparing cheeses made from raw milk and raw milk after elimination of the microflora, the R cheeses received a higher score for the pungent attribute. Similarly, acid, sour or rancid characteristics were also generally higher in these cheeses. It is likely that these sensory attributes are related to the presence of volatile and FFAs (Curioni and Bosset, 2002;

Raw Milk Cheeses

Gomez-Ruiz et al., 2002). In general, R cheeses are characterised by more 'strong' attributes, such as animal, garlic, spicy, sulphur and unclean. All these characteristics of R cheeses are linked to the notion of higher maturity, expressed from a sensory point of view, but also revealed by physico-chemical patterns, i.e., a greater degree of proteolysis, a higher content of most volatile compounds, and sometimes greater lipolysis (Fig. 2). The distribution of milder attributes, such as fruit, milk or sweet, differs with the study; they can be characteristic of cheeses made either from R or P milk. In Idiazabal cheese, Ordonez et al. (1999) found a relationship between the sweet taste and the amounts of free proline and asparagine, which were higher in P cheeses. Fruity notes may be linked to some methyl ketones such as 2-nonanone (Gomez-Ruiz et al., 2002) or esters (Ortigosa et al., 2001; Gomez-Ruiz et al., 2002). Milky notes are characteristic of diacetyl and acetoin (GomezRuiz et al., 2002). The relationship between bitterness and the presence of the microflora depends on the variety of cheese. When differences were observed in relation to the milk treatment, R semi-hard cheeses were more bitter than P (Buchin etal., 1998; Shakeel-Ur-Rehman etal., 2000a) or MF cheeses (Buchin et al., 2002). In contrast, hard cheeses made from R milk were less bitter than those made from MF or P milk (Bouton and Grappin, 1995; Beuvier et al., 1997; Mendia etal., 1999; Ginzinger et al., 1999a). On the one hand, it seems that the presence of the indigenous microflora is involved,

Axis 3 14 %

2heptanone 2,3peb.tar ~edione i I Ax~s 1 9 dia~tyl ~ 43% 9 aceto.~ ! '..... 2pentan#ne 3m/~ butana ...... 3me 2penta.aOne// ..............................9........heptan/e "Hk

Figure 2 Distribution of volatile compounds and flavour attributes (additional variables, italicized and boldfaced) within R ( . ) and P (O) semi-hard Morbier-type cheese using principal component analysis C2, C3, C5: acetic, propionic, valeric acids (from Buchin et al., 1998).

337

because P and MF cheeses were similar and differed from R cheeses. On the other hand, the heat treatment is likely to play a role. In the study by Beuvier et al. (1997), where R, R MF and P + indigenous microflora (PR) milks were processed, the most bitter cheeses were P and PR. Bitterness is attributed mainly to the presence of hydrophobic peptides, resulting from the hydrolysis of caseins, mostly ORS1- and 6-. Bitterness in cheese results from the balance between the production of bitter peptides by the action of rennet (preferentially in semi-hard cheeses), plasmin (preferentially in hardcooked cheeses), bacterial proteinases and peptidases, and their further degradation by bacterial peptidases. The role of the respective proteolytic systems and their interactions are known to differ according to the cheese variety (Bergere and Lenoir, 1997). In Bergk/ise, a Swisstype cheese, Ginzinger etal. (1999a) found more hydrophobic peptides in P cheeses, which were also more bitter than R cheeses. This distribution of peptides was confirmed in Cheddar by Lau et al. (1991). According to Gomez et al. (1997), bitterness of peptide origin is more likely to be masked by other flavour components in R than in P cheeses. Bergere and Lenoir (1997) pointed out that other components such as indole, amino acids, amines, amides, long-chain ketones or monoglycerides could contribute to the bitter taste of cheese. Thus, Ordonez et al. (1999) found a relationship between bitterness and the amounts of arginine and aromatic amino acids in Idiazabal, though no differences in bitterness were found between R and P cheeses. It is likely that the presence of the R milk microflora affects the flavour characteristics in two ways: on the one hand, acceleration of ripening by faster metabolic pathways, and, on the other hand, the occurrence of a greater variety of metabolic pathways, specific to particular strains of bacteria, which is also influenced by the microbial diversity. The acceptability of R cheeses is also dependent on the cheese variety. Cheddar cheese made from raw milk is, in general, of lower quality than that made from pasteurised milk (Johnson et al., 1990b; McSweeney et al., 1993). In the study of Shakeel-Ur-Rehman et al. (2000b), R Cheddar cheese received higher flavour scores than P cheeses, but this was dependent on the ripening temperature, as higher temperatures (8 ~ instead of 1 ~ led to defects in R cheeses after 6 months. In the study by Morgan et al. (2001), soft goat's milk cheeses had more flavour defects when made from raw than from pasteurised milk, in relationship to microflora and lipolysis levels. Moreover, although the 'goat' flavour of these cheeses is linked to the liberation of particular fatty acids (Le Quere et al., 1996), no differences were found in this attribute.

338

Raw Milk Cheeses

According to Klantschitsch et al. (2000), the quality of Raclette cheese in relation to raw milk is related to ripening temperature and time; to avoid flavour and openness defects, R cheeses should be ripened for less than 90 days at 11 ~ or 60 days at 14 ~ whereas P or MF milk cheeses can be ripened at 17 ~ for 90 days. These differences in acceptability are of course related to the speed of ripening, since the presence of the native microflora accelerates biochemical transformations in the cheese. The difference in maturity, and hence in the occurrence of defects, is more perceptible in soft or semi-hard cheeses, because of their high moisture content; biochemical activities are favoured by the presence of water. Thus, besides the elimination of pathogens, pasteurisation is useful in this type of cheese to obtain a longer shelf-life by slowing the ripening and delaying the occurrence of flavour defects. The consumer of these cheeses is used to the milder flavour provided by pasteurised milk, and may regard the stronger flavour of R cheeses as a defect. It is likely that these varieties of cheese made from raw milk would be appreciated mostly by 'connoisseurs'. Conversely, Swiss-type cheeses, hard Italian cheeses (Johnson et al., 1990b; Bouton and Grappin, 1995), or hard Spanish ovine cheeses, like Idiazabal (Ordonez et al., 1999; Chavarri et al., 2000), are preferred when made from raw milk. Because of their low moisture content, hard cheeses ripen more slowly than soft or semi-hard ones. The presence of the natural microflora in the raw milk may have a lesser influence on the speed of ripening and on the shelf-life of these cheeses. The use of raw milk does not induce defects, and may even reduce some, e.g., bitterness. Moreover, the more complex flavour provided by raw milk may be appreciated by the consumer of hard cheeses. The loss of microflora and, to a lesser extent, of native milk enzyme activities, in pasteurised milk, affects the typical flavour of these cheeses. In Idiazabal cheese, the level of the sensory scores was related to the level of lipolysis, the less lipolysed cheeses being rated 'rather mild', suggesting that this cheese requires a minimum level of lipolysis to develop its characteristic flavour (Chavarri et al., 2000). In hard Italian cheeses such as Romano, Parmesan or Asiago, the inhibition of milk lipase (LPL) in pasteurised milk may be detrimental to the development of typical flavour (Johnson et al., 1990b). In goats' milk cheeses, the preservation of LPL activity can be important for the development of the 'goat' flavour, linked to the liberation of typical goat-flavoured fatty acids from glycerides (Le Qu~r~ et al., 1996). According to Ordonez et al. (1999) and Chavarri et al. (2000), the characteristic Idiazabal flavour is related to the extent of proteolysis. In Swiss-type cheeses, Bouton and Grappin (1995) found a relationship between the extent of primary proteolysis

and the flavour intensity, whereas the typical flavour was related to the concentration of propionic acid. The presence of the raw milk microflora contributes to the sensory diversity of raw milk cheeses. This has been supposed by Shakeel-Ur-Rehman et al. (200Oh) for Cheddar cheese, and shown in Swiss-type cheese models by Beuvier et al. (1997) and Demarigny et al. (1997). There is a higher heterogeneity in the sensory characteristics of cheeses when the native microflora was retained than when it was removed from milk (Fig. 3). The diversity of R cheeses is likely to depend on the level but also on the nature of the strains present in the microflora. Whether the strains in themselves have different metabolic potentialities or interfere by affecting the activity of starter bacteria has not yet been elucidated. Nevertheless, Bouton and Grappin (1995) have shown an interaction between the composition of starter mixtures and the raw milk microflora in the biochemical transformations and sensory characteristics of Swiss-type cheeses. Thus, whatever the mechanisms involved, the preservation of the microbial diversity in raw milk seems to contribute to the diversity of cheeses such as Swiss-type cheeses, particularly Comte. This diversity in the sensory characteristics is a point of major interest in the production of PDO cheeses. Texture

The texture of cheeses is the macroscopic expression of the structure of the cheese matrix, i.e., its composition and organisation. The texture is formed during two

Axis 2 17%

ABxis 1

{

.~_

~ I _ C N

Acid

Pungent) C3 Salted Aroma 9 1 6 2

P+bact

y-CN

Figure 3 Distribution of physico-chemical, microbiological and flavour criteria (additional variables, italicized and boldfaced) within raw (R), pasteurised (P), microfiltered (MF) and pasteurised + microorganisms contained in retentate (P + Bact) milk using principal component analysis. MesoLb: mesophilic lactobacilli; Entero:enterococci; PAB: propionibacteria; C2: acetic acid; C3: propionic acid; iC5: isoavaleric acid; PTA: PTA-soluble N (from Beuvier et aL, 1997).

Raw Milk Cheeses

stages of cheese processing: manufacturing and ripening. The events that occur during these two steps are different in nature. During manufacture, the cheese matrix is formed. It begins with coagulation of the milk, where the caseins organise themselves into a network, entrapping fat globules, water pockets and gas bubbles. The initial structure of the network is thus determined by the composition of the milk, and also by the technological conditions of coagulation: renneting parameters and work in the vat which influence the moisture content of the curd. The network is then modified by acidification due to the fermentation of lactose, that begins in the vat and continues in the mould. Acidification influences the extent of mineralisation of the caseins, and thus their hydration as well as their interactions. During ripening, changes occur in the matrix through the influence of the loss of water and proteolysis. Proteolysis begins with coagulation in the vat; this is essentially primary proteolysis, i.e., internal hydrolysis of casein molecules, by the coagulant or indigenous enzymes of milk, such as plasmin. Secondary proteolysis occurs essentially during ripening, by the action of peptidases of microorganisms. Proteolysis weakens the structure of the casein matrix. It can be easily supposed that removal of the native microflora from raw milk may alter the texture of subsequent cheeses by two major mechanisms. On the one hand, the heat treatment of the milk used to destroy the microflora may alter the structure of the casein matrix by denaturation of whey proteins or the loss of water, or modify the proteolysis patterns by denaturation, activation or modified retention of enzymes. On the other hand, the elimination of most of the indigenous microflora, either by heating or microfiltration, may modify the biochemical changes in cheeses, in particular proteolysis (Grappin and Beuvier, 1997). Among all the articles in which R, P or MF cheeses were compared, few deal with cheese texture. Some work resulted in no differences related to the treatment of milk: no clear differences between R/P/MF milks (McSweeney et al., 1993) and R/P milks (Shakeel-UrRehman et al., 1999) in Cheddar cheese, no sensory textural differences between R/MF milk Swiss-type cheeses (Bouton and Grappin, 1995) or rheological differences between R/P Bergk~se cheese (Ginzinger et al., 1999a). The comparison of R and P cheeses from a texture point of view is difficult, because of the differences in the behaviour of milk during the coagulation step, due to the heat treatment. Depending on the cheesemaking procedures, contradictory findings have been reported, in terms of moisture, on the compositional differences of

339

cheeses (Lau etal., 1990; Buffa etal., 2001b), or pH (Buffa et al., 2001b). Texture differences are thus difficult to interpret. The results of Beuvier et al. (1997) seem to indicate that in Swiss-type cheeses, sensory texture characteristics of R cheeses are influenced by both the heat treatment of milk and the activity of the indigenous microflora. They showed in a comparison of R, P, MF and P cheeses to which the indigenous microflora contained in microfiltration retentate had been added, that R cheeses had a firmer and more granular texture. Proteolysis appears to be the main factor responsible for differences in texture between R and P cheeses. According to Shakeel-Ur-Rehman et al. (2000a), the increase in chymosin retention and in plasmin activity by heat-treatment of milk is the major cause of texture differences between R and P Cheddar cheeses ripened at 1 or 8 ~ While the temperature influenced all texture descriptors, milk treatment influenced only rubberiness (P > R) and graininess ( R > P). They attributed the texture characteristics mostly to differences in water-soluble N (WSN) (Shakeel-Ur-Rehman et al., 2000b), in which enzymes such as chymosin or plasmin have more influence than the activity of the indigenous microflora. Gaya et al. (1990) found a lower fracturability, elasticity and hardness in Manchego cheese made from R ewes' milk than in P cheeses, whatever the ripening time (2 or 4 months) and the ripening temperature (between 8 and 16 ~ They attributed these differences to higher secondary proteolysis in R cheeses, measured by pH 4.6-, TCA- and PTA- soluble N. Buffa et al. (2001b) studied the rheological characteristics of goats' milk semi-hard cheeses made from R or P milk. R cheeses were firmer, less fracturable, and more cohesive than P ones. These characteristics were attributed to the levels of moisture and WSN: the lower the moisture and more intact the caseins, the less the fracturability and deformability. Fracture stress was higher for R cheeses, i.e., a lower fracturability than the P cheeses. This parameter was correlated with the levels of moisture and WSN: the less the moisture and more intact the caseins, the less the fracturability. Fracture strain, which describes the deformability of cheese, was higher for R cheeses, but only at one day. It could be due to the higher pH of these cheeses at this stage, water being partly absorbed to hydrate the negative charges formed in caseins with high pH values. This parameter has the same correlation with moisture and WSN as previously- deformability decreases when the hydration of proteins decreases and when elastic structural elements disappear. The microstructure of R cheeses was more regular, with a closed protein matrix, and smaller and more uniform fat globules, whereas

340

Raw Milk Cheeses

P cheeses had an open structure with irregular cavities. As a consequence, differences in colour were observed. Rosenberg etal. (1995) measured the viscoelastic characteristics, G' (storage modulus) and G" (loss modulus), of Cheddar cheeses. These parameters were higher in R cheeses than in P cheeses ripened for 8 months. In P cheeses, they were found to be related to the extent of proteolysis; a higher G' signified a higher elastic behaviour of the matrix with the accumulation of proteolysis products. The authors explained this observation by the binding of water by the ionic groups liberated by the cleavage of peptide bounds. This relation with the extent of proteolysis was not observed in R cheeses, maybe because of different proteolytic activities during the ripening of these cheeses, as revealed by differences in peptide composition. Mendia et al. (1999) found more graininess and firmness and less creaminess and elasticity in R ewes' milk Idiazabal cheeses than in P cheeses. These differences were attributed to the slower maturation of P cheeses. This was confirmed by the fact that the differences diminished with increases in ripening time, and were thought to be linked to the moisture content. For certain types of cheese consumed mainly in a melted form, such as Raclette, it is more interesting to evaluate the texture characteristics of the cheese after melting. Melting properties were evaluated in Raclette cheeses made from R, P or MF milk and mixtures of the three types of milk in different proportions (Klantschitsch et al., 2000). R cheeses had a longer consistency than P/MF cheeses after 90 days ripening. According to the authors, this is related to the proteolysis patterns, proteolysis 'in width', pH 4.6 N/TN (lower in MF) leading to longer consistency and higher viscosity, proteolysis 'in depth' (NPN/TN) leading to shorter consistency. The viscosity did not differ between the cheeses. The firmness of melted cheese was also higher in R than in P/MF cheeses after 90 days ripening, with a score indicating insufficient melting quality. Fat separation increased more rapidly with ripening time in R than in P cheeses. Softening and dropping points were in the range for good melting quality in all cheeses ripened at 11 or 14 ~ but only in the MF cheeses ripened at 17 ~ The effect of the microflora on the melting quality of Raclette is dependant on the ripening temperature and time; a high temperature (17 ~ is detrimental when using raw milk, whereas, in the case of microfiltered milk, it is useful to accelerate ripening. In all these studies, the lack of microbial investigations made it difficult to establish a relationship between the microbial populations, whether of indigenous or starter origin, and the characteristics of texture. Nevertheless, when the observed differences were

attributed to the secondary proteolysis, microbial activity was involved.

Conclusion

Microbial communities play an essential role in the control of sensory qualities of cheese. They are more diverse and complex in R cheeses for which milk undergoes no treatment to reduce the microflora. They contribute to the development of a typical cheese taste and flavour. Diversity of the sensory qualities is a specific feature of R cheese. Elimination of the raw milk microflora by pasteurisation or microfiltration definitively leads to different cheeses from a sensorial point of view. Still, is it necessary to have raw milk that is sufficiently rich in terms of quantity and diversity of microorganisms? As outlined at the beginning, the improvement in hygienic practices on farms has led to a 'clean' raw milk, with low microbial counts (Odet, 1999). Raw milk with a low level of microbes could induce a reduction in sensorial diversity of cheese due to a reduction of microbial diversity. Indeed, Dasen et al. (2003) have observed that the strain diversity of mesophilic lactobacilli in raw milk experimental Cheddar cheese was close to that observed in industrial Cheddar cheese manufactured with pasteurised milk. The former was made from raw milk with a total of around 10 000 cfu m1-1. The fact that raw milk tends to be more and more microbiologically 'clean' implies that there is a risk that the sensorial differences between R and P cheeses will be erased. Some experiments in progress, particularly in France, aim to evaluate dairy farming practices, including milking practices, on the raw milk microflora in terms of quantity and diversity. Recently, Michel et al. (2001) observed links between milking practices and the bacteriological quality of milk, showing that it is possible to manage the microbial quality of milk on the farm to promote the technologically 'useful' microflora, while maintaining pathogens at a low level. This is a good way to keep the natural microflora in R cheese production, in terms of quantity and diversity, in order to preserve their sensorial diversity. To add selected microorganisms could enhance the aroma of cheese, but the cheese would have a more uniform flavour, a characteristic which is not sought by both the producers and the consumers, because diversity of flavour is considered a special feature of traditional R cheeses (Grappin and Beuvier, 1997). Otherwise, according to Montel (2002), microbial communities may play a key role in the microbiological safety of R cheese. This potential role is supported by several studies in which cheeses or milk, with a more complex microflora, were less contaminated by

Raw Milk Cheeses

L. monocytogenes than those with a less diversified flora (Brouillaud-Delattre et al., 1997; Eppert et al., 1997). Thus, well-monitored microbial diversity, from farm to cheese, by acting as a barrier against pathogens, may be a trump card for cheese safety (Montel, 2002). According to Stanton et al. (1998), cheeses, because of their high fat content and their texture, could offer protection to the living microorganisms contained within them, especially at the m o m e n t of their passage into the gastrointestinal tract of the consumer. More and more studies demonstrate the beneficial effects on health of strains of microorganisms and give hope for other discoveries in R cheeses, which are rich in microorganisms (Bouton, 2001; Moreau and Vuitton, 2002). The preservation of the microbial diversity in raw milk, essential to obtain cheeses with greater sensorial diversity, more and more appreciated by (European) consumers, potentially useful to fight against pathogens and potentially useful for health, is a challenge for milk and cheese producers, and researchers, to take up over the next years.

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Developments in Dairy Chemistry, Vol. 2, RE Fox, ed., Applied Science Publishers, London. pp. 195-239. Demarigny, Y., Beuvier, E., Dasen, A. and Duboz, G. (1996). Influence of raw milk microflora on the characteristics of Swiss-type cheeses. I. Evolution of microflora during ripening and characterization of facultatively heterofermentative lactobacilli. Lait 76,371-387. Demarigny, Y., Beuvier, E., Buchin, S., Pochet, S. and Grappin, R. (1997). Influence of raw milk microflora on the characteristics of Swiss-type cheeses. II. Biochemical and sensory characteristics. Lait 77, 151-167. Donnelly, C.W. (2001). Factors associated with hygienic control and quality of cheeses prepared from raw milk: a review. Bulletin 369. International Dairy Federation, Brussels. pp. 16-27. Eliskases-Lechner, E, Ginzinger, W., Rohm, H. and Tschager, E. (1999). Raw milk flora affects composition and quality of Bergkase. 1. Microbiology and fermentation compounds. Lait 79, 385-396. Eppert, I., Lechner, E., Mayr, R. and Scherer, S. (1995). Listeria and coliforms in soft cheeses made from raw and pasteurized milk. Arch. Lebens. 46, 85-88. Eppert, I., Valdes-Stauber, N., Gotz, H., Busse, M. and Scherer, S. (1997). Growth reduction of Listeria spp. caused by undefined industrial red smear cheese cultures and bacteriocin-producing Brevibacterium linens as evaluated in situ on soft cheese. Appl. Environ. Microbiol. 63, 4812-4817. Estepar, J., del Mar Sanchez, M., Alonso, L. and Mayo, B. (1999). Biochemical and microbiological characterization of artisanal "Pefiamellera" cheese: analysis of its indigenous lactic acid bacteria. Int. Dairy J. 9, 737-746. Fernandez-Garcia, E., Carbonell, M. and Nunez, M. (2002). Volatile fraction and sensory characteristics of Manchego cheese. 1. Comparison of raw and pasteurized milk cheese. J. Dairy Res. 69,579-593. Fortina, M.G., Ricci, G., Acquati, A., Zeppa, G., Gandini, A. and Manachini, EL. (2003). Genetic characterization of some lactic acid bacteria occurring in an artisanal protected denomination origin (PDO) Italian cheese, the Toma Piemontese. Food Microbiol. 20,397-404. Fox, RE and Stepaniak, L. (1993). Enzymes in cheese technology. Int. Dairy J. 3,509-530. Frau, M., Massanet, J., Rossello, C., Simal, S. and Canellas, J. (1997). Evolution of free amino acid content during ripening of Mahon cheese.J. Food Chem. 60,651-657. Freitas, C. and Malcata, EX. (2000). Microbiology and biochemistry of cheeses with Appelation d'Origine Protegee and manufactured in the Iberian Peninsula from ovine and caprine milks. J. Dairy Sci. 83,584-602. Gallmann, P. and Puhan, Z. (1982). Beeinflussung der Kasequalit~t durch ausgew~ihlte Mikroorganismen der Rohmilchflora. Schweiz. Milchwirt. Forsch. 11, 64-69. Garcia, M.C., Rodrfguez, M.J., Bemardo, A., Tornadijo, M.E and Carballo, J. (2002). Study of enterococci and micrococci isolated throughout manufacture and ripening of San SimOn cheese. Food Microbiol. 19, 23-33. Gaya, P., Medina, M., Rodriguez-Marin, M.A. and Nunez, M. (1990). Accelerated ripening of ewes' milk Manchego

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Rychlik, M. and Bosset, J.O. (2001). Flavour and off-flavour compounds of Swiss Gruyere cheese. Evaluation of potent odorants. Int. Dairy J. 11,895-901. Ryser, E.T. and Marth, E.H. (1987). Fate of Listeria monocytogenes during the manufacture and ripening of Camembert cheese. J. Food Prot. 50,372-378. Saboya, L.V. and Maubois, J.L. (2000). Current developments of microfiltration technology in the dairy industry. Lait 80, 541-553. Sarantinopoulos, P., Kakantzopoulos, G. and Tsakalidou, E. (2002). Effect of Enterococcus faecium on microbiological, physicochemical and sensory characteristics of Greek Feta cheese. Int. J. Food Microbiol. 76, 93-105. Saric, Z., Luthi-Peng, Q.-Q. and Puhan, Z. (2002). Quality aspects of Travnicki cheese made from raw and pasteurised cow and goat milk. Milchwissenschaft 57, 631-634. Schneller, R., Good, P. and Jenny, M. (1997). Influence of pasteurized milk, raw milk and different ripening cultures on biogenic amine concentrations in semi-soft cheeses during ripening. Z. Lebensm. Unters. Forsch. A. 204,265-272. Shakeel-Ur-Rehman, McSweeney, P.L.H. and Fox, RE (1999). A study on the role of the indigenous microflora of raw milk on the ripening of Cheddar cheese. Milchwissenschaft 54, 388-392. Shakeel-Ur-Rehman, Banks, J.M., Brechany, E.Y., Muir, D.D., McSweeney, P.L.H. and Fox, RE (2000a). Influence of ripening temperature on the volatiles profile and flavour of Cheddar cheese made from raw or pasteurised milk. Int. Dairy J. 10, 55-65. Shakeel-Ur-Rehman, Banks, J.M., McSweeney, RL.H and Fox, P.E (2000b). Effect of ripening temperature on the growth and significance of non-starter lactic acid bacteria in Cheddar cheese made from raw or pasteurised milk. Int. DairyJ. 10, 45-53. Shakeel-Ur-Rehman, McSweeney, P.L.H., Banks, J.M., Brechany, E.Y., Muir, D.D. and Fox, RE (2000c). Ripening of Cheddar cheese made from blends of raw and pasteurised milk. Int. Dairy J. 10, 33-44. Skie, S. and Ardo, Y. (2000). Influence from raw milk flora on cheese ripening studied by different treatments of milk to model cheese. Lebensm. Wiss. Technol. 33, 499-505. Sousa, M.J. and Malcata, X. (1997). Ripening of ovine milk cheeses: effects of plant rennet, pasteurization, and addition of starter on lipolysis. Food Chem. 59,427-432. Stanton, C., Gardiner, G., Lynch, P.B., Collins, J.K., Fitzgerald, D. and Ross, R.P. (1998). Probiotic cheeses. Int. Dairy J. 8, 491-496. Thierry, A. and Maillard, M.B. (2002). Production of cheese flavour compounds derived from amino acid catabolism by Propionibacterium freudenreichii: a review. Lait 82, 17-32. Urbach, G. (1993). Relations between cheese flavour and chemical composition. Int. Dairy J. 3,389-422. Urbach, G. (1995). Contribution of lactic acid bacteria to flavour compound formation in dairy products. Int. Dairy J. 5,877-903.

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Biochemistry of Cheese Ripening: Introduction and Overview P.L.H. McSweeney, Department of Food and Nutritional Sciences, University College, Cork, Ireland

Introduction As discussed in 'Cheese: An Overview', Volume 1, rennetcoagulated cheeses are ripened (matured) for a period ranging from 2 weeks (e.g., Mozzarella) to 2 or more years (e.g., Parmigiano Reggiano or extra-mature Cheddar) during which the flavour and texture characteristic of the variety develop. Ripening usually involves changes to the microflora of the cheese, including death and lysis of the starter cells, development of an adventitious non-starter microflora and, in many cheeses, growth of a secondary microflora (e.g., Propionibacterium freudenreichii subsp, shermanii in Swiss cheese, moulds in mould-ripened varieties and a complex Gram-positive bacterial microflora on the surface of smear-ripened cheeses). The metabolic activity of the secondary microflora often dominates flavour development, and in some cases, e.g., whitemould cheeses, the texture, of varieties in which they grow. The microbiology of cheese during ripening is discussed in 'The Microbiology of Cheese Ripening', Volume 1. As discussed in 'Rheology and Texture of Cheese', Volume 1, ripening usually involves the softening of cheese texture, as a consequence of the hydrolysis of the casein matrix, changes in the waterbinding ability of the curd and changes in pH (which may cause other changes such as the migration and precipitation of calcium phosphate). The flavour of cheese curd immediately after manufacture is rather bland and indeed it can be difficult to differentiate the flavours of certain varieties at this stage. During ripening, cheese flavour develops due to the production of a wide range of sapid compounds by the biochemical pathways described below. Volatile flavour compounds are of particular importance to cheese flavour and are discussed in 'Sensory Character of Cheese and its Evaluation', Volume 1. Quantification of the volatile flavour compounds of cheese are described in 'Instrumental Techniques', Volume 1. Biochemical reactions which occur in cheese during ripening are usually grouped into four major categories: (1) glycolysis of residual lactose and catabolism of lactate, (2) catabolism of citrate, which is very important in certain varieties, (3) lipolysis and the

catabolism of free fatty acids and (4) proteolysis and the catabolism of amino acids (Fig. 1). These reactions are discussed in 'Metabolism of Residual Lactose and of Lactate and Citrate', 'Lipolysis and Catabolism of Fatty Acids in Cheese', 'Proteolysis in Cheese during Ripening', 'Catabolism of Amino Acids in Cheese during Ripening', Volume 1. Since the biochemistry of cheese ripening is complex, the purpose of this chapter is to present an overview of the principal biochemical pathways which contribute to cheese ripening and to discuss the role of the principal ripening agents in cheese and the acceleration of cheese ripening. Aspects of cheese ripening common to many varieties are discussed in 'Metabolism of Residual Lactose and of Lactate and Citrate', 'Lipolysis and Catabolism of Fatty Acids in Cheese', 'Proteolysis in Cheese during Ripening', 'Catabolism of Amino Acids in Cheese during Ripening', Volume 1; ripening of specific varieties is discussed in the relevant chapters in Volume 2.

Glycolysis of Residual Lactose and Catabolism of Lactate Since cheeses are fermented dairy products, the metabolism of lactose to lactate is essential in the manufacture of all varieties. Cheese curd contains a low level of residual lactose which is metabolised rapidly early in ripening to lactate which may be catabolised subsequently via a range of pathways. Catabolism of lactate probably occurs in all cheeses and is particularly important in surface mould-ripened varieties (e.g., Camembert) and in Swiss cheese. These reactions were reviewed by Fox et al. (1990, 1993) and McSweeney and Sousa (2000) and are discussed in detail in 'Metabolism of Residual Lactose and of Lactate and Citrate', Volume 1. The pathway through which lactose is metabolised depends on the starter type (see 'Starter Cultures: General Aspects', Volume 1; Cogan and Hill, 1993; Fox et al., 2000; McSweeney and Sousa, 2000; Broome et al., 2003). The final step in the glycolysis of lactose is the conversion of pyruvate to lactate which is catalysed by lactate dehydrogenase (LDH). Depending on

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

Copyright 9 2004 Elsevier Ltd All rights reserved

348

Biochemistry of Cheese Ripening: Introduction and Overview

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Figure 1 General overview of the biochemical pathways which operate in cheese during ripening.

the type of LDH (D- or L-LDH) in the cell, D- (e.g.,

Lb. delbrueckii subsp, bulgaricus), L- (e.g., Lactococcus, Sc. thermophilus) or D/L- (e.g., Lb. helveticus) lactate is the end product of glycolysis which converts 1 mol of lactose to 4 mol of lactate with the production of 4 mol of ATR Unlike most lactic acid bacteria (LAB), Leuconostoc spp. use the phosphoketolase pathway to metabolise lactose; the end products of this pathway are lactate, ethanol and CO2 and thus differ from that of the glycolytic pathway9 Although essential for cheese manufacture, the metabolism of lactose to lactate is essentially complete at the end of manufacture or during the early stages of ripening. Most lactose in milk is lost in the whey and that which is retained in the curd is metabolised rapidly after drainage. However, the activity of the starter is greatly reduced at the end of manufacture or soon thereafter due to the combination of low pH, high NaC1 and lack of a fermentable carbohydrate. The inhibition of acid production is particularly abrupt in dry-salted varieties (e.g., Cheddar) where NaC1 concentration reaches equilibrium much faster than in brine-salted cheeses. Fresh cheese curd contains a low level of lactose which, in the case of Cheddar cheese, is reduced to trace levels within one

month of ripening by the (albeit reduced) activity of the starter or by the action of the non-starter lactic acid bacteria (NSLAB). Lactate contributes to the flavour of cheese, particularly early during maturation, but the major effect of acidification on flavour development is indirect since, together with the buffering capacity of the curd, it influences pH and thus the growth of the secondary flora and the activity of ripening enzymes. Lactate is an important substrate for a range of reactions which contribute positively or negatively to cheese ripening. L-Lactate, produced by Lactococcus, can be racemised to DL-lactate by the NSLAB flora in Cheddar and Dutch-type cheeses. DL-Lactate is less soluble than k-lactate, resulting in the formation of Ca-D-lactate crystals which appear as white specks on the surface of the mature cheese. Lactate can also be metabolised to acetate and CO2 by some members of the NSLAB flora, although this oxidative pathway is relatively minor in cheese due to its low oxidationreduction (redox) potential (c. - 2 5 0 mV) and is limited by the availability of 02. Late gas blowing is a defect in certain hard and semi-hard varieties caused by the anaerobic catabolism of lactate to butyrate and H2

Biochemistry of Cheese Ripening: Introduction and Overview

by Clostridium tyrobutyricum. This problem can be overcome by good hygiene, addition of NaNO3 or lysozyme or by the physical removal of the spores by bactofugation or microfiltration. However, catabolism of lactate is particularly important in Swiss and surface mould-ripened cheeses. In the former, lactate is catabolised by Propionibacterium freudenreichii subsp, shermanii to propionate, acetate, H20 and CO2. Propionate and acetate contribute to the flavour of Swiss cheese; CO2 migrates through the curd to points of weakness where it collects to form the large eyes characteristic of Swiss-type cheese. The oxidative catabolism of lactate to H20 and CO2 by Penicilliurn camemberti at the surface of Camembert and Brie-type cheeses is of great indirect importance to their ripening. The catabolism of lactic acid causes a large increase in the pH of the surface of these cheeses which leads to a pH gradient from the surface to the core and to the migration of lactate towards the surface. The high pH at the surface causes precipitation of calcium phosphate, which, in turn, causes the migration of calcium and phosphate to the cheese surface. These changes lead to the characteristic softening of surface mould-ripened cheese which, when mature, have an almost liquid-like consistency. Oxidative metabolism of lactate is also of significance at the surface of smear-ripened cheeses (e.g., Tilst or Limburger) where, early in ripening, yeasts deacidify the surface which encourages the growth of the Gram-positive bacteria characteristic of the surface. Oxidative metabolism of lactate probably also occurs in Blue cheese but its effect is less important than in surface mouldripened cheese since P. roqueforti is distributed throughout the cheese and thus gradients do not develop across the cheese mass.

Lipolysis and Metabolism of Fatty Acids Studies in which milk fat was replaced with other lipids have demonstrated that milk fat is essential for the development of the flavour of Cheddar and probably all other ripened cheeses. As in all high-fat foods, lipids present in cheese can undergo hydrolytic or oxidative degradation; the latter is generally considered not to be important in cheese, primarily due to its low redox potential. Lipolysis in cheese during ripening is discussed in detail in 'Lipolysis and Catabolism of Fatty Acids in Cheese', Volume 1. As discussed by McSweeney and Sousa (2000) and Collins et al. (2003a), lipases in cheese originate from a number of sources. Milk contains an indigenous lipoprotein lipase (LPL), which contributes to lipolysis in cheese during ripening. Lipoprotein lipase activity is more important in cheese made from raw milk than

349

in that made from pasteurised milk since the enzyme is extensively inactivated by pasteurisation. Rennet paste, used as coagulant in certain Italian cheese varieties, contains a potent lipase, pregastric esterase, which is responsible for lipolysis in cheeses such as Provolone and the Pecorino varieties. Lactic acid bacteria are weakly lipolytic, but their enzymes have been shown to contribute to the low level of lipolysis characteristic of Cheddar cheese (Collins etal., 2003b). Likewise, Pr. Jreudenreichii subsp, shermanii possesses a lipase which, together with enzymes from the thermophilic starter organisms, contributes to the low level of lipolysis in Swiss cheese. Penicillium roqueforti produces potent extracellular lipases which are responsible for the extensive lipolysis characteristic of Blue cheese. P. camemberti and the complex Gram-positive surface microflora of smear cheeses also produce extracellular lipases which contribute to lipolysis in surface-bacterial or white mould-ripened varieties. The level of lipolysis in cheese is determined using various non-specific techniques (e.g., solvent extraction and titration of the fatty acids with alcoholic KOH or by the formation of coloured Cu soaps) or by quantitation of individual fatty acids, usually by gas chromatography (see Collins et al., 2003a). Fatty acids have a direct impact on the flavour of many cheese varieties. In particular, C4-C10 acids are strongly flavoured. Levels of fatty acids vary considerably between varieties. Many internal bacterially ripened varieties (e.g., Edam, Swiss and Cheddar) contain low levels of fatty acids (c. 200-1000 mg kg-1). Very high levels of fatty acids are found in Blue cheese (c. 30 000 mg kg-1). In addition to their direct role in cheese flavour, fatty acids are important precursors for the production of other volatile flavour compounds during ripening (Fig. 2). Fatty acid esters are produced by reaction of fatty acids with an alcohol; ethyl esters are most common in cheese. Thioesters are formed by reaction of a fatty acid with a thiol compound formed via the catabolism of sulphur-containing amino acids. Fatty acid lactones are cyclic compounds formed by the intramolecular esterification of hydroxyacids; 7- and 8-1actones contribute to the flavour of a number of cheese varieties. The principal class of volatile flavour compounds in Blue cheese is n-methyl ketones (alkan2-ones) which are produced from fatty acids by partial ]3-oxidation. n-Methyl ketones may be reduced to the corresponding secondary alcohols. Fatty acid catabolism is summarised in Fig. 2 and is discussed in detail in 'Lipolysis and Catabolism of Fatty Acids in Cheese', Volume 1. Volatile fiavour compounds in cheese, including those derived from fatty acids, are usually quantified using gas chromatography-mass spectrometry (GC-MS; see 'Instrumental Techniques', Volume 1).

350

Biochemistry of Cheese Ripening: Introduction and Overview

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Proteolysis and Catabolism of Amino Acids Proteolysis is the most complex, and in most varieties, the most important biochemical event which occurs during cheese ripening. Proteolysis has been discussed in reviews by Grappin et al. (1985), Rank et al. (1985), Fox (1989), Fox et al. (1993, 1994, 1995), Fox and McSweeney (1996), McSweeney and Sousa (2000) and Sousa et al. (2001) and is covered in detail in 'Proteolysis in Cheese during Ripening', Volume 1. Proteolysis is very important for cheese texture by hydrolysing the para-casein matrix which gives cheese its structure and by increasing the water-binding capacity of the curd (i.e., to the new ot-carboxylic and or-amino groups produced on cleavage of peptide bonds). Proteolysis may indirectly affect texture by increasing pH through the production of NH3 following amino acid catabolism.

Peptides may have a direct impact on cheese flavour (some are bitter) or they may provide a brothy background flavour to cheese. However, recent research has indicated that the major role of proteolysis in cheese flavour is in the production of amino acids which act as precursors for a range of catabolic reactions which produce many important volatile flavour compounds (see McSweeney and Sousa, 2000; Yvon and Rijnen, 2001). In most cheese varieties, the initial hydrolysis of caseins is caused by the coagulant and to a lesser extent by plasmin and perhaps somatic cell proteinases (e.g., cathepsin D) which result in the formation of large (water-insoluble) and intermediate-sized (watersoluble) peptides which are subsequently hydrolysed by the coagulant and enzymes from the starter and non-starter flora of the cheese. The production of

Biochemistry of Cheese Ripening: Introduction and Overview

small peptides and amino acids is caused by the action of microbial proteinases and peptidases, respectively. Preparations of selected aspartyl proteinases are used to coagulate milk. Chymosin (EC 3.4.23.4) is the principal proteinase (88-94%) in traditional calf rennets, the remainder being pepsin (EC 3.4.23.1) (Rothe et al., 1977). Although, the principal role of the coagulant in cheesemaking is to coagulate milk, some activity is retained in the curd, depending on factors such as coagulant type, cooking temperature and pH at drainage, and contributes to proteolysis in many varieties (Creamer et al., 1985). Plasmin (fibrinolysin; EC 3.4.21.7) is the dominant indigenous proteinase in milk and is produced from its inactive precursor, plasminogen, by a system of plasminogen activators (PA). Inhibitors of plasmin and of PA are also present in milk. Plasmin, which is optimally active at pH 7.5 and 37 ~ is most active in high-cook cheeses due to denaturation of inhibitors and increased activation of plasmin and in cheeses in which the pH increases during ripening (e.g., Blue cheese or the surfaces of white-mould and smearripened varieties). Plasmin is most active on [3-casein, hydrolysing it at three sites to produce the y-caseins and some proteose peptones. Milk contains somatic (white blood) cells, which contain lysosomes, which in turn, contain many proteolytic enzymes. To date, cathepsin D (see review by Hurley et al., 2000) and cathepsin B (Magboul et al., 2001) have been confirmed in milk. Lactic acid bacteria (Lactococcus, Lactobacillus, Streptococcus) possess very comprehensive proteolytic systems that have been studied extensively and reviewed (e.g., Fox and McSweeney, 1996; Kunji et al., 1996; Law and Haandrikman, 1997; Christensen etal., 1999). Lactic acid bacteria possess a cell envelope-associated proteinase (PrtP or lactocepin), 3-4 intracellular proteinases, intracellular oligoendopeptidases (PepO, PepF), a number of aminopeptidases (PepN, PepC, PepG, PepA, PepL), a pyrolidone carboxyl peptidase (PCP), a dipeptidylaminopeptidase (PepX), a proline iminopeptidase (PepI), an aminopeptidase P (PepP), a prolinase (PepR), a prolidase (PepQ), general dipeptidases (PepV, PepD, PepDA) and a general tripeptidase (PepT). They also possess oligopeptide, di/tripeptide and amino acid transport systems (Fig. 3). This proteolytic system is necessary to enable the LAB to grow to high numbers in milk (109-1010 cfuml-1), which contains only low levels of small peptides and amino acids. PrtP contributes to the formation of small peptides in cheese, probably by hydrolysing larger peptides produced from Otsl-casein by chymosin or from [3-casein by plasmin, whereas the aminopeptidases, dipeptidases and tripeptidases (which are intra-

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351

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cellular) are responsible for the release of free amino acids after the cells have lysed. Non-starter lactic acid bacteria, although present initially at low numbers (5% or >2% for pediococci and lactobacilli, respectively (Thomas and Crow, 1983). Racemization of lactate in a Cheddar cheese inoculated with pediococci was complete after ---19 days, while it required ---3 months in a control cheese with a much lower number of NSLAB, especially pediococci (Thomas and Crow, 1983). The racemization of L-lactate is probably not significant from the favour viewpoint. However, Ca-lactate may crystallize in cheese, causing undesirable white specks, especially on cut surfaces (Pearce et al., 1973; Severn et al., 1986; Dybing et al., 1988). Such crystals are harmless, but they may cause consumers to reject cheese as being mouldy or containing foreign bodies (Dybing et al., 1988). The solubility of Ca-DL-lactate is lower than that of pure Ca-L-lactate (Thomas and Crow, 1983; Dybing et al., 1988) and hence racemization of lactate favours the development of crystals in cheese. Dybing etal. (1988) calculated that the amount of available lactate in cheese can potentially create enough Ca-lactate pentahydrate to exceed its

364

Metabolism of Residual Lactose and of Lactate and Citrate

solubility only slightly at 0 ~ Thus, crystal formation is favoured if microbial metabolism increases the concentration of D- relative to L-lactate, due to the lower solubility of Ca-DL-lactate. Crystal growth requires nucleation centres which may be bacterial cells, microcrystals of calcium phosphate or undissolved CaCO3. Increased levels of residual lactose, which favour the growth of NSLAB, can facilitate production of Ca-lactate crystals (Pearce et al., 1973; Sutherland and Jameson, 1981). Likewise, factors which increase the release of casein-bound Ca (e.g., low pH or high salt which causes the ion-exchange of Na + for Ca2+; Dybing et al., 1988) or reduce the solubility of Ca-lactate (e.g., a lower ripening temperature) favour crystal formation.

Oxidation of lactate

Lactate can be metabolized by LAB, depending on strain, to acetate, ethanol, formate and CO2 (see Fox et al., 2000). Pediococci, if present in cheese together with high concentrations of 02, produce 1 mol of acetate and 1 mol of CO2 and consume 1 mol of 02 per mol of lactate utilized (Thomas et al., 1985). The pH optimum for oxidation is 5-6 and depends on the lactate concentration. The concentration of lactate in cheese exceeds that required for optimal oxidation, and lactate is not oxidized until all sugars have been exhausted. However, the oxidation of L-lactate to acetate occurs to a very limited extent in cheese wrapped in film due to the low level of 02 available. The oxidative activity of suspensions of starter and NSLAB isolated from cheese on lactose, lactate, citrate, amino acids and peptides was studied by Thomas (1986). Starter bacteria were active mainly on lactose, with low activity on enzyme-hydrolysed casein; Lb. casei oxidized citrate, while Lb. plantarum, Lb. brevis and P. pentosaceus oxidized lactose, peptides, L- and D-lactate, but not citrate. These results suggest that the oxidation of lactate to acetate in cheese depends on the NSLAB population and on the availability of 02, which is determined by the size of the block and the oxygen permeability of the packaging material (Thomas, 1987). Acetate, which may also be produced by starter bacteria from lactose (Thomas et al., 1979), citrate or from amino acids by starter bacteria and lactobacilli (Nakae and Elliott, 1965), is usually present at high concentrations in most, or all, cheeses and is considered to contribute to cheese flavour, although a high concentration may cause off-flavours (Aston and Dulley, 1982).

Oxidative metabolism of lactate in surface mould-ripened varieties

The catabolism of lactate is very extensive in surface mould-ripened varieties, e.g., Camembert and Brie. The concentration of lactate in these cheeses at day 1 is ---1.0%, produced mainly or exclusively by the mesophilic starter, and hence, presumably, is L-lactate. Secondary organisms quickly colonize and dominate the surface of these cheeses (Addis et al., 2001), initially Geotrichum candiclum and yeasts (e.g., Kluyveromyces lactis, Debaryomyces hansenii and Saccharomyces cerevisiae; Gripon, 1993), followed by a dense growth of Penicillium carnemberti (Mollimard et al., 1995) and, particularly in traditional manufacture, by low numbers of Gram-positive organisms similar to those found on the surface of smear-ripened cheeses, which do not colonize the cheese surface until the pH has increased to >5.8 (see 'Surface Mould-ripened Cheeses', Volume 2). G. candidurn and P. camemberti rapidly metabolize lactate to CO2 and H20, causing an increase in pH. Deacidification occurs initially at the surface, resulting in a pH gradient from the surface to the centre and causing lactate to diffuse outwards. When the lactate has been exhausted, P. carnemberti metabolizes proteins, producing NH3 which diffuses inwards, further increasing the pH. The concentration of calcium phosphate at the surface exceeds its solubility at the high pH and precipitates as a layer of Ca3(PO4)2 on the surface, thereby causing a calcium phosphate gradient within the cheese, resulting in its outwards diffusion; reduction of the concentration of calcium phosphate in the interior helps to soften the body of the cheese (Fig. 3). In addition to softening the texture, changes to the cheese matrix may influence cheese flavour by changing the rates of migration or release of flavour compounds (Engel et al., 2001). The elevated pH stimulates the action of plasmin, which, together with residual coagulant, is responsible for proteolysis in this cheese rather than proteinases secreted by the surface microorganisms, which, although very potent, diffuse into the cheese to only a very limited extent, although peptides or other low molecular weight compounds produced by them at the surface may diffuse into the body of the cheese (Sousa and McSweeney, 2001; Churchill et al., 2003). The combined action of increased pH, loss of calcium (which affects to the integrity of the protein network) and proteolysis are necessary for the very considerable softening of the body of Brie and Camembert (see Noomen, 1983; Lenoir, 1984; Karahadian and Lindsay, 1987; Sousa and McSweeney, 2001). Changes which occur in Camembert-type cheese during ripening are indicated in Fig. 3.

M e t a b o l i s m of Residual Lactose and of Lactate and Citrate

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the hydrolysis of these peptides (after internalization into the cell) to amino acids is catalysed by peptidases. Many different peptidases from LAB have been characterized biochemically and genetically (see Kun]i et al., 1996; Law and Haandrikman, 1997; Christensen et al., 1999; Siezen et al., 2002). The biochemical properties of the peptidases from cheese-related bacteria characterized to date are shown in Table 1. While the role of some of these peptidases (e.g., endopeptidases) is the degradation of oligopeptides to shorter peptides, exopeptidases function to release one or two amino acids at a time from short peptides. Based on their substrate specificity, peptidases are classified into different groups, as shown in Fig. 11.

Endopeptidases Several endopeptidases have been reported in lactococci and lactobacilli (Table 1), most of which are monomeric metallopeptidases. On the basis of substrate specificity, LAB appear to possess three types of endopeptidases (Monnet etal., 1994). PepO is a monomeric metallopeptidase with a molecular mass of ---70 kDa. It is capable of efficiently hydrolysing Metenkephalin, bradykinin, substance P, glucagon, oxidized B-chain of insulin and several casein fragments but not di-, tri- or tetra-peptides. PepO was the first endopeptidase for which the gene was sequenced (Mierau et al., 1993). The pepO gene is located immediately downstream of the genes for the oligopeptide transport system, indicating that the two systems are physiologically linked (Tynkkynen et al., 1993). Another oligopeptidase, designated PepE specifically cleaves the Phe--Ser bond in bradykinin and was purified from Lc. lactis subsp, lactis NDCO 763; its gene (pepF) was cloned and sequenced (Monnet et al., 1994). This enzyme is a monomeric metallopeptidase of "~70 kDa and is capable of hydrolysing peptides containing 7-17 amino acids with broad specificity but not smaller or larger peptides. PepF is unable to hydrolyse Metenkephalin, which is a good substrate for PepO.

A gene (pepE) encoding a thiol-dependent endopeptidase has been isolated from Lb. helveticus CNRZ32 (Fenster etal., 1997). The deduced amino acid sequence of PepE showed high homology with PepC from Lb. delbrueckii subsp, lactis DSM7290 (Klein et al., 1994a), Lb. helveticus CNRZ32 (Fernandez et al., 1994; Vesanto et al., 1994), Sc. thermophilus CNRZ302 (Chapot-Chartier et al., 1994) and Lc. lactis subsp, cremoris AM2 (Chapot-Chartier et al., 1993). Fenster et al. (1997) isolated and characterized recombinant PepE; the general properties of this enzyme indicated that it was different from the other metallo-endopeptidases characterized from LAB.

Di- and tripeptidases Tripeptidases (PepT) purified from LAB are generally di- or tri-meric metallopeptidases (Table 1) with broad specificity, capable of hydrolysing tripeptides with acidic, basic or neutral N-terminal amino acid residues. A broad-specificity general dipeptidase, PepV, which hydrolyses only dipeptides, is found in LAB (Kunji et al., 1996; Law and Haandrikman, 1997). A number of dipeptidases with similar properties have been purified and characterized from strains of Lactococcus and Lactobacillus (see Table 1). Most of the dipeptidases isolated from LAB are monomers with a molecular mass in the range 40-55 kDa (Table 1). With the exception of a dipeptidase from Lb. helveticus 53/7, which was reported to have a thiol catalytic mechanism (Vesanto et al., 1996), all the dipeptidases characterized to date are metallopeptidases (Table 1). All dipeptidases of LAB show broad specificity and are capable of hydrolysing all dipeptides except those containing a proline residue. Carboxypeptidases Carboxypeptidases are exopeptidases which catalyse the hydrolysis of peptides from the C-terminal. No carboxypeptidase activity has been detected in lactococci but some activity towards N-terminal-blocked

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Proteolysis in Cheese during Ripening

411

Endopeptidases (PepO, PepF)

Exopeptidases Aminopeptidases (PepN, PepA, PepC, PepL)

Iminopeptidase (Pepl)

1.0,..0..Q_ Pyrolidonyl carboxylylpeptidase (PCP)

X-Prolyldipeptidylaminopeptidase(PepX)

Carboxypeptidase

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Dipeptidases (PepV, P e p D )

Prolidase(PepQ)

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|

Tripeptidase (PepT)

Figure 11 Schematic representation of the action of peptidases found in lactic acid bacteria.

peptides has been reported in strains of lactobacilli (Abo-Elnaga and Plapp, 1987; E1 Soda et al., 1987a,b). There are no reports on the purification and characterization of a carboxypeptidase from Lactobacillus or other LAB.

Aminopeptidases The most thoroughly studied exopeptidase from LAB is the general aminopetidase, PepN. In most strains studied, this enzyme is a monomeric metallopeptidase of 85-98 kDa. PepN is a broad specificity aminopeptidase; in addition to p-nitroanilide (pNA) derivatives of amino acids, the enzyme is capable of hydrolysing a wide range of peptides differing in both size and

amino acid composition (Arora and Lee, 1992; Miyakawa et al., 1992; Tan et al., 1992a,b; Niven et al., 1995; Sasaki et al., 1996). Substrates with a hydrophobic or basic amino acid residue at the N-terminal are hydrolysed preferentially. The ability to hydrolyse peptides containing hydrophobic amino acids suggests its potential as a debittering enzyme. The addition of PepN from Lc. lactis subsp, cremoris Wg2 was found to be effective in reducing the bitterness of tryptic digests of [3-casein (Tan et al., 1993). The manufacture of cheese using PepN-negative mutants resulted in increased bitterness (Baankreis, 1992). Generally, PepN does not hydrolyse substrates with Glu, Asp or Pro at the N-terminal or dipeptides containing Pro

412

Proteolysis in Cheese during Ripening

(Tan etal., 1991; Arora and Lee, 1992; Miayakawa et al., 1992; Tan et al., 1993). However, PepNs from Lb. delbrueckii subsp, bulgaricus B14 (Wohlrab and Bockelmann, 1993) and Lb. helveticus SBT 2171 (Sasaki et al., 1996) hydrolysed Pro-containing substrates. Specificity studies indicated that the PepN from Lc. lactis subsp, cremoris Wg2 was active on oligopeptides with a preference for peptides with six amino acid residues (Niven et al., 1995). PepC in LAB is a metal-independent general aminopeptidase (Kunji et al., 1996; Table 1). PepCs from Lactococcus and Lactobacillus strains characterized so far are muhimeric thiol aminopeptidases which are inhibited by p-chloromercuribenzoate and iodoacetamide (Neviani et al., 1989; Wohlrab and Bockelmann, 1993; Fernandez de Palencia et al., 1997). In both cases, the subunit molecular mass of the enzyme is ---40-50 kDa. PepC shows broad specificity, with particularly high activity on synthetic substrates containing a hydrophobic amino acid but exhibits little activity on peptides with positively charged amino acid residues (Neviani et al., 1989; Wohlrab and Bockelmann, 1993; Fernandez de Palencia et al., 1997; Mistou and Gripon, 1998). A gene (pepG) encoding a novel cysteine aminopeptidase and with a high degree of similarity to PepC has been identified in Lb. delbrueckii subsp, lactis DSM7290 by Klein et al. (1997). These authors over-expressed the pepG gene in E. coli and compared the enzyme to PepC; although both enzymes were structurally related, they had different substrate specificities. Lactococcal glutamyl/aspartyl aminopeptidase (PepA) is a muhimeric metallopeptidase with a subunit molecular mass of 38-43 kDa (Table 1). PepA is a narrow-specificity peptidase which releases only an Nterminal Glu or Asp from di-, tri- and oligo-peptides with up to ten amino acid residues (Exterkate and de Veer, 1987; Niven, 1991; Bacon et al., 1994). Glutamate is a well-recognized flavour enhancer and therefore the role of PepA in the development of flavour in cheese may be of great importance. Studies on mature Cheddar cheese have shown that glutamate is important for Cheddar cheese flavour (McGugan et al., 1979; Aston and Creamer, 1986; Engels and Visser, 1994; Fox et al., 1994). However, the precise role of PepA in the development of cheese flavour is unclear. Under certain conditions, the N-terminal glutamyl residue of a peptide can undergo spontaneous intramolecular cyclization, forming an N-terminal 2-pyrrolidone5-carboxylic acid (PCA; pyroglutamate residue) (Law and Haandrikman, 1997). An N-terminal PCA residue has been found in bitter peptides produced from [3casein by the lactocepin of Lc. lactis subsp, cremoris HP (Visser etal., 1983). Pyrrolidone carboxylyl peptidase

(PCP) is an aminopeptidase capable of releasing a PCA residue from peptides and proteins (Kunji et al., 1996). This enzyme is present in lactococcal strains and has been partially characterized from Lc. lactis subsp, cremoris HP (Baankreis, 1992). Two serine peptidases with a molecular mass of 25 and 80kDa and PCAp-nitroanilide hydrolase activity were identified in Lc. lactis subsp, cremoris HP using non-denaturing gel electrophoresis (Baankreis, 1992). The presence of more than one leucyl aminopeptidase in LAB has been reported (Atlan et al., 1989; Blanc et al., 1993; Banks et al., 1998). A gene encoding a specific leucyl aminopeptidase (pepL) in Lb. delbrueckii subsp, lactis DSM 7290 has been cloned and sequenced (Klein et al., 1995). PepL has a molecular mass of 35 kDa (Table 1) and it preferentially hydrolyses dipeptides (and some tripeptides) with an N-terminal leucyl residue. Sequence alignments of PepL with prolinases from Lb. helveticus and B. coagulans and an iminopeptidase from Lb. delbrueckii subsp, lactis and Lb. delbrueckii subsp, bulgaricus showed 46, 21.5, 25.5 and 25.5% homology, respectively. Two aminopeptidases, with characteristics similar to PepL, were purified from Lb. sake IATA115 and Lb. curvatus DPC2024 by Sanz and Toldra (1997) and Magboul and McSweeney (1999b), respectively. The former was a monomer with a molecular mass of 35-36 kDa and maximum activity at pH 7.5 and 37 ~ while the latter was a dimer with a subunit molecular mass of---32 kDa and optimum activity at pH 7.0 and 40 ~ The 20 N-terminal amino acid residues of the PepL from Lb. curvatus DPC2024 showed 50, 80 and 95% homology with PepL from Lb. delbrueckii subsp. lactis DSM 7290 (Klein et al., 1995), the prolinase from Lb. helveticus CNRZ32 (Dudley and Steele, 1994) and the prolinase from Lb. rhamnosus 1/6 (Varmanen et al., 1998), respectively.

Proline-specific peptidases Caseins, the major proteins in bovine milk, are rich in the imino acid, proline. Because of its unique structure, specialized peptidases are required to hydrolyse peptide bonds involving proline, thus making peptides accessible to the action of other peptidases (see review by Cunningham and O'Connor, 1997). Several proline-specific peptidases with distinct substrate specificities have been found in LAB. X-Prolyl dipeptidyl aminopeptidase (PepX) is a peptide hydrolase capable of releasing X-Pro and sometimes X-Ala dipeptides from the N-terminal of oligopeptides. Due to its unique specificity, PepX is the best characterized of the proline-specific peptidases. The enzyme has been demonstrated in several genera of LAB and isolated from a number of strains and

Proteolysis in Cheese during Ripening characterized (Table 1). All PepXs purified from LAB have a serine catalytic mechanism and most are dimeric proteins with a native molecular mass of 117-200 kDa (Table 1); however, a high molecular mass endopeptidase (---350 kDa) with PepX activity and able to hydrolyse Otsl-casein was isolated and characterized by Stepaniak et al. (1998a). Increasing the proportion of pepX-negative mutants in a starter culture reduced the organoleptic quality of the resultant cheese but did not increase bitterness (Baankreis, 1992). Meyer and Spahni (1998) studied the role of PepX from Lb. delbrueckii subsp, lactis by using PepXnegative mutants. This enzyme influenced proteolysis and the sensorial characteristics of Gruyere cheese but it was not essential for the growth of the microorganism in milk (Meyer and Spahni, 1998). Proline iminopeptidase (PepI) catalyses the release of an N-terminal proline residue from di-, tri- and oligo-peptides. PepI from Lc. lactis subsp, cremoris HP (Baankreis and Exterkate, 1991) is the only iminopeptidase that has been purified from Lactococcus. This enzyme is a dimeric metallopeptidase with a native molecular mass of 110 kDa (Table 1). In contrast, the iminopeptidases purified from Lb. helveticus LHE-511 (Miyakawa et al., 1994b) and Lb. casei subsp, casei LLG (Habibi-Najafi and Lee, 1995) were monomeric thiol peptidases which were slightly inhibited by the serine protease inhibitor phenylmethyl sulphonyl fluoride. The molecular mass of the enzymes from Lb. helveticus and Lb. casei was estimated as 70 and 46 kDa, respectively. In addition to these two iminopeptidases, a PepI was purified from Lb. delbrueckii subsp, bulgaricus CNRZ 397 by amplification and expression of the gene in E. coli (Gilbert et al., 1994). The purified enzyme was characterized as a trimeric serine peptidase with a subunit molecular mass of 33 kDa (Table 1). Prolinase (PepR) is a specific dipeptidase which hydrolyses dipeptides with the sequence Pro-X. PepR from Lb. helveticus CNRZ32 was purified and biochemically characterized by Shao et al. (1997) and found to have a relatively broad specificity. The PepR from Lb. rhamnosus 1/6 (Varmanen et al., 1998), in addition to its prolinase activity, hydrolysed the aminopeptidase substrates, Pro-[3NA, Leu-[3NA and Phe-[3NA. Prolidase (PepQ) is an X-Pro-specific dipeptidase. With the exception of PepQ from Lb. helveticus CNRZ32, which is a homodimer with a subunit molecular mass of 45 kDa, most PepQs characterized to date are monomeric metallopeptidases with a native molecular mass of ---42 kDa. These enzymes hydrolysed most X-Pro dipeptides with the exception of Gly-Pro and Pro-Pro (Kaminogawa et al., 1984; Femandez-Espki et al., 1997b; Morel et al., 1999). However, PepQs isolated from Lc. lactis subsp. cremoris AM2 (Booth et al., 1990a) and Lb. delbrueckii

413

subsp, lactis DSM7290 (Stuckey et al., 1995), hydrolysed di- and tripeptides that did not contain Pro, in addition to Pro-X dipeptides. Aminopeptidase P (PepP) is a specific aminopeptidase that catalyses the removal of the N-terminal amino acid from oligopeptides having the sequence X-Pro-Pro-(X)n or X-Pro-(X)n (Kunji et al., 1996). The enzyme has been purified from strains of Lactococcus and is a monomeric metallopeptidase with a molecular mass of 41-43 kDa (Table 1). Provided that the peptide contains the above sequences, PepP is capable of releasing the N-terminal amino acid from oligopeptides up to 11 residues long. This enzyme also hydrolyses peptides with Ala in the penultimate position but at a slower rate (McDonnell et al., 1997).

Enzymes from Secondary Starter Microorganisms Enzymes of LAB play an important role in the secondalT proteolysis in internal-ripened cheese varieties, and hence contribute significantly to the development of flavour and aroma. In mould-ripened, smear-ripened and Swiss-type cheeses, microorganisms other than LAB play a pivotal role in the development of characteristic flavour and texture. The ripening of these cheese varieties involves complex biochemical reactions, which are discussed in detail in Volume 2. While the enzymes of LAB have been well studied and characterized, there have been fewer studies on organisms associated with mould-ripened or smear-ripened cheese varieties or on enzymes from Propionibacterium freudenreichii subsp. shermanii. The microbial flora of surface mould-ripened and blue-veined cheese, such as Camembert and Roquefort, includes yeasts (e.g., Kluyveromyces lactis, Saccharomyces spp. and Debaryomyces hansenii), moulds (Geotrichum candidum, Penicillium spp.), lactococci, lactobacilli, micrococci, staphylococci, coryneform bacteria and coliforms. Penicillium spp. are major components of the microflora and their enzymes play an important role in cheese ripening. Proteolytic systems of P camemberti and P roqueforti are somewhat similar; both synthesize an aspartyl proteinase, a metalloproteinase, an acid carboxypeptidase and an alkaline aminopeptidase ('Surface Mould-ripened Cheeses' and 'Blue Cheese', Volume 2). The aspartyl proteinase from P camemberti hydrolyses Otsl-casein faster than [3-casein or K-casein (Gripon, 1993). Acid proteinases of P. camemberti and P roqueforti have similar action on [3-casein and hydrolyse Lys97mVa198, Lys99mGlu100 and Lys29~Ile30 bonds at a faster rate than other bonds in [3-casein (Le Bars and Gripon, 1981; Trieu-Cuot et al., 1982). Metalloproteinases of

414

Proteolysis in Cheese during Ripening

both species have similar properties and have a pH optimum in the range 5.5-6.0. Chrzanowska et al. (1995) purified an aspartic proteinase from the culture filtrate of P. camemberti by a two-step purification procedure. The proteinase had a molecular mass of 33.5 kDa and an optimum pH of 3.4 on haemoglobin. The enzyme showed specificity towards peptide bonds containing an aromatic or hydrophobic amino acid residue in the B-chain of insulin. Besides these proteinases, P. roqueforti has a carboxypeptidase, which has an optimum pH of 3.5 and releases acidic, basic or hydrophobic amino acids (Gripon, 1993). Geotrichurn candidum also synthesizes extracellular and intracellular proteinases, but the contribution of these enzymes to cheese ripening is less than that of enzymes from Penicillium spp. (Gripon, 1993). The bacterial microflora of surface cheeses, such as Tilsit, Limburger, MOnster or Taleggio at the beginning of ripening is dominated by yeasts and moulds, which are acid and salt tolerant, but at the end of ripening, bacteria of the genera Brevibacterium, Arthrobacter, Micrococcus, Staphylococcus and Corynebacterium dominate (Eliskases-Lechner and Ginzinger, 1995; Valdes-Stauber et al., 1997; 'Bacterial Surface-ripened Cheeses', Volume 2). Growth of B. linens on the cheese surface is thought to play an important role in the development of the characteristic colour, flavour and aroma of smear surface-ripened cheese varieties (Rattray and Fox, 1999) and hence, its enzymes have been characterized. Extracellular enzymes of B. linens include proteinases, aminopeptidases and esterases, the biochemical properties of which vary because of wide inter-strain differences within the species. Brezina et al. (1987) partially purified four extracellular proteinases from B. linens, with pH and temperature optima of 5.0-8.0 and 50 ~ respectively. Hayashi et al. (1990) purified five extracellular proteinases from B. linens F (designated A, B, C, D and E), having a molecular mass of 37, 37, 44, 127 and 325 kDa, respectively, as determined by size exclusion chromatography (SEC). Proteinases A and B were stable at 35 ~ for 1 h and had a temperature optimum of 40 ~ while proteinases C, D and E were stable at 45 ~ for 1 h and had a temperature optimum of 50 ~ All five proteinases were optimally active at pH 11.0 and were serine proteinases. The production of multiple forms of the extracellular proteinases by B. linens ATCC 9172 is a result of aggregation of subunits and autocatalytic degradation (Buchinger et al., 2001). An extracellular serine proteinase partially purified from a strain of B. linens (Laktoflora 200), had a molecular mass of 52-55 kDa, as determined by SDSPAGE, and pH and temperature optima of 7.0-8.5 and

45 ~ respectively (Juh~isz and Sk~irka, 1990). A thermostable proteinase was partially purified from B. linens IDM 376; it had molecular mass of 18.5 kDa and pH and temperature optima of 7.5 and 67.5 ~ respectively, on azocasein (Clancy and O'Sullivan, 1993). An extracellular serine proteinase purified from B. linens ATCC 9174 had a molecular mass of 126 kDa, as determined by SEC and was optimally active at pH 8.5 and 50 ~ (Rattray et al., 1995). It hydrolysed Otsl-casein at His8mGln9, Ser161mGly162 and either Glnlr2mTyr173 or Phe23m Phe24 (Rattray et al., 1996) and [3-casein at Serls~Ser19, Glu20~Glu21, Gln56~Sers7, Gln72~Asn73, Leu77~Thr78, Alal01~ Met102, Phe119~Thr120, Leu139mLeu140, Ser142~Trp143, His145~Gln146, Gln167~Ser168, Gln175~Lys176, Tyr180~Pro181 and Phe190~Leu191 (Rattray et al., 1997). One of the five extracellular enzymes of B. linens ATCC 9172 was purified to homogeneity by Tomaschov~i et al. (1998) using ion-exchange chromatography and native preparative PAGE. The enzyme had nearly identical properties to the serine proteinase of /3. linens ATCC 9174 purified by Rattray etal. (1995). Its molecular mass was estimated to be 56 kDa by SDSPAGE and pH and temperature optima were 8.0 and 50 ~ respectively. B. linens also produces extracellular aminopeptidases, intracellular peptidases and proteinases. Sorhaug (1981) reported the presence of intracellular dipeptidase activity in six strains of B. linens. The presence of three extracellular aminopeptidases in B. linens (Laktoflora 200), having pH and temperature optima of 7.0-9.0 and 30 ~ respectively, was reported by Brezina et al. (1987). Two extracellular aminopeptidases, designated A and B, with a molecular mass of estimated to be 150 and 110 kDa, respectively, and pH and temperature optima of 9.3 and 40 ~ respectively, were purified from B. linens F by Hayashi and Law (1989). Ezzat et al. (1993) reported the presence of cell wall proteinases and dipeptidase activities in B. linens CNRZ 944. The authors partially purified the cell wall proteinase, which had maximum activity at pH 6.5 and 40 ~ An intracellular aminopeptidase from B. linens ATCC 9174, with a molecular mass of 59 kDa, as determined by SDSPAGE, and 69 kDa by SEC, was reported by Rattray and Fox (1997). The enzyme was optimally active at pH 8.5 and 35 ~ Curtin et al. (2002) showed aminopeptidase, dipeptidase and tripeptidase activities in brevibacteria, corynebacteria, staphylococci and brachybacteria, isolated from smear surface-ripened cheeses, Tilsit and Gubeen. Species of the genus Arthrobacter are major components of the microflora of surface mould-ripened cheeses, such as Brie and Camembert and red-smear

Proteolysis in Cheese during Ripening 415 cheeses. However, the enzymes of Arthrobacter have not been well studied. Smacchi et al. (1999a) purified two extracellular serine proteinases from A. nicotianae 9458, with molecular masses of about 53-55 and 70-72 kDa, as determined by SDS-PAGE. The enzymes were optimally active at 55-60 and 37 ~ respectively. Both enzymes were optimally active in the pH range of 9.0-9.5 and preferentially hydrolysed [3-casein over Otsl-casein. An extracellular PepI from A. nicotianae 9458 with a molecular mass of about 53 kDa, was purified and characterized by Smacchi et al. (1999b). The enzyme was optimally active at 37 ~ and 8.0. Some Micrococcus spp. are very proteolytic and produce extracellular proteinases and intracellular proteinases and peptidases (Fox et al., 1993). Nath and Ledford (1972) reported that extracellular proteinases from certain micrococci preferentially hydrolysed Otslcasein; production of extracellular proteinase was also reported by Garcia de Fernando and Fox (1991). Bhowmik and Marth (1989) purified and characterized an aminopeptidase, with broad substrate specificity, from M. freudenreichii ATCC 407. Propionibacterium spp. are weakly proteolytic, but they are highly peptidolytic, especially on proline-containing peptide bonds, thus contributing to the characteristic flavour of Swiss-type cheeses (see 'Cheese with Propionic Acid Fermentation', Volume 2). Biochemical characteristics of peptidases from propionic acid bacteria have been reviewed by Gagnaire et al. (1999). A PepX with a molecular mass of 84 kDa and pH and temperature optima of 7.0 and 40 ~ respectively, was purified and characterized from P. freudenreichii subsp, shermanii NCDO 853 by Fernandez-Espla and Fox (1997). Endopeptidases have been isolated from P. freudenreichii subsp, shermanii and characterized (Table 1) (Tobiassen et al., 1996; Stepaniak et al., 1998b).

Patterns of Proteolysis in Cheese The pattern of proteolysis in many varieties may be summarized as follows: the caseins are hydrolysed initially by residual coagulant activity retained in the curd and by plasmin (and perhaps other indigenous proteolytic enzymes) to a range of large and intermediate-sized peptides which are hydrolysed by proteinases and peptidases from the starter LAB, NSLAB and perhaps secondary microflora to shorter peptides and amino acids. However, the pattern and extent of proteolysis varies considerably between varieties due to differences in manufacturing practices (particularly cooking temperature), which cause differences in moisture content, residual coagulant activity, activation of plasminogen to

plasmin, and possibly the development of a highly proteolytic secondary microflora and ripening time. The extent of proteolysis (i.e., the degree to which the caseins and peptides therefrom are hydrolysed and measured by the development of water- or pH 4.6soluble N) in cheese varies from very limited (e.g., Mozzarella) to very extensive (e.g., Blue) and is summarized for many varieties in Table 2. The pattern of proteolysis (i.e., the relative concentrations of different peptides and amino acids) is very variable and is essentially unique to a particular variety. The differences in soluble N content are due to differences in moisture content, temperature and pH, length of ripening, cooking temperature and pH at draining (Fox and McSweeney, 1996) and is mainly due to the action of chymosin and to a lesser extent of plasmin (Fox and McSweeney, 1997). A short ripening period ( - 3 weeks) and extensive denaturation of chymosin during the high temperature ( - 7 0 ~ stretching step during the manufacture of Mozzarella cheese explain the low level of soluble N, whereas extensive proteolysis is characteristic of Blue cheese and some smear-ripened varieties, caused by the action of chymosin, plasmin and proteinases from their characteristic secondary microflora. In addition, differences in the action of these proteolytic agents cause differences in peptide profiles. Primary proteolysis is similar during the ripening of most cheeses; chymosin hydrolyses the Phe23--Phe24 bond of Otsl-casein (Hill et al., 1974; Caries and Ribadeau-Dumas, 1985) except in cheeses that are cooked at a high temperature ( - 5 5 ~ e.g., Swiss cheese), in which plasmin is the principal proteolytic agent. In blue-veined cheeses, after sporulation, enzymes from P. roqueforti hydrolyse Otsl-CN (f24-199) and other peptides, changing the peptide profile (Gripon, 1993). Analysis of the water-insoluble fraction of various cheeses by urea-PAGE gives insight into the differences in peptide profile between cheeses (Fig. 12). In many cheeses, Otsl-casein is hydrolysed faster than [3-casein (Sousa et al., 2001). In Blue-veined cheeses, both Ors1- and [3-caseins are completely hydrolysed at the end of ripening. In Swiss-type cheeses, [3-casein is hydrolysed faster than Otsl-casein, with concomitant increases in y-caseins, indicating a role of plasmin and denaturation of chymosin during cooking. However, Ot~l-CN (f24-199) is produced slowly in Swiss cheese, indicating either the survival of some chymosin during cooking or the activity of indigenous milk acid proteinase, cathepsin D (Gagnaire et al., 2001), which has specificity similar to chymosin (Hurley et al., 2000a). In the case of Camembert-type cheese, about - 2 0 % of total N is soluble at pH 4.6 (Khidr, 1995) (Table 2) and the pattern of proteolysis is similar to Cheddar cheese (Fig. 12). During the ripening of Mozzarella

416

Proteolysis in Cheese during Ripening Table 2 Soluble N as % of total nitrogen in different cheese varieties Cheese

Age

SN/'IN %

References

Mozzarella

25 days

4-5

Quarg Gouda

4 weeks 6 weeks 24 weeks 1 month

Somerset al. (2002) O'Reilly et al. (2002) Guinee et al. (1998) Mara and Kelly (1998) Messens et al. (1999) Exterkate and Alting (1995) Michalski et aL (2003) Sousa and McSweeney (2001) Khidr (1995)

Swiss Feta

16 weeks 2-6 months

--~12 12-13 23-25 Surface 15-17 Core 9-12 Surface---20 Core ---12 16-17 17-20

Mahon Cheddar



II

C

OH

I CH--CH 3 I CH3 o~-Keto-isovalerate

Figure 8 Transamination of the branched-chain amino acids to their corresponding c~-keto acids.

active on leucine, although it was also, but less, active on the aromatic amino acids (Yvon et al., 1997), while that of Lc. lactis subsp, cremoris B78 catalysed the transamination of valine, isoleucine and leucine (Engels, 1997). A branched-chain aminotransferase from Lc. lactis subsp, cremoris NCDO 763 was characterised by Yvon et al. (2000). The enzyme catalysed the transamination of the three branched-chain amino acids and was active under cheese-ripening conditions, although it had pH and temperature optima of 7.5 and 35-40 ~ respectively. Since the enzyme has a role in the degradation of isoleucine and valine, as well as in the transamination of leucine and methionine and was active under conditions similar to those found in cheese during ripening, the authors concluded that the enzyme was involved in flavour development. The branched-chain aminotransferase of Lc. lactis LM0230 has been cloned and sequenced (Atiles et al., 2000). The enzyme has broad specificity, being active on isoleucine, leucine, valine, methionine and phenylalanine.

Lb. paracasei subsp, paracasei LCO 1 produces at least one aminotransferase, capable of transaminating branched-chain amino acids, which was most active on isoleucine and leucine (Hansen et al., 2001). Responsesurface methodology showed that leucine concentration had a negligible effect on aminotransferase activity, while too high a concentration of ot-ketoglutarate could inhibit the enzyme. Ayad et al. (2001a) studied the effects of combining selected lactococci on flavour formation in milk. A chocolate-like flavour was produced by a combination of Lc. lactis subsp, cremoris NIZO131157 and Lc. lactis subsp, cremoris SKl l0. The authors speculated that this flavour was due to branched-chain aldehydes produced from branched-chain amino acids. Subsequently, Ayad etal. (2001b) studied the flavour-generating ability of wild lactococci isolated from artinsanal and non-dairy sources (fermented raw goats', sheep's and cows' milk, as well as from soil, grass, silage and the udder) in milk and in a cheese model. The authors believed that these wild

Catabolism of Amino Acids in Cheese during Ripening

strains may be able to produce more flavour compounds in cheese than the industrial strains currently used in cheesemaking. The majority of wild strains produced different flavours from industrial strains. Methylated alcohols and methylated aldehydes, probably produced from branched-chain amino acids, were the main volatile compounds formed. It was concluded that wild strains could be used for the development of new cheeses or to alter the flavour of existing types of cheese. However, since the non-dairy wild strains had no proteolytic activity, they would be unable to grow in and acidify cheese milk and would have to be combined with industrial starters. The catabolism of leucine by propionic acid bacteria was investigated by Thierry et al. (2002). P.freudenreichii catabolised leucine to ot-ketoisocaproic acid, but only if ot-ketoglutarate was present. The bacterium also converted ot-ketoisocaproic acid to isovaleric acid via oxidative decarboxylation by ot-ketoacid dehydrogenase activity yielding an acyl-CoA derivative which was then converted to the acid. The authors noted that the catabolism of branched-chain amino acids by P. freudenreichii was different to the catabolism of branched-chain amino acids by lactococci.

Dearninases There are two types of deamination involving redox reactions (Hemme et al., 1982), differing according to the nature of hydrogen acceptor: 9 Dehydrogenases (EC 1.4.1) which utilise NAD + as the co-enzyme. The general reaction catalysed by these enzymes is: L-amino acid + H20 + NAD + ---* ot-keto acid + NH4 + + NADH These reactions can produce compounds such as pyruvic acid and ot-ketoglutaric acid from alanine and glutamic acid, respectively. 9 Oxidases which use oxygen as hydrogen acceptor. L-amino acid oxidases (EC 1.4.3.2) produce ot-keto acids according to the following reaction: L-amino acid + 02--+ ot-keto acid + NH3 + H202 L-amine oxidases (EC 1.4.3.6) according to the reaction:

form aldehydes

Amine + O2--+ aldehyde + NH3 + H202 Ammonia, a product of these deamination reactions, is an important constituent of the flavour of cheeses such as Camembert, Gruyere and Comte and

449

contributes to an increase in pH during ripening (McSweeney and Sousa, 2000). Microorganisms from the smear surface have deaminating ability, e.g., G. candidum (see Fox and Wallace, 1997), while B. linens produces large quantities of ammonia from serine, glutamine, asparagine and threonine. However, most strains of coryneform bacteria from smear cheese were found to have low deaminating activity except on serine, glutamine and asparagine (Hemme et al., 1982). Williams et al. (2001) studied the deaminating ability of LAB isolated from mature Cheddar. Deaminase activity was not widespread in the isolates but this may have been due to the insensitivity or lack of specificity of the assay method used.

Decarboxylases Decarboxylation is the conversion of an amino acid to the corresponding amine with the removal of CO2. Decarboxylases generally have an acid pH optimum (---pH 5.5) and usually require PLP as a coenzyme (Hemme et al., 1982). Amines generally have strong and often unpleasant aromas, as evident in certain smearripened cheese types (Fox and McSweeney, 1996). In addition, many amines (e.g., tyramine, histamine, tryptamine, putrescine, cadaverine and phenylethylamine) cause adverse physiological effects ('biogenic amines'; see 'Toxins in Cheese', Volume 1). The relative concentration of amines in cheese depends on the type of cheese and its microflora (McSweeney and Sousa, 2000). The relative concentration of some amines does not compare with that of the parent amino acid, which may be due to differences in the rates of conversion of amino acids (Adda et al., 1982). Most amines in cheese can be formed by decarboxylation, as is the case with the production of tyramine from tyrosine and histamine from histidine. However, the formation of secondary and tertiary amines cannot be explained readily (Fox and McSweeney, 1996). Joosten (1988) studied factors that affect the concentrations of biogenic amines formed in cheese. It was observed that in Gouda cheese, a higher pH, combined with a storage temperature of 21 ~ caused an increase in concentration of histamine, as did low saltin-moisture. Starter type and pasteurisation of milk did not appear to affect the formation of histamine. The role of non-starter bacteria in the formation of biogenic amines in cheese was examined by Joosten and Northolt (1987) who investigated the decarboxylase activity of bacteria including lactobacilli, enterococci, enterobacteriaccae and pediococci. Some strains of lactobacilli could form biogenic amines in cheese. Since the number of enterococcal cells required to produce significant amounts of tyramine is rarely reached

450

Catabolism of Amino Acids in Cheese during Ripening

in cheese, these bacteria are not important for amine formation in Dutch cheese, although this may not be true for certain artisanal cheeses for which enterococci are a major part of the starter. The authors concluded that non-starter lactobacilli were the most important agents in Dutch cheese for the formation of biogenic amines. This is in agreement with the findings of Broome et al. (1990) who reported that the concentrations of tyramine and histamine in cheeses inoculated with lactobacilli were twice as high as in control cheeses, indicating that decarboxylases of lactobacilli have a role in their production. Novella-Rodriguez et al. (2002a) studied the effect of defined-strain starters on the production of amines in goats' milk cheese during ripening. The main amines found were tyramine (94.59 mg kg- 1), putrescine and tryptamine. The effect of high hydrostatic pressure on the production of amines in goats' milk cheese was studied by Novella-Rodriguez et al. (2002b) who found maximum production of amines when the cheeses were treated at 50 MPa for 72 h; rates of production were lower when cheeses received higher pressure treatments (400 MPa for 5 min or 400 MPa for 5 min followed by 50 MPa for 72 h) and in the untreated control cheeses. In addition to being involved in the production of amines, B. linens is able to reduce the amounts of histamine and tyramine in cheese during ripening (Leuschner and Hammes, 1998). During the four weeks of ripening of Munster cheese, B. linens reduced the histamine and tyramine content by 55-70%. Degradation of amines occurs at the surface of the cheese but the concentration of amines on the surface and interior differed only slightly after inoculation with B. linens LTH456. It was suggested that the concentration gradient was removed by diffusion of amines, leading to a decrease in the concentration of biogenic amines in the interior of the cheese. Lactobacilli used as cheese starter adjuncts were incubated by Gummalla and Broadbent (1999) in a defined medium containing L-tryptophan under carbohydrate starvation (CS), or under near-cheese ripening conditions (a chemically defined medium containing 4% salt, at pH 5.2). The specific activity of the tryptophan decarboxylases from Lb. casei strains was lower than those of the corresponding enzymes from Lb. helveticus strains. Generally, activity in either strain did not vary significantly with time or incubation conditions. Twenty-two Lb. plantarurn strains and seven strains of Lb. casei had no decarboxylase activity on methionine (Amarita et al., 2001). The combined effects of temperature, pH and salt on the growth of E. faecalis EF37, its proteolytic activity and its ability to produce biogenic amines were studied by

Gardini et al. (2001) who observed that 2-phenylethylamine accounted for more than half of the total content of biogenic amines. The production of biogenic amines was found to be independent of the incubation temperature and in general, was very low at the higher NaC1 concentration and was increased by lower pH. Roig-Sagues et al. (2002) studied the ability of 694 strains of bacteria isolated from Spanish artisanal cheeses to produce histamine and tyramine. Tyramineforming activity (mainly by enterococci and some other LAB) was found more frequently than histamine-forming activity, which was formed mainly by enterobacteria, but also by small numbers of other LAB. Most of the tyramine-forming strains of LAB were isolated from cheeses containing the highest levels of tyramine. However, histamine-forming LAB were generally isolated from samples with a low level of histamine. The amount of tyramine found in the samples was significantly higher than that of histamine. The distribution of aromatic L-amino acid decarboxylases in 326 bacteria (four species of E. coli, Erwinia herbicola, Serratia plymuthicum, two species of Proteus, Alcaligenes faecalis, Bacillus natto, Achrombacter hartlebii, 11 species of Micrococcus, one Staphylococcus, three Sarcina spp., Brevibacterium ammoniagenes, Bacterium cadaveris and three Pseudomonas spp.) was studied by Nakazawa etal. (1977). Micrococcaceae were observed to have the highest decarboxylase activity on L-tryptophan, S-hydroxy-L-tryptophan and L-phenylalanine. The amino acid decarboxylase of M. percitreus was reported by this author to be involved in synthesis of aromatic amines such as dopamine and tyramine. A histidine decarboxylase, which did not require PLP as a coenzyme, has been purified from Lactobacillus 30a (Chang and Snell, 1968). The substrates of the enzyme were found to have a heterocyclic nitrogen atom at the same position relative to its alanyl side chain which may be important in the formation of the enzyme-substrate complex. Jetten and Sinskey (1995) studied a decarboxylase isolated from a strain of Corynebacterium glutamicum with activity on oxaloacetate. The enzyme, which catalysed the decarboxylation of oxaloacetate only, a key intermediate in carbon metabolism, had optimum activity between pH 7.0 and 7.5. A glutamate decarboxylase was isolated from Lb. brevis IFO 12005 by Ueno et al. (1997) and was found to be a dimer. Temperature and pH optima were 30 ~ and 4.2, respectively. The enzyme could not decarboxylate any other amino acid assayed. Lucas and Lonvaud-Funel (2002) purified the tyrosine decarboxylase of Lb. brevis lOEB 9809. The enzyme had features typical of pyridoxal phosphate-dependent amino acid decarboxylases although this enzyme was

Catabolism of Amino Acids in Cheese during Ripening

not related by sequence homology to any known tyrosine decarboxylase.

Catabolism of Other Amino Acids Goux et al. (1995) investigated aspartate catabolism in an effort to understand ammonia generation by E. coli. It was reported that arginine may be an intermediate in aspartate catabolism, and may also be an intermediate for ammonia production from aspartate during nitrogen-limited growth. Hayashi et al. (1993) compared an aspartate aminotransferase with the aromatic amino acid aminotransferase of E. coli. Both enzymes were composed of two identical "--43.5 kDa subunits, and contained one molecule of PLP per subunit. An aspartate aminotransferase isolated from Lc. lactis LM0230 was cloned and characterised by Dudley and Steele (2001). It was determined using homologous recombination that a mutation in the Asp biosynthetic pathway prevented this strain from growing in milk. According to Kaneoke et al. (1993), at least seven I_-arginine degradation pathways are known, and in some species, more than one of these pathways can be operational. These authors studied the arginine oxygenase pathway in two coryneforms, Arthrobacter globiformis IFO 12137 (ATCC 8010) and B. helvolum IFO 12073. This pathway involves four enzymes and produces succinate from L-arginine (Fig. 9). This pathway in the coryneforms studied is not identical to other pathways reported, e.g., the pathways of Pseudomonas aeruginosa and Streptococcus faecalis. E. coli utilises the ammonia-producing succinyl transferase pathway for arginine catabolism, and to a lesser degree, the arginine decarboxylase pathway (Schneider et al., 1998).

Arginine

Agmatine

Citrulline

N-carbamoylputrescine

.12 Ornithine

~

Putrescine

Figure 9 Pathways of arginine metabolism in bacteria (Arena and Manca de Nadra, 2001). (1) Arginine deiminase, (2) Catabolic ornithine transcarbamylase, (3) Arginine decarboxylase, (4) Agmatine deiminase, (5) Agmatinase, (6) N-carbamoylputrescine hydrolase, (7) Ornithine decarboxylase, (8) Anabolic system.

451

Lactic acid bacteria isolated from wine can catabolise arginine by at least two pathways (Arena et al., 1999). The arginine deiminase pathway produces orthinine, CO2 and NH3 via three enzymatic reactions. The enzymes involved in this pathway are arginine deiminase (EC 3.5.3.6), catabolic ornithine transcarbamoylase (EC 2.1.3.3) and carbamate kinase (EC 2.7.2.2) (Champomier Verges et al., 1999). Alternatively, the arginase-urease pathway leads to the production of urea. Arginine deiminase, ornithine transcarbamoylase and carbamate kinase from the sourdough microorganism, Lb. sanfranciscencis CB1, were isolated by de Angelis et al. (2003). The enzymes had acidic pH optima and were optimally active at 30-37 ~ Interestingly, arginine has been proposed as a possible growth substrate for the secondary microflora of Swiss cheese (Laht et al., 2002); calculations showed that ATP available from the metabolism of arginine to ornithine was theoretically sufficient to support the growth of non-starter bacteria to populations of 108 cfu g-1 The catabolism of threonine, asparagine, arginine and glutamate in cheese has attracted some study. Aminotransferase or dehydrogenase activities catabolise glutamate to produce ot-ketoglutarate, while y-aminobutyrate is formed from glutamate by the action of a decarboxylase. Threonine is converted to acetaldehyde and glycine (McSweeney and Sousa, 2000). The specific pathways for the catabolism of other amino acids (e.g., glycine, alanine and serine) by cheese-related microorganisms have attracted little attention.

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Chang, G.W. and Snell, E.E. (1968). Histidine decarboxylase of Lactobacillus 30a. II. Purification, substrate specificity and stereospecificity. Biochemistry 7, 2005-2012. Collin, J.C. and Law, B.A. (1989). Isolation and characterisation of the L-methionine-y-demethiolase from Brevibacterium linens BCDO739. Sci. Aliment. 9,805-812. Curtin, A.C., Gobbetti, M. and McSweeney, P.L.H. (2002). Peptidolytic, esterolytic and amino acid catabolic activities of selected bacterial strains from the surface of smear cheese. Int. J. Food Microbiol. 76, 231-240. de Angelis, M., Curtin, A.C., McSweeney, P.L.H., Faccia, M. and Gobbetti, M. (2002). Lactobacillus reuteri DSM 20016: purification and characterization of a cystathionine gamma-lyase and use as adjunct starter in cheesemaking. J. Dairy Res. 69, 255-267. de Angelis, M., Mariotti, L., Rossi, J., Servilli, M., Fox, P.E, Rollan, G. and Gobbetti, M. (2003). Arginine catabolism by sourdough lactic acid bacteria: Purification and characterization of the arginine deiminase pathway enzymes from Lactobacillus sanfranciscensis CB1. Appl. Environ. Microbiol. 68, 6193-6201. Demarigny, Y., Berger, C., Desmasures, N., Gueguen, M. and Spinnler, H.-E. (2000). Flavour sulphides produced from methionine by two different pathways by Geotrichum candidum. J. Dairy Res. 67, 371-380. Dias, B. and Weimer, B. (1998a). Conversion of methionine to thiols by lactococci, lactobacilli and brevibacteria. Appl. Environ. Microbiol. 64, 3320-3326. Dias, B. and Weimer, B. (1998b). Purification and characterisation of L-methionine y-lyase from Brevibacterium linens BL2. Appl. Environ. Microbiol. 64, 3327-3331. Dias, B. and Weimer, B. (1999). Production of volatile sulfur compounds in Cheddar cheese slurries. Int. Dairy J. 9, 605-611. Dobric, N., Limsowtin, G.K.Y., Hillier, A.J., Dudman, N.P.B. and Davidson, B.E. (2000). Identification and characterisation of a cystathionine [3/y-lyase from Lactococcus lactis subsp, cremoris MG1363. FEMS Microbiol. Lett. 182, 249-254. Dudley, E.G. and Steele, J.L. (2001). Lactococcus lactis LM0230 contains a single aminotransferase involved in aspartate biosynthesis, which is essential for growth in milk. Microbiology 147,215-224. Dunn, H.C. and Lindsay, R.C. (1985). Evaluation of the role of microbial Strecker-derived aroma compounds in uncleantype flavors in Cheddar cheese. J. Dairy Sci. 68, 2859-2874. Dwivedi, C.M., Ragin, R.C. and Uren, J.R. (1982). Cloning, purification and characterisation of [3-cystathionase from Escherichia coll. Biochemistry 21, 3064-3069. Engels, W.J.M. (1997). Flavour Formation in Cheese-like Systems: Role of Methionine-degrading Enzymes from Lactococci. PhD Thesis, University of Waginengen, The Netherlands. Ferchichi, M., Heroine, D., Nardi, M. and Pamboukdjian, M. (1985). Production of methanethiol from methionirle by Brevibacterium linens CNRZ 918. J. Gen. Microbiol. 131, 715-723. Fernandez, M., van Doesburg, W., Rutten, G.A.M., Marugg, J.D., Alting, A.C., van Kranenburg, R. and Kupers, O.P.

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Kaneoke, M., Shiota, K., Kusunose, M., Shimizu, E. and Yorifuji, T. (1993). Function of the arginine oxygenase pathway in utilization of L-arginine-related compounds in Arthrobacter globiformis and Brevibacterium helvolum. Biosci. Biotechnol. Biochem. 57, 814-820. Klein, N., Maillard, M.-B., Thierry, A. and Lortal, S. (2001). Conversion of amino acids into aroma compounds by cell-free extracts of Lactobacillus helveticus. J. Appl. Microbiol. 91,404-411. Laber, B., Clausen, T., Huber, R., Messerschmidt, A., Egner, U., Muller-Fahrnow, A. and Pohlenz, H.D. (1996). Cloning, purification and crystallisation of Escherichia coli cystathionine [3-1yase. FEBS Lett. 379, 94-96. Laht, T.M., Kask, S., Elias, P., Adamberg, K., Paalme, T. (2002). Role of arginine in the development of secondary microflora in Swiss-type cheese. Int. Dairy J. 12, 831-840. Lamberet, G., Auberger, B. and Bergere, J.L. (1997a). Aptitude of cheese bacteria for volatile S-methyl thioester synthesis. I. Effect of substrate and pH on their formation by Brevibacterium linens GC171. Appl. Microbiol. Biotechnol. 47, 279-283. Lamberet, G., Auberger, B. and Bergere, J.L. (1997b). Aptitude of cheese bacteria for volatile S-methyl thioester synthesis. II. Comparison of coryneform bacteria, Micrococcaceae and some lactic acid bacteria starters. Appl. Microbiol. Biotechnol. 48,393-397. Lee, C.W. and Desmazeaud, M.J. (1985). Utilisation of aromatic amino acids as nitrogen sources in Brevibacterium linens: an inducible aromatic amino acid aminotransferase. Arch. Microbiol. 140,331-337. Lee, C.W. and Richard, J. (1984). Catabolism of L-phenylalanine by some microorganisms of cheese origin. J. Dairy Res. 51, 461-469. Lee, C.W., Lucas, S. and Desmeaud, M.J. (1985). Phenylalanine and tyrosine catabolism in some cheese coryneform bacteria. FEMS Microbiol. Lett. 26, 201-205. Leuschner, R.G.K. and Hammes, W.P. (1998). Degradation of histamine and tyramine by Brevibacterium linens during surface ripening of Munster cheese. J. Food Prot. 61, 874-878. Lockwood, B.C. and Coombs, G.H. (1991). Purification and characterisation of methionine -y-lyase from Trichomonas vaginalis. Biochem. J. 279,675-682. Lucas, P. and Lonvaud-Funel, A. (2002). Purification and partial gene sequence of the tyrosine decarboxylase of Lactobacillus brevis lOEB 9809. FEMS Microbiol. Lett. 211, 85-89. Martinez-Cuesta, C., Ruquena, T. and Pelaez, C. (2002). Effect of bacteriocin-induced cell damage on the branched-chain amino acid transamination by Lactococcus lactis. FEMS Microbiol. Lett. 217, 109-113. McSweeney, P.L.H. and Sousa, M.J. (2000). Biochemical pathways for the production of flavour compounds in cheese during ripening: a review. Lait 80,293-324. Molimard, P. and Spinnler, H.E. (1996). Compounds involved in the flavor of surface mould-ripened cheeses: origins and properties. J. Dairy Sci. 79, 169-184. Nakazawa, H., Sano, K., Kumagai, H. and Yamada, H. (1977). Distribution and formation of aromatic L-amino

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acid decarboxylase in bacteria. Agric. Biol. Chem. 41, 2241-2247. Nakayama, T., Esaki, N., Lee, W.J., Tanaka, I., Tanaka, H. and Soda, K. (1984). Purification and properties of k-methionine y-lyase from Aeromonas sp. Agric. Biol. Chem. 48, 2367-2369. Nierop Groot, M.N. and de Bont, J.A.M. (1998). Conversion of phenylalanine to benzaldehyde initiated by an aminotransferase in Lactobacillus plantarum. Appl. Environ. Microbiol. 64, 3009-3013. Nierop Groot, M.N. and de Bont, J.A.M. (1999). Involvement of manganese in conversion of phenylalanine to benzaldehyde by lactic acid bacteria. Appl. Environ. Microbiol. 65, 5590-5593. Novella-Rodrtguez, S., Veciana-Nogues, M.T., Roig-Sagues, A.X., Trujillo-Mesa, A.J. and Vidal-Carou, M.C. (2002a). Influence of starter and nonstarter on the formation of biogenic amine in goat cheese during ripening. J. Dairy Sci. 85, 2471-2478. Novella-Rodriguez, S., Veciana-Nogues, M.T., Saldo, J. and Vidal-Carou, M.C. (2002b). Effects of high hydrostatic pressure treatments on biogenic amine contents in goat cheeses during ripening. J. Agric. Food Chem. 50, 7288-7292. Rijnen, L., Bonneau, S. and Yvon, M. (1999a). Genetic characterisation of the major lactococcal aminotransferase and its involvement in conversion of amino acids to aroma compounds. Appl. Environ. Microbiol. 65, 4873-4880. Rijnen, L., Delacroix-Buchet, A., Demaizieres, D., Le Quere, J.L., Gripon, J.C. and Yvon, M. (1999b). Inactivation of lactococcal aromatic aminotransferase prevents the formation of floral aroma compounds from aromatic amino acids in semi-hard cheese. Int. Dairy J. 9,877-885. Rijnen, L., Courtin, P., Gripon, J.-C. and Yvon, M. (2000). Expression of a heterologous glutamate dehydrogenase gene in Lactococcus lactis highly improves the conversion of amino acids to aroma compounds. Appl. Environ. Microbiol. 66, 1354-1359. Roig-Sagues, A.X., Molina, A.P. and Hernandez-Herrerok, M.M. (2002). Histamine and tyramine-forming microorgamisms in Spanish traditional cheeses. Eur.. Food Res. Technol. 215, 96-100. Schneider, B.L., Kiupakis, A.K. and Reitzer, LJ. (1998). Arginine catabolism and the arginine succinyltransferase pathway in Escherichia coll. J. Bacteriol. 180, 4278-4286. Seefeldt, K.E. and Weimer, B. (2000). Diversity of sulfur compound production in lactic acid bacteria. J. Dairy Sci. 83, 2740-2746. Shakeel-Ur-Rehman and Fox, RE (2002). Effect of added ot-ketoglutaratic acid, pyruvic acid or pyridoxal phosphate on proteolysis and quality of Cheddar cheese. Food Chem. 76, 21-26. Smacchi, E. and Gobbetti, M. (1998). Purification and characterisation of cystathionine y-lyase from Lactobacillus fermentum DT41. FEMS Microbiol. Lett. 166, 197-202. Smit, G., van Hylckama Vlieg, J.E.T., Smit, B.A., Ayad, E.H.E. and Engels, W.J.M. (2002). Fermentative formation of flavour compounds by lactic acid bacteria. Aust. J. Dairy Technol. 57, 61-68.

Soda, K., Tanaka, H. and Esaki, N. (1983). Multifunctional biocatalysis: methionine y-lyase. Trends Biochem. Sci. 8, 214-217. Tamman, J.D., Williams, A.G., Noble, J. and Lloyd, D. (2000). Amino acid fermentation in non-starter Lactobacillus spp. isolated from Cheddar cheese. Lett. Appl. Microbiol. 30, 3 70-3 74. Tanaka, H., Esaki, N., Yamamoto, T. and Soda, K. (1976). Purification and characterisation of methionase from Pseudomonas ovalis. FEBS Lett. 66,307-311. Tanaka, H., Esaki, N. and Soda, K. (1985). A versatile bacterial enzyme: k-methionine y-lyase. Enzyme Microbiol. Technol. 7,530-537. Thierry, A., Maillard, M.-B. and Yvon, M. (2002). Conversion of k-leucine to isovaleric acid by Propionibacterium freudenreichii TL34 and ITGP23. Appl. Environ. Microbiol. 68,608-615. Ueno, Y., Hayakawa, K., Takahashi, S. and Oda, K. (1997). Purification and characterization of glutamate decarboxylase from Lactobacillus brevis IFO 12005. Biosci. Biotechnol. Biochem. 61, 1168-1171. Ummadi, M. and Weimer, B.C. (2001). Tryptophan catabolism in Brevibacterium linens as a potential cheese flavor adjunct. J. Dairy Sci. 84, 1173-1182. Weimer, B., Dias, B., Ummadi, M., Broadbent, J., Brennand, C., Jaegi, J., Johnson, M., Miliani, E, Steele, J. and Sisson, D.V. (1997). Influence of NaC1 and pH on intracellular enzymes that influence Cheddar cheese ripening. Lait 77, 383-398. Weimer, B., Seefeldt, K. and Dias, B. (1999). Sulfur metabolism in bacteria associated with cheese. Antonie van Leeuwenhoek 76, 247-261. Williams, A.G., Noble, J. and Banks, J.M. (2001). Catabolism of amino acids by lactic acid bacteria isolated from Cheddar cheese. Int. Dairy J. 11,203-215. Williams, A.G., Noble, J., Tammam, T., Lloyd, D. and Banks, J.M. (2002). Factors affecting the activity of enzymes involved in peptide and amino acid catabolism in nonstarter lactic acid bacteria isolated from Cheddar cheese. Int. Dairy J. 12,841-852. Yamagata, S., D'Andrea, R.J., Fujisaki, S., Isaji, M. and Nakamura, N. (1993). Cloning and bacterial expression of the CYS3 gene encoding cystathionine y-lyase of Saccharomyces cerevisae and the physiochemical and enzymatic properties of the protein. J. Bacteriol. 175, 4800-4808. Yvon, M. and Rijnen, L. (2001). Cheese flavour formation by amino acid catabolism. Int. Dairy J. 11,185-201. Yvon, M., Thirouin, S., Rijnen, L., Fromentier, D. and Gripon, J.C. (1997). An aminotransferase from Lactococcus lactis initiates conversion of amino acids to cheese flavor compounds. Appl. Environ. Mirobiol. 63, 414-419. Yvon, M., Berthelot, S. and Gripon, J.C. (1998). Adding ot-ketoglutarate to semi-hard cheese curd highly enhances the conversion of amino acids to aroma compounds. Int. Dairy J. 8,889-898. Yvon, M., Chambellon, E., Bolotin, A. and Roudot-Algaron, E (2000). Characterisation and role of the branched chain aminotransferase (BcatT) isolated from Lactococcus lactis subsp, cremoris NCDO763. Appl. Environ. Microbiol. 66, 571-577.

Sensory Character of Cheese and its Evaluation C.M. Delahunty, Department of Food and Nutritional Sciences, UniversityCollege Cork, Ireland M.A. Drake, Department of Food Science, Southeast Dairy Foods Research Center, North Carolina State University, Raleigh, North Carolina, USA

Introduction A remarkable variety of cheeses are made in all parts of the world where milk is produced. Cheeses are consumed for their highly regarded nutritional value, and enjoyed for their complex and varied eating quality. The sensory characteristics of cheeses, which determine their eating quality, are properties that are perceived by the human senses, predominantly during consumption. These properties can be described as appearance characteristics, flavour characteristics and texture characteristics. However, cheeses are complex foods, produced using milk from different animals, by many different techniques, and are presented in a variety of sizes, shapes, packages or coatings. Some cheeses are produced in small quantities, such as farmhouse types, sold in local markets and consumed by a relatively small number of people. Others are produced in large quantities in very large automated facilities, may find their way to markets in many different countries and are consumed by very many people. Some cheeses are ripened or matured for years before they are consumed; others are consumed young or unripened. Cheeses may have moulds of different types growing on their surface, may be pierced to allow blue moulds grow within the cheese, or include ingredients such as herbs and/or spices. This considerable diversity in cheesemaking practice, and the number of stages that any single cheese undergoes during its production, results in a wide variety of cheeses each of which has complex sensory characteristics. Sensory evaluation of cheese is absolutely necessary to determine the relative merits of cheesemaking procedures and the influence of measured composition on specific sensory characteristics of cheese. Sensory evaluation is also needed to determine the influence of sensory characteristics on the eating quality of cheese and its consumer acceptability. However, the complexity of cheese presents a considerable challenge for its sensory evaluation. This chapter will focus on human perception of sensory characteristics, on the advantages and disadvantages of sensory evaluation methods, on the intensity

and quality of the sensory characteristics of cheeses, and on the relationships between cheesemaking, cheese composition, cheese sensory characteristics and consumer acceptability of cheese.

A Definition of Sensory Character Sensory characteristics of cheeses are human responses to perceptions of stimuli that are experienced with the cheeses, and can generally be described using terms defined within the categories of appearance, flavour and texture. Sensory characteristics result from interactions of the human sensory modalities of vision, touch, olfaction, gustation and mouthfeel with stimuli induced by rheological, structural and chemical components of the cheese. Sensory characteristics are perceived by consumers when they observe, manipulate, smell and take cheese into the mouth for consumption, and are subsequently expressed as a behavioural response using actions or descriptive terms. A majority of sensory characteristics are complex and are stimulated by the association of many different properties of the cheese, with different sensory modalities acting together. It is this complexity, or component balance, that hinders attempts to adequately represent cheese sensory character using instrumental or chemical analyses. In addition, and unfortunately from the sensory scientists' point of view, consumers differ from one another. Sensory perception, and particularly its communication, differs between individuals as a result of physiological, psychological, social and cultural differences.

Sensory Characteristics and Cheese Preferences Cheese quality has been defined for many years by manufacturers as cheese produced reliably and economically (Muir et al., 1995a). In the past, limited choices were available to consumers and as a result of this

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Sensory Character of Cheese and its Evaluation

limited experience, the consumer's palate was less discerning. Today, cheese markets are international, and cheesemakers compete openly for consumers, offering them an eve>widening choice. Cheese consumers are more affluent and many have tasted or regularly consume a diversity of cheese types, leading them to become increasingly discerning. These consumers now define the quality standard for cheeses, which is ultimately determined by eating quality. The eating quality of cheese, or a consumer's liking for cheese, is an integrated response. The stimuli are the sensory characteristics, perceived before and during consumption. However, the response is influenced by other individual consumer-related factors that include sensory abilities, past experiences with cheese, what is expected from a cheese and when and where it will be consumed. Expectations are based on experience, but are created for a specific product by marketing, packaging and familiarity. Finally, liking for, and satisfaction with, a cheese is determined by the context in which it is consumed, and its appropriateness for that context (e.g., would one wish to consume Epoisses for breakfast?). Eating quality determines consumer acceptability and willingness to repeat purchase. Highly regarded eating quality is not that found in a cheese with no defects, but that which offers unique and appealing characteristics consistently. The producer of the cheese with the most acceptable sensory characteristics, if he is aware of this and can ensure that the market presentation of his cheese matches its sensory character, will have an advantage in the market. The concepts of 'healthy eating' and 'conscientious eating' (e.g., vegetarian and vegan diets) and environmentally friendly eating are now increasingly important to consumers. To meet these consumers' expectations, cheese producers are challenged to produce new, wholesome products that taste as good as traditional alternatives. This task is proving difficult as dietary guidelines for healthy eating may recommend reducing the intake of ingredients that provide desirable sensory character, such as fat or salt. The production of reduced- and low-fat cheeses to replace traditional types is an example of such consumer-driven product development. However, the majority of new low-fat cheeses do not meet the sensory quality requirements of discerning consumers (Mistry, 2001). This is because fat is not just a provider of desirable sensory character, but it is also important for cheese texture and body, for the development of compounds responsible for flavour and for the release of flavour compounds during consumption. It will be difficult to improve the eating quality of these cheeses unless eating quality is understood better.

Cheesemaking and the Variety of Sensory Character The sensory characteristics of a cheese at the time of its consumption reflect the milk from which it was produced (e.g., a goats' milk cheese is distinct from a cows' milk cheese), the processes used in its production and the physical and the chemical changes that occurred during maturation (e.g., proteolysis breaks down proteins to amino acids during cheese maturation, which may subsequently act as substrates for the formation of volatile compounds (see 'Catabolism of Amino Acids in Cheese during Ripening', Volume 1)). Milk from the cow, sheep, goat, buffalo or other animals can be used as raw material, and its qualities are determined by breed, diet and stage of lactation. Treatment of milk before cheese production, particularly pasteurisation, can kill micro-organisms and reduce enzyme activity that could otherwise contribute to the development of sensory character during maturation. During cheese production, the coagulant used to form curds, the amount of salt added, the type of starter culture and the use of adjunct cultures will determine sensory characteristics. Finally, the maturation time and the temperature of maturation may be varied. The sensory characteristics of different types of cheese, and the potential variety that may be achieved, are determined by the choices the producer makes at each of the stages in production. Sensory characteristics of many different cheeses are described in the literature and in specialist cheese books. However, the sensory characteristics of relatively few types have been defined, standardised and measured objectively using sensory science methods. Lack of objective knowledge makes it difficult to compare accurately the sensory characteristics of different cheese types, but more importantly, as the cause of sensory characteristics is only partially known, it is difficult to compare accurately cheese appearance, texture and flavour research carried out in different laboratories. Tables 1 and 2 present terms used to describe the appearance, texture and flavour characteristics of cheeses that have been defined and standardised in an objective way. Table 3 presents terms used for descriptive sensory analysis by other researchers, but that have not been defined and standardised adequately. Similar terms are used in many cases even though each descriptive language referenced was developed independently by different research groups. In addition, in many cases, similar terms have been used to describe dominant characteristics of different cheese types. This comparison suggests that even though a remarkable variety of cheese types are produced, that potentially exhibit a wide variety of sensory characteristics, it should

Sensory Character of Cheese and its Evaluation

457

Table 1 Terms used to describe the appearance and texture of cheese using descriptive analysis methods. Terms in this list were developed and defined by trained panels, and in many cases standard materials that help to illustrate the term are provided. Cheeses studied were low-fat, full-fat and smoked Swiss, Cheddar and Gouda (Adhikari etaL, 2003), natural and processed cheeses (Drake etaL, 1999a; Gwartney etaL, 2002), ten different types of cheese (Lawlor and Delahunty, 2000), Cheddar and Camembert (Cooper, 1987) and Mozzarella cheeses (Brown et aL, 2003)

Term Appearance Chalky Colour/colour intensity

Mottling Mouldy Open/openness Shiny

Texture Adhesiveness Chewy Cohesiveness Creamy/creaminess

Crumbly/crumbliness

Crustiness Curdiness Degree of breakdown Dry Firm/firmness

First-bite sticky Fracturability at first bite Grainy

Hardness Mealy Moist Mouth-coating Oily Rate of recovery Residual mouthfeel Residual smoothness of mouth coating

Definitiona

Resembling chalk in appearance The colour of Cheddar ranging from pale yellow to orange, the palest yellow representing the start of the scale The colour of cheese ranging from white to orange The evenness of colour shading within the cheese sample, with the most uniform coloured cheese being free from mottling, marbling or any other deficiencies in colour The degree of mouldiness/visible mould growth in the cheese structure The extent to which the interior of the cheese (that is the cut surface) is open, this encompasses cracks, pinholes, irregular-shaped holes and any other openings The extent to which the surface of the cheese is shiny, glossy, moist or sweaty-looking, as opposed to looking matt or dull The degree to which the chewed mass sticks to mouth surfaces, evaluated after five chews Requiring a good deal of mastication, toffee-like texture. Degree of chewing needed to break up the cheese The degree to which the chewed mass holds together, evaluated after five chews The extent to which the texture has broken down to a creamy semi-liquid texture, assessed between tongue and palate during mastication The feeling associated with heavy whipping cream (e.g., >30% fat content) The extent to which the cheese structure breaks up in the mouth, assessed during the first 2-3 chews The feeling in the mouth when the sample falls apart quickly in mouth during mastication The force required to break through the crust of the cheese when taking the first bite, assessed using the front teeth The extent to which a curdy or mealy texture is perceived in the mouth during mastication The amount of breakdown that occurs in the sample as a result of mastication, evaluated after five chews The degree of dryness or moistness sensed in the mouth during mastication Ranging from soft to firm. The extent of resistance offered by the cheese, assessed during the first five chews using the front teeth The force required to squeeze a cube (1.5 • 1.5 • 1.5 cm) of cheese flat between the first finger and thumb The amount of force required to take the first bite of cheese, assessed using the front teeth The amount of force required to completely bite through the cheese, assessed using the molars Sticky sensation experienced during the first bite Completely bite through the sample with the molars and evaluate the degree to which the sample fractures The extent to which granular structures are formed as the sample breaks down, perceived in the second half of chewing The feeling of coarse particles in the mouth during mastication The force required to bite the sample (first bite) The feeling in mouth when the sample breaks down in small pieces and it is difficult to gather them for swallowing The perceived moisture content of the cheese. Ranging from dry to moist The extent to which the cheese has a moist or wet texture around the palate during mastication The extent to which the cheese coats the palate and teeth during mastication The degree of coating on the tongue and the palate during mastication Oily, fatty, greasy mouth-feel of any kind Depress sample between thumb and first finger 30%, evaluate the speed or rate at which the sample returns to its original shape The degree of 'bittiness' or graininess in the mouth just before swallowing The degree of smoothness felt in the mouth after expectorating the sample

continued

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Sensory Character of Cheese and its Evaluation

Table 1 continued Term

Definition a

Rubbery/rubberiness

The extent to which the cheese returns to its initial from after biting, assessed during the first 2-3 chews The degree of springiness experienced while biting the sample Of the nature of slime, soft, glutinous or viscous substance, soft, moist and sticky The smoothness of the cheese against the palate as it breaks up during mastication The degree to which the chewed mass surface is smooth, evaluated after five chews Yielding easily to pressure, easily moulded, pliable, easily spreadable Depress sample between thumb and first finger 30%, evaluate the total amount of recovery of the sample The stickiness of the cheese against the palate and around the teeth during mastication Overall sensation of stickiness during mastication The mouthfeel associated with consuming very viscous fluids like heavy whipping cream or honey

Slimy Smooth/smoothness Softness Springiness Sticky/stickiness Viscous

a The precise wording of some definitions has been changed to allow the use of consistent language in this table. However, the meaning of each definition is unchanged.

Table 2 Terms used to describe the flavour of cheese using descriptive analysis methods. Terms in this list were developed, defined and referenced using standard materials by trained panels. Cheeses studied were: Cheddar (Murray and Delahunty, 2000b; Drake et aL, 2001), low-fat, full-fat and smoked Swiss, Cheddar and Gouda (Adhikari et al., 2003), aged natural cheese of many types (Heisserer and Chambers, 1993), ewes' milk cheese (B~.rcenas et al., 1999) and cheese flavours (Stampanoni, 1994) Term

Definition a

S t a n d a r d b, c

Acid/yoghurt, acidic

The taste on the tongue associated with acids (citric, lactic... ) A sour, tangy, sharp, citrus-like taste. The fundamental taste sensations of which lactic and citric acids are typical Flavours indicating age in Cheddar cheese

0.35-0.86 g lactic acid/100 g Ricotta Fermented milk Natural yoghurt Citric acid (0.2% in water)

Age Ammonia Animal, animalic Astringent

Balanced

Bell pepper Biting

Bitter

Blue Brine

Brothy

The combination of aromatics reminiscent of farm animals and barnyards The complex of drying, puckering, shrinking sensations in the oral cavity causing contraction of the body tissues A mouth-drying and harsh sensation Mellow, smooth, clean. In equilibrium, wellarranged or disposed, with no constituent lacking or in excess Aroma associated with freshly cut green peppers The slightly burning, prickling and/or numbness of the tongue and/or mouth surface Fundamental taste sensation of which caffeine or quinine are typical A chemical-like taste The combination of aromatics associated with the saturated brine used during traditional ewes' milk cheesemaking Aromatics associated with boiled meat or vegetable stock soup

Aged Cheddar cheese (1 yr or older) Ammonia solution (0.25% in water) 4-Methyl-octanoic acid (2% in PG d) 1-Phenyl-2-thiourea (5000 mg/kg in PG) Alum (0.1% in water) Tea, six bags soaked in watere for 3 h Tannic acid (0.05% in water) Mild Cheddar

Methoxy pyrazines (5 #g/kg) Freshly cut bell pepper Horseradish sauce

Caffeine (0.02, 0.06 or 0.08% in water) Tonic water, quinine (0.01% in water) Octan-2-one (1% in PG) Ewes' milk cheese brine at room temperature

Canned potatoes Low-sodium beef broth cubes Methional (20 mg/kg)

Sensory Character of Cheese and its Evaluation

459

Table 2 continued Term

Definition a

Butter milk Buttery

B

Fatty, buttery tasting, of the nature of, or containing butter The aromatics commonly associated with natural, fresh, slightly salted butter Aroma rising from butter at room temperature

Butyric, butyric acid

Capric acid Caramel

Caseinate Catty Cheddary

Cheese rind Cooked, cooked milk

Cottage cheese Cowy/phenolic

Creamy

Dairy fat Dairy sour Dairy sweet Decaying animal Diacetyl Earthy Fatty Faecal Fermented Fermented fruity / winey

Flavour intensity Free-fatty acid Fresh fish

Sour flavour, similar to baby vomit The aromatics reminiscent of baby vomit; is sour and cheesy m

The taste and aromatics associated with burnt sugar or syrup; toffee made from sugar that has been melted further Aroma associated with tom-cat urine The taste and aromatics associated with typical Cheddar Typical aroma and taste of sharp/mature Cheddar cheese m

Aromatics associated with cooked milk The combination of sweet, brown flavour notes and aromatics associated with heated milk m

Aromas associated with barns and stock trailers, indicative of animal sweat and waste Fatty, creamy tasting, of the nature of, or containing cream The oily aromatics reminiscent of milk or dairy fat The sour aromatics associated with dairysoured products The sweet aromatics associated with fresh dairy products The aromatics reminiscent of decaying animal material Aromatics associated with diacetyl m

Aroma associated with complex protein decomposition The combination of aromatics reminiscent of red wine in general; it is sweet, slightly brown, overripe and somewhat sour The overall intensity of flavour in the sample, from mild to strong Aromatics associated with short chain fatty acids The aromatics associated with fresh fish

S t a n d a r d b, c

Pasteurised butter milk Unsalted butter Lightly salted butter Pasteurised cooking butter Diacetyl (1% in PG) Diacetyl in vaseline oil (several concentrations) Butyric acid, 2500 mg/kg in vaseline oil =SSf. 2 ml SS + cotton in 60-ml flask Butyric acid (10 000 mg/kg in PG) Butyric acid (1% in PG) Capric acid (pure) Condensed milk 3-Hydroxy-2-methyl-4-pyrone (2% in PG)

Sodium caseinate powder 2-Mercapto-2-methyl-pentan-4-one (20 mg/kg) Processed cheese Mature Cheddar cheese

Cheese rind (Tilsit mild, pasteurised full fat) Skim milk heated to 85 ~ for 30 min Evaporated milk UHT milk 3.6% fat, cooked for 10 min Cottage cheese 25% fat p-Cresol (160 mg/kg), bandaids

Mascarpone cheese ,y-Decanolactone (0.1% in PG) UHT Cream 35% fat Whipping cream Unsalted butter Sour cream Vitamin D milk Dimethyl disulfide (bottom notes only; 10 000 mg/kg in PG) Diacetyl (20 mg/kg) Geosmin (0.001% in PG) Palm kernal fat Indole, skatole (20 mg/kg) Fermented milk, 12% fat Burgundy cooking wine

Butyric acid (20 mg/kg) Elodea

(an aquatic plant) growing in water continued

460

Sensory Character of Cheese and its Evaluation

Table 2

continued

Term

Definitiona

Standard b, c

Fruity

The taste and aromatic blend of different fruity identities The aromatics associated with different fruits

Goaty

The aromatics reminiscent of wet animal hair; it tends to be pungent, musty and somewhat sour

Canned fruit salad (in syrup) trans-2-Hexenal (10 000 mg/kg in PG) Canned fruit cocktail juice Fruit of the forest yoghurt Ethyl butyrate (0.1% in PG) trans-2-Hexenal. 300 mg/kg in vaseline oil = SS. 3 ml SS + cotton in 60-ml flask Fresh pineapple Ethyl hexanoate (20 mg/kg) Hexanoic acid (5000 mg/kg in PG)

Green-grass Methyl ketone / blue Milkfat /lactone

Milky Mouldy, mouldy/musty

Mushroom

u

Aroma associated with blue-vein cheeses Aromatics associated with milkfat

The aromatics commonly associated with ewes' raw milk The combination of tastes and aromatics generally associated with moulds; they usually are earthy, dirty, stale, musty and slightly sour Aromas associated with moulds and/or freshly turned soil The taste and aromatics associated with raw mushrooms

Musty

Aroma of a damp room or very old book

Nutty

The aromatics reminiscent of several dry fruits such as pecans, walnuts and hazelnuts The non-specific nut-like taste and aromatics characteristic of several different nuts, e.g., peanuts, hazelnuts and pecans The nut-like aromatic associated with different nuts

Overall intensity

Strength of the stimuli perceived by the nose Strength of global stimuli originated by the volatiles released during mastication and perceived on the olfactory receptors via the retronasal route

Oxidised

Aroma associated with oxidised fat The fruity aromatic associated with pineapple

Pineapple

cis-3-Hexenol(1% in PG) 2-Octanone (40 mg/kg) Fresh coconut meat Heavy cream 5-Dodecalactone (40 mg/kg) Ewes' milk raw Pasteurised milk, 3.6% fat 2-Ethyl-l-hexanol (10 000 mg/kg in PG) 2-Ethyl-l-hexanol (40 mg/kg) Stilton cheese 2,4,6 Trichloroanisole (1% in PG)

Button mushroom (raw) Brown mushrooms (chopped, raw) 1-Octen-3-ol (0.5% or 1% in PG) 3-Octanol (10 000 mg/kg in PG) 3-Octanol. 5-10 mg/kg in vaseline oil = SS. 3 ml SS + cotton in 60 ml flask Cola infusion in ethanol (pure) Damp room Very old book Wheat germ 2 g Walnuts + 2 g hazelnuts, minced in 60-ml flask (mixed particulates to be sampled) Mixed crushed nuts 2-Acetyl-pyridine (0.01% in PG) Lightly toasted unsalted nuts Unsalted wheat thins Roasted peanut oil extract Roasted peanuts, ground hazelnuts, ground almonds, 1:1:1 1000-73 nut base by Givaudan-Roureg (10% in PG) 4 g cheese aroma/100 ml of pasteurised ewes' milk 0.5-3.5 g cheese aroma/100 g Quark 91549-24 by Givaudan Roureg 91483-24 by Givaudan Roure 91428-24 by Givaudan Roure 91125-73 by Givaudan Roure 10418-71 by Givaudan Roure 2,4 Decadienal, 20 mg/kg 4-Pentenoic acid (10 000 mg/kg in PG) Canned pineapple chunks

Sensory Character of Cheese and its Evaluation

461

Table 2 continued Term

Definition a

Standardb, c

Prickle/bite

Chemical feeling factor of which the sensation of carbonation on the tongue is typical A bland, shallow and artificial taste. Made by melting, blending and frequently emulsifying other cheeses

Soda water

Processed

Propionic acid Pungent

Rancid

Rennet Rosy/floral Salty

Sauerkraut Scorched Sharp

Smokey, smoky

Soapy

Sour

Soya sauce

m

A physically penetrating sensation in the nasal cavity. Sharp smelling or tasting, irritating Irritative, burnt and/or penetrating sensation in the interior of the mouth

The taste and aroma associated with sour milk and oxidised fats. Having the rank unpleasant aroma or taste characteristic of oils and fats when no longer fresh The aromatics associated with natural lamb rennet Aroma associated with flowers Fundamental taste sensation of which sodium chloride is typical Fundamental taste sensation elicited by salts Fundamental taste sensation produced by aqueous solutions of several products such as sodium chloride The aromatics associated with fermented cabbage Aroma associated with extreme heat treatment of milk proteins The total impression associated with the combination of aromatics that are sour, astringent and pungent Total impression of penetration into the nasal cavity The perception associated with aged and ripened cheeses, from flat to sharp The penetrating, dark brown, acrid aromatic of charred wood Aroma and taste of hickory-smoked ham The penetrating smoky taste and aromatics, similar to charred wood Tainted by exposure to smoke Perception of any kind of smoke odour (hickory, apple, cherry, mesquite or artificial flavouring) A detergent-like taste and smell. Similar to when a food is tainted with a cleansing agent Fundamental taste sensation elicited by acids Fundamental taste sensation of which lactic and citric acids are typical The aromatics that are reminiscent of soy sauce; they are sour, slightly brown and pungent

Cheese strings (a processed cheese snack) Propionic acid (1% in PG) A ratio of 1 part sour cream to 0.68 parts horseradish sauce Danish blue cheese Ammonia (1% in PG) 0.5 g cayenne/100 ml water, boiled in water for 5 min, 1.5 ml of filtration/10 g Quark Cheese stored at 21 ~ for 4 days Butyric acid (0.1% in PG)

Natural lamb rennet (33% NaCI) 2-Phenethylamine, 20 mg/kg Sodium chloride (0.25, 0.5, 0.75 or 1% in water) Pecorino Romano sheep cheese, 1200 mg NaCI/100 g Quark

Dimethyl disulfide (top notes only; 10 000 mg/kg in PG) Milk heated to 121 ~ for 25 min Propionic acid (100 000 mg/kg in PG) 5000 mg/kg of propionic acid in Vaseline oil = SS. 2 ml SS + cotton in 60 ml flask

Oil of cade Hickory smoked ham Applewood cheese Guaiacol (0.5% in PG) Guaiacol in vaseline oil (several concentrations) Liquid smoke flavouring. 40 #1 + cotton in 60-ml flask Lauric acid (pure) Mellow processed Cheddar Citric acid (0.08% in water) Lactic acid (0.05 and 0.085% in water) Soya sauce

continued

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Table 2 continued Term

Spicy/pungent Strength Sulfur Sweaty

Sweet

Toasty Umami Vinegary Waxy, waxy/crayon

Whey Yeasty

S t a n d a r d b, c

Definition a m

The overall intensity of aroma and flavour, the degree of mildness and maturity Aromatics associated with sulphurous compounds The aromatics-associated reminiscent of perspiration-generated foot odour; sour, stale, slightly cheesy and is found in unwashed gym socks and shoes Fundamental taste sensation of which sucrose is typical Fundamental taste sensation elicited by sugars Fundamental taste sensation produced by aqueous solutions of several products such as sucrose or fructose The combination of sweet aromatics produced after food toasting or cooking Chemical feeling factor elicited by certain peptides and nucleotides Aroma described as acidic, fermented and sweaty by the panelists The sweet aromatic that is associated with waxed paper or wax candles Aromatics associated with medium chain fatty acids Aromatics associated with Cheddar cheese whey Aromatics associated with fermenting yeast

Yoghurt

Valeric acid (1% in PG) English blue Stilton cheese Boiled mashed egg. H2S bubbled through water; struck match Isovaleric acid (10 000 mg/kg in PG) Isovaleric acid (0.1% in PG) Isobutyric acid (5% in PG) Cheese stored at 30 ~ for 3 h Sucrose (1,3, 4 or 5% in water) Condensed milk 1.2 g sucrose/100 g Quark

Cooked condensed milk Ciclotene (several concentrations in water) Monosodium glutamate (1% in water) Combination of acetic, butyric and propionic acids Decanoic acid (pure) Capric acid, lauric acid or decanoic acid (100 mg/ml) Fresh Cheddar whey Whey powder Raw yeast dough Yeast in 3% warm sucrose water Yoghurt, 3.2% fat

a The precise wording of some definitions has been changed to allow the use of consistent language in this table. However, the meaning of each definition is unchanged. b Units of measurement are changed to a standard format where possible. c Publications referenced often provided brand names of food standards used. Brand names are not provided in this table as it is recognised that many of these will only be of interest to readers in their country of origin. In addition, as some publications referenced are now more than 10 years old, products may have changed. d Propylene glycol. e Volume of water not given in publication referenced. f Stock solution. g Codes refer to commercially available flavour mixtures that can be provided by Givaudan Roure, Switzerland.

be possible to develop and standardise a terminology that can be used universally, and for all cheese types, eventually leading to a much-improved understanding about the eating quality of cheese.

The Human Senses and the Sensory Properties of Cheese Cheese appearance

Humans are highly visual creatures and allow vision to dominate other sensory modalities. Vision is the perception of shape and texture, size and distance, brightness,

colour and movement. Appearance characteristics of cheese are assessed visually, usually prior to consuming the cheese, or when preparing the cheese for consumption by cutting or spreading. Appearance characteristics include colour, presence of eyes or holes (or openness), mould, rind, and visual texture (Tables 1 and 3). In addition, appearance includes a cheese's market image (e.g., size, shape, packaging), as most cheese is purchased in this form (Murray and Delahunty, 2000a). Appearance characteristics create sensory expectations, or expectations of how the cheese will 'taste', and as vision can dominate other sensory modalities, visual aspects of cheese can often have a strong influence on

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Sensory Character of Cheese and its Evaluation

the perception of other characteristics that, general experience has taught us, are related (even if they may not be physically related). For example, many consumers believe that a coloured cheese is more intensely flavoured than its uncoloured equivalent (Bogue et al., 1999). Cheese texture

Texture can be defined as the attribute of a cheese resulting from a combination of physical properties, including size, shape, number, nature and conformation of the constituent structural elements, that are perceived by a combination of the senses of touch (tactile texture), vision (visual texture) and hearing (auditory texture). For example, the 'softness' of a cream cheese can be assessed visually upon cutting the cheese, by proprioceptive sensations when spreading the cheese, and finally by tactile sensations in the mouth during consumption. During mastication and consumption, texture perception occurs in the superficial structures of the mouth, around the roots of the teeth and in the muscles and tendons. Cheese texture characteristics frequently described include firmness, rubberiness, crumbliness, graininess, cohesiveness and adhesiveness (Tables 1 and 3). Cheese flavour

Flavour is most often defined as the integrated perception of olfactory, taste and chemesthesis (or trigeminal) stimuli. Flavour perception begins prior to consumption when a consumer can smell a cheese, but is finally perceived during consumption when compounds that stimulate the olfactory system in the nose, the taste system in the mouth and the trigeminal system in the mouth and nose are released from the cheese and become available to receptors. A large number of flavour characteristics have been described in cheese. Some that have been defined and standardised for application in descriptive sensory evaluations are listed in Table 2. Smell or aroma is usually the first aspect of flavour encountered by a consumer. The stimuli for smell are air-borne compounds of volatile substances that allow them to travel from their source to the olfactory receptors, where perceptions are created that are endowed with distinctive smells. Volatile stimuli are released from cheese into the air, and may be delivered to the nose orthonasally, often consciously, by sniffing (e.g., when one opens a cheese package or removes a trier from the cheese for evaluation). Volatile compounds may also be released into the buccal cavity air during consumption, where they are delivered to the nose retronasally without any conscious effort. Many hundreds of different volatile

compounds, each with a distinctive aroma character, have been identified in cheese, and these provide the largest contribution to the diversity of cheese flavours. Compounds identified in cheeses include fatty acids, methyl, ethyl and higher esters, methyl ketones, aliphatic and aromatic hydrocarbons, short- and longchain alcohols, aromatic alcohols, aldehydes, amines, amides, phenols and sulphur compounds (Maarse and Visscher, 1996). Much of what we commonly refer to as 'taste' is incorrectly localised smell detection. The significant contribution of aroma to flavour can be easily demonstrated if one pinches the nose shut whilst eating, effectively blocking air circulation through the nasal passages. Then, a familiar cheese, e.g., Cheddar, will not be recognised, and can easily be confused with one that would otherwise be easily distinguished, e.g., Gruyere. Taste is another aspect of flavour. Tasting occurs in the oral cavity, primarily on the tongue, but also on the soft palate. The primary stimuli for taste are nonvolatile compounds, and these must make contact with the taste receptors. This contact creates perceptions that endow four distinctive taste qualities, referred to as sweet, salty, sour and bitter. A fifth taste, 'umami', has been accepted more recently, particularly in Japan and other cultures where it is the most familiar and the most easily perceived. Compounds that contribute directly to cheese taste include lactic acid (sour), sodium chloride (salty), mineral salts of potassium, calcium and magnesium (salty) and free amino acids and peptides of varying types (sweet, bitter, umami) (Warmke et al., 1996; Engel et al., 2000). The last aspect of flavour is chemesthesis. This term is used to describe the sensory system responsible for detecting chemical irritants. Detection is more general than that of taste and smell and occurs primarily in the eyes, nose and mouth. The perception is closely related to the somato-sensory characteristics of pain and temperature change, and provokes a strong behavioural response. The fizz of carbon dioxide (CO2), the cooling sensation of menthol and the burning sensation of chilli are perhaps the best examples of how chemical irritation can provide additional character that is very much desired in a wide range of food products. With regard to cheese, the pungency, the prickle/bite and the sharpness of mature Cheddar are examples of perceived chemical irritation (Table 2). Sensory interactions

Cross-modal sensory interactions also occur, adding complexity to the perception and description of sensory character. Consumers rarely distinguish between stimuli of different sensory modalities (unless trained to do so), and generally describe the integrated sensation as 'taste'.

Sensory Character of Cheese and its Evaluation

However, the factors that cause apparent cross-modal sensory interactions are not always the same and can be difficult to comprehend. A first cause of apparent sensory interactions when perceptible components of a cheese are studied together can be interactions between the components of the cheese prior to introduction to the senses. For example, changing the fat content or salt content of a cheese can influence the physical chemistry of the cheese matrix dramatically, changing the partition coefficients of volatile compounds, and therefore releasing volatiles from the cheese matrix (Delahunty and Piggott, 1995). As a cheese matures, the protein composition changes significantly due to proteolysis, and this may change the binding ability of the cheese for specific volatile compounds (Delahunty and Piggott, 1995). A second cause of sensory interaction is termed a halo effect, and is caused by learning to place greater reliance on one sensory modality over another to make behavioural decisions. This effect was referred to in the context of appearance, as it is most obvious by the dominance, or bias, of the visual sense over the taste or olfactory sense. It can be demonstrated by confusing familiar colour and flavour combinations, or by varying colour intensity beyond expectation (Clydesdale, 1993). With regard to cheese, an influence of added colour on consumers' perception of flavour has been reported (Bogue et al., 1999). A true cross-modal sensory interaction is one where the function of one sense (e.g., threshold measures, concentration-response functions) is changed by stimulation of another sense. This type of interaction can occur at receptor level, where one component blocks access to the receptor by another (e.g., increasing viscosity may coat the tongue and reduce access of tastants to taste receptors (Lynch et al., 1993)), or where stimulation by both components results in neural convergence as receptor sites are in close proximity and are served by the same nerve (e.g., capsaicin desensitisation reduces perceived taste intensity (Karrer and Bartoshuk, 1995)). The extent of these types of interactions in cheese, and their effect, is not known. Taste-aroma interactions are also observed and appear to be true interactions even though the physiology of the senses of olfaction and taste is independent. In this case, interaction is believed to occur centrally at a cognitive level where stimulus integration takes place (Stevenson et al., 1999). Taste-odour interactions have been observed in many different types of food and are easily demonstrated in model food studies (Noble, 1996). When volatile compounds are introduced to the oral cavity in the absence of taste-active compounds, they are generally perceived to be of low intensity and are described as bland in character. In cheese, it is most likely that the flavour impact of specific volatile compounds will be pronounced (and become familiar) only

467

when perceived in combination with appropriate tasteactive compounds, such as lactic acid, mineral salts, free amino acids and bitter peptides typically present in cheese (Frank and Byram, 1988). In addition, variations in taste quality and intensity, for example an increase in sourness (i.e., acidity), or an increase in bitterness, will affect how aroma is perceived (although volatile composition may be unchanged) and give the impression that overall flavour has changed considerably. Flavour-texture interactions are also observed widely. The precise nature of many of these interactions is not known, although structural components, such as proteins, can bind volatile compounds; rheology and structure can also influence mass transfer of non-volatile and volatile compounds to the surface of a cheese bolus where they will be released and become available for perception; fat can coat the receptor surface of the tongue, effectively blocking taste transduction (Lynch et al., 1993) and finally, interactions may occur at a cognitive level during perception integration in a way similar to taste-odour interactions (Weel etal., 2002). Texture-flavour interactions can also be influenced by individual consumer physiology, such as mastication behaviour and saliva flow rate and volume.

Sensory Methods Used to Evaluate Cheese Many reported studies on cheesemaking, cheese composition and cheese microbiology had the objective of controlling or improving sensory characteristics such as appearance, flavour and texture. However, it is difficult to compare the success of these studies as the final sensory character was often measured inappropriately. In many studies, judgements of overall sensory quality (i.e., a grade of 'good' or 'bad'), rather than objective measurements of the perceived intensity of specific sensory characteristics, were carried out to determine the influence of cheese composition, counts of micro-organisms, or control of a cheesemaking variable on flavour or texture quality. Although standard procedures may be followed, e.g., International Dairy Federation standards (IDE 1997), quality judgements are biased by the individual(s) who makes them. In addition, and of greater importance, traditional quality judgements do not allow the application of statistical analyses that would enable relationships between cheese study variables and specific sensory characteristics to be determined. The unaware reader of the literature can very easily confuse measurements of overall sensory quality with descriptions of sensory difference, as it is often reported, for example, that a specific cheesemaking procedure produced cheeses that 'tasted' similar, when in fact they were judged to be of similar quality (i.e., had no defects). Cheeses judged to be of similar quality by the same judge may differ

468

Sensory Character of Cheese and its Evaluation

significantly in sensory characteristics (Delahunty and Murray, 1997). The American Society for Testing and Materials (ASTM) Committee E-18 on Sensory Evaluation of Materials and Products has defined sensory evaluation as 'a scientific discipline used to evoke, measure, analyse and interpret reactions to the characteristics of foods and materials as they are perceived by the senses of sight, taste, touch and hearing'. A key distinction between sensory evaluation and other chemical and instrumental analytical techniques, is that different techniques can be used to evoke, measure and interpret sensory characteristics that have very different objectives and outcomes. Sensory evaluation can be carried out to determine whether cheeses exhibit defects or other undesirable characteristics, whether a difference in overall sensory character can be detected between two or more cheeses, whether specific differences in sensory characteristics can be perceived, to quantify the intensity of one or more sensory characteristics, to quantify the onset, maximum intensity and decline of a sensory characteristic, and to determine whether consumers find the cheeses to be acceptable or not, based on their sensory characteristics. The distinctions in sensory evaluation methodology can be broadly classified as quality scoring, discrimination testing, descriptive testing, time-intensity testing and consumer acceptability testing, respectively. There are some excellent texts that outline sensory tests in detail (Piggott, 1988; Stone and Sidel, 1993; Lawless and Heymann, 1998; Meilgaard et al., 1999).

Grading and quality scoring The manufacture of cheese of consistent quality is extremely difficult due to the number of production factors that ultimately contribute to eating quality (see 'Factors that Affect the Quality of Cheese', Volume 1). In addition, cheeses are susceptible to a large number of defects that can originate in milk, transfer to the cheese curd during making and storage, result from microbial contamination or develop during maturation if the composition at manufacture is not controlled. However, to maintain consumer confidence and loyalty towards a cheese, it is very important to control its quality. In addition, as consumers are becoming more brand-conscious, they become less-accepting of variations in sensory characteristics that traditionally would not be considered defects, and expect to find a cheese with near-identical appearance, flavour and texture in the package each time. To test instrumentally for all possible flavours and structural properties that contribute to eating quality would be an extremely laborious task, and may not achieve success. For example, many compounds that contribute to flavour are present

in concentrations below the detection limit of even the most sophisticated instruments. Quality scoring, grading or judging against specified defects on standardised scorecards (Bodyfelt et al., 1988) is the traditional and still most widely used type of formal sensory evaluation in the cheese industry. Cheese grading is carried out to classify the potential of a cheese to develop a satisfactory character during maturation, and to maintain quality at the point of sale. Grading standards generally specify a scoring system, where top grade is awarded a maximum score, and points are deducted when defects are found. For example, the IDF provides standard scorecards for cheese, and specifies a scale that ranges from 5, representing the highest possible quality, to 0, representing the lowest possible quality (IDE 1997). Each point deducted from the scale is supported by a list of defects that merit the deduction. The defect list that accompanies each score on the scale aims to provide objectivity to the evaluation. The US cheese grading system and the American Dairy Science Association (ADSA) cheese-judging ballot operate in a similar manner (Bodyfelt et al., 1988). Tables 4 and 5 show the United States Department of Agriculture (USDA) standards for grades of Cheddar cheese, effective since 1956, which provide guidelines for the award of four g r a d e s - AA, A, B or C. Table 6 shows the ballot used by the ADSA to judge Cheddar cheese quality. McBride and Muir (1999) recently reviewed grading systems used for Cheddar cheese in Australia, United Kingdom, United States, Canada, the IDF and New Zealand. In addition, chapters in recent textbooks by Kosikowski and Mistry (1997) and Robinson and Wilbey (1998) review in detail methods of cheese grading and defects found in cheese. Kosikowski and Mistry (1997) described the sequence of cheese quality judgement. One or more expert evaluators, who have detailed product knowledge built up over many years and maintain a standard in memory of what the ideal product is in terms of sensory characteristics, carry out this evaluation. These experts have the ability to relate their recognition of specific defects to the cause of that defect and to weight the influence of each defect at different levels of severity and how they detract from overall product quality. The overall exterior of a cheese is first judged to determine if it appears deformed or soiled in any way. The rind or surface is judged next as it may be discoloured, cracked or irregular. Internal appearance is judged following cutting, or directly from a cheese trier, as it may have holes, cracks, spots or other opening defects, and colour may be uneven, mottled or dull. Odour, which may be uncharacteristic in many ways, is judged prior to placing a cheese in the mouth,

Sensory Character of Cheese and its Evaluation

469

Table 4 Specifications for Grade AA and Grade A Cheddar cheese (United States Department of Agriculture, Agricultural Marketing Service, Dairy Division)

Detailed specifications for US Grade AA Fresh or current

Medium cured

Cured or aged

(a) Flavour : Fine and highly pleasing. May be lacking in flavour development or may possess slight characteristic Cheddar cheese flavour. May possess a very slight feed flavour, but shall be free from any undesirable flavours and odours.

Fine and highly pleasing. Possesses a moderate degree of characteristic Cheddar cheese flavour. May possess a very slight feed flavour but shall be free from any undesirable flavours and odours.

Fine and highly pleasing characteristic Cheddar cheese flavour showing moderate to well-developed degrees of flavour or sharpness. May possess a very slight feed flavour but shall be free from any undesirable flavours and odours.

A plug drawn from the cheese shall be firm, appear smooth, waxy, compact, close, flexible and translucent, but may have a few mechanical openings if not large and connecting. May be slightly or not entirely broken down. May possess not more than one sweet hole per plug but shall be free from other gas holes.

A plug drawn from the cheese shall be firm, appear smooth, waxy, compact, close, and translucent but may have a few mechanical openings if not large and connecting. Should be free from curdiness and possess a cohesive velvet-like texture. May possess not more than one sweet hole per plug but shall be free from other gas holes.

Shall have a uniform, bright attractive appearence; practically free from white lines or seams. May be coloured or uncoloured, but if coloured it should be medium yellow-orange.

Shall have a uniform, bright attractive appearance; practically free from white lines or seams. May show numerous tiny white specks. May be coloured or uncoloured, but if coloured it should be medium yellow-orange.

Bandaged and paraffin dipped. Shall possess a sound, firm rind with a smooth bandage and paraffin coating adhering tightly but may possess very slight mould under bandage and paraffin, and the following other characteristics to a slight degree: Soiled surface and surface mould. The cheese shall be even and uniform in shape. Rindless. Same as for current, except very slight mould under wrapper or covering permitted.

Bandaged and paraffin dipped. Shall possess a sound, firm rind with a smooth bandage and paraffin coating adhering tightly but may possess the following characteristics to a slight degree: Soiled surface and mould under bandage and paraffin; and surface mould to a definite degree. The cheese shall be even and uniform in shape. Rindless. Same as for medium.

(b) Body and texture: A plug drawn from the cheese shall be firm, appear smooth, compact, close and should be slightly translucent, but may have a few small mechanical openings. The texture may be definitely curdy or may be partially broken down if more than 3 weeks old. Shall be free from sweet holes, yeast holes and gas holes of any kind. (c) Colour : Shall have a uniform, bright attractive appearance; practically free from white lines or seams. May be coloured or uncoloured but if coloured it should be a medium yellow-orange.

(d) Finish and appearance: Bandaged and paraffin-dipped. Shall possess a sound, firm rind with a smooth bandage and paraffin coating adhering tightly but may possess soiled surface to a very slight degree. The cheese shall be even and uniform in shape. Rindless. The wrapper or covering shall be practically smooth, properly sealed with adequate overlapping at the seams or by any other satisfactory type of closure. The wrapper or covering shall be neat and adequately and securely envelop the cheese. May be slightly wrinkled but shall be of such character as to protect fully the surface of the cheese and not detract from its initial quality. Shall be free from mould under wrapper or covering and shall not be huffed or lopsided.

continued

470

Sensory Character of Cheese and its Evaluation

Table 4

continued

Detailed specifications for US Grade A Fresh or current

Medium cured

Cured or aged

(a) Flavour: Shall possess a pleasing flavour. May be lacking in flavour development or may possess slight characteristic Cheddar cheese flavour. May possess very slight acid, slight feed but shall not possess any undesirable flavours and odours.

Shall possess a pleasing characteristic Cheddar cheese flavour and aroma. May possess a very slight bitter flavour and the following flavours to a slight degree: Feed and acid.

Shall possess a pleasing characteristic Cheddar cheese flavour and aroma with moderate to well-developed degrees of flavour or sharpness. May possess the following flavours to a slight degree: Bitter, feed and acid.

A plug drawn from the cheese shall be reasonably firm, appear reasonably smooth, waxy, fairly close and translucent but may have a few mechanical openings if not large and connecting. May be slightly curdy or not entirely broken down. May possess not more than two sweet holes per plug but shall be free from other gas holes. May possess the following other characteristics to a slight degree: Mealy, short and weak.

A plug drawn from the cheese should be fairly firm, appear smooth, waxy, fairly close and translucent but may have a few mechanical openings. Should be free from curdiness. May possess not more than two sweet holes per plug but shall be free from other gas holes. May possess the following other characteristics to a slight degree: Crumbly, mealy, short, weak and pasty.

Shall have a uniform, bright attractive appearance. May have slight white lines or seams. May be coloured or uncoloured but if coloured, it should be a medium yellow-orange.

Shall have a uniform, bright attractive appearance. May have slight white lines or seams and numerous tiny white it should be a medium specks. May be coloured or uncoloured, but if coloured, it should be a medium yellow-orange.

Bandaged and paraffin dipped. Shall possess a sound, firm rind with the bandage and paraffin coating adhering tightly but may possess very slight mould under bandage and paraffin and the following other characteristics to a slight degree: Soiled surface, surface mould, rough surface, irregular bandaging, lopsided and high edges. Rindless. Same as for current, except very slight mould under wrapper or covering permitted.

Bandaged and paraffin dipped. Shall possess a sound, firm rind with the bandage and paraffin coating adhering tightly but may possess the following characteristics to a slight degree: Soiled surface, rough surface, mould under bandage and paraffin, irregular bandaging, lopsided and high edges; and surface mould to a definite degree. Rindless. Same as for medium.

(b) Body and texture: A plug drawn from the cheese shall be firm, appear smooth, compact, close and should be slightly translucent but may have a few mechanical openings if not large and connecting. May possess not more than two sweet holes per plug but shall be free from other gas holes. May be definitely curdy or partially broken down if more than 3 weeks old. (c) Colour: Shall have a fairly uniform, bright attractive appearance. May have slight white lines or seams or be very slightly wavy. May be coloured or uncoloured but if coloured, it should be a medium yellow-orange. (d) Finish and appearance: Bandaged and paraffin dipped. Shall possess a sound, firm rind with the bandage and paraffin coating adhering tightly, but may possess the following characteristics to a very slight degree: Soiled surface and surface mould; and to a slight degree: Rough surface, irregular bandaging, lopsided and high edges. Rindless. The wrapper or covering shall be practically smooth, properly sealed with adequate overlapping at the seams or by any other satisfactory type of closure. The wrapper or covering shall be neat and adequately and securely envelop the cheese. May be slightly wrinkled but shall be of such character as to fully protect the surface of the cheese and not detract from its initial quality. Shall be free from mould under the wrapper or covering and shall not be huffed but may be slightly lopsided.

Sensory Character of Cheese and its Evaluation

471

Table 5 Specifications for Grade B and Grade C Cheddar cheese (United States Department of Agriculture, Agricultural Marketing Service, Dairy Division)

Detailed specifications for US Grade B Fresh or current

Medium cured

(a) Flavour: Should possess a fairly pleasing Should possess a fairly pleasing charactercharacteristic Cheddar cheese flavour, but istic Cheddar cheese flavour and aroma. may possess very slight onion and the May possess very slight onion and the following flavours to a slight degree: Acid, following flavours to a slight degree: Flat, flat, bitter, fruity, utensil, whey-taint, yeasty, yeasty, malty, old milk, weedy, barny and malty, old milk, weedy, barny and lipase; lipase; the following to a definite degree: feed flavour to a definite degree. Feed, acid, bitter, fruity, utensil, and whey-taint.

(b) Body and texture: A plug drawn from the cheese may possess the following characteristics to a slight degree: Coarse, short, mealy, weak, pasty, crumbly, gassy, slitty and corky; the following to a definite degree: Curdy open, and sweet holes. (c) Colour: May possess the following characteristics to a slight degree:Wavy, acid-cut, mottled, salt spots, dull or faded; and definitely seamy. May be coloured or uncoloured but if coloured, may be slightly unnatural

(d) Finish and appearance: Bandaged and paraffin dipped. Shall possess a reasonably firm sound rind, but may possess very slight mould under bandage and paraffin. The following characteristics to a slight degree: Soiled surface, surface mould, defective coating, checked rind, huffed, weak rind, and sour rind; and to a definite degree: Rough surface, irregular bandaging, lopsided and high edges.

Rindless. The wrapper or covering shall be fairly smooth and properly sealed with adequate overlapping at the seams or by other satisfactory type of closure. The wrapper or covering shall be fairly neat and adequately and securely envelop the cheese. May be definitely wrinkled but shall be of such character as to protect the surface of the cheese and not detract from its initial quality. Shall be free from mould under wrapper or covering but may be slightly huffed and slightly lopsided.

Cured or aged

Should possess a fairly pleasing characteristic Cheddar cheese flavour and aroma, with moderate to well-developed degrees of flavour or sharpness. May possess very slight onion and the following flavours to a slight degree: Flat, yeasty, malty, old milk, weedy, barny, lipase and sulfide; the following to a definite degree: Feed, acid, bitter, fruity, utensil, and whey-taint.

A plug drawn from the cheese may possess the following characteristics to a slight degree: Curdy, coarse, gassy, slitty, and corky; the following to a definite degree: Open, short, mealy, weak, pasty, crumbly, and sweet holes.

A plug drawn from the cheese may possess the following characteristics to a slight degree: Gassy, slitty, the following to a definite degree: Open, sweet holes, short, mealy, weak, pasty and crumbly.

May possess a very slight bleached surface; and the following characteristics to a slight degree: Wavy, acid-cut, mottled, salt spots, dull or faded and definitely seamy. May be coloured or uncoloured but if coloured, may be slightly unnatural.

May possess the following characteristics to a slight degree: Wavy, acid-cut, mottled, salt spots, dull or faded; and definitely seamy. May be coloured or uncoloured but if coloured, may be slightly unnatural.

Bandaged and paraffin dipped. Shall possess a reasonably firm sound rind, but may possess the following characteristics to a slight degree: Surface mould, mould under bandage and paraffin, checked rind, huffed, weak rind, and sour rind; the following to a definite degree: Soiled surface, rough surface, irregular bandaging, lopsided, high edges and defective coating.

Bandaged and paraffin dipped. Shall possess a reasonably firm sound rind, but may possess the following characteristics to a slight degree: Checked rind, huffed, weak rind, and sour rind; the following to a definite degree: Soiled surface, surface mould, mould under bandage and paraffin, rough surface, irregular bandaging, lopsided, high edges and defective coating. Rindless. Same as for medium.

Rindless. Same as for current, except slight mould underwrapper or covering permitted.

continued

472

Sensory Character of Cheese and its Evaluation

Table 5 continued Detailed specifications for US Grade C Fresh or current

Medium cured

Cured or aged

(a) Flavour: May possess the following flavours to a slight degree: Sour, metallic, onion; and to a definite degree: Acid, flat, bitter, fruity, utensil, whey-taint, yeasty, malty, old milk, weedy, barny, and lipase; feed flavour to a pronounced degree.

May possess the following flavours to a slight degree: Onion and sulfide; and to a definite degree: Flat, sour, metallic, sour, metallic, yeasty, malty, old milk, weedy, barny and lipase; and to a pronounced degree: Feed, acid, bitter, fruity, utensil, and whey-taint.

May possess slight onion and the following flavours to a definite degree: Flat, yeasty, malty, old milk, weedy, barny, lipase and sulfide; and to a pronounced degree: Feed, acid, bitter, fruity, utensil and whey-taint.

(b) Body and texture: A plug drawn from the cheese may possess the following characteristics to a definite degree: Curdy, coarse, corky, crumbly, mealy, short, weak, pasty, gassy, slitty, pinny; and to a pronounced degree: Open and sweet holes. The cheese shall be sufficiently compact to permit the drawing of a plug.

A plug drawn from the cheese may be slightly curdy and may possess the following other characteristics to a definite degree: Coarse, corky, gassy, slitty and pinny; and to a pronounced to a pronounced degree: Open, sweet holes, short, weak, pasty, crumbly and mealy. The cheese shall be sufficiently compact to permit the drawing of a plug.

A plug drawn from the cheese may possess the following characteristics to a definite degree: Gassy, slitty, pinny; and to a pronounced degree: Open, sweet holes, short, weak, pasty, crumbly and mealy. The cheese shall be sufficiently compact to permit the drawing of a plug.

(c) Colour: May have a slight bleached surface and possess the following other characteristics to a definite degree: Wavy, acid-cut, mottled, salt spots, dull or faded; and seamy to a pronounced degree. May be coloured or uncoloured but if coloured, may be definitely unnatural. The colour shall not be particularly unattractive.

May possess the following characteristics to a definite degree: Wavy, acid-cut, mottled, salt spots, bleached surface, dull or faded; and seamy to a pronounced degree. May be coloured or uncoloured but if coloured may be definitely unnatural. The colour shall not be particularly unattractive.

Same as for medium.

Bandaged and paraffin dipped. May possess very slight rind rot and the following other characteristics to a slight degree: Cracks in rind; soft spots and wet rind; and to a definite degree: Surface mould, mould under bandage and paraffin, huffed; and to a pronounced degree: Checked rind, weak rind, sour rind and huffed; and to a pronounced degree: Soiled surface, rough surface, defective coating, irregular bandaging, lopsided and high edges. Rindless. Same as for current, except definite mould under the wrapper or covering permitted.

Bandaged and paraffin dipped. May possess the following characteristics to a slight degree: Rind rot, cracks in rind; and to a definite degree: Checked rind, weak rind, sour rind, wet rind, soft spots and huffed; and to a pronounced degree: Rough surface, soiled surface, surface mould, mould under bandage and paraffin, defective coating, irregular bandaging, lopsided and high edges. Rindless. Same as for medium.

(d) Finish and appearance: Bandaged and paraffin dipped. May possess the following characteristics to a slight degree: Cracks in rind, soft spots and wet rind; and mould under bandage and paraffin; and to a definite degree: Soiled surface, surface mould, defective coating, checked rind, weak rind, sour rind, and huffed; and to a pronounced degree: Rough surface, irregular bandaging, lopsided and high edges. Rindless. The wrapper or covering shall be fairly smooth and properly scaled with adequate overlapping at the seams or by other satisfactory type of closure. The wrapper or covering shall adequately and securely envelop the cheese. May be definitely soiled and wrinkled but shall be of such character as to protect the surface of the cheese and not detract from its initial quality. May have slight mould under the wrapper or covering and may be definitely huffed and lopsided.

Sensory Character of Cheese and its Evaluation

473

Table 6 American Dairy Science Association ballot for judging the quality of Cheddar cheese. A score of 10 is awarded if the judge cannot find fault with the flavour of the cheese. A score of 5 is awarded if a judge cannot find fault with the body and texture of the cheese. When scores of 9 or less, or 5 or less, for flavour or body and texture, respectively, are awarded, the cause for deduction of marks should be indicated

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and usually immediately upon opening a packed cheese, cutting a coated cheese or removing a trier plug from a cheese. Flavour judgement is made next, when a sample of cheese is placed in the mouth, chewed and moved around and then expectorated. As for odour, numerous uncharacteristic flavours may be detected in defective cheese, and in addition a cheese that is over-salty or very bitter may be considered defective. Finally, but sometimes simultaneously, body and texture are judged. Defects such as over-hardness, crumbliness, mealy and sticky are judged, most often by working a cheese between the thumb and the fingers. Table 7 presents a list of cheese sensory quality characteristics, which are mostly defects recognised internationally and are described in the IDF standard (IDE 1997). It should be noted that a characteristic considered to be a defect in one cheese type may be very much desired in another (e.g., the acceptable hardness of Parmigiano-Reggiano would be considered a defect in Cheddar), and therefore judges must take this into account, and evaluate based on their experience of each cheese type individually. In addition, it may be that a characteristic found in the same cheese type produced in two different countries may be considered defective in one country, but acceptable in another. This will be related to the experience of the cheese consumer in each country, which can be very different. However, cheeses produced in automated facilities today are much less likely to suffer from significant defects due to improved hygiene practices at

all stages of milk handling and cheesemaking, beginning on the farm. In addition, control over cheesemaking has improved significantly in recent years. Cheese grading or quality scoring provides a rapid and simple way quickly to assess overall sensory quality, but does not adequately take into account so-called 'non-quality' related differences in sensory characteristics that give the cheese of individual producers, or regions of production, a distinctive taste. Traditional 'quality criteria' are changing as product ranges expand (e.g., to include low-fat cheeses); variety of cheeses is much greater, and differentiation is increasingly made by purposely developing distinctive sensory characteristics, such as those now given to cheeses by the use of adjunct cultures. Sensory characteristics that are not traditionally considered defects, but which can also differ from one cheese to another, are now also important in determining eating quality for the discerning consumer. What is a negative attribute to one consumer may be a desirable attribute to another consumer. Also, although the characteristics that expert judges seek are those that their market demands, their assessments do not always coincide with those of consumers. It is now well documented that the consumer and the expert opinions of quality often differ. For example, McBride and Hall (1979) found that consumers' preferences among twelve cheeses, ranging from poor to good quality, were not correlated with their official grade scores. Finally, the current cheese-grading practice does not measure accurately the intensity of a given defect, and

Table 7 Terms used by cheese graders to describe sensory characteristics of cheeses that determine quality with particular emphasis on defects (IDF, 1997; Robinson and Wilbey, 1998) Exterior appearance Rind/surface

Appearance interior: Openings

Appearance interior: Colour

Consistency, body and texture

Flavour, odour and taste

Concave, convex, deformed (bulged), dirty, oblique, soiled, too flat, too high, vaulted (blown) Corroded, cracked, discoloured, dry, fatty, holes, incorrect mould, irregular mould, mould under covering, rotten, rough, smear under covering, smeary, speckled, spots of mould, thick, thin, too little mould, too little smear, too much smear, wet, wrinkled Blown, close, collapsed, cracks, distorted, foreign material, foreign mould, glossy openings, hoop side mould, many holes near the surface, nesty openings, no holes, not typical, pin-holed, spots of putrification, too few, too large, too many, too small, uneven, unevenly mouldy Bleached near the surface, bright, brownish, dirty, discoloured, grey, marbled, mottled, natural, pale/dull, red colour near the surface, speckled, streaky, strong, two-coloured, unevenly coloured, weak, yellow Brittle, chalky, close, coarse, crumbly, curdy, dripping, dry, elastic, firm, flaky, friable, gassy, granular (grainy), greasy, gritty, gummy, hard, harsh, hoop side sift, layered, leathery, long, lumpy, mealy, pasty, runny, rough, short, smeary, smooth, soft, soggy, spongy, springy, squeaky, sticky, stringy, thin (watery), tight, tough, uneven, wet Acid, alcoholic, ammoniacal, aromatic, bitter, bland, burnt, buttery, butyric acid, chemical, clove, cooked, cowy, creamy, ethereal, feedy, fermented, fishy, flat, flowery, foreign flavour, foul, foetid, fruity, garlic, goaty, harsh, malty, metallic, mild, mouldy, musty, musty-flat, nutty, off, oily, oniony, over-ripe, pale, peardrop, putrid, rancid, resinous, rich, ripe, sandy, salty, sharp, soapy, sour, spicy, stale, strong, superfine, sulphide, sweaty, sweet, tangy, tallowy, uncharacteristic, unclean, weedy, yeasty

Sensory Character of Cheese and its Evaluation

therefore further statistical analyses that determine the extent to which cheeses differ, and that mathematically relate composition to defect intensity, are not appropriate. It is important to note that there are still industry situations where grading or quality scoring may be appropriate due to a large number of products that must be assessed in a short period of time. However, these sensory tools were not designed to be quantitative or representative of the entire cheese sensory profile and are not ideal tools for research or marketing. Discrimination tests

Sensory discrimination tests differ from quality scoring tests in that they involve direct comparisons of cheeses to determine whether there is either an overall difference between them or whether they differ for a specific and designated characteristic. The most commonly used discrimination tests include the Paired Comparison (ISO, 1983a), Duo-Trio (ISO, 1991), Triangle (ISO, 1983b) and Ranking tests (I50, 1988). In the Paired Comparison test, two cheeses are presented for comparison with one another and assessors are asked whether they differ; generally, a difference for one specific sensory characteristic is tested. In the Duo-Trio test, assessors are asked which of the two products is the most similar to a third reference product, allowing a common reference to be used again and again. This test has obvious advantages for quality control, although it is not possible to maintain a consistent cheese reference over time. In the Triangle test, assessors are presented with three cheeses and asked to choose which is the most different from the other two. In the Ranking test, four to six cheeses are generally presented for comparison of a single-designated attribute, and the assessor is asked to rank them in order of increasing intensity of that attribute. In best practice, the assessors are forced to make a choice each time for all discrimination tests, thus eliminating response bias. Whether a difference exists or not is determined statistically, based on the number of choices a panel of assessors makes for each cheese in the test, using binomial tables or Chi-squared tests. Therefore, discrimination tests are the most objective and the most sensitive of sensory tests. An additional advantage of these tests is that they do not require well-trained assessors. The only requirement is that all assessors are reasonably sensitive and recognise and understand the designated attribute in a common way. W h e n compared with the traditional quality scoring methods, these discrimination procedures are by far better suited to application to research problems, they follow good sensory evaluation principles and do not encounter problems in scaling and statistical analyses.

475

For this reason, their principles should now be added to quality scoring methods in an attempt to introduce comparability between the scores of one judge and another. It is also common practice to carry out discrimination tests on cheeses to determine whether a difference exists prior to further testing by more costly methods that aim to describe and quantify differences. Descriptive analyses

A majority of scientists who study cheese are interested in understanding the fundamental reasons why a cheese 'tastes' as it does, not just whether the cheese is acceptable, and for this purpose quality control sensory methods are of little value. Descriptive sensory analysis refers to a collection of techniques that seek not only to discriminate between the sensory characteristics of a range of cheeses, but also to determine a quantitative description of all the sensory differences that can be identified. For example, Figs la and lb illustrate quantitative differences in perceived flavour, measured using descriptive analysis, between two hard Swiss cheeses and two Blue cheeses, respectively. All cheeses may be profiled in this way, providing objective and reproducible sensory descriptions of cheeses and providing a basis for determining what characteristics are influenced by changes in cheesemaking practice or composition, and also what characteristics are important for consumer acceptance. The most commonly used descriptive analysis methods for all food types include the Flavour Profile Method (Cairncross and Sj6strom, 1950), Texture Profile Method (Brandt et al., 1963), Quantitative Descriptive Analysis (QDA) T M (Stone et al., 1974), the Spectrum T M method (Meilgaard et al., 1999), Quantitative Flavour Profiling (Stampanoni, 1993a,b), and Free-Choice Profiling (Langron, 1983; Thompson and MacFie, 1983). A review of descriptive sensory analysis, which details advantages, disadvantages and applications of each of the methods referred to above was published recently by Murray et al. (2001). Each descriptive method has three stages to its implementation. The first involves selecting a panel to conduct the sensory evaluations, the second, establishing terminology or a vocabulary, by which to describe a products' sensory characteristics and the third, quantifying these sensory aspects. However, for each method, the process is somewhat different. In the cheese industry, as there is a strong tradition of judging that is linked to extensive knowledge of cheese, then it is a wise approach that seeks to build on this knowledge rather than to reinvent the wheel. If the investment in descriptive sensory testing is for the long term, then the Spectrum T M method, or a similar one, is preferable.

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Using this method, a group of cheese experts develop and define a descriptive language using a series of universal intensity scales upon which assessors score their perceptions (Drake et al., 2001; Drake and Civille, 2003). The sensory panels that will use the method, often at more than one research site, are then extensively trained. When trained, individual assessors must be able to discriminate between cheeses using each attribute in the descriptive language, repeat their assessments and agree with other panel members on the size and the direction of differences in cheese attributes. The advantages of this descriptive analysis technique are that one panel can be trained readily on several cheese types since one intensity scale is used, different types of cheese can be compared directly and panel scaling is less prone to drift with time (Drake and Civille, 2003). In addition, this approach is objective and allows comparison of results between panels, between laboratories, and from one time to another. For example, if one wishes to study the maturation of a cheese over time, then one must ensure that the differences observed in the results between 3, 6 and 9 months are related specifically to changes that occur in the cheese and not to changes in the performance of the sensory panel. If a cheese type is to be evaluated not very often, or the sensory panel available will not specialise in cheese only, or resources are limited, then the QDA T M approach may be preferable. Using this method, the panel of assessors develop and define the language themselves whilst tasting a wide range of the test cheeses (Murray and Delahunty, 2000b). Assessors must agree with other panel members on the meaning of terms in the descriptive vocabulary and repeat their assessments, but are not required to agree on how to use the attribute scales to rate intensity. When this method is used instead of the Spectrum T M method, it is more difficult to compare the results from one study with those from another in absolute terms. Free-Choice Profiling (FCP) is another useful descriptive analysis method (Williams and Langron, 1984). This method allows the use of untrained assessors, or consumers, to profile the sensory characteristics of cheese. Each assessor may use an individual descriptive vocabulary that they have developed themselves, and which they then readily understand, and data are analysed using Generalised Procrustes Analysis (GPA; Arnold and Williams, 1986). Free Choice Profiling has been used to describe Cheddar cheese (Jack et al., 1993; O'Riordan et al., 1998), Parmigiano-Reggiano (Parolari et al., 1994) and ewes' milk cheeses (Barcenas et al., 2003). The advantages of FCP are that accurate discrimination between cheeses in terms of perceived sensory characteristics can be achieved in a very short

477

time and at a relatively little cost, and that discrimination is based on a large selection of informative words that consumers use and with which they are familiar with. The main disadvantage is that it is difficult to correlate perceived intensity of sensory characteristics obtained in this way, as they are too numerous and imprecise, and there is no consensus vocabulary. To obtain improved accuracy, sensory panels used for descriptive analyses generally comprise of 10-12 assessors instead of a smaller number of experts (with the exception of FCP where 15-20 assessors are needed). These assessors are screened for sensory acuity and relative interest (Stone and Sidel, 1993). A panel or group of individuals is used as factors such as age, saliva flow and onset of fatigue vary between assessors. Assessors also vary in sensitivity to particular stimuli, and it is highly probable that they also vary in their concentration-response functions (Lawless et al., 1994; Williams, 1994). In addition, temporary illness or psychological bias can cause day-to-day changes in sensory ratings. The key point of objective descriptive analysis is that it should be reproducible and independent of consumer preferences. Unlike traditional quality methods that use scorecards, there is no judgment of 'good' or 'bad' as this is not the purpose of the evaluation. The trained sensory panel operates as an instrument and generates quantitative data analogous to instrumental data. As with any instrument, replication is required. Two guidelines have been published dealing with cheese texture (Lavanchy et al., 1994) and the aroma and flavour of cheese (B~rodier et al., 1997a). These guidelines are very valuable as they define descriptive vocabularies, and then detail a procedure for evaluation of each characteristic, including the use of universal scales that are standardised at a number of points with common food references. In addition, they provide translations of many descriptive characteristics of cheese in Spanish, French, Italian, English and German. However, it is important to note that sensory lexicons or languages are not finite and will continue to evolve with time, usage and application. Time-intensity sensory analyses The sensory methods discussed above do not account for the dynamics of flavour release from cheeses that occurs during their consumption. Nor do they account adequately for changes to cheese texture, which occur progressively during mastication and breakdown of a cheese in the mouth. When using conventional sensory procedures, particularly descriptive analyses, assessors 'time-average' their responses to arrive at a single intensity value. This looses much useful information such as

478

Sensory Character of Cheese and its Evaluation

rate of onset of stimulation, time and duration of maximum intensity, rate of decay of perceived intensity, time of extinction and total duration of the entire process (Lee and Pangborn, 1986). To determine most details about sensory characteristics, changes in sensory character that occur during cheese consumption (which can take up to 30 s for a 'bite-sized' piece) can be measured using time-intensity methodology (Lee and Pangborn, 1986), or in the case of texture, using progressive profiling (Jack et al., 1994). Time-intensity methods are useful for the study of new cheese types, such as low-fat cheeses, as the reduction in fat content not only influences sensory character development, but also the breakdown of the cheese in the mouth during consumption and the rate of release of compounds that contribute to flavour. For example, in a study of Cheddar cheese flavour, the time taken to reach maximum intensity for 'sharpness', 'bitterness' and 'astringency' was consistently longer in reduced-fat than in full-fat Cheddar and, more importantly, the rate of flavour release was greater (Shamil et al., 1991/92). Temporal differences in perception indicate an altered flavour balance, caused by reducing the fat content of the cheese, which may be important in consumer acceptability. Delahunty et al. (1996a) showed that a 'fruity' note, which might be considered an off-flavour (Aston et al., 1985; Urbach, 1993), became a dominant flavour characteristic sooner during consumption and at a much greater intensity in a low-fat Cheddar-type cheese than in the full-fat equivalent. Delahunty et al. (1996b) also demonstrated that improved relationships between volatile composition and perceived sensory characteristics could be achieved by relating time-intensity sensory data with dynamic volatile compound release data. Jack et al. (1994) found that the texture of Cheddar cheese was perceived to be relatively coarse and crumbly earlier in the chewing sequence, but became increasingly smooth and creamy as chewing progressed. In addition, other more subtle or specific cheese-dependent changes occurred as breakdown in the mouth progressed. It was hypothesised that knowledge of these dynamic changes in texture character is important for understanding consumer acceptability. Consumer acceptability testing

Trained sensory panels should not be asked to express a preference as their expert knowledge will introduce bias. To determine the eating quality of cheese, a naive consumer panel or subjective assessors are used. Ideally, these assessors will be regular consumers of the product type under test or represent the target market for the product. Such consumers bring their subjective experience to this test, for although their preferences

will be based on the sensory characteristics tested, they will be referring to past eating experience. In addition, when one considers that the target market may be children, elderly consumers, consumers in another country or consumers from a culture virtually unknown to the producer, then it becomes clear that the internal expertise in a company or organisation cannot hope to predict acceptability adequately. Consumer acceptability testing makes use of rating scales that measure relative dislikes and likes (e.g., the ninepoint hedonic scale (Peryam and Girardot, 1952)), discrimination tests based on preference (e.g., paired preference, ranked preference) or just right scales that ask a consumer how they feel about the designated sensory characteristic. It is recommended that a minimum of 50-60 targeted consumers be used for consume> sensory testing, and a greater number than this if one expects segmentation of preferences (MacFie and Hedderly, 1993). One of the biggest challenges in consumer research is the clarification of consumer language. Consumers may use terms that are ambiguous, have multiple meanings, are associated with 'good' or 'bad' or are combinations of several terms. Integrated terms, such as 'creamy', are often used by consumers to represent a combination of positive attributes. Determining exactly what attribute or attributes 'creamy' refers to (flavour or texture or mouthfeel) have been the subject of many studies relating consumer and trained sensory panels (Mela, 1988; Elmore et al., 1999; Bom Frost et al., 2001). Dacremont and Vickers (1994a,b), who used concept matching to clarify consumer perception of Cheddar cheese flavour, found that the concept of Cheddar cheese flavour is a consumer concept and probably varies widely among consumers, as does Cheddar cheese flavour itself. However, the number of consumers questioned was small and further studies with larger consumer groups, and with demographic information, including types (brand, age) of Cheddar cheese normally consumed, would provide additional clarification.

Influence of Cheesemaking Variables on Sensory Character During the past ten years or so, there have been numerous reports of the application of descriptive sensory analysis to determine accurately the influence of cheesemaking variables, e.g., maturation time and temperature, starter culture or use of adjunct cultures, on the sensory characteristics of cheese (Table 3). Studies of Cheddar cheese maturation have found that, overall, the intensity of odour, flavour and aftertaste is determined by the length (Piggott and Mowat,

Sensory Character of Cheese and its Evaluation

1991; Muir and Hunter, 1992a) and the temperature of maturation (Hannon et al., 2003). However, flavours such as milky/buttery and creamy decrease in intensity, while flavours such as sour, bitter, rancid and pungent increase in intensity (Piggott and Mowat, 1991; Muir and Hunter, 1992a; Hannon et al., 2003). Some textural changes, e.g., firmness, are controlled by the cheesemaking procedure and cheese composition, whereas mouth-coating character is related to maturation time (Piggott and Mowat, 1991; Muir and Hunter, 1992a). Hort and Le Grys (2001), who also studied Cheddar, found that springiness decreased, and crumbliness and creaminess increased as maturation progressed. Banks et al. (1993) and Fenelon et al. (2000) used descriptive analysis to determine the sensory properties of low-fat Cheddar cheese, and to compare these with the sensory properties of full-fat Cheddars. Fenelon et al. (2000) found that there were some differences in flavour characteristics related to fat content that were present regardless of the age of cheese. Fullfat cheeses were consistently more buttery, creamy and caramel-like. Adhikari et al. (2003) found that low-fat and full-fat Swiss cheeses, and low-fat Cheddar cheeses were dry and crumbly. Factory and farmhouse Cheddars have also been compared using descriptive sensory analysis (Muir etal., 1997a; Murray and Delahunty, 2000c); farmhouse cheeses were found to have a greater diversity in sensory characteristics. In addition, cheeses produced from pasteurised milk were found to be clearly different from those produced from unpasteurised milk, with the unpasteurised milk cheeses being more diverse in sensory character and more intensely flavoured (Grappin and Beuvier, 1997; Muir et al., 1997a; Murray and Delahunty, 2000c). Numerous studies have used descriptive sensory analysis to address the role of specific adjunct cultures or starter culture enzyme systems in Cheddar cheese flavour (Drake et al., 1996, 1997; Muir et al., 1996; Delahunty and Murray, 1997; Lynch et al., 1999; Banks et al., 2001; Broadbent et al., 2002). Muir et al. ( 1 9 9 6 ) demonstrated that starter culture type and adjunct determined the sensory character of cheese. However, they also found direct and interactive effects of composition. More recently, O'Riordan and Delahunty (2003a,b) found that starter culture type led to consistent differences in sensory characteristics between Cheddar cheeses, but that composition led to significant variation within batches of cheese made using the same starter culture. Delahunty and Murray (1997) also demonstrated differences between Cheddar cheeses based on starter culture type, although these cheeses were awarded the same grade score (Fig. 2). Descriptive sensory analysis has been used to determine the impact of yeast extract and milk standardisation

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PC1 (43%)

Figure 2 Two-dimensional representation of the result of Principal Components Analysis of descriptive analysis sensory data for Cheddar cheese produced using three different starter cultures (coded $1-$3). Grade scores for flavour, awarded by an expert cheese grader, are also illustrated close to each cheese code (range 37-39).

with milk protein concentrate on reduced-fat Cheddar cheese flavour (Shakeel-ur-Rehman et al., 2003a,b,c) and of smoking parameters on cheese flavour (Shakeelur-Rehman et al., 2003d). There have been many studies of cheese types other than Cheddar, and to discuss them all would be impossible within the scope of this chapter. Of most interest are studies of Comte cheese using a flavour-descriptive vocabulary developed by B~rodier et al. (1997b; published in French). This lexicon has been used to identify naturally existing cheese geo-regions within France (Monnet et al., 2000). In addition, Virgili et al. (1994) used descriptive analysis to study the sensory-chemical relationships in Parmigiano-Reggiano cheese. Descriptive analysis of cheese texture has been conducted recently on a variety of cheeses, on cheeses of different fat contents and on fat replacers (Drake and Swanson, 1996; Drake et al., 1999a; Lobato-Calleros et al., 2001; Madsen and Ardo, 2001; Gwartney et al., 2002). In these studies, descriptive sensory analysis was used to differentiate cheeses and/or the impact of various treatments. A sensory texture language, like a cheese flavour language, is also not necessarily finite. The language will continue to be refined, particularly as additional cheeses are studied or as additional instrumental studies are conducted. The texture languages used by Drake et al.

480 Sensory Character of Cheese and its Evaluation (1999a) and Gwartney et al. (2002) were merged into one complete language by Brown et al. (2003).

Towards a Universal Cheese-Sensory Language As mentioned previously, some of the key advantages of using descriptive sensory vocabularies with definitions and references are the ability to communicate accurately results between multiple research groups or to reproduce research results at different sites. Hirst et al. (1994) compared the evaluation of cheese between trained British and Norwegian panels using independently developed sensory languages. Cross-cultural differences were attributed to the observed discrepancies in term usage and sample differentiation. More recently, ring trials at seven sites across the European Union were conducted and a core sensory language for evaluation was developed (Hunter and McEwan, 1998; Nielsen and Zannoni, 1998). While similar patterns of differentiation among samples by panels that use different languages are expected (particularly if the vocabularies are comprehensive and the panellists highly trained), standardised language with definitions and references improves communication, cross panel validation and subsequent application of descriptive analysis results to instrumental or consumer data. Further, other sources of variation potentially exist in comparing panel results at different sites within the same country using the same language. Drake et al. (2002) reported on the performance of three descriptive panels trained at different sites by different panel leaders on a previously developed and standardised cheese descriptive language (Drake et al., 2001). Panels were able to communicate accurately attribute differences between cheeses. However, differences were observed between these panels in scale usage and attribute recognition. These differences were attributed to the differences in panel leadership and the duration of panellist training. In a similar study, Martin et al. (2000) compared odour profile results of two panels. Language, scale and method of presentation were standardised. Results obtained from the two panels were similar. However, differences between attribute intensities were reported and were attributed to differences in the experience and/or perception of individual panellists. As with the conclusions of Drake etal. (2002), strong panel-leader interaction was recommended as a means of rectifying these differences, along with regular feedbacks between the two panels. As referred to previously, Tables 1, 2 and 3 present terms used for descriptive sensory analysis by different research groups for a wide variety of cheeses. In many cases similar terms have been used to describe dominant

characteristics of different cheese types, suggesting that it could be possible to develop and standardise a terminology that can be used universally and for all cheese types. The will to achieve this objective is much needed.

Relating S e n s o r y Characteristics to C o n s u m e r Preferences Preference mapping is a generic term given to a collection of techniques, which have emerged in recent years to quantify, analyse and interpret consumer preferences for products. A premise can be made that the preferences of a group of consumers of sufficient size (60 or more) will discriminate between comparable products based on intrinsic sensory differences, and that the degree and direction of discrimination will reflect the number and the intensity of sensory differences that can be perceived. Therefore, by simply quantifying and analysing preference, or acceptance for the range, a preference map reflecting sensory differences can be drawn. The preferences of individual consumers can be represented as a map loading, and areas of minimum and maximum preference can be identified. In addition, segmentation techniques, when used in tandem, can illustrate opportunities for a selection of optimised products within the same range (or sensory space). Analysis of consumer preference data in this way is referred to as internal preference mapping (McEwan, 1995; Schlich, 1995). When consumer preference evaluation of a set of cheeses is followed by the application of descriptive analysis to the same set of cheeses, this allows multivariate statistical analysis, e.g., using Partial Least Squares Regression (PLSR; Martens and Martens, 1986), and relation of descriptive properties that describe exactly what attributes are perceived and at what levels with the extent and direction of consumer preferences. This additional analysis facilitates interpretation of the internal preference map, and is referred to as external preference mapping (McEwan, 1995; Schlich, 1995). These techniques provide a powerful research tool for market analysis and new product development. One can extend the preference map by seeking technical extensions, or relationships between preferences, sensory characteristics and physical and chemical properties of products. One can also extend the preference map by seeking behavioural extensions, or by determining characteristics of the consumers and how they have developed their preferences and make their choice decisions. Preference mapping has been conducted with many products, including cheese (McEwan etal., 1989; Lawlor and Delahunty, 2000; Murray and Delahunty, 2000a,c; B~ircenas et al., 2001). Recently, Young et al.

Sensory Character of Cheese and its Evaluation 481 (2003) conducted preference mapping of Cheddar cheeses using consumers at two different locations (Oregon and North Carolina, USA). Seven Cheddar cheeses with distinct descriptive sensory properties were selected. Six distinct consumer clusters were identified, indicating a wide variability in consumer preferences even among one cheese type. Analysis of the consumer concept of 'aged cheese flavour' and 'young cheese flavour' indicated that consumers could differentiate between young and aged Cheddar cheeses and that these concepts were consistent with descriptive panel language. However, the consumer concept of 'Cheddar flavour' varied widely and was not pinpointed to specific descriptive cheese flavour terms. Lawlor and Delahunty (2000) conducted preference mapping with a diverse range of cheese types, and also found wide variability in consumer preferences. Although a Blue Shropshire cheese, described as coloured, mouldy and crumbly, was the least liked overall (162 consumers), it was preferred by two of seven segments of the consumer sample, representing 50% of the total questioned. On the other hand, a Gruyere cheese, described as fruity, sweet and firm, was preferred overall, but was the first choice of only one segment with 10 consumers.

Relating Sensory Perception to Chemical Components and Instrumental Measurements Relating defined sensory flavour and/or texture to specific instrumental tests or chemical compounds is an important and expanding area of research. Cheese flavour chemistry and texture analyses are addressed in detail in 'Cheese Flavour: Instrumental Techniques' and 'Rheology and Texture of Cheese' of Volume 1, but sensory characteristics of cheeses cannot be addressed without brief attention to this subject. Relating sensory perception to instrumental measurements is important because in certain cases an instrumental test would be more cost-effective and/or convenient than sensory testing. However, more importantly, establishment of key relationships between sensory perception and flavour chemistry or rheology provides the potential to link cheese flavour or texture to the technology of cheese production; this is a key issue in providing a consistent and high-quality product to the discerning consumer. Relating sensory language and chemical volatile compounds represents a challenge for several reasons. The relative concentration of a compound in a cheese is not necessarily a measure of its sensory impact due to different sensory thresholds and the effects of the food matrix on retention and release. The sensitivity and selectivity of the extraction technique must also be taken into account (Delahunty and Piggott, 1995).

Finally, only a small percentage of the volatile components in a food are odour-active (Friedrich and Acree, 1998; see also 'Cheese Flavour: Instrumental Techniques', Volume 1). Establishing these relationships is time-consuming and tedious. To use flavour as an example, extensive and relevant instrumental volatile analysis must be conducted, followed by gas chromatography-olfactometry (GC-O) and quantitative analysis to pinpoint volatiles of interest. On the sensory side, descriptive analysis with a defined and anchored language is required. Sensory threshold testing to confirm that key volatile compounds are above detection thresholds must be conducted, followed by descriptive sensory analysis of compounds in model systems across the concentration range found in the cheese to confirm the sensory response (Drake and Civille, 2003). It should also be noted that the perception of the cheese flavour is an integrated response to numerous mixed stimuli, including volatile compounds, nonvolatile compounds and structural properties. The perception of this stimulation is multi-modal, but simultaneous, and therefore very complex. Panelists tasted water-soluble extracts of Comte cheese to identify fractions, which had particular tastes, in an attempt to clarify the effect of peptides and amino acids on flavour (Salles etal., 1995). Preininger et al. (1996) used an unripened cheese matrix to evaluate both volatile and non-volatile flavour components of two Swiss cheese samples. A similar study was conducted on Emmental cheese and reduced-fat Cheddar cheeses (Rychlik etal., 1997; Suriyaphan etal., 1999). Suriyaphan etal. (2001) identified key chemical volatile components of cowy/barny and earth/bell pepper sensory perceived flavours in selected aged British Farmhouse cheeses. In this study, sensory properties were identified by descriptive sensory analysis, aroma volatiles were quantified by gas chromatography-mass spectrometry (GC-MS) and aroma properties described by GC-O. Suspected key volatiles were selected from GC-O data based on aroma properties and flavour dilution values. The selected aroma components were subsequently incorporated into mild (bland) cheese across the concentration range encountered in the Farmhouse cheeses and evaluated by descriptive analysis. Studies such as these provide convincing evidence of the contribution of particular compounds to flavour. Model systems have not as yet provided insights into the role of compound mixtures and the role of compounds at sub-threshold levels. These are complex issues and will require extensive future research. An alternative approach to determining the influence of composition on sensory character is to use multivariate statistical techniques, such as PLS, to determine

482

Sensory Character of Cheese and its Evaluation

relationships between compositional data and quantitative descriptive sensory data. This technique has the advantage of enabling comparison of all mathematically possible combinations of compositional variables with perceived intensity of one or more sensory characteristics, following theoretically the principle of the component balance theory (Mulder, 1952). The validity and value of relationships determined in this way will depend on the amount and type of compositional data collected, and the accuracy of both the compositional and the sensory data. Lawlor et al. (2001, 2002, 2003) determined predictive models using this technique for numerous flavour and texture attributes described in a wide variety of cheese types. Many studies have also been conducted to explore the relationships between sensory properties, compositional measurements and instrumental measurements of cheese texture (Wium et al., 1997; Bachmann et al., 1999; Drake etal., 1999b; Antoniou etal., 2000; Benedito etal., 2000; Truong etal., 2002) and to devise instrumental methods to assess more accurately or predict sensory properties of cheese (Sorensen and Jepsen, 1998; Breuil and Meullenet, 2001; Meullenet and Finney, 2002). Lawlor et al. (2001, 2002, 2003), using PLS, determined relationships between gross composition and perceived texture for a wide variety of cheeses, and found a number of consistent relationships. In particular, it was found that firmness was positively correlated with protein and mineral salt content, and negatively correlated with moisture and pH. Both hand and mouth terms can be used for sensory analysis of cheese texture (Drake et al., 1999c). In general, empirical texture tests and large-strain tests (compression) have been shown to correlate well with sensory bite terms (firmness, elasticity) although the correlation varies with cheese type, instrumental test and specific sensory term and definition. More recently, Brown et al. (2003) demonstrated specific knowledge gaps in relating sensory chewdown terms to rheological tests. Sensory rigidity and resiliency terms were correlated with rheological tests. However, chewdown terms such as 'degree of breakdown', 'cohesiveness', 'adhesiveness', 'smoothness of mass' and 'smoothness of mouth coating' were not related to instrumental tests. Additional work is needed to investigate the role that fundamental rheological tests can play in differentiating and relating to these important sensory texture parameters in cheese.

Conclusions The sensory characteristics of cheese determine the eating quality of cheese and consumer acceptability. The appearance, flavour and texture of cheese are extremely complex, not simply due to the very wide

diversity of cheese types that are produced, but also the many stages that any cheese goes through during its production and ripening. The complex composition and structure of cheese stimulate each of the human sensory modalities at approximately the same time, resulting in an integrated perception that a consumer responds to during and after cheese consumption. The dairy industry, including cheese production and marketing, has relied on outdated grading and judging methods for quality control and product development for many years. While these methods still have use, objective descriptive analysis techniques are increasingly being applied in cheese quality research in parallel with innovative studies of cheesemaking, cheese composition and consumer acceptability of cheese. Advances in the application of objective sensory science techniques have improved understanding of the relationships between these factors and the sensory attributes of cheese. However, direct comparison of research findings between different laboratories working with the same cheese type, and between studies on different types of cheese, will not be possible until such time as a universal language to describe cheese sensory character is defined and standardised.

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Muir, D., Banks, J.M. and Hunter, E.A. (1995a). Sensory properties of cheese, in, Proceedings of the 4th Cheese Symposium, T.M. Cogan, P.E Fox and R.P. Ross, eds, National Dairy Products Research Centre, Fermoy. pp. 25-31. Muir, D.D., Hunter, E.A., Banks, J.M. and Horne, D.S. (1995b). Sensory properties of hard cheese: identification of key attributes. Int. Dairy J. 5, 157-177. Muir, D.D., Hunter, E.A., Banks, J.M. and Home, D.S. (1995c). Sensory properties of hard cheese: changes during maturation. Food Res. Int. 28, 561-568. Muir, D.D., Hunter, E.A. and Watson, M. (1995d). Aroma of cheese. 1: sensory characterisation, Milchwissenschaft 50, 499-503. Muir, D.D., Banks, J.M. and Hunter, E.A. (1996). Sensory properties of Cheddar cheese: effect of starter type and adjunct. Int. Dairy J. 6,407-423. Muir, D.D., Banks, J.M. and Hunter, E.A. (1997a). A comparison of flavour and texture of Cheddar cheese of factory or farmhouse origin. Int. Dairy J. 7,479-485. Muir, D.D., Hunter, E.A., Watson, M. (1997b). Aroma of cheese. 2. Contribution of aroma to overall flavour. Milchwissenschaft 52, 85-88. Mulder, H. (1952). Taste and flavour forming substances in cheeses. Neth. Milk DairyJ. 6, 157-168. Murray, J.M. and Delahunty, C.M. (2000a). Mapping preference for the sensory and packaging attributes of Cheddar cheese. Food Qual. Pref. 11,419-435. Murray, J.M. and Delahunty, C.M. (2000b). Selection of standards to reference terms in a Cheddar cheese flavour language. J. Sensory Studies 15, 179-199. Murray, J.M. and Delahunty, C.M. (2000c). Consumer preference for Irish farmhouse and factory cheeses. Ir. J. Agric. Food Res. 39,433-449. Murray, J.M., Delahunty, C.M. and Baxter, I. (2001). Descriptive sensory analysis: a review. Food Res. Int. 34, 461-471. Nielsen, R.G. and Zannoni, M. (1998). Progress in developing an international protocol for sensory profiling of hard cheese. Int. J. Dairy Technol. 31, 57-64. Noble, A.C. (1996). Taste-aroma interactions. Trends Food Sci. Technol. 7,439-444. Ordohez, A.I., Ibafmz, EC., Torre, P. and Barcina, Y. (1998). Application of multivariate analysis to sensory characterization of ewes' milk cheese. J. Sens. Stud. 13, 45-55. O'Riordan, P.J. and Delahunty, C.M. (2003a). Characterisation of commercial Cheddar cheese flavour. 1: traditional and electronic nose approach to quality assessment and market classification. Int. Dairy J. 13,355-370. O'Riordan, P.J. and Delahunty, C.M. (2003b). Characterisation of commercial Cheddar cheese flavour. 2: study of Cheddar cheese discrimination by composition, volatile compounds and descriptive flavour assessment. Int. Dairy J. 13,371-389. O'Riordan, P.J., Delahunty, C.M., Sheehan, E.M. and Morrissey, P.A. (1998). Comparisons of volatile compounds released during consumption of a complex food by different consumers with expressions of perceived flavor determined by free-choice profiling. J. Sens. Stud. 13, 435-459.

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Papademas, P. and Robinson, R.K. (2001). The sensory characteristics of different types of halloumi cheese as perceived by tasters of different ages. Int. J. Dairy Technol. 54, 94-99. Parolari, G., Virgili, R., Panari, G. and Zannoni, M. (1994). Development of a vocabulary of terms for sensory evaluation of Parmigiano-Reggiano cheese by flee-choice profiling. Ital. J. Food Sci. 3, 317-324. Peryam, D.R. and Girardot, N.E (1952). Advanced taste test method. Food Eng. 24, 58-61. Piggott, J.R. (1988). Sensory Analysis of Foods, 2nd edn, Elsevier Applied Science, Barking, Essex. Piggott, J.R. and Mowat, R.G. (1991). Sensory aspects of maturation of Cheddar cheese by descriptive analysis. J. Sens. Stud. 6, 49-62. Preininger, M., Warmke, R. and Grosch, W. (1996). Identification of the character impact flavour compounds of Swiss cheese by sensory studies of models. Z. Lebensm. Unters. Forsch. 202, 30-34. Roberts, A.K. and u Z.M. (1994). A comparison of trained and untrained judges evaluation of sensory attribute intensities and liking of Cheddar cheeses. J. Sens. Stud. 9, 1-20. Robinson, R.K. and Wilbey, R.A. (1998). Cheesemaking Practice, R Scott 3rd edn., Aspen Publications, Inc., Gaithesburg, MD. Rychlik, M., Warmke, R. and Grosch, W. (1997). Ripening of Emmental cheese wrapped in foil with and without addition of Lactobacillus casei ssp. casei. III. Analysis of character impact flavour compounds. Lebensm. Wiss. Technol. 30,471-478. Salles, C., Septier, C., Roudot-Algaron, E, Guillot, A. and t~tievant, P.X. (1995). Sensory and chemical analysis of fractions obtained by gel permeation of water soluble Comte cheese extracts. J. Agric. Food Chem. 43, 1659-1668. Schlich, P. (1995). Preference mapping: relating consumer preferences to sensory or instrumental measurements, in, Bioflavour 95, P. Etievant and P. Schreier, eds, INRA, Paris. pp. 135-150. Shakeel-ur-Rehman, Farkye, N. and Drake, M.A. (2003a). Effects of standardization of whole milk with dry milk protein concentrate on the yield and ripening of reduced-fat Cheddar cheese. J. Dairy Sci. 86, 1608-1615. Shakeel-ur-Rehman, Farkye, N. and Drake, M.A. (2003b). Reduced-fat Cheddar cheese from a mixture of cream and liquid milk protein concentrate. Int. J. Dairy Technol. 56, 94-98. Shakeel-ur-Rehman, Farkye, N., Vedamuthu, E. and Drake, M.A. (2003c). A preliminary study on the effect of adding yeast extract to cheese curd on proteolysis and flavor development of reduced fat Cheddar. J. Dairy Res. 70, 99-103. Shakeel-ur-Rehman, Farkye, N. and Drake, M.A. (2003d). The ripening of smoked Cheddar cheese. J. Dairy Sci. 86, 1910-1917. Shamil, S., Wyeth, L.J. and Kilcast, D. (1991/92). Flavour release and perception in reduced-fat foods. Food Qual. Pref. 3, 51-60.

Sorensen, L.K. and Jepsen, R. (1998). Assessment of sensory properties of cheese by near-infrared spectroscopy. Int. DairyJ. 8,863-871. Stampanoni, C.R. (1993a). Quantitative flavour profiling: an effective tool in flavour perception. Food Marketing Technol. February, 4-8. Stampanoni, C.R. (1993b). The quantitative profiling technique. Perfum. Flavour. 18, 19-24. Stampanoni, C.R. (1994). The use of standardized flavor languages and quantitative flavor profiling technique for flavoured dairy products, J. Sens. Stud. 9, 383-400. Stevenson, R.J., Prescott, J. and Boakes, R. (1999). Confusing tastes and smells: how odours can influence the perception of sweet and sour tastes. Chem. Senses 24, 627-635. Stone, S. and Sidel, J.L. (1993). Sensory Evaluation Practices, 2nd edn, Academic Press, Inc., London. Stone, H., Sidel, J., Oliver, S., Woolsey, A. and Singleton, R.C. (1974). Sensory evaluation by quantitative descriptive analysis. Food Technol. 28, 24-34. Suriyaphan, O., Drake, M.A. and Cadwallader, K.R. (1999). Identification of volatile off-flavors in reduced-fat Cheddar cheeses containing lecithin. Lebensm. Wiss. Technol. 32,250-254. Suriyaphan, O., Drake, M.A., Chen, X.Q. and Cadwallader, K.R. (2001). Characteristic aroma components of British Farmhouse Cheddar cheese. J. Agric. Food Chem. 49, 1382-1387. Thompson, D.M.H. and MacFie, H.J.H. (1983). Is there an alternative to descriptive sensory assessment?, in, Sensory Quality in Food and Beverages: Definition, Measurement and Control, A.A. Williams and R.K. Atkin, eds., Ellis Horwood Ltd, Chichester. pp. 96-107. Truong, V.D., Daubert, C.R., Drake, M.A. and Baxter, S.R. (2002). Vane rheometry for textural characterization of Cheddar cheese: correlation with other instrumental and sensory measurements. Lebensm. Wiss. Technol. 35, 305-314. Urbach, G. (1993). Relations between cheese flavour and chemical composition. Int. Dairy J. 3,389-422. Virgili, R., Parolari, G., Bolzoni, L., Barbieri, G., Mangia, A., Careri, M., Spagnoli, S., Panari, G. and Zannoni, M. (1994). Sensory chemical relationships in ParmigianoReggiano cheese. Lebensm. Wiss. Technol. 27,491-495. Warmke, R., Belitz, H.D. and Grosch, W. (1996). Evaluation of taste compounds of Swiss cheese (Emmentaler). Z. Lebensm. Unters. Forsch. 203,230-235. Weel, K.G.C., Boelrijk, A.E.M., Alting, A.C., van Mil, P.J.J.M., Burger, J.J., Gruppen, H., Voragen, A.G.J. and Smit, G. (2002). Flavor release and perception of flavored whey protein gels: perception is determined by texture rather than by release. J. Agric. Food Chem. 50, 5149-5155. Wendin, K., Langton, M., Caous, L. and Hall, G. (2000). Dynamic analyses of sensory and microstructural properties of cream cheese, Food Chem. 71,363-378. Williams, A.A. (1994). Flavour q u a l i t y - understanding the relationship between sensory responses and

Sensory Character of Cheese and its Evaluation

chemical stimuli. What are we trying to do? The data, approaches and problems. Food Qual. Pref. 5, 3-16. Williams, A.A. and Langron, S.P. (1984). Use of free-choice profiling for evaluation of commercial ports. J. Sci. Food Agric. 35,558-568.

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Wium, H., Gross, M. and Qvist, K.B. (1997). Uniaxial compression of UF-Feta cheese related to sensory texture analysis. J. Text. Stud. 28,455-476. Young, N., Drake, M.A., Lopetcharat, K. and McDaniel, M. (2004). Preference mapping of Cheddar cheeses. J. Dairy Sci. 87, 11-i9.

Cheese Flavour: Instrumental Techniques J.-L. Le Qu~r~, Institut National de la Recherche Agronomique (INRA), Unite Mixte de Recherche sur les Ar6mes (UMRA), Dijon, France

Introduction The sensory properties of food are important determinants in the choice of foodstuffs by the consumer, and flavour plays a prominent role in this context. Flavour may be defined as the combination of taste and odour, sensations of pain, heat and cold (chemesthesis or trigeminal sensitivity), and tactile sensation. Sensory analysis is clearly the most valid means of measuring flavour characteristics. Applied to cheese flavour, sensory evaluation is a prominent descriptive tool which is used widely in dairy science and industry (Issanchou et al., 1997; see also 'Sensory Character of Cheese and its Evaluation', Volume 1). However, determining flavour also means analysing volatile compounds that are sensed in the nose at the olfactory receptors either via the orthonasal (odour) or retronasal (aroma) routes when foods are eaten, non-volatile compounds sensed on the tongue (taste), and compounds perceived as mouthfeel and texture. Instrumental analyses of flavour have been used primarily to analyse volatile components. The main reason for this is the major importance of aroma in the overall flavour of a food, as is easily demonstrated by the difficulties encountered by subjects attempting to identify a particular flavour if the air flow through the nose is prevented, and the fact that volatile components are more amenable to conventional instrumental analysis than non-volatile compounds. Therefore, since the early studies published in the 1960s and the 1970s (Dumont and Adda, 1972, and references cited therein), instrumental methods have concentrated on identification of aroma compounds (Mariaca and Bosset, 1997). Only recently, some significant efforts have been made to develop instrumental procedures to characterise non-volatile components in cheese which are responsible for cheese taste (Salles et al., 1995a; Salles and Le Qu~r~, 1998; Engel et al., 2000a,b; Le Quere and Salles, 2001). Instrumental analysis of aroma volatiles has been the subject of important specialised treatises (for the most recent literature on the subject, see Ho and Manley, 1993; Marsili, 1997; Mussinan and Morello, 1998; Stephan etal., 2000; van Ruth, 2001a; Reineccius, 2002, and specifically for instrumental analysis of

volatiles in milk and dairy products see Delahunty and Piggott, 1995; Mariaca and Bosset, 1997). Therefore, the part of this chapter that will be devoted to the analysis of cheese volatiles will focus on particular techniques adapted to the particular characteristics of cheese. Cheese flavour components result from the principal biochemical degradation pathways: glycolysis, lipolysis and proteolysis (see 'Biochemistry of Cheese Ripening: Introduction and Overview', 'Metabolism of Residual Lactose and of Lactate and Citrate', 'Lipolysis and Catabolism of Fatty Acids in Cheese', 'Proteolysis in Cheese during Ripening' and 'Catabolism of Amino Acids in Cheese During Ripening', Volume 1). The aroma compounds produced are mainly hydrophobic, or lipophilic, and consequently they tend to concentrate in the cheese fat according to their water/fat partition coefficient. Instrumental analysis of cheese volatiles must, therefore, consider, as a first step, an extraction method suitable for separating these volatiles from the cheese fat matrix. However, no single method yields a 'true' picture of a food aroma (Reineccius, 2002), and isolation and analysis of aroma remain challenging (Teranishi, 1998). Moreover, not only may the extraction step lead to artefacts, but the total volatile content in most cases is very difficult to relate to the flavour profile determined by a panel in sensory evaluation. Therefore, it appears much more efficient to concentrate efforts on the identification of those compounds that are really relevant to flavour. As no universally suitable extraction method exists, it appears essential to choose a method that yields an extract representative of the sensory properties of the food (Abbott et al., 1993; Eti~vant et al., 1994; Eti~vant and Langlois, 1998). Once this extraction method has been chosen, the next steps involve various forms of gas chromatography among which gas chromatography-olfactometry (GC-O) plays a prominent role in determining the key volatile compounds that contribute significantly to the flavour of the food (Leland et al., 2001), and gas chromatography-mass spectrometry (GC-MS), which is essential for the identification of those key odorants. Water-soluble extracts (WSE) from cheese have strong flavours (Biede and Hammond, 1979; McGugan

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Cheese Flavour: InstrumentaITechniques

et al., 1979; Aston and Creamer, 1986). Such extracts contain some volatile compounds (Le Quere et al., 1996; Engels et al., 1997; Le Quere and Salles, 2001), partly extracted by water according to their water/fat partition coefficient, although flavour compounds are generally more lipophilic than hydrophilic. However, the water extract mainly contains non-volatile compounds. This non-volatile, water-soluble fraction is composed of mineral salts, lactic acid, lactose, amino acids and peptides and has characteristic taste properties (Salles et al., 1995a). Amino acids and small peptides are considered to be mainly responsible for the taste characteristics of water-soluble extracts (McGugan et al., 1979; Aston and Creamer, 1986), their flavour impact being modulated by interaction with calcium and magnesium ions (Biede and Hammond, 1979). Moreover, it has been recognised for a long time that water-soluble, low molecular weight and mainly hydrophobic peptides, which accumulate during ripening as a result of proteolysis, are responsible for bitterness in cheese (Lowrie and Lawrence, 1972; Schalinatus and Behnke, 1975; Furtado, 1984; Lemieux and Simard, 1992). Some fundamental studies on model compounds have characterised the tastes of amino acids and low molecular weight peptides (Salles et al., 1995a and references cited therein); studies conducted on tastes in casein hydrolysates were reviewed by RoudotAlgaron (1996). However, until recently and apart from bitterness, no clear sensory data were obtained on water-soluble extracts from cheese. Although several hundred peptides have been isolated and identified from various types of cheese, only a few small peptides, that are suspected to be responsible for particular tastes, were isolated from the water-soluble fractions of various cheeses and identified (Salles et al., 1995a and references cited therein). However, no direct correlations between these peptides and the organoleptic properties of the fractions have been demonstrated, apart from bitterness. In fact, the watersoluble fraction of cheese generally has a very complex composition, and separation and identification of individual compounds are difficult. Moreover, most analytical techniques require the use of non-food-grade solvents or buffers that make sensory evaluation of sub-fractions difficult or impossible. Part of this chapter will focus on recent advances made to study and identify the taste-active components present in the water-soluble fraction of cheese. A general procedure for the preparation of fractions involves an extraction of grated cheese by water followed by a fractionation scheme, generally adapted from the fractionation protocol used to isolate cheese nitrogen fractions in the study of proteolysis in cheese during ripening (Fox et al., 1994; McSweeney and

Fox, 1997). However, as sub-fractions have to be evaluated sensorially to assess their relative sensory impact and try to link it to their chemical composition, a suitable eluent has to be used in the chromatographic steps. Water (Roudot-Algaron et al., 1993; Salles et al., 1995a; Molina et al., 1999) or water-food-grade ethanol mixtures (Lee and Warthesen, 1996a,b) have been used for this purpose in combination with gel permeation chromatography (GPC) or high-performance liquid chromatography (HPLC). The final identification step generally involves mass spectrometry (MS) and tandem mass spectrometry (MS/MS) of nitrogenous compounds isolated using HPLC, either in a standalone mode or coupled with a mass spectrometer (HPLC-MS) (Roudot-Algaron etal., 1993, 1994b; Sommerer et al., 2001). A specific method for the isolation of small peptides from cheese has been described (Sommerer et al., 1998a). As already outlined for cheese aroma, the relationships between all flavour compounds identified in a food and sensory perception experienced by consumers when eating this food are still not entirely clear. In fact, it is particularly difficult to predict a flavour perception as it is still not known how the various components combine to produce an overall sensory impression. Moreover, interactions between taste and aroma (Noble, 1996) and interactions of trigeminal sensations with taste and aroma (Green, 1996) occur and play an important role in overall flavour perception. However, methods that allow direct analysis of flavour molecules released in the mouth during consumption have been developed in recent years (Taylor and Linforth, 1996; Roberts and Taylor, 2000). Development of instrumental techniques and data obtained recently for volatile and non-volatile flavour compounds in cheese will be presented which may explain the link between flavour perception and cheese composition. Finally, specific instrumental techniques have been developed for the analysis of the complete flavour of cheese. The methods currently used in the quality control of food flavour are still usually based on sensory evaluation by a panel of experts. These panels are able to monitor the quality of a particular food, to detect defects and to compare samples for classification purposes. Nevertheless, obtaining results rapidly at low cost using instruments could be desirable. The so-called 'electronic noses' based on gas sensor technology, despite some important drawbacks for some of them (Schaller et al., 2000a), are theoretically able to perform some classification tasks (Schaller etal., 1998), and some applications for the analysis of cheese have been developed (Mariaca and Bosset, 1997; Schaller et al., 1999). However, two other global

Cheese Flavour: InstrumentaITechniques

analysis methods based on mass spectrometry seem more powerful and reliable for purposes of classification. One of these methods analyses total headspace using a mass spectrometer, without any prior GC separation (Vernat and Berdague, 1995). This method is often referred to as a mass-based electronic nose. Alternatively, headspace sampling may be replaced by solidphase microextraction (SPME) of food volatiles (Marsili, 1999). Both sampling methods, followed directly by mass spectrometry, have found applications for the rapid characterisation of cheese (Schaller et al., 2000b; Peres et al., 2001, 2002a). The second method is pyrolysis }nass spectrometry (Aries and Gutteridge, 1987), where a small food sample is pyrolysed at up to 500 ~ The resulting volatile fraction, characteristic of the flavour but also of the matrix composition, is analysed by a mass spectrometer. As with the other rapid instrumental methods for classification, a pattern or fingerprint is obtained for each sample, and extensive data treatment, either by conventional multivariate statistics or artificial neural networks, allows the construction of maps useful for classification and quality control purposes (Peres et al., 2002b).

Characterisation of Aroma (Volatiles) Sample treatment

Volatile aroma compounds in cheese, like in other foodstuffs, are hydrophobic, generally distributed in a heterogeneous manner throughout the matrix and present at low or even traces ( 10), modulated by their hydrophobicity (Molimard and Spinnler, 1993). A high-performance size-exclusion chromatographic method has also been described for the purification of aroma compounds from organic extracts of fat-containing food (Lubke et al., 1996). The method was applied

A

I IF i I

E m

E

m

Figure 2 Dialysis cell with solvent recycling device. A, B, cell compartments; C, round-bottom flask containing solvent to distil; D, condenser; E, magnetic stirrers; F, dialysis membrane.

Cheese Flavour: InstrumentaITechniques

successfully to the clean-up of a dichloromethane extract from goat cheese (L(ibke et al., 1996). The main interest in this size-exclusion chromatographic method is the limited number of injections necessary and the reduced final volume of the fractions, which in terms of final useful concentration, appeared significantly quicker and gave rise to less thermally induced artefacts and to reduced losses of the most volatile components than any other distillation method (Lobke et al., 1996). Headspace methods, either static or more often dynamic, also called 'purge-and-trap' methods, are popular techniques used to isolate volatiles from cheese. Although direct analysis of the equilibrium headspace would appear to be an ideal method to study aroma compounds, in terms of sensory representativeness and ease of use, static headspace techniques have severe limitations in terms of sensitivity, being restricted to the most volatile and abundant components (Mariaca and Bosset, 1997; Reineccius, 2002). Dynamic headspace, or 'purge-and-trap', methods are basically pre-concentration and enrichment techniques. They use stripping of the volatiles from the cheese samples, sometimes dispersed in water, with an inert gas. The volatiles are concentrated in a cold trap or adsorbed onto an inert support (adsorbing polymer, generally of the Tenax | type) and analysed by subsequent thermal desorption or elution by a suitable solvent (Mariaca and Bosset, 1997; van Ruth, 2001a; Reineccius, 2002). Although dynamic headspace methods minimise artefacts developed or introduced during sampling (van Ruth, 2001a), distortion of the aroma profile may result from the trapping of aromas (Reineccius, 2002), especially when polymeric adsorbents are used. However, despite the drawback of relatively poor sensitivity compared to other extraction methods, the main advantages of dynamic headspace techniques are the small amount of sample needed to perform the analysis (c. 20 g) and its speed (Le Quere and Molimard, 2002). The technique, even though it favours the isolation of the most volatile flavour compounds (Reineccius, 2002), has been applied widely to the analysis of cheese volatiles (see for example Arora et al., 1995; Canac-Arteaga et al., 1999a,b, 2000; Larrayoz et al., 2001; Rychlik and Bosset, 2001a,b). Recent comprehensive reviews on the technique include Wampler (1997) and Pillonel et al. (2002). A comparative study on the advantages of the use of dynamic headspace with cheese samples in the 'dry' form or in 'dispersed suspension' in water has been published recently (Larrayoz et al., 2001). The 'dry' method allowed the extraction of a greater number of compounds and in larger quantities, but a few compounds were extracted better using the 'suspension' technique (Larrayoz et al., 2001). Simultaneous distil-

493

lation extraction (SDE) was also used in this study and compared to dynamic headspace analysis. As expected, the authors concluded that the techniques were complementary; dynamic headspace extracted more highly volatile compounds and SDE was more efficient for phenols, free fatty acids, lactones and heavier aldehydes, ketones, alcohols and esters (Larrayoz et al., 2001). Interference from water in dynamic headspace that could be detrimental to the efficiency of the technique has been discussed in detail by Canac-Arteaga et al. (1999a,b, 2000) and Pillonel et al. (2002). Solid-phase microextraction, first developed for the extraction of volatile organic compounds in water, has been applied recently to the isolation of aroma compounds from food (Harmon, 1997; Pillonel et al., 2002; Reineccius, 2002). Solid-phase microextraction partitions analytes between a liquid or a vapour phase and a thin solid-phase adsorbent, of which there are several choices in terms of polarity and film thickness, coated on inert fibres, generally associated with a syringe which serves as a direct injection device (Harmon, 1997). The method, which is an equilibrium one, can be performed either in the direct extraction mode (immersion of the fibre in the sample matrix, generally in an aqueous solution or suspension) or in a headspace configuration. It can be automated very easily, but the extraction of the solutes depends on polarity, volatility, partition coefficients, sample volume, temperature and the nature of the adsorbentcoating material. Therefore, the technique exhibits a certain degree of selectivity, but with the advantages of sensitivity, ease of use, no solvent and small sample volume (Harmon, 1997; Pillonel et al., 2002; Reineccius, 2002). Solid-phase microextraction, used for the first time for the analyses of cheese volatiles by Chin et al. (1996), has since been used in some significant applications on cheese aroma (Dufour et al., 2001; Pillonel et al., 2002 and references cited therein). Analysing volatiles directly by immersing the fibre in highly complex matrices (as cheese) could damage the fibre, and SPME is, therefore, used almost always in the headspace mode. Comparison of direct SPME and headspace SPME of Camembert volatiles obtained after cryo-trapping of the aqueous phase under vacuum showed only a slight reduction in sensitivity using headspace SPME compared to direct SPME (Jaillais et al., 1999). The water-soluble extract (WSE) of cheese has been described for a long time as possessing a strong flavour (Biede and Hammond, 1979; Aston and Creamer, 1986; Engels and Visser, 1994; Salles et al., 1995a). Besides non-volatile materials responsible for taste, WSE also contains volatile compounds responsible for its intense aroma. Thus, water-soluble extracts of various

494

Cheese Flavour: InstrumentaITechniques

cheeses, obtained by direct extraction with water followed by various centrifugation steps (Le Quere et al., 1996; Engels et al., 1997; Engel et al., 2002c) or by pressing to obtain an aqueous phase called 'cheese juice' (Salvat-Brunaud etal., 1995; Thierry etal., 1999), have been investigated for their volatile components. To be analysed using gas chromatography, WSEs were either extracted with a suitable solvent (Le Quere et al., 1996), submitted to dynamic headspace analysis (Engels et al., 1997; Thierry et al., 1999) or fractionated using nanofihration as the final membrane-filtration step (Engel et al., 2002c).

Representativeness As already outlined, because there is no universally applicable method, none of the extraction techniques described above yields an aroma isolate that truly represents either qualitatively or quantitatively the aroma profile of a food (Reineccius, 2002). This fact explains the frequently observed discrepancies between aroma analysis of a food extract and sensory analysis of the food itself. Therefore, the flavour analyst must choose the isolation procedure best suited to address the problem faced: determination of the complete aroma profile, identification of key odorants or off-flavours, monitoring aroma changes with time in foods or prediction of sensory properties (Reineccius, 2002). When the ultimate aim of a particular study is the identification of the compounds that are important for flavour (the key odorants), the most reliable results will be obtained if the odour of the extract resembles closely that of the food itself (Etievant et al., 1994; Etievant and Langlois, 1998). Different sensory methods, which necessitate a trained sensory panel, can be used to check the sensory representativeness of the food extract odours (Etievant et al., 1994). When an estimation of the relative importance of key constituents in a single sample is required, a similarity test is preferred. The panellists are asked to score the similarity of the odour of the extracts obtained by different methods to the odour of the food itself used as reference on an unstructured 10 cm scale. This approach was applied to three French and Swiss hardtype cheeses by Etievant et al. (1994) and Guichard (1995). It was shown that the distillates obtained at a pressure in the range 10-100 Pa had odours more similar to those of the cheeses than the distillates obtained at a lower pressure (10 mPa). This result means that strongly absorbed and less volatile flavour compounds, extracted only at lower pressure, may not be important for the odour of these cheeses. Similar results were obtained for extracts of Camembert cheese, showing clearly that the second step (molecular distillation operated under a high vacuum) is not necessary to obtain a representative distillate of the cheese odour.

When applied to goat milk cheese, this approach indicated that the best extract was obtained by a direct water extraction of the cheese volatiles (Le Quere et al., 1996). This result could perhaps be explained by the chemical and hydrophilic nature of the free fatty acids identified as key odorants of goat milk cheese (Le Quere et al., 1996; Salles and Le Qu~r~, 1998; Le Quer~ and Salles, 2001). A key point in these evaluations of representativeness is the choice of a suitable matrix for testing the olfactory character of the extracts. For cheese, the best results have been obtained when the extracts are added to an emulsion, i.e., a matrix similar to cheese in terms of fat composition (Etievant et al., 1994). Since, generally, a combination of techniques should be used to obtain a reasonably complete view of an aroma profile (Reineccius, 2002), it is noteworthy that sensory evaluation of headspace or SPME extracts by 'direct GC-olfactometry' (i.e., without a chromatographic column) has been demonstrated recently (Lecanu et al., 2002; Rega et al., 2003).

Identification of volatile aroma compounds using hyphenated GC techniques As aroma molecules are essentially volatile, the techniques used to analyse them are usually based on separation using high resolution gas chromatography (HRGC). Substantial progress has been made in this field during the last 20 years and several stationary phases are available which allow almost all separation problems to be overcome. Combined with universal or selective detectors, HRGC is clearly a fundamental technique, essential for all aroma identification studies. A comprehensive review on the use of HRGC for the analysis of milk and dairy products is available (Mariaca and Bosset, 1997). Other interesting comments on qualitative, including multidimensional GC (Wright, 1997), and quantitative aspects may be found in Marsili (1997), van Ruth (2001b) and Reineccius (2002). Among the hyphenated techniques that are coupled to HRGC, the one that uses the human nose as a detector and known as gas chromatography-olfactometry (GC-O, sometimes referred to as 'GC-sniffing'), has received considerable attention during the past 20 years in aroma research (see for example Blank, 1997; Leland et al., 2001; Reineccius, 2002). The selectivity of this specific detector is based only on the odorous properties of the individual compounds separated by HRGC. As the most abundant volatiles may have little, if any, odour of significance in a food (Mistry et al., 1997), GC-sniffing has been an invaluable tool for identifying target compounds in aroma extracts that are always very complex. The primary aim of this technique is to discriminate the odorous compounds from the many background volatile components. The so-called 'aromagram'

Cheese Flavour: InstrumentaITechniques

constructed from the chromatogram obtained by simply smelling a GC effluent (Blank, 1997; Reineccius, 2002) constitutes an interesting interface with sensory analysis, as odour descriptors sensed at the GC sniffing port can be compared to the descriptors generated by a sensory panel. This method is particularly efficient for identifying off-flavours. Selection of key odorants or character-impact compounds in a food is another objective of GC-sniffing. Quantitative approaches (the true GC-olfactometry) based on odour detection thresholds or on odour intensity have been developed and are the subject of specialised treatises (Mistry et al., 1997; Leland et al., 2001; van Ruth, 2001b; Reineccius, 2002). Three different methods have been developed for GC-O: dilution analyses based on determination of detection thresholds, detection frequency methods and intensity measurement methods. Original dilution methods, CHARM (for Combined Hedonic Aroma Measurement) analysis developed by Acree and co-workers (Acree et al., 1984) and Aroma Extract Dilution Analysis (AEDA) developed by Grosch and co-workers (Ullrich and Grosch, 1987) are essentially screening methodologies since the methods, based only on detection threshold determinations, violate certain sensory rules and psychophysical laws (Reineccius, 2002 and references cited therein). They can be used to determine those odorous compounds that are most likely to contribute to the odour of a food. Originally developed by McDaniel et al. (1990), the odour-specific magnitude estimation (OSME) method is basically a crossmodal technique aimed at measuring the perceived odour intensity of eluting volatiles. In OSME and other cross-modality matching methods (Guichard etal., 1995; Eti~vant et al., 1999), results are not based on odour detection thresholds, and only one concentration of the extract is evaluated by a panel, unlike dilution methods where several dilutions of the extract are evaluated. Results can be subjected to statistical analysis and more consistent results are obtained when panellists are trained (Callement et al., 2001). The detection frequency methods, originally developed by Roozen and co-workers (Linssen et al., 1993), and referred to as nasal impact frequency (NIF) or surface nasal impact frequency (SNIF) since the work of Chaintreau and co-workers (Pollien et al., 1997), also use a group of assessors who simply have to note when they detect an odour in a single GC run (i.e., also at only one concentration). Those GC peaks being detected as odorous by the greatest number of assessors are considered to be the most important. Not being based on real odour intensities, the method has important drawbacks, especially when all the odorous compounds are present above their sensory threshold for all the assessors (Reineccius, 2002).

495

There is no perfect GC-sniffing method for finding key odorants in foods. Each of the methods described above has its advantages and weaknesses. Only two studies have compared the methods in terms of performance (Le Guen et al., 2000; van Ruth and O'Connor, 2001). In both cases, the results obtained with the different techniques were found to be very similar and well correlated. Finally, the choice of a GC-O method depends on the objective of the study, on the quality of the panel and on the time scheduled for the analyses (Le Guen et al., 2000). Dilution techniques are clearly time-consuming, intensity methods require a trained panel (Le Guen et al., 2000; Callement et al., 2001) while detection frequency methods are the least demanding but also the least precise (Le Guen et al., 2000). The aim of any GC-O experiment is to determine the relative odour potency of volatiles present in an aroma extract or fraction and to prioritise compounds for identification. This identification step is done mainly through the use of another hyphenated technique that couples HRGC to mass spectrometry (GC-MS). For difficult identifications, GC coupled with Fourier transform infrared spectroscopy (GC/FTIR) provides an interesting complement to GC-MS (Le Quire, 2000). Mass spectrometry is also used for quantification purposes through the use of a stable isotope dilution assay (Milo and Blank, 1998; Blank et al., 1999 and references cited therein). Such a precise quantitation is required for the determination of odour activity values (OAVs) generally calculated when using AEDA (Grosch, 1994). Odour activity values, calculated as the ratio of concentrations to odour thresholds, despite their limitations in terms of psychophysical validity (Mistry et al., 1997), give a good indication of the respective contributions of key odorants to the aroma of foods. They are the basis of the first attempts at using recombination studies to validate impact odorants sensorially in model cheeses (Grosch, 1994). Aroma-recombination studies are the important last step in sensorially verifying the analytical data obtained by GC-O and for quantification of key odorants of food (Mistry etal., 1997). Either bland unripened cheese (Grosch, 1994; Preininger etal., 1996; Kubickova and Grosch, 1998a) or specially designed odourless model cheese systems (Smitet al., 1995; Salles et al., 1995b) have been used to incorporate potential key odorants. Thus, the importance of methional, 4-hydroxy-2,5-dimethyl-3(2H)-furanone and 2-ethyl-4-hydroxy-5-methyl-3(2H)-furanone, acetic acid and propionic acid was confirmed as key compounds for the aroma of Emmental-type cheese (Preininger et al., 1996). The branched-chain volatile fatty acids, 4-methyloctanoic and 4-ethyloctanoic acids, were confirmed to be essential for the typical goaty note of goat cheese (Le Quere et al., 1996) and their

496 Cheese Flavour: InstrumentaITechniques retronasal aroma thresholds were determined in a cheese model (Salles and Le Quere, 1998; Le Quere and Salles, 2001; Salles et al., 2002). Finally, the odour profile of the aroma model built with a set of 11 potent odorants identified in a GC-O study of an extract from Camembert cheese (Kubickova and Grosch, 1997, 1998b), with four additional volatile compounds identified by headspace-GC-O, has been found to resemble closely the aroma of genuine French Camembert cheese (Kubickova and Grosch, 1998a; Grosch et al., 2001). The GC-O methods that have been developed during the past 20 years, combined with either aroma extracts, headspace or even SPME (Dufour et al., 2001), have facilitated the identification of potent odorants in various cheeses, including Swiss (Preininger and Grosch, 1994; Rychlik et al., 1997; Rychlik and Bosset, 2001a,b), Cheddar (Arora etal., 1995; Christensen and Reineccius, 1995; Dufour et al., 2001), ParmigianoReggiano (Qian and Reineccius, 2002a,b), Blue (Le Quere et al., 2002; Qian et al., 2002), Mozzarella (Moio et al., 1993), Grana Padano (Moio and Addeo, 1998) and Gorgonzola (Moio et al., 2000) cheeses.

Characterisation of Sapid (Non-Volatile) Flavour C o m p o u n d s Water-soluble extracts (WSE) of cheese The water-soluble extract (WSE) of cheese has been reported to possess a strong flavour (Biede and Hammond, 1979; McGugan etal., 1979; Aston and Creamer, 1986). Apart from some water-soluble volatile components responsible for aroma, a WSE of cheese contains mainly non-volatile components that have been considered to be responsible for the taste of cheese (McSweeney, 1997). It has been recognised for a long time that bitterness, which can limit cheese acceptability if too intense, is due to an excessive concentration of low molecular weight and mainly hydrophobic peptides, which accumulate during ripening as a result of proteolysis (Lemieux and Simard, 1992; McSweeney, 1997). Amino acids and small peptides were hypothesised to be mainly responsible for the basic taste of cheese (McGugan etal., 1979; Aston and Creamer, 1986; Engels and Visser, 1994), their flavour impact being supposedly influenced by their interaction with calcium and magnesium ions (Biede and Hammond, 1979). However, the exact role of medium- and smallsize peptides and free amino acids in cheese flavour has not been clearly demonstrated, although it is likely that they contribute to the background flavour of cheese (McSweeney, 1997). In fact, until recently and apart from bitterness, no clear sensory data have been available for

WSEs of cheese and no direct correlations between specific nitrogen-containing compounds and organoleptic properties of fractions have been demonstrated. Among the mineral salts present in the WSE of cheese, the compound responsible for the salty taste is almost always supposed to be NaC1 (McSweeney, 1997). The taste of most high molecular weight salts is known to be bitter rather than salty (McSweeney, 1997 and references cited therein). Acid taste is caused by H30 + and the principal acid in cheese is lactic acid. However, total lactate concentration does not seem to be a good index of cheese acidity as the pH may increase during ripening caused by the production of ammonia (McSweeney, 1997). Moreover, the perception of acidity in cheese was hypothesised to be influenced by the concentration of NaC1 (Stampanoni and Noble, 1991), and no correlation between acid taste and either cheese pH or the amount of lactic acid was found for the flavour of Swiss cheese (Biede and Hammond, 1979), while the acid flavour correlated positively with the levels of triand tetra-peptides and with amino acids (Biede and Hammond, 1979). It has also been hypothesised that short- and medium-chain fatty acids might contribute to the acid taste of cheese (McSweeney, 1997). Although this assumption seems reasonable for short chain acids (e.g., formic, acetic or propionic), their principal contribution to cheese flavour is to its aroma in the unionised form (RCOOH) (Le Qu~r~ et al., 1996; Salles and Le Quere, 1998; Qian and Reineccius, 2002b).

Extraction, separation, identification of sapid compounds in relation to their sensory properties The study of taste-impact compounds in cheese, or more precisely in its water-soluble fraction, involves the study of soluble low molecular weight material (i.e., small peptides, amino acids, organic acids, minerals, etc.) dispersed in a very complex mixture. As it is necessary to assess the relative sensory impact of potential taste-active compounds, a fractionation scheme suitable for subsequent sensory evaluation is needed, and non-food-grade solvents or buffers must be rejected. Commonly used procedures involve extraction of grated cheese with water, possibly completed by precipitation of caseins and large peptides at pH 4.6, leading to edible fractions with good recovery of nitrogenous compounds (Kuchroo and Fox, 1982). The fractionation scheme that follows is generally adapted from the fractionation protocol used for isolating cheese nitrogen fractions for the study of proteolysis (Fox et al., 1994; McSweeney and Fox, 1997). The following steps (Fig. 3) involve ultrafihration using membranes with 1, 3 or 10 kDa molecular weight cutoff or precipitation with 70% ethanol (Cliffe et al.,

Cheese Flavour: InstrumentaITechniques

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1993). The ultrafiltered water-soluble or 70% ethanolsoluble extracts are then subjected to gel filtration chromatography (Fig. 3). Sephadex G10 (Engels and Visser, 1994; Roudot-Algaron et al., 1994a; Engels et al., 1995; Molina et al., 1999), G15 (Roudot-Algaron et al., 1993; Warmke etal., 1996; Kubickova and Grosch, 1998a), G25 (Cliffe et al., 1993; Salles etal., 1995a), or Toyopearl HW-40S (Salles et al., 1995a, 2000; Sommerer et al., 1998a, 2001) media have been used for this purpose, using pure water (generally), 0.01 M NaC1 (Engels and Visser, 1994), or aqueous 0.5 M acetic acid (Warmke etal., 1996; Kubickova and Grosch, 1998a) as eluent. The fractions obtained by gel permeation chromatography may be evaluated sensorially (Fig. 3) after freeze-drying and re-dissolution in water, possibly with pH adjustment. Alternatively, liquid chromatographic methods involving Sep Pak C18 cartridges eluted with a stepwise water-ethanol gradient (Engels and Visser, 1994; Engels et al., 1995) or HPLC

using a water/food-grade ethanol gradient (Lee and Warthesen, 1996a,b) have been used instead of gel filtration. This fractionation scheme was developed originally in order to identify small hydrophobic peptides supposedly responsible for taste characteristics such as bitter or umami (Mojarro-Guerra et al., 1991; Cliffe et al., 1993; Roudot-Algaron et al., 1993, 1994a). A dedicated liquid chromatographic purification method has been developed to isolate and identify oligopeptides from the WSE of goat milk cheese (Sommerer et al., 1998a, 2001). Systematic sensory evaluation of the final fractions allows target fractions to be determined that possess interesting tastes, and physicochemical assessment of these key fractions should permit the identification of those compounds that are really relevant to the flavour of cheese (Engels and Visser, 1994; Salles etal., 1995a). Using this approach, some recent studies have been dedicated to the taste of the WSE of various cheeses.

498

Cheese Flavour: InstrumentaITechniques

Low molecular weight peptides, with two to four amino residues, were identified in Vacherin Mont d'Or (Mojarro-Guerra et al., 1991). As there was not enough natural material available for sensory evaluation, commercially available analogous synthetic peptides were used in sensory experiments. The dipeptides tested were dissolved in tap water at a rather high concentration (50 mg/100 mL) and were found to be essentially bitter. However, neither quantitative nor threshold data were estimated and the importance of these peptides for the overall taste of the cheese was only an hypothesis (Mojarro-Guerra et al., 1991). In a study on Cheddar cheese, Cliffe et al. (1993) found bitter fractions in material thought to be large hydrophobic peptides while lower molecular weight fractions with savoury notes were thought to be small, more hydrophilic peptides and amino acids. The flavour of the WSE of Comt~ cheese was the subject of substantial efforts in the early 1990s. A great variety of small peptides was identified in these extracts (Roudot-Algaron et al., 1993, 1994a,b). Some of them were found to be essentially bitter (Roudot-Algaron et al., 1993), y-glutamyl dipeptides were found to be sour (Roudot-Algaron etal., 1994a), but all the identified compounds, including non-peptide material (Roudot-Algaron et al., 1993; Salles et al., 1995a), were found at a concentration much lower than their threshold values. Although possible synergistic effects between several molecules found at concentrations below individual threshold values cannot be a priori eliminated, these observations suggest that these components alone could not affect cheese flavour (Salles et al., 1995a). Umami taste was clearly identified in a fraction and easily explained by a substantial amount of monosodium glutamate which was found at a concentration ten times above its threshold value, while the concentrations of the other amino acids were all well below their thresholds (Sales et al., 1995a). Following the same methodology, Grosch and co-workers evaluated the taste compounds of Emmental cheese (Warmke et al., 1996). The contribution of individual free fatty acids, free amino acids, minerals, biogenic amines, lactic and succinic acids, and ammonia was estimated on the basis of taste activity values (TAVs), a concept analogous to the odour activity values (OAVs), and defined as the ratio of concentration to taste threshold. From these results, acetic and propionic acids were confirmed to be important contributors to the taste of Emmental cheese. Glutamic acid was the major taste compound in the fraction containing free amino acids while all the ions investigated might be involved in the taste of Emmental, as were also biogenic amines (tyramine and histamine), ammonia, lactic and succinic acids (Warmke et al., 1996).

However, taste evaluation of mixtures of compounds conducted in tap water suggested that the characteristic taste compounds of Emmental are acetic, propionic, lactic, succinic and glutamic acids, each in the undissociated form and/or as ammonium, sodium, potassium, magnesium and calcium salts, as well as chlorides and phosphates analogues (Warmke et al., 1996). A study conducted on a model based on unripened Mozzarella-type cheese confirmed the importance of acetic, propionic, lactic, succinic and glutamic acids, and sodium, potassium, calcium, magnesium, ammonium, phosphate and chloride ions to the taste of Emmental cheese (Preininger et al., 1996). The same approach applied to Camembert led to the conclusion that the important taste contributors for Camembert are acetic, butyric, 3-methylbutyric, caprylic and succinic acids, monosodium glutamate, ammonia and NaC1 (Kubickova and Grosch, 1998a). It was also found that the biogenic amine, cadaverine, and the rare amino acids, ornithine and citrulline, when present, are likely to contribute to the bitter taste of Camembert (Kubickova and Grosch, 1998a). The above results clearly indicated that only low molecular weight compounds found in the WSE contribute significantly to the taste of cheese, while small peptides do not seem to be key flavour compounds, as was previously hypothesised. A study on goat milk cheese led to the same conclusions (Sales and Le Quere, 1998; Salles et al., 2000; Le Quere and Sales, 2001). The taste of the various goat milk cheeses investigated was essentially due to mineral salts and lactic acid. Fractions rich in small peptides and free amino acids were found to be essentially tasteless when evaluated either in water (Sales et al., 2000) or in a model cheese (Sales and Le Quere, 1998; Le Quere and Sales, 2001). In a comparative study on cheeses made from cows', ewes' or goats' milk, Molina et al. (1999) concluded that, even though differences were found in the intensity and predominance of individual tastes in the fractions of the cheeses made from the milk of the three species, it was difficult to correlate the peptide pattern and the free amino acid content of cheese with the sensory evaluation of the fractions. However, synergistic effects on taste have been demonstrated between peptides, amino acids and mineral salts (Wang et al., 1996) and interactions between tastes in mixtures may exist (Breslin, 1996). Therefore, it appeared interesting to generalise the evaluation of model mixtures of compounds that have been identified and quantified in the WSE of cheese (Warmke et al., 1996; Kubickova and Grosch, 1998a). Moreover, fractionation of the WSE by gel filtration has two main limitations: poor resolution and the necessity of

Cheese Flavour: InstrumentaITechniques

tedious repetitive steps in order to obtain sufficient peptide material for sensory evaluation. To clarify the putative effect of the small water-soluble peptides on the taste of cheese, it was therefore necessary to develop a new isolation procedure. Nanofiltration using ionisable membranes with a molecular weight cut-off of 500 Da was used by Sommerer et al. (1998b). A nanofiltrate was prepared from the 1-kDa permeate obtained by ultrafiltration of the WSE (Fig. 3). A large proportion of mineral salts and a substantial proportion of amino acids were thus eliminated from the nanofiltration retentate in which the majority of small peptides were concentrated (Sommerer et al., 1998b). This relatively pure and edible peptide-containing fraction could be used in sensory analysis, after incorporation into a bland model cheese system (Salles etal., 1995b), on its own or with the addition of putative synergistic effectors such as mineral salts or amino acids (Sommerer et al., 1998b). Using omission tests (see Engel et al., 2002a,b, and references cited therein for a comprehensive review), it was shown that small peptides have no effect on the taste of goat milk cheese, and no additive or synergistic effects were found between those peptides and salts or amino acids (Sommerer et al., 1998b). This unexpected result has been confirmed after complete physicochemical assessment of the WSE from goats' milk cheese has allowed the development of a model mixture that was validated sensorially (Engel et al., 2000a). Using omission tests, the relative impact of WSE components on goat cheese taste has been determined (Engel et al., 2000b). Among the main taste characteristics of the WSE from goats' milk cheese (salty, sour and bitter), saltiness was explained by additive effects of Na +, K +, Ca 2+ and Mg 2+, sourness was due to synergism between NaC1, phosphates and lactic acid, and bitterness resulted entirely from CaCI2 and MgCI2. Amino acids, lactose and peptides had no significant impact on the taste properties of the WSE of goats' milk cheese (Engel et al., 2000b). The same procedure was applied recently to a specially selected bitter Camembert cheese (Engel et al., 2001a,b,c) and confirmed that the WSE from cheese contained taste-active compounds, the impact of which could be modulated by an effect of the cheese matrix (Engel et al., 2001a). Sourness of Camembert WSE was explained by an enhancing effect of NaC1 on the acid taste due to the concentration of H30 +, saltiness was due to NaC1 whereas bitterness was mainly due to the bitter peptides found in the fraction with a molecular weight in the range 500-1000 Da (Engel et al., 2001b). The intense proteolytic activity of the strain of Penicillium camemberti, specially selected to develop bitterness in this case, has been demonstrated to be responsible for the accumula-

499

tion of small (MW < 1000 Da) bitter peptides during ripening (Engel et al., 2001c).

Dynamic Methods for Flavour Characterisation Even if the 'best' extraction and identification methods are used, poor correlations are often found between the overall levels of flavour components (volatile and non-volatile) and sensory perception experienced by a consumer. In other words, it is not enough to know the exact composition of food in terms of flavour compounds to understand perfectly the perception of its flavour. In fact, the perception of flavour is a dynamic process (Piggott, 2000). During the consumption of food, the concentration of aroma compounds at the olfactory epithelium and of sapid compounds at the taste buds varies with time. Flavour components are released progressively from the food matrix during chewing. Kinetics of the release of flavour depends on the nature of the food matrix composition and of individual mastication pattern. Sensory methods, such as time-intensity, have been used to study the dynamicand time-related aspects of flavour perception (Piggott,

2000). Release of volatiles in vivo

Techniques which measure volatiles directly in the mouth or in the nose have been developed to obtain physico-chemical data that reflect the pattern of aroma molecules released from food and that are effectively present at the olfactory receptors during consumption (Linforth and Taylor, 1993; Taylor and Linforth, 1994). Among the various approaches aimed at sampiing aroma from the nose (nose-space), the collection of expired air samples on Tenax | traps (Fig. 4) provided the first robust results (Linforth and Taylor, 1993; Taylor and Linforth, 1994). When applied to

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Figure 4 Collection and analysis of expired air by Tenax trapping and GC-MS (reproduced from Roberts and Taylor (2000), with permission from the American Chemical Society).

500

Cheese Flavour: InstrumentaITechniques

Cheddar cheese (Delahunty et al., 1994), the 'buccal headspace' method demonstrated that, despite a similar composition of volatiles found with conventional headspace analysis, some cheeses, depending on their fat content, released a different balance of volatiles during consumption (Delahunty et al., 1996a). Gas chromatography-olfactometry of buccal headspace showed a number of volatile compounds which have been suspected to contribute primarily and most likely to Cheddar cheese flavour (Delahunty et al., 1996b). It was presumed that the buccal headspace extract was representative of the aroma compounds that a consumer perceives during consumption (O'Riordan and Delahunty, 2001). By overlapping the sampling time periods, release curves can be constructed and temporal changes reflecting relative concentrations of volatiles at a particular moment during consumption can be determined (Linforth et al., 1996). When applied to Cheddar cheese, 'temporal buccal headspace' results, obtained on an accumulated 'time-concentration' basis (four time periods: 15, 30, 45 and 60 s of cheese consumption), were correlated with sensory time-intensity data (Delahunty et al., 1996c). Time-course data confirmed the results of conventional analysis while providing improved sensory predictions from the instrumental results (Delahunty et al., 1996c). Mastication behaviour using electromyography and saliva production rates of individuals have also been measured during consumption of Cheddar cheese (Delahunty et al., 1998; O'Riordan et al., 1998). Combined to nose-space analysis and sensory evaluation using free choice profiling, these authors demonstrated that although there were differences in chewing styles and saliva production rates, the assessors' individual nose-space profiles were very similar for all Cheddar cheeses examined (Delahunty et al., 1998). Partial least-squares regression analysis allowed the most important flavour differences between cheeses to be predicted from the volatiles released during consumption (O'Riordan et al., 1998). Recently, atmospheric pressure ionisation-mass spectrometry (API-MS) has been developed to monitor aroma release during chewing (Taylor et al., 2000). Air from the nose (nose-space) is sampled directly into the API-MS source through an interface (Fig. 5), making real time breath-by-breath analysis possible (Linforth etal., 1996; Taylor and Linforth, 1996). Therefore, by combining time-intensity sensory studies with nose-space analysis, it is now possible to relate temporal parameters of aroma release to perception (Linforth etal., 2000). The method, reviewed in detail in specialised treatises (Roberts and Taylor, 2000; Taylor, 2002), has been applied recently to soft French

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cheeses (Salles etal., 2003). Three French mouldripened soft cheeses (Brie made from pasteurised milk, Camembert made from pasteurised milk and from raw milk) were evaluated by a panel of 15 assessors (Salles et al., 2003). Retronasal aroma profiles made by citation frequency of attributes revealed four main descriptors for the three cheeses. The sulphury note (cabbage/cauliflower/vegetable) was particularly intense for the Camembert cheeses, while the buttery/creamy note was important for the three cheeses studied; the mushroom attribute was less intense in the Camembert cheeses, and ammonia was perceived in all cheeses but was found particularly difficult to score by the panellists (Salles et al., 2003). Therefore, the three main aroma notes (sulphury, buttery and mushroom) were selected for subsequent time-intensity (TI) scoring (15 assessors evaluated each attribute, with three replicates of each cheese). Gas chromatography-olfactometry of the dynamic headspace sampling of the three cheeses allowed odour-active compounds to be identified, amongst which sulphur compounds (methanethiol, dimethylsulphide (DMS), S-methylthioacetate, dimethyldisulphide (DMDS), 2,4-dithiapentane, dimethyltrisulphide, 2,3,5-trithiahexane and dimethyltetrasulphide) could be related to the sulphury attribute scored by the panellists. However, API-MS nose-space experiments allowed the detection of only six compounds of which three contained sulphur ones (DMS, 5-methylthioacetate and DMDS). Simultaneous TI scoring of the sulphury note allowed a perfect superposition of the time-intensity curve with the release of the sulphur compounds (Fig. 6). The most significant perception and flavour release parameters allowed the three cheeses to be well discriminated by principal component analysis (PCA), showing a good agreement between perception scored by assessors and consistency in their release of aroma compounds while eating cheeses (Salles et al., 2003). Another PCA analysis showed a positive correlation for the sulphury note between the perception parameters derived from the TI curves and parameters derived from the aroma

Cheese Flavour: InstrumentaITechniques

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Figure 6 Flavour release from Camembert cheese for one assessor. Simultaneous time-intensity scoring of the sulphury/ cabbage attribute and API-MS analysis of S-methyl thioacetate, dimethyldisulphide (DMDS) and dimethylsulphide (DMS) in the nose-space.

release curves (Salles et al., 2003), as suggested by the characteristic curves presented in Fig. 6 for one panellist within one session. Non-volatiles in vivo

Development of methods to study flavour release has concentrated mainly on the volatile fraction, while only a limited number of studies have been devoted to the release of non-volatile compounds in the mouth. Conductivity measurements have been used to relate the release of salt during chewing to Cheddar cheese texture (Jack et al., 1995), and a similar approach with additional in-mouth measurement of pH has been used with a variety of foodstuffs, including Cheddar cheese (Davidson etal., 1998). However, in these approaches, the sensors available for in vivo measurements only give the best estimate for salt (non-specific to sodium) and acid release. Saliva sampling using cotton buds coupled to a direct liquid mass spectrometry procedure has been described to study the rate of release of sucrose (Davidson et al., 1999). Panellists were instructed to take a swab from a specific location on the tongue at different times during mastication using a cotton bud. The weight of saliva swabbed was measured and sucrose concentration was monitored using liquid-API-MS after extraction by a methanolwater solution (Davidson et al., 1999). A continuous

501

sampling technique using a motor-driven ribbon placed across the tongue while a panellist chews a food sample has also been described (Davidson et al., 2000). At the end of the eating process, the ribbon was cut into 5 cm lengths after estimation of the saliva weight adsorbed on the ribbon, each piece representing a certain time. Non-volatile components were extracted from the pieces of ribbon with a solvent and their concentration determined by direct liquid phase API- or electrospray (ES)-MS (Davidson et al., 2000). Temporal release of sucrose and glucose from biscuits, of sodium from potato crisps, of sucrose, glucose and fructose, citric and malic acids from fresh orange and finally minerals (sodium, calcium and potassium) from Cheddar cheese was monitored successfully (Davidson et al., 2000). The cotton bud technique has been applied recently to a model processed cheese in which aroma and non-volatiles compounds consistent with literature data had been incorporated (Pionnier et al., 2003). As it was demonstrated that with certain foodstuffs the increased frequency of sampling affected the chewing pattern (Davidson et al., 2000), each panellist produced only one saliva sample per mastication, at a time-consuming cost, however. Using ES-MS in negative ionisation mode, time-course release curves for minerals (sodium, calcium, magnesium and potassium), amino acids (leucine, phenylalanine, glutamic acid), organic acids (citric, lactic, propanoic and butyric) and phosphoric acid have been obtained (Pionnier et al., 2003). As a typical example, Fig. 7, shows release curves from cheese for phenylalanine, glutamic acid, leucine, phosphoric and lactic acids obtained for one assessor. The first conclusion that could be stressed from the analyses of the release curves is that individual physiological parameters (mainly mastication behaviour and salivation rate) are related more closely to the temporal release of taste compounds than to their physico-chemical properties (Pionnier et al., 2003). Model mouth systems

A number of mechanical devices which mimic in more or less detail the processes that occur in the mouth during eating 'model mouths' have been developed (Piggott, 2000 and references therein). These are often variants of dynamic headspace analysis, but their aim is to obtain time-resolved samples containing volatiles as similar as possible to those present during actual eating. The various parameters like temperature, air flow, mastication rate and addition of artificial saliva can be varied to study their effects on volatile flavour release. The main advantages of model mouths are the large quantities of food samples that can be handled, overcoming some sensitivity problems encountered

502 Cheese Flavour: InstrumentaITechniques

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---- critical strain, the structure of the cheese is altered via the breaking of bonds between structural elements, which are stressed beyond their elastic limit. Eventually, when the stress-bearing structural casein matrix has fractured, the cheese is said to flow. At short time scales and low r, most hard cheese varieties are essentially elastic, whereas after a long time, they flow, albeit very slowly, and do not recover to their original shape on removal of the stress. Failure to appreciate this characteristic can often lead to loss of shape (e.g., manifested by bulging, inclined surfaces) during storage, distribution and retailing, especially if cheeses of different consistencies are laid haphazardly upon each other. A stress relaxation test generally entails the instantaneous application of a constant deformation or strain, e (typically 0.10-0.20), by compression of the cheese sample between two parallel plates of a texture analyser (e.g., TA HDi Texture Analyser, Stable Micro Systems, Godalming, England; Instron Universal Testing Instrument (UTM); Instron Corporation, Massachusetts, USA.). On the application of e, or increases instantaneously to oro (i.e., zero-time value) but decays exponentially with time (t) (Shama and Sherman, 1973). The resultant or-time curve is used to determine the stress relaxation time, t, which may be defined as the time required for or to decrease to a fraction of Oro, e.g., t at which or = O-o/e, where e is the base of the natural logarithm. In a variation on such a test, Emmons et al. (1980) compressed full-fat (35%) and reduced-fat (17%) Cheddar cheeses, having a common level of moisture-in-non-fat-substance, at a constant speed to a strain of 0.2 and held the strain for 1 min. They

Rheology and Texture of Cheese

0.3

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Recovery after removal of stress

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0.2 s (U .,,-, 00

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Non-recoverable strain J I, ,/ 0

50

100

150

200

250

300

Time, s

Figure 9 Creep-relaxation curve for mature Maasdammer cheese (fat, 29%, w/w, protein, 28%, w/w). A stress of 3700 Pa was applied to a cheese disc (diameter, 40 mm; height, 2.27 mm), placed between the parallel plates of a controlled strain rheometer (TA Carrri-Med csl2500) at 20 ~ and removed after 180 s. The curve is divided into regions indicating elastic, viscoelastic and viscous behaviour.

showed that the initial compression slope (or modulus of deformability), the relaxation slope and the residual force (after 1 min) were much higher for reduced-fat cheese, made from milk with or without homogenisation, than for full-fat cheese. Mechanical models of c h e e s e theology From its creep and stress-relaxation behaviour (Fig. 9), it can be inferred that cheese is a viscoelastic material. It exhibits elastic and viscous characteristics, but unlike true elastic or viscous materials, the relationship between stress and strain depends on the magnitude and the duration of the applied stress or strain. On the application of a low stress, that is sufficiently small so as not to induce permanent damage or fracturing (breaking of bonds between the structural elements) of the microstructure, for short times, cheese behaves as an elastic solid. However, a low stress applied over a relatively long time scale results in an increasing strain, a gradual failure of the structure and an eventual flow. Hence, the relationship between r (or o9 and T (or ~) is linear only at very low r and short time scales. The T at which linearity between r and 3' is lost is referred to as the critical strain (i.e., at the end of the linear viscoelastic range), which for most solidlike foods, including cheese, is relatively small, e.g., 0.02-0.05 (Walstra and van Vliet, 1982). The modelling of cheese rheology begins with simple relationships such as Hooke's Law for small displacements in the elastic region. In the region beyond the elastic limit, sometimes referred to as the elastoplastic region (i.e., where recovery following deformation is partial on removal of stress), modelling the rheology of cheese requires more complex models.

Mechanical models have been used to simulate creep and relaxation effects in materials (Rao, 1992; Tanner, 2000). The viscoelastic behaviour of cheese may be simulated by various mechanical models that contain different arrangements of dashpots (representing the fluid element) and springs (representing the elastic element) in series and/or in parallel. A simple model consisting of a spring in parallel with a dashpot is referred to variously as a Kelvin or Voigt element (Whorlow, 1992) or Kelvin-Meyer solid (Tanner, 2000) (Fig. 10). In contrast, a Maxwell element consists of a spring in series with a dashpot, which gives an exponentially decaying response to a suddenly applied constant strain (Fig. 11). Several models have been based on multiple Kelvin bodies in series, or Maxwell bodies in parallel, to simulate creep and stress relaxation, respectively, in viscoelastic solids (Whorlow, 1992); elements with a spectrum of time constants are employed in these models to approximate viscoelastic

(7

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Time

Figure 10 Kelvin model and its response to constant applied stress.

520

Rheology andTexture of Cheese

the force-displacement equations (Whorlow, 1992; Steffe, 1996).

Applied strain

of these models

Large strain deformation

Time Figure 11 A Maxwell model and its stress relaxation response to a constant applied strain.

behaviour (Fig. 12). Subramanian and Gunasekaran (1997b) showed that a model consisting of eight Maxwell elements could simulate the shear modulus over a wide dynamic range in low amplitude oscillation (0.1-20 Hz). Ma et al. (1996) showed that a six-element Kelvin model could simulate creep compliance in full-fat and reduced-fat Cheddar cheese. The Burgers body, which consists of a combination of Maxwell and Kelvin elements in series (Fig. 13), affords a close approximation to both the creep and stress relaxation behaviour of cheese. The mechanical representation of these models provides an intuitive guide to the nature of viscoelasticity and a simulation of rheological behaviour based on

(a)

(b)

rl Applied strain

9

89

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Figure 12 (a) Series of three Kelvin elements with a spectrum of time constants, which may be used to simulate creep and (b) A combination of Maxwell elements with a spectrum of time constants, which may be used to simulate relaxation behaviour in a viscoelastic solid.

Definitions and terminology Large strain measurement implies permanent deformation and measurement of non-linear rheological characteristics which are related to deformation of the microstructure. In contrast to linear viscocelastic deformation where applied strains are generally > 1). Consideration of the forces that are applied to cheese from manufacture to consumption, indicates a very broad range of deformation. In some situations, the strains are of a relatively low magnitude and do not result in visible damage (e.g., during ripening, transport, retailing), while in others the strain results in fracture (e.g., during portioning) or complete disintegration of the cheese mass (e.g., comminution, as in shredding, grating, grinding, as for example in the preparation of cheese ingredients and in the manufacture of processed cheese products and cheese powders). Hence, in the current context, large strain deformation is arbitrarily subdivided into two regions, i.e., large strain deformation-elastoplastic (LSD-E; e.g., strains ---0.1-0.5; Fig. 14), where deformation does not result in fracture and the structure can partially recover, and large strain deformation-fracture (LSD-F; 0.3-0.9), where the cheese mass undergoes fracture or disintegrates and cannot recover. In the following discussion, the LSD-E and LSD-F regions will be treated jointly (Fig. 14). Measurement using texture analyser Large strain deformation testing of cheese usually involves the application of strains (e.g., ~ "-- 0.8) that result in fracture, by compression of the cheese sample

I Typical linear ] visco-elastic limit j I I

j_ Applied strain

I

( Typical extentof "~ |compression to which | I cheese is subjected in| | chewing and in | (poiitTypical fracture I L compression testing ) '

Time

0

0.40

0.80 Strain, A U L

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o

Figure 14 Range of strain in compression tests on cheese.

1.0

Rheology andTexture of Cheese

between two parallel plates of a texture analyser (Culioli and Sherman, 1976; Dickinson and Goulding, 1980; Creamer and Olson, 1982; Tunick et al., 1991; Guinee et al., 1996; Fenelon and Guinee, 2000; Truong et al., 2002). The cheese sample is placed on a base plate and is compressed at a fixed rate (typically 20 mm/min -1) to a pre-determined level (e.g., 75% of its original height) by the mobile plate (cross-head). However, the rate of compression used in various studies has differed widely, e.g., 5-500 mm/min -1 (Table 3). The force (F) developed during compression is recorded as a function of distance (or displacement); alternatively, the force may be converted to o-and the displacement to g. The resultant o-versus g curves for a range of hard rennet-curd cheeses (Fig. 15) typically show a number of distinct regions and enable the determination of a number of rheological parameters: 9 A-B; or increases proportionally with ~. The slope of this linear region defines the compression modulus, E (i.e., E = o-/~), which is of little practical significance in relation to cheese behaviour during processing or consumption, where strains are > >0.05. However, in the commercial grading of cheese, E may be an indication of springiness (e.g., where a grader sensorically monitors the resistance to small deformation, as in pressing the thumb into the outside of the cheese block; the force applied during this hand deformation is typically 18 N or o---- 40 kPa). 9 B-C, o-increases less than proportionally with ~. The slightly lower slope of the curve in this region compared to that in A-B is probably due to the formation of microcracks that do not spread throughout the sample but which allow some stress to be dissipated; 9 C-D, the slope of the o-/~ curve decreases markedly. The cheese begins to fracture at C, as cracks grow and spread throughout the entire sample at an increasing rate. Eventually, at D the rate of collapse of the stress-bearing para-casein matrix overtakes the build-up of o-within the matrix through further compression and a peak or, denoted as the fracture stress, is reached. The fracture stress, o-f, and strain, ~f, are measures of the stress and strain, respectively, required to cause complete fracture of the sample. Strength, or fracturability, is defined as the stress required to fracture the sample (at D), while toughness, or fracture work, is defined as the area under the curve up to the point of fracture. 9 D-E, o- decreases with further compression due to the collapse of the stress-bearing structure. The decrease in o-may be attributable to: (i) shattering of the samples into pieces that spread over the base plate, resulting in an increased surface area and (ii) the probable loss of contact between some

521

of the pieces of cheese and the base plate which results in dissipation of stress energy stored within the individual pieces. E-E cr increases as the cross-head begins to compress the fragmented pieces of cheese. The o- at the end of the compression (point F) is a measure of firmness, as judged in the first bite of mastication (Sherman, 1969; van Vliet, 1991a). The various quantities obtained from the o - ~ curve and their interpretation are given in Table 2. The application of a strain to a segment of cheese (e.g., cube or cylinder) and monitoring the resultant cr by a texture analyser, as above, is a typical method for measuring the large strain deformation behaviour of cheese. However, many variations of both the procedure of stress or strain application, and the levels, are possible. A so-called apparent elastic modulus can be calculated at a strain well below the fracture point, e.g., ~ --- 0.1, as the ratio between or and ~. A preferred term for this parameter is modulus of deformability, as the deformation in question may include some plastic flow (Ak and Gunasekaran, 1995; Johnston, 2000). However, such a parameter needs to be interpreted with caution as some apparent initial deformation may occur before complete contact is made between the compression plate and the sample surface, an occurrence that could lead to erroneous values. Fracture and work of fracture. Rheological behaviour over such a range of ~ in the form of shear or compression, can be explored in several ways, such as applying a gradually increasing ~, a fixed ~, a defined o followed by its removal, a gradually increasing ~ up to a point followed by its reversal. Stress-strain cycles, often referred to as bites (analogous to compression between the molar teeth during mastication), may be repeated at interval(s) or applied in a given sequence (e.g., pre-test compression). Depending on the level of applied strain, cheese exhibits a combination of rheological behaviours, such as non-linear elastic (e.g., region B-C, Fig. 15), sometimes referred to as viscoelastic, or inelastic (e.g., region D-E, Fig. 15), sometimes referred to as plastic behaviour.

Rheological Measurements in Cheese: Sensoric Methods The methods used to assess the rheological characteristics of cheese may be broadly classified as sensoric or instrumental, where instrumental methods can be categorised further as empirical or fundamental. The aim of sensoric methods, which are performed routinely by cheese graders, is to acquire an impression

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0.1), especially >el, the distribution of stress and strain within the sample depends on sample dimensions, as the sample may be deformed into an irregular shape, due to fracturing, barreling and squeezing. Squeezing flow is an intrinsic aspect of large strain uniaxial compression of cheese, i.e., as sample height is reduced, the cheese spreads in a lateral direction (Fig. 24). This implies that shearing takes place within the sample and that frictional shear forces occur at the points of contact between the sample and the compression plates. Friction can be reduced by lubrication of the contact surfaces with mineral oil or grease; in contrast, surface friction can be increased by the use of emery paper, or the surfaces can be bonded using glue, both of which eliminate slippage as a result of cheese 'sweating'. Because lubrication allows lateral movement at the contact surfaces during compression, the sample shows a slight tendency towards an hour-glass shape, as opposed to the relatively large barreling effect. Lubrication can reduce the stresses in squeezing flow by as much as 50% and increase the observed ef from - 0 . 4 5 to 0.55, in the case of Gouda cheese at 20 ~ with an aspect ratio (i.e., height/width) of unity and a cross-head speed of 500 mm/min (Culioli and Sherman, 1976). However, the frictional effect increases with cross-head speed. At a low cross-head speed (5 mm/min), lubrication decreased of b y - 2 0 % in Cheddar cheese where the aspect ratio was 0.35, with the effect becoming more pronounced (of the order of 20-30%) at e > ef (Casiraghi et al., 1985). In contrast, the bonding of the cheese surfaces to the compression plates (e.g., using cyanoacrylate ester adhesive) had relatively little effect on of, ef and O'max. A similar trend was found for Mozzarella and processed cheese spread (Casiraghi et al., 1985). At low compression plate speeds ( < 2 0 mm/min), friction had only a negligible effect in Gruyere and processed Mozzarella cheese with aspect ratios near unity (Charalambides et al., 2001). However, at aspect ratios 0.1; cf. Fig. 9).

Effect of Sample Temperature on Large Strain Deformation Characteristics in Cheese Early research showed that increasing the temperature of Gouda cheese in the range 10-20 ~ reduced the value of el, o-f and O'max, as measured by compression to 80% using the Instron UTM (Culioli and Sherman, 1976). While o'f in Cheshire and Leicester cheeses decreased exponentially as the temperature was increased from 0 to 40 ~ the effect on fracture strain depended on the type of cheese; fracture strain for Cheshire cheese increased by ---2 over the range of temperature, while fracture strain for Leicester cheese was not affected by temperature (Dickinson and Goulding, 1980). Molander et al. (1990) reported a similar trend for o-f and O'max in 4-week-old Brie between 5 and 20 ~ however, in contrast to the results of Culioli and Sherman (1976), ef increased slightly on raising the temperature. The discrepancy between the latter studies in relation to strain may be attributable to differences in the degree of fat coalescence, proteolysis and therefore fat separation and slippage. On heating cheese to a temperature (30-60 ~ greater than those (e.g., 4-25 ~ normally associated with retailing, domestic refrigeration and consumption, compression results in squeezing flow behaviour (Ak and Gunasekaran, 1995), i.e., stress increases with strain as the cheese is squeezed between the plates and no fracture point is observed. The deformability modulus (initial slope of the stress-strain curve) showed an Arrhenius type of characteristic, decreasing exponentially with temperature from 18 kPa at 30 ~ to 3 kPa at 60 ~ Such a trend is expected, as milkfat is essentially fully liquid at 30 ~ (Norris et al., 1973). Indeed, heating cheese to 60 ~ in the absence of an applied stress generally results in flow of the part-molten cheese mass to an extent dependent on cheese type and heating time.

Techniques for Measurement of Viscosity In some situations, cheese products may occur in 'liquid' form, either in the course of processing or in their usage. Typical examples are processed cheese, cheese dips and cheese sauces. The viscosity of these products may be measured by a number of instruments, e.g., the

Rheology andTexture of Cheese

Bostwick consistometer, which has been used to give an empirical measurement of viscosity of a soft processed cheese spread (Rosenthal, 1999). In the latter instrument, a sample of the material being tested is placed in a cell and released by opening a simple guillotine slide gate, allowing the product to flow horizontally across a scale marked in centimeters. The length of flow in a given time period (usually 30 s), known as the Bostwick number, is taken as a measure of viscosity. Alternatively, viscosity can be measured under defined shear or low amplitude stress or strain in a rheometer, using different geometries such as concentric cylinders, a cone and plate, or parallel plates. Online measurements of viscosity of cheese products may be important, e.g., as an early measure of indicating the susceptibility of a formulation to 'creaming' (see 'Pasteurized Processed Cheese and Substitute/Imitation Cheese Products', Volume 2). A range of commercial on-line viscometers are available for measuring viscosity in a continuous flow situation.

Terminology Applied to Cheese Texture Cheese texture may be defined as a composite sensory attribute resulting from a combination of physical properties that are perceived by the senses of touch (including kinaesthesis and mouth-feel), sight and hearing (Brennan, 1988). Thus, cheese texture is directly measurable only by sensory analysis. Sensory analysis requires definition and classification of textural attributes or descriptors. Descriptors applied to cheese texture have been grouped into mechanical, geometrical and other characteristics (Fig. 31). The mechanical characteristics are sensed as forces on the teeth, tongue and the mouth

generally during eating, and by hearing in the case of fracture, whereas geometrical characteristics are mostly sensed visually but may also be partly sensed by touch. The other characteristics are 'mouth-feel' qualities, described subjectively by terms such as hard, soft, firm, springy, crumbly, adhesive, moist or dry. These terms are thought to have significance in relation to consumer appeal and satisfaction (Szczesniak, 1963a). The mechanical characteristics, in turn, have been divided into five primary parameters and three secondary parameters (Table 5, Fig. 31). The secondary parameters are considered to be composed of various intensities of hardness and cohesiveness. The geometrical parameters are divided into two classes, i.e., those related to particle size and hardness, and those related to particle shape and orientation. Experience shows that panelists found hardness relatively easy to sense but that adhesiveness was much more difficult to judge (Halmos, 2000). Sensory texture terms, as distinct from rheological terms, have linguistic boundaries, i.e., they are susceptible to different interpretation in different languages (Lawless et al., 1997; Bourne, 2002). Some texturerelated characteristics can be measured by machines and these are not bound by language. These characteristics include hardness, cohesiveness, adhesiveness, elasticity, viscosity, brittleness, chewiness and gumminess, definitions for which are given in Table 5. The measurements give objective quantifiable data, provided the measurement conditions are well defined. Relationships between cheese texture and rheology

The Texture Profile Analysis (TPA) method, involving instrumental measurement using double bite compression, was developed to imitate the compressing action 9 visual appearance 9 sampling and slicingcharacteristics 9 spreading, creaming characteristics, pourability

Initial perception { (before placing in mouth)

9 analyticalcharacteristics

9 particle size, shape and size distribution 9 oil content; size, shape and size distribution of oil

Primary characteristics Initial perception on palate

particles 9 elasticity, cohesion 9 viscosity 9 adhesion (to palate)

l Secondary characteristics I

Mastication (high shearing

9 9 9 9 9

I Ze.iar characteristics

stress) Residual

masticatory impression

Figure 31

f

533

9

hard, soft brittle, plastic, crisp, rubbery, spongy smooth, coarse, powdery, lumpy, pasty creamy, watery, soggy sticky, tacky

9 greasy, gummy, stringy 9 melt down properties on palate

Classificationof food texture into primary, secondary or tertiary characteristics, based on Sherman (1969).

534

Rheology andTexture of Cheese

Table 5

Classification of the mechanical characteristics of cheese into primary and secondary parameters a

Primary parameters

Secondary parameters

Hardness - the force necessary to attain a given deformation Cohesiveness - strength of internal bonds making up the body of the product Elasticity- the rate at which a deformed material returns to its original form after the deforming force is removed Viscosity- rate of flow per unit force Adhesiveness - the work necessary to overcome the attractive forces between the surface of a food and surface of other materials with which it comes in contact, e.g., the teeth, palate and tongue

Brittleness - the force at which the material fractures Chewiness - the energy required to masticate a solid food, e.g., some cheeses such as Mozzarella, to a state ready for swallowing Guminess - the energy required to disintegrate a semi-solid food, e.g., some cheeses such as ripe Camembert, to a state ready for swallowing

a Modified from Szczesniak (1963a), Bourne (1978).

of molar teeth while masticating food in the mouth (Szczesniak, 1963a; Peleg, 1976; Bourne, 1978). Classification of the mechanical attributes of cheese texture, as described above, was designed with the aim of integrating sensory data for foods evaluated by trained panels, with texture-profile data obtained on the same foods using compression testing. For this purpose, objective rheological parameters, some of which correspond in name to the sensory-determined parameters, were defined (Table 5) and are known as TPA parameters (see 'Texture profile analysis'). While this classification system has been modified, the textural descriptors and their interpretation as devised by this classification scheme (Table 5) are still widely used in textural evaluation of food (Brennan, 1988; Drake et aI., 1999). Sherman (1969) proposed an alternative classification of food texture (Fig. 31). The characteristics contributing to the texture of cheese, and other foods, during eating have been classified as primary, secondary (e.g., adhesiveness) or tertiary (e.g., firmness) (Fig. 31). The primary characteristics, from which all others are derived, include the food's composition, its micro- and macro-structure, and its molecular properties. The secondary and tertiary categories of textural properties include many characteristics which are directly related to the rheological properties as it is subjected to various stresses and strains during eating, e.g., hardness, brittleness and adhesiveness (Sherman, 1969). According to this classification, the secondary characteristics are associated with initial perception in the mouth, i.e., upon contact with tongue, palate and teeth prior to mastication. Sherman (1969) claimed that the main characteristics sensed at this stage are elasticity (E), viscosity (r/) and adhesion to the palate, where elasticity is understood as the tendency to recover its shape after removal of the stress. Two of those characteristics, namely elasticity and viscosity, can together be represented by the Burgers mechanical model (see 'Cheese texture').

Texture profile analysis (TPA)

A system of rheological parameters (e.g., firmness, elasticity) related to texture and known as TPA was developed (Fig. 32; Table 6; Friedman et al., 1963). The rheological measurements were originally carried out using the General Foods Texturometer (see 'Imitative tests'), using double-bite compression (Bourne, 1978). Texture profile analysis parameters were later calculated from measurements using uniaxial doublebite compression at constant speed, using texture analysers including the Instron UTM (Breene, 1975; Bourne, 1978; Lee etal., 1978) and the texture analyser (TA series from Stable Micro Systems) (Halmos, 1997; Meullenet and Gross, 1999). Use of TPA to evaluate cheese texture

Szczesniak (1963b) found a curvilinear relationship between TPA hardness and an organoleptic rating of hardness. Casiraghi et al. (1989), working with five different Italian cheese varieties, including Grana Padano and Italico, showed that sensory hardness was highly correlated with instrumental hardness.

1st compression stroke

1

2nd compression stroke

b

0

Time

Figure 32

Typical stress trend during a double-bite compression test, from which TPA parameters are calculated (see Table 5).

Rheology andTexture of Cheese

Table 6

535

Texture profile analysis (TPA) parameters and physical definitions a

Terminologyb

Physical definition (TPA term)

Units

Fracturability Firmness Springiness (or elasticity) Cohesiveness Gumminess Chewiness Adhesiveness

Stress (or sometimes, force) to fracture point, H1 (Fig. 32) Stress (or sometimes, force) at a given deformation Percentage of deformation which is recovered between the first and second bites

Pa, kPa Pa or kPa

Area of second bite over area of the first bite (A2/A1) in Fig. 32 Hardness • Cohesiveness Hardness • Cohesiveness x Springiness Work necessary to pull the plunger (or compression plate) away from the sample (Area 3 in Fig. 32)

m

Pa, kPa Pa, kPa J/m3

a Sources: Bourne (1978), van Vliet (1991a), Szczesniak (1963a), Yang and Taranto (1982). b Fracturability was originally known as brittleness (Bourne, 1978), and firmness as hardness (Szczesniak, 1963a).

Green et al. (1985) found significant correlations between five sensory attributes (firmness, springiness, crumbliness, graininess and stickiness) and instrumental parameters (of and ef ). Hennequin and Hardy (1993) reported that TPA-hardness, i.e., force at 70% compression, also had a high correlation with sensory hardness (r = 0.78, n = 19, P < 0.001) for four soft cheeses. Halmos (2000) compared sensory and instrumental measurements of hardness, cohesiveness and adhesiveness of six cheeses with a wide range of texture (including Havarti, Swiss and Romano). The sensory measurements increased with the corresponding instrumental readings, apart from one parameter for Romano cheese, for which the cohesiveness as measured instrumentally was ranked higher than the corresponding sensory measurement. The significant correlations, which were characteristic of the overall study, confirm the value of objective measurements in support of sensory measurements. However, the deviation in the trend for the Romano cheese highlights the complexity of textural (i.e., tactile sensory) characteristics as compared with instrumental measurements. Antoniou et al. (2000) performed sensory and TPA analyses on 15 French cheeses (Munster, Emmental, Roquefort, Beaufort, Camembert, Reblochon, Pont l'Eveque, Brie de Meaus, Tomme de Savoie, Valencay, St Nectaire, Pyrenees Brebis, Blue d'Auvergne, Comte Vieux and Fourme de Salers). The cheeses fell into three compositional groups on the basis of moisture (means 34, 45 and 51%, w/w). This grouping carried through to the results of mechanical and sensory analysis. The mechanical (TPA) terms which were most significant in differentiating the groups were: force at 10% deformation, relaxation force (after holding sample for - 1 2 s at 10% compression), force at 80% deformation (hardness), fracture force and adhesiveness. The most significant sensory terms were: hardness, fracturability and chewiness. Some of the mechanical parameters were highly correlated with

each other (e.g., force at 10% deformation, fracture force and hardness). Likewise, some of the sensory parameters were inter-correlated, e.g., hardness with adhesiveness. In agreement with previous studies (Green et al., 1985; Casiraghi et al., 1989; Hennequin and Hardy, 1993; Halmos, 2000), sensory parameters were highly correlated with mechanical parameters, e.g., mechanical hardness with sensory hardness. It is noteworthy that the 10% compression measurements (a level of deformation that is mostly recoverable) predicted cheese texture (i.e., as judged in sensory terms) better than the 80% compression tests. Despite the significant correlations between some sensory textural parameters and rheological measurements, instrumental analysis of texture, e.g., using texture analysers, is not considered a complete substitute for sensory evaluation (see Halmos, 2000), because of several factors: complexity of mastication, differences between individuals in the perception of texture, effect of time of day upon perception of texture, and others. While instrumental methods alone cannot be relied upon to determine consumer acceptance, their value resides in their ability consistently to enable small changes in physical characteristics, which contribute to texture, to be quantified. Use of instrumental shear deformation to evaluate cheese texture

Three techniques for large strain shear deformation testing have been described (see 'Large strain shear measurements'). Truong et al. (2002) compared instrumental textural measurements on Cheddar cheese, as obtained using vane rheometry (shear), uniaxial compression (single bite) or TPA (double bite), with the corresponding sensory texture measurements. Instrumental texture maps of ten commercial Cheddar cheeses, generated by the vane method and by compression testing, clearly separated the cheeses and showed similar distribution patterns. Highly significant

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correlations were found between vane parameters and TPA parameters (i.e., by uniaxial compression), and between TPA parameters and sensory texture parameters (by mouth). Correlations between vane parameters and sensory parameters were significant, but not as highly significant as between sensory and TPA parameters. The higher correlation between sensory texture and TPA texture could be due to the fact that TPA parameters were developed in conjunction with compression (i.e., General Foods Texturometer), while no corresponding texture-related parameters have been developed for torsional techniques, such as the vane method.

Conclusions The rheological properties of cheese have a large influence on its texture and behaviour during size reduction, and hence, its suitability as an ingredient (see 'Cheese as an Ingredient', Volume 2). Many factors influence the rheological properties, including m a n u facturing procedure, variety, composition and biochemical changes during ripening. The latter parameters have a major influence on the degree of hydration, or aggregation, of the para-casein matrix, which is the major structural element controlling deformation on the application of a stress. Many m e t h o d s are available for measuring the theological properties of cheese; some measure within the linear viscoelastic range to yield precise rheological quantities. In contrast, rheological m e a s u r e m e n t s made u n d e r large strain or stress yield quantities which are more empirical in nature, but which are typically related to the stresses and strains experienced during c o n s u m p t i o n and size reduction.

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Guinee, T.P., O'Callaghan, DJ., Mulholland E.O. and Harrington, D. (1996). Milk protein standardization by uhrafiltration for Cheddar cheese manufacture. J. Dairy Res. 63,281-293. Guinee, T.P., Fenelon, M.A., Mulholland, E.O., Kennedy, B.T., O'Brien, N. and Reville, W.J. (1998). The influence of milk pasteurization temperature and pH at curd milling on the composition, texture and maturation of reduced fat Cheddar cheese. Int. J. Dairy Technol. 51, 1-90. Guinee, T.P., Auty, M.A.E. and Mullins, C. (1999). Observations on the microstructure and heat-induced changes in the viscoelasticity of commercial cheeses. Aust. J. Dairy Technol. 54, 84-89. Guinee, T.P., Auty, M.A.E., Mullins, C., Corcoran, M.O. and Mulholland, E.O. (2000a). Preliminary observations on effects of fat content and degree of fat emulsification on the structure-functional relationship of Cheddar-type cheese. J. Text. Stud. 31,645-663. Guinee, T.P., Auty, M.A.E. and Fenelon, M.A. (2000b). The effect of fat content on the rheology, microstructure and heat-induced functional characteristics of Cheddar cheese. Int. Dairy J. 10, 277-288. Guinee, T.P., Feeney, E.P., Auty, M.A.A. and Fox, RE (2002). Effect of pH on calcium concentration on some textural and functional properties of Mozzarella cheese. J. Dairy Sci. 85, 1655-1669. Hall, D.M. and Creamer, L.K. (1972). A study of the submicroscopic structure of Cheddar, Cheshire and Gouda cheese by electron microscopy. NZ J. Dairy Sci. Technol. 7, 95-102. Halmos, A.L. (1997). Food texture and sensory properties of dairy ingredients. Food Aust. 49, 169-173. Halmos, A.L. (2000). Relationships between instrumental texture measurements and sensory attributes, in, Hydrocolloids - Part 2, Nishinari, K., ed., Elsevier, Amsterdam. pp. 431-444. Hennequin, D. and Hardy, J. (1993). Evaluation instrumentale et sensorielle de certaines proprietes texturales de fromage/t pate molle. Int. Dairy J. 3,635-647. Home, D.S., Banks, J.M., Leaver, J. and Law, A.J.R. (1994). Dynamic mechanical spectroscopy of Cheddar cheese, in, Cheese Yield and Factors Affecting its Control. Special Issue No. 9402. International Dairy Federation, Brussels. pp. 507-512. Hort, J. and LeGrys, G. (2000). Rheological models of Cheddar cheese texture and their application to maturation. J. Text. Stud. 31, 1-24. Hwang, C.H. and Gunasekaran, S. (2001). Measuring crumbliness of some commercial Queso Fresco-type Latin American cheeses. Milchwissenschaft 56,446-450. Imoto, E.M., Lee, C.-H. and Rha, C. (1979). Effect of compression ratio on the mechanical properties of cheese. J. Food Sci. 44,343-345. Innocente, N., Pittia, R, Stefanuto, O. and Corradini, C. (2002). Correlation among instrumental texture, chemical composition and presence of characteristic holes in a semi-hard Italian cheese. Milchwissenschaft 57, 204-208.

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Glossary Cauchy strain. See Engineering strain. Compliance. Symbol J, is the ratio of strain to stress. In the elastic region, J = 1/G', where G' is shear (or storage) modulus. Cox-Merz rule. This rule states that the (steady) viscosity versus shear rate curve is virtually identical to the viscosity versus frequency curve, determined by dynamic oscillation. Creep. The response to a constant applied (normal or shear) stress. Creep can be expressed in terms of strain or compliance. Creep compliance. The ratio of strain, y(t), resulting from an applied constant stress, Zc, to the stress, i.e., 7(t)/~'c. Creep modulus. The inverse of creep compliance, i.e.,

~-d~,(t). Deformability modulus. Slope of the stress-strain curve in an approximately linear region, typically up to a strain of ~0.10. Elastic material behaviour. An elastic deformation is one where the material recovers fully u p o n removal of applied stress without time dependency, i.e., recovery is instantaneous and complete u p o n removal of stress. Elastoplastic material behaviour. W h e n the stress in the material exceeds a certain limit, irreversible deformation results with negligible time dependency, i.e., partial recovery is instantaneous upon removal of stress; also k n o w n as elastoplastic deformation. Engineering strain. Deformation relative to original sample dimension, i.e., AL/Lo, is called engineering strain, or Cauchy strain, or strain. Engineering stress. The ratio between applied force, F, and original sample area, Ao, is k n o w n as engineering stress or stress. Fracture work. See Toughness. Kelvin element. Also k n o w n as a Voigt element or a Kelvin-Meyer solid. This is a mechanical model consisting of a spring in parallel with a dashpot. A number of such elements in series, with a spectrum of time constants, can be used to simulate creep compliance. Kinematic viscosity. This is the ratio between dynamic viscosity and density. Units: m2/s = 10 4 stokes. Linear behaviour. If one measured parameter varies in proportion to another, e.g., stress in proportion to a range of applied strain, their behaviour is described as linear and a modulus may be defined as the ratio between the parameters, e.g., Young's modulus. Linear viscoelastic deformation. Cheese and other organic materials exhibit a combination of elastic and viscous behaviour at low strains, i.e., they recover their shape upon removal of applied stress, but not instantly. The elastic and viscous effects can be determined using

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Rheology andTexture of Cheese

low-amplitude oscillatory rheometry. At low amplitudes of oscillation there is a constant relationship between the elastic and viscous components of complex modulus. Consequently, displacements of this type are referred to as linear viscoelastic deformation. Loss modulus. The ratio between the out-of-phase component of shear stress and shear strain (G" = r"lT) in a dynamic oscillatory measurement; also referred to as viscous modulus. Maxwell element. This is a mechanical model consisting of a spring in series with a dashpot. A combination of such elements in parallel, with a spectrum of time constants, may be used to simulate relaxation behaviour in a viscoelastic material. Modulus of deformability. See Deformability modulus. Poisson effect. W h e n a sample is compressed it bulges in the lateral direction, i.e., the cross-section increases with compression; this is the Poisson effect. The ratio between lateral strain and longitudinal strain is k n o w n as Poisson's ratio. Poisson's ratio equals 0.5 in the absence of a volume change, and is less than 0.5 for a compressible material. Shear modulus. The ratio between the in-phase components of shear stress and shear strain (G' = r'/T) in a dynamic oscillatory measurement; also referred to as storage, elastic, or in-phase, modulus. Storage modulus. See Shear modulus. Strength. The m a x i m u m stress a material withstands before it breaks (i.e., fractures) or flows (i.e., becomes plastic). Stress. See Engineering stress. Stress relaxation m o d u l u s . The stress that is required to maintain a constant deformation is observed, as a function of time (i.e., in a stress relaxation test). The ratio of shear stress to strain is k n o w n as stress relaxation modulus, or relaxation modulus. The relaxation modulus depends on the applied strain if the strain exceeds the limit of linear viscoelasticity. Thus, G(t, T) = r(t)/% Stress relaxation test. This test involves an initial application of (a normal or shear) strain at a constant rate up to a pre-determined level of strain and then measuring

the decay of stress as a function of time while holding the sample at constant strain; also k n o w n as a step strain transient test. Toughness. The work required to fracture; this is measured as the area under a force-deformation curve up to the point of fracture (Fig. 23). True strain. The accumulated strain during the applied loading, e' = In (ULo), where In is the natural logarithm, L is the sample length under load, and Lo is the original sample length, is k n o w n as the true strain, Hencky strain or natural strain. This is applicable where the strain is large and sample cross-section changes appreciably under the load. True strain is not used very much in cheese rheology. True strain can be related to engineering strain, e, using, e' = In (1 + e). True stress. The ratio between applied force, F, and actual area of cross-section, A', is termed true stress. Thus, O'true FIA', where A' is the actual area, taking the Poisson effect into account. Uniaxial compressive strength. The apparent stress at fracture, i.e., Fo/Ao, where Fo is the compression force at fracture and Ao is the initial cross-sectional area of the sample. Viscoelastic material behaviour. Where rheological behaviour can be resolved into elastic and viscous components, e.g., as represented by a Maxwell model. Viscoplastic material behaviour. In contrast to elastic behaviour, this is a time-dependent and irreversible deformation that occurs when a certain stress level has been exceeded, i.e., strain does not respond instantaneously to applied stress, but instead strain keeps on growing while the stress is applied and does not return to zero upon removal of stress; also referred to as viscous material behaviour. Viscosity or dynamic viscosity. Coefficient of dynamic viscosity, 77, is the ratio between shear stress and shear rate. = rl~ where r is shear stress and ~ is shear rate. Units: Pa.s or N.s/m 2 = 10 poise. Viscous modulus. See Loss modulus. Young's modulus. The ratio between normal stress and engineering strain (E = ole). =

Growth and Survival of Microbial Pathogens in Cheese C.W. Donnelly, Department of Nutrition and Food Science The University of Vermont, Burlington, USA

Introduction Cheesemaking evolved centuries ago as a means of preserving raw milk via fermentation. Selection of the beneficial natural flora in milk, such as lactobacilli, streptococci and lactococci, or direct addition of these as starter cultures, preserves products and in many instances allows competition with bacterial pathogens. However, cheeses can become contaminated with pathogens as a result of their presence in the raw milk used for cheesemaking and subsequent survival during the cheesemaking process. Alternatively, bacterial pathogens can contaminate cheese via post-processing contamination if sanitation and other measures in the processing plant are not sufficient to prevent re-contamination (Linnan et al., 1988; Johnson et al., 1990a). The characteristics of the specific cheese variety will dictate the potential for growth and survival of microbial pathogens, with ripened soft cheeses presenting a higher risk for growth and survival of pathogens than aged hard cheeses where a combination of factors, including pH, salt content and aw, interact to render cheeses microbiologically safe. Although cheeses have been linked with documented outbreaks of food-borne illness, epidemiological evidence collected from around the world confirms that this occurs infrequently (Johnson et al., 1990a; Ahekruse et al., 1998; De Buyser et al., 2001). This chapter will provide an overview of factors which affect growth and survival of microbial pathogens in cheese.

Factors that Influence the Safety of Cheese The pathogens, Salmonella enterica, listeria monocytogenes, Staphylococcus aureus and enteropathogenic E. coli (ETEC) pose the greatest risk to the safety of cheese 0ohnson et al., 1990a; De Buyser et al., 2001; Leuschner and Boughtflower, 2002). If active lactic acid starter cultures are used, Staph. aureus is considered to be a low-risk pathogen (Johnson et al., 1990a). However, in traditional cheeses where active starter cultures are not used, Staph. aureus may pose a significant risk for toxin production in cheese if numbers are sufficiently high. The factors that contribute to

the safety of cheese with respect to pathogenic bacteria include milk quality, starter culture or native lactic acid bacterial growth during cheesemaking, pH, salt, control of aging conditions and chemical changes that occur in cheese during aging (Johnson et al., 1990c). Other technologies (e.g., use of starter cultures that produce substances inhibitory to pathogens) may provide opportunities to add additional barriers to the growth of bacterial pathogens. It is particularly important for the producers of raw milk cheeses to have a documented and systematic approach to ensure product safety. Pathogens in raw milk

S. enterica, L. monocytogenes, Staph. aureus and ETEC are associated with raw milk. E. coli 0157:H7 can readily contaminate raw milk on the farm with contamination levels of 4.2-10% and 2% reported in the US and Canada, respectively (D'Aoust, 1989; Padhye and Doyle, 1991). Over 70 cases of E. coli infection, characterized by bloody diarrhea, haemolytic uremic syndrome (HUS) and kidney failure, have been traced to the consumption of raw milk (Martin et al., 1986; Borczyk et al., 1987; Bleem, 1994) with a few additional cases in England linked to yoghurt (Morgan et al., 1993). E. coli 0157:H7 was first characterized in 1982 during epidemiological investigations of two outbreaks which occurred in North America. Cattle are thought to be the principal reservoir for this important human pathogen, and in investigations where food has been identified as the vehicle of transmission, ground beef is the product most frequently linked to human illness. Shere et al. (1998), in a longitudinal study of E. coli dissemination on four Wisconsin dairy farms, identified contaminated animal drinking water as the most probable vehicle for infection of animals and a potential intervention point for on-farm control of dissemination of this pathogen. Since shedding of this pathogen by cattle is intermittent, re-inoculation from an environmental source rather than colonization of the pathogen is the more likely explanation than intermittent shedding.

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

Copyright 9 2004 Elsevier Ltd All rights reserved

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Growth and Survival of Microbial Pathogens in Cheese

S. enterica serovars Enteritidis, Typhimurium and Dublin have been associated with food-borne disease outbreaks involving raw milk and milk products (Maguire et al., 1992; Cody et al., 1999; Villar et al., 1999; De Valk etal., 2000). A 1987 FDA survey revealed the presence of salmonella in 32 of 678 (4.7%) samples of raw milk obtained from bulk-tank trucks in Wisconsin, Michigan and Illinois, with 10 of 16 (62.5%) collection sites also testing positive (McManus and Lanier, 1987). Salmonella spp. were isolated from 26 of 292 (8.9%) of farm bulk tank samples collected in eastern Tennessee and southwest Virginia (Rohrbach et al., 1992). Wells et al. (2001) examined recovery of salmonella from faecal samples obtained from dairy cows in 91 herds from 19 US states. Salmonella spp. were recovered from 5.4% of the samples. Recovery levels from cows on farms with less than 100 animals were much lower (0.6%) than those from farms with over 100 cows, where recovery levels were 8.8%. The incidence of Salmonella spp. in milk samples would be expected to occur at a much lower frequency than in faecal samples. Most farmstead cheesemakers maintain small dairy herds, where the lower incidence data would apply. S. enterica serotype Typhimurium definitive type (DT) 104 emerged in the UK as an important source of human infection in the late 1980s (Threlfall et al., 1996). Subsequent outbreaks of human illness traced to dairy sources were reported in Vermont (Friedman et al., 1998), Nebraska, California (Cody et al., 1999) and Washington State (Villar et al., 1999). This organism is notable because it is resistant to multiple antibiotics. Two outbreaks of S. enterica subsp, enterica serotype Typhimurium DT104 infection were recently linked to the consumption of Mexican-style soft cheese manufactured from raw milk (Cody et al., 1999; Villar et al., 1999). Aceto et al. (2000) conducted a survey to assess the herd prevalence of 5. enterica subsp, enterica serotype typhimurium DT 104 in Pennsylvania dairy herds. Of 51 farms surveyed, 11 were positive for salmonella species and 4 for 5. typhimuriurn, 2 of which were DT-104 positive. S. enterica serovar Dublin is present in dairy cattle and was identified as the most invasive of the salmonella bacteria for humans in studies conducted in Denmark (Lester et al., 1995). Beckers et al. (1987) and Lovett et al. (1987) estimate that extremely low levels of L. monocytogenes (0.5-1.0 ml) exist in commercial bulk-tank raw milk. Listeria is inactivated by pasteurization, and contamination of processed dairy products is therefore most likely a function of post-pasteurization contamination from the dairy plant environment. In fact, numerous surveys document the presence of listeria within the dairy plant environment, including floors in coolers, freezers, processing rooms, particularly entrances, cases

and case washers, floor mats and foot baths and the beds of paper fillers (Charlton et al., 1990; Klausner and Donnelly, 1991). Pritchard et al. (1994), in a study of dairy processing facilities, found that processing plants near a farm had a significantly higher incidence of listeria contamination than those without an on-site dairy farm. Arimi et al. (1997) demonstrated the link between on-farm sources of listeria contamination (dairy cattle, raw milk and silage) and subsequent contamination of dairy-processing environments. These investigators subjected listeria strains collected from farms and dairy processing environments over a 10-year period to strain-specific ribotyping using the automated Riboprinter T M microbial characterization system. A total of 388 listeria isolates from 20 different dairy processing facilities were examined along with 44 silage, 14 raw-milk bulk tank and 29 dairy cattle isolates. These 475 isolates included 93 L. monocytogenes, 362 L. innocua, 11 L. welshimeri, 6 L. seeligeri, 2 L. grayii and 1 L. ivanovii strains. Thirty-seven different listeria ribotypes (RTs) comprising 16 L. monocytogenes (including five known clinical RTs responsible for food-borne listeriosis), 12 L. innocua, 5 L. welshimeri, 2 L. seeligeri, 1 L. ivanovii and 1 L. grayii were identified. Greatest diversity was seen among the isolates from dairy-processing facilities with 14 of 16 (87.5%) L. monocytogenes RTs (including 5 clinical RTs), and 19 out of 21 (90.5%) non-L, monocytogenes RTs detected. Sixty-five of the ninety-three L. monocytogenes isolates belonged to the group of the five clinical RTs, which included one RT unique to dairy-processing environments, two RTs common to dairy-processing environments and silage, and one RT common to dairyprocessing environments, silage, raw milk and dairy cattle with the last RT appearing in dairy-processing environments, silage, raw-milk bulk tanks and dairy cattle. The finding of eight L. rnonocytogenes and twelve non-L, monocytogenes RTs common to both dairyprocessing and farm environments clearly implicates the farm as a natural reservoir for listeria RTs capable of entering dairy-processing facilities. These findings, which support the link between on-farm sources of listeria contamination (dairy cattle, raw milk and silage) and subsequent contamination of dairy-processing environments, stress the importance of farm-based Hazard Analysis and Critical Control Points (HACCP) programmes for controlling listeria. This work also showed that two important clinical L. monocytogenes ribotypes which were previously identified as RT 19092 and RT 19161 and epidemiologically linked to listeriosis cases involving pasteurized milk and turkey frankfurters were recovered from dairy-processing facilities A and B for 12 and 3 months, respectively, with L. innocua RT 19094 also present in these same two facilities for at least five years.

Growth and Survival of Microbial Pathogens in Cheese

Abou-Eleinin et al. (2000) analysed 450 goats' milk samples obtained from the bulk tanks of 39 goat farms for listeria spp. over a 1-year period. Modified versions of the USDA-FSIS (McClain and Lee,1989) and FDA (Lovett et al., 1987) protocols were used for recovery of listeria. Overall, 35 (7.8%) samples yielded listeria, with L. monocytogenes identified in 17 of the 35 (3.8%) listeria-positive samples and L. innocua in 26 (5.8%) of samples. Eight milk samples contained both L. monocytogenes and L. innocua. Milk samples from 18 of the 39 (46.2%) farms were positive for listeria at least once during the year-long study. Five different listeria RTs were identified from 34 selected L. rnonocytogenes isolates, 2 of which were deemed to be of clinical importance. Isolation rates of listeria were markedly higher during the winter (14.3%) and spring (10.4%), compared to autumn (5.3%) and summer (0.9%). Similar trends have been previously reported for cows' milk (Reaet al., 1992; Ryser, 1999). Milk quality

Raw-milk quality is important in producing all cheeses, but particularly for those made from raw milk. Low bacterial counts and low somatic cell counts are the key indicators of milk quality, and as their numbers increase, there is a higher risk for contamination of milk and cheese with pathogens. Monitoring and controlling bacteria and somatic cell counts in milk should be components of a HACCP programme to ensure product safety. As rapid, cost-effective methods become available for detection of bacterial pathogens in raw milk, the use of specific pathogen testing could become part of a HACCP programme. In general, when rawmilk bacteria and somatic cell counts are high, there will be other negative impacts on cheese quality that may reduce consumer acceptability and cheese yield. In most artisanal cheesemaking, the time from milking to cheesemaking is very short and in some cases the milk is made into cheese immediately on the farm without cooling. Minimizing the time from milk collection to the initiation of cheesemaking reduces the opportunity for the growth of undesirable bacteria in raw milk. Conversely, when milk is cooled and held in transport, the opportunity for pathogen growth, particularly growth of psychrotrophic pathogens, is increased. The European Community Directives 92/46 and 92/47 (Anonymous, 1992) contain regulations for the hygienic production and placing on the market of raw milk, heat-treated milk and milk-based products. These regulations establish hygienic standards for raw-milk collection and transport that focus on issues such as temperature, sanitation and microbiological standards, enabling the production of raw milk of the highest

543

possible quality. Raw cows' milk must meet quality standards, e.g., a standard plate count at 30 ~ of < 100 000 cfu/ml and somatic cell counts of -64.5 ~ (148.2 ~ for 16.2 sec (Line et al., 1991) which is similar to that required for most salmonella except S. sen ftenberg. Much of the aged raw milk cheese produced in the US is subjected to some form of heat treatment, generally thermization. This treatment generally consists of heat treatment at 55 ~ for a period ranging from 2 to 16 sec. The specific impact of this heat treatment combined with the interactive effects of salt and pH during subsequent ripening on pathogens such as listeria, salmonella and E. coli has not been well explored.

Extrinsic and intrinsic parameters in cheese which dictate microbial growth

Growth of microbial pathogens in cheese is dictated by extrinsic and intrinsic parameters. The important intrinsic parameters include moisture content, pH and acidity, nutrient content, redox potential, presence of antimicrobial compounds, either those occurring naturally or those which are added as food preservatives, e.g., NOs, and the presence of competitive microflora (ICMSE 1986). All of these factors dictate the potential for bacterial pathogens to grow, persist or decline in cheeses. Extrinsic parameters include factors such as type of packaging/packaging atmosphere, time and temperature of storage and holding conditions, processing steps, product history and traditional use. The interaction of these factors dictates the potential for microbial growth in cheese. Depending on the cheese variety, intrinsic parameters such as pH may serve to enhance or inhibit the growth of bacterial pathogens. Ryser and Marth (1987a) studied the behaviour of L. monocytogenes in Camembert cheese. The high moisture content and the neutral pH of this surface-ripened cheese facilitate growth and survival of pathogens such as listeria. Growth of listeria in Camembert cheese was found to parallel the increase in cheese pH during ripening and reached a final population of 106-108 per g. This contrasts with Blue cheese, where listeria failed to grow and decreased in number during

Growth and Survival of Microbial Pathogens in Cheese

56 days of storage (Papageorgiou and Marth, 1989). These authors suggested that Penicilliurn roqueforti may produce bacteriocins against L. monocytogenes. In hard cheese varieties like Colby and Cheddar, L. monocytogenes populations decline during aging, with survival strongly influenced by the moisture content and the pH (Ryser and Marth, 1987b; Yousef and Marth, 1990). Cheeses such as Camembert and Feta have nearly identical composition in terms of moisture content, water activity, % salt-in-water and ripening temperature. However, fully ripened Camembert has a pH of 7.5 versus Feta which has a pH of 4.4 that prevents the growth of listeria. Cheeses made from raw milk

In the US and other parts of the world, the manufacture of cheese from raw milk is a topic which is being revisited from the perspective of microbiological safety. Pasteurization of milk prior to cheesemaking is but one step that may reduce the risk of the presence of pathogenic bacteria in cheese. Current US regulations which govern the use of raw, heat-treated and pasteurized milk for cheesemaking were promulgated in 1949 (Anonymous, 1950; 21 CFR Part 133). One of the two options can be selected by cheesemakers to assure the safety of cheesepasteurize milk destined for cheesemaking or hold cheese at a temperature of not less than 1.7 ~ (35 ~ for a minimum of 60 days. Recent research has shown that S. typhimurium, E. coli 0157:H7 and L. monocytogenes can survive well beyond the mandatory 60-day holding period in Cheddar cheese prepared from pasteurized milk (Reitsma and Henning, 1996; Ryser, 1998). In a referral to the National Advisory Committee on Microbiological Criteria for Foods in April 1997, the FDA asked if a revision of policy requiring a minimum 60-day aging period for raw-milk hard cheeses was necessary. The FDA, in its communication, noted that such a duration may be insufficient to provide an adequate level of public health protection. The FDA cited numerous studies and outbreak investigations documenting the presence of listeria, salmonella and E. coli 0157:H7 in raw milk. Of particular concern was the report by Reitsma and Henning (1996) detailing the survival of E. coli 0157:H7 in aged Cheddar cheese. The FDA did note, however, that there was 'limited epidemiological evidence that food-borne illness results from consumption of raw-milk hard cheeses that have been aged for 60 days', citing work by Fabian (1947), D'Aoust et al. (1985) and Johnson et al. (1990b) in support of this claim. Groups outside of the US have recently expressed concern about the safety of raw-milk cheeses. The Institute of Food Science and Technology (IFST, 2000) in the UK issued a position statement drawing attention to the

545

potential public health hazards posed by pathogenic bacteria in cheeses made from raw milk. The IFST indicates that these hazards apply particularly to soft and semi-soft cheeses (IFST, 2000). Codex Alimentarious is presently recommending a 'combination of control measures' (including pasteurization) to achieve the appropriate level of public health protection (Groves, 1998). In a comprehensive review of all outbreaks of human illness associated with the consumption of aged rawmilk cheese, in the majority of instances, confounding parameters other than use of raw milk contributed to pathogens being present in the product at the time of consumption (Donnelly, 2001). Further, in challenge studies which examine the fate of pathogens in aged cheese, confounding factors can also explain the appearance of pathogens following 60 days of aging. Such confounding parameters in actual outbreaks or challenge studies involve the use of pasteurized versus raw milk in cheesemaking trials, inadequate development of acidity during cheesemaking, a low salt level, contamination by ill employees during manufacture, temperature abuse of milk designed for cheesemaking and environmental contamination during cheesemaking.

P r e v i o u s R e v i e w s on t h e S a f e t y of R a w Milk C h e e s e s

Two comprehensive reviews have been published regarding outbreaks of human illness linked to consumption of cheese. Johnson et al. (1990b) conducted a comprehensive review of the epidemiological literature during the 40-year period, 1948-1988. These authors identified only six outbreaks of illness transmitted by cheese produced in the US during this period. Post-pasteurization contamination was the most frequent causative factor in these outbreaks. Improper pasteurization equipment and/or procedures were implicated in only one outbreak each in the US and Canada, and use of raw milk was a factor in one outbreak in each of these countries. No outbreaks were linked to hard Italian cheese varieties such as Parmesan, Romano and Provolone. In rare instances, Swiss and Cheddar cheeses were linked to food-poisoning outbreaks. Factors other than pasteurization cited by Johnson et al. (1990b) as contributors to cheese safety include milk quality and management, lactic starter management, pH, salt, controlled aging conditions and natural inhibitory substances in the raw milk. These authors proposed three actions to improve the safety of raw milk cheeses: (1) Establish a guideline for minimum heat-treatment of milk for cheesemaking, e.g., 64.4 ~ (148~ for 16sec or equivalent with adequate process control, (2) Evaluate current safety

546

Growth and Survival of Microbial Pathogens in Cheese

technology and practices used for cheese manufacture and (3) Evaluate technologies not currently used in cheese manufacture for safety potential (Johnson et al., 1990c). Altekruse et al. (1998) reviewed all cheese-associated outbreaks reported to the Centers for Disease Control and Prevention (CDC) during the period 1973-1992. These authors noted the infrequency of large, cheeseassociated outbreaks reported during this period and suggested that improvement of cheesemaking methods and process control have resulted in cheese being a safer product. There were 32 cheese-associated outbreaks, 11 of which could be attributed to contamination at the farm, during manufacturing or during processing. Of the 11 outbreaks attributed to contamination prior to distribution, 5 were associated with the consumption of Mexican-style soft cheese versus only one outbreak linked to Cheddar cheese. It is notable that no outbreaks reported to the CDC during 1973-1992 were associated with raw milk cheese that was aged for a minimum of 60 days. The authors indicated that salmonella, E. coli 0157:H7 and L. monocytogenes may survive the aging process. However, the literature reference for survival of listeria points to Camembert cheese (Ryser and Marth, 1987a), and the authors failed to note the rapid decline of listeria populations in aged Cheddar cheese as documented by Ryser and Marth (1987b). Altekruse et al. (1998) suggest that aging alone may not be a sufficient pathogen control step to eliminate salmonella, listeria and E. coli 0157:H7 from cheese. Outbreaks involving Cheddar cheese

In 1976, seven lots of Cheddar cheese manufactured from pasteurized milk were contaminated with S. heidelberg and were responsible for 339 confirmed cases of illness and an additional 28 000-36 000 cases of illness (Fontaine et al., 1980). The cheese involved was aged for less than 60 days, and improper pasteurization was cited as the cause of the outbreak. Follow-up with the first few patients led epidemiologists to suspect cheese eaten in Mexican-style restaurants as the vehicle of infection. Seven lots of Cheddar cheese produced from pasteurized milk by a Kansas manufacturer and purchased from a single Denver distributor were identified as the potential sources of contamination. The epidemic began in July in two widely separated Colorado cities, Denver and Pueblo. Levels of S. heidelberg in these cheeses were estimated to be 0.36-1.8 per 100 g. The pH of contaminated cheese was 5.6, which may have been a factor in this outbreak. Poor manufacturing practices coupled with inadequate control programmes at the cheese plant were cited as causative factors in this outbreak. The Kansas State Health Department had

recorded 25 instances of non-compliance with good manufacturing practices by that particular food-processing plant. The Kansas Board of Agriculture required that raw milk contain 105 per g), which may indicate poor starter activity (Johnson et al., 1990b) or contamination through handling. It is difficult to understand how D'Aoust et al. (1985) could support their concluding statement in this article 'Although pasteurization of milk used in cheesemaking increases the safety of the finished product, use of heat-treated (unpasteurized) milk in the manufacture of medium and old Cheddar cheese and survival of salmonella during prolonged periods of refrigerated storage raises legitimate doubts of the safety of current manufacturing practices.' In the data presented, pasteurization did not result in the unequivocal safety of mild Cheddar cheese. An evaluation of the pasteurization process, described by Johnson et al. (1990b), indicated that the employee in charge of the process manually overrode the electronic controls, which shut down the pasteurizer while milk continued to flow through the unit and into the vat. The pasteurizer was shut down after filling three vats and later restarted to fill the next three vat series. The first and the third vats of each three vat sequence tested positive for salmonella, except for the first vat of the day and the middle vat of each three vat series which consistently tested negative. This pattern only occurred when raw milk which included milk from the cow shedding S. typhirnurium was used. Bezanson et al. (1985) subsequently subjected outbreak strains to molecular analysis by biotyping, antibiotic resistance patterns, plasmid restriction and endonuclease analyses and revealed that two genetically distinct organisms were the aetiologic agents in this outbreak. These studies revealed the existence of a double infection, indicating that the incriminated cheese likely had two sources of contamination. S. typhimurium phage type 10 subgroup I strains were identified among cultures from raw milk and cattle associated with the incriminated dairy. S. typhimurium phage type 10 subgroup I and II strains were recovered from individuals employed at the dairy along with their family members. S. typhirnuriurn subgroup I and II strains were present in cheese curd samples obtained from the plant as well as from a consumer pack obtained from a distributor. Cheese plant workers from whom both subgroup I and II strains were cultured were involved in the production and/or packaging of Cheddar cheese, raising questions about the possibility of contamination of the cheese by ill workers. Salmonella were confirmed in a cheese-trim bucket. Plant inspections revealed that employees used their bare hands to transfer cheese to a forming machine, and an employee tested

547

positive for S. typhimurium. It is likely that this incriminated cheese was also responsible for an outbreak of illness reported at the same time in Ontario linked to S. typhimurium phage type 10 biotype 4 (D'Aoust et al., 1985). Hedberg et al. (1992) reported on a multi-state outbreak of S. javiana and S. oranienburg linked to the consumption of contaminated Mozzarella cheese and shredded cheese products. Cases were more likely to have consumed cheese manufactured at a single cheese plant or cheese shredded at processing plants that also shredded cheese from the single plant, than matched controls. The outbreak strains were isolated from 2 of 68 unopened 16-oz blocks of Mozzarella cheese. Inspections revealed deficiencies in plant sanitation and cleaning, and equipment was not routinely cleaned and sanitized between shredding different types of cheese from different manufacturers. However, no deficiencies in pasteurization were identified. Cheese-manufacturing equipment was found to be susceptible to environmental contamination and contamination by aerosols. Investigators believed that the contaminated Mozzarella cheese sent to four processing plants for shredding, crosscontaminated other cheese products at those plants. It is most likely that the cheese was contaminated from environmental sources or from infected production workers. Four outbreaks occurring in the late 1990s were reported in the UK, although detailed epidemiologic data on these outbreaks is lacking. An outbreak of E. coli 0157:H7 (phage type 8, Verotoxin gene 2) infection involving 22 cases was reported in Scotland in 1994. This outbreak was associated with the consumption of raw-milk cheese (Anonymous, 1997a). A December 1996 outbreak of salmonella gold-coast which occurred in England and Wales was linked to the consumption of a brand of mild, coloured, Cheddar cheese produced in August and September 1996 in Somerset, England. Phosphatase tests and examination of recording chart records from the pasteurizer indicated that pasteurization had failed at the plant on several occasions (Anonymous, 1997b). An outbreak of infection caused by E. coli 0157:H7 (phage type 21/28 VT2) was reported in 1999 in north-east England (Anonymous, 1999a,b). The vehicle of infection was Cotherstone cheese, a rawmilk cheese, manufactured in small quantities and distributed to specialty cheese shops in England. Samples from the dairy herd, slurry and environmental samples from the cheese manufacturing facilities were negative for E. coli 0157:H7. In March of 1999, a large outbreak of infection was reported in England and Wales due to consumption of contaminated milk from a single dairy. An outbreak of E. coli 0157:H7 infection was reported which was linked to the consumption of fresh cheese

548

Growth and Survival of Microbial Pathogens in Cheese

curd, which was held for < 60 days, from a dairy plant in Wisconsin (Durch et al., 2000). Nineteen of 55 laboratory-confirmed patients had purchased cheese curds from an unrefrigerated display at the cheese plant. To be legal, cheese curds must be manufactured from pasteurized milk. Vats of raw-milk Cheddar cheese were inadvertently used to make fresh curds, which were incorrectly labelled as 'pasteurized' Cheddar cheese curd. A comprehensive risk assessment would consider, among other factors, the degree to which the consuming population is exposed to risks associated with the consumption of aged raw-milk cheeses. Cheddar cheese is produced worldwide and is therefore considered an important variety of hard cheese. The USDA, National Agricultural Statistics Service, reports that Cheddar cheese was the most popular variety of cheese produced and consumed in the US in 1999, with a production level of 2.8 billion pounds (1.2 million tonnes) or 35.4% of the total cheese produced (Anonymous, 1999c). Given that a large amount of this cheese is produced from raw or heat-treated milk, the high degree of exposure (consumption) of this product coupled with the low incidence of disease outbreaks attests to the safety of aged cheese made from raw and heat-treated milk. Table 1 summarizes outbreaks involving Cheddar cheese which have occurred since 1976. Listed in this table are confounding parameters which contributed to the presence of pathogens in the finished product and the subsequent onset of human illness.

Challenge Studies Reitsma and Henning (1996) examined the survival of E. coli 0157:H7 during the manufacture and ripening of Cheddar cheese. E. coli 0157:H7 was inoculated at two levels into pasteurized milk, 1 x 103 cfu/ml and 1 cfu/ml. The organism showed a sharp decrease in numbers over the 158-day testing period. Treatment 1 (1000 cfu/ml) showed a 2-log CFU/g reduction after 60 days of ripening; however, E. coli 0157:H7 was still present even after 158 days of ripening when viable cells were detected in four of five replicates. Treatment 2 (1 cfu/g) showed a reduction to < 1 cfu/g in 60 days, with no viable E. coli 0157:H7 detected at 158 days. As the authors state, 'the results of this study cannot predict the behaviour of heat-injured cells which could result from the pasteurization of naturally contaminating E. coli.' Further, the low salt-in-moisture content (SM) and absence of natural inhibitors present in raw milk create an artificially protective environment for E. coli 0157:H7 in pasteurized milk. The SM determines the water activity, which, in turn, dictates the potential for growth of a micro-organism in the cheese environment. The SM in that study ranged from 2.75 to

3.76% with a mean of 3.25%, whereas in normal Cheddar, the SM ranges from 4 to 6%. The low SM could have affected the results in the study of Reitsma and Henning (1996) and the authors recommend further research with Cheddar containing a higher SM to determine if similar results would be obtained with an SM more commonly encountered in Cheddar cheese. NaC1 is an important inhibitor of microbial growth in cheese. The major roles of NaC1 in Cheddar cheese are to check lactic acid fermentation after an optimum peak has been attained, reduce moisture through syneresis of the curd, suppress the growth of spoilage micro-organisms and create physical changes in cheese proteins which influence cheese texture, protein solubility and protein conformation (Fox et al., 2000; 'Salt in Cheese: Physical, Chemical and Biological Aspects', Volume 1). While there are no state or federal standards for the amount of salt added to Cheddar cheese, variations in salt content from 0.8 to 2% are common. The minimum aw (adjusted with NaC1) for the growth of E. coli is 0.950 (Fennema, 1985). Further, most raw milk receives some form of heat treatment, albeit subpasteurization. The combination of heat, salt and natural inhibitors could provide barriers to the survival of E. coli 0157:H7. The experimental design used by Reitsma and Henning (1996) failed to consider these potential safeguards. It is plausible that the use of pasteurized milk for cheesemaking provides E. coli 0157:H7 with a more protective environment than raw milk, thus heat treatment could create more of a problem to food safety. The authors state 'The low number of outbreaks seem to indicate that pathogens in cheese are not a major problem.' The authors further state 'treatment 1 (1000 cfu/ml) would not likely be encountered in industry because of co-mingling of milk from several or many farms, thus creating a dilution effect.' Concern is expressed about the authors' concluding statement 'The current requirement for ripening of Cheddar cheese will not assure consumers of a safe product if the cheese is made from raw milk and a pathogen such as E. coli 0157:H7 is present in the cheese at the beginning of ripening.' This statement is contradicted by the authors' own data which show that E. coli 0157:H7 present at 60 cfu/g in curd after salting was reduced to < 1 cfu/g after 60 days, even in the artificially low SM of cheeses in the study. A subsequent study by Zhang and Henning (1999) described mathematically the decline of E. coli populations during cheese ripening. The authors inoculated pasteurized whole milk with E. coli biotype 1 at populations of 100-1000/ml. The authors used a complete factorial design to investigate the effects of high- and low-level environmental factors such as moisture (34-40%), pH (5.1-5.6), temperature (4-13 ~ and salt

Growth and Survival of Microbial Pathogens in Cheese

549

Table 1 Data from outbreak investigations involving aged raw milk cheese and confounding parameters which contribute to the presence of pathogens

Number of cases

Cheese type

Confounding parameter

339 confirmed; 28 00036 000 suspected

Cheddar made from pasteurized milk

1. Raw milk did not meet standards 2. Raw milk stored 1-3 days in holding tank - no refrigeration 3. Milk filtered after pasteurization 4. Cheese pH, 5.6 5.25 instances of noncompliance with GMP Milk traced to single farm; lack of co-mingling 1. Employee manually shut down pasteurizer 2. Group II type shed by workers 1. Deficiencies in cleaning and sanitation 2. Equipment not routinely cleaned and sanitized between shredding of different cheese types from different makers 3. Cheese equipment susceptible to contamination from environment/aerosols 4. Cheese contaminated by infected workers 5. No deficiencies in pasteurization Incorrectly labelled as a pasteurized product

Date

Location

Isolate

1976

Colorado

Salmonella heidelberg

1980-1982

Ontario

Salmonella muenster

1984

4 Canadian Atlantic Provinces and Ontario

Salmonella typhimurium phage type 10, group I and II

>2700 confirmed cases

Cheddar made from pasteurized and/or heattreated milk

1989

Multistate (Minnesota, Wisconsin, Michigan, New York)

Salmonella javiana and Salmonella oranienburg

164

Shredded cheese

1999

Raw-milk Cheddar

E. coil 0157:H7

concentration (0.8-1.7%) on survival of E. coli. Temperature and pH were found to have the most significant impact on survival, and there was no significant interaction among the four parameters studied. Salt concentration within the ranges used in this study (0.8-1.7%) was found to have no impact on survival of E. coll. Teo and Schlesser (2000) examined the survival of three groups of bacteria in raw-milk Cheddar cheese during cheesemaking and ripening; naturally occurring

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Nutritional Aspects of Cheese

Table 3

Mineral content of selected cheeses, mg/100 g (Holland et aL, 1989)

Cheese type Brie Caerphilly Camembert Cheddar (normal) Cheddar (reduced-fat) Cheshire Cottage cheese Cream cheese Danish blue Edam Emmental Feta Fromage frais Gouda Gruyere Mozzarella Parmesan Processed cheese* Ricotta Roquefort Stilton

Na

K

Ca

Mg

P

Fe

Zn

700 480 650 670 670 550 380 300 1260 1020 450 1440 31 910 670 610 1090 1320 100 1670 930

100 91 100 77 110 87 89 160 89 97 89 95 110 91 99 75 110 130 110 91 130

540 550 350 720 840 560 73 98 500 770 970 360 89 740 950 590 1200 600 240 530 320

27 20 21 25 39 19 9 10 27 39 35 20

390 400 310 490 620 400 160 100 370 530 590 280 110 490 610 420 810 800 170 400 310

0.8 0.7 0.2 0.3 0.2 0.3 0.1 0.1 0.2 0.4 0.3 0.2 0.1 0.1 0.3 0.3 1.1 0.5 0.4 0.4 0.3

2.2 3.3 2.7 2.3 2.8 3.3 0.6 0.5 2.0 2.2 4.4 0.9 0.3 1.8 2.3 1.4 5.3 3.2 1.3 1.6 2.5

8 38 37 27 45 22 13 33 20

* Variety not specified.

1961) showed that the incorporation of dairy products in the diet greatly reduced the development of dental caries in rats. Reynolds and Johnson (1981) confirmed these findings. Later work (Jenkins and Ferguson, 1966; Weiss and Bibby, 1966) indicated that if enamel is treated with milk in vitro and subsequently washed, the solubility of the enamel is greatly reduced. This effect was attributed to the high levels of calcium and phosphate in milk (Jenkins and Ferguson, 1966) or to casein adsorption onto enamel surfaces (Weiss and Bibby, 1966). Reynolds and del Rio (1984) reported that both casein and whey proteins significantly reduced the extent of caries, with the former exerting the greater effect. Further evidence for the protective effect of casein was provided in a study on rats fed with caseinenriched chocolate (Reynolds and Black, 1987). Calcium and phosphate appear to become available under the acidic conditions of the plaque and reduce demineralization of enamel (Reynolds, 1997; Reynolds et al., 1999). Concentrates containing various levels of whey protein, calcium and phosphate but negligible amounts of casein, significantly reduced the incidence of dental caries in rats (Harper et al., 1987). Thus, there is evidence that milk proteins, calcium and phosphate all exert an anticariogenic effect. Guggenheim et al. (1999) reported that micellar casein inhibits oral colonization by the cariogenic Streptococcus sobrinus and dental caries in rats. Vacca-Smith et al. (1994) demonstrated that K-casein can reduce the adherence

of the cariogenic Sc. mutans to hydroxyapatite (the mineral of enamel). Rugg-Gunn et al. (1975) provided the first evidence that the consumption of cheese had an anticariogenic effect in humans. They showed that the consumption of Cheddar cheese after sweetened coffee or a sausage roll increased plaque pH, possibly due to increased salivary output. Similar effects were reported by Imfeld et al. (1978) who used a more sophisticated continuous wire telemetry procedure to monitor variations in plaque pH. The effect of eating patterns on dental caries in rats was investigated by Edgar et al. (1982). Rats fed additional meals of cheese while on a high-sucrose diet, developed fewer smooth surface carious lesions and exhibited increased salivary output (which buffers acid formed in plaque) and a reduction in the number of Sc. mutans. Harper et al. ( 1 9 8 3 ) suggested that the cariostatic effect of cheese in rats is due to its calcium phosphate and/or casein; the fat or lactose appeared to exert little influence. Further work by Rosen et al. (1984) on the effect of cheese, with or without sucrose, on dental caries and the recovery of inoculated Sc. mutans in rats indicated beneficial cariostatic effects of cheese consumption but little effect on Sc. mutans numbers. These data suggest that the cariostatic effects of cheese may not be directly related to effects on Sc. mutans. Work on the protective effects of four cheese varieties in an in vitro demineralization system suggested that most, but not all, of the protective

Nutritional Aspects of Cheese

effects of cheese could be explained by mass action effects of soluble ions, particularly calcium and phosphate (Jenkins and Harper, 1983). The effect of Cheddar cheese on experimental caries in humans was investigated by Silva et al. (1986) using an 'intraoral cariogenicity test' (ICT). Demineralization and consequent reduction in the hardness of enamel monitored in this test is assumed to be typical of the early stage of the development of caries. Enamel slabs were polished and their initial micro-hardness determined using a Knoop Indenter. The slabs were then wrapped in Dacron and fastened on a prosthetic applicance made specifically for each subject to replace a missing lower first permanent molar. The subjects chewed 5 g of cheese immediately after rinsing their mouths with 10% (w/v) sucrose. Chewing cheese immediately after sucrose rinses resulted in a 71% reduction in demineralization of the enamel slabs, raised plaque pH but caused no significant change in the microflora of plaque compared with controls. Silva et al. (1987) investigated the effects of the water-soluble components of cheese on human caries using the I CT procedure and an experimental protocol which avoided salivary stimulus caused by chewing cheese. An average reduction of 55.7% in the cariogenicity of sucrose was reported, indicating the presence of one or more water-soluble anticariogenic components in cheese. Further evidence that cheese may inhibit dental caries in the absence of saliva was provided by Krobicka et al. (1987); rats that had their saliva-secreting glands surgically removed developed fewer and less-severe lesions when fed with cheese in addition to a cariogenic diet when compared to appropriate controls. Trials on human subjects have confirmed that the consumption of hard cheese leads to significant rehardening of softened enamel surfaces (Jenkins and Hargreaves, 1989; Gedalia etal., 1991). Jensen and Wefel (1990) showed that processed cheese was both antiacidogenic and enamel-protective in human subjects fed with processed cheese four times a day for one month. Saliva flow is greatly reduced in individuals who receive head and neck irradiation for malignancies. These individuals are at high risk of developing dental caries. Sela et al. (1994) reported that hard cheese consumption by these individuals was effective in controlling caries. Moynihan et al. (1999) noted that the concentration of calcium in plaque was significantly higher in human subjects fed with cheese-containing meals than in control subjects fed with meals without cheese. The beneficial effects of cheese were observed even when it was incorporated into other foods, e.g., pasta with cheese sauce. Epidemiological studies (Pappas et al., 1995a,b) indi-

579

cate that high intake of cheese is negatively associated with root caries in elderly populations, many of whom are at high risk for such lesions. While more research is needed to define the precise mechanism(s) involved in the cariostatic effects of cheese, there is ample evidence to support the consumption of cheese as an anticaries measure (Herod, 1991; Kashket and DePaola, 2002). The most plausible mechanisms for the protective effect of cheese appear to be related to the mineralization potential of the casein-calcium phosphate of cheese, to the stimulation of saliva flow induced by its texture and/or flavour, the buffering effects of cheese proteins on acid formation in dental plaque and the inhibition of cariogenic bacteria.

References Brown, A.J. and Jessup, W. (1999). Oxysterols and atherosclerosis (review). Atherosclerosis 142, 1-28. Burrell, A. (1996). Economic Aspects of Milk Production in the EU. Eurostat Statistical Document, Eurostat, Luxembourg. Chin, S.E, Liu, W., Storkson, J.M., Ha, Y.L. and Pariza, M.W. (1992). Dietary sources of conjugated dienoic isomers of linoleic acid, a newly recognised class of anticarcinogens. J. Food Cornp. Anal. 5,185-197. Dreizen, S., Dreizen, J.G. and Stone, R.E. (1961). The effect of cow's milk on dental caries in the rat. J. Dent. Res. 40, 1025-1028. Edgar, W.M., Bowen, W.H., Amsbaugh, S., Monell-Torrens, E. and Brunelle, J. (1982). Effects of different eating patterns on dental caries in the rat. Caries Res. 16, 384-389. FAO Food Balance Sheets (1995-1999). http://apps.fao.org. Fitzgerald, R.J. and Meisel, H. (2003). Milk protein hydrolysates and bioactive peptides, in, Advanced Dairy Chemistry, Vol. 1, Proteins, Fox, RE and McSweeney, P.L.H., eds., Kluwer Academic/Plenum Publishers, New York. pp. 675-697. Fritsche, J. and Steinhart, H. (1998). Amounts of conjugated linoleic acid (CLA) in German foods and evaluation of daily intake. Z. Lebensm. Unters. Forsch. 206, 77-82. Gedalia, I., Ionat-Bendat, D., Ben-Mosheh, S. and Shapira, L. (1991). Tooth enamel softening with a cola type drink and rehardening with hard cheese or stimulated saliva. J. Oral. Rehabil. 18, 501-506. Gobbetti, M., Stepaniak, L., DeAngelis, M., Corsetti, A. and Di Cagno, R. (2002). Latent bioactive peptides in milk proteins: proteolytic activation and significance in dairy processing. Crit. Rev. Food Sci. Nutr. 42,223-239. Guggenheim, B., Schmid, R. and Aeschlimann, J.M. (1999). Powdered milk micellar casein prevents oral colonization by Streptococcus sobrinus and dental caries in rats: a basis for the caries-protective effect of dairy products. Caries Res. 33,446-454. Ha, Y.L., Grimm, N.K. and Pariza, M.W. (1987). Anticarcinogens from fried ground beef: heat-altered derivatives of linoleic acid. Carcinogenesis 8, 1881-1887.

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Nutritional Aspects of Cheese

Ha, Y.L., Storkson, J. and Pariza, M.W. (1990). Inhibition of benzo[alpyrene-induced mouse forestomach neoplasia by conjugated dienoic derivatives of linoleic acid. Cancer Res. 50, 1097-1101. Harper, D.S., Osborn, J.C. and Clayton, R. (1983). Cariostatic potential of four cheeses evaluated in a programmedfed rat model. J. Dent. Res. 62, 283 (abstr.). Harper, D.S., Osborn, J.C., Clayton, R. and Hefferren, J.J. (1987). Modification of food cariogenicity in rats by mineral-rich concentrates from milk. J. Dent. Res. 66, 42-45. Hayes, K.C., Pronczuk, A., Lindsey, S. and Diersen-Schade, D. (1991). Dietary saturated fatty acids differ in their impact on plasma cholesterol and lipoproteins in non-human primates. Am. J. Clin. Nutr. 53, 491-498. Heaney, R.P. (1991). Evaluation of Publically Available Scientific Evidence Regarding Nutrient-Disease Relationships. 3. Calcium and Osteoporosis. Life Sciences Research Office, Federation of American Societies for Experimental Biology, Rockville Pike, MD. Herod, E.L. (1991). The effect of cheese on dental caries. Aust. DentalJ. 36, 120-125. Holland, B., Unwin, I.D. and Buss, D.H. (1989). Milk Products and Eggs: The Fourth Supplement to McCance and Widdowson's The Composition of Foods, 4th edn, Royal Society of Chemistry/Ministry of Agriculture, Fisheries and Food, Cambridge, UK. Imfeld, T.H., Hirsch, R.S. and Muhlmann, H.R. (1978). Telemetric recordings of interdental plaque pH during different meal patterns. Br. Dent. J. 139,351-356. Ip, C., Chin, S.E, Scimeca, J.A. and Pariza, M.W. (1991). Mammary cancer prevention by conjugated dienoic derivatives of linoleic acid. Cancer Res. 51, 6118-6124. Jenkins, G.N. and Ferguson, D.B. (1966). Milk and dental caries. Br. Dent. J. 120, 472-477. Jenkins, G.N. and Hargreaves, J.A. (1989). Effect of eating cheese on Ca and P concentrations of whole mouth saliva and plaque. Caries Res. 23, 159-164. Jenkins, G.N. and Harper, D.S. (1983). Protective effect of different cheeses in an in vitro demineralization system. J. Dent. Res. 62,284 (abstr.). Jensen, M.E. and Wefel, J.S. (1990). Effects of processed cheese on human plaque pH and demineralization and remineralization. Am. J. Dent. 3,217-223. Jiang, J., Bjorck, L. and Fonden, R. (1997). Conjugated linoleic acid in Swedish dairy products with special reference to manufacture of hard cheeses. Int. Dairy J. 7, 863-867. Kashket, S. and DePaola, D.P. (2002). Cheese consumption and the development and progression of dental caries. Nutr. Rev. 60, 97-103. Keys, A. (1984). Serum cholesterol response to dietary cholesterol. Am. J. Clin. Nutr. 40,351-359. Kim, H.D., Lee, J.H., Shin, Z.I., Man, H.S. and Woo, H.J. (1995). Anticancer effects of hydrophobic peptides derived from a cheese slurry. Food Biotechnol. 4, 268-272. Krobicka, A., Bowen, W.H., Pearson, S. and Young, D.A. (1987). The effects of cheese snacks on caries in desalivated rats.J. Dent. Res. 66, 1116-1119.

Lee, K.N., Kritschevsky, D. and Pariza, M.W. (1994). Conjugated linoleic acid and atherosclerosis in rabbits. Atherosclerosis 118, 19-25. Leonarduzzi, G., Sottero, B. and Poli, G. (2002). Oxidized products of cholesterol: dietary and metabolic origin and proatherosclerotic effects (review). J. Nutr. Biochem. 13, 700-710. Lin, H., Boylston, T.D., Chang, M.L., Luedecke, L.O. and Shultz, T.D. (1995). Survey of the conjugated linoleic acid contents of dairy products. J. Dairy Sci. 78, 2358-2365. Ma, D.W.L., Wierzbicki, A.A., Field, C.J. and Clandinin, M.T. (1999). Conjugated linoleic acid in Canadian dairy and beef products. J. Agric. Food Chem. 47, 1956-1960. MacDonald, H.B. (2000). Conjugated linoleic acid and disease prevention: a review of current knowledge. J. Am. Coll. Nutr. 19, 111S-118S. McNamara, D.J. (1987). Effects of fat-modified diets on cholesterol and lipoprotein metabolism. Ann. Rev. Nutr. 7, 273-290. Meisel, H. (1993). Casokinins as inhibitors of angiotensinconverting-enzyme, in, New Perspectives in Infant Nutrition, Sawatzki, G. and Renner, B., eds., Thieme, Stuttgart. pp. 153-159. Meisel, H. (1998). Overview on milk protein-derived peptides. Int. Dairy J. 8,363-373. Meisel, H., Goepfert, A. and Gunther, S. (1997). ACE inhibitory activities in milk products. Milchwissenschaft 52,307-311. Morrissey, R.B., Burkholder, B.D. and Tarka, S.M. (1984). The cariogenic potential of several snack foods. J. Am. Dent. Assoc. 109,589-591. Moss, M. and Freed, D. (2003). The cow and the coronary: epidemiology, biochemistry and immunology. Int. J. Cardiol. 87,203-216. Mougios, V., Matsakas, A., Petridou, A., Ring, S., Sagredos, A., Melissopoulou, A., Tsigilis, N. and Nicolaidis, M. (2001). Effect of supplementation with conjugated linoleic acid on human serum lipids and body fat. J. Nutr. Biochem. I2, 585-594. Moynihan, P.J., Ferrier, S. and Jenkins, G.N. (1999). Carlostatic potential of cheese: cooked cheese-containing meals increase plaque calcium concentration. Br. Dent. J. 187, 664-667. Norat, T. and Riboli, E. (2003). Dairy products and colorectal cancer: a review of possible mechanism and epidemiological evidence. Eur. J. Clin. Nutr. 57, 1-17. O'Brierl, N.M. and O'Connor, T.P. (1993). Milk, cheese and dental caries. J. Soc. Dairy Technol. 46, 46-49. Olson, N.E and Johnson, M.E. (1990). Light cheese products: characteristics and economics. Food Technol. 44 (10), 93-96. Pappas, A.S., Joshi, A. and Palmer, C.A. (1995a). Relationship of diet to root caries. Am. J. Clin. Nutr. 61,423S-429S. Pappas, A.S., Joshi, A. and Belanger, A.J. (1995b). Dietary models for root caries. Am. J. Clin. Nutr. 61,417S-422S. Pihlanto-Leppala, A. (2002). Bioactive peptides, in, Encyclopedia of Dairy Sciences, Roginski, H., Fuquay, J.W. and Fox, P.F., eds., Academic Press, London. pp. 1960-1967.

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Recker, R.R., Bammi, A., Barger-Lux, M.J. and Heaney, R.R (1988). Calcium absorbability from milk products, an imitation milk and calcium carbonate. Am. J. Clin. Nutr. 47, 93-95. Renner, E. (1987). Nutritional aspects of cheese, in, Cheese: Chemistry, Physics and Microbiology, Vol. 1, General Aspects, Fox, RE, ed., Elsevier Applied Science, London. pp. 345-363. Reynolds, E.C. (1997). Remineralization of enamel subsurface lesions by casein phosphopeptide-stabilized calcium phosphate solutions. J. Dent. Res. 76, 1587-1595. Reynolds, E.C. and Black, C.L. (1987). Reduction of chocolate's cariogenicity by supplementation with sodium caseinate. Caries Res. 21,445-451. Reynolds, E.C. and del Rio, A. (1984). Effect of casein and whey protein solutions on caries experience and feeding patterns of the rat. Arch. Oral Biol. 29,927-933. Reynolds, E.C. and Johnson, I.H. (1981). Effect of milk on caries incidence and bacterial composition of dental plaque in the rat. Arch. Oral Biol. 26,445-451. Reynolds, E.C., Black, C.L. and Cai, E (1999). Advances in enamel remineralization: casein phosphopeptide-amorphous calcium phosphate. J. Clin. Dent. 10, 86-88. Rosen, S., Min, D.B., Harper, D.S., Harper, W.J., Beck, E.X. and Beck, EM. (1984). Effect of cheese, with or without sucrose, on dental caries and recovery of Streptococcus mutans in rats. J. Dent. Res. 63,894-896. Rugg-Gunn, A.J., Edgar, W.M., Geddes, D.A.M. and Jenkins, G.N. (1975). The effect of different meal patterns upon plaque pH in human subjects. Br. Dent. J. 139, 351-356. Sela, M., Gedalia, I. and Shah, L. (1994). Enamel rehardening with cheese in irradiated patients. Am. J. Dent. 7, 134-136. Shaw, J.H., Ensfield, B.J. and Vv~ollman, D.H. (1959). Studies on the relation of dairy products to dental caries in caries-susceptible rats. J. Nutr. 67, 253-273. Silva, M.D. de A., Jenkins, G.N., Burgess, R.C. and Sandham, H.J. (1986). Effect of cheese on experimental caries in human subjects. Caries Res. 20, 263-269. Silva, M.E de A., Burgess, R.C., Sandham, H.J. and Jenkins, G.N. (1987). Effects of water-soluble components of cheese on experimental caries in humans. J. Dent. Res. 66, 38-41. Smacchi, E. and Gobbetti, M. (1998). Peptides from several Italian cheeses inhibitory to proteolytic enzymes of lactic

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acid bacteria, Pseudomonas fluorescenes ATCC948 and to the angiotensin I-converting enzyme. Enzyme Microbiol. Technol. 22,687-694. Stanton, C. and Devery, R. (2002). Formation and content of cholesterol oxidation products in milk and dairy products, in, Cholesterol and Phytosterol Oxidation Products: Analysis, Occurrence and Biological Effects, Guardiola, E, Dutta, R C., Codony, R. and Savage, G.R, eds., AOCS Press, Champaign, IL. pp. 147-161. Stepaniak, L., Fox, RE, Sorhaug, T. and Grabska, J.J. (1995). Effect of peptides from the sequence 58-72 of [3-casein on the activity of endopeptidase, aminopeptidase and X-proplyl-dipeptidyl aminopeptidase from Lactococcus. J. Agric. Food Chem. 43,849-853. Surgeon General (2000). Oral Health in America: A Report of the Surgeon General. US Department of Health and Human Services, Rockville, MD. Vacca-Smith, A.M., Wuyckhuyse, B.C., Tabak, L.A. and Bowen, W.H. (1994). The effect of milk and casein proteins on the adherence of Streptococcus mutans to saliva-coated hydroxyapatite. Arch. Oral Biol. 39, 1063-1068. Voorrips, L.E., Brants, H.A.M., Kardinaal, A.EM., Hiddink, G.J., van den Brandt, RA. and Goldbohm, R.A. (2002). Intake of conjugated linoleic acid, fat, and other fatty acids in relation to postmenopausal breast cancer: the Netherlands Cohort Study on Diet and Cancer. Am. J. Clin. Nutr. 76, 873-882. Weiss, M.E. and Bibby, B.G. (1966). Effects of milk on enamel solubility. Arch. Oral Biol. 11, 49-57. Zhang, D. arid Mahoney, A.W. (1989a). Effect of iron fortification on quality of Cheddar cheese. J. Dairy Sci. 72, 322-332. Zhang, D. and Mahoney, A.W. (1989b). Bioavailability of iron-milk protein complexes and fortified Cheddar cheese. J. Dairy Sci. 72, 2845-2855. Zhang, D. and Mahoney, A.W. (1990). Effect of iron fortification on Cheddar cheese. J. Dairy Sci. 73, 2252-2258. Zhang, D. and Mahoney, A.W. (1991). Iron fortification of process Cheddar cheese. J. Dairy Sci. 74,353-358. Zlatanos, S., Laskaridis, K., Feist, C. and Zagredos, A. (2002). CLA content and fatty acid composition of Greek Feta and hard cheeses. Food Chem. 78,471-477.

Factors that Affect the Quality of Cheese P.F. Fox, Department of Food Science, Food Technology and Nutrition, University College, Ireland T.M. Cogan, Dairy Products Research Centre, Teagasc, Fermoy, Ireland

Introduction As discussed in 'Cheese: An Overview', Volume 1 and 'Diversity of Cheese Varieties: an Overview', Volume 2, the manufacture of cheese exploits either of two properties of the casein system: precipitation/coagulation at the isoelectric pH (4.6), which is exploited in the production of fresh, acid-coagulated cheeses ( - 2 5 % of total cheese production), or by limited proteolysis using rennets which specifically hydrolyse the micellestabilizing protein, K-casein, following which the rennetaltered micelles coagulate in the presence of Ca 2+ at a temperature > 2 0 ~ usually 3 0 - 3 5 ~ ( ' 7 5 % of cheese production). Most acid-coagulated cheeses are consumed fresh (unripened) whereas the vast majority of rennet-coagulated cheeses are ripened for a period ranging from - 3 weeks to > 2 years. Although there are recognizable differences between the unripened curds for different cheeses, mainly with respect to moisture content and texture, the characteristic differences between the 1000 or so varieties of cheese develop during ripening. The quality of acid-coagulated cheeses is subject to some variation but the fact that they are consumed fresh and that no modifications are required after manufacture, makes them relatively easy to produce with consistent quality (see 'Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels', Volume 1 and 'Acid- and Acid-Rennet-Curd Cheeses: Part A Quark, Cream Cheese and Related Varieties', Volume 2). In contrast, the characteristic quality of rennet-coagulated cheeses develops mainly during ripening and frequently depends on the growth of a secondary microflora, which are not readily reproducible. During ripening, a complex array of microbiological, biochemical and chemical reactions occur and therefore there are many opportunities for problems to develop. In this chapter, the quality aspects of rennet-coagulated cheeses will be considered. Some of the principal areas of cheese science through which cheese quality can be improved through research will be discussed. Cheese is the quintessential convenience f o o d - it can be consumed as it is without preparation, can be

used as a sandwich filler, grated or diced and used as a condiment or as a component of several cooked dishes. At least 50% of cheese is used, at home or in the factory, as an ingredient or component of other foods. The principal applications of cheese as an ingredient are discussed in 'Cheese as an Ingredient', Volume 2. The use of cheese as a food ingredient is increasing and for many of these applications, the principal criterion of quality depends on the functionality of the cheese, which depends mainly on the physico-chemical properties of the proteins. When used as an ingredient in food applications, cheese is expected to perform one or more functions and there is considerable commercial interest in producing cheese products with functional properties tailor-made for particular applications. Cheese may be used as an ingredient in several forms: 9 natural cheese: sliced, diced or grated; 9 processed cheese products; 9 dried cheese products: - dried, grated natural cheese (a traditional product); - cheese powders. 9 enzyme-modified cheese products, which may be produced from mild cheese or fresh cheese curd or from blends of enzyme-treated casein and fats and are used mainly as high-intensity cheese flavours. There are several aspects to the quality of cheese; some are applicable to all cheese products and applications, others are significant for specific cheeses. Probably the most important aspects of cheese are: 9 9 9 9 9 9

safety from a public health viewpoint nutritional flavour texture functionality appearance, e.g., - Cheese must conform to the expected characteristics of the variety. There are obvious differences between the principal families of cheese but it may not be so easy to differentiate between cheeses claimed to

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

Copyright 9 2004 Elsevier Ltd All rights reserved

584

Factors that Affect the Quality of Cheese

belong to similar varieties, e.g., Gouda and Edam, Cheddar and Gloucester, Brie and Camembert. The borderlines are blurred and within each variety, a certain degree of variability is tolerated and acceptable. - Reproducibility/reliability - consumers expect that a product will be reproducible and consistent between batches and over time with respect to flavour, texture, appearance and functionality, especially for the principal, 'mass-produced' varieties; some variability is tolerated, perhaps even expected, in artisanal cheeses.

Milk Selection Pre-treatment Standardization

Cheesemilk Addition of: starter culture (acidification) colour (optional) CaCI2 (optional) Coagulation [rennet or acid (produced in situ or pre-formed) or heat/acid]

Production of Rennet-Coagulated Cheese The production of rennet-coagulated cheese can be divided into two phases:

Coagulum (gel) Cut coagulum Stir Heat Acidification (rennet-coagulated cheeses) Separation of curds from whey

9 Conversion of milk to curd 9 Conversion of curd to cheese The key operations are summarized in Fig. 1. The production of good-quality cheese depends on a good milk supply from the chemical and microbiological viewpoints. Raw milk is a rather variable commodity and is subjected to a range of processes aimed at modifying, standardizing and optimizing the cheesemaking properties of milk. Given a good milk supply, the first objective is to produce curd of the desired chemical composition with the desired microflora. Unless these criteria are met, the curd will not evolve into cheese with the characteristic flavour, texture and functionality during ripening. The ripening of cheese, and hence its quality, is due to the activity of micro-organisms and enzymes from four or five sources: 9 9 9 9 9

Milk Rennet Primary starter Secondary cultures Adventitious (non-starter) bacteria

It is reasonable to expect that it should be possible to produce premium-quality cheese consistently by controlling these agents; however, in spite of considerable research and quality control efforts, it is not yet possible to do so. A very wide and diverse range of factors interact to affect the composition of cheese curd and hence the quality of the final cheese; an attempt to summarize these is shown in Fig. 2, which follows the same sequence as Fig. 1. Some of these factors/agents can be manipulated easily and precisely while others are more difficult, or perhaps impossible, to control. Indeed, the precise influence of many of the factors included

Curds Acidification Special operations (e.g., cheddaring, stretching) Salting (some varieties) Moulding Pressing (some varieties)

Fresh cheese

~

Salting (most varieties) Ripening (most rennet-coagulated cheeses)

Mature cheese Figure 1 General protocol for cheese manufacture (from Fox et al., 2000).

in Fig. 2 on cheese ripening and quality are not known precisely and many of the factors are interactive. It is possible to apply the principles of Hazard Analysis Critical Control Point (HACCP) analysis to cheese production and it is hoped that this article may stimulate efforts to apply HACCP principles to cheesemaking. The principal items in Fig. 1 and 2 will be discussed individually in the following sections.

Milk Supply It is well recognized that the quality of the milk supply has a major impact on the quality and consistency of the resultant cheese. Three aspects of quality must be considered: microbiological, enzymatic and chemical.

Species Breed Stage of lactation Plane of nutrition Animal health

Composition cell c o u n t

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Figure 2 Interaction of compositional and technological factors that affect the quality of cheese. 585

586

Factors that Affect the Quality of Cheese

Microbiological

Three aspects of the microbiology of cheese milk are of interest/concern: 9 Public health 9 Off-flavours and spoilage 9 Desirable bacteria

Public health aspects As a product of animal origin, milk may become contaminated with pathogenic micro-organisms, and this, clearly, is of great concern. Previously, the principal pathogens of concern in milk were Mycobacterium boris and Brucella abortus, but in developed dairying countries today, these pathogens have been largely or entirely eliminated from the dairy herd. Today, a wide range of pathogens are of concern, mainly Listeria monocytogenes, enterotoxigenic strains of E. coli, e.g., E. coli O157 H7, Shigella, Erwinia, Campylobacter, Staphlyococcus, Salmonella spp. and M. paratuberculosis. Many of these micro-organisms do not grow in milk which simply acts as a vector. In cheese, these pathogens die off under the rather hostile conditions in well-made cheese which has a relatively low pH (5.3), a relatively high salt content (5-10% sah-inmoisture; S/M) and perhaps bacteriocins. For this reason, Public Health Authorities in many counties require that cheese made from raw milk be aged for 60 days, although this practice may not be fully effective. Alternatively, cheese must be made from pasteurized milk or the cheese itself must be pasteurized, as in processed cheese. Cheese, the pH of which does not decrease at the desired rate or to the desired extent during manufacture (e.g., due to bacteriophage infection or contamination with antibiotics) or if the pH increases substantially during ripening, e.g., surface mould- or smear-ripened cheese, are most at risk. High-moisture, fast-ripening cheeses are at a greater risk of harbouring pathogens than low-moisture, slowripening varieties. A considerable amount of cheese is made from raw milk, especially in France, Germany and southern European countries, but there is increasing pressure in northern Europe and North America to produce all cheese from pasteurized milk. There have been very few reported outbreaks of food poisoning from the consumption of raw milk hard, long-ripened cheese varieties. In all outbreaks for which adequate data are available, mitigating circumstances, e.g., lack of a starter culture or poor starter activity, have also been involved. Clearly, raw milk cheese is safe if adequate precautions are taken. However, it should be remembered that most raw milk cheeses are high-cooked, hard

or extra-hard v a r i e t i e s - many of these cheeses, e.g., Parmigiano Reggiano, Grana Padano, Swiss Emmental and Gruyere de Comte, are cooked at --~55 ~ the cooked curds are transferred while hot to moulds with a capacity of 20-60 kg curd and consequently, the curds cool s l o w l y - most of these 'raw milk' cheeses are in fact pasteurized, as indicated by a negative alkaline phosphatase test. High-moisture, raw-milk cheeses are of more concern but most of these have a low initial pH (4.6) and appear to be safe. It is probably significant that raw-milk cheeses are made on a small/very small scale from very fresh milk from healthy cows. For further discussion on pathogens in cheese, see 'Growth and Survival of Microbial Pathogens in Cheese', Volume 1. It is unlikely that it will be possible to produce raw milk guaranteed free of pathogenic bacteria. However, milk with very low numbers of pathogens can be produced from healthy animals and any pathogens that do enter milk can be: 9 killed (pasteurization or novel alternatives); 9 removed (bactofugation or microfihration (MF); see 'Application of Membrane Separation Technology to Cheese Production', Volume 1); 9 prevented from growing or killed, e.g., by use of low pH, selected bacteriocin-producing starters. To date, efforts to remove pathogens from cheese milk have concentrated on adequate pasteurization. There is ongoing research on alternative methods and it is likely that work in this area will continue and probably expand. Off-flavours and spoilage A second beneficial effect of pasteurization is the killing of spoilage micro-organisms, e.g., coliforms, pseudomonads and yeasts. In countries with a developed dairy industry, the quality of the milk supply has improved markedly during the past 30 y e a r s - total bacterial counts (TBC) are now usually 20 ~ (in cheesemaking, at 30-35 ~ It has been proposed (Andreeva et al., 1992; Gustchina et al., 1996) that chymosin normally exists in an inactive conformation but is activated when the substrate binds in the active site cleft of the enzyme. It has been suggested that the sequence--H.P.H.P.HB (residues 98-102 of K-casein) is responsible for this conformational change. This sequence occurs in the K-casein of cow, goat, sheep and buffalo but not in the K-casein of the mare, camel, pig, rat or human, in which the corresponding sequence is ..HPRPH.., ..RPRPR.., ..RPRPH.., ..HPINR. and ..RPNLH.., respectively (Iametti et al., 2001; Martin etal., 2003). Therefore, one would expect that calf chymosin would not coagulate the milk of the mare, camel, pig, human or rat. There have been few studies on the coagulation of non-bovine milk by calf chymosin. The commercial use of calf rennet in cheesemaking from sheep, goat or buffalo milk indicates that calf chymosin can hydrolyse the K-casein in these milks, as expected from the above hypothesis. Calf chymosin can also coagulate porcine milk (Fox, 1975b); in fact, porcine milk is coagulated by calf rennet at 4 ~ whereas bovine milk is not, due to the nature of the non-enzymatic secondary phase. Some investigators have reported that camel milk is not coagulated by calf rennet but Farah (1993) reported that it is coagulated slowly to a weak gel. The status of mares' milk with respect to K-casein remained unclear until very recently. Ochirkhuyag et al. (2000) reported that equine milk does not contain K-casein and that the micelle-stabilizing function is played by [3-casein; however, Malacarne et al. (2002) reported that it contains a low level (20 ~ this is referred to as the secondary phase of rennet coagulation. Renneted bovine milk does not coagulate 6.5. Low-cooked, low-pH, highmoisture cheese, e.g., Camembert, retains ---30% of the added chymosin activity; Cheddar retains "--6% and Emmental "--0%. Everything else being equal, increased retention of the coagulant in cheese curd results in greater initial hydrolysis of C~sl-casein; however, the significance of this variable on the flavour and texture of cheese has not been studied thoroughly. It has been suggested that the activity of chymosin in cheese curd is the limiting factor in cheese ripening; however, excessive rennet activity leads to bitterness. Proteolysis in cheese during ripening is discussed later; there have been relatively few studies on the significance of chymosin activity to cheese quality, an aspect which appears to warrant further research. Considering the importance of proteolysis in the ripening and quality of cheese and the significance of the coagulant thereto, studies on various factors that affect the retention of the coagulant in cheese curd appear warranted, e.g., 9 the adsorption of chymosin on casein micelles and the apparent lack of adsorption of fungal proteinases; 9 stability of various rennets under various conditions of temperature, pH and other factors.

Starter The second key reaction in cheesemaking is acidificat i o n - the pH of all rennet-coagulated cheeses should decrease to a value in the range 4.6-5.2 within a few

days of manufacture, or in some varieties, at the end of curd manufacture (5-6 h). Acidification at the appropriate rate and time is an essential and characteristic feature of cheesemaking- it is, in fact, a sine qua non. Among the important consequences of acidification are: 9 activity of the coagulant; 9 survival and retention of coagulant in the curd; 9 firmness of the coagulum, which affects the loss of fat and protein in the whey on cutting and hence reduces the yield of cheese; 9 syneresis of the curds and hence the composition of the cheese; 9 solubilization of colloidal calcium phosphate (CCP), which has a major effect on the texture, meltability and stretchability of the cheese; 9 inhibition of the growth of undesirable bacteria, most importantly pathogenic and food poisoning bacteria; 9 the activity of various enzymes in the cheese during ripening and consequently the rate of ripening and the quality of the cheese. Originally, acidification was due to the production of lactic acid from lactose by adventitious LAB. Acidification of some cheese varieties still depends on the activity of the adventitious microflora but most cheeses now are acidified using selected LAB added to the cheesemilk as a culture (starter). The idea of using starter cultures was introduced in ---1870 in Denmark. The cultures used today in cheesemaking can be divided into two groups: 1. Mesophilic - with an optimum growth temperature of "- 28 ~ 2. Thermophilic- which grow optimally at --~42 ~ Mesophilic cultures are used for cheese curds which are cooked at a temperature 1.4%

Premium quality

MNFS 52-56% MNFS 50-57%

S/M 4.0-6.0% S/M 4.0--6.0%

Gilles and Lawrence (1973) Composition of cheeses was determined at 14 days and related to quality of mature Cheddar cheese.

Moisture < 38%

pH < 5.4

l Fox (1975a) Relationship between the quality and composition ._o!10-week-old Cheddar cheese.

MNFS 52-54%

S/M 4.2-5.2%

Pearce and Gilles (1979) Composition of cheeses was determined at 14 days and related to quality of Cheddar cheese.

Figure 6 Relationships between composition (determined at various stages during ripening) and the quality of mature Cheddar cheese (moisture-in-non-fat substances (MNFS) fat-in-dry-matter (FDM), and salt-in-moisture (S/M)).

604

Factors that Affect the Quality of Cheese

O'Connor (1971) found that the flavour, texture and total score of Cheddar were significantly correlated with % NaC1 and particularly with pH; moisture content had less effect on cheese quality. Salt content and pH were strongly correlated with each other, as were salt and moisture. Based on the results of a study on experimental and commercial cheeses in New Zealand, Gilles and Lawrence (1973) proposed a grading (selection) scheme which has since been applied commercially in New Zealand to young (14 day) Cheddar cheese. The standards prescribed for Premium grade cheese were: pH: 4.95-5.10; % S/M: 4.0-6.0%; MNFS: 52-56%; FDM: 52-55%. The corresponding values for First Grade cheeses were: 4.85-5.20%, 2.5-6%, 50-57% and 50-56%; young cheeses with a composition outside these ranges were considered unlikely to yield goodquality mature cheese. Quite wide ranges of FDM are acceptable; Lawrence and Gilles (1980) suggested that since relatively little lipolysis occurs in Cheddar cheese, fat content plays a minor role in determining cheese quality but if FDM is below about 48%, the cheese is noticeably more firm and less attractive in flavour. Pearce and Gilles (1979) reported that the grade of young (14-day-old) cheeses produced at the New Zealand Dairy Research Institute was most highly correlated with moisture content; the optimum compositional ranges were: MNFS: 52-54%; S/M: 4.2-5.2%; pH: 4.95-5.15. Fox (1975a,b) reported a weak correlation between grade and moisture, salt and pH for Irish Cheddar cheeses but a high percentage of cheeses with compositional extremes was downgraded, especially those with low salt (38%) or high pH (>5.4). Salt concentration seemed to exercise the strongest influence on cheese quality and the lowest percentage of down-graded cheeses can be expected in the salt range 1.6-1.8% (S/M: 4.0-4.9%); apart from the upper extremes, pH and moisture had little influence on quality in the sample studied. High salt levels tend to cause a curdy texture, probably due to insufficient proteolysis; a pasty body, often accompanied by off-flavours, is associated with low salt and high moisture levels. In the same study, the composition of extra-mature Cheddar cheeses was found to vary less and the mean moisture content was 1% lower than that of regular cheeses. A very extensive study of the relationship between the composition and quality of nearly 10 000 cheeses produced at five commercial New Zealand factories was reported by Lelievre and Gilles (1982). As in previous studies, considerable compositional variation was evident but was less for some factories than others. While the precise relationship between quality

and composition varied between plants, certain generalizations emerged: 9 within the compositional range suggested by Gilles and Lawrence (1973) for 'premium' quality cheese, composition does not have a decisive influence on grade, which decreases outside this range; 9 composition alone does not provide an exclusive basis for grading; 9 MNFS was again found to be the principal factor affecting quality; 9 within the recommended compositional bands, grades declined marginally as MNFS increased from 51 to 55% and increased slightly as S/M decreased from 6 to 4; pH had no consistent effect within the range 4.9-5.2 and FDM had no influence in the range 50-57%. 9 there were specific intra-plant relationships between grade and composition; therefore, each plant should determine the optimum compositional parameters pertinent to it. The results of the foregoing investigations indicate that high values for moisture and pH and a low salt content lead to flavour and textural defects. The desired ranges suggested by Gilles and Lawrence (1973) appear to be reasonable, at least for New Zealand conditions, but within the prescribed zones, composition is not a good predictor of Cheddar cheese quality. Presumably, several other factors, e.g., starter, NSLAB, activity of indigenous milk enzymes, relatively small variations in cheese composition and probably other unknown factors, influence cheese quality but become dominant only under conditions where the principal determinants, moisture, salt and pH, are within appropriate limits. Although the role of calcium concentration in cheese quality has received occasional mention, its significance has been largely overlooked. Lawrence and Gilles (1980) pointed out that the concentration of calcium in cheese curd determines the cheese matrix and, together with pH, indicates whether proper procedures were used to manufacture a specific cheese variety. As the pH decreases during cheese manufacture, CCP dissolves and is removed in the whey. The whey removed after cooking comprises 90-95% of the total whey expressed during cheesemaking and under normal conditions contains ---85% of the calcium and "--90% of the phosphorus lost from the cheese curd. Thus, the calcium content of cheese reflects the pH of the curd at whey drainage; there are strong correlations between the calcium content of cheese and the pH at 1 or 14 days and the amount of starter used (see Lawrence et al., 1984). Since the pH of cheese increases during ripening, the pH of mature

Factors that Affect the Quality of Cheese 605 cheese may be a poor index of the pH of the young cheese. Therefore, calcium concentration is probably a better record of the history of a cheese with respect to the rate of acidification than the final pH. Reduction in calcium phosphate concentration by excessively rapid acid development also reduces the buffering capacity of cheese and hence the pH of the curd will fall to a lower value for any particular level of acid production. No recent work on the level and significance of calcium in Cheddar cheese appears to be available. The calcium content of cheese has a major effect on its meltability and stretchability, e.g., pasta-filata cheese does not stretch well, or not at all, until the pH falls below ---5.4. Biologically acidified Mozzarella has poor stretchability and meltability immediately after manufacture but these properties improve during the early stages of ripening and are optimal after about 2-3 weeks; functionality deteriorates on continued ripening due to proteolysis. In contrast, directly acidified cheese is functional immediately after manufacture. The difference in behaviour is due to the lower calcium concentration in the directly acidified cheese owing to the faster decline in pH to ---5.6. Under such conditions, much of the CCP dissolves and is removed in the whey at drainage; the concentration of calcium per unit of protein, which is very important for cheese functionality, in biologically and chemically acidified Mozzarella cheese was 27.7 and 21.8 mg/g, respectively (Guinee et al., 2002). There is little published information on the relationships between composition and quality for other cheese varieties. However, it is very likely that similar factors affect the quality of all cheeses more or less to the same extent.

Ripening T e m p e r a t u r e Ripening temperature has a major influence on the rate of ripening and quality of cheese. Traditionally, cheese was ripened in caves or cellars at a relatively constant temperature. This practice is still widespread for some varieties but artificially refrigerated rooms are now used by large-scale manufacture. The ripening temperature is fairly characteristic of the variety, e.g., Cheddar, 6-8 ~ Gouda, 12-14 ~ ParmigianoReggiano, 18-20 ~ Emmental, 6 ~ for ---2 weeks, then at 22 ~ for 4-6 weeks to allow the propionic acid bacteria to grow rapidly and produce adequate CO2 for good eye development, then at ---4 ~ for several months to complete ripening; Camembert, 14 ~ for --~2 weeks to induce the growth of P. camemberti, then at 4 ~ for 2-4 weeks.

Ripening can be accelerated by increasing the ripening temperature but all reactions, desirable and undesirable, are accelerated and an unbalanced flavour or off-flavour may develop. Ripening at an elevated temperature is normally considered with the objective of accelerating ripening (see Fox et al., 1996b). Cheese flavour can probably be modified by manipulating temperature; however, this is rarely practised except for Swiss-type cheeses. The rate at which the curd is cooled after moulding has a major effect on the growth of starter LAB and NSLAB. The curds for most cheeses are moulded immediately after cooking and acidification occurs mainly in the moulds. Hence, the rate at which the curd cools in the moulds has a major effect on starter growth and rate of acid development, and is strongly affected by the size of the cheese and ambient temperature. The effect of cooling on starter growth is particularly noticeable for high-cooked cheeses, e.g., Swiss and Grana types. The thermophilic starters used for these cheeses do not grow at the cook temperature but begin to grow as the curd cools in the moulds. For consistency, it is important to control the ambient temperature. For Cheddar-type cheeses, acidification is almost complete at moulding. Traditionally, the moulded cheeses were pressed overnight at ambient temperature and the cheeses cooled close to ambient during this period, although ambient temperature probably varied significantly with season. In modern practice, the cheeses exit the Wincanton tower at ---36 ~ and are packaged and stacked on pallets (5 • 10 cheeses ---1 tonne) and transferred to ripening rooms. The cheeses at the centre of the pallet do not decrease to ambient (store) temperature for about 4 weeks and this causes considerable variation in the number and probably the type of NSLAB, and hence in the quality of the cheese. Many factories now cool the packaged cheese in a cooling tunnel overnight before stacking on pallets. If the cheese is cooled to " & 2 - r

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ffJ

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~ C ~ mc ~ = 5o ~ "= ~ :~

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6

Diversity of Cheese Varieties: An Overview

Classification scheme for cheeses according to Walter and Hargrove (1972)

1.

Very hard (grating) 1.1 Ripened by bacteria: Asiago (old), Parmesan, Romano, Sapsago, Spalen

2.

Hard

2.1 2.2 3.

Ripened by bacteria, without eyes: Cheddar, Granular, Caciocavallo Ripened by bacteria, with eyes: Emmental, Gruyere

Semi-soft

3.1 3.2

Ripened principally by bacteria: Brick, MC~nster Ripened by bacteria and surface micro-organisms: Limburger, Port du Salut, Trappist 3.3 Ripenedprincipally by blue mould in the interior: Roquefort, Gorgonzola, Danablu, Stilton, Blue Wensleydale

4.

Soft

4.1 Ripened:Bel Paese, Brie, Camembert, Hand, Neufchatel 4.2 Unripened: Cottage, Pot, Baker's, Cream, Ricotta, Mysost, Primost

Volume 2). Cheddar and British territorial varieties (for which the curds are often textured and dry-salted) are classified as hard or semi-hard internal bacterially ripened cheeses ('Cheddar Cheese and Related Drysalted Cheese Varieties', Volume 2). Internal bacterially ripened cheeses with eyes are further sub-divided on the basis of moisture content into hard varieties (e.g., Emmental; 'Cheese with Propionic Acid Fermentation', Volume 2) in which the eyes are formed by CO2 produced on fermentation of lactate by Propionibacterium freudenreichii subsp, shermanii or semi-hard (e.g.,

Edam and Gouda; 'Gouda and Related Cheeses', Volume 2) in which a few small eyes develop due to the formation of CO2 by fermentation of citrate by the LAB. Pastafilata cheeses (e.g., Mozzarella; see 'Pasta-Filata Cheeses', Volume 2) are characterized by stretching in hot water which texturizes the curd. White-brined cheeses, including Feta and Domiati ('Cheese Varieties Ripened in Brine', Volume 2), are ripened under brine and have a high salt content and, consequently, they are grouped together as a separate category within the group of internal bacterially ripened cheeses. Soft cheese varieties are usually not included in the group of internal bacterially ripened cheeses because they have a characteristic secondary microflora which has a major effect on the characteristics of these cheeses. Mould-ripened cheeses are subdivided into surface mould-ripened varieties (e.g., Camembert or Brie; 'Surface Mould-ripened Cheeses', Volume 2) in which ripening is characterized by the growth of Penicillium camemberti on the surface, and internal mould-ripened cheeses ('Blue Cheese', Volume 2) in which P. roqueforti grows throughout the cheese. Smear-ripened cheeses ('Bacterial Surface-ripened Cheeses', Volume 2) are characterized by the development of a complex microflora consisting of yeasts and, later, bacteria (particularly coryneforms) on the cheese surface during ripening. The classification scheme of Fox et al. (2000) is not without inconsistencies. For example, cheeses made from the milk of different species are grouped together (e.g., Roquefort and Gorgonzola are both Blue cheeses

Classification of cheese according to source of milk, moisture content, texture and ripening agents* 1.

Cow's milk

1.1

Hard (55%; very low or no scald) Bs or Sm

Bel Paese (I) Maroilles (F)

Sm

Ns

Un, Ac

Brie (F) Camembert (F) Carre d'est (F) Neufchatel (F) Chaource (F)

Colwich (UK) Lactic (UK) Bondon (F)

Coulommier (F) York (UK) Cambridge (UK) Cottage (UK) Quarg Petit Suisse (F) Cream (UK)

Pr, propionic acid bacteria; Ns, normal lactic acid starter of milk flora; Bs, smear coat (Brevibacterium linens and other organisms) Sm, surface mould (R camemberti); Bv, blue-veined internal mould (R roqueforti); Ac, acid-coagulated; Un, normally unripened, fresh cheese. a Modified from Scott (1986).

but the former is made from sheep's milk and the latter from cows' milk). Of course, the scheme can be readily modified by subdividing relevant categories to indicate the type of milk used. The subdivision between hard and semi-hard cheeses is somewhat arbitrary and overlaps. Most varieties lose moisture during ripening by evaporation from the surface, i.e., develop a rind. Several varieties, e.g., Pecorino Romano and Montasio, are consumed after various lengths of ripening and hence may be classified as semi-hard, hard or extra hard, depending on age of cheese at consumption. There is also some cross-over between categories. Gruyere is classified as an internal bacterially ripened

variety with eyes but it is also characterized by the growth of a surface microflora, while some cheeses classified as surface-ripened (e.g., Havarti and Port du Salut) are often produced without a surface microflora and thus are, in effect, soft, internal bacterially ripened varieties. Fox et al. (2000) considered pasta-filata and high-salt varieties as separate families because of their unique technologies (stretching and ripening under brine, respectively) but they are actually ripened by the same agents as other internal bacterially ripened cheeses. However, the scheme of Fox et al. (2000) is a useful basis for classification; the arrangement of topics within this volume largely follows this scheme.

8

D i v e r s i t y of C h e e s e Varieties: A n O v e r v i e w

Major omissions from the scheme of Fox et al. (2000) are processed cheeses, cheese-based products (cheese powders, enzyme-modified cheese), cheese analogues and cheese substitutes. Processed cheese products represent ---14% of world cheese production and thus surpass the production of most natural varieties except Cheddar, Gouda, Mozzarella and Camembert. None of the classification schemes referred to above includes processed cheeses - it would seem reasonable to include them as a separate category. From the discussion in 'Pasteurized Processed Cheese and Substitute/Imitation Cheese Products', Volume 2, it will be apparent that this is a very diverse group of products with respect to raw material, process technology and composition. One could also argue that each class of the other cheese-based products, which are described in 'Cheese as an Ingredient', Volume 2, warrants inclusion and of course this can be accommodated readily. It must be remembered that the dried and enzyme-modified cheeses are very heterogeneous groups. Although cheese analogues may not be considered to be authentic cheese products, there seems to be no

valid reason for their exclusion. They are usually based on dry rennet casein into which lipids and water are emulsified or absorbed, respectively. Their production involves many of the operations used for other types of cheese, e.g., rennet coagulation, cooking, syneresis (as for natural rennet-coagulated cheeses), heating and emulsification, packaging (as for processed cheese). Since they are not ripened, it seems reasonable to classify cheese analogues as 'processed unripened cheese'. The principal among such cheeses at present is analogue pizza cheese. A modified version of the classification scheme of Fox et al. (2000) is shown in Fig. 1, incorporating processed cheese, cheese-derived products and cheese analogues. Probably the most comprehensive classification scheme for cheese developed to date is that of Ottogalli (1998, 2000a,b, 2001) which organizes cheeses into three main groups (indicated by the Latin words: 'Lacticinia' (milk-like), 'Formatica' (shaped), 'Miscellanea' (miscellaneous; Table 7). The Lacticinia group includes products which are produced from milk, cream, whey or

Cheese Analogues

tt

"l

Enzyme-ModifiedCheese

~

~.

Acid-Coagulated

Ricotta

I

Cottage, Cream, Quarg

i

Dried Cheeses

Heat/Acid Coagulation

Cheese

Processed Cheese

Rennet-Coagulated

9

Concentration/CrystallizatiOnMysost

Most varieties of cheese may be processed

Natural Cheese

I Surface-ripened

Mould-ripened

Internal bacterially ripened

Havarti Limburger MOnster Port du Salut Trappist Taleggio Tilsit

I Surface mould

(usually P. camemberti ) Brie Camembert

Internal mould (P. roqueforti) Roquefort Danablu Stilton

Cheeses with eyes Hard Grana Padano Parmesan Asiago Sbrinz

Cheddar Cheshire Graviera Ras

Caerphilly Mahon Monterey Jack

Swiss-type (Lactate metabolism by Propionibacterium spp.) Emmental Gruyere Maasdam

High-salt varieties Domiati Feta

Dutch-type (Eyes caused by citrate metabolism)

Pasta-filata varieties Mozzarella Kashkaval Provolone

Edam Gouda

The diversity of cheese. Cheese varieties are classified into super-families based on the method of coagulation and further sub-divided based on the principal ripening agents and/or characteristic technology (modified from Fox et al., 2000).

Diversity of Cheese Varieties: An Overview

9

Classification of cheeses according to Ottogalli (1998, 2000a,b, 2001)

~5

Farn~

Class

Description

Examples

Yoghurt-like product, but with loss of some whey

Lebneh (Middle East); Fromage Blanc (Switzerland, France); Sauer-milchk&se, Quarg (Germany) Queso Blanco (Latin America); Cottage (UK, USA); Quarg (Germany); Tvorog (Poland) Whey cheese (UK); Ricotta (Italy); Manouri (Former Yugoslavia); Brunost, Getost (Norway) Whey cheese (UK); Ricotta (Italy); Ziger (Germany); Mysost (Norway) Mascarpone (Italy) Skyr (Iceland); Karish (Egypt); Buttermilk Quark (Germany); Aoules (Algeria); Kolostrumkase (Germany); Sa Casada (Italy), Armada (Spain)

Milk coagulated by addition of organic acid

c O. .m

Acid addition and heating of whey (goat or ewe) Acid addition and heating of whey (cow) Acid addition and heating of cream Acid addition and heating of buttermilk

o rL. LL

Acid addition and heating of colostrum or beestings Acid-rennet coagulation I

Rennet-acid coagulation c .m

c O.

I

I

~ c o II

Goat or sheep

~~Fresh-kneaded or plastic or stretched cheeses

r -g .~ gg O.

Coagulum cut into cubes and/or flakes cooked, drained, washed and water cooled Rindless, very short ripening phase Thin rind, short ripening ( ~'~ "~- L_"

Ewes' or goats'

~o~ N

9"~

m ~

~, ..=

~9 E I-r I

E

Untextured, usually semi-cooked and pressed

C 0 (I) "O

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Washed curd (eyes caused by citrate metabolism or by heterolactic bacteria) Same as F1 but from goats' or ewes' milk

u~ E - ~ ~ 0 ._0 C~ ~.C_

c

c

9

0

Kneaded curds ('pasta filata') Propionic cheeses. Big round eyes

Textured (and dry salted) curd c- ~ .& E II

(0--

Smeared rind

Buxton Blue, Stilton, Dovedale (UK); Gorgonzola (Italy); Danablu, Mycella (Denmark); Bergader (Germany); Gammelost (Norway); Adelost (Sweden); Bleu d'Auvergne, Bleu de Causses, Bleu de Gex, Bleu de Laqueille, Fourme d'Aubert (France); Cashel Blue (Ireland) Bleu de Bresse (France); Cambozola (Germany) Roquefort (France); Cabrales (Spain); Kopanisti (Greece); Castelmagno, Murianengo (Italy) Montasio, Raschera, Bettelmatt (Italy); Pinzgauer (Austria); Beaumont, Laguiole, Murol (France); Raclette (Switzerland); Trappisten (Germany) Edam, Gouda (The Netherlands); Fontal (Italy); Mimolette (France); Blarney (Ireland) Serra (PR); Orduna, Mahon (Spain); Ossau-lraty (France); Pecorini: Pecorino Toscano, Canestrato (Italy); Altemburger (Germany) Caciocavallo (Italy); Ostwepock, Kasseri (Greece); Oaxaca (Mexico) Maasdamer (The Netherlands); Fol Epi (France); Jarlsberg (Norway); Samsoe (Denmark); Pategras, Colonia (Argentina) Lancashire, Colby (UK). Leiden (The Netherlands), Monterey (USA) Fontina (Italy); Tilsit (Germany); Appenzeller (Swtzerland); Stinking Bishop (UK)

Diversity of Cheese Varieties: An Overview

11

continued

Class

Fam~ c 0 if)

~ c-

c 9

I

9

0

.=

9r-- II E ~- -b- o

~~ -8=8 =o.

Description

Examples

Untextured, usually cooked and pressed

Asiago d'Allevo, Grana (Italy); Reggianito (Latin America); Sbrinz (Switzerland) Edam, Gouda (The Netherlands) Pecorino Romano, Pecorino Sardo (Italy); Kefalotiri (Greece); Manchego, Idiazabal (Spain); Ras (Egypt) Provolone (Italy); Parenica (Russia); Kashkaval (Bulgaria); Kasar peyniri (Turchia) Emmental (Switzerland, France); Svembo, Danbo (Denmark); Kefalograviera (Greece) Cantal (France); Cheddar, Cheshire, Derby, Single Gloucester, Double Gloucester (UK); Monterey (USA) Gruyere (Switzerland, France); Puzzone di Moena (Italy); Tete de Moine (Switzerland)

Washed curd, long ripened Same as G1 but goats' or ewes' milk Kneaded curds ('pasta filata')

0

~_o ii

Cheeses with eyes

e-, -d_J .~ x

-r--

m

I

I-

o x o

(D O') O 0 c cO 0 .@.., O) 0 ::t: "~

~-~

~. .-=

Textured (and dry salted) curd ('Cheddaring') Smeared rind The microbial coat causes the development of strong aroma Melted Smoked Grated or fractionated Mixed with other ingredients (fruit, vegetables, spices)

Ripened or kept under particular conditions, i.e., 'Pickled cheeses'

"0O

E 0 0 e-

o

Obtained using special technologies (i.e., ultrafiltration, sterilization or finished cheese) Products similar to cheese and with non dairy ingredients

Processed cheese, Spread cheese, Sottilette TM Oak-smoked Cheddar (United Kingdom) 'Grating cheeses' Friesan Clove cheese (NL); Sage Derby (UK); Kummelkas~, K&se mit Champignons (Germany); Sapsago (Switzerland); Ciboulette (France) Devon Garland (United Kingdom); Bruss (Italy); Kopanisti (Greece); Tupi (Spain); Fromage fort (France) PhiladelphiaTM (USA); BelgioiosoTM (Italy)

'Imitation cheeses', Filled cheeses

1Index of maturation (IM) = soluble N • 100/total N. 2Index of lipolysis (IL) = free fatty acids • 100/total fat.

buttermilk by coagulation with acid (lactic or citric), with or without a heating step. However, a small amount of rennet is often used to increase the firmness of the coagulum (e.g., Quarg and Cottage cheese). The Lacticinia group contains one class (A) comprised of seven families. Family A1 includes yoghurt-like products from which some whey is removed. Family A2 contains somewhat similar products but from which a large volume of whey is removed and acid is added. Families A3 and A4 are whey cheeses produced by the combination of heat and acid (e.g., Ricotta) while cheeses in Families A5, A6 and A7 are similar to other products in the Lacticinia group except that they are made from cream, buttermilk or colostrum, respectively.

The second group, Formatica (Table 7), contains most cheese varieties, all of which are coagulated by rennet. This is a large heterogeneous collection of varieties which are divided into 6 Classes (B-G), based essentially on the moisture content and the extent of ripening, and 31 families. Classes B and C include fresh cheeses and varieties with a short ripening period, respectively. The cheeses in Class D are soft surface-ripened varieties with a surface growth of moulds or smear bacteria. Blue cheeses are grouped in Class E while Classes F and G contain semi-hard and hard/extra-hard varieties, respectively. The third group of cheeses, Miscellanea (Table 7), is a heterogeneous collection of varieties and includes

12

Diversity of Cheese Varieties" An Overview

processed, smoked, grated and pickled cheeses, cheeses containing non-dairy ingredients (fruit, vegetables, spices), cheese analogues and cheeses made using ultrafiltration technology. The scheme of Ottogalli (1998, 2000a,b, 2001) takes into consideration the technological, chemical, microbiological and organoleptic characteristics of different cheese varieties, with the objective of a better classification of cheeses and related fermented dairy products into distinct categories. Chemical indices, which were given particular importance in the development of this classification scheme, included index of maturation (IM = soluble N • 100/total N, which can range from 1-2 to 60-70% although data for many cheeses are not available), lipolytic index (LI = free fatty acids • 100/total fat, which can range from 1-2 to 15-20%, although data for many cheeses are lacking) and fat:protein ratio (high fat = 2-5, medium fat = 1.2-1.5, low fat = 20 000 L) volumes of milk. The operating principles of the design are illustrated in Fig. 2. Switching from the vertical to the horizontally mounted vats simplified the construction required to process the larger milk volumes. The essential difference between the three horizontal OST models (III, IV and V) is in the design of the cutting/stirring mechanisms. The knife in the OST III vat is thicker and its cutting/stirring speed is

27

limited to 6 rev/min. In comparison, the knife in the OST IV vat is thinner and has 'stay-sharp' qualities that reputedly reduce fat and fines losses to the whey. The construction and design of the OST V knife frames was revised to meet the latest hygiene requirements and to improve cheesemaking performance. In early 2002, Tetra Tebel delivered the thousandth vat of the series (OST III-OST V). OST vats have been installed in 35 countries and this vat type is used to make a range of cheese types, including semi-hard (Edam, Gouda, St Paulin, Havarti), hard (Cheddar, Emmental, Romano, Monterey Jack, Egmont, etc.) and low-moisture Mozzarella (Pizza type). The Damrow double-O vat

The vertical Damrow vat was developed in 1972 and has had two updates (Fig. 3). This vertical design was to become Damrow's 'proven standard', and to date

OST IV cheese vat. 1. Combined cutting and stirring tools, 2. Strainer for whey drainage, 3. Frequency-controlled motor drive, 4. Jacket for heating, 5. Manhole, 6. CIP nozzle. Courtesy of Tetra Pak, Sweden.

28

General Aspects of Cheese Technology

Damrow Double-O cheese vat. Courtesy of Damrow Inc., USA.

900 are in use worldwide. Although used to make a range of cheese types, the vertical Damrow vats were used almost exclusively in the New Zealand cheese industry in the early days of mechanisation to produce Cheddar and other dry-salt cheeses. Easily recognised with its 'double OO' configuration, the vertical Damrow vat has two vertical knife arrangements that were used both to cut and stir the curd. Capacity ranges between ~ 1000 and 22 700 1. The Damrow horizontal vat

The horizontal double OO Damrow (DOH) was Damrow's second-generation vat. The design was patented in 1994 and improved upon in 1997, 1999 and 2000 (Fig. 4). Superior draining capability, improved yield and a hot water or steam dimple jacket are characteristics of this vat type. To date, 49 DOH vats are in service in Canada, USA and New Zealand. Three vat sizes are available: ~ 1 6 000 1, ~ 1 8 000 1 and ~ 3 0 000 1. The Scherping horizontal cheese vat

(HCV)

The first dual-barrelled horizontal cheese vat was developed by Scherping Systems in 1988. Of interest are the unique design of the vat's 'counter-rotation', dual agitator, the cutting and stirring system and the

staggered design of the knife arrangement of the thirdgeneration model (see Fig. 5). The unique 'interlocking' action and the lower speed required by the two counter-rotating agitators in both cutting and stirring modes are claimed to reduce losses and to give a more uniform curd particle size distribution. A study on cutting similar to that of Johnston et al. (1991) was undertaken on the Scherping HCV by McLeavey (1995). Since 1998, 328 of the patented HCVs have been built mainly for US customers; HCVs have been installed in one plant in New Zealand. The most popular capacities are 25 000 and 30 000 1. As would be expected in a mostly American market, consumer cheeses made using HCVs are Americanstyle Cheddar, Colby, Swiss, Co-jack and Monterey Jack cheeses and the Italian-style Mozzarella, Asiago and Parmesan cheeses. Cheeses for further processing, such as the fat-free, reduced-fat or low-moisture barrel Cheddar and Swiss barrel cheeses are also made in HCVs. Scherping Systems, now a Carlisle company, has now developed and is producing the fully automated thirdgeneration HCV incorporating new counter-rotating agitators, dual curd outlets for more effective emptying and changes to the knife configuration of previous HCVs.

General Aspects of Cheese Technology

29

Damrow DOH horizontal cheese vat. Courtesy of Damrow Inc., USA.

The A P V CurdMaster

The first APV CurdMaster was produced in 1993 and its design is based on the Protech CurdMaster and the Damrow Double-O vat design, as shown in Fig. 6. As with the Damrow Double-O vat, each of the two knife panels of the APV CurdMaster is hung-off centrally located axes within each 'barrel'. However, the light stainless steel knives are mounted vertically in a stag-

gered formation across each panel, and the stirring blades are made of polypropylene. APV Denmark decided to concentrate on the DoubleO design because there were several advantages. The Double-O design allows: 9 for variable degrees of filling from 40 to 100%; 9 all shaft seals to be located above product level;

Scherping horizontal cheese vat. Courtesy of Scherping Systems, USA.

30

General Aspects of Cheese Technology

APV CurdMaster cheese vat. Courtesy of Invensys APV, UK.

9 efficient horizontal and vertical mixing; 9 minimal air entrapment after predraw or reduced fill levels. In addition, APV modified the attachment of the bottom of the vat to its support frame (floating bottom) to avoid welds cracking during heating and cooling. A 5 ~ incline and two outlets instead of one for more rapid and efficient emptying, staggered stay-sharp knives, polypropylene agitators and whey predraw during agitation are other modifications made by APV. Since 1993, APV Denmark, now part of the Invensys APV group of companies, has sold 146 APV CurdMaster vats to 56 customers throughout Europe and Latin America. The capacity ranges from 6000 to 30 000 1. Cheese types made using the APV CurdMaster include Danbo, Raclette, Mozzarella, Gouda, Edam,

Emmental, Tilsit, Blue, Feta, Maasdam, Cagliata, Provolone, Norvegia, Manchego, Camembert, Pecorino, Grana, Cheddar, Havarti, Port Salut and Parmesan. It is interesting to note that many of the cheeses listed are curd-washed varieties. Continuous processes

There have been various attempts to replace the batch vat process by continuous systems. Two systems warrant brief mention. An innovative system using uhrafihration technology and a sequential coagulation system was developed jointly by the CSIRO in Australia and APV, the process being named Sirocurd. Two commercial plants were developed and these successfully produced Cheddar-types cheese, with the benefits of increased yield from the uhrafihration stage (Jameson, 1987); however, the Sirocurd equipment is not now in operation.

General Aspects of Cheese Technology

The other system, which is still widely used, is the Alpma continuous coagulator. A diagram of this equipment is shown in Fig. 7. The system incorporates the use of a continuous belt, which is formed into a trough to hold the milk. This trough is then subdivided by a series of plates to effectively form mini-vats. As the belt moves, the vats also move along and the same processes that occur in a batch vessel are carried out on the belt, via the use of cutting tools, stirrers and other tools that are incorporated along the length of the belt. Partial whey drainage and water addition can also be incorporated, with the main curd/whey separation occurring at the end of the belt. Cooking is difficult with this system, which is therefore more suitable for the production of soft to semi-hard cheese types. Gentle treatment of the curd and evenness of particle size result in uniformity and continuity of output. These coagulators are in use worldwide, producing a wide range of cheese varieties from fresh curd to Havarti. Post vat stages - dry-salted types

Processing options here depend largely on whether the curd undergoes further development and handling as curd particles, followed by dry-salting and block formation, or whether the final cheese block is formed immediately, followed by subsequent brining for salt uptake. As shown in Fig. 1, distinctive processes are involved. The processes described here apply to hard cheese varieties such as Cheddar, Colby, Egmont and stirredcurd cheeses.

Oewheying The vats are emptied by pumping out their contents of curds and whey. This process is commonly described as running or draining the vat. Correct pump selection

31

is of vital importance as the curd can potentially be damaged, generating large quantities of fine particles that are lost into the whey stream. Large, slowly revolving, positive rotary lobe pumps are a common option, with the Sine | pump, which uses a specially formed impeller, becoming increasingly popular because of its gentle operation and low curd damage. During emptying of the vats, the stirrers remain in operation to ensure mixing of the vat contents. For the whole cheesemaking process to be effectively continuous, despite the batch vat stage, it is necessary for there to be a number of vats, e.g., eight vats operating and emptying in sequence to provide a continuous flow of curd. Even with this system, there is variation in acidity and composition between the curds that first leave the vat and those that leave towards the end. This effect can be minimised on multi-vat plants by overlapping vat emptying using dual pumps. The ratio of curd to whey also varies as the vat is emptied, with a higher proportion of curd at the start. The pump speed is controlled to increase during vat emptying to provide a uniform flow of curd to the next stage of the process. Primary separation of the curds and whey is achieved by pumping the curds/whey mixture from the cheese vat over a specially designed dewheying screen. This is normally parabolic in shape, fitted with horizontally oriented wedge wires, to maximise the efficiency of the separation process with minimal curd damage. The whey passes through the screen and the curd is transported to the next stage. The feed to the screen is designed to provide an even, gentle flow across its width; this is often achieved by the use of a weir feed arrangement. An example of the system used is illustrated at the top of Fig. 8, the Alfomatic cheesemaker. The whey that is removed through the screen is

Alpma coagulator. 1. Belt infeed, 2. Spacing plate insertion station, 3. Milk infeed, 4. Spacing plate in the coagulator, 5. Spacing plate transport, 6. Spacing plate extraction, 7. Curd-releasing station, 8. Curd cutter, longitudinal, 9. Curd cutter, crosswise, 10. Open syneresis sector, 11. Belt discharge, 12. Spacer plate cleaning. Courtesy of Alpma, Germany.

32

General Aspects of Cheese Technology

Alfomatic cheesemaker. 1. Whey screen, 2. Whey sump, 3. Agitator, 4. Conveyors (variable speed), 5. Agitators (optional) for stirred curd, 6. Chip mill. 7. Dry-salting system. Courtesy of Tetra Pak, Sweden.

collected and pumped to a tank prior to separate processing operations to produce a wide range of products. Initial processing operations include clarification to remove casein fines, centrifugal separation to recover fat and pasteurisation or thermisation to reduce the microbiological activity.

Drying (draining) the curd Commercial plants almost universally use a belt system for this next part of the process. Specially designed slotted plastic or stainless steel conveyor belts are used. These are usually fitted with peg-stirring devices mounted above the belts to agitate the curds in order to facilitate whey drainage and to prevent clumping of the curds. Residence times of 10 min are common. This belt often forms the first part of a cheese-texturing belt system. An example of these is the Alfomatic shown in Fig. 8.

Texturing (cheddaring) or stirring For varieties such as Cheddar, a traditional step in manufacturing protocol is the cheddaring stage, during which the curd is allowed to knit together, to flow and stretch and to develop a cooked chicken meat-type of structure. In the small open-vat process, cheddaring is achieved by heaping the drained curd along the sides of the vat and allowing it to fuse together. The fused mass is then cut into blocks of 10-20 cm and these are turned every 15-40 min over a period of 90-120 min to encourage flow and stretch to develop the desired

structure. There have been numerous attempts to replace this highly manual, labour-intensive process by a fully mechanised system. One such system is the cheddaring tower, a version of which was developed in New Zealand and is still available from Invensys APV. An example of this system is shown in Fig. 9. Essentially, the towers are cylindrical holding tubes, changing to a rectangular discharge section. Incorporated into their structure is a whey drainage system. Holding times of 1-2 h can be achieved with a capacity of up to 5000 kg curd/h. Large blocks of curd are guillotined from the column of curd as it exits from the base of the tower and fed into a curd mill. In the newer plants, a belt system has become very popular, typically with two belts running at different speeds to provide stretch, flow and inversion of the curd mass, and also to provide the desired holding time. Capacities of 12 000 kg of curd/h are possible. Examples of such equipments are the Alfomatic (Fig. 8), the Cheddarmaster (Fig. 10) and the Scherping draining conveyor (Fig. 11). These belt systems are totally enclosed in stainless steel housings. This provides a hygienic environment, and also the facility for in-place cleaning and maintenance of temperature. The belts are made of plastic or stainless steel and are generally not perforated, unlike the draining belts described earlier. The belts that are available for the cheddaring/holding stage can also be fitted with peg stirrers mounted

General Aspects of Cheese Technology

33

above the belt to facilitate the manufacture of stirred curd varieties, e.g., Cheshire and Egmont, on the same equipment. Similarly, the speed of the conveyors can be adjusted to provide the desired residence times.

Milling (size reduction) Following the texturing or cheddaring stage, the curd mass has fused into a solid structure. For the incorporation of salt in the next stage, it is necessary to reduce the solid mass to curd fingers (chips) of approximately 1.5 • 1.5 • 8 cm. This is achieved by the use of curd mills, of which there are a number of types. Most operate by using a rotating cutting tool, which cuts the curd mass in two directions using a blade and a comb. Prevention of fine particle generation is an important feature of the design. For stirred curd varieties, where little curd fusion has occurred, the mill still operates to break up any lumps that have formed. The mill is located at the base of the tower in a cheddaring tower system, or at the end of a conveyor belt in the more common belt systems.

Dry-salting and mellowing

APV cheddaring tower, with guillotine and mill at base. Courtesy of Invensys APV, UK.

Salting the curds is a vital part of the cheesemaking process. Salt has very important roles in flavour enhancement and in the control of microbiology, final cheese pH and moisture content. A detailed discussion on salting is given in 'Salt in Cheese: Physical, Chemical and Biological Aspects', Volume 1. Critical factors include the application of the correct ratio of salt to

APV Cheddarmaster belt system. Courtesy of NZMP Whareroa, New Zealand. (See Colour plate 2.)

34

General Aspects of Cheese Technology

Scherping cheese curd draining conveyor. Courtesy of Scherping Systems, USA.

curd, even uptake of the salt and controlled loss of moisture. The level of salt required will vary according to the type of cheese being manufactured. There are two components to the salting p r o c e s s - the application of the salt (salting) and the subsequent mixing, uptake and associated moisture loss (mellowing). There has been a range of equipment designs to achieve satisfactory salting, with variable success. Simpler styles have included belt systems in which the quantity of curd being conveyed is measured by means of a fork sensing curd depth, with dry salt then being air-conveyed and distributed across the belt by a reciprocating boom. The quantity of salt is varied in proportion to the curd flow and is metered by a funnel and salt wheel device in a dry area of the plant. Better control can be achieved by using load cells on the belt to weigh the curd flow. Twin-salting booms are another alternative, each applying a proportion of the salt. A widely used system is the trommel or drum salter, in which the curd flow is directed over a weighing belt and then into a rotating drum into which the salt stream is directed. This provides accurate measurement and good mixing. However, if this system is to be used in conjunction with a belt plant, the curds must be conveyed from the belt to the salter and returned to the next belt. An example of such a system is shown in Fig. 12. A variation on this concept involves the use of an auger conveyor instead of the rotating drum to provide mixing of the salt and curds, as they are conveyed back onto the mellowing belt.

The mellowing belt provides a holding time of 10-20 min to allow the applied dry salt to be mixed, dissolved and absorbed by the curd, at the same time as moisture is expelled. The belts are equipped with peg stirrers to encourage mixing and moisture loss, and they are also enclosed to maintain temperature. An alternative to the belt system is the use of finishing/sahing vats or tables, which are suitable for stirredcurd varieties. In these, the curds/whey mixture is pumped from the vat into these batch tanks, which allow whey drainage, holding time and pH drop, salt addition and mellowing, all in one vessel. An example is the Damrow enclosed finishing vat shown in Fig. 13.

Pressing~block formation- general discussion This process is common to most cheese varieties, exceptions being particulate cheeses such as Cottage cheese. Block formation involves the conversion of granular, particulate curd into a solid block of cheese. The degree of compression required and the techniques used vary according to the cheese type. For example, close-textured hard cheeses such as Cheddar require the application of considerable pressure and air removal to form appropriate blocks. Other varieties, such as Blue cheese, require little compression and pressure in order to produce an open texture enabling air penetration and mould growth. Varieties such as Gouda and Edam require preliminary block formation while submerged in the whey prior to further compression. A vital component of block formation during the history of cheesemaking has been the cheese hoop or

General Aspects of Cheese Technology 35

Figure 12 Trommel salting system. Courtesy of NZMP Edendale, New Zealand. (See Colour plate 3.) mould. Although its use has been superseded by blockformers in the large-scale production of dry-salted cheese, it is still a vital component of many other plants and also small-scale dry-salt plants. The cheese hoop or mould is a specialised container designed to hold and form the curd into the desired shape, permitting the further loss of whey and the application of pressure and vacuum, if so desired. The moulds were made originally of wood, with the inner shape being that of the final cheese. They were cylindrical or rectangular and had holes drilled through the sides, base and lid to permit whey drainage. They were often lined with cloth (hence the term cheesecloth) to provide a porous barrier between the curd and the walls to allow whey drainage. An early option was the use of metal, especially for rectangular blocks, and the use of telescopic lids and bases to permit compression of the blocks under applied external pressure. This system is still in use for small-scale operations, with stainless-steel moulds and synthetic cloths providing improved hygiene. A major technological development has been the introduction of plastic moulds. These may range from a simple plastic or metal tube with appropriate perforations, for a variety such as Camembert, to which no external pressure is applied, to a highly sophisticated micro-perforated, grooved, muhi-mould for Gouda. This technology has eliminated the need for cheesecloths, as drainage is via the grooves and the micro-porous holes. Hygiene is

maintained through an appropriate cleaning process, which may include ultrasonics. The desired cheese surface effect may be achieved by selecting an appropriate surface grooving. A major recent advance has been the introduction of welded plastic moulds, eliminating the use of metal screws as in earlier types. The Dutch company, Laude bv, has been at the forefront of developments in this field, and examples of its products are shown in Fig. 14. The appropriate pressing regime to be applied to the curd contained in the mould depends on the cheese type and is discussed separately. There is a risk in the application of too much pressure initially, which results in surface closure and poor subsequent whey removal.

Pressing/blockforming of dry-salted cheese For dry-salted cheeses, the next stage of the process is the conversion of the salted chips of curd into a solid block. The traditional process involved the use of hoops or moulds into which the curd was weighed and then compressed, often overnight, by externally applied pressure using hydraulic rams, commonly in horizontal gang presses. This system is still in use in small-scale plants, and developments in this area are discussed in more detail under brine-salted cheeses. The universal system adopted in large-scale dry-sah plants involves the use of blockformers, of which there are a number of varieties. Wincanton Engineering in the UK patented the original development over 25 years ago.

36

General Aspects of Cheese Technology

Damrow enclosed finishing vat. Courtesy of Damrow Inc., USA.

Plant capacity requirements usually mean that several blockformers are necessary and it is therefore important for reasons of product uniformity that an even feed is supplied to each blockformer. This may be achieved by using devices such as curd distribution tanks, which provide mixing of the curd from the mellowing belt and even distribution of the curd to the suction tubes feeding the blockformers. An example of these is shown in Fig. 15. All blockforming towers operate on a similar principle of using vacuum to draw curd into the top of the tower. The curd column is then subjected to further vacuum as it progresses down the tower. The internal side walls are perforated to facilitate whey and air removal, and the height of the towers (6-9.5 m) provides compression by gravity. As the curd travels down the tower, it is converted from individual curd par-

ticles into a fused column. This is discharged at the base via a guillotine arrangement, which produces blocks of cheese of a uniform shape and weight, typically 18-20 kg. The operation of the tower is illustrated in Fig. 16. The typical residence time in the towers is 30 min. Weight control is effected by adjustments to the platform height in the guillotine section. All the major equipment suppliers produce blockformers with variations in detail. Some of the more recent developments include extending the height to increase capacity and the provision of two different vacuum stages, as in the Tetra TwinVac Blockformer | This permits the use of a higher vacuum in the lower column, which is effectively separated from the upper column by a plug of curd, permiting the use of a lower transport vacuum in the upper section and a higher throughput.

General Aspects of Cheese Technology

37

Laude block mould. Courtesy of Laude by, The Netherlands. (See Colour plate 4.)

There are a number of variations of blockformers, producing differently shaped and sized blocks from 10-kg cylinders to 290-kg blocks. The type used depends on the product's end-use. A recent innovation by Cryovac | has been the introduction of bag loaders at the base of the towers, which automatically fit cheese bags to the discharge channels to receive the cheese blocks from the tower. The same company also supplies gusset stretchers to help present the bagged cheese in the appropriate form to the vacuum-sealing device. This equipment has removed another repetitive manual operation from the process. An example of blockformers fitted with bag presenters is shown in Fig. 17. The packing of the cheese is important as it plays a role during curing and storage, in the final cheese shape and appearance and in protection from the environment. The formed cheese blocks are discharged from the pressing towers into muhi-layered plastic bags. These are conveyed to a vacuum-sealing chamber where air is removed from the bag which is heatsealed. The gas and water permeability properties of the bag and the level of vacuum applied vary according to the cheese type. Prevention of moisture loss and prevention of mould growth are key factors for Cheddartype cheeses. The curd is still warm (typically 33 ~ as it exits the blockformers and is quite plastic. Therefore, the vacuum-sealed block requires the support of a carton

while cooling to maintain its desired shape and finish. Cartoning operations are normally fully automated with a variety of carton styles in use, ranging from a shoebox style with a separate base and lid to a wraparound one-piece type.

Ripening and storage This is a highly complex topic, which is the subject of several other chapters in this book (see 'Biochemistry of Cheese Ripening: Introduction and Overview', 'Metabolism of Residual Lactose and of Lactate and Citrate', 'Lipolysis and Catabolism of Fatty Acids in Cheese', 'Proteolysis in Cheese during Ripening', 'Catabolism of Amino Acids in Cheese during Ripening', 'Sensory Character of Cheese and its Evaluation' and 'Instrumental Techniques', Volume 1). Cheese is essentially a complex matrix of protein, fat and carbohydrate, containing a range of enzymes and microorganisms. Their activities produce the changes that convert the young or green cheese into the desired final product, primarily through proteolysis, lipolysis and glycolysis. The primary objective of the cheesemaking process is to produce a material with the desired characteristics for ongoing changes during curing and storage. Factors such as salt content, pH and moisture content are of critical importance. The primary controllable factors after the young cheese has been made are the time and the temperature of storage. During ripening, changes in flavour and texture

38

General Aspects of Cheese Technology

which 40 or more blocks are stacked and shrinkwrapped. This format is suitable if the cheese is to be used for manufacture into processed cheese. Alternatively, the cheeses in cartons may be stacked on a pallet, or the cheeses in carton bases may be placed in bulk bins. These are strapped and tension is applied to help maintain shape and finish. This format is suitable for cheese intended for the precutting trade, where the large blocks are cut and repacked into consumer packs. Robots are normally used for these assembly operations. A typical assembly is shown in Fig. 19.

Ripening (curing). This involves the transfer of the palletised product to controlled-temperature storage rooms where the pallets are assembled onto racks. Typical temperatures are 8-10 ~ for a period of 35 days or so. Temperature and time after this stage will depend on the desired end-use for the product. For example, if a more rapid maturation is required, the temperature may be elevated to 15 ~ for 1 month. If a slower rate is required, a temperature of 2 ~ may be used. Once the desired degree of ripening has been achieved, the product is transferred to reducedtemperature storage to reduce the rate of further change. Storage. In this stage, the objective is for minimal change in product characteristics with time. This is achieved primarily through controlling the temperature. Freezing of the product is an option if the enduse for the product is processed cheese. Curd distribution tank. Courtesy of NZMP Stirling, New Zealand. (See Colour plate 5.)

occur. From a technological point of view, several stages can be identified- initial cooling, curing or ripening and controlled storage. The particular regime used depends on the cheese type and its intended use. Initial cooling of dry-salted cheese. This serves two

purposes. Firstly, a reduction in the temperature of the cheese curd causes the fat to solidify and the cheese to become firm and maintain its shape. Secondly, a sharp drop in temperature prevents the rapid growth of undesirable non-starter lactic acid bacteria, which could otherwise use residual lactose and produce undesirable gas and flavour defects. A reduction in temperature to 16 ~ within 12-16 h of manufacture is achieved by the use of open-rack stacking of the cheese blocks, which are then conveyed into a blast chiller, using air at 2-8 ~ Openrack stacking is necessary to permit good air flow and heat transfer. The rapid chillers operate on a first-in/firstout basis. An example is shown in Fig. 18. Following the rapid cooling operation, the cheeses are stacked into the form required for their long-term curing and storage. This may be a cartonless pallet on

Despatch The process described thus far is for the production of bulk blocks of cheese, typically weighing 20 kg. This product has many end-uses, such as an ingredient for many food products that contain cheese, conversion to grated cheese or processed cheese or cutting as natural cheese into consumer-size blocks. The uses of cheese as a food ingredient and as processed cheese are the subject of separate chapters ('Pasteurized Processed Cheese and Substitute/Imitation Cheese Products', 'Cheese as an Ingredient', Volume 2). The preparation and packaging of cheese for domestic consumers involves the use of a wide range of sophisticated equipment and packaging technologies, the detail of which is beyond the scope of this chapter. Typical steps involve cutting the cheese blocks into the appropriately sized smaller blocks, followed by packaging in appropriate laminated material, under either vacuum or a modified atmosphere. Post vat stages - hard/semi-hard brine-salted types

Post vat processing of the cheese curd differs considerably for cheeses that are essentially formed into their final block shape on leaving the vat, as these generally require

General Aspects of Cheese Technology

Blockformer operating principles. Courtesy of Tetra Pak, Sweden.

Blockformers with bag presenters. Courtesy of NZMP Edendale, New Zealand. (See Colour plate 6.)

39

40

General Aspects of Cheese Technology

Dewheying For many varieties, partial whey removal occurs during the vat stage of processing, when the agitators are stopped for a period, allowing the curds to sink, and a whey-removal screen is lowered into the vat and the required amount of whey is drawn off. This is replaced by hot water, which serves to cook the vat contents and also to dilute the lactose and lactic acid content of the remaining whey. Further whey may be removed in the same way before the curds/whey mixture is pumped from the vat.

Pre-pressing

Rapid cooling tunnel. Courtesy of NZMP Hautapu, New Zealand. (See Colour plate 7.)

immersion in brine to achieve salt uptake. There are also processing differences depending on whether the cheeses are hard/semi-hard or soft and possibly mould-ripened. These differences are summarised in Fig. 1.

The presence of eyes or holes in the cheese is an important characteristic of several major cheese types, such as Gouda, Edam and Emmental. An important feature of the curd block formed for such cheese is the absence of air from within the block, and instead the presence of microscopic wheyfilled cavities in which micro-organisms can grow and produce gas, in particular CO2, which can ultimately form the characteristic round eyes (Martley and Crow, 1996; Kosikowski and Mistry, 1997). For the appropriate curd characteristics, the curds are formed into blocks below the surface of the whey prior to curds/whey separation, in contrast to the procedure with dry-sahed cheeses such as Cheddar. This process is known as pre-pressing. As block formation occurs prior to salting, an alternative salting technique, brine salting, also becomes necessary. To reduce the volume of material to be handled during block formation, some whey is removed using the vat sieve or strainer prior to pumping out the curds/whey

Robot stacking of cheese blocks. Courtesy of NZMP Hautapu, New Zealand. (See Colour plate 8.)

General Aspects of Cheese Technology

mixture to the pressing stage. An early development of a mechanised system to achieve the objective of pressing under the whey involved the use of prepressing vats, as illustrated in Fig. 20. The curds/whey mixture is pumped into a rectangular vat, and perforated metal or plastic plates are placed above the vat contents, and then lowered below the whey to the curd layer, which is supported by a woven plastic belt at the base of the vat. This layer is then compressed by the application of hydraulic pressure to the plates and a solid curd mass is formed. The whey is then removed, and the curd layer is conveyed from the base of the vat through the now-open end and is cut into appropriately sized curd blocks by cutting tools prior to being placed in moulds for further pressing and formation. More advanced systems use a semi-continuous prepressing blockforming system of which the Casomatic | equipment produced by Tetra Pak Tebel is a widely used example. A diagram illustrating the working principles is shown in Fig. 21. Buffer tanks are used to store the curds/whey mixture pumped from the cheese vat; they are essential to provide an evenly mixed feed to the pressing system. The curds/whey mixture in the ratio of about 1:4 is then pumped to the top of the column, which is about 3 m in height, with a total unit height of 5.5 m. The column is filled and the curds settle below the whey to a height of about 2 m. Whey is removed from the column via three whey drainage bands; a controlled rate of removal is

41

critical for the formation of a block of the correct density at the base of the column. The curd block is formed in a dosing chamber and is cut from the column above by means of a guillotine. The dosing chamber then moves forward and discharges the formed block into a mould or hoop for further pressing and formation. Several variations using the same operating principle are available to produce blocks of various shapes and sizes from 1 to 20 kg, with discharge of multiple blocks from one column being possible. Exchangeable perforated drainage columns within a common jacket can be used, as in the Casomatic | MC model. Cheese types with irregular holes or eyes, also known as granular, e.g., Parmesan, can also be handled using equipment such as the Casomatic | Pressing under the whey is not required, and curds/whey separation can be achieved by the use of rotating sieves or strainers placed above the columns, discharging curd into the column for initial block formation.

Pressing Having formed the curd into the final cheese block by moulding in the pre-pressing stage, further pressing of the block is necessary. This provides a further period for ongoing acid development and pH and texture change, and assists final whey expulsion, shape formation and also surface texture for subsequent rind formation, where appropriate. Simple vertical pressing systems are suitable for small-scale operation, where the cheese moulds are loaded into

Pre-pressing vat. 1. Pre-pressing vat, 2. Curd distributors or CIP nozzle (2a), 3. Unloading device, 4. Conveyor. Courtesy of Tetra Pak, Sweden.

42

General Aspects of Cheese Technology

programme. Again, simultaneous loading and unloading of the pressing bays are practised. An example of a conveyor pressing system is shown in Fig. 23. Pressing times and pressures vary with the cheese variety and block size. It is important that there is a gradual increase in pressure, as the application of too much pressure at the start can cause closure of the surface and prevent whey removal. A typical programme for 10 kg Gouda cheese is 1 bar (0.1 MPa) for 20 min, followed by 2 bar for 40 min. For cheeses such as Emmental where blocks of 30-100 kg are common, a specialised system has been developed by Tetra Pak Tebel; it incorporates a specialised mould-filling system that can also incorporate pressing, with a further external press equipped with inverting facilities to help improve cheese quality and uniformity. Another automated system for blocks up to 700 kg is available. Once the required pressing operation has been completed and the desired pH drop has been achieved, the cheese blocks are removed from the moulds and are conveyed to the next stage of brining. The used moulds and lids are returned to the system via a cleaning process.

Brining

Casomatic operating principles. 1. Curd/whey mixture inlet, 2. Column with sight glass, 3. Perforated whey discharge, 4. Interceptor, 5. Whey balance tank, 6. Cutting and discharge system, 7. Mould, 8. Pawl conveyor, 9. Whey collecting chute. Courtesy of Tetra Pak, Sweden.

the press and the appropriate pressure regime is applied by lowering hydraulic rams. For larger-scale operations, trolley presses, tunnel presses or conveyor presses are used. With trolley presses, the cheese moulds are placed on a trolley, which is then fed into a tunnel equipped with a series of individual vertical rams. These are subsequently lowered to apply the appropriate pressure to the batch of cheese. Automatically fed tunnel presses operate by automatically loading cheese into the tunnel, followed by the pressing programme for the whole batch. Simultaneous loading and unloading is possible. An example is the APV SaniPress system shown in Fig. 22. The conveyor press is another option, with the cheese moulds being loaded onto a conveyor system, where the blocks are assembled into groups. Each block or pair of blocks has an individual hydraulic ram and each group has its own individual pressing

Cheeses that have been formed into blocks under the whey cannot be salted prior to moulding and pressing. The application of dry salt to the cheese surface is one technique that is used for some cheeses, such as Blue, but for many cheeses brine-salting is simpler, provides greater uniformity and is less labour-intensive. Many cheeses that have traditionally been made using brinesalting can in fact be made using the simpler and cheaper dry-salting technology described already for Cheddar-type cheeses. However, eye development is not usually attempted, with the major objective being to produce the appropriate typical flavour and texture. As already mentioned, there is a detailed discussion of salting in 'Salt in Cheese: Physical, Chemical and Biological Aspects', Volume 1. Brine-salting basically involves the immersion of the cheese block into a brine bath. The brine is a solution about 19-21%, w/w, of NaC1. It should also contain an appropriate level of CaC12, e.g., 0.2%, w/w, to prevent leaching of calcium from the cheese. Its pH should be close to the cheese pH (typically 5.2-5.3) and its temperature should be 10-14 ~ As the brine is used, its salt concentration must be maintained as salt moves into the cheese and water/whey moves out, causing dilution. Also, the brine will become contaminated with cheese particles, whey proteins and undesirable bacteria. Filtration (including membrane filtration), centrifugal clarification and pasteurisation can be used to maintain brine quality. If properly cared

General Aspects of Cheese Technology

43

APV SaniPress tunnel pressing system. Courtesy of Invensys APV, UK. (See Colour plate 9.)

for, the same brine can be used for many years (Bylund, 1995b; Kristensen, 1999). The time required for adequate salt uptake in the brine depends on the size of the cheese block and the

desired final salt level. For example, a small 250 g Camembert may require only a few hours, whereas a 10 kg Gouda may require 2 days. Brining systems can be a simple tank in which the cheese is placed once it has

Conveyor pressing system, with Casomatics in foreground. Courtesy of NZMP Lichfield, New Zealand. (See Colour plate 10.)

44

General Aspects of Cheese Technology

been removed from its mould. Alternatively, a more continuous system, known as the serpentine or surface brining system, may be used, where the cheeses are floated in brine channels to holding pens for the required period. As the surface of the cheese is above the brine, periodic spraying of the surface with brine or forced dipping of the cheese below the surface is required to achieve even salt uptake. Another option for brine application is the TrayBrine System from APV (Fig. 24). Here, the cheeses are placed on plastic trays, which are stacked and connected to a brine distribution system. The brine flows down over the cheese surface, is recirculated for the required period and is then recovered. A common method of brining for large-scale operations is the deep brining technique, where the cheeses are floated onto shelves on racks which are then progressively submerged below the brine surface. Ideally, the racks should be emptied and the loading sequence reversed midway through the brining process to ensure the first-in/firstout principle for consistent salt uptake. An example of a deep brining system is shown in Fig. 25. In addition to the vital effect of providing salt uptake for control of the microbiology and flavour of the cheese, brining also provides a rapid cooling effect,

reducing the cheese temperature to a value close to that of the brine within several hours. This helps control the growth of undesirable bacteria in a similar fashion to the rapid cooling step used in Cheddar production.

Ripening Once the cheese has been brined for the required period, it is floated to the discharge point and removed from the brine via a conveyor. Its surface may be rinsed with a brine solution to remove any foreign matter and is then air-dried with a blower or air knife. Thereafter, packing and curing depend on the intended market. Rindless cheeses, which are very commonly produced for bulk markets, especially if they are to be used subsequently as ingredients, are packed into appropriate laminated plastics bags under vacuum. They are then put into cartons and are stacked on pallets and transported to the appropriate curing and storage conditions. If eye development is required, several stages of temperature change will be used, e.g., for Emmental, 3-4 weeks at 10 ~ followed by 6-7 weeks at 22-25 ~ for eye development, and storage/curing at 8 ~ for several months. For Gouda, conditions may be several weeks at 10-12 ~ followed

APV tray brining system. Courtesy of Invensys APV, UK. (See Colour plate 11 .)

General Aspects of Cheese Technology

45

Deep brining system. Courtesy of NZMP Lichfield, New Zealand. (See Colour plate 12.)

by 3-4 weeks at 12-18 ~ followed by several months at 10-12 ~ (Bylund, 1995b). If eye development is desired, as gas production is necessary, appropriately permeable laminated bags must be used to permit gas transport. If rinded cheeses are being produced, control of the humidity in the curing rooms is important (usually about 85-90%) to prevent undue moisture loss. Coloured wax coatings may also be applied to provide protection for the cheese. Some varieties, such as Parmesan and Emmental, require frequent turning during curing to maintain the desired shape. Mechanised systems, such as revolving shelf rails, are available for all the material-handling operations such as inversion of the final cheeses. As already discussed under Cheddar types, curing and maturation are a combination of time and temperature conditions, with the additional influence of humidity for cheeses that are not packed in plastic film.

Despatch The cheeses have the same multiple end-uses as already described for dry-salted varieties. However, as the brinesalting system tends to be more expensive, these products are more typically directed at the retail consumer market, requiring appropriate cutting and packaging. Post vat stages - brine-salted, soft mould-ripened

Cheeses such as Camembert and Blue fall into this category. Technological advances and automation have been applied to these varieties and ultrafiltration has had a major impact, as numerous advantages, includ-

ing yield improvement, can be obtained. The use of ultrafiltration is discussed in detail in 'Application of Membrane Separation Technology to Cheese Production', Volume 1. Discussion of these cheeses commences at the vat stage in Fig. 1. Uniformity of milk, starter and coagulant activity are of critical importance for the uniformity of syneresis, which is essential for these varieties (Pointurier and Law, 2001). The normal operations of coagulation, cutting, stirring and acid development occur in the vat. The milk entering the vat may have been pre-ripened with starter culture and is likely to include the mould spores for later development. However, because of the high moisture content, which changes rapidly with time due to syneresis, it is not practical or desirable to use large vats for the production of Camembert types, in particular, as the curd composition of the material first being discharged would be very different from that discharged 30 min later. Hence, curd formation in small vats of up to 300 1 is necessary, so that the contents may be discharged rapidly into multi-moulds where curds/whey separation (dewheying) occurs. This is combined with moulding and may be done by tipping the vats directly into the moulds or by using a specialised portioning system such as the APV Contifiller, illustrated in Fig. 26. Also illustrated here is the use of multiple small vats on a semi-automated line and handling systems for the filled moulds. The batch-continuous production system is necessary to obtain a uniform fill of curds/whey mixture into the moulds, as this is the determinant of the final cheese size and weight.

46

General Aspects of Cheese Technology

1. Curdmaking 2. Curd draining and filling 3. Stacking of mould batteries (A) and trays (B) 4. Turning of mould stacks

5. Acidification lines 6. Destacking 7. Transfer/turning of cheese from mould batteries to trays

8. Transport to climate room (A) and from brining (B) 9. Turning/emptying 10. Washing of mould batteries (A) and trays (B)

Process line for soft cheese with Contifiller. Courtesy of Invensys APV, UK.

The development of various systems, such as the ~11Pril'l,.., ,.,,..~.l~ nar"s ruP' 'c' ~c l~ i~ APcrrihPA i n m n r ~ , ,..,,..,.,.,.rlPtailhyu R p r l r a n r - ] (1987) and Pointurier and Law (2001). Some earlier systems include the use of micro-pans, which produce just enough curd for one mould. The Alpma continuous coagulator, already described in the vat stage section under continuous processes, has special application for these soft cheeses, being effectively a continuous series of small vats. The multi-moulds used to form the cheese may be in two sections to provide sufficient volume for the initial fill. The upper layer can be removed once initial block formation has occurred. The filled moulds can be stacked automatically and conveyed to the initial ripening rooms for further acid development, followed by brining in tanks for about 30 min, and then ripening for about 10 days in high humidity rooms for mould development. Frequent turning of the cheese is necessary during the first few days to ensure even block formation. This can be automated in larger plants. Final wrapping is done in air-permeable material and despatch follows. Variations such as dry-salting the cheese by surface application, may be used for Blue cheese. A feature of these mould-ripened cheeses is that a very open texture may be necessary to allow oxygen penetration for mould growth. Hence the cheeses are

not pressed by the application of any external pressurei11~t

J ""

"

crr~rit,r 8 ..... )'

ir

11~r-]

Fnr

ehwPr

r

~r

R111~

~xTh~,rp

internal mould growth is desired, the passage of air is facilitated by spiking holes through the cheese with special needles. Smear-ripened cheeses are another type within both the semi-hard and the soft categories. The key process is the application and growth of a smear culture, predominantly Brevibacterium linens, on the surface of the cheese during ripening. Various mechanised brushing systems are available for smear application, which is usually repeated several times during ripening, where control of humidity and temperature is critical. Post vat stages - fresh cheeses

Cottage cheese falls into the soft/fresh category but is unusual in that the final product consists of curd particles packed in the final container with the appropriate dressing. Specialised equipment has been developed to mechanise and automate the production of this highly popular product. An example of this equipment is the O-vat by Tetra Pak Tebel. Quark, cream cheese and similar products also fit here but their manufacture is very different and is not described in detail (see 'Acid- and Acid/Rennet

General Aspects of Cheese Technology

Curd Cheeses: Part A Quark, Cream Cheese and Related Varieties, Part B Cottage Cheese, Part C Acidheat Coagulated Cheeses', Volume 2). Following the formation of the coagulum in special ripening vats, the whey is separated using a specially designed centrifugal separator. The product is then blended with appropriate additional components, e.g., cream, and then filled directly into the final container. Post vat stages- pasta filata

Pasta-filata

cheeses are those varieties for which the curd has been worked or stretched and moulded at an elevated temperature before cooling. This process imparts a unique and characteristic fibrous structure that influences both the ripening and the functional profiles of the final cheese. Mozzarella is probably the best known of the Pastafilata cheeses, which are mainly Italian in origin. However, the category also includes cheeses such as Provolone, Scamorza, Caciocavallo, Kashkaval and Pizza cheese. Composition, particularly moisture level, and fresh versus ripened textures are characteristics that define the various varieties. The increase in popularity of the pizza in its various forms (from the thin-based traditional Italian pizza, with few or no toppings except Mozzarella and cooked in a wood-fired oven, to the American-style thick pan-based pizza, with a myriad of toppings and cooked rapidly in an impinge> type oven) has focussed attention on low-moisture Mozzarella or Pizza cheese (see 'Pasta-Filata Cheeses', Volume 2). del Prato (2001) discusses the various varieties of Pasta-filata cheeses and the traditional processes and purpose-built equipment to make them. However, another manufacturing option is to use existing equipment and to add on a cooker/stretcher and a cooling operation at the end of the curdmaking stage of the existing process. This has been the case in the development of New Zealand's Mozzarella industry. New Zealand produces only low-moisture part-skim (LMPS) Mozzarella and has adapted its Mozzarella-make procedure so that the existing Cheddar vats and curd-handling and cheddaring systems can be used to produce Mozzarella curd for stretching and subsequent cooling. Hence, the Pasta-filata process is included as a branch of the dry-salt Cheddar-type process in Fig. 1. Dry-salting can also partially or completely replace brining. Equipment designed to perform the stretching operation incorporates two essential components: cooking and stretching (the mechanical treatment of the curd following cooking). The cooking phase is where the Pasta-filata curd is transferred to the hot water section of a cooker/stretcher.

47

At this point, the curd is immersed, heated and worked by single- or twin-screw augers. The temperature of the water is determined by the temperature of the curd entering the stretcher, the curd flow rate and the target temperature of the cooked curd. Typical water temperature varies between 60 and 75 ~ with the cooked curd temperature varying between 55 and 65 ~ The mechanical treatment of the cooked curd influences the final cheese structure, composition and functionality. Moisture can be expelled or further incorporated. Salt and other ingredients can also be added at this point. Mechanical treatment or mechanical conditioning of the cooked curd is usually achieved by further working by single- or twin-screw augers or by 'dipping' arms in a relatively moisturefree environment. Following mechanical working, the curd may be extruded into a mould and immersed in chilled brine for cooling and salt uptake. Packaging and despatch follow, with shredding being a common option for pizza use. Almac s.r.l., Modena, Italy, Stainless Steel Fabricating, Wisconsin, USA and Construzioni Meccaniche E Technologia S.p.A (CMT), Italy, are examples of companies that manufacture a range of Pasta-filata processing equipment, including cooker/stretchers. Their equipment is described in the following sections. A l m a c s.r.L

Almac s.r.1, has been producing systems for making Pasta-filata cheese since the 1980s. They manufacture essentially three standard systems: for the production of high-moisture Mozzarella, for the production of Pizza cheese (low-moisture Mozzarella) and for the production of the ripened Pasta-filata cheeses (Provolone, Kashkaval and Kasseri). Turnkey design starts at curd draining and each system includes cheddaring (curd ripening), cooking/stretching, moulding and cooling (including pre-hardening and hardening), brining and packaging. Almac s.r.1, has an extensive range of cooker/stretchers with various capacities, built to handle a range of curd textures, depending on the type of Pasta-filata cheese to be made. An example is shown in Fig. 27. All the larger capacity cooker/stretchers use twin screws to convey the cut curd through the cooking section and all use the 'dipping arm' technology to condition the curd following cooking. All product contact surfaces are coated with a non-stick agent. A minimum quantity of water is used during the cooking phase to ensure high yields. Almac s.r.1, supplies Mozzarella cooker/stretchers to customers throughout Italy, other European countries and to Australia, Canada, Iran, Ecuador, Argentina, Brazil, the USA, Venezuela and Eygpt.

48

General Aspects of Cheese Technology

Almac cooker/stretcher. Courtesy of Almac, Italy. (See Colour plate 13.)

Stainless Steel Fabricating, Inc. qt.inloqq

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equipment for producing mainly low-moisture Mozzarella (American Pizza cheese) and Provolone. Stainless Steel Fabricating can provide cooker/stretchers, moulders and chilling-brining systems. It is a family-owned business, operating for the last 35 years and supplying Mozzarella equipment to Mozzarella manufacturers in North America, South America, Europe, Asia, Africa, Australia and New Zealand. Five models, ranging in capacity from 25% of the DM of the forage used must be produced on the same farm where the cheese is manufactured; ->75% of the DM of the forage used must be produced

Gross chemical composition of the principal extra-hard cheese varieties (average data)

Cheese

Moisture (%)

Total protein (Nx6.38) (%)

Fat(%)

Ash(%)

Soluble N/Total N (%)

Grana Padano Parmigiano Reggiano Asiago Montasio Pecorino Romano Pecorino Siciliano Pecorino Sardo Fiore Sardo Canestrato Pugliese Castel mag no Fossa Sbrinz Mah6n Manchego Roncal Idiazabal Kefalotyri

32.0 30.8 34.0 32.0 31.0 31.5 31.0 26.5 34.5 35.0 32.0 31.0 31.7 35.5 29.4 33.2 35.0

33.0 33.0 29.0 26.0 28.5 32.5 27.2 30.0 26.5 26.0 27.0 31.0 26.9 24.0 24.7 23.3 26.6

27.0 28.4 31.0 34.0 29.0 28.0 35.0 32.5 30.0 33.0 35.0 32.0 32.6 33.6 38.8 37.8 28.7

4.9 4.6 5.0 n.a. 8.5 n.a. n.a. n.a. n.a. 5.0 n.a. 5.0 6.8 4.6 4.8 4.0 3.9

34.0 32.0 28.5 26.5 22.5 26.5 24.0 25.5 30.0 26.5 32.0 31.5 31.1 25.9 26.2 29.0 24.5

n.a., data not available; From various sources.

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5.3) to waxy (pH 5.3-5.1) to mealy (pH ~6%) produces a firm-textured cheese which is drier and ripens at a slow rate (Van Slyke and Price, 1952), whereas under-salting (i.e., an S/M < ~4%) results in a pasty cheese with abnormal ripening and flavour characteristics. Such factors as enzyme activity and the conformation of ors1- and [3-caseins in salt solutions (Fox and Walley, 1971), solubility of protein breakdown products, hydration of the protein network (Guerts et al., 1974) and interactions of calcium with the para-caseinate complex in cheese (Guerts et al., 1972) are all influenced by salt concentration. Effect of protein, fat and moisture

In dry-salted cheeses, water, fat and casein are present in roughly equal proportions by weight, together with small amounts of NaC1 and lactic acid. As protein is considerably more dense than either water or fat, it occupies only about one-sixth of the total volume. Nevertheless, the protein matrix is largely responsible for the rigid form of the cheese. Any modification of the nature or the amount of the protein in the cheese will modify its texture. Thus, reduced-fat Cheddar (17% fat) is considerably more firm and more elastic than full-fat Cheddar (35% fat), even when the level of MNFS in the cheese are the same (Emmons et al., 1980). This difference was explained by the presence in the reduced-fat cheese of about 30% more protein matrix, which must be cut or deformed in texture assessments, but such a large reduction in fat must also affect the texture of the cheese. Fat in cheese exists as physically distinct globules, dispersed in the aqueous protein matrix (Kimber et al., 1974). In general, increasing the fat content results in a slightly softer cheese (Bryant et al., 1995), as does an increase in moisture content, because the protein framework is weakened as the volume fraction of protein molecules decreases. However, relatively large variations in the fat content are necessary before the texture of the cheese is affected significantly (Lawrence and Gilles, 1980). Commercial cheese with a high FDM usually has a high MNFS (Lawrence and Gilles, 1986) and this causes a decrease in firmness. An inverse relationship between the fat content and cheese hardness has been reported (Whitehead, 1948; Baron, 1949; Fenelon and Guinee, 2000).

88

Cheddar Cheese and Related Dry-salted Cheese Varieties

Effect of ripening

Considerable changes in texture occur during ripening as a consequence of proteolysis (Hort and Le Grys, 2000, 2001). The rubbery texture of 'green' cheese changes relatively rapidly as the framework of Otsl-casein molecules is cleaved by the residual coagulant (Creamer and Olson, 1982; Johnston et al., 1994; Watkinson et al., 2001). A group of Cheddar cheeses examined over a period of nearly a year increased in hardness and decreased in elasticity with the age of the cheese, the greatest changes occurring during the first 30 days (Baron, 1949). Watkinson et al. (1997) measured proteolysis of ors1- and [3-caseins, and the strain at fracture (a measure of shortness (Gunasekaran and Ak, 2003)) as a function of ripening time. These results showed that the strain at fracture increased initially, probably as curd fusion continued, and then decreased continuously for the 400 days of the experiment. In part, this latter rheological (or textural) change is caused by the loss of structural elements, but another feature of proteolysis is probably important (Creamer and Olson, 1982): as each peptide bond is cleaved a molecule of water is incorporated into the resulting polypeptides and, in addition, two new ionic groups are generated and each of which will compete for the available water in the system. Thus, the water previously available for solvation of the protein chains becomes tied up by the new ionic groups, making the cheese more firm and less easily deformed. This change, in combination with the loss of an extensive protein network, gives the observed effect. Clearly, the change in texture during ripening depends upon the extent of proteolysis, which, for any individual cheese, is determined by the duration and temperature of maturation. The main factor that influences the rate of proteolysis appears to be S/M (Fox and Walley, 1971; Pearce, 1982; Fox, 1987). A direct relationship between S/M and residual protein was established whereas the correlation between moisture and residual protein was relatively weak. A cheese with a low S/M value has a higher rate of proteolysis and is correspondingly softer in texture than a cheese with a high S/M. The concentrations of residual rennet and plasmin in the cheese, together with the starter and non-starter proteinases present, are the important factors that determine the rate of proteolysis (Lawrence etal., 1983; C.J. Coker, T.M. Dodds, S.P. Gregory, K.A. Johnston and L.K. Creamer, unpublished results, 2000).

Cheese ripening is essentially the slow controlled decomposition of a rennet-induced coagulum of the constituents of milk to produce flavour (taste and aroma)

and textural changes. The final targeted flavour profiles and textures of ripened Cheddar and related dry-salted cheese varieties are variable as defined by different endcustomer requirements and traditional cultural flavour expectations. At the young end of the age range is cheese used solely as a source of intact casein for processed cheese, which has minimal flavour and textural change from the fresh curd. A low coagulant concentration, a low storage temperature, high S/M, short storage time or combinations of these are the main parameters used to achieve this end-use. At the other extreme are the strong flavoured Cheddar cheeses ripened for 12-24 months or more. During ripening, there are many changes and the ripening processes responsible are understood in general terms but many of the details are still being investigated. A vocabulary of sensory attributes has been developed to describe Cheddar (Muir and Hunter, 1992), and has been modified to include five odour, ten flavour and five textural attributes (Muir et al., 1995). Using this vocabulary with an experienced panel in combination with data analysis, the similarities and differences between Cheddar and 13 other hard cheeses popular in the United Kingdom have been described (Muir et al., 1995). The medium and vintage Cheddars stand out in a number of respects. In a similar analysis of 34 different Cheddars, a diversity of flavours was shown (Muir e t a l . , 1997). Cheddars made from raw milk were more intensely flavoured ,,,,,a ~,,,4 ,,t,,~,,,~l n . . . . . . . . . . ., ~,~ farmhouse cheeses It showing wide variations in composition and being associated with atypical flavour and texture. There is a significant correlation between the levels of proteolysis products and the extent of flavour development. Hydrolysis of the casein network, specifically e~sl-casein, by the coagulant appears to be responsible for the initial changes in the coagulum matrix (Creamer and Olson, 1982). The level of chymosin retained in the curd is pH dependent (Lawrence et al., 1983; Creamer et al., 1985). In fresh milk, plasmin, the indigenous alkaline milk proteinase, is associated with the casein micelles but it dissociates at low pH (Richardson and Pearce, 1981" Farkye and Fox, 1990). The activity of plasmin in cheese is reported to be dependent on cooking temperature (Farkye and Fox, 1990) as well as on pH and the salt and moisture contents of the cheese (Richardson and Pearce, 1981" Farkye and Fox, 1990). The role of plasmin in Cheddar cheese flavour has yet to be elucidated but it has been reported that the rate and extent of characteristic flavour development in Cheddar cheese slurries appeared to be related directly only to the degradation of [g-casein (Harper et al., 1971). Therefore, plasmin may well prove to be an enzyme of considerable importance in the development of cheese flavour. . . . . . .

Cheddar Cheese and Related Dry-salted Cheese Varieties

As the original casein network is broken down, ideally a desired balance of flavour and aroma compounds is formed. However, the precise nature of the reactions that produce flavour compounds and the way in which their relative rates are controlled are poorly understood. This has been due firstly to the lack of knowledge of compounds that impart typical flavour to Cheddar cheese, and secondly to the complexity of the cheese microflora as the potential producers of flavour compounds. Any organism that grows in the cheese, whether starter, adventitious non-starter lactic acid bacteria (NSLAB) or adjunct culture and any active enzyme that may be present, such as chymosin or plasmin, will have an influence on the subsequent cheese flavour (Fig. 11). Research in New Zealand has shown that if the growth of starter and NSLAB is limited (Fryer, 1982; Lawrence et al., 1983) and if as little chymosin as possible is used (Lawrence and Gilles, 1971; Lawrence et al., 1972), the flavour that develops in Cheddar cheese is likely to be acceptable to most consumers. This section is an attempt by the present authors to summarize what they consider to be relevant to flavour development in Cheddar. Since the last version of this section (Lawrence et al., 1993), more details have been published; however, the last word on the flavour of Cheddar cheese is still to come. For more

Basic structure for Cheddar (pH and mineral content)

Ripening conditions within cheese (Moisture-in-casein; salt-in-moisture; lactose; temperature)

Residual rennet, plasmin and starter activity

Non-starter activity " " " " " ,,

~ SSSSS

s

89

details on the biochemistry of cheese ripening, refer to '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. Effect of milk-fat

It is well accepted that Cheddar cheese made from skim milk does not develop a characteristic flavour. Cheese with an FDM greater than 50% developed a typical flavour whereas cheese with an FDM less than 50% did not (Ohren and Tuckey, 1969). In this study, when a series of batches of cheese were made from milk of increasing fat content (from 0 to 4.5%), the quality of the flavour improved as the fat content increased. However, if the fat content was increased above a certain limit, the flavour was not further improved. Substituting vegetable or mineral oil for milk-fat still resulted in a degree of Cheddar flavour (Foda et al., 1974). This suggests that the water-fat interface in cheese is important and that the flavour components are dissolved and retained in the fat. Clearly, although milk proteins and lactose are the most likely sources of many of the flavour precursors in Cheddar cheese, the fat plays an important but not yet defined role; in part, the lack of understanding is due to the more limited fat modifications. The extent of lipolysis has been calculated to vary between 0.5 and 1.6% over time in good quality Cheddar (Perret, 1978). A number of fatty acids, keto acids, methyl ketones, esters and lactones in Cheddar are likely to have been derived from milk-fat; some are at concentrations to impact on flavour, but others contribute only to a background flavour (Urbach, 1995; McSweeney and Sousa, 2000). The residual activity after pasteurization of the indigenous milk lipase and the relatively low lipase/esterase activities of the starter and NSLAB are likely to be important in the hydrolysis of milk-fat to free fatty acids because of their flavour potency. The quality of the milk is probably a factor in excessive lipolysis in off-flavoured Cheddar (Perret, 1978). The catabolism of free fatty acids to other flavour compounds, by implication of their presence, occurs but the mechanisms are ill-defined.

i

Effect of proteolysis Acceptable Cheddar flavour

Off-flavours

The main factors that determine the development of flavour in Cheddar cheese.

As described earlier, the consequence of proteolysis of casein represents the most important biochemical ripening event in Cheddar, causing major texture changes and in addition making important contributions to both aroma and taste (Fox, 1989; Fox and

90

Cheddar Cheese and Related Dry-salted Cheese Varieties

McSweeney, 1996). A further consequence of proteolysis may be the release of flavour components that were previously bound to the protein (McGugan et al., 1979). The products of proteolysis include small- and intermediate-sized peptides and free amino acids and contribute at least to a background flavour (McSweeney and Sousa, 2000), or make a significant contribution to flavour intensity. It has been suggested (McGugan et al., 1979; Aston and Creamer, 1986) that the importance of low levels of such non-volatile compounds as peptides, amino acids and salts has been under-rated in the past. This view is supported by the highly significant correlations found between the levels of proteolysis products and the extent of flavour development (Aston et al., 1983). The level of phosphotungstic acid-soluble amino nitrogen was found to be a reliable indicator of flavour development. Above certain limits, however, the level of peptides results in bitterness. Cheddar cheeses made using temperatureinsensitive starter strains were found to become bitter because large numbers of starter cells contributed excessive levels of proteinases. These released bittertasting peptides from high molecular weight peptides that had been produced mainly as a result of chymosin action (Lowrie and Lawrence, 1972). The subject of bitterness, the single most common defect in Cheddar cheese, has been extensively reviewed (Crawford, 1977; Fox, 1989).

quality of the cheese decreased. Some amino acids such as phenylalanine and the branched amino acids yield Strecker degradation products, which in excess cause unclean flavour defects in Cheddar (Dunn and Lindsay, 1985).

for reactions that produce a range of flavour compounds (McSweeney and Sousa, 2000). Recent studies using gas chromatography-olfactometry and related techniques have identified key aroma components of Cheddar cheese (O'Riordan and Delahunty, 2001; Zehentbauer and Reineccius, 2002). Some of these (dimethyl sulphide, methional, dimethyl trisulphide and 3-methylbutanal) are likely to originate from amino acids (Urbach, 1995). Several reports strongly implicate the volatile sulphur compounds, specifically methanethiol, in Cheddar cheese flavour (Green and Manning, 1982; Lindsay and Rippe, 1986), but an Australian report (Aston and Douglas, 1983) concluded that none of these sulphur compounds is a reliable indicator of flavour development. However, it is conceivable that, although the volatiles do not make a measurable contribution to the intensity of Cheddar flavour, they may still be an essential factor in the quality of the flavour (McGugan et al., 1979). This is supported by the finding (Manning et al., 1983) that the quality of blocks of Cheddar cheese decreased, and off-flavours increased, with a decrease in block size. Headspace analysis showed that the concentrations of HzS and CH3SH, compounds that are extremely susceptible to oxidation, decreased as the

bitterness, which mask or detract from cheese flavour, are produced. A reduction in unpleasant flavour is associated with improved perception of the Cheddar flavour (Lowrie and Lawrence, 1972; Lowrie et al., 1974). The increase in the use of direct vat inoculum (DVI) cultures in Europe for the manufacture of cheese has led to greater usage of Lc. lactis subsp. lactis strains of starter. Because these strains have a greater tendency than Lc. lactis subsp, cremoris strains to produce bitterness in cheese, bitterness is more common with the use of DVI cultures than with bulk cheese starter (Heap, personal observation). During, or soon after, the manufacture of Cheddar curd, the starter viability decreases and is 7 ~ under anaerobic conditions with lactate as C-source. The redox potential will then be reduced from - - 1 3 0 mV to < - 2 0 0 mV, which may be used as an indicator for BAE Gouda cheese, as a brined cheese, is especially vulnerable to BAE the more so for larger cheese loaves. The normal eye formation is limited and too large holes are easily detected as a defect. Because of the serious nature of the defect, much research has been undertaken to find ways to reduce the number of spores in milk and to prevent their germination and growth in cheese. Factors studied include: bactofugation of the milk, addition to

133

the milk of nitrate, hydrogen peroxide or other oxidising substances, or lysozyme, the use of a nisin-producing starter, the salt content and pH of the cheese, cheese ripening temperature and amount of (undissociated) lactic acid (for relevant literature information, see van den Berg et al., 1980, 1988; Stadhouders et al., 1983c). The technique of microfihration is practically not used for Gouda cheese (see 'Bactofugation'). Nitrate may be used effectively to prevent BAF and has been used for this purpose for about 170 years. The mechanism of inhibition requires the presence of xanthine oxidase (EC 1.2.3.2), which reduces nitrate to nitrite (Galesloot, 1961). Nitrite is considered to delay the germination of spores for a certain period after brining (but the actual mechanism may well be more complicated, according to Stadhouders et al., 1983a). Later on, the inhibitory action is taken over by NaC1 when it has become evenly distributed throughout the cheese and if it is present at a sufficient concentration. If nitrite is the only factor involved in the initial inhibition, it must be very effective since it is present at only a very low concentration. The formation of nitrite was a reason to investigate the presence of nitrosamines in Gouda cheese. From the results of Goodhead et al. (1976), the existence of this danger appears to be not likely. Since xanthine oxidase is a milk enzyme, its inactivation by pasteurisation will increase between 72 and 82 ~ (see Fig. 2). In this way the effectiveness of nitrate declines. High numbers of coliform bacteria and some strains of mesophilic lactobacilli will degrade nitrate to nitrite during the first weeks, increasing the risk of BAE In cheese, nitrite is supposed to be degraded slowly eventually to NO and N2 that may diffuse outside the cheese. At a given curing temperature, usually about 14 ~ the combined effect of several factors determines whether growth of BAB is possible or not. Important factors promoting growth are a large number of spores in the cheese milk, a low content of undissociated lactic acid (hence usually a high pH), a low nitrate content in the cheese and a low level of NaC1 in the cheese moisture. The rate at which salt becomes homogeneously distributed throughout the cheese mass, its final concentration and the initial nitrate content of the cheese are, therefore, crucial. For example, a cheese with a high pH requires a higher than normal final salt concentration to inhibit growth. Since the pH of the cheese is increased by BAE growth conditions for the organism then become more favourable and consequently the rate of fermentation is accelerated. It is the experience of the authors that germination of the first spores may occur in cheese very soon. Therefore, the presence of nitrate on the first day is necessary and even a later start of brining by some

134

Gouda and Related Cheeses

hours will increase the incidence of BAE An L- or Otype starter makes the cheese somewhat less-sensitive to BAF in comparison with the use of the DL-type starter, probably because of the difference in production of acetate (Stadhouders, 1990). Low numbers of spores in the cheese milk, which also can be achieved by bactofugation, permit the amount of nitrate to be reduced considerably. If one wishes to produce cheese without nitrate addition, the critical number of spores in milk capable of causing the BAF is extremely low, ---5 spores/1 milk as found by van den Berg et al. (1988). To this end, effective double bactofugation of the milk is certainly necessary when making standard 12 kg Gouda cheese. Critical numbers of spores at different nitrate concentrations are also given in this study, e.g., ---250 spores/1 need 2.5 g nitrate/100 1 milk and ---10 000 spores/l need 15 g nitrate/100 1 milk. The latter number of spores may occur in the winter season. If then, only 2.5 g nitrate/100 1 milk may be used, single bactofugation with sludge sterilisation will be necessary under north-west European conditions. Lysozyme has often been proposed as an alternative to nitrate. Its use is somewhat more expensive than bactofugation for a similar protective effect. At the amount recommended (e.g., 500 IU/ml of cheese milk), it is usually less effective than nitrate, according to experience with Gouda cheese (Stadhouders et al., 1986). In comhinalion with qinale harlnfllg~fiem thiq amount may be used. Some spores are quite resistant to lysozyme, whereas others are readily inhibited or are even more sensitive to lysozyme than to nitrate (Lindblad, 1990). Nisin shows antimicrobial activity against a broad spectrum of Gram-positive bacteria, such as Bacillus, Clostridium, Listeria and Staphylococcus spp. Currently, it is used in a wide range of foods and beverages, such as processed cheeses. However, for normal cheese manufacturing it cannot be used because it is lost in the whey and inhibits many starter bacteria. Recently, defined nisin-producing starter cultures were selected and have begun to be marketed; these could be used to manufacture good-quality Gouda-type cheese. Starters which produce nisin in situ during cheese manufacture give a very strong protection against spores of Clostridium tyrobutyricum and Staphylococcus aureus bacteria (Meijer et al., 1998). Lactobacilli Growth of mesophilic normal or salt-tolerant lactobacilli may cause flavour and texture defects, especially in mature cheese. Even when initially present at small numbers, e.g., 10/ml of cheese milk, some strains of common lactobacilli (Lb. plantarum, Lb. casei, Lb. brevis) may grow slowly in cheese to more than

2 • 107/g in 4-6 weeks (Stadhouders et al., 1983c), causing gassy and putrid flavours and an excessively open texture. Probably, amino acids are used as a carbon source. The organisms are killed by adequate pasteurisation of milk, e.g., 15 s at 72 ~ In industrial practice, continuously working curd-drainage machines were often an important source of contamination but improved designs, minimising 'dead spots', make longer standing times possible. However, growth on surfaces of tanks should also be considered. Especially when the salting of cheese is carried out in brine of reduced strength, there is a risk of defects caused by salt-tolerant lactobacilli, some strains being able to survive even in the presence of >15% NaC1. Furthermore, they differ from normal lactobacilli by their continuing growth in cheese and their active amino acid metabolism, causing phenolic, putrid, mealy and H2S-like flavours in 4-6-month-old cheeses. Some strains also produce excessive quantities of CO2, causing the formation of holes, either eyes or cracks according to the consistency of the cheese (Stadhouders et al., 1974). More than 103 of these gas-forming lactobacilli per ml of brine is considered to be dangerous. The lactobacilli may enter the cheese by penetrating the rind during brining, this being facilitated if the cheese is insufficiently pressed and the rind not wellclosed (Hup et al., 1982). Of course, contamination of the cheese milk with these bacteria must be prevented. If ...... 1. k ~ ; . . (e.g., . . . . . . . . . j . . . . ~,. . . . . . . . . . . . . y acid (pH 90% lactate. The streptococci produce only L-lactic acid, whereas Lb. delbrueckii subsp. lactis converts lactose entirely to D-lactate. Both isomers are produced by Lb. helveticus. Lactose is fully hydrolysed within 4-6 h after addition of the lactic starters, and the lactic acid fermentation is completed after 24 h. Galactose from lactose breakdown is not utilised by the streptococci, but is metabolised by the lactobacilli. To avoid undesired fermentations, no residual galactose should remain after the lactic fermentation. During cheese ripening, the proteinases and peptidases of lactobacilli play a major role in the breakdown of casein. Some decades ago, Lb. helveticus was a major component of starter cultures in the manufacture of Swiss Emmental. Due to its intensive peptidolytic activity, which promotes late fermentation, it has been replaced by Lb. delbrueckii subsp, lactis. Streptococci play a minor role in proteolysis. In areas where the cheese milk is collected twice daily, it is quite common to add a mesophilic culture of lactococci (Lactococcus lactis) to the evening milk to pre-ripen it. In the production of semi-hard cheeses with a propionic acid fermentation, mesophilic species are also used.

Cheese: Chemistry, Physics and Microbiology, Third edition - Volume 2: Major Cheese Groups ISBN: 0-1226-3653-8 Set ISBN: 0-1226-3651-1

Copyright 9 2004 Elsevier Ltd All rights reserved

142

Cheeses with Propionic Acid Fermentation

Traditional Swiss Emmental cheese. (See Colour plate 16.)

Facultatively heterofermentative non-starter lactobacilli are purposely used in the Swiss artisanal cheese industry to slow down the propionic acid fermentation. They ferment hexoses almost exclusively to lactic acid. This group of micro-organisms contains, among others, Lb. casei and Lb. rhamnosus which are indigenous to raw milk. During cheese ripening, they grow by utilising citrate which is found in the fresh unripened cheese. Starting from 9 mmol/kg citrate in the cheese curd, native facultatively heterofermentative lactobacilli utilise approximately 3 mmol and those added as adjunct cultures metabolise all available citrate to formic acid, acetic acid and CO2 (Table 4).

about 1000 1 of milk). Propionic acid fermentation begins about 30 days after the start of manufacture at about 20-24 ~ for roughly 7 weeks and then continues at a slower rate at 10-13 ~ In cheeses ready for consumption, about 108-109 cfu/g of propionic acid bacteria are present. Propionibacteria are Gram-positive, non-motile, non-sporulating and appear under the microscope as short rods which grow at low oxygen concentrations only (anaerobic to aerotolerant), and occur naturally in the rumen and intestine of ruminants, in soil and in silage (Fig. 2). Strain diversity of the natural propionibacterium flora is great which, fortunately, has not been influenced by the wide use of commercially available cultures (Fessler, 1997). They are sensitive to salt and grow optimally at a pH between 6 and 7 (maximum 8.5, minimum 4.6). The optimal growth temperature is 30 ~ but growth occurs also at 14 ~ They develop well in cheese from low numbers, but do not grow in milk (Piveteau et al., 2000). The propionibacterial metabolism in cheese is rather complex and not yet fully understood (Crow et al., 1988" FrOhlich-Wyder et al., 2002). Three different metabolic pathways (Fig. 3) have been described for the utilisation of lactate as an energy source and aspartate as an electron acceptor, both of which are available in cheese (Brendehaug and Langsrud, 1985; Crow and Turner, 1986; Crow, 1986b). In the presence nf a~partale, the [ermentntinn of lnrtnto i~ rn,,plocl with the fermentation of aspartate to succinate and no propionate is produced. Consequently, more lactate is fermented to acetate and CO2 than to propionate. The role of pathway B (formation of succinate by fixation

Propionic acid fermentation

Nowadays, selected propionibacteria of the species P. freudenreichii are used in the manufacture of cheeses with propionic acid fermentation in order to achieve the characteristic eyes and nutty flavour. For Emmental cheeses, the inoculum size is very small (only a few hundred colony forming units (cfu) per vat containing

Scanning electron micrograph of a culture of Propionibacterium freudenreichfi (Source: Swiss Federal Dairy Research Station, CH-3003 Berne).

Cheeses with Propionic Acid Fermentation

143

(A) Classical propionic acid fermentation: 3 mol lactate ~

2 mol propionate + 1 mol acetate + 1 mol CO 2 + l m o l A T P

(B) Formation of succinate during propionic acid fermentation by CO2-fixation: 3 mol lactate ~

(2 - x) mol propionate + 1 mol acetate + (1 - x) mol CO 2 + x mol succinate

Wood-Werkman pathway

(C) Fermentation of aspartate to succinate during propionic acid fermentation: 3 mol lactate + 6 mol aspartate ~

3 mol acetate + 3 mol CO 2 + 6 mol succinate + 6 mol NH 3 + 3 mol A T P

Metabolic pathways for the utilisation of lactate by propionic acid bacteria according to Crow and Turner (1986) and Sebastiani and Tschager (1993).

of CO2) is certainly of minor importance, but it has not yet been clarified (Sebastiani and Tschager, 1993). Propionibacterial strains can differ markedly in their aspartase activity (Richoux and Kerjean, 1995). In the manufacture of Emmental cheese, the use of cultures with differing aspartase activity leads to different products (Wyder et al., 2001). Tables 1 and 2 show clearly the characteristics of Emmental cheeses made with propionibacteria with either strong or weak aspartase activity. Propionibacteria with weak aspartase activity are able to metabolise not more than 100 nmol aspartate per minute in vitro (Fr0hlich-Wyder et al., 2002). These strains metabolise lactate mainly by the classical pathway (A) and deaminate only little aspartate (Fig. 3). Strains with high aspartase activity are able to metabolise up to 8 0 0 n m o l aspartate per minute in vitro. During the ripening of Swiss-type cheese, aspartate is metabolised rapidly and L-lactate is used preferentially (Crow, 1986a; Piveteau et al., 1995). As an effect, usually all available aspartate is metabolised to succinate (Fig. 4) and lactate, preferentially the isomer L, is metabolised to propionate, acetate and CO2 (Tables 1 and 4). A comparison with other traditional cheese varieties from Switzerland, which do not undergo a propionic acid fermentation, reveals that the content of aspartate is always much lower and that of succinate much higher in Emmental cheeses (Sieber et al., 1988). A strong aspartase activity is generally coupled with a stronger growth rate of propionibacteria, leading to higher counts and higher concentrations of propionate, acetate and CO2 (Table 1). Piveteau et al. (1995) showed that the growth rate and yield of propionibacteria in whey can be enhanced by the addition of aspartate. Yet it is not possible to answer the question whether aspartase activity is the cause or just an indicator. The appearance of Emmental cheese is greatly affected by the aspartase activity of the propionibacteria used. Figure 5 shows clearly the outer appearance

of different Emmental cheeses. The number and size of eyes and the height of loaves are greater for cheeses made with a culture with strong aspartase activity (Table 2) as a result of increased CO2 release (Table 1). The storage time for the cheeses in the warm room may be shortened by up to 10 days (Fr0hlich-Wyder et al., 2002). Such cheeses are more prone to late fermentation which is not desired when the cheeses are ripened for a longer time (Bachmann, 1998a). Late fermentation is a resumption of the propionic acid fermentation during maturation. The intensity of taste, odour and aroma is also more pronounced compared to cheeses made with propionibacteria of low aspartase activity (Table 2). The main reason appears to be the higher concentrations of free short chain acids produced through fermentation as well as the free fatty acids, n-butyric and n-caproic acids, released by lipolytic activity of propionibacteria (Table 1). Thus, propionibacteria with strong aspartase activity accelerate the ripening process. This is a 15'I

lo'

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1

2

3

4

5

6

9

E

o~

5

0

0

7

Asp + Asn (mmol/kg) Linear regression, with a 95% confidence interval, of succinate and sum of aspartate (Asp) and asparagine (Asn) in 6-month-old Emmental cheese (T, propionibacteria with high aspartase activity; A, propionibacteria with weak aspartase activity) (Wyder et al., 2001).

144

Cheeses with Propionic Acid Fermentation

Mean values of metabolites, proteolytic parameters and propionibacterial counts in Emmental cheese (6 and 12 months) made with propionibacteria with weak or strong a s p a r t a s e activity (Wyder et aL, 2001 )

Emmental cheeses at 6 months Parameter

Weak (N = 10)

Lactate a L(+)-Lactate a pH Free S C A a Acetate a Propionate a n-Butyrate a n-Caproate a Succinate a 002 a Propionibacteriab Total nitrogenc WSN a TCASN a Free a m i n o acids a Aspartate a Asparagine a

57.4 31.1 5.75 114.4 48.4 60.1 1.1 0.4 4.0 27.6

_ 10.5 _ 9.3 _ 0.02 +_ 5.2 ___ 1.3 ___4.4 _+ 0.2 +_ 0.1 _+ 0.6 _ 1.6 nd 3.17 _ 0.06 693.4 _ 33.5 469.2 _ 46.6 169.02 _ 23.72 2.219 +_ 0.861 2.863 +_ 1.100

Strong (N = 8) 45.3 17.0 5.79 126.0 53.1 67.1 1.2 0.5 11.9 33.6 3.20 720.4 470.4 165.58 0 0.125

_+ 17.4 _+ 8.9 +_ 0.02 _+ 5.2 _+ 5.1 _ 10.2 _+ 0.1 _+ 0.1 _+ 1.7 +_ 2.0 nd __ 0.07 _ 26.1 _+ 40.1 +_ 30.20 +_ 0 _ 0.237

Emmental cheeses at 12 months t-test

Weak (N = 10)

ns ** ** ns * ns ns * *** *** ns ns ns ns *** ***

47.0 25.4 5.63 117.4 47.6 63.2 1.7 0.5 5.1 6.7 3.16 901.0 682.6 266.92 4.834 1.886

+_ 8.5 _+ 8.1 _+ 0.06 +_ 5.9 _+ 0.6 _ 4.2 _+ 0.9 _+ 0.1 ___2.8 nd _ 0.9 _ 0.08 _ 28.3 _+ 50.3 +_ 34.51 +_ 0.585 _ 0.494

Strong (N = 8) 11.3 2.9 5.73 148.1 58.7 83.6 1.7 0.7 17.7 8.4 3.21 926.3 687.3 246.86 0.588 0.054

_+ 6.7 _+ 2.4 _ 0.02 _+ 5.0 _+ 1.7 _ 3.6 _+ 0.1 +_ 0.1 +_ 2.5 nd _ 0.3 __ 0.06 _ 28.7 _+ 46.8 +_ 22.95 _+ 0.097 _+ 0.154

t-test *** *** *** *** *** *** ns ** *** *** ns ns ns ns *** ***

a mmol/kg. b log CFU/g. c mol/kg. SCA, Short Chain Acids; WSN, Water-soluble N; T C A S N , 12% TCA-soluble N; nd, not determined; ns, not significant. *p < 0.05; **p < 0.01; ***p < 0.001.

combined effect of aspartate metabolism and of the lncreaseu numt)er of proplonlDacterla." - :' ....... " For the application of propionibacterial cultures in cheese production, their ability to utilise aspartate must be taken into consideration. Excessive aspartase activity has hidden dangers, such as late fermentation, as mentioned above; a moderate activity, however, may influence the quality of Emmental cheese positively, e.g., improving openness, increasing the intensity of flavour and reducing the maturation time. _9

.

.

.

.

.

.

.

.

.

_1

.

.

.

.

.

.

.

1

.

.

.

Interactions

.

In Emmental cheese, interactions between propionibacteria and factors such as the type of lactic acid bacteria, season of milk production (feeding) and proteolysis have a major impact on the propionic acid fermentation. Nowadays, it is easy to control the propionic acid fermentation during the ripening of Emmental cheese. Since the introduction of starter lactic acid bacteria in the 1970s, of facultatively heterofermentative lactobacilli in 1989 and of propionibacteria cultures with weak aspartase activity in 1996, the defect of late fermentation has been practically eliminated in Switzerland. Nevertheless, it is still possible to produce Emmental cheese with eyes made to measure (Fig. 6) - large eyes are achievable with the use of Lb. helveticus together with a strongly aspartase-positive propionibacteria culture. Small eyes are obtained through the use of facultatively heterofermentative lactobacilli together with a weakly aspartase-positive propionibacteria culture. Feeding season

E m m e n t a l c h e e s e (12 m o n t h s old) m a d e with prop i o n i b a c t e r i a with strong (no. 2 6 - 2 7 ) or w e a k (no. 25) aspartase activity ( W y d e r et al., 2001).

Cheeses made from milk produced during the hayfeeding season (winter) are, from experience, more prone to the defect of late fermentation during ripening than cheeses made from milk produced during the grass-feeding season (summer). Cheese producers

Cheeses with Propionic Acid Fermentation

145

Sensory and quality parameters of Emmental cheese (6 and 12 months) made with propionibacteria with weak or strong aspartase activity (mean values and t-test)

Emmental cheeses of 12 months

Emmental cheeses of 6 months Parameter (Index)

Weak (N = 10)

Strong (N = 8)

t-test

Weak (N = 10)

Strong (N = 8)

t-test

Openness (1-6) Number of eyes (0-5) Size of eyes (1-5) Texture (1-6) Firmness (2-8) Maturity (2-8) Intensity of taste (1-6) Intensity of odour (0-7) Intensity of aroma (0-7) Sweetness (0-7) Saltiness (0-7) Sourness (0-7) Bitterness (0-7) Height of cheese (cm)

5.3 4.7 4.9 5.4 4.9 4.4 4.3 3.0 3.1 2.3 1.9 2.0 1.8 19.1

4.6 5.3 5.8 5.5 4.6 5.3 4.7 3.3 3.5 2.2 2.3 2.2 1.7 21.3

* * ** ns ns * ns ns *** ns ** ns ns *

4.6 4.4 4.5 5.3 4.6 6.5 4.5 3.6 3.7 2.5 2.3 2.6 1.8 18.1

4.6 5.4 5.8 5.0 4.7 6.8 4.4 3.5 3.8 2.4 2.5 2.8 1.9 20.6

ns *** *** ns ns ns ns ns ns ns ns ns ns **

+_ 0.6 _ 0.6 _ 0.3 +_ 0.4 _ 0.4 _+ 0.8 _ 0.5 _+ 0.3 _+ 0.2 _ 0.2 + 0.3 _+ 0.2 +_ 0.4 _ 1.5

+_ 0.6 _ 0.4 +_ 0.6 +_ 0.3 _ 0.5 _+ 0.6 _+ 0.3 ___0.3 _+ 0.2 _+ 0.1 _ 0.2 _ 0.2 + 0.4 +_ 1.7

_+ 0.6 _+ 0.6 _ 0.5 _+ 0.7 _+ 0.6 _+ 0.5 _+ 0.6 _ 0.3 _+ 0.4 _+ 0.3 _+ 0.2 _+ 0.3 _+ 0.4 _+ 1.8

_ 0.8 _ 0.3 _ 0.6 _+ 0.6 _+ 0.4 ___0.4 _+ 0.5 _+ 0.3 _ 0.3 _+ 0.2 _+ 0.3 _ 0.4 _+ 0.2 _+ 1.0

ns, not significant. *p < 0.05. **p < 0.01. ***p < 0.001: index indicate the range of appreciation (lowest number = lowest possible score; highest number = highest possible score).

generally observe a slightly slower rate of acidification of the winter milk, resulting in a higher content of water and thus a higher content of lactate in the cheese after 24 h and consequently a lower pH (Table 3). A low pH leads to a slower propionic acid fermentation, since the optimum pH range for propionibacteria is 6-7. Only with proteolysis, a change in pH can be anticipated. Thus, a higher number of propionibacteria is needed in order to start the propionic acid fermentation under this disadvantageous pH. This may be the cause for higher propionibacteria counts which lead to more lactate consumption and therefore more

propionic acid and CO2 production (Table 4). Due to a higher water content, proteolysis is also enhanced. As mentioned above, this is advantageous for the pH but also for the liberation of amino acids, such as asparagine and aspartate, which are substrates for the metabolism of aspartase-positive propionibacteria (Fr6hlich-Wyder et al., 2002). Facultatively heterofermentive lactic acid bacteria Facultatively heterofermentative non-starter lactobacilli are used in the Swiss artisanal cheese industry to slow down the propionic acid fermentation (Sollberger and

X-rays of 180-day-old Emmental cheese produced with strong aspartase-positive propionibacteria and Lb. helveticus (left) or with weak aspartase-positive propionibacteria and facultatively heterofermentative lactobacilli (right) (from Fr6hlich-Wyder et aL, 2002).

146

Cheeses with Propionic Acid Fermentation

Water, lactate, pH and proteolytic parameters for Emmental cheese (Fr6hlich-Wyder et al., 2002)

Factor

Feeding Grass Hay Propionibacteria Weak Strong

Water

Lactate

(g/kg)

(mmol/kg)

TN

WSN

NPN

Free AA

pH

(g/kg)

(% TN)

(% WSN)

(mmol/kg)

N

1d

180 d

1d

180 d

180 d

180 d

180 d

180 d

180 d

16 16

372.5 373.9

326.2 331.4

125.6 131.3

26.9 25.5

5.83 5.72

46.5 44.7

23.2 25.0

61.9 64.6

175.5 199.1

16 16

372.9 373.5

328.2 329.4

128.3 128.6

34.5 17.9

5.77 5.78

45.5 45.6

24.6 23.6

63.1 63.2

196.8 177.8

16 16

372.9 373.5

328.9 328.7

128.9 127.9

51.2 1.2

5.76 5.79

45.6 45.6

24.5 23.7

63.3 63.0

191.9 182.7

16 16

372.8 373.6

328.5 329.1

127.9 129.0

25.4 26.9

5.78 5.77

45.6 45.6

24.0 24.2

64.5 61.8

196.0 178.5

-

. . . . . . . . . *** *. . . . . -

Lb. casei

Added Not added Lb. helveticus

Added Not added ANOVA Feeding Propionibacteria

. -

Lb. casei Lb. helveticus

.

.

.

.

.

.

-

.

.

.

.

**

-

** * -

-, not significant. * p < 0.05. **p < 0.01. ***p < 0.001. TN, total N; WSN, water soluble N; NPN, non-protein N; AA, amino acids.

Free short-chain acids (FSCA), succinate and citrate in mmol/kg, as well as propionibacteria (PAB) and facultatively heterofermentative lactobacilli (FHL) in log cfu/g in 180-day-old Emmental cheese (n = 16 for each factor level) (Fr6hlich-Wyder et aL, 2002) Factor

Feeding Grass Hay Propionibacteria Weak Strong

C1

C2

C3

C4

C6

FSCA

Succinate

Citrate

FHL

PAB

1.7 2.4

41.5 51.6

75.8 88.7

0.87 1.17

0.32 0.36

120.4 144.4

9.8 10.4

4.2 3.3

7.24 7.23

8.12 8.03

2.3 1.8

43.3 49.8

76.6 87.9

1.03 1.02

0.32 0.36

123.8 141.0

4.2 15.9

3.8 3.7

7.30 7.17

7.56 8.59

3.5 0.6

47.3 45.8

68.6 95.9

1.05 0.99

0.33 0.35

121.1 143.7

9.3 10.8

0.2 7.4

7.53 6.94

7.95 8.20

2.2 1.9

47.3 45.9

82.0 82.5

1.02 1.02

0.34 0.34

133.0 131.8

10.2 9.9

3.6 3.9

7.30 7.17

7.97 8.18

Lb. casei

Added Not added Lb. helveticus

Added Not added ANOVA Feeding Propionibacteria

. .

Lb. casei Lb. helveticus

*** .

. . . . . . . . . . . .

. ***

.

.

m

-

.

.

-

.

-, not significant. *p < 0.05. **p < 0.01. ***p < 0.001. C1, formate; C2, acetate; C3, propionate; C4, butyrate; C6, caproate.

m

a

m

B

m

C h e e s e s with P r o p i o n i c A c i d F e r m e n t a t i o n

Wyder, 2000). Jimeno et al. (1995) found growth inhibition of propionibacteria in cheese of up to 80% compared to the control without facuhatively heterofermentative lactobacilli (Lb. casei and Lb. rhamnosus). As a consequence, less propionic acid is produced. The observed inhibition could not be reproduced in co-cultures, suggesting that bacteriocin production is not responsible for this effect. Citrate metabolism most probably plays the key role, since citrate-negative mutants were shown to inhibit propionibacteria much less than the corresponding citrate-positive strains (Jimeno, 1997). Lb. rhamnosus also produces small but appreciable amounts of diacetyl which has a lethal effect on propionibacteria. Acetate and formate seem to have an inhibitory effect on the growth of propionibacteria. In addition, the metabolism of citrate, which takes place before the propionic acid fermentation, leads to the release of the complexed copper. The ratio of citrate and copper plays an important role in the observed inhibition (Perez et al., 1987). However, the mechanism of inhibition is not yet conclusively clarified. Since the introduction of cultures of facuhatively heterofermentative non-starter lactobacilli in Switzerland in 1989, the defect of late fermentation has decreased considerably. Propionibacteria with differing aspartase activity are not inhibited in the same way, a fact already known by cheesemakers. A weak aspartase-positive culture together with Lb. casei requires a prolonged period in the warm room for Emmental cheese while a strong aspartase-positive culture without the addition of facuhatively heterofermentative lactobacilli leads to a shorter stay. Thus, propionibacteria with weak aspartase activity are inhibited much more than propionibacteria with strong aspartase activity. The question arises as to whether the weakly aspartase-positive propionibacteria are more sensitive to formate and acetate. The interaction in Fig. 7 shows that both cultures produce approximately the same amount of propionic acid after 180 days of maturation, but with the addition of Lb. casei, the propionibacteria with weak aspartase activity produce much less propionic acid. This is why propionibacteria with strong aspartase activity are generally more prone to provoke late fermentation. Lb. helveticus

Proteolysis is very important for the development of the texture and flavour characteristics of Emmental cheese. Intensified proteolysis generally leads to accelerated ripening of the product which is desired as long as no adverse effect on the storage quality is encountered. In Emmental cheese production, strong proteolysis, together with intense propionic acid fermentation, may,

147

100 -

Icn

90 -

m O

E 80E c-

70-

-"

Prop96

---1- Prop90

O .m O

a_ 6 0 50-

no

yes

I

I

Addition of Lb. casei Two-way interaction between propionibacteria with different aspartase activity and Lb. casei for propionate in 180day-old Emmental cheese (Prop96, weak aspartase activity; Prop90, strong aspartase activity) (Fr6hlich-Wyder et al., 2002).

however, be the primary cause of late fermentation (Bachmann, 1998a; Baer and Ryba, 1999). The texture which becomes shorter and crumbly during proteolysis shows a loss of elasticity and the cheese can develop cracks because of excessive CO2 production. Several investigations have shown that thermophilic lactic acid bacteria, especially Lb. delbruechii and Lb. helveticus, can stimulate the growth of propionibacteria (Perez et al., 1987; Piveteau et al., 1995; Chamba, 2000; Kerjean et al., 2000). Baer (1995) found poor growth of propionibacteria in milk alone or with added rennet, but good growth in the presence of lactic acid bacteria alone or with added rennet. It was concluded that the growth of propionibacteria depends on the presence of free amino acids or small peptides. In later work, Baer and Ryba (1999) found that propionibacteria clearly prefer free amino acids to peptides. They concluded that the growth of propionibacteria, and thus the intensity of propionic acid fermentation and the risk of late fermentation, is correlated with the amount of free amino acids. In fact, Lb. helveticus is responsible for the liberation of a larger quantity of small peptides in Emmental cheese (Table 3, NPN, % of WSN). Piveteau et al. (1995) described the liberation of a heat-resistant stimulatory compound by Lb. helveticus which might be an aspartate or a peptide containing it. In contrast, the absence of nutrients is not the reason why propionibacteria fail to grow in milk when inoculated at NaC1 > protein > fat > moisture (Zaki, 1990). The textural characteristics of fresh Domiati (UF or traditional) cheese are significantly different. Ultrafiltration-Domiati is harder and more adhesive than the traditional cheese, while the latter is more chewy

Cheese Varieties Ripened in Brine

and gummy (Gomaa, 1990). Both types of cheese increase in hardness, adhesiveness and gumminess during the early stages of ripening, followed by a decrease in these parameters after 3 months of ripening in brine. However, traditional Domiati is more elastic than UFDomiati throughout ripening (Gomaa, 1990). It seems that the increase in the textural parameters during early ripening is related to the decrease in moisture and pH, leading to a firmer texture. During the latter stages, changes in texture are related more to changes in the protein matrix, due to proteolysis, particularly of Otsl-casein, and the loss of Ca. The textural parameters of Domiati are also related to the method of storage, i.e., in pouches without brine or in brine in cans. Cheese stored in pouches is significantly harder, more cohesive and gummy than cheese stored in brine (Gomaa, 1990). Also, the hardness of Domiati made from milk supplemented with whey protein concentrate (WPC) decreases as the level of WPC is increased (Gomaa, 1990). The hardness of flesh UF-Domiati can be controlled by changing the homogenization pressure, heat treatment and pH of the pre-cheese (A1-Khamy, 1988). Increasing the homogenization pressure and heat treatment, and reducing the pH of the pre-cheese increase the hardness of flesh UF-Domiati cheese (A1-Khamy, 1988). Electron microscopy of ultra-thin sections of Domiati (Abd E1-Salam and E1-Shibiny, 1973; Hofi et al., 2001) indicates that the internal structure of fresh cheese is a framework of spherical casein aggregates held together by bridges and occluding fat. On storage in brine, the casein aggregates dissociate into smaller spherical particles, forming looser structure. Differences have been observed in the microstructure of traditional and UF-Domiati (Hofi et al., 2001). The protein matrix of UF-Domiati is characterized by denser and bigger protein aggregates in which whey proteins are included with casein in the protein matrix. Additional proof that changes occur in the microstructure of Domiati cheese during storage was provided by scanning electron microscopy (Kerr et al., 1981; Zaki, 1990). The high salt content has little effect on the morphological characteristics of the surface of the cheese and fat globules per se are unlikely to be changed during storage. Most of the changes occur in the protein matrix. In fresh Domiati, hydrophobic interactions between casein molecules seem to be dominant and overcome the repulsive forces from the negatively charged protein matrix due to the relatively high pH (>5.8) of the cheese. The partial exchange of Na + for Ca 2+ weakens the strong interactions in the casein aggregates.

233

Microbiology Micro-organisms present Lactic acid bacteria are predominant in Domiati; lactococci grow during early storage and later lactobacilli (Naguib et al., 1974; Shehata et al., 1984). Salt-tolerant enterococci are the predominant cocci (94.5% of isolated cocci) in ripened Domiati (Hemati et al., 1998). Enterococcus faecalis, E. faecium, Lc. lactis subsp, lactis, Lc. lactis subsp, cremoris, Lb. casei, Lb. plantarum, Lb. brevis, Lb. fermentum, Lb. delbruekii subsp, lactis, Lb. alimentarius, Leuconostoc mesenteroides subsp, cremoris, Brevibacterium linens and Propionibacterium jensenii have been found in Domiati cheese (Naguib, 1965; Shehata et al., 1984; E1Zayat et al., 1995). Yeasts of the genera Trichosporon,

Saccharomyces, Pichia, Debaryomyces, Hansenula, Torulopsis, Endomycopsis and Cryptococcus are also found in Domiati (Ghoniem, 1968; Seham et al., 1982). Effect of manufacturing and ripening on cheese micoflora Raw milk Domiati generally has a higher bacterial count than cheese made from pasteurized milk during the first month of ripening, but cheeses made from both milks have similar counts thereafter (Naguib etal., 1974). The total microbial count increases rapidly to a maximum after a week of storage and then declines. Lactococci behave similarly, but disappear after 2-3 months of ripening. Lactobacilli reach a maximum after 2-4 weeks and then decrease gradually (Helmy, 1960; Naguib et al., 1974). The high salt content of the cheese milk reduces the total microbial and groups counts in Domiati (Shehata etal., 1984). Micrococci and lactobacilli are equally important in Domiati with a high salt content (Helmy, 1960). Starters Traditionally, starters are not used in the manufacture of Domiati cheese. Several attempts have been made to isolate salt-tolerant organisms from ripened Domiati for use as starters. These include Enterococcus faecalis, Pedicoccus spp., Lb. mesenteroides and Lb. casei (E1Gendy etal., 1983). The enterococci isolated from Domiati cheese have high esterolytic and autolytic activities and they can grow well in a medium with 9.5-10.0% NaC1 (Hemati et al., 1997). They are considered to be suitable starters for Domiati made from pasteurized milk. Survival of harmful organisms The presence of coliforms in Domiati is related to the level of salt added to the cheese milk. Not less than 9.5% NaC1 should be added to milk to suppress the growth of coliforms in Domiati made from raw milk (E1-Sadek and Eissa, 1956; Hegazi, 1972).

234

Cheese Varieties Ripened in Brine

Campylobacter spp. are present in Domiati, but C. jejuni has not been detected (E1-Nokrashy et al., 1998). However, added C. jejuni can survive for 21 days in Domiati made with or without Lb. casei as starter. Listeria monocytogenes remains viable in Domiati depending on the pH, NaC1 content and storage temperature. Storage in brine for 60 days at 20-25 ~ is recommended to ensure product safety (Tawfik, 1993). Aeromonas spp. (A. caviae, A. hydrophila, A. sobria) are found in Domiati (El-Prince, 1998). Clostridium spp. are found in Domiati made from pasteurized milk without the addition of starters. The species isolated are predominantly CI. tyrobutyricum and CI. perfringens (Naguib and Shauman, 1973). Bacillus cereus has been isolated from Domiati (E1Nawawy et al., 1981). Staphylococcus aureus can tolerate up to 15% NaC1 in Domiati but its enterotoxin has not been detected in this cheese (Ahmed et al., 1983). Salmonella typhi can survive for up to 16 days in Domiati made from milk containing 10% NaC1 (Naguib et al., 1979). Defects Early blowing is the principal defect in Domiati cheese, particularly that made from raw milk. It is characterized by the formation of gas holes in the cheese, a spongy texture and blowing of the tins. This defect arises from two factors: gas-forming yeasts or coliforms (Hegazi, 1972; E1-Shibiny etal., 1988) or electrolytic corrosion of tins by NaC1 and developed acidity (Abo Elnaga, 1971).

Introduction

One of the most famous cheeses ripened in brine is, undoubtedly, Feta, which has been produced in Greece since Homeric time (Anifantakis, 1991). Feta is the principal cheese produced in Greece and, in most cases, 'Feta' is synonymous with cheese in Greece. Feta represents over 50% of the total cheese consumed in Greece. The name Feta, which means 'slice' in Greek, has probably come from the original shape of the cheese or from the property which allowed it to be sliced without falling apart. Over the past 30-40 years, the name Feta has acquired an important trade value and, nowadays, it is used to designate many cheeses ripened in brine, which are made from different kinds of milk, using various technologies, even uhrafihrated cows' milk. Of course, the flavour and other sensory qualities of these cheeses does not equate to those of the original Feta cheese.

Manufacture

Milk The most suitable milk for the manufacture of Feta is sheep's milk, but also mixtures of sheep's milk with not more than 30% goats' milk are used. Milk is filtered and standardized to about 6% fat. The ratio of casein to fat is usually 0.7-0.8:1. The pH of the milk should be >-6.5. Heat treatment The majority of cheese milk for Feta is pasteurized (72 ~ 2 1 5 15-20s or 6 5 ~ However, in small enterprises and on farms, the cheese milk is either processed raw or receives a thermal treatment lower than pasteurization. Following heat treatment, the milk is cooled to 32-34 ~ and, if pasteurized, a 40% solution of CaC12 is added at a level of 200 ml/100 kg milk. Starter culture Starters used are a combination of lactic acid bacteria. A yoghurt culture (Streptococcus thermophilus and Lactobacillus delbrueckii subsp, bulgaricus, 1:1) or 24 h-old yoghurt was used traditionally, but have been gradually replaced partly by other commercial cultures capable of a higher acidification rate, e.g., Lactococcus lactis subsp, lactis and Lb. delbrueckii subsp, bulgaricus (1:3), Lc. lactis subsp, lactis and Lc. lactis subsp. cremoris. The culture is added to the cheese milk at a level of 0.5-1.0% (v/v) and incubated for 20-30 min before the addition of rennet. Coagulation Coagulation is performed at 32-34 ~ The quantity of the coagulant is regulated so that the coagulum is ready for cutting in 45-50 min. In large- and mediumsized factories, commercial calf rennet is used. In small enterprises and in mountainous areas, the traditional rennet (rennet paste) made from the abomasa of unweaned lambs and kids is used commonly alone or in combination with commercial calf rennet. Cutting and draining The coagulum is cut crossways into cubes of 2-3 cm and left for about 10 min for partial whey exudation. Then, the curds are ladled into perforated moulds, gradually in order to assist draining. The gradual transfer of the curds leads to the formation of small, almond-shaped openings in the cheese mass, which is a characteristic of the structure of Feta cheese. Moulds are cylindrical of various dimensions when the cheese is to be packed in barrels and rectangular (23 • 23 • 20-25 cm) when it is to be packed in tinplated cans (tin cans). The curds are left to drain in the moulds at 14-16 ~ without pressing for 2-3 h

Cheese Varieties Ripened in Brine

and the moulds are then inverted and left for another 2-3 h to complete draining.

Salting When the curd is firm enough, the mould is removed and the curd is cut into two (23 • 11.5 cm) or four (11.5 • 11.5 cm) pieces, which are placed close together on a salting table, the surface of which has already been sprinkled with coarse cooking salt (particles of the size of rice grains). The upper surface of the pieces is also sprinkled with salt which penetrates slowly into the curd mass. Every 12 h, the cheese pieces are inverted and the surface is dry-salted again. This procedure is repeated until the cheese contains about 3.0-3.5% salt. Following salting, the cheese blocks remain on the table for a few more days until a slime of bacteria, yeast and some moulds starts to develop on the surface. Dry salting and slime formation are essential for the development of characteristic Feta flavour during ripening. Before packaging, the slime is washed off from the surface of the cheese using a soft brush and water or brine. Nowadays, in large factories, moulding, draining and salting are performed mechanically. The curds are transferred by gravity to the moulds. The moulds on a belt conveyor pass under a special outlet of the cheese vat and are filled automatically by gravity (no pumps are used). After about 2 h, the palettes supporting the moulds are inverted to complete draining. Then, the curd is cut to the dimensions of the final cheese and dry-salted. Next morning, the cheese pieces are layered in tin-plated cans. The bottom of the can and the surface of each layer of cheese are sprinkled with coarse salt (rice grain size). After about 2 days, the cheese pieces are packed in the final container (tin-plated can).

Packaging Wooden barrels (kegs) were the traditional containers for Feta. However, handling a filled barrel (--~50 kg) is difficult. Nowadays, Feta is packaged mostly in tinplated cans weighting "--19 kg (net weight of cheese: ---16 kg), making the transportation easier and more economical. The cost of the barrels is also higher than that of tin-plated cans but the cheese develops a stronger and spicier flavour than when packed in tin-plated cans.

Ripening Cheese pieces are tightly packed in the tin-plated cans, allowing little space between them. Brine (6-8% NaC1 in water) is added to the container to fill the space between pieces and to cover the surface of the cheese. Usually, the ratio of brine to cheese is 1:8 (v/w). Cheeses are kept at 16-18 ~ until the pH reaches 4.4-4.6 and the moisture decreases to less than 56% (pre-ripening period, usually 2-3 weeks). From time to time, the lid of the container is untightened to per-

235

mit escape of the gases produced by fermentation and inspection of the level of brine, which must always cover the cheese surface. This is a usual practice with cheese ripened in barrels. If not covered by brine, the surface of the cheese becomes dry, its colour changes (from snow white to ivory or even light yellow) and the growth of yeasts and moulds is possible. After the pre-ripening period, the cans of cheese are transferred to a cold room (4-5 ~ to complete ripening. Feta is permitted to be sold at not less than 2 months postmanufacture (Greek Food Code, 1998). A good quality Feta cheese may be stored, always in brine, for up to 1 year at 2-4 ~ Yield and gross composition

The average dry matter of sheep's milk is 18-20% (Alichanidis and Polychroniadou, 1996) and a cheese yield of about 25% is expected for Feta cheese (Anifantakis, 1991; Mallatou etal., 1994). However, the yield varies with the percentage of goats' milk added to sheep's milk and, also, with the season, because the composition of the milk varies with season. The compositional provisions of the Greek Food Code (1998) for Feta cheese are: maximum moisture, 56% and minimum FDM, 43%. Analyses of 60 market samples (60-180-day-old) produced throughout the cheesemaking period in four major factories showed that the average composition (g/100 g) of Feta cheese is: moisture, 54.2; FDM, 50.82; protein, 17.23; salt-in-cheese moisture, 6.27. The average pH is 4.58 (Michaelidou, 1997). Biochemistry of Feta cheese ripening

The breakdown of the main cheese constituents (protein, fat and lactose) by the action of many enzymes involved in cheese ripening is of importance, since it greatly influences the texture and flavour of the mature cheese.

Proteolysis The most complicated event during cheese ripening is, undoubtedly, proteolysis. Proteolysis in cheese is mediated by the concerted action of many proteolytic enzymes, derived from various sources. The contribution of each enzyme depends, amongst other factors, on their relative concentration and on the environment of each cheese. One of the key points for the successful manufacture of Feta cheese is the high acidification rate exerted by starter cultures and the consequent significant drop in pH from about 6.5 to 5.0 in 6-8 h during coagulation and draining, and to about 4.8 after 18-20 h from the beginning of manufacture. This ensures that more rennet is retained in Feta than in some other cheeses

236

Cheese Varieties Ripened in Brine

(Samal et al., 1993; van den Berg and Exterkate, 1993). Furthermore, the pH of Feta (about 4.5) is favourable for the proteolytic activity of chymosin and, also, is close to the pH optimum of the indigenous milk acid proteinase, cathepsin D. However, only a small part ("-8%) of the activity of this enzyme survives pasteurization (Larsen et al., 2000), and its role is expected to be of some importance only in Feta made from raw milk. Even so, since this enzyme has many of the same cleavage sites in Ors1- and [~-caseins as chymosin (Larsen et al., 1996), its activity would be overshadowed by the far higher activity of chymosin in Feta cheese. The activity of the dominant indigenous milk proteinase, plasmin, differs substantially between cheese varieties (Sousa et al., 2001). No data are available for plasmin activity in Feta. The absence of a curd-cooking step during Feta cheese manufacture, the relatively low pH and the high salt content are conditions which do not favour either the conversion of plasminogen to plasmin or the activity of the enzyme itself. Bands in the y-casein region on the electrophoretograms of Feta cheese, indicating some plasmin activity, are in most cases not strong and their intensity does not change during ripening. Proteolysis is not very intense in Feta cheese. Only about 15-18% of the TN of the cheese is soluble in water (WSN) after 60 days of ripening, reaching a value of up to 20-25% in well-ripened cheese in 120-180 days post-manufacture (Fig. 2). The main reason for this relatively low proteolysis is the short ripening period (2-3 weeks) at 16-18 ~ after which the cheese is transferred to a cold room (---4 ~ where

all biochemical reactions, including proteolysis, are slowed down. Additionally, the activity of many proteolytic enzymes, other than chymosin, is not favoured by the low pH of Feta cheese. It is worth noting that the water-soluble fraction of Feta (and other similar cheeses) contains not only the hydrolytic products of caseins (peptides and amino acids), which are soluble in water, but also some whey proteins (mainly [3-1actoglobulin and ot-lactalbumin), which remain in the curd after draining. On the other hand, as the cheese matures in brine, some of the peptides and amino acids, as well as some of the whey proteins, diffuse into the brine. Consequently, the level of WSN measured (e.g., by the Kjeldahl method) is either overestimated at the beginning of the ripening period, due to the presence of the whey proteins, or underestimated later on, due to diffusion (Katsiari et al., 2000a). Because of the diffusion process, the TN of the cheese decreases continuously during ripening and storage (Alichanidis et al., 1984; Katsiari and Voutsinas, 1994; Katsiari et al., 2000a). The rate of proteolysis in Feta cheese is high during the first 15-20 days, when the cheese is in the warm room (Fig. 2) but slows down when the cheese is transferred to the cold room (4 ~ Large amounts of low molecular weight nitrogenous compounds are produced during ripening in the warm room; at the end of this period about 60% of the WSN is soluble in 12% trichloroacetic acid (TCA-SN). The composition of this fraction changes continuously; during further ripening, it is enriched in very small peptides (130 days) or holding for a shorter time (e.g., 70 days) at higher storage temperature (e.g., 10-15 ~ (Guinee, 2002a). A more fundamental approach was described by Apostolopoulos (1994), who used lubricated squeezing flow to determine the elongational viscosity of melted LMMC at 65 ~ which can be used as a measure of the ability of the cheese to stretch and form strings. Cavella et al. (1992) used a spinning test method to objectively evaluate the stretchability of Mozzarella cheese. Horizontal (Ak etal., 1993) and vertical (Ak and Gunasekaran, 1995a) uniaxial extension methods have also been used to measure the elongational properties of LMMC. From the data presented in these reports, it appears that the horizontal method is more sensitive

260

Pasta-Filata Cheeses

than the vertical method to changes in the stretching behaviour of the cheese during 1 month of ageing.

Oiling-off. Oiling-off is caused by the release of free oil from the body of melted cheese. Excessive oilingoff results in pools of liquid fat at the surface and throughout the body of the melted cheese, giving the cheese a greasy appearance and mouthfeel that are generally regarded as undesirable. However, a moderate release of free oil contributes to desirable melting characteristics by creating a hydrophobic film on the cheese surface during baking, giving the surface a desirable sheen and, more importantly, slowing down evaporative loss of moisture. Excessive dehydration during melting, as occurs when insufficient free oil is released, results in the formation of a tough skin on the cheese surface that inhibits flow and scorches readily (Rudan and Barbano, 1998; Rudan et al., 1999). Free oil has been measured empirically by two different approaches: melting a disk of cheese on a filter paper and then measuring the area of the oil ring that diffuses into the filter paper; or melting and centrifuging the cheese to recover the free oil (Kindstedt and Rippe, 1990; Kindstedt and Fox, 1991). In general, oiling-off of Mozzarella cheese has been shown to increase with increasing fat content (Kindstedt and Rippe, 1990; Rudan et al., 1999), decreasing salt content (Rippe and Kindstedt, 1989; Kindstedt et al., 1992) and increasing time of storage and level of proteolysis (e.g., Tunick et al., 1993, 1995; Yun et al., 1993b,d,e, 1995a, 1998; Barbano et al., 1994; Renda et al., 1997; Hong et al., 1998; Poduval and Mistry, 1999). Furthermore, the release of free oil from Mozzarella cheese was reduced substantially when the milk or the cream fraction of the milk was homogenized before cheesemaking (Tunick, 1994; Rudan et al., 1998; Poduval and Mistry, 1999). Homogenization results in a much finer dispersion of fat within the cheese structure, as observed by SEM, which limits the ability of fat globules to coalesce and flow on melting. The use of a twin-screw extruder to stretch Mozzarella cheese also reduced oiling-off to a negligible level, presumably because the high shear mixing of the extruder produces a finer dispersion of the fat within the cheese structure (Apostolopoulos et al., 1994). Free oil was reduced by the addition of buttermilk solids to the cheese milk, presumably due to phospholipid-mediated enhancement of emulsification (Poduval and Mistry, 1999). Browning. Mozzarella cheese that contains both reducing sugars (i.e., lactose and galactose) and proteolysis products is susceptible to non-enzymatic (Maillard) browning reactions at high temperatures, such as that which occur during pizza baking. The browning potential of Mozzarella cheese has been evaluated

objectively by reflectance colourimetry after heating the cheese under various conditions (Johnson and Olson, 1985; Oberg et al., 1992; Barbano et al., 1994; Mukherjee and Hutkins, 1994). After heating and cooling, the cheese may be analysed for three colour indices, L '~ (light to dark), a ~ (red to green) and b '~ (yellow to blue), from which an evaluation of the intensity of browness can be made. Reduced browning potential in LMMC has been associated with lower galactose levels and the use of galactose-fermenting starter cultures (Johnson and Olson, 1985; Matzdorf etal., 1994; Mukherjee and Hutkins, 1994). Conversely, LMMC made from milk fortified with non-fat dry milk solids showed increased browning, presumably due to higher levels of lactose and galactose in the cheese (Yun et al., 1998). Directly acidified Mozzarella shows very little browning, presumably due to the absence of proteolysis products of starter culture origin (Oberg et al., 1992). Cultured Mozzarella cheese has generally been reported to increase in browning potential during ageing (Oberg et al., 1991; Barbano et al., 1994; Merrill et al., 1994; Yun et al., 1998). Presumably, increased browning is caused by the accumulation of proteolysis products and/or galactose released by non-galactose fermenting starter bacteria during ageing. Age-related changes in structure and function

Newly manufactured cultured LMMC generally melts to a tough, fibrous, chewy consistency that has limited ability to stretch and flow. Typically, it takes several weeks of storage at refrigerated temperatures before cultured LMMC attains its optimum melting characteristics (Kindstedt, 1995). Therefore, much research has been aimed at elucidating the age-related changes in the structure and function of Mozzarella cheese. However, it is important to recognize that the initial structure and functional properties of Mozzarella may vary substantially depending on the chemical composition of the cheese. Fat plays a particularly important role in the initial structure and function because the amount of fat determines the extent to which the paracasein fibers are interrupted by fat-serum columns (see Fig. 4). As the fat content of Mozzarella decreases, the volume fraction of the casein matrix increases and the para-casein strands become thicker with fewer inclusions of fat-serum channels between them (Merrill et al., 1996; McMahon et al., 1999). The abundance and size of the fat-serum channels influence the melting characteristics of the cheese because the channels act as a low viscosity lubricant which facilitates the displacement of adjacent planes of para-casein during heating (Guinee, 2002b). Consequently, cultured Mozzarella cheese with a reduced fat content initially

Pasta-Filata Cheeses

melts to a tougher, more chewy (higher apparent viscosity) and less flowable (lower meltability) consistency than Mozzarella made by the same process but with a higher fat content (Rudan et al., 1999). Furthermore, the distance separating the fat-serum channels from one another increases with decreasing fat content (Merrill et al., 1996), which restricts the ability of liquid fat globules in adjacent channels to flow and coalesce with one another to form pools of free oil. Consequently, the fat remains more finely dispersed on melting and the proportion of total fat that is released as free oil decreases with decreasing fat content (Rudan et al., 1999). The level of casein-associated calcium in the newly made cheese also plays a critical role in the initial structure and function of the cheese, as demonstrated by several recent studies in which different strategies to vary casein-associated calcium were used. Metzger et al. (2000, 2001a,b) used pre-acidification to vary the total calcium content of low-fat Mozzarella while holding other aspects of composition nearly constant. They reported that the level of water-insoluble (i.e., casein-associated) calcium decreased as the total calcium content decreased, which resulted in para-casein fibers that were less highly crosslinked with calcium and more highly solvated, the latter being evidenced by less serum expressed on centrifugation. Consequently, cheeses with less total calcium (and therefore less casein-associated calcium) had lower hardness, apparent viscosity and post-meh chewiness values immediately after manufacture, indicative of a softer cheese before heating and a less fibrous and chewy melted consistency. Several researchers (Kindstedt et al., 2001; Cortez et al., 2002; Ge et al., 2002) used a post-manufacture method to change the pH of cultured LMMC while holding other aspects of composition nearly constant. Increasing the cheese pH in the range of c. 5.0-6.5 caused a progressive increase in the amount of waterinsoluble (i.e., casein-associated) calcium and in the apparent viscosity of the cheese. Furthermore, changes in both calcium distribution and apparent viscosity were reversible when the pH of the cheese was reversed (Ge et al., 2002). These results, in combination with those reported by Metzger et al. (2001a,b), indicate that the initial cheese pH and the total calcium content independently affect the level of casein-associated calcium and, therefore, the initial structure and functional properties of Mozzarella cheese. Guinee et al. (2002) came to a similar conclusion by using direct acidification to simultaneously vary the pH and total calcium content of Mozzarella cheese. They observed that when the calcium level was typical, i.e., 28-30 mg/g protein, higher cheese pH, in the range 5.3-5.8, resulted in higher apparent viscosity, longer melt time,

261

and reduced flowability and stretchability. However, at a relatively low calcium level (e.g., 21 mg/g protein), LMMC with a high pH (i.e., 5.8) had functionality flow, stretch and apparent viscosity, at 1 day, similar to that of the control LMMC after storage at 4 ~ for 12-20 days. Furthermore, a lower total calcium content resulted in less serum expressed on centrifugation and a high degree of swelling of the para-casein fibers at the microstructural level immediately after manufacture, as observed by CSLM. From the results of the above studies, it may be concluded that initial cheese pH, in combination with the total calcium content, largely determines the amount of casein-associated calcium in the initial cheese structure. Casein-associated calcium, in turn, influences the amount of calcium crosslinking and solvation of the para-casein fibers and thus the initial cheese structure and functional characteristics. Less calcium crosslinking and greater solvation enable adjacent planes of para-casein to be displaced more readily during melting, resulting in greater meltability and stretchability and lower apparent viscosity and chewiness. Thus, the initial melting characteristics of Mozzarella cheese can vary widely, depending on the amount of caseinassociated calcium present in the cheese immediately after manufacture. During the first few weeks after the manufacture of cultured LMMC, it is well documented that meltability, stretchability and oiling-off increase, and the apparent viscosity, melt time and hardness decrease, as discussed earlier. These fairly dramatic functional changes are influenced by proteolysis that occurs concurrently during ageing, and proteolysis is clearly one of the driving forces behind the age-related changes in structure and function. For example, when proteolysis in LMMC was reduced by stretching at high temperature (i.e., cheese temperature at exit = 66 ~ the usual changes in hardness, meltabilty and apparent viscosity occurred more slowly (Yun et al., 1994a; Kindstedt et al., 1995b). Conversely, increasing the rate of proteolysis by using a more proteolytic coagulant or by storing LMMC at a higher temperature resulted in a faster decrease in the melt time and/or apparent viscosity and a faster increase in meltability (flowability) during ageing (Yun et al., 1993c,d; Guinee et al., 2002). However, proteolysis is not solely responsible for functional changes during ageing. Considerable interest has also been directed towards changes in the serum phase of Mozzarella cheese and elucidating their effects on structure and function (Kindstedt and Guo, 1998; McMahon etal., 1999). Several investigators have reported that the amount of serum expressed from cultured LMMC by centrifugation or pressing decreased from levels equivalent to c. 20-40% of the total cheese moisture immediately after manufacture

262

Pasta-Filata Cheeses

to no expressible serum after 2-3 weeks of ageing (Guo and Kindstedt, 1995; Kindstedt, 1995; Kindstedt et al., 1995b; Guo et al., 1997; Guinee et al., 2001, 2002; Kuo etal., 2001b). Thus, the water-holding capacity of cultured LMMC increases steeply during the first weeks after manufacture. Consistent with these results, data obtained using pulsed nuclear magnetic resonance suggest that a redistribution of water from a more- to less-mobile state occurs in cultured LMMC during the first 10 days of storage (Kuo et al., 2001b). McMahon et al. (1999) further demonstrated that the redistribution of water and the resulting increase in the water-holding capacity of Mozzarella cheese involved entrapped bulk water, whereas the amount of unfreezable (i.e., chemically bound) water did not change. The mechanism by which bulk water is redistributed has been elucidated using a couple of different approaches. Several studies have shown that intact caseins, especially [3-casein, and calcium are present in the expressible serum from cultured LMMC, and that their concentrations increase as the amount of serum decreases during storage (Guo and Kindstedt, 1995; Kindstedt et al., 1995b; Guo et al., 1997). These data suggested that a progressive dissociation of calcium and caseins from, and association of water with, the para-casein matrix occur over time. Guo et al. (1997) also observed that the solvation and solubilization of the para-casein matrix occurs much more slowly when

cultured LMMC contains no added salt (NaCI), as evidenced by higher amounts of expressible serum and lower concentrations of intact caseins in the serum obtained from the unsalted cheese. These investigators postulated that age-related changes in the water-holding capacity of cultured LMMC result in part from a NaCl-mediated process of swelling and solubilization of the para-casein matrix at the microstructural level. Furthermore, they suggested that the presumed microstructural swelling may be analogous to the swelling phenomenon known as 'soft rind defect' that occurs at the macrostructural level (Guo and Kindstedt, 1995; Guo et al., 1997). 'Soft rind defect' occurs when cheese is exposed to dilute salt brine (i.e., 108 cfu/g) and a high coliform count (105 cfu/g). Soluble N of 30-day-old cheese was 18.2-28.4% of total N, and FAAs reached 3.7-4.5 g/kg (Casaha et al., 2001).

Cheeses Made from Ewes' and Goats' Milk

Cacioricotta cheese is made traditionally by heating goats' milk at 95 ~ cooling it to 40 ~ and adding a Sc. thermophilus culture. Use of lower doses of rennet and variable amounts of mesophilic lactic cultures increased the yield of 15-day-old cheese from 7.39 to 7.88% on a DM basis, probably due to reduced proteolysis (0.38% NPN instead of 0.47%; Caponio et al., 2001). Lipolysis was also retarded by the modified technology, which improved the palatability of the cheese. The use of thermized milk in the manufacture of farm-made goat-milk cheese has been studied in order to improve its microbiological quality (Clementi et al., 1998). Reduced proteolysis was found in thermized milk cheese compared with raw-milk cheese. Thermization of milk reduced the 'goaty' taste and led to a slightly more bitter and salty flavour, a harder texture and a more intense white colour. High-pressure homogenization (HPH) of goats' milk at 1000 bar (100 MPa) has been compared with pasteurization and thermization in the manufacture of soft cheese (Guerzoni et al., 1999). High-pressure homogenization of milk reduced counts of most microbial groups by at least 2 log cycles. Fresh curd yields were 16.0% for raw milk, 20.7% for thermized milk, 20.3% for pasteurized milk and 32.0% for HPH milk. Lipolysis was favoured in cheeses from HPH milk, with 6.89 mg FFAs/kg compared to 5.25 mg FFAs/kg in raw-milk cheese. Proteolysis was also enhanced in cheeses made from HPH milk, which received the highest overall score from panellists. Portuguese goat cheeses

Goats' milk production in Portugal was 35 000 tonnes in 2001. The only PDO cheese in Portugal made exclusively from goats' milk is Cabra Transmontano, although other PDO cheeses such as Picante da Beira Baixa, Amarelo da Beira Baixa and Rabacal are manufactured from a mixture of goats' and ewes' milks. The production of goats' milk cheese was 1295 tonnes in 2001, and the production of cheese from mixed ewes' and goats' milks, 4791 tonnes. Cabra Transmontano is a hard cheese made from raw Serrana goats' milk, which is coagulated at 35 ~ with animal rennet. The coagulum is cut manually into irregular pieces and pressed by hand. Cheeses (fiat cylinders) are dry-salted and ripened at 5-18 ~ and 70-85% RH for a minimum of 60 days. Ripe cheese weighs 0.6-0.9 kg. No scientific information is available on this cheese variety (Freitas et al., 2000). Raba~;al cheese is manufactured with variable proportions of ewes' and goats' raw milks, although a 2:1 ratio is considered to be optimal (Delgado, 1993). Milk is coagulated at 30 ~ with animal rennet in

291

45-60 min and the coagulum is cut by hand to irregular grains. Cheeses (fiat cylinders) are pressed manually, dry-salted and ripened at 10-15 ~ and 7 0 - 8 5 % RH for 20 days. Ripe cheese weighs 0.3-0.5 kg. Sensory studies of this cheese variety describe its peculiar aroma and flavour as milky, floral and acid (Freitas et al., 2000). Picante da Beira Baixa may be manufactured from goats' or ewes' raw milk or their mixture, a 2:3 ratio being common. Milk at 28-30 ~ is coagulated with animal rennet in 40-50 min. The coagulum is cut into 1-1.5 cm cubes and pressed by hand. Cheeses (fiat cylinders) are dry-salted, stacked in groups of two or three and turned frequently. Ripening takes place at 10-18 ~ and 70-80% RH for 120-180 days. Ripe cheese weighs 0.4-1.0 kg. Picante cheese has high counts of staphylococci, up to 106 cfu/g, and coliforms, up to 108 cfu/g during the first week. Coliform counts decreased by 5-6 log cycles, and staphylococci counts by 3-4 log cycles, after 180 days in spite of an increase in pH from 4.5 to 5.2 in 9-day-old cheeses to 5.8-5.9 in ripe cheeses (Freitas et al., 1995). The predominant microbial species were identified by Freitas et al. (1996). Water-soluble N in ripe cheeses was 25-29% of total N, and NPN was 87-92% of soluble N. Residual Ors- and [~-caseins in ripe cheeses were 7-64% and 44-81%, respectively. The proportion of goats' to ewes' milk had no significant effect on cheese sensory characteristics (Freitas et al., 1997). Free amino acids of Picante cheese manufactured from different proportions of goats' and ewes' milks, animal or thistle rennets and salting once or twice were investigated by Freitas et al. (1999). The highest amount of FAAs was in cheese made using a mixture of goats' and ewes' milk (ratio of 1:4), animal rennet and salted once. Amarelo da Beira Baixa is a cheese variety similar to Picante, weighing 0.6-1.3 kg, with a straw to dark yellow rind (Freitas et al., 2000). Spanish goat cheeses

Spain is the third largest producer of goats' milk in the European Union, with 320 000 tonnes in 2001. Most of it is mixed with cows' and/or ewes' milks for the production o f - 2 0 non-PDO traditional cheese varieties, or new varieties such as Iberico cheese, manufactured from a mixture of milks of the three species with a minimum of 30% goats' milk. Technological aspects of Spanish goat cheeses have been reviewed (Franco et al., 2001). Twenty-eight varieties are made exclusively from goats' milk, although only four are PDO cheeses. In 2001, the production of PDO Majorero cheese was 352 tonnes, the production of PDO Ibores cheese began that year with 45 tonnes, and

292

Cheeses Made from Ewes' and Goats' Milk

the production of PDO Murcia and Palmero cheeses began in 2002. Majorero cheese is made in Fuerteventura, one of the Canary islands, from raw or pasteurized goats' milk. Coagulation with animal rennet takes place at 28-32 ~ in 60 min, after which the coagulum is cut to 1-cm-size grains and the whey is drained out. Cheeses are pressed, dry- or brine-salted, and ripened for 20-90 days at 12-18 ~ and a low RH. The surface is rubbed with oil, paprika or both during ripening. The shape is fiat cylindrical, and the weight 1-6 kg. In 90-day-old raw-milk cheese the DM was 83% and pH 5.44 (Fontecha et al., 1990). Two days after manufacture, coliforms and staphylococci reached 106-107 and 104-105 cfu/g, respectively, and after 90 days were less than 101 cfu/g. In 60-day-old cheese, residual Ors- and [3-caseins were 27% and 76%, respectively, and NPN was 19.0% of total N. Total FFAs reached 32.0 g/kg in 90-day-old cheese. Pasteurized milk cheese had a DM content of 61% and a pH of 5.46 on day 90 (Martin-Hern~indez et al., 1992). Residual %- and [3-caseins on day 60 were reduced to 47 and 81%, respectively, and NPN was 16.6% of total N. Total FFAs reached 6.11 g/kg in 90-day-old cheese, a much lower value than that of raw-milk cheese. Palmero, Tenerife and Conejero are traditional goat cheeses similar to Majorero made from raw milk, to which a Lc. lactis starter may be added, in different Canary islands. Tenerife cheese is a farm-house variety made from raw milk coagulated with animal rennet at 28-32 ~ in 30-60 min, of fiat cylindrical shape and weighing 0.9-1.2 kg, with an annual production close to 1500 tonnes. The DM increases slightly during ripening (46% after 2 days to 49% after 60 days) and the pH declines from 4.93 on day 2 to 4.64 on day 30, and remains constant during the second month of ripening. Coliform counts decreased from 107 cfu/g in 2-day-old cheese to 103-104 cfu/g in 60-day-old cheese, while S. aureus counts in 2-day-old cheese were 103 cfu/g and less than 10 cfu/g in 60-day-old cheese (Z~irate et al., 1997). Ibores cheese is made in Extremadura from raw milk, to which a Lc. lactis starter may be added. Milk is coagulated at 28-32 ~ in 60-90 min, generally with animal rennet. The coagulum is cut to medium-size (1-2 cm) grains. Cheeses of fiat cylindrical shape, weighing 0.7-1.2 kg, are pressed for 3-8 h, dry- or brine-salted and ripened for a minimum of 60 days. Seasonal differences have been recorded for pH, with higher values for cheeses made in winter than for those made in spring (Mas and Gonz~ilez Crespo, 1993). Cheese ripened for 60 days had a pH of 5.18, a DM of 59%, ---21% pH 4.6-soluble N as % of total N and ---10%

TCA-soluble N. In 60-day-old cheese, coliforms were 103-104 cfu/g, and coagulase-positive staphylococci less than 10 cfu/g. Lc. lactis subsp, lactis, E. faecium, Leuc. mesenteroides subsp, dextranicum and Lb. casei were the most abundant species within their respective genera (Mas et al., 2002). A total of 29 volatile compounds have been identified in Ibores cheese, including five ketones, five alcohols, two aromatic hydrocarbons, ten esters, four terpenes and one aldehyde (Sabio and Vidal AragOn, 1996). Murcia cheese is made from pasteurized milk. It may be fresh, ripened or 'al vino' (wine-cured). In fresh cheese manufacture, the milk is coagulated at 35-38 ~ in 30-60 min, the coagulum is cut and stirred, and cheeses are pressed for 2-4 h. After brinesalting, cheeses (fiat cylinders weighing 0.3-1.5 kg) are held at 4 ~ For ripened cheese manufacture, milk is coagulated at 32-33 ~ in 45-60 min with animal rennet. The coagulum is cut, stirred and heated to 35-37 ~ Cheeses (fiat cylinders weighing 1-2 kg) are pressed, brine-salted for 12h and ripened at 12-14~ and 75-85% RH for at least 21 days. Murcia cheese 'al vino' is made from washed curd. Cheeses are immersed in red wine for 30 min at the beginning of ripening, for 15-30 min on day 7, for 15-30 min on day 14, and on day 21 for a time depending on rind characteristics (Franco et al., 2001). There is no scientific information available on Murcia cheese. Gredos cheese, also called Tietar or La Vera, is farm-made from raw milk, coagulated with animal rennet at 25-30 ~ in 1.5-2.5 h. The coagulum is cut to rice-grain or smaller size, left to settle, scooped into moulds and pressed by hand. Cheeses, of flat cylindrical shape and weighing 0.8-1.2 kg, are dry-salted and ripened for 15 days at 8-10 ~ and 80-90% RH. If not consumed as fresh cheese, they are immersed in olive oil and held for 45-60 days at 8-10 ~ The pH declines from 6.27 on day 4 to 4.64 on day 45, while DM increases from 38% on day 4 to 45% on day 60. Most microbial groups reach maximum numbers after 15 days of ripening, with coliform counts of 105-106 cfu/g and coagulase-positive staphylococci counts of 102-103 cfu/g at that time. In 60-day-old cheese, coliform counts had decreased by 4 log cycles and coagulase-positive staphylococci by 2 log cycles. Residual ors- and [3-caseins were 22% and 40%, respectively, and NPN was 14.9% of total N in 60-day-old cheese (Medina et al., 1992). Cendrat del Montsec is made from raw milk inoculated with 3% Lc. lactis starter, coagulated at 15-20 ~ in 20 h using animal rennet. The coagulum is not cut, but scooped into cylindrical moulds where whey drains spontaneously for 6 - 7 h , and afterwards, cheeses weighing 1.5 kg are slightly pressed for 24 h.

Cheeses Made from Ewes' and Goats' Milk

Cheeses are dry-salted, and after 5 days are covered with oak ash. Ripening takes place at 10-15 ~ and 90-95% RH for 9 weeks. The pH increases from 4.02 in 1-day-old cheese to 4.40 in 63-day-old cheese, and the DM increases during this time from 46 to 53%. In ripe cheese, Ors-casein is almost completely degraded, but 50% [3-casein remains unaltered (Carretero et al., 1994). Due to the low pH value, coliforms and S. aureus were at low numbers (101-102 cfu/g) at the end of the ripening period (Mor-Mur et al., 1992). Valdeteja is farm-made from raw milk coagulated at 35 ~ with animal rennet in 105-120 min. The coagulum is cut to 1-cm-size grains, moulded and pressed for 12 h. Cheeses, fiat cylinders weighing 0.8-1.2 kg are dry-salted and ripened at 10-15 ~ and 70-80% RH for 30 days. During ripening, the pH declines to 5.1 on day 2 and 4.5 on day 10, and remains unchanged until day 30, while the DM is 48% on day 2 and increases to 62% on day 30. The acidity index of the fat increased from 0.89 on day 2 to 1.46 on day 30. Only 4-5% NPN of total N was found in 30-day-old cheese (Carballo et al., 1994). Armada cheese is farm-made from raw milk, to which a small amount of whey from the previous day is added, coagulated with animal rennet at 30 ~ in 60 min. The coagulum is cut, left to settle, cut again to a smaller size and scooped into cloths which are hung for 48 h. Afterwards, the curds are kneaded intensely by hand, transferred to new cloths, hung for a further 72 h and salt is added. The curds are kneaded again and moulded to cheeses, 20 cm in diameter and 20 cm high, which are wrapped in cloths and hung to ripen at 10-15 ~ and 70-85% RH for 60-120 days. During ripening, the pH declines to a minimum of 4.31-4.68 on day 7, increasing later to 4.89-5.25 on day 120, while the DM increases from 49-57% on day 7 to 75-82% on day 120 (Tornadijo et al., 1993). The NPN is 5.0% of total N on day 7 and increases to only 7.3% by day 120, whereas residual Ors- and [3-caseins were 93 and 98%, respectively, on day 120. Total FAAs increased from 2.1 g/kg on day 7 to 3.6 g/kg on day 120, and total FFAs increased in the meantime from 5.9 g/kg to 44.5 g/kg (Fresno et al., 1997). Cameros cheese is made from raw or pasteurized milk, coagulated at 32 ~ in 60 min with animal rennet. The coagulum is cut by hand, moulded in plastic baskets and slightly pressed for 8-12 h. Cheeses are dry-salted and ripened for up to 60 days at 12-14 ~ and 70-80% RH. Raw- and pasteurized-milk cheeses have been studied. A pH of 4.52-4.89 was reached on day 5, decreasing to 4.49-4.65 on day 30, followed by an increase to 4.70-4.98 on day 60. The DM was 51-56% on day 5 and increased to 79-83% on day 60. Proteolysis was slight, with only 5.0-7.7% NPN

293

of total N on day 60. In raw-milk cheese, coliform counts were less than 10 cfu/g on day 60, but numbers of S. aureus were close to 106 cfu/g on days 5-15 and still over 103 cfu/g on day 30 (Olarte etal.,

2000). Recently, extensive studies on the effects of highpressure treatment on the microbiological (Capellas etal., 1996; Buffa etal., 2001b), physico-chemical (Trujillo et al., 1999; Capellas et al., 2001; Buffa et al., 2001a; Saldo et al., 2002) and textural (Saldo et al., 2000; Buffa et al., 2001c) characteristics of goats' milk cheeses have been carried out. High-pressure treatments of cheeses at 400-500 MPa improved microbiological quality, enhanced proteolysis and resulted in a more fluid-like texture. Lipolysis in cheeses made from high-pressure-treated milk was similar to that in raw-milk cheeses, and higher than lipolysis in pasteurized-milk cheeses. Cheeses made from high-pressure-treated milk were, like raw-milk cheeses, firmer and less fracturable than pasteurizedmilk cheeses.

More than 100 cheese varieties, many of them protected by a Designation of Origin, are made from ewes' or goats' milk in Europe. This rich heritage, dating in some cases from the Middle Ages, should be maintained for cultural and socio-economic reasons. Farming of ewes and goats and transformation of their milks into cheeses contribute to the sustainable development of many regions, mostly in Mediterranean countries. The peculiar flavour and texture typical of ewes' or goats' milk cheeses can be explained partly by compositional differences in caseins and fat, distinct from those of cows' milk. In raw-milk cheeses, a diverse microbiota composed of adventitious LAB (Cogan et al., 1997), but also of bacteria other than LAB, yeasts and moulds, contribute to their distinct sensory characteristics. In order to maintain the traditional characteristics of these cheese varieties, there is a need to preserve the biological diversity involved in the ripening process of ewes' and goats' milk cheeses, by the use of authoctonous lactic starters and mould cultures in their manufacture. Recent studies on ewes' and goats' milk cheeses have considerably enlarged our knowledge of their microbiology, chemistry and texture. However, current scientific information on many varieties, some of major economic importance, is still scarce and research for the better understanding and improving of their manufacture and ripening is needed.

294

Cheeses Made from Ewes' and Goats' Milk

Albenzio, M., Corbo, M.R., Shakeel-Ur-Rehman, Fox, P.E, De Angelis, M., Corsetti, A., Sevi, A. and Gobbetti, M. (2001). Microbiological and biochemical characteristics of Canestrato Pugliese cheese made from raw milk, pasteurized milk or by heating the curd in hot whey. Int..]. Food Microbiol. 67, 35-48. Ambrosoli, R., Di Stasio, L. and Mazzoco, P. (1988). Content of Otsl-casein and coagulation properties in goat milk. J. Dairy Sci. 71, 24-28. Amigo, L., Recio, I. and Ramos, M. (2000). Genetic polymorphism of ovine milk proteins: its influence on technological properties of m i l k - a review. Int. DairyJ. 10, 135-149. Anifantakis, E. (1986). Comparison of the Physico-chemical Properties of Ewes' and Cow's Milk. In Bulletin 202, International Dairy Federation, Brussels. pp. 42-53. Anifantakis, E., Kehagias, C., Kotouza, E. and Kalantzopoulous, G. (1980). Frozen stability of sheep's milk under various conditions. Milchwissenschaft 35, 80-82. Arizcun, C., Barcina, Y. and Torre, P. (1997). Identification of lactic acid bacteria isolated from Roncal and Idiaz~ibal cheeses. Lait 77,729-736. Battistotti, C. and Corradini, C. (1993). Italian cheese, in, Cheese: Chemistry, Physics and Microbiology, Vol. 2, 2nd edn, P.E Fox, ed., Chapman & Hall, London. pp. 221-243. Bizzarro, R., Torri-Tarelli, G., Giraffa, G. and Neviani, E. (2000). Phenotypic and genotypic characterization of lactic acid bacteria isolated from Pecorino Toscano cheese. Ital. J. Food Sci. 3,303-316. Brennand, C.P., Ha, J.K. and Lindsay, R.C. (1989). Aroma properties and thresholds of some branched-chain and other minor volatile fatty acids occurring in milkfat and meat lipids.J. Sens. Stud. 4, 105-120. Buffa, M., Guamis, B., Pavia, M. and Trujillo, J.A. (2001a). Lipolysis in cheese made from raw, pasteurized or highpressure-treated goats' milk. Int. Dairy J. 11, 175-179. Buffa, M., Guamis, B., Royo, C. and Trujillo, A.J. (2001b). Microbiological changes throughout ripening of goat cheese made from raw, pasteurized and high-pressuretreated milk. Food Microbiol. 18, 45-51. Buffa, M., Trujillo, J.A., Pavia, M. and Guamis, B. (2001c). Changes in textural, microstructural, and colour characteristics during ripening of cheeses made from raw, pasteurized or high-pressure-treated goats' milk. Int. Dairy J. 11,927-934. Bustamante, M., Ch~ivarri, E, Santisteban, A., Ceballos, G., Hernandez, I., Miguelez, M.J., Aranburu, I., Barr6n, L.J.R., Virto, M. and De Renobales, M. (2000). Coagulating and lipolytic activities of artisanal lamb rennet pastes. J. Dairy Res. 67,393-402. Campos, R., Guerra, R., Aguiar, M., Ventura, O. and Camacho, L. (1990). Chemical characterization of proteases extracted from wild thistle (Cynara cardunculus). Food Chem. 35, 89-97. Capellas, M., Mor-Mur, M., Sendra, E., Pla, R. and Guamis, B. (1996). Populations of aerobic mesophiles and inoculated E. coli during storage of fresh goat's milk cheese treated with high pressure. J. Food Prot. 59,582-587.

Capellas, M., Mor-Mur, M., Sendra, E. and Guamis, B. (2001). Effect of high pressure processing on physico-chemical characteristics of fresh goats' milk cheese (Mat6). Int. Dairy J. 11,165-173. Caponio, E, Pasqualone, A. and Gomes, T. (2001). Apulian Cacioricotta goat's cheese: technical interventions for improving yield and organoleptic characteristics. Eur. Food Res. Technol. 213, 178-182. Carballo, J., Fresno, J.M., Tuero, J.R., Prieto, J.G., Bernardo, A. and Martfn-Sarmiento, R. (1994). Characterization and biochemical changes during the ripening of a Spanish hard goat cheese. Food Chem. 49, 77-82. Carbonell, M., Nufiez, M. and Fernandez-Garcia, E. (2002). Evolution of the volatile components of ewe raw milk La Serena cheese during ripening. Correlation with flavour characteristics. Lait 82, 683-698. Carretero, C., Trujillo, A.J., Mor-Mur, M., Pla, R. and Guamis, V. (1994). Electrophoretic study of casein breakdown during ripening of goat's milk cheese. J. Agric. Food Chem. 42, 1546-1550. Casaha, E., Noel, Y., Le Bars, D., Carre, C., Achilleos, C. and Maroselli, M.-X. (2001). Caracterisation du fromage Bastelicaccia. Lait 81,529-546. Chavarri, E, Bustamante, M.A., Santisteban, A., Virto, M. and De Renobales, M. (1999). Changes in free fatty acids during ripening of Idiazabal cheese manufactured at different times of the year. J. Dairy Sci. 82,885-890. Clementi, E, Cenci Goga, B.T., Trabalza Marinucci, M. and Di Antonio, E. (1998). Use of selected starter cultures in the production of farm manufactured goat cheese from thermized milk. Ital. J. Food Sci. 10, 41-56. CNIEL (2002). Banque de donnees statistiques, http://www. maison-du-lait, corrgScrip ts/public/sta t. asp ?Language = FR. Cogan, T.M., Barbosa, M., Beuvier, E., Bianchi-Salvadori, B., Cocconcelli, PS., Fernandes, I., Gomez, M.J., G6mez, R., Kalantzopoulos, G., Ledda, A., Medina, M., Rea, M. and Rodriguez, E. (1997). Characterization of the lactic acid bacteria in artisanal dairy products. J. Dairy Res. 64, 409-421. Coni, E., Bocca, B. and Caroli, S. (1999). Minor and trace element content of two typical Italian sheep dairy products. J. Dairy Res. 66, 589-598. Dahl, S., Tavaria, EK. and Malcata, EX. (2000). Relationships between flavour and microbiological profiles in Serra da Estrela cheese throughout ripening. Int. DairyJ. 10, 255-262. Deiana, P., Fatichenti, E, Farris, G.A., Mocquot, G., Lodi, R., Todesco, R. and Cecchi, L. (1984). Metabolization of lactic and acetic acids in Pecorino Romano cheese made with a combined starter of lactic acid bacteria and yeast. Lait 64, 380-394. Deiana, P., Rossi, J., Caredda, M., Cattina, A.P. and Smacchi, E. (1997). Secondary microflora in Pecorino Romano cheese. Sci. Tecn. Latt.-Cas. 48,487-500. Delgado, M.G.H. (1993). Physico-chemical characterization of Rabacal cheese. Via Lactea 3, 64-66. Emaldi, G.C. (1987). Goat cheeses. Sci. Tecn. Latt.-Cas. 38, 46-53. Engel, E., Nicklaus, S., Septier, C., Salles, C. and Le Quere, J.L. (2000). Taste active compounds in a goat cheese watersoluble extract. 2. Determination of the relative impact of

Cheeses Made from Ewes' and Goats' Milk

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299

logical characteristics of ewes' milk cheese manufactured with extracts from flowers of Cynara cardunculus and Cynara humilis as coagulants. J. Agric. Food Chem. 48, 451-456. Voutsinas, L.P., Katsiari, M.C., Pappas, C.P. and Mallatou, H. (1995). Production of brined soft cheese from frozen ultrafiltered sheep's milk. Part 2: Compositional, physicochemical, microbiological and organoleptic properties of cheese. Food Chem. 52, 235-247. Xanthopoulos, V., Polychroniadou, A., Litopoulou-Tzanetaki, E. and Tzanetakis, N. (2000). Characteristics of Anevato cheese made from raw or heat-treated goat milk inoculated with a lactic starter. Lebens.-Wiss. Technol. 33, 483-488. Z~irate, V., Belda, E, PCrez, C. and Cardell, E. (1997). Changes in the microbial flora of Tenerife goats' milk cheese during ripening. Int. Dairy J. 7, 635-641. Zerfiridis, G.K. (1999). G r e e c e - a country of diverse milk production but short in dairy products. Milchwissenschaft 54, 424-426.

Acid- and Acid/Rennet-curd Cheeses Part A: Quark, Cream Cheese and Related Varieties D. Schulz-Collins, Arrabawn Co-op, Nenagh, Co. Tipperary, Ireland B. Senge, Technische Universit&t Berlin, Faculty of Process Sciences, Department of Food Rheology, Berlin, Germany

Fresh cheeses are unripened cheeses, which are manufactured by the coagulation of milk, cream or whey using acid, a combination of acid and rennet or a combination of acid and heat. Fresh cheeses are ready for consumption immediately after production. In most countries and cultures, there is some traditional form of flesh cheese. With increased globalisation and tourism, the various regional types of fresh cheese have begun to spread outside their regions of origin. Cream cheese, Cottage cheese, Quark or Tvorog, Fromage frais and Ricotta are among the better-known types. Quark (in German-speaking countries) or Tvorog (in Eastern European countries) is essentially a milk protein paste. It is milky white to faintly yellowish in colour; smooth, homogeneously soft, mildly supple and elastic in body; mildly acidic and clean in flavour. Due to the high moisture content (--~82%, w/w), the shelf-life is limited to 2-4 weeks at < 8 ~ There should be no appearance of whey, dryness or graininess, bacteriological deterioration, over-acidification or bitter flavour during storage (Kroger, 1980; Siggelkow, 1984; Guinee et al., 1993). Hot-pack Cream cheese ('Soft Cheese' in the UK; 'Fresh Cheese' in Germany) is a creamy-white, slightly acid-tasting product with a mild diacetyl flavour; its consistency ranges from brittle, especially for double Cream cheese (DCC), to spreadable for single Cream cheese (SCC). Cream cheese, which is very popular in North America, has a shelf-life of "-3 months < 8 ~ (Guinee et al., 1993). Hard and brittle structures can be obtained only in high-fat Cream cheese (55-60%, w/w, fat-in-dry matter (FDM); Walenta et al., 1988). Quark and Cream cheeses can be consumed plain or in sweet or savoury dishes. Most fresh cheeses are very versatile and particularly suitable for processing into fresh cheese preparations or various dishes (e.g., cheesecakes, sauces, desserts).

Fresh cheeses can be divided into various categories, e.g., by the method of coagulation- acid, acidrennet, acid-heat, etc., their consistency- paste, grainy or gel-like, or raw m a t e r i a l - milk or whey (Fig. 1). In comparison to most ripened cheeses, fresh cheeses are generally low in dry matter (DM) and, hence, low in fat and protein and high in lactose/lactate (Table 1). As most of the calcium is solubilised during the acid coagulation and removed with the whey, fresh cheeses are much lower in calcium than rennet-curd cheeses. Classification and definition of cheeses are, in most countries, controlled by a codex or law, as done in Germany (Table 2) with the Kaseverordnung (Cheese order; Anon, 1986). German Quark is defined as containing at least 18%, w/w, DM, at least 12%, w/w, protein and a maximum 18.5%, w/w, whey protein in the total nitrogen content; products with a DM < 18%, w/w, are to be labelled as Frischktise (Fresh Cheese; Anon, 1986). In other countries, definitions can be less stringent or nonexistent. Often, only total moisture and protein contents are specified, as for, e.g., Kwark or Verse kaas (Quark or Fresh cheese) in The Netherlands, i.e., moisture maximum 87%, w/w, and protein minimum 60%, w/w, of non-fat DM (Anon, 1994b). American Cream cheese (>33%, w/w, fat, 45%, w/w, DM), Neufchatel (20-33%, w/w, fat, 35%, w/w, DM) and German Double Cream (fresh) cheese (26.4-38.3%, w/w, fat, 44%, w/w, DM) are similar in composition and comparable to Petit Suisse or Fromage frais/t la cr~me cheeses of France (Anon, 1986; Kosikowski and Mistry, 1997). World cheese production experienced a low of --~14 million tonnes in 1992/1993 due to the crisis in the former USSR. In 1995-1996 an upward trend started again and world production increased to 15.4 million tonnes in 1999. When cheese production was analysed for 26 countries that accounted for ---80% of the world production in 2001 (Table 3), the most distinct tendency is the remarkable upward trend for fresh cheese, which increased by 38% (from 2 660 000 tonnes in

Cheese: Chemistry, Physics and Microbiology, Third edition - Volume 2: Major Cheese Groups ISBN: 0-1226-3653-8 Set ISBN: 0-1226-3651-1

Copyright 9 2004 Elsevier Ltd All rights reserved

302

Quark,

Cream

Cheese

and

Related

Varieties

and a c i d - c u r d

Acid/rennet-

cheese

varieties

I

I Fresh c h e e s e s

I I

Standard varieties

[

I

-- Paste-like consistency --Quark and quark-related

-

[

Mainly acid coagulated varieties

Baker's cheese - Topfen -Tvorog, Tvarog, Twarogow - Fromage Frais -- Labneh, Labaneh - Buttermilkquark - Petit Suisse - Neufchatel --Ymer -- Chakka, Shirkhand -- Skyr - - Queso Blanco - Cream cheese -Double Cream cheese -

Other varieties

Ripened

acid-curd

cheeses

Harzer Mainzer Olmuetzer Quargel Topfkaese

~ Cottage cheese Acid-heat coagulated t Ricotta, Ricottone Mascarpone Queso Blanco, Queso Fresco

Gel-like consistency

L Layered white cheese (Schichtkaese) Fresh cheese varieties.

1990 to 3 662 000 tonnes in 1999). In i999, 32% of the total cheese production was fresh cheese, compared to 30% 10 years ago (Sorensen, 2001). One reason for the steady increase in output of fresh cheese is that the ingredient sector is becoming more and more important. Major producers are the US (with a large ingredient sector) and the E U - in which Germany, France and Italy produce the highest levels, although Spain and Denmark have also experienced a large increase. Neither The Netherlands nor New Zealand and Australia have a fresh cheese output of importance on the world market (Anon, 1994a; Sorensen, 2001). Of the total production of fresh cheeses in the EU, approximately 47% is produced in Germany, 35% in France and 13% in Italy. In Germany and France, fresh cheese constitutes 47% and 33% of total cheese production, respectively. In Europe, the per capita consumption of fresh cheese is highest in Germany (8.7 kg/year in 1999), followed by France, Poland and Iceland. Almost half of the fresh cheese consumed in Germany is Quark (4.0 kg/year); the balance is Cream, Cottage and other fresh cheeses. Especially high growth rates have been observed for fresh cheese preparations con-

taining fruit or herbs (Richarts, 2001). Fresh cheese consumption is also very high in the Middle-East (e.g., in Israel, 12.3 kg/year in 1998). In Eastern European markets, particularly in Russia and Poland, Tvorog-type cheeses represent up to two-thirds of total cheese consumption (Rouyer, 1997). Poland and Russia are amongst the biggest Tvorog producers in Europe. Mann (1978a,b, 1982, 1984, 1987, 1994, 1997, 2000) has been following and reviewing the world literature on the manufacture, composition and utilisation of Quark and related products for almost the last three decades. The production of fresh acid- or acid/rennet-curd cheeses typically involves the addition of a starter culture and a relatively small amount of rennet to skim milk. Under these conditions, the milk undergoes slow quiescent acidification resulting in the formation of a gel at a pH value near the isoelectric pH of casein (typically 4.8-4.6). The gel is then stirred and concentrated by one of the several techniques, such as centrifugation or ultrafiltration (UF), which involve removal of whey or permeate. The resulting product might be cooled and packaged directly (e.g., Speisequark) or further processed (e.g., heat-treated Quark desserts, Fig. 2).

Quark, Cream C h e e s e and Related Varieties

303

Approximate composition (%, w/w) of various fresh cheeses

Variety (German) Skim Quark (German) Single Cream cheese (German) Double Cream cheese American Cream cheese Neufchatel Labneh Skyr Ymer Lactofil Buttermilk Quark Whole milk Ricotta Part skim milk Ricotta Mascarpone Cottage cheese Baker's cheese Cebreiro cheese

Dry matter

Fat

Protein

Lactose and lactate

pH

> 18

12

3-4

4.6

39

19.5

n.a.

3.5

4.6

44

26.4-38.3

n.a.

2-3

4.6

>45 >35 22-26 18.5-20.5 14.5 16 15 28-41

>33 20-33 7-10 0.2-0.4 3.5 5 0.75-0.95 13-17

n.a. n.a. 7-10 12.5-16.0 5-6 5-6 9-10 11.3-1 8

2-3 2-3 --~4.2 3.6-3.8 n.a. n.a. 3.5-3.6 3.0

4.6 4.6 4.0-4.2 4.6 4.6 4.6 4.5-4.7 5.7-5.8

25

8

12

3.6

5.8

45-55 21 26 30-35

45-55 4.5 0.2 15-17

7-8 12.5 19 --~12

n.a. 2.6 3-4 n.a.

5.8 n.a. 4.6 4.55

Compiled from Anon, 1986; Tamime and Robinson, 1988; Jelen and Renz-Schauen, 1989; Modler and Emmons, 1989; Lehmann et aL, 1991; Guinee et aL, 1993; Kessler, 1996; Kosikowski and Mistry, 1997; Ozer et aL, 1999; Boone, 2001a,b; Fernandez-Albalat et aL, 2001.

Compositional specification of fresh cheeses according to German regulations

Fat category (Fettstufe)

German Quark Dry matter (%) Protein (%) Fat in dry matter (%) Fat, absolute (%)

Skim

Quarterfat a

Halffat

Threequarter fat

Fat

Fullfat

Cream

Double Cream

>18.0 b >12.0 c ' 1.0 c,

5.97

(1)

E

a_ 0.5

pH 6.33

0

~

p

I 0

1

H

I

6.68

I

2 3 Time (h)

Permeability of rennet-induced skim milk gels as a function of time after renneting at 30 ~ and various pH values (redrawn from Walstra, 1993).

German-type Quark and East European Tvorog are usually produced using small amounts of rennet in order to improve the draining characteristics of the curd, to reduce casein fines and increase curd firmness (Jelen and Renz-Schauen, 1989). Rennet is also known to produce bitter peptides by its proteolytic activity; therefore, a high rennet concentration can lead to bitterness in Quark (B~iurle et al., 1984; Sohal et al., 1988; Shah et al., 1990). The typical rennet concentration used in commercial Quark manufacture, depending on rennet type and strength, is 2-20 ml of standard strength rennet per 1000 1 of milk (Table 4). The rennet action enhances the destabilisation and aggregation of the casein micelles during acidification, i.e., the ratio between aggregation and disaggregation

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309

310

Quark, Cream Cheese and Related Varieties

forces during the early stages of acidification is increased (Guinee et al., 1993). If Quark is produced using acidification only, the coagulum is cut at pH 4.7-4.5 whereas the acid-rennet gel can be cut at a higher pH (4.8-4.9). The shift of the maximum storage modulus of a pure acid-induced gel from pH 4.5 to 4.7-4.8 for an acid-rennet gel is explained by the higher isoelectric point of the para-K-casein in comparison to the K-casein (Roefs et al., 1990). Therefore, over-acidification can be prevented by rennet addition. If the heat treatment is severe (i.e., 95 ~ for 5 min), there are no perceptible differences in UF Quark made with or without rennet (Sachdeva et al., 1993) as the casein is less susceptible to rennet hydrolysis when complexed with denatured whey protein. Ultrafiltration- or Thermoquarks are usually heated in the region 88-95 ~ for 3-6 min. If rennet is used in the process to obtain a firmer product, a lower heat treatment is used, i.e., 88 ~ for 3 min. Much firmer gels are produced when a small amount of rennet is added at the beginning of acidification as the enzymatic reaction is accelerated by lowering the pH (Fig. 7; Lehmann et al., 1991; Schkoda, 1998; Herbert et al., 1999; Schulz et al., 1999; Lucey et al., 2000; Schulz, 2000; Tranchant et al., 2001). The addition of rennet at the beginning of acidification induces a coarser network (Bishop et al., 1983; Roefs et al., 1990; Schkoda, 1998; Lucey et al., 2000; Schkoda et al., 2001a). Particle strands of stirred acid-rennet gels are thicker than in stirred acid gels, i.e., casein micelles partially fuse together (Roefs, 1986; Roefs et al., 1990; Schkoda, 1998). Casein particles in acidset curd are smaller than from enzyme-acid-set curd throughout various stages of Cottage cheese produc-

tion, i.e., after cutting, cooking and draining (Bishop et al., 1983). Rennet gels at pH 6.7 (tan 6 = 0.60) are more viscous-like than acid gels at pH 4.6 (tan ~ = 0.27) or acid-rennet gels at pH 4.6 (tan 6 = 0.26). As tan 6 is related to the relaxation of bonds in the gel during its deformation, rennet-induced gels are more prone to syneresis (Roefs, 1986; Roefs et al., 1990; Walstra, 1993). Therefore, syneresis of combined gels increases strongly with increasing rennet concentration (Roefs, 1986; Walstra, 1993; Schkoda, 1998; Schkoda et al., 2001a). However, Lucey et al. (2001) found more syneresis in low-rennet-GDL gels (10 ml of a double strength calf rennet/1000 1 of milk) than in high-rennet-GDL gels (150 ml/1000 1) for heated and unheated milks. Various parameters of rennet gels, acid gels and acid-rennet gels are compared in Table 5. In addition to the milk coagulation initiated by acidification alone, two other distinct types of coagulation profiles (Fig. 7) were identified, depending on the relative contribution of acidification versus renneting to initial gel formation (Dalgleish and Home, 1991; Schulz et al., 1999; Schulz, 2000; Tranchant, 2000; Kelly and O'Kennedy, 2001; Tranchant et al., 2001). Up to a certain, i.e., 'critical', rennet concentration, gel formation and firming due to acidification and renneting occur simultaneously, i.e., truly combined acidification and renneting (Schulz, 2000; Tranchant et al., 2001). This 'critical' rennet concentration was found to be 1.7-2.2 ml rennet per 1000 1 of milk (see Table 4 for rennet strength) at 30-40 ~ and a renneting pH of 6.4-6.6 (Dalgleish and Horne, 1991; Schulz et al., 1999; Tranchant et al., 2001). The highest gel firming rate and final complex viscosity of acid-rennet gels were found at this critical rennet concentration (Schulz etal.,

c

200

6.5

180 160 EL

6.0

E 140 v

120 g

~00

~"

80

~. 8

"r

5.5

6o

b

40 20 0 200

a 400

600

800

1000

5.0 4.5 1200

Time (min) Time-related change in complex viscosity (m) and pH ( ) of skim milk treated with a mesophilic starter culture and rennet (Fromase 220TL, DSM Food Specialties B.V., Dortmund) at different levels (ml rennet/1000 I milk: a, 0; b, 0.5; c, 2; d, 9; e, 20) during slow and quiescent acidification at 31 ~ (redrawn from Schulz, 2000).

Quark,

Properties of skim milk gels obtained by renneting (aged for about 1 h), by acidification (aged for 6-16 h) or by combined renneting and acidification. All gels were formed by slow quiescent acidification using GDL at 30 ~

pH Elastic modulus, G' at = 0.01 rad s -1 (Pa) Loss tangent, tan 8 at ~o = 0.01 rad s -1 Fracture stress (Pa) Fracture strain ( - ) Permeability B (l~m2)

Rennet gel

Acid gel

Combined gel

6.65 32

4.6 20

4.6 800

0.55

0.27

0.27

10 3.0 0.25

100 1.1-1.5 0.15

300 0.7 0.28

Data from Roefs et aL, 1990; Walstra, 1993; Lucey et al., 2000.

1999). Rennet concentrations higher than the 'critical' value lead to a distinct local maximum in gel consistency followed by a local minimum, i.e., sequential formation of rennet gel and acidification (Schulz, 2000; Tranchant et al., 2001). Rennet addition shortens the clotting time, and gelation occurs at a higher pH (Noel, 1989; Noel et al., 1991; Schkoda, 1998; Lucey et al., 2000; Schulz, 2000). There is a linear relationship between the inverse of rennet concentration and the clotting time as well as the time to reach the local viscosity maximum for the combined acidification-renneting method (Schulz, 2000). On increasing the rennet concentration in the range 0-20 ml/1000 1, the pH at aggregation and the local maximum of the complex viscosity increased

Cream

Cheese

and Related Varieties

311

from pH 5.44 to 6.31 and 5.05 to 5.38, respectively (Fig. 8; Schulz et al., 1999; Schulz, 2000; Fromase 220TL, DSM Food Specialties B.V., Dortmund). However, the local viscosity minimum and final viscosity maximum occured at the same pH, i.e., pH 4.95 and 4.45, respectively, when rennet was added at pH 6.45 and 30 ~ The complex viscosity of the local maximum is constant, i.e., does not change with rennet concentration. After the local maximum, the complex viscosity at all points, e.g., inflection points, local minimum and absolute (final) maximum in the viscosity curve (Fig. 4), decreases at rennet concentrations above the critical rennet concentration (Schulz, 2000). Noel et al. (1991) observed an initial increase of the storage modulus at the local maximum and then a slight decrease. Not only are acid-rennet gels firmer, but also the apparent viscosity of stirred products like Quark is higher than in those made by acidification alone. Syneresis of Quark produced by acid-rennet coagulation is also higher than in purely acid-fermented Quarks (Shah et al., 1990; Schkoda et al., 2001a). Heat treatment

Heat treatment of milk has very different effects on acid, rennet and acid-rennet coagulation and gels. Heat treatment of milk at > 70 ~ causes denaturation of the whey proteins, ot-lactalbumin and [3-1actoglobulin, some of which may complex with micellar K-casein by hydrophobic and disulphide intermolecular interactions (Smits and van Brouwershaven, 1980; Law et al., 1994; Lucey 1995; Singh, 1995). The yield of Quark can be increased by heat denaturation of the whey proteins (Puhan and Pltieler, 1974; RA

6.5 6.3

Enzymatic hydrolysis

.---------

6.1

A

5.9 5.7

Initial aggregation

IP1

-r{3. 5.5 5.3 5.1 4.9

Max1 /

~

4.7 4.5

I 2

Microsyneresis

Acid gel formation I I I 4 6 8 Rennet concentration (ml/1000 I)

IP3

I 10

Phases during combined acid-rennet coagulation of skim milk as a function of rennet concentration. The skim milk was heat-treated at 72 ~ for 30 s; gelation at 31 ~ was initiated by a mesophilic starter culture and rennet (Fromase 220TL, DSM Food Specialties B.V., Dortmund) at pH 6.45. The pH was measured at various points obtained from the complex viscosity/time curve (as in Fig. 4). RA, rennet addition; A, aggregation (initial increase of complex viscosity); IP1 and IP3, inflection points 1 and 3; Max1, local maximum (redrawn from Schulz, 2000).

312

Quark, Cream Cheese and Related Varieties

Sheth et al., 1988; Shah et al., 1990; Kelly and O'Donnell, 1998; Mara and Kelly, 1998) as exploited in the Thermoprocess. However, in the Thermoprocess, a higher level of rennet and a secondary heating prior to whey separation are necessary to enhance whey expulsion to give the desired DM content. The resulting Quark is softer and creamier than the traditional separator-type Quark (Dolle, 1977, 1981; Ott, 1977). In particular, when rennet is used in Quark production, the heat treatment must be lower than in yoghurt production. The degree of hydration and apparent viscosity of stirred acid-rennet gels (7%, w/w, protein) increase markedly up to a degree of whey protein denaturation of 30% when heated to 80 ~ with only a slight further increase at higher levels of whey protein denaturation (Schkoda, 1998). The optimum degree of whey protein denaturation for fresh cheese is 75-80%. If it is too low, the consistency of the product will be too soft, and not creamy; however, over-denaturation, i.e., at a temperature >120 ~ leads to protein aggregation which causes sandiness in the fresh cheese (B~urle et al., 1984; Schkoda and Kessler, 1997a,b). Depending on the manufacturing method used, the heat treatment is usually in the range 88-95 ~ for 3-6 min for Thermo- or UF Quarks (e.g., B/iurle et al., 1984; ROckseisen, 1987; Sachdeva et al., 1993; Rogenhofer et al., 1994). Several authors have observed spontaneous syneresis in combined gels made from unheated milk (Schulz, 2000; Lucey et al., 2001; Tranchant et al., 2001). Confocal scanning laser micrographs of acid-rennet gels made from unheated milk showed much larger pores than acid-rennet gels made from heated milk. This was

6.5

confirmed by permeability measurements (Lucey et al., 2001). Acid-rennet gels made from unheated milk are extremely prone to spontaneous whey separation, possibly due to considerable rearrangements of aggregated particles at an early stage of the gelation process (Schulz, 2000; Lucey et al., 2001). In pure acid coagulation, gelation occurs more rapidly and at a higher pH with increasing heat treatment (Heertje et al., 1985; Banon and Hardy, 1992). During combined acidification and renneting of low-heated milk (74 ~ for 30 s) or high-heated milk (86 ~ for 6 min), the pH at which aggregation begins remained constant at 6.3 but the pH for the local maximum (Maxl in Fig. 4) decreased from 5.6 to 5.0 (Fig. 9; Schulz, 2000). The initial increase in complex viscosity of acid-rennet gels is reduced by the heat treatment of milk (Lucey et al., 2000; Schulz, 2000). As this stage relates to the secondary phase of rennet coagulation (Schulz, 2000), this confirms the findings that this phase is more adversely affected by heat treatment than the enzymatic phase of rennet coagulation. Acid-rennet gels made from heated milk are firmer than those from unheated milks because the casein is crosslinked by denatured whey proteins and the local maximum/minimum are less pronounced due to reduced (micro-) syneresis (Lucey et al., 2000; Schulz, 2000). The maximum tan is smaller in gels from heated milks compared to unheated milks (---0.43 and 0.51, respectively), indicating that the proteins undergo fewer large-scale rearrangements (Lucey et al., 2000). Confocal micrographs indicate that the pores are much smaller and

RA A

Enzymatic hydrolysis

6.3 6.1

Initial aggregation

5.9 5.7 "T" T 5.5

IP1

~Maxl

Q.

5.3 5.1

Micro~ s y n e r e s i s ~

Rennet gel formation IP3

4.9 Acid gel formation

4.7 4.5

1:0

1 "1 Ratio Low-heatedhigh-heated skim milk

0:1

Phases during combined acid-rennet coagulation of skim milk as a function of the ratio of low-heat-treated skim milk (74 ~ for 30 s) and high-heat-treated skim milk (86 ~ for 6 min) in the milk blend used for gelation; gelation was initiated at 31 ~ by a mesophilic starter culture and 9 ml/1000 I rennet (Fromase 220TL, DSM Food Specialties B.V., Dortmund) at pH 6.45. The pH was measured at various points obtained from the complex viscosity/time curve (as in Fig. 4). RA, rennet addition; A, aggregation (initial increase of complex viscosity); IP1 and IP3, inflection points 1 and 3; Max1, local maximum (redrawn from Schulz, 2000).

Quark, Cream Cheese and Related Varieties

there appears to be more interconnectivity of the network in acid-rennet gels made from heated milk than those from unheated milk (Lucey et al., 2000).

313

The clotting time is also reduced at lower pH values during combined acidification and renneting (van Hooydonk et al., 1986a; Noel et al., 1991; Schulz et al., 1999; Schulz, 2000). There are discrepancies over the following stages of the coagulation process. Nod et al. (1991) investigated the effect of renneting pH in the range 5.98-6.62 up to the local minimum. At low rennet concentrations, the clotting time decreases markedly with decreasing pH whereas at high rennet concentrations the clotting time is independent of the renneting pH. The complex viscosity of acid-rennet gels at the local maximum (Maxl in Fig. 4) increases with decreasing pH for all rennet levels. Schulz et al. (1999) and Schulz (2000) found no difference in the final viscosity for renneting pH between 6.6 and 5.8. However, the initial aggregation reactions, i.e., due mainly to rennet, are affected by the renneting pH. If the rennet is added at a pH below 6.0, the typical local maximum and minimum are less pronounced as the two processes of acidification and renneting occur simultaneously. The pH values for clotting (pH 6.40-5.63), inflection point 1 (pH 5.65-5.17) and local maximum (pH 5.16-5.02) are directly related to the renneting pH (pH 6.6-5.8) whereas the pH values for the local minimum (pH 5.0), inflection point 3 (pH 4.80--4.85) and the final maximum (pH 4.45--4.50) are influenced solely by the acidification and not by the pH at renneting (Fig. 10). At pH values >5.9, the pH at which the rennet is added does not affect the magnitude of the complex viscosity ~/* of acid-rennet gels at the local maximum, local minimum and final maximum (Schulz, 2000). No information is

pH at renneting In Quark manufacture, rennet is rarely added simultaneously with the culture, but after 60-90 min when the pH is around 6.3. The correct moment of rennet addition and the effect on structural properties is based mainly on empirical experience. The pH value at which rennet is added (Table 4) varies from the natural pH to 6.00, and is mainly around 6.30-6.45. During rennet coagulation alone, the clotting time is markedly reduced at lower pH values as the pH is very important for the enzymatic activity of the rennet, with an optimum at pH 6.0 (Mehaia and Cheryan, 1983; van Hooydonk et al., 1986a; Zoon et al., 1989; Fox and Mulvihill, 1990; Dalgleish, 1992). With decreasing pH, the aggregation of micelles starts at a lower conversion of K-casein to para-K-casein (70% at pH 6.7 compared to 30% at pH 5.6) and the rate of aggregation and gel formation increases (van Hooydonk et al., 1986a). This is due mainly to the higher calcium ion activity at low pH values; the rate of aggregation is doubled by reducing the pH from 6.8 to 6.3 (Dalgleish, 1992). A lower pH possibly also leads to a faster rearrangement of strands and fusion of micelles, resulting in a faster increase in the storage modulus (G') directly after the onset of gelation and the earlier attainment of a plateau value of the storage modulus (Zoon et al., 1989).

RA 6.5

A

6.0 Initial aggregation -r

IP1

5.5

O.

~ 5,0

~

Rennet gel formation ~

Max1 Microsyneresis

-

IP3

Acid gel formation 4.5

5.8

, 6.0

I

I

I

6.2

6.4

6.6

pH at rennet addition Phases during combined acid-rennet coagulation of skim milk as a function of pH at rennet addition. The skim milk was heat-treated at 72 ~ for 30 s; gelation was initiated by a mesophilic starter culture and 9 ml/1000 I rennet (Fromase 220TL, DSM Food Specialties B.V., Dortmund) at 31 ~ The pH was measured at various points obtained from the complex viscosity/time curve (as in Fig. 4). RA, rennet addition; A, aggregation (initial increase of complex viscosity); IP1 and IP3, inflection points 1 and 3; Max1, local maximum (redrawn from Schulz, 2000).

314

Quark, Cream Cheese and Related Varieties

available on how the renneting pH affects the rheological and syneretic properties of the final product. Rate of gelation Culture addition and acidification profile are normally such that the milk has reached pH 6.3 after 1.5 h (pH for rennet addition) and pH 4.5-4.6 after about 16 h (German-type Quark). American-style Cream cheese or Quark is fermented in a shorter period of time, i.e., 5-6 h (Kosikowski and Mistry, 1997) or 8-9 h (Sohal et al., 1988). High rates of acid gelation lead to coarser networks with a greater tendency to syneresis. The rate of gelation increases with increasing rate of acidification, increasing temperature and increasing casein concentration (Heertje et al., 1985; Hammelehle, 1994). Incubation temperature For Quark-type products, either the cold method (22-24 ~ or warm method (28-31 ~ can be used. The amount of starter added is normally adjusted so that Quark can be separated the following morning, i.e., 16 h coagulation with an optional rennet addition after 60-90 min. The higher the temperature for acid gelation, the higher is the pH at which clotting and gelation begins during acidification (Heertje et al., 1985; Kim and Kinsella, 1989; Banon and Hardy, 1992). Increasing temperature also causes an increase in the maximum rate of coagulation due to an increase in the frequency of thermal collision between casein micelles (Kim and Kinsella, 1989). The coagulation rate of casein has a Q10 of 2-5 under various conditions (Walstra and Jennes, 1984). In acid gels, higher gelation temperatures result in a greater permeability coefficient, indicating the presence of larger pores and, therefore, increased susceptibility to syneresis (Lucey et al., 1997). Microscopic investigations show a coarser network at higher temperatures (Heertje et al., 1985; Rods, 1986). These effects at increased incubation temperatures may be attributed to a higher ratio of aggregation to dissaggregation forces during the early stages of acidification owing to decreased casein dissociation from the micelles, a reduction in repulsive forces due to increased hydrophobicity and a faster rate of acidification which is subject to the type of bacterial culture (Guinee et al., 1993). There is no information available on the effect of incubation temperature on the acid-rennet coagulation. Level and type of gel-forming protein Fermented milk gels and rennet curds are particle gels, networks built up of casein micelles or marginally modified micelles (Roefs, 1986; Home, 1998). The level and nature of proteins in the fresh cheese milk mainly determine the structure of the product. The manufacture of fresh cheeses involves a step to increase protein concentration (e.g., 12%, w/w, protein for Quark). Quark pro-

duced using the standard separator method incorporates a maximum of 15%, w/w, whey proteins; this type of Quark is generally described as firm, dry and sour. Thermoquark or UF Quark may contain all the whey proteins present in milk and is creamier, smoother, softer and often milder (Lehmann etal., 1991; Ottosen, 1996; Schkoda and Kessler, 1996; Hinrichs, 2001). The level of whey proteins in Quark is also regulated by law in Germany (> 12%, w/w, protein of which a maximum of 18.5%, w/w, is whey protein; Anon, 1986). For a gel with a given protein concentration, the final gel strength at 30 ~ and pH 4.6 increases up to a ratio of 1.5/10.5 whey protein/casein and decreases at a ratio 2.0/10 (Kelly and O'Kennedy, 2001). The proportion of pre-denatured whey protein required to give the desired synergism is substantially lower in the fresh cheese model compared to 2.5/10 in the model yoghurt system studied by O'Kennedy and Kelly (2000). The firmness of fresh cheeses increases with increasing total protein content (Korolczuk and Mahaut, 1991a; Mahaut and Korolczuk, 1992; Ozer et al., 1999). For a given protein type and degree of gel fineness, high levels of gel-forming protein result in a denser (i.e., greater number of strands of equal thickness per unit volume), more highly branched network which has a greater degree of overlapping of strands and a narrower pore size (Harwalker and Kalab, 1980; Modler and Kalab, 1983; Modler et al., 1983; Ozer et al., 1999). Increasing the protein concentration in skim milk by nanofihration from 3.5 to 7.0%, w/w, increases gel firmness, apparent viscosity, serum-holding capacity, solvation and fineness of the gel network; the rate of increase of the apparent viscosity over the protein range is slightly higher for acid-rennet gels than for acid gels (Schkoda, 1998; Schkoda et al., 2001a). Undenatured whey proteins do not participate in texture formation in acid-type fresh cheese (Korolczuk and Mahaut, 1991a,b; Mahaut and Korolczuk, 1992). For milk heated at 72 ~ for 15 s, increasing the whey protein content (from 19.6 to 25.6%, 32.9% and 41.4%, w/w, of total protein), by the addition of spraydried UF protein concentrate, reduced cheese viscosity substantially. However, as the heat treatment of the milk was increased to 92 ~ for 15 s or 92 ~ for 60 s, starting at a higher initial viscosity (i.e. at 19.6%, w/w, whey protein of total protein) increasing the whey protein content caused smaller decreases in cheese viscosity (Mahaut and Korolczuk, 1992). Factors which lead to an increase in the effective protein concentration include: (i) fortification with proteins, as often practised in the production of Fromage frais or Cream cheese by the addition of protein powders to either the milk or Cream cheese after separation;

Quark, Cream Cheese and Related Varieties

(ii) high heat treatment which causes the co-precipitation of denatured whey proteins onto the casein micelles and which therefore participate in gel formation; (iii) combining high-temperature heating and membrane technology to retain the denatured and aggregated whey proteins; (iv) homogenising of the fat-containing milk, as practised in Cream cheese production, which results in the incorporation of proteins in the fat globule membrane. Calcium chloride

Progressive solubilisation of salts bound to the casein leads to almost complete demineralisation at pH 5.00 (Heertje etal., 1985; van Hooydonk et al., 1986b; Dalgleish and Law, 1989). This suggests that the addition of CaCI2 to milk during flesh cheese production is not justified. If milk has been subjected to a high heat treatment, 500-800 ml of a liquid CaC12 solution (33%, ww) per 1000 1 milk can be added to improve its rennet coagulation properties (Spreer, 1998). The effect of CaC12 on the process of combined acidification and renneting is difficult to establish as it decreases the pH and, therefore, accelerates the rennet action (Walstra, 1993; Schkoda, 1998; Schulz, 2000). The viscosity of stirred acid-rennet gels is higher when CaCI2 is added (Schkoda, 1998). Schulz (2000) did not observe an effect of CaC12 on acid-rennet coagulation when the rennet was added at pH 6.45. Gastaldi et al. (1994) established the effect of calcium on combined acidification and renneting in the range of 10-30 ml rennet/1000 1 (for rennet specification see Table 4). No difference was found between calcium-free and calcium-enriched milk (6.25 mM) at 10 ml rennet/1000 1. At a rennet concentration >20 ml rennet/1000 1, the clotting time and pH were reduced by calcium, i.e., calcium affects the acid-rennet gelation only when >10 ml rennet/1000 1 are added and the gelation becomes more like rennet coagulation. Noel (1989) also found that the clotting time remained constant for various calcium concentrations (0-400 mg/hg). The storage modulus of the local maximum decreased with increasing calcium concentration (40-400 mg/hg), whereas storage modulus of the local minimum increased slightly (0-160 mg/~g) and then decreased (Noel, 1989).

The majority of acid- and acid/rennet-curd flesh cheeses are produced by acid (and rennet) coagulation, separation of the curd from the whey, various heating and homogenising steps. Fresh cheese preparations are blended with different ingredients (Fig. 2).

315

Q u a r k - traditional batch methods

Batch separation of curd from whey was done originally by draining and pressing the curd in filter bags. This process produces a granular textured Quark with a smooth mouthfeel and is still used for Farmhouse cheeses or Quarks with very high DM, up to 27-33%, w/w (Kroger, 1980; Dolle, 1991; Kessler, 1996). Semiautomated processes are the Berge-process (an oscillating suspended cloth method; Ramet, 1990; Kosikowski and Mistry, 1997) and the 'Schulenberg processor' (specially constructed double bottom Quark vat; Jelen and Renz-Schauen, 1989). Q u a r k - original (standard) separator process

Skim milk is pasteurised (72 ~ for 40 s), cooled to 28-30 ~ and coagulated with a mesophilic culture and a small amount of rennet within ---16 h. Rennet (--2-20 ml standard strength rennet/1000 1 of milk) is usually added approximately 90 min after culture addition at a pH around 6.3. The coagulated skim milk is then stirred for ---10-15 min and passed through a tubular strainer to remove larger particles. After separation (34-40 ~ the Quark is cooled, optionally blended with cream or other condiments and packed. The whey discharged from the separator still contains nearly all, i.e., -0.65%, w/w, whey proteins and 0.2%, w/w, NPN (Siggelkow, 1984; Ramet, 1990; Dolle, 1991; Lehmann et al., 1991; Senge, 2002a). Whey proteins in the native, undenatured state do not gel under the heating and acidification conditions used in standard separator Quark production. Various methods have been developed to increase the whey protein content of Quark and reduce losses in the whey. Early methods recovered the whey proteins from the whey and incorporated them either into the Quark or the following day's cheese milk (Centriwhey and Lactal processes, uhrafihration of whey). In the Centriwhey Process, the Quark whey is heated to 95 ~ to precipitate the whey proteins which are concentrated to 12%, w/w, DM by centrifugation and then added back to the cheese milk for the next batch of Quark (Dolle, 1977, 1981; Kroger, 1980; Jelen and RenzSchauen, 1989). In the Westfalia Lactal process, the heat-precipitated whey proteins are allowed to settle, and by partial decanting of the supernatant, a whey concentrate of 7-8%, w/w, solids is obtained. This is further concentrated in a Quark separator into whey Quark (17-18%, w/w, solids) which is added to regular Quark at a level of 20%, w/w (Dolle, 1977; Kroger, 1980; Jelen and Renz-Schauen, 1989). Uhrafiltration can also be used to concentrate whey instead of separators (Herbertz, 1982; Kn~pfer, 1982; Kreuder and Liebermann, 1983).

316

Quark, Cream Cheese and Related Varieties

Q u a r k - Thermo process (Westfalia)

The milk is pasteurised at 95-96 ~ for 2-3 min to denature and co-precipitate the whey proteins onto the caseins. The resulting finer milk coagulum after fermentation requires a further heat treatment at --~60 ~ for 3 min (so-called thermisation) in order to enhance aggregation and improve sedimentation characteristics. The stirred curd is then cooled to separation temperature (Dolle, 1977; Ott, 1977; Kroger, 1980; Siggelkow, 1984; Jelen and Renz-Schauen, 1989; Ramet, 1990; Lehmann et al., 1991). The majority of Quark in Germany is produced by this process. Q u a r k - filtration methods

Filtration technology can be used at different stages during the manufacture of Quark-type products, e.g., filtration of the acid whey, (partial) filtration of the sweet milk or filtration of (partially) acidified milk. The yield is higher than for Thermoquark as all whey proteins are incorporated. However, the structure is different from conventional Quark as UF Quark is generally softer, smoother and creamier. This can be an advantage if consumed as such; however, for cheese-cakes or desserts, the higher firmness of conventional Quark and Cream cheese is more desirable. When full filtration to final cheese solids was carried out before acidification, the sensory attributes of the resulting products were described as impaired due to bitterness contributed by the slower rate of acidification, failure to reach the desired pH and the high calcium content (Dolle, 1977; Kroger, 1980; Kreuder and Liebermann, 1983; Btturle et al., 1984; Mann, 1984; Patel et al., 1986). Labneh produced by culturing UF milk retentate was also not satisfactory (Tamime et al., 1989b). This problem has been overcome by UF of partially (pH 5.7-5.95) or fully (pH 4.8-4.6) acidified milk. Low-protein fresh cheeses, like Ymer and Lactofil (--6%, w/w, protein), are easily produced by ultrafiltering milk (Tamime and Robinson, 1988; Nakazawa et al., 1991; Kosikowski and Mistry, 1997). To produce UF Quark, acidified skim milk (pH 4.6) is heated to around 40 ~ and ultra- or micro-filtered to the desired DM content, cooled, optionally homogenised and packed (e.g., Btturle etal., 1984; Siggelkow, 1984; Dieu etal., 1990; Korolczuk and Mahaut, 1991a,b; Rogenhofer etal., 1994; Ottosen, 1996). The UF method gives complete recovery of whey proteins (native or denatured); however, NPN in the milk (-0.2%, w/w), is lost in the permeate. As native whey proteins are not retained during microfiltration, the curd is usually heat-treated (thermisation) before separating the curd form the whey (Dieu et al., 1990). Thermisation of the curd (60 ~ for 5 min)

before ultrafiltration also considerably reduces the development of stale, bitter and metallic flavours (Sachdeva et al., 1993; Rogenhofer et al., 1994). Ultrafiltration is carried out around 40-45 ~ in order to maintain good calcium solubility so as to remove calcium in the permeate (Ottosen, 1996). Quark and Labneh, ultrafiltered at higher temperatures, are described as gritty, granular and coarse (B~urle et al., 1984; Tamime et al., 1991a,b; Sachdeva et al., 1993). The viscosity of fresh cheeses produced by filtration is lower than of those manufactured by traditional technologies. In Germany, UF Quark is used only for Speisequarkzubereitungen (Quark preparations), as the possible slightly bitter flavour at the end of the shelf-life in plain Speisequark is not satisfactory. Several studies have been conducted to investigate the effect of the following during the manufacture of Quark using filtration methods: milk heat treatment, full (pH 4.6) or partial (pH 6.0) acidification of skim milk and type and configuration of membranes (Sachdeva etal., 1992a,b; Sharma etal., 1992a,b; Sharma and Reuter, 1993). Ultrafiltration using mineral membranes was found to be best for making Quark by UF from fully acidified skim milk (Sharma et al., 1992a; Sharma and Reuter, 1993). Recently, pilot-scale filtration methods have been developed by partially pre-concentrating the acidified milk in order to reduce the amount of acid whey. In the FML process (Forschungszentrum for Milch und Lebensmittel, Weihenstephan), skim milk is nanofiltered 2fold to 7%, w/w, protein and then fermented. The coagulum is stirred and concentrated by either ultrafiltration or separation. A separator needs to be adapted to the higher viscosity of the retentate coagulum in comparison to unconcentrated fermented skim milk. The texture of the final product is between that of conventional UF fresh cheese and of Thermoquark (Schkoda and Kessler, 1996, 1997a,b). Mucchetti et al. (2000) confirmed the findings of Schkoda and Kessler by nanofiltering milk 2.1-fold. In another method (Aubios process, Hannover), the skim milk is pre-concentrated 1.7-fold to 5.4%, w/w, protein (or up to 2.2-fold without causing bitterness) using microfiltration, producing a product which is similar to Thermoquark (Hulsen, 2002). A special combination of starter cultures is needed for the fermentation of retentates as more lactic acid must be formed than in unconcentrated milk. Pfalzer and Jelen (1994) enriched cheese milk with 25% sweet whey UF retentate containing 12%, w/w, DM and 4%, w/w, protein for an experimental Thermoquark-type fresh cheese produced using cheesecloth bags without significantly affecting the quality of the final product.

Quark, Cream Cheese and Related Varieties

Table 6 summarises the yield and whey protein recovery for the various methods. Q u a r k - recombination technology

Recombination technology is used to only a limited extent for the manufacture of Quark and related types. Fresh cheeses low in DM, like Frornage frais, can be produced by a method similar to yoghurt, i.e., skim milk is fortified with various milk proteins to approximately 14%, w/w, DM and then fermented. Labneh (a concentrated yoghurt with 23%, w/w, DM) can also be produced by direct recombination; fermentation at 23%, w/w, DM takes about 5-6 h in comparison with 3.5 h at 16%, w/w, DM (Ozer et al., 1999). Further treatments of the acid or acid/rennet gel

After fermentation, the gel is broken up by agitators and pumped through a sieve to the separators. Stirring the gel leads to breakage of the matrix strands, with the extent of breakage depending on the severity of the agitation. This non-Newtonian shear-thinning dispersion can be described rheologically by the Power-law model (Senge, 2002a). Increasing the temperature (25-50 ~ lowers the activation energy for aggregate interaction within the broken strands and facilitates the process of subsequent whey separation. A high pH (>4.6) at whey separation results in large losses of nitrogenous compounds in the whey (more casein fines) owing to greater physical damage to the softer gel. Any

317

factors which increase gel firmness at separation (e.g., rennet addition, higher level of gel-forming protein), will make it less susceptible to breakage for a given degree of shear and, therefore, reduce the amount of casein fines. Cooling of the gel to a temperature of Bingham (BH)

r-

1"0 4- 1 7 B H 5,

115

3.39

1.0

0.974

21.06

135

2.37

1.0

0.998

81

1.35

0.5

0.998

6.13

102

0.73

0.5

0.984

83

17.87

0.64

0.999

3.57

131

3.12

0.94

0.998

4.59

linear plastic, 2-dimensional Casson (CA)

Herschel-Bulkley

(HB)

r = ",Jroro+ ~/r/CA + 5'0 non-linear plastic, 2-dimensional r = 1"0 + K 5'n non-linear plastic, 3-dimensional

r, shear stress (Pa); r0, yield value (Pa) 5', shear rate s-l" r/, viscosity (Pa s)" K, consistency factor (Pa sion coefficient; s, standard deviation of shear stress (Pa) (Data from Senge, 2002a).

Cream cheese is produced from standardised (Double Cream Cheese (DCC), 8-12%, w/w, fat; Single Cream Cheese (SCC) 3.0-5.0%, w/w, fat), homogenised, pasteurised (72-75 ~ for 15-90s) milk or cream. Homogenisation is important for the following reasons: (i) it reduces fat loss on subsequent whey separation; (ii) it brings about, via coating of fat with casein and whey protein, the conversion of naturally emulsified fat globules into pseudo-protein particles which participate in gel formation on subsequent acidification. The incorporation of fat by this means into the gel structure gives a smoother and firmer curd (similar to yoghurt manufacture) and therefore is especially important for the quality of cold-pack Cream cheese for which the curd is not further treated (Guinee et al., 1993). Following pasteurisation, the milk is cooled (20-30 ~ inoculated at a level of 0.8-1.2%, with a D-type starter culture (Lactococcus lactis subsp, lactis, Lc. lactis subsp, cremoris and citrate-positive Lc. lactis subsp, lactis) and held at this temperature until the desired pH of 4.5-4.8 is reached. The resulting gel is agitated gently, optionally cooled to 10-12 ~ in order to prevent over-acidification, heated (80 ~ for up to 20 min) and deaerated. The curd is then concentrated by methods similar to those used for Quark, i.e., traditional method using bags, separator methods or UF methods. The acidified high-fat curd for DCC is obtained by centrifugal separ-

ation at 70-85 ~ 1996b).

12.7

5.11

sn); n, flow index; r, regres-

or UF at 50-55 ~

(Sanchez et al.,

Whey separation using separators

For separator-produced SCC, milk is standardised to 3.0-5.0%, w/w, fat. The specific weight of the cheese mass is greater than that of the whey and is separated outwards in the centrifuge during separation. The whey contains 0.2-0.5%, w/w, fat which can be reduced subsequently to ~-0.1%, w/w, by separation in a milk separator designed for this purpose (Lehmann et al., 1991). For separator-produced DCC, the milk is standardised to 8-12%, w/w, fat, giving a fat-protein mixture which has a lower specific density than that of the whey. At a fat content of 7%, w/w, the specific weights are too close to be separated by centrifugation (Dolle, 1991; Lehmann et al., 1991; Spreer, 1998). Whey separation using UF

Owing to the thick, viscous consistency of Cream cheese, concentration by UF necessitates a two-stage process (stage one: standard modules with centrifugal/positive displacement pumps; stage two: high-flow modules with positive displacement pumps) in order to maintain satisfactory flux rates and to obtain the correct DM level (Guinee etal., 1993). For fresh cheese, UF is normally carried out around 40-45 ~ for Cream cheese, 50-55 ~ can be used to improve throughput and reduce viscosity during concentration (Ottosen, 1996).

322

Quark, Cream Cheese and Related Varieties

Recombination technology

Recombination methods for experimental Cream cheesetype products include steps of combining a cheese base (e.g., dry Cottage cheese, Bakers cheese curd or fermented skim milk concentrate at pH 4.8-5.0) with emulsifying salts, bulking agents (e.g., buttermilk powder, corn syrup solids) and various gums (e.g., carrageenan or guar gum), followed by various heating, mixing and homogenisation steps (e.g., Baker, 198i; Crane, 1992). The advantage of these methods is that Cream cheese products can be formulated precisely to meet legal requirements without excess solids or butterfat. Another type of all-dairy Cream-type cheese is made by blending Ricotta cheese or Queso Blanco with a high-fat (58%, w/w) sour cream. The most critical aspect is proper dispersion of the large pieces of curd with the liquid components. The blend is standardised with cultured buttermilk, if necessary, pasteurised, homogenised and hot-packed. The final product has ---59%, w/w, moisture, 30%, w/w, fat and a pH of 5.29-5.55, depending on the cheese base used (Modler et al., 1985; Kakib and Modler, 1985a,b). Further treatments of curd after separation

In most cases, the curds are heat-treated (70-95 ~ mixed with salt (0.5-2.0%, w/w) and stabilisers (mainly hydrocolloids), homogenised (two-stage at 15-25 MPa at 65-85 ~ and either hot-packed or cold-packed after cooling to 10-20 ~ in a scraped-surface heat exchanger (Walenta etal., 1988; Sanchez etal., 1996b). Locust bean gum (0.30-0.35%, w/w), carrageenan (0.15%, w/w), xanthan gum, tara gum and sodium alginate are the most widely used stabilisers for hot-pack Cream cheese (Hunt and Maynes, 1997; Kosikowski and Mistry, 1997). Guar gum on its own gives a high processing viscosity, a soft body and undesirable texture. A synergistic effect of n-carrageenan on the gelling properties of tara gum was observed in Cream cheese (Hunt and Maynes, 1997). The extent of 'creaming' (emulsification and thickening) is influenced by the degree of heat and shear and the duration of cooking and has a major influence on the consistency of the final product. Increasing the holding time and shear during cooking generally results in a firmer product with an increasingly brittle texture (Walenta et al., 1988; Guinee et al., 1993). Static cooling of hot-pack cheeses gives a firmer texture than dynamic cooling (cold-pack cheeses; Jaupert and Vesperini, 1989; Mahaut, 1990; Sanchez et al., 1994b). The cheese firmness is already reduced by lowering the filling temperature from 85 to 75 ~ (Walenta et al., 1988). Cold-pack Cream cheese has a somewhat spongy, aerated consistency and a coarse appearance (Guinee et al., 1993).

The main particles structuring DCC, i.e., milk fat globules and milk proteins, undergo several thermal treatments, and therefore large temperature fluctuations and shear stresses during processing. Such technological treatments change the structure of particles (size, shape, state of aggregation) and physico-chemical properties (charge density and hydration of milk proteins, solid/liquid milk fat ratio and milk fat globule stability). The resulting micro- and macro-structural arrangements of particles, as well as the nature of interactions, mainly determine the texture and stability of DCC (Sanchez et al., 1996b). Jaupert and Vesperini (1989), Mahaut (1990), Sanchez et al. (1994a,b, 1996a,b,c), Sanchez and Hardy (1997) investigated the effects of processing parameters on the structure and stability of DCC. Cream cheese becomes firmer and more elastic after heating and homogenisation, and softer and more viscous after mixing and cooling. TEM and SEM show that the rheological changes during manufacture are correlated with aggregation (during heating and homogenisation) and disruption (during cooling) of milk fat globule/casein complexes. Dispersion of the fat globule clusters, formed on homogenisation, after cooling and aggregation of milk fat globules during storage causes structural instability to occur in Cream cheese. The following stages for structuring and destructuring processes occurred during the manufacture of experimental DCC (Sanchez et al., 1996c; Sanchez and Hardy, 1997): 9 Starting curd after centrifugal separation: Casein-fat

globule aggregates are first produced during curd formation. 9 Blending with water, salt and heat-denatured whey proteins: The casein-fat globule aggregates are destroyed

during blending of curd with ingredients. Fat globule destabilisation (e.g., coalescence) occurs during this stage. 9 Heat treatment: The broken aggregates reaggregate on heat treatment, but with extensive fat globule aggregation and coalescence. Leakage of oil and creaming can occur at the outlet of the heat exchanger. 9 Homogenisation: Emulsification of the system occurs during high-pressure homogenisation with strong clustering of fat globules, caused by a 'polymerbridging' mechanism. When homogenisation pressure is increased from 0 to 50 MPa, the firmness of DCC increases due to a stronger structural organisation within the cheese (Sanchez et al., 1994a). 9 Cooling: Homogenisation clusters are destroyed by high shear stresses developed in the heat exchanger during cooling to 20 ~ This effect is even more pronounced w h e n the final curd temperature is

Quark, Cream Cheese and Related Varieties

below 20 ~ (Sanchez et al., 1994b). The cooling rate and final temperature are the main factors for instability. An increase in curd cooling rate leads to a softer cheese with weaker structural organisation (reduced storage and loss moduli). Rheological and syneretic aspects of Cream cheese

TEM and cryo-SEM studies showed that DCC is structured mainly by compact casein/milk fat globule aggregates occluding large whey-containing areas, as well as partly coalesced milk fat globules. No rigid protein matrix was observed because of stirring and homogenisation of the curd during manufacture (Kahib et al., 1981; Kal~ib and Modler, 1985a,b; Sanchez et al., 1996b). The corpuscular structure seems to be responsible for good spreadability with a high moisture content facilitating the mobility of the corpuscular constituents during spreading (Kahib and Modler, 1985a,b). Double Cream cheese exhibits viscoelastic-plastic rheological behaviour with an unusual flow curve; the shear stress in the ascending shear rate curve shows, depending on the manufacturing stages, two or three peaks. The first peak is commonly referred to as the static yield value (Sanchez et al., 1994b, 1996c). The complex rheological behaviour of DCC indicates a 3-dimensional gel-like structure (Sanchez et al., 1994a). Double Cream cheese shows timedependent flow behaviour (partially thixotropic) and dynamic viscoelastic properties similar to those of non-chemically cross-linked polymers or pharmaceutical creams (Sanchez et al., 1994a,b, 1996c). Senge (2002b) also measured various cream cheese types in the oscillation and rotation mode.

Fresh cheese preparations (Frischk~isezubereitungen) are blends of Quark, Cream cheese or Cottage cheese with cream and up to 30%, w/w, fruit or vegetable preparations or up to 15%, w/w, of fruits, spices, herbs or other seasoning (KOseverordnung, Cheese order; Anon, 1986). A foamy consistency can be obtained by admixing nitrogen. Fresh cheese preparations can be heat-treated to increase shelf-life and may contain stabilisers. The components can be blended by either continuous in-line mixing or batch mixing (Lehmann et al., 1991; Kosikowski and Mistry, 1997; Spreer, 1998).

Acid-curd cheeses (Sauermilchkase), typical examples of which are Harzer, Mainzer or Olmatzer Quargel cheese, have a very strong flavour and odour, a white

323

to slightly yellow colour and a slightly brittle texture (Spreer, 1998). There are mould-ripened cheeses and yellow cheeses which have been treated with a 'smear' of red culture (Brevibacterium linens). Ripened acidcurd cheeses are generally produced in specialised plants which buy the acid Quark from dairies. The acid Quark used for these cheeses is produced by acid coagulation (cold: 1-2% mesophilic starter at 22-27 ~ in 15-20 h or warm: 2-5%, thermophilic culture at 40-45 ~ in 1.5-3.0 h). The coagulum is cut and cooked at 35-45 ~ while stirring until the granules are 2-4 cm in size. Whey is drained using filter bags or decanters. Acid Quark has a DM content >32%, w/w (pH --~4.6) and is granulated in a Quark mill and chilled below 10 ~ (Kessler, 1996; Bruckert, 1998; Spreer, 1998). In the manufacture of the cheese, 2-3%, w/w, of salt and caraway seeds are added, as well as NaHCO3 and CaCO3 (0.5-1.0%, w/w) to influence ripening (acceleration, neutralisation). The mass is mixed, milled, moulded into bars and spread on racks and then subjected to the following processes: drying at 18 ~ for 15 h; sweating at 20-25 ~ and 90% relative humidity for 2-3 days, ripening at 12-16 ~ and 90% relative humidity for 3 days and further ripening at 10-15 ~ After sweating, the cheeses are washed with salt water plus a culture of B. linens or sprayed with a culture consisting of Penicillium candidum and P. camemberti. The cheese ripens from the outside to the centre and is ready for distribution when up to 25% of the cheese mass is ripened, i.e., when the outside has a translucent, yellow appearance, even though the interior (75% of the cheese mass) is still white, dry, hard and crumbly and unripened. Cooked cheese (Kochk~ise, Topfk~ise), also manufactured from Quark, is a different type of cheese, usually with 20%, w/w, FDM. During ripening at 18-22 ~ for 3-6 days, the surface turns greasy and the pH increases to 5.5-6.0. Water, butterfat, salt, NaHCO3 and CaCO3 and caraway seeds are added to the cheese blend which is heated in a jacketed vessel at 70-110 ~ for 10-20 min. The hot cheese is filled into cups or pots (Kessler, 1996; Spreer, 1998).

Layered cheese (Schichtkiise)

German Layered cheese has a firm coagulated (gel-like) consistency with a middle layer which is higher in fat and therefore darker in colour. One-third of the cheese milk can be standardised to a higher fat level and/or colouring may be added. Standardisation of fat content must be carried out in the cheese milk, as it cannot be done in the drained curd without destroying the firm

324

Quark, Cream Cheese and Related Varieties

coagulated structure. Layered cheese is classified as a quarter-fat cheese (10%, w/w, FDM, Table 2), even though the middle layer has a higher fat content. The fermentation temperature is about 25 ~ the amount of rennet added is slightly higher than for Quark in order to shorten the gelation time by 3-4 h. The strong gel is cut into cubes (approximately 2 • 2 cm) and the curd then transferred into moulds starting with a lowfat white layer, followed by the yellow high-fat layer and finished with a low-fat white layer (Spreer, 1998).

Mascarpone Mascarpone, a high-fat ('--50%, w/w, fat) firm, spreadable fresh cheese with a mild buttery, slightly tangy flavour, is produced by direct acidification with an organic acid, instead of lactic acid bacteria, to a pH of about 5.0-5.8. Cream with 30%, w/w, fat is heated to 80-95 ~ organic acid added slowly and stirred for 10 min. Whey drainage takes about 16-24 h. To increase shelf-life, Mascarpone is usually heat-treated. No stabilisers are necessary due to the high fat content (Kessler, 1996).

The authors wish to thank Tim Guinee (Dairy Products Research Centre, Fermoy, Ireland) and Liam Gallagher (Dairygold Cooperative Society, Mitchelstown, Ireland) for useful comments on the manuscript.

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Quark, Cream Cheese and Related Varieties

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Sebastiani, H., Gelsomino, R. and Walser, H. (1998). Cultures for the improvement of texture in quarg. Proc. Syrnp. Texture of Fermented Milk Products and Dairy Desserts, Vicenza, May 1997. International Dairy Federation, Brussels. pp. 78-92. Senge, B. (2002a). Rheologische Besonderheiten von Frischkase - Konsequenzen zur Optimierung der Prozesstechnik. Dtsch. Molkereizeitung 14, 26-33. Senge, B. (2002b). Materialwissenschaftliche Untersuchung von past6sen Milchprodukten. Unpublished results. Technical University Berlin. Senge, B., Krenkel, K. and Ringer, C. (1998). Rheologische Charakterisierung von Speisequark. Dtsch. Milchwirtschaft 49, 65-67. Shah, N., Jelen, P. and Ujvarosy, S. (1990). Rennet effects and partitioning of bacterial cultures during quarg cheese manufacture. J. Food Sci. 55,398-400. Sharma, D.K. and Reuter, H. (1993). Quarg-making ultrafiltration using polymeric and mineral membrane modules: a comparative performance study. Lait 73,303-310. Sharma, D.K., Reuter, H., Prokopek, D. and Klobes, H. (1992a). Ultrafiltration of heated, acidic and coagulated skim milk with different modules. Part 3. Mineral membrane modules. Kieler Milchwirtsch. Forschungsberichte 44, 35-46. Sharma, D.K., Reuter, H., Prokopek, D. and Klobes, H. (1992b). Ultrafiltration of heated, acidic and coagulated skim milk with different modules. Part 4. Hollow fibres modules. Kieler Milchwirtsch. Forschungsberichte 44, 47-54. Sheth, H., Jelen, P., Ozimek, L. and Sauer, W. (1988). Yield, sensory properties and nutritive value of Quarg produced from lactose-hydrolysed and high heated milk. J. Dairy Sci. 71,2891-2897. Siggelkow, M.A. (1984). Modern methods in quarg production for consumer sale. Dairy Ind. Int. 49 (6), 17-21. Singh, H. (1995). Heat-induced changes in casein, including interactions with whey proteins, in, Heat induced Changes of Milk, Fox, P.E, ed., Special Issue 9501, International Dairy Federation, Brussels. pp. 86-104. Smits, P. and van Brouwershaven, J.H. (1980). Heat-induced association of [~-lactoglobulin and casein micelles. J. Dairy Res. 47, 313-325. Sohal, T.S., Roehl, D. and Jelen, p. (1988). A survey for quarg acceptance by Canadian consumers. Can. Inst. Food Sci. Technol. J. 21, 312-315. Sorensen, H.H. (1995). The world market for cheese. Bulletin 307. International Dairy Federation, Brussels. Sorensen, H.H. (2001). The world market for cheese. Bulletin 359. International Dairy Federation, Brussels. Spreer, E. (1998). Cheese manufacture, in, Milk and Dairy Product Technology, Marcel Dekker, Inc., New York, Basel. pp. 245-319. Tamime, A.Y. and Robinson, R.K. (1988). Fermented milks and their future trends. Part II. Technological aspects. J. Dairy Res. 55,281-307. Tamime, A.Y., Davies, G., Chehade, A.S. and Mahdi, H.A. (1989a). The production of 'Labneh' by ultrafiltration: a new technology. J. Soc. Dairy Technol. 42, 35-39.

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Tamime, A.Y., Kalab, M. and Davies, G. (1989b). Rheology and microstructure of strained yoghurt (Labneh) made from cow's milk by three different methods. Food Microstruct. 8, 125-135. Tamime, A.Y., Davies, G., Chehade, A.S. and Mahdi, H.A. (1991a). The effect of processing temperatures on the quality of Labneh made by ultrafiltration. J. Soc. Dairy Technol. 44, 99-103. Tamime, A.Y., Kal~ib, M. and Davies, G. (1991b). The effect of processing temperatures on the microstructure and firmness of labneh made from cow's milk by the traditional method or by ultrafiltration. Food Struct. 10, 345-352. Tamime, A.Y., Kalab, M. and Davies, G. (199 lc). Microstructure and firmness of Labneh (high solids yoghurt) made from cow's, goat's and sheep's milks by a traditional method or by ultrafiltration. Food Struct. 10, 37-44. Tranchant, C.C. (2000). Coagulation Behaviour of Differently Acidified and Renneted Milk and the Effects of Pre-treatment of Milk. PhD Thesis, University of Guelph, Guelph, Ontario. Tranchant, C.C., Dalgleish, D.G. and Hill, A.R. (2001). Different coagulation behaviour of bacteriologically acidified and renneted milk: the importance of fine-tuning acid production and rennet action. Int. Dairy J. 11, 483-494. Tscheuschner, H.-D. and Nimbs, H. (1993). Milch und Milchprodukte, in, Rheologie der Lebensmittel, Weipert, D., ed., Behr's Verlag, Hamburg. pp. 503-542.

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Acid- and Acid/Rennet-curd Cheeses Part B: Cottage Cheese N.Y. Farkye, Dairy Products Technology Center, California Polytechnic State University, San Luis Obispo, CA 93407

Cottage cheese is a soft, unripened, mild acid cheese with discrete curd particles of relatively uniform size. Creamed Cottage cheese is dry-curd Cottage cheese covered with a cream dressing. The specific origin of Cottage cheese is unknown. However, as the name implies, it was produced originally in homes (cottages) but industrial Cottage cheese production began in the USA in --1916 (Reidy and Hedrick, 1970). Cottage cheese is classified into different groups, subgroups, types, classes and styles (Table 1).

By definition, Cottage cheese and dry-curd Cottage cheese shall comply with the US Food and Drug Administration's Standards of Identity 21 CFR Part 122.128 for Cottage cheese or 21 CFR Part 133.129 for dry-curd Cottage cheese (see Table 2). Reducedfat, light and fat-free Cottage cheese or dry-curd Cottage cheese shall comply with 21 CFR Part 101.62 for nutrient claims for fat. Codex Alimentarius official standard (Codex Stan C-16) for Cottage cheese and creamed Cottage cheese (Codex Alimentarius, 1968) lists the raw material for manufacture as pasteurized bovine skim milk, and the following authorized ingredients: harmless lactic acid and aroma-producing bacteria, rennet or other suitable coagulating agent, CaC12 (maximum of 200mg/kg milk), NaC1 and water. Dairy ingredients allowed in cream dressing (creaming mixture) are: cream, skim milk, condensed milk, non-fat dry milk and dry milk protein. Other permitted ingredients in the cream dressing are: harmless lactic acid- or aroma-producing bacteria, chymosin or other suitable milk-clotting enzyme, NaC1, lactic acid, citric acid, phosphoric acid, hydrochloric acid, glucono-8-1actone (maximum level, 10 g/kg), sodium caseinate, ammonium caseinate, calcium caseinate, potassium caseinate. In addition, the following stabilizing agents are permitted: carob bean gum, guar gum, calcium sulphate, carrageenan or its salts, furcelleran or its salts, gelatine, lecithin, alginic acid or its salt, propylene glycol ester of alginic acid, sodium

carboxymethyl cellulose. Permitted carriers for stabilizers are sugar, dextrose, corn syrup solids, dextrine, glycerine and 1,2-propylene glycol. The limitations for ingredient use are as follows: (1) the weight of solids (including caseinates) added singly or in combination should not exceed 3% (w/w) of the cream dressing mixture and (2) the stabilizing solids, including carrier shall not exceed 0.5% (w/w) of creaming mixture.

Cottage cheese is produced by acid coagulation of pasteurized skimmilk or reconstituted extra low-heat skimmilk powder (RSM). The minimum heat treatment given to skimmilk or RSM for Cottage cheese manufacture is the minimum allowable pasteurization temperature • time of 62.8 ~ • 30 min or 71.7 ~ • 15 s. In a survey of seven Cottage cheese plants in California, Rosenberg et al. (1994) found that the average milk pasteurization temperature used for manufacture is 74-75 ~ Excessive heat treatment of milk (i.e., higher pasteurization temperature and/or longer time) results in a soft coagulum from which it is difficult to expel whey. The skimmilk or RSM used for Cottage cheese manufacture must be of good microbial quality and have a high dry matter (DM) content to ensure good quality and yield of cheese. The differences in DM content of milk from different breeds influence the yield and quality of Cottage cheese. Cottage cheese curds made from Friesian skimmilk (8.7% DM) are more fragile than curds made from Jersey skimmilk (9.8% DM) (Mutzelburg et al., 1982). According to Mutzelburg etal. (1982), when the DM content of Friesian skimmilk was increased to at least 9%, by the addition of Na citrate (0.1%, w/w) or Na caseinate (0.25-0.55%, w/w), curd formation improved during Cottage cheese manufacture. Reconstituted extra low-heat skimmilk powder can be used for Cottage cheese manufacture immediately after reconstitution without holding (Flanagan et al., 1978; White and Ryan, 1983). Increasing DM (8-20%) in RSM increases the moisture-adjusted (80%) Cottage cheese yield by 17.1-31.2%. However, using RSM containing >10.5% DM is not economical because the

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330

Cottage C h e e s e

Groups, types, classes and styles of Cottage cheese

Groups A B Sub-groups 1 2 Types I II III Classes 1 2 Style A B

Culture acidified Chemically acidified Normal shelf-life (14 days and over) Extended shelf-life (21 days and over) Dry-curd Cottage cheese Low-fat Cottage cheese Cottage cheese Unflavoured Flavoured (with nuts, fruit condiments) Small curd (0.635 cm) Large curd (0.953-1.27 cm)

additional yield advantage is offset by the extra cost of ingredients (White and Ryan, 1983), suggesting that 10.5% DM in skimmilk or RSM is optimal for Cottage cheesemaking. Also, cheese manufacturing time is increased when skimmilk containing >10.5% DM content is used because of its high buffering capacity. Emmons and Beckett (1984a) reported that it takes longer than 75 min (normal cooking time) to reduce the pH of skimmilk with a high DM content from 6.6 to 4.8 or lower when conventional bulk starter is used at a level of 5% (w/w). There are conflicting reports on the use of lactosehydrolysed skimmilk for Cottage cheese manufacture. Gyuricsek and Thompson (1976) reported that when >90% of the lactose in skimmilk is hydrolysed before Cottage cheesemaking, the manufacturing time is reduced by 135min because the starter bacteria ferment glucose better than lactose. The shortened manufacturing time results in reduced curd shattering and consequently increase in yields. However, Fedrick and Houliman (1981) found that the use of lactosehydrolysed skimmilk did not affect setting time, yield or quality of Cottage cheese.

The mode of setting (incubation) or acidifying milk for Cottage cheese manufacture depends on whether cultured or direct-acid Cottage cheese is being made. For cultured Cottage cheese, acidification is done by harmTable 2

Standards of Identity for Cottage cheese variants

Cheese type

Fat (%)

Moisture(%)

pH

Dry-curd Cottage cheese Cottage cheese

300 nm in diameter) is surrounded by an outer lining, 30-50 nm thick with a void space 50-80 nm wide that separates the lining and the core. Harwalkar and Kalab (1988) suggest that a pH of 5.2-5.5 is critical for the development of the core-and-lining structure because in this pH range, casein micelles have optimal voluminosity or hydrodynamic volume, a high percentage of non-sedimantable casein and little or no colloidal calcium phosphate. They suggest that the heat-induced interaction between [3-1actoglobulin and K-casein, enhanced by the presence of calcium ions, results in the development of filamentous appendages. Caseins, particularly [3-casein, which dissociate from the micelles during heat treatment, precipitate on the filamentous appendages to form a lining and leave an annular space between the casein core and the lining formed. Dissociation of caseins occurs on heating milk to high temperatures (Fox et al., 1967). The intensity of the core-and-lining structure in Queso Blanco increases with heat treatment of the cheese milk. The average diameter of casein particles in Queso Blanco made from milk coagulated at 62.8~ is 0.1 bLm compared to 0.5-5 b~m when coagulation is at 96-98 ~ (Kalab and Modler, 1985). Kalab et al. (1988) also observed coreand-lining structures in Paneer made from cow and buffalo milks.

Acid-heat Coagulated Cheeses

Queso Blanco made without rennet has a unique functionality- it has good slicing properties (Siapantas and Kosikowski, 1967) and resists melting when fried (Chandan et al., 1979). However, Queso Blanco made with rennet has excellent melting properties (Siapantas and Kosikowski, 1973). The small indistinguishable casein particles in Queso Blanco permits its use as an ingredient in the manufacture of cheese spreads free of grittiness (Modler et al., 1985, 1989). The texture, and hence the sliceability, of Queso Blanco is influenced by the moisture content of the cheese (Chandan etal., 1979) and the age of the cheese (Torres and Chandan, 1981b). Parnell-Clunies et al. (1985b,c) reported that the hardness of Queso Blanco increased linearly over time (17 days at 5 ~ but decreased with increasing moisture in the range 50-54%. Farkye et al. (1995) studied the textural properties of Queso Blanco made with acetic, citric or lactic acid and reported that texture profile analysis (TPA) hardness, fracturability, chewiness and gumminess were highest for cheese made with acetic acid and lowest for that made with lactic acid. They also found that TPA springiness and cohesiveness of Queso Blanco were independent of acid type, and that all the textural parameters except cohesiveness increased with age of cheese up to 7 weeks at 5 ~ Traditionally, Queso Blanco is consumed fresh because the nature of the processing conditions allows for very little biochemical changes during storage. However, Torres and Chandan (1981b) reported that lactobacilli or exogenous lipases can be added to the dry curd before salting and pressing to improve the flavour of the cheese during ripening (12 weeks at 10 ~ The rate of increase in non-protein nitrogen during the 12 weeks ripening period was slight (0.23%) but greater in cheese containing added lactobacilli than in cheese without (0.17%). Treatment with lipase increases the concentration of free fatty acids in cheese >300-fold. Major volatile compounds contributing to the flavour and aroma of Queso Blanco include acetaldehyde, acetone, ethyl, isopropyl and butyl alcohols and formic, acetic, propionic and butyric acids (Siapantas, 1967). Unlike most cheese varieties, the pH of Queso Blanco decreases from approximately 5.2 to 4.9 during ripening. The fermentation of residual lactose by heat-stable indigenous bacteria in milk that survive cheesemaking or by post-manufacture contaminating bacteria (Torres and Chandan, 1981b), or perhaps the dissociation of residual coagulating acid may account for the decrease in the pH of Queso Blanco during storage.

345

Information on the microbiological quality of Queso Blanco made in the US or Canada by methods described above is limited, even though poor keeping qualities of such cheeses made by different methods have been reported (Arispe and Westhoff, 1984a,b). In commercial Venezuelan Queso Blanco made without exogenous acids or starter bacteria, micro-organisms enumerated include Salmonella, Escherichia coli, Staphylococcus aureus, Bacillus cereus, Clostridium perfringes, Lactobacillus plantarum, Lb. casei, yeasts and moulds (Arispe and Westhoff, 1984b). Those cheeses were made under poor sanitary conditions and had a pH > 5.3. The high heat-acid treatment of milk, together with the low pH of the cheese and the presence of undissociated coagulating acid prevent the growth of spoilage organisms during refrigerated storage of Queso Blanco made in North America. Glass et al. (1995) reported differences in the efficacy of different organic acids and a bacteriocin-type product in the control of L. monocytogenes in Queso Blanco-type cheese. Siapantas (1967) reported that storage of Queso Blanco at a high temperature (>26 ~ results in butyric acid fermentation due to the growth of spore-forming bacteria in the milk used for manufacture.

Ricotta is an unripened soft cheese that originated from Italy. In Latin American and the Hispanic communities in North America, Ricotta is known as Requeson. The USDA specifies three types of Ricotta cheese: 1. Whole milk R i c o t t a - manufactured from whole milk, and the finished product shall contain not more than 80.0% moisture and not less than 11.0% milk fat. 2. Part-skim Ricotta - manufactured from milk with a reduced fat content, and the finished product shall contain not more 80.0% moisture and less than 11.0% but not less than 6.0% milk fat. 3. Ricotta (Ricottone) from whey or s k i m m i l k manufactured from skimmilk, whey or a blend of these products and the finished product shall contain not more than 82.5% moisture and less than 1.0% milk fat. Whole milk or part-skim Ricotta is a soft creamy cheese and has a pleasant and slightly sweet or caramel flavour whereas Ricottone has a slightly sweet, bland flavour. Typically, Ricotta is made from whey containing

346

Acid-heat Coagulated Cheeses

5-20% whole milk, skimmilk or non-fat dry milk (NFDM; Shahani, 1979). However, to produce Ricotta with desirable curd handling characteristics, it is necessary to add at least 5 parts of whole milk to 95 parts of whey, or 1 part NFDM to 99 parts of whey (Shahani, 1979). Traditionally, the starting material used for the manufacture of Ricotta cheese is whey resulting from Mozzarella cheese production. At present, Ricotta can be made from almost any type of sweet whey, provided the initial titratable acidity of the whey is -6.0. The best initial titratable acidity of whey for Ricotta cheese manufacture is 0.13-0.14% lactic acid (True, 1973). The use of whey concentrates containing up to 36% DM as starting material for Ricotta cheese manufacture has been reported (Nilson and Streiff, 1978). In the traditional method, whey or whey and milk blends are heated to 40-45 ~ and NaC1 is added. The mixture is heated continuously in large open kettles to 80-85 ~ A slow heating rate produces a better coagulum than rapid heating (True, 1973). Then, a suitable food-grade acid is added to reduce the pH to 6.0, thereby inducing coagulation. The coagulated curds float to the surface and are scooped off and placed in perforated hoops to drain and cool. In industrial methods, the whey is first neutralized to pH >6.5 (6.9-7.1) with a 25% (w/v) solution of NaOH. pH manipulation minimizes protein aggregation and produces a more cohesive coagulum (Modler and Emmons, 1989b). The neutralized whey is heated to 65-70 ~ then, whole milk or skimmilk equal to 5-25% of the whey volume is added and heating of the whey/milk mixture is continued to 75-80 ~ Cream may be added at this stage. Next, NaC1 (0.5%, w/v) is added and heating continued to 85-95 ~ Alternately, CaC12 may be added. NaC1 dehydrates the whey proteins and has a destabilizing effect on bovine serum albumin. Similarly, calcium destabilizes the whey proteins. Then, dilute food-grade acetic or citric acid is added for coagulation and curd formation. Typically, --1.5% (v/v) of dilute (---3.85%) acetic acid is needed to clot the whey/milk mixture. The curds are left in the hot whey for about an hour to increase in firmness and enhance whey drainage. The curds, which float on the surface of the whey, are ladled off. Alternately, the whey may be drained from the bottom, leaving the curds in the vat or kettle. Optimal coagulation occurs at pH 5.6-5.8 to give maximum yield (Weatherup, 1986). Approximately 5 kg of fresh Ricotta is obtained from 100 kg whey to which 5 kg of whole milk has been added. True (1973) obtained 30-39 g Ricotta cheese from 750 ml of whey; the highest yield was from whey heated to - 8 8 ~

Table 1 cheese

Proximate composition of different types of Ricotta

Ricotta cheese varieties

Component Moisture (g/100 g) Fat (g/100 g) Protein (g/100 g) Carbohydrate (g/100 g) Ash (g/100 g) Energy (kcal/100 g)

Part-skim

Whey (Ricottone)

72 13 11 3

74.5 8 11.5 5

77 2.5 16 3.5

1 174

1 138

1.0 100

Whole milk

Kosikowski (1967) describes the following procedure for the manufacture of whole milk Ricotta. Whole milk is adjusted to pH 6.0 or titratable acidity of 0.30-0.31% lactic acid, preferably with lactic starter, before heating. During heating, NaC1 (1.86 g/kg milk) is added. Also, stabilizer (0.23 g ~ g milk) is added to prevent foaming of the milk during heating. When the temperature of the milk reaches "-76 ~ a wideblade spatula is passed through the milk to observe the initiation of curd formation. Heating is continued to 80 ~ The floating curd is left undisturbed for about 10 min. Then, the curd is moved gently away from the wall of the vat or kettle towards the centre. This is continued for about 15 min and the curd is ladled from the top. The remaining whey is subjected to a second precipitation by heating to 85 ~ and adding granular citric acid (0.12 g&g milk) to give a pH of 5.4. The curd is ladled off. The curds from the primary and secondary precipitations are cooled and packaged. The use of ultrafiltration techniques to improve the yield of Ricotta cheese has been demonstrated (Maubois and Kosikowski, 1978). Also, a continuous manufacturing process for whole milk Ricotta cheese, with yields of 14.45-15.11kg/100kg milk, was reported (Modler, 1984, 1988; Modler and Emmons, 1989b). The typical composition of whole milk and partskim Ricotta, and Ricottone are given in Table 1.

Shelf-Life of Ricotta Ricotta has a relatively short shelf-life- about 3 weeks if properly packaged and stored at 4 ~ or lower (True, 1973), although Kosikowski (1967) reported a shelf-life of 70 days for whole milk. Ricotta cheese is packaged under vacuum, gas flushed and stored at "--4 ~

Acid-heat Coagulated Cheeses

Arispe, I. and Westhoff, D. (1984a). Manufacture and quality of Venezuelan white cheese. J. Food Sci. 49, 1005-1010. Arispe, I. and Westhoff, D. (1984b). Venezuelan white cheese: composition and quality. J. Food Protect. 47, 27-35. Bringe, N.A. and Kinsella, J.E. (1990). Acidic coagulation of casein micelles: mechanisms inferred from spectrophotometric studies. J. Dairy Res. 57,365-375. Chandan, R.C. (1991). Cheeses made by direct acidification, in, Feta and Related Cheeses, Robinson, R.K. and Tamine, A.Y., eds, Ellis Horwood, New York. pp. 229-252. Chandan, R.C., Marin, H., Nakrani, K.R. and Zehner, M.D. (1979). Production and consumer acceptance of Latin American white cheese. J. Dairy Sci. 62, 691-696. Farkye, N.Y., Prasad, B.B., Rossi, R. and Noyes, O.R. (1995). Sensory and textural properties of Queso Blanco-type cheese influenced by acid type. J. Dairy Sci. 78, 1649-1656. Fox, K.K., Harper, M.K., Holsinger, V.H. and Pallansch, M.J. (1967). Effect of high-heat treatment on stability of calcium casein aggregates in milk. J. Dairy Sci. 50,443-450. Gastaldi, E., Laguade, A. and Tarodo de la Fuente (1996). Micellar transition state in casein between pH 5.5 and 5.0. J. Food Sci. 61, 59-64. Glass, K.A., Bhanu Prasad, B., Schlyter, J.M., Uljas, H.E., Farkye, N.Y. and Luchansky, J.B. (1995). Effects of acid type and Aha TM 2341 on Listeria monocytogenes in Queso Blanco type of cheese. J. Food Prot. 58, 737-741. Harwalkar, V.R. and Kalab, M. (1980). Milk gel structure. XI. Electron microscopy of glucono-8-1actone-induced skim milk gels. J. Texture Stud. 11, 35-49. Harwalkar, VR. and Kalab, M. (1981). Effect of acidulants and temperature on microstructure, firmness, and susceptibility to syneresis of skimmilk gels. Scanning Electron Microsc. III, 503-513. Harwalkar, V.R. and Kalab, M. (1988). The role of [3-1actoglobulin in the development of the core-and-lining structure of casein particles in acid-heat induced milk gels. Food Microstruct. 7, 173-179. Hill, A.R., Bullock, D.H. and Irvine, D.M. (1982). Manufacturing parameters of Queso Blanco made from milk and recombined milk. Can. Inst. Food Sci. Technol. J. 15, 47-53. Hirschl, R. and Kosikowski, EV. (1975). Manufacture of Queso Blanco using whey concentrates. J. Dairy Sci. 58, 793 (abstr.). Kalab, M. and Modler, H.W. (1985). Development of microstructure in a cream cheese based on Queso Blanco cheese. Food Microstruct. 4, 89-98. Kalab, M., Gupta, S.K., Desai, H.K. and Patil, G.R. (1988). Development of microstructure in raw, fried, and fried and cooked Paneer made from buffalo, cow and mixed milks. Food Microstruct. 7, 83-91. Kosikowski, EV. (1967). The making of Ricotta cheese. Proc. 4th Annual Marschall Invitational Italian Cheese Seminar, Madison, WI. pp. 1-7. Kosikowski, EV. (1982). Cheese and Fermented Milk Foods, 2nd edn, Edward Bros, Inc., Ann Arbor, MI.

347

Maubois, J.L. and Kosikowski, EV. (1978). Making Ricotta Cheese by ultrafiltration principles. J. Dairy Sci. 61, 881-884. Mistry, C.D., Singh, S. and Sharma, R.S. (1992). Physicochemical characteristics of Paneer from cow milk by altering salt balance. Aust. J. Dairy Technol. 47, 23-27. Modler, H.W. (1984). Continuous Ricotta manufacture. Mod. Dairy 63 (4), 10-12. Modler, H.W. (1988). Development of a continuous process for the production of Ricotta cheese. J. Dairy Sci. 71, 2003-2009. Modler, H.W. and Emmons, D.B. (1989a). Production and yield of whole milk Ricotta manufacture by a continuous process. I. Materials and methods. Milchwissenschaft 44, 673-676. Modler, H.W. and Emmons, D.B. (1989b). Production and yield of whole milk Ricotta manufactured by a continuous process. II. Results and discussion. Milchwissenschaft 44, 753-757. Modler, H.W., Poste, L.M. and Butler, G. (1985). Sensory evaluation of an all-dairy fermented cream-type cheese produced by a new method. J. Dairy Sci. 68, 2835-2839. Modler, H.W., Yiu, S.H., Bollinger, U.K. and Kalab, M. (1989). Grittiness in a pasteurized cheese spread: a microscopic study. Food Microstruct. 8, 201-210. Nilson, K.M. and Streiff, P. (1978). Comparison of whey Ricotta cheese manufactured from whey and whey concentrates. Proc. 15th Marschall Invitation Cheese Seminar, Madison, WI. pp. 1-12. Parnell-Clunies, E.M., Irvine, D.M. and Bullock, D.H. (1985a). Heat treatment and homogenization of milk for Queso Blanco (Latin American white cheese) manufacture. Can. Inst. Food Sci Technol. J. 18, 133-136. Parnell-Clunies, E.M., Irvine, D.M. and Bullock, D.H. (1985b). Composition and yield studies for Queso Blanco made in pilot plants and commercial trials with dilute acidulant solutions. J. Dairy Sci. 68, 3095. Parnell-Clunies, E.M., Irvine, D.M. and Bullock, D.H. (1985c). Textural characteristics of Queso Blanco. J. Dairy Sci. 68, 789-793. Pyne, G.T. and McGann, T.C.A. (1960). The colloidal phosphate of milk. II. Influence of citrate. J. Dairy Res. 27, 9-17. Rao, K.V.S.S., Zanjpad, P.N. and Mathur, B.N. (1992). Paneer technology- a review. Indian J. Dairy Sci. 45 (6), 281-291. Sawyer, W.H. (1969). Complex between [3-1actoglobulin and K-casein: a review. J. Dairy Sci. 52, 1347-1355. Shahani, K.M. (1979). Newer techniques for making and utilization of Ricotta cheese. Proc. 1st Biennial Marschall International Cheese Conference, Madison, WI. pp. 77-87. Siapantas, L.G. (1967). Biochemical Changes in "Queso Blanco" Cheese during Storage at High Temperatures. IDM Potential for Developing Countries. PhD Thesis, Cornell University Press, Ithaca, NY. Siapantas, L.A. and Kosikowski, EV. (1965). Acetic acid preparation phenomenon of whole milk for Queso Blanco cheese. J. Dairy Sci. 48, 764 (abstr.). Siapantas, L.G. and Kosikowski, EV. (1967). Properties of Latin American white cheese influenced by glacial acetic acid. J. Dairy Sci. 50, 1589.

348

Acid-heat Coagulated Cheeses

Siapantas, L.G. and Kosikowski, EV. (1973). The chemical mode of action of four acids and milk acidity in the manufacture of Queso Blanco. J. Dairy Sci. 56, 631. Torres, N. and Chandan, R.C. (1981a). Latin American white cheese: a review. J. Dairy Sci. 64, 552-559. Torres, N. and Chandan, R.C. (1981b). Flavor and texture development in Latin American white cheese. J. Dairy Sci. 64, 2161-2169. True, L.C. (1973). Effect of various processing conditions on the yield of whey Ricotta cheese. Proc. l Oth Marschall Invitational Cheese Seminar, Madison, WI. pp. 1-11.

United States Department of Agriculture (1978). Cheese Varieties and Descriptions. Agric. Handbook 54. Washington, DC. pp. 99-100. Vishweshwaraiah, L. and Anantakrishnan, C.P. (1985). A study on technological aspects of preparing Paneer from cow's milk. AsianJ. Dairy Res. 4 (3), 171-176. Weatherup, W. (1986). The effect of processing variables on the yield and quality of Ricotta. Dairy Ind. Int. 5 (8), 41-45. Weigold, G.W. (1958). Development of a factory method for the manufacture of Queso Del Pias. Milk Prod. J. 49 (10), 16-17, 25.

Pasteurized Processed Cheese and Substitute/Imitation Cheese Products T.P. Guinee, Dairy Products Research Centre, Teagasc, Moorepark, Fermoy, Co. Cork, Ireland M. Cari~, University of Novi Sad, Faculty of Technology, Bulevar Cara Lazara 1, Serbia and Montenegro M. Kah~b, Agriculture and Agri-Food Canada, Food Research Program, Guelph, Ontario, Canada

The products in this group differ from natural cheeses in that they are not made directly from milk (or dehydrated milk), but rather from various ingredients such as skim milk, natural cheese, water, butter oil, casein, caseinates, other dairy ingredients, vegetable oils, vegetable proteins and/or minor ingredients. The two main categories, namely pasteurized processed cheese products (PCPs) and analogue cheese products (ACPs), may be subdivided further depending on the composition and the types and levels of ingredients used (Fig. 1). The individual categories will be discussed separately below.

Introduction

Pasteurized PCPs are cheese-based foods produced by comminuting, melting and emulsifying into a smooth homogeneous molten blend, one or more natural cheeses and optional ingredients using heat, mechanical shear and (usually) emulsifying salts (ES). Optional ingredients permitted depend on the product type, i.e., whether processed cheese, processed cheese food (PCF) or processed cheese spread (PCS), and include dairy ingredients, vegetables, meats, stabilisers, ES, flavours, colours, preservatives and water (Tables 1 and 2). Cheese, as an ingredient of PCPs, ranges from a minimum of 51% in pasteurized PCSs and PCFs to "-095% in pasteurized processed cheese (Code of Federal Regulations, 1986; Fox et al., 1996). Attempts to increase the shelf-life of cheese during the early twentieth century were inspired by the possibility of increased cheese trade, via the production of more stable transportable products, and by the existence of heated cheese dishes such as Swiss Fondue, Welsh Rarebit and Kochktise. Many of the early approaches

were unsuccessful; the heat-treated cheeses were unstable, undergoing oiling-off and moisture-exudation during cooling and storage. In 1911, Swiss workers, Walter Gerber and Fritz Stettler, produced a stable heat-treated Emmental cheese, known as Schachtelk~ise, by the addition of a 'melting salt', sodium citrate, to the comminuted cheese before processing (i.e., heating and shearing; Meyer, 1973). Subsequently, it was found that other cheeses (e.g., Cheddar) could be also processed to form stable products by the addition of other 'melting salts' (e.g., sodium phosphates) or blends of different ES. The 'melting salts' were gradually referred to as ES when their function became known, i.e., mediation of the processes of protein hydration and emulsification of free fat during processing. Initial successes were followed by numerous patents for different melting salt blends and later for the inclusion of food ingredients other than cheese. Processed cheese products are used in many applications, in both the raw and the heated forms. The suitability for particular applications depends primarily on the textural and the flavour characteristics of the unheated cheese and the cooking properties of the heated cheese. In the unheated form, it may be used as a table product with a spectrum of consistencies ranging from firm, elastic and sliceable to creamy, smooth and spreadable. The variations in consistency make it suitable for a range of uses, e.g., substitute for natural sliceable or shredded cheese (e.g., on bread, crackers or in sandwiches), table spread, sauces and dips. Processed cheese products are also used as an ingredient in several cookery applications, e.g., as slices in burgers, in toasted sandwiches, pasta dishes, au-gratin sauces or cordon-bleu poultry products. Processed cheese products may be also be dried, as cheese powders, which are then dry-blended with other ingredients in the preparation of formulated foods such as dry

Cheese: Chemistry, Physics and Microbiology, Third edition - Volume 2: Major Cheese Groups ISBN: 0-1226-3653-8 Set ISBN: 0-1226-3651-1

Copyright 9 2004 Elsevier Ltd All rights reserved

350

P a s t e u r i z e d P r o c e s s e d C h e e s e and S u b s t i t u t e / I m i t a t i o n C h e e s e P r o d u c t s

Pasteurized processed and analogue cheese products

Pasteurized process cheese products

Analogue cheese products

I

9 manufactured by blending, heating and shearing mixtures of ingredients, mainly of dairy origin 9 natural cheese must be >51% (w/w) of the final product

Categories - Processed cheese - Processed cheese food - Processed cheese spread - Blended cheese -Blended cheese spread

9 manufactured by blending, heating and shearing mixtures of ingredients of dairy and/or vegetable origin 9 not necessary to include natural cheese 9 natural cheese may be added at a low level (e.g., 5% ) to impart cheesy flavour or to comply with a particular customer specification

Categories - Dairy analogue - Part-dairy analogue - Non-dairy analogue

Generalized classification scheme for pasteurized processed and analogue cheese products; the analogue cheeses may be either substituted or imitated depending on the nutritional equivalence compared to natural cheese (Analogue cheese products, ACPs).

soup or sauce mixes, ready-prepared meals, snack coatings (see 'Cheese as an Ingredient', Volume 2). The production of pasteurized processed cheese in different countries is shown in Table 3. Global production of PCPs, based on available information, is estimated to be "--2.0 million tonnes/annum, which is equivalent to ---13% of natural cheese production. Production in the EU15 increased steadily at a rate of ---1% per annum during the period 1996-2000, i.e., at a rate lower than that for natural cheese (1.6%) over the same period, but has increased by 2.7% per annum for the 1999-2000 period (ZMP, 2001). Factors contributing to the continued growth of PCPs include: 9 Their versatility as foods which offer wide variety in flavour, texture (e.g., elasticity, firmness, spreadability, sliceability), cooking attributes (e.g., degrees of flowability, browning, viscosity), size and shape of the final product and overall consumer appeal made possible by differences in formulation and processing conditions, condiment addition and packaging technology (Mann, 1970, 1972, 1974, 1975, 1978a,b, 1981, 1986, 1987, 1990, 1993, 1997; Price and Bush, 197ara,b; Abou-E1-Nour, 2001; Subak and Petranin, 2001).

9 Their popularity with children of different ages owing to their safe ingestable consistency (for infants), mild flavours and their packaging (colour, caricatures, strength, ease of opening, size) and shape (e.g., triangles, fingers, cartoon characters) which is generally attractive and convenient for lunch boxes. 9 Their nutritive value (e.g., especially as a source of calcium and protein) as a food for children. 9 Their ability to meet special dietary needs if fortified with vitamins and minerals (Zhang and Mahoney, 1991; Sukhinina et al., 1997), which is technologically easy in the manufacture of PCPs. 9 Their adaptability as an ingredient with properties customized to the needs of several sectors of the food formulation and assembly industries (e.g., manufacturers of cheese powders, cheese-flavoured coated snacks, soups, cheese-meat products, prepared meals). 9 Their convenience of use in the culinary and food service sectors, especially the fast food trade, and the home because of their excellent preservation (stability), consistent tailor-made functionality (e.g., cooking properties), convenient portion size and packaging (e.g., as slices for the beef burger and sandwiches trade).

Pasteurized Processed Cheese and Substitute/Imitation Cheese Products

351

Optional ingredients permitted in pasteurized processed cheese productsa,b

Ingredient type

Main function~effect

Examples

9 Standardization of composition 9 Contributes to flavour, texture and cooking characteristics 9 Standardization of composition 9 Assist in 'creaming' (thickening of blend during manufacture) and formation of product 9 Contribute to texture and rheological (e.g., fracturability, hardness) and cooking properties 9 Low-cost filler; may affect texture

Cream, anhydrous milk fat, dehydrated cream, butter

Dairy Ingredients Milk fat

Milk proteins

Lactose Cheese base

Stabilizers

Acidifying agents Flavourings

Flavour enhancers Condiments Sweetening agents Colours Preservatives

9 Substitute for young cheese 9 Similar in behaviour to milk proteins, it contributes to thickening during manufacture, texture and cooking properties 9 Assist the formation of a physico-chemically stable product 9 Impart desired texture and cooking characteristics 9 Assist control of the pH of final product 9 Impart flavour to processed cheese foods and spreads, especially where much young cheese, cheese base, or milk proteins are used 9 Accentuate flavour 9 Affect appearance, flavour and texture, and product differentiation 9 Increase sweetness, especially in products targeted to young children 9 Impart desired colour 9 Retard mould growth; prolong shelf-life

Casein, caseinates, whey proteins, milk protein concentrates (ultrafiltered milk and microfiltered milk preparations), co-precipitates, skim milk powder Whey powder, skim milk powder, whey permeate powder Typically, high dry-matter milk solids (=60%, w/w) prepared by evaporation of milk ultrafiltrates to which starter culture and rennet have been added Emulsifying salts: sodium phosphates and sodium citrates Hydrocolloids: carrob bean gum, guar gum, xanthan gum, sodium carboxymethylcellulose, carageenan Food-grade organic acids, e.g., lactic, acetic, citric, phosphoric Enzyme-modified cheese, starter distillate, wood smoke extracts, spices NaCI, yeast extract Sterile preparations of meat, fish, vegetables, nuts and/or fruits Sucrose, dextrose, corn syrup, hydrolysed lactose Annato, paprika, artificial colours Nisin, potassium sorbate, Ca- or Na- propionate

a The ingredients permitted depend on product type, category, regulations in the region of manufacture. b The effects of different ingredient types are discussed in detail in 'Influence of various parameters on the consistency and cooking characteristics of PCPs and ACPs' and 'Properties of ES important in cheese processing'.

9 Low cost relative to natural cheese due to the incorporation of low-grade natural cheese, off-cuts and cheaper non-cheese milk solids (e.g., skim milk powder, whey, casein and caseinates). Casein and fat in cheese are generally more expensive, on a weight basis, than casein and fat in the form of ingredients such as casein powders and butter oil. 9 Relatively long shelf-life, good physico-chemical stability (e.g., compared to natural cheeses in which fat and/or moisture separation sometimes occur on prolonged storage) and absence of waste (e.g., compared to natural cheeses with rind or surface mould or smear). This makes them easy to use in the food service and food formulation assembly sectors. 9 The developments in manufacturing technology, emulsifying salt blends and functional dairy ingredients which facilitate the manufacture of consistent quality products with customized quality attributes, shape, size and appearance (e.g., processed cheese

slices with holes similar to eye cheeses; Polkowski, 2002). Classification of PCPs

There are various types of PCPs, with standards of identity (relating to composition and levels and types of permitted ingredients) that vary somewhat from country to country. Hence, in the UK there are two categories of PCPs, namely processed cheese and cheese spread (as specified by the Cheese and Cream Regulations, 1995, SI 1995/3240, HMSO, London) whereas in Germany there are four categories, viz., Schmelzk/~se (processed cheese), Schmelzk~isezubereitung (processed cheese preparation), Kasezubereitung (cheese preparation) and K/isekomposition (cheese composition), as detailed in the Deutsche K/iseverordnung of 12 November 1990. Currently, the IDF, under the auspices of the Codex Alimentarius Commission, a

352

Pasteurized Processed-Cheese and Substitute/Imitation Cheese Products

Ingredients and composition specifications of different categories of pasteurized (processed) cheese productsa, b

Compositional specifications

Product category

Permitted ingredients

Pasteurized blended cheese

Cheese; cream, anhydrous milk fat, dehydrated cream (in quantities such that the fat derived from them is less than 5%, w/w, in finished product); water; salt, food-grade colours, spices and flavourings (other than those which simulate the flavour of cheese, and wood smoke extracts); mould inhibitors (sorbic acid, potassium/sodium sorbate at levels _ 1.5%, w/w; cf., Briozzo et al., 1983; Tanaka et al., 1986; Leistner and Russell, 1991; Eckner et al., 1994; Rajkowski et al., 1994; Ter Steeg et al., 1995) without affecting the product quality otherwise. 9 Using ES with bacteriostatic properties. 9 Good manufacturing practice, minimization of manual handling of product, avoiding post-processing contamination and reducing storage temperature (Ter Steeg et al., 1995; Palmas et al., 1999).

Flavour effects. It is generally recognized that sodium citrates impart a 'clean' flavour while phosphates may impart off-flavours described as soapy (especially orthophosphates), chemical or salty (Meyer, 1973; Gupta et al., 1984). Pyrophosphates may cause bitterness if added at a level of 2%, w/w (Templeton and Sommer, 1936); potassium citrates also tend to cause bitterness (Templeton and Sommer, 1936; Meyer, 1973).

Bacterial spoilage can be effectively eliminated by the use of UHT processing (sterilization), which destroys heat-resistant spores such as Cl. butyricum, Cl. tyrobutyricum, Cl. sporogenes, in combination with hot filling at 85-95 ~ to eliminate post-pasteurization contamination (Sch~r and Bosset, 2002).

Influence of various parameters on the consistency and cooking characteristics of PCPs and ACPs

Processed cheese products are consumed directly as table products or as ingredients in certain cooking applications (Guinee, 2002a). As a table product, different PCPs offer a spectrum of consistencies ranging from firm, elastic and sliceable to creamy, smooth and spreadable. The variation in consistency makes PCPs suitable for a range of uses, e.g., substitute for natural sliceable or shredded cheese (e.g., on bread, crackers or sandwiches), table spread, sauces or dips. When consumed as table products, PCPs are subjected to various stresses and strains in the form of shearing (e.g., during spreading, mastication), cutting (e.g., during slicing and ingestion) or compression (e.g., during chewing). The rheological properties of the PCPs characterize its response (e.g., degree of spread,

Pasteurized Processed Cheese and Substitute/Imitation Cheese Products

fracture, crumbling, springiness) to the applied stresses or strains (see 'Rheology and Texture of Cheese', Volume 1) and have a major impact on the textural and sensory characteristics (see 'Sensory Character of Cheese and its Evaluation', Volume 1). Processed cheese products are also used as an ingredient in several cookery applications, e.g., as slices in burgers, toasted sandwiches, pasta dishes, au gratin sauces and cordon-bleu poultry. A key aspect of the cooking performance of cheese is its heat-induced functionality, which is a composite of different attributes, including softening (melting), stretchability, flowability, apparent viscosity and tendency to brown (see 'Cheese as an Ingredient', Volume 2). Hence, the textural properties of the unheated PCP and cooking characteristics of the heated product are major factors affecting quality. Consequently, numerous investigations have been undertaken on the effects of different factors on the rheological characteristics of PCPs. Although some discrepancies exist between studies, probably as a consequence of inter-study differences in factors other than that being investigated (e.g., formulation and processing conditions), definite trends are evident. The quality of PCPs is influenced by many factors, including: the type and level of ES, the composition and degree of maturity of the natural cheese used, the type and level of optional ingredients, the processing conditions and the interactions between the different factors. These are summarized in Table 6 and are discussed briefly below.

Processing time Processing conditions can vary markedly. As discussed in 'Principles of manufacture of PCPs', the heat and shear applied during processing contribute to hydration of the para-casein and other ingredients and to emulsification of free fat/oil. They do this by aiding: 9 the mixing and the uniform distribution of all ingredients throughout the blend; 9 the dissolution of the ES and their interaction with the para-casein (in the cheese) or casein aggregates (as in added milk-protein ingredients such as milk powders, caseinates, caseins); 9 destruction of the structure of the natural cheese being processed (by promoting aggregation and dehydration of the para-casein matrix and by destruction of the milk fat globule membrane in the natural cheese); 9 dispersion of free (non-globular) fat/oil and moisture; 9 transformation of the structure, e.g., from a paracasein gel with occluded fat globules and moisture (as in cheese), or from a protein aggregate/precipitate

371

in the case of added milk protein ingredients, to a concentrated o/w emulsion. Increasing processing time and shear (speed of mixing) is generally accompanied by an increase in the DE, as reflected by an increase in the number, and reduction in the mean diameter, of the emulsified fat globules (Rayan et al., 1980; Kimura et al., 1986; Tatsumi et al., 1989). The increases in casein hydration and DE result in a progressive thickening of PCPs with holding time at a temperature in the range 70-90 ~ (cf. Swiatek, 1964; Rayan et al., 1980; Kalab et al., 1987). The thickening, referred to as creaming or creaming effect in the industry, may be attributed to the ongoing interaction of the ES with the casein and the consequent increases in para-casein hydration and DEE. Creaming is desirable, especially in high-moisture PCS, as it imparts the desired viscous consistency to the molten blend for filling/packing (which prevents splashing) and gives a thick, creamy-bodied final product; in such products, the lack of an adequate creaming gives a thin runny consistency. However, extending the holding time (e.g., due to a delay or stoppage of packaging lines) of the molten product at 70-90 ~ can result in a defect known as over-creaming. In PCPs, over-creaming manifests itself as the development of a short, stiff, heavy, pudding-like consistency and dull appearance; this development may not become obvious until the product has cooled. In block PCPs and slices, it is reflected by the appearance of an 'orangepeel'-like surface and development of an over-firm and heavy pudding-like (coarse) structure which leaks free moisture and exudes beads of free oil (through the 'surface dimples'), especially on cooling. Over-creaming is highly undesirable in practice as it creates problems in pumping/filling (e.g., clogging of filling heads, excessive stand-up in packages) of the product and causes a deterioration in the end product quality, e.g., loss of spreadability, loss of surface sheen, non-uniform greasy appearance (in slices/blocks), loss of cooking properties. In experimental studies, increasing the processing time from 0 to 40 min at 70-82 ~ resulted in progressive increases in the elasticity and the firmness of the unheated PCPs and a decrease in the flowability of the melted PCPs, to an extent dependent on the ES type (Rayan et al., 1980; Harvey et al., 1982; Tatsumi et al., 1991). In contrast to these results, Swenson et al. (2000) found that increasing the processing time at 75 ~ from 0 to 20 min resulted in a decrease in firmness and an increase in the flowability of fat-free PCPs. These results may suggest the absence of a creaming process in the fat-free PCPs and highlight the importance of fat content and degree of fat emulsification to the creaming process.

372

Pasteurized Processed Cheese and Substitute/Imitation Cheese Products

General effects of various parameters on the textural characteristics and heat-induced flowability of pasteurized processed cheese productsa,b,c,d,e,f,g

Firmness

Elasticity

Spreadability

Heat-induced flowability

t 4,

f 4,

NA NA

f 4,

4, f

4, 1'

4, f

4, f

f f t

t t NA

4, 4, NA

4, 4, 4,

NA NA NA t NA NA

NA NA NA NA NA NA

NA NA NA NA NA t

4, 4, 4, 4, t NA

4, 4,

4, 4,

t t

NA t

t t

t t

~ ~

,~

Formulation Emulsifying salt concentration Increasing level in the range 0.0-0.5%, w/w Increasing level in the range 0.5-3.0%, w/w Cheese Increasing degree of proteolysis Increasing content of intact casein Substitution of rennet-curd cheese by: Reworked processed cheese Cheese base Acid-heat coagulated cheeses Dairy ingredients Whey proteins Total milk proteins Milk ultrafiltrates Calcium co-precipitate Calcium caseinate Skim milk powder

Composition of PCP Increasing moisture content Increasing pH

Processing conditions Increasing temperature Holding time at maximum temperature

a Modified from Guinee (2002a). b The general effects of the different parameters, as summarized from a review of the published literature, are presented. However, the precise effects of changing any parameter may depend on the particular formulation, processing conditions and the effects of their interaction. c NA, data not available, data limited, or conflicting data from which no general trends emerge. d Arrows, magnitude of factor (e.g., firmness) increases 1' or decreases ~. e Rework refers to pasteurized processed cheese product that is not packaged for sale; it is obtained from the 'left-overs' in cookers and filling machines, damaged packs and batches that have 'over-creamed' (thickened) and are too viscous to pump or fill. f Cheese base refers to milk ultrafiltrate which is diafiltered, inoculated with starter culture (and sometimes with rennet also) until the pH reaches ---5.2-5.8, pasteurized and concentrated to a dry matter content of - 6 0 % , w/w. g See text for more detail on effects ('Blend ingredients: cheese base (CB), ultrafiltered milk retentate (UFMR), cheeses from high heat-treated milks and whey proteins').

Several factors may contribute to over-creaming. Prolonged holding at a high temperature is conducive to aggregation and dehydration of para-casein. Hence, Csok (1982) reported that on holding a cooked processed cheese at 95 ~ the bound water increased to a maximum (e.g., at - 1 5 min) and decreased thereafter (Fig. 14). The initial increase may be attributed to increased solution of the ES (not fully solubilized at the end of the heating step) and calcium sequestration, while the eventual decrease may reflect aggregation of the paracaseinate on prolonged holding at the high temperature. In agreement with the above hypothesis of casein dehydration and aggregation, Bowland (1997), using image analysis of light micrographs, concluded that the level of protein incorporated into the matrix of PCP

increased with creaming time (holding time at the cooking temperature). Moreover, Tatsumi et al. (1991) reported that the level of water-insoluble N in the PCP increased with holding time at 80 ~ and that there was a significant inverse relationship between the holding time and the flowability of the cooked PCP. The increased degree of protein aggregation is consistent with the increase in firmness and elasticity that occurs with processing time (Rayanet al., 1980). Moreover, microstructural analyses of a processed cheese food (PCF) showed that the number and the area of electron-dense zones in a very firm product, cooked to 85 ~ and held for 5 h, was markedly higher than in the control, which was cooled after 3 min at 85 ~ (Kalab et al., 1987). The electron-dense zones may correspond to regions of

Pasteurized P r o c e s s e d C h e e s e and Substitute/Imitation C h e e s e P r o d u c t s

1.65 '7" c O c

.g1.35 m O u')

if) ID t-O

1.05

-O U) if) C) O

0.75 . cm (1) .4-.,

0.45 0

10 20 Holding time, min

30

Changes in the level free water (A) and total bound water (A) in pasteurized processed cheese as a function of processing time at 95 ~ (redrawn from Csok, 1982).

strand overlap and/or reflect areas with a relatively high degree of aggregation and fusion of the paracaseinate particles. The release of moisture and free oil during over-creaming of block PCPs also suggests that the process coincides with the onset of protein dehydration, emulsion destabilization and phase inversion. It is noteworthy that at a micro-structural level, clumping and coalescence of fat globules was evident in the PCF held for 5 h at 85 ~ but was absent, or markedly less, in fresh PCF held for 3 min at 82 ~ (Kal~ib et al., 1987). Another factor contributing to the over-creaming with time is the increase in the degree of fat emulsification (Rayan et al., 1980; Kalab et al., 1987). For a given protein-to-fat ratio in PCPs, increasing the DE leads to an increase in the surface area-to-volume ratio of the emulsified fat globules, which may be considered to behave as structure-building pseudo-protein particles. These particles are expected to increase the firmness of the PCP (see 'Micro-structure of PCPs and ACPs'). This hypothesis concurs with the positive correlation between the DEE and the firmness or elasticity, and the inverse relationship between the DE and the flowability of PCPs (Rayan et al., 1980; Cari~ et al., 1985; Savello et al., 1989). While increasing the DE beyond the critical emulsification point (where all the 'available' protein in the system is not sufficient to cover the available fat surface) maximizes the surface area of emulsified fat particles, it may also lead to free fat separation, especially in high-fat PCPs.

373

Processing temperature and shear According to Meyer (1973), processing at a temperature >95 ~ results in a decrease in product firmness. This coincides with observations in practice where UHT treatment, as in continuous processing, frequently gives PCPs which are more fluid than those processed at a lower temperature. The effect of temperatures >95 ~ may be attributable to thermal hydrolysis of polyphosphate ES, a consequent reduction in paracasein hydration and DE, and/or an increase in the rapidity of, and in the degree of, thermal-induced para-casein aggregation (which would reduce the extent of hydration and viscosity). However, tee et al. (1981) observed a positive relationship between the firmness of processed Emmental and the processing temperature in the range 80-140 ~ The effects of increased processing temperature are less clear when whey proteins are present in the PC blend. These undergo thermal denaturation and complex with para-K-casein at the high processing temperature (Jelen and Rattray, 1995; Singh, 1995). This denaturation may in turn lead to aggregation/pseudo-gelation on cooling the formed PCP (Doi et al., 1983a,b, 1985) to an extent which would be expected to increase with processing temperature. In this case, while a thin consistency may be observed in the kettle, the product may firm up more than usual on cooling. In contrast to Lee et al. (1981), Swenson et al. (2000) found that increasing the processing temperature from 70 to 90~ gave a significant increase in flowability and decrease in the spreadability of fatfree PCP; the firmness was highest at 70~ and lowest at 80 ~ Blend ingredients: ES Numerous studies have compared the effects of different ES blends on the texture and cooking properties of PCPs and ACPs (Templeton and Sommer, 1936; Swiatek, 1964; Thomas etal., 1980; Harvey etal., 1982; Gupta et al., 1984; Cavalier-Salou and Cheftel, 1991; Sutheerawattananonda and Bastian, 1998; Swenson et al., 2000; Abdel-Hamid et al., 2000a,b). Discrepancies between the various results may be due to inter-study differences in cheese (type, age and composition), blend pH, quantity of ES, processing conditions, moisture content and other compositional parameters and assessment methodology. However, general trends emerge showing that orthophosphates, citrates and sodium aluminium phosphates give relatively soft processed cheeses, which generally undergo a slight oiling-off ('sweating') on heating and have desirable melting properties (i.e., good flowability, moistness and surface sheen). In contrast, condensed phosphates generally give harder processed cheeses,

374

Pasteurized Processed Cheese and Substitute/Imitation Cheese Products

which show little, or no, oiling-off on heating and have poor melting properties (little or no flow, skin formation and crusting, dull and dry surface appearance). Overall, the flowability and oiling-off on cooking of PCPs or ACPs made with the different ES show the following general trend: sodium aluminium phosphate trisodium citrate (slightly) > disodium orthophosphate > >sodium tripolyphosphates ~ tetrasodium pyrophosphates > higher chain sodium polyphosphates. Generally, the opposite effect is observed with firmness. The above trends reflect the greater calcium sequestration and hydration effects of the condensed phosphates (Table 5) which affords them better emulsification and hence structural-forming properties. It is noteworthy that the DE is positively correlated with firmness and elasticity of the unheated PCP or ACP and inversely correlated with flowability of the heated products (Rayan et al., 1980). In contrast to the above, Lazaridis and Rosenau (1980) noted the following trend for the effect of ES on the flowability of a melted PCP made from a chemically-acidified curd (50%, w/w, moisture; pH 5.5): Na3PO4 > Na2HPO4 > trisodium citrate > sodium aluminium phosphate (kasal). This trend was probably due to the very low calcium sequestering ability of the latter two ES at pH 5.5 (see 'Characteristics of different ES in the manufacture of PCPs and ACPs'). Blend ingredients: cheese As cheese is a major blend constituent in PCPs, it is expected that both the cheese type and the degree of maturity would have major effects on the texture, flavour and cooking characteristics of the final product. The results of the few published studies, the authors' experience and the undocumented evidence from experienced manufacturers suggest that the following are important criteria: type (variety), composition (e.g., contents of moisture, fat, protein and Ca; pH; Thomas et al., 1980; Shimp, 1985; Salam, 1988; Marshall, 1990), age and level of proteolysis (Sood, and Kosikowski, 1979; Thomas et al., 1980; Lazaridis et al., 1981; Mahoney et al., 1982) and flavour. Proteolysis is inversely related to the level of intact casein (Fenelon and Guinee, 2000; Feeney et al., 2001; Guinee et a/.,2001). The pH, intact casein content and calcium-to-casein ratio are expected to influence the degree of casein hydration during processing, and in turn the DE, degree of casein aggregation and elasticity of the final product. However, there is very little direct experimental evidence to clearly demonstrate relationships between the various attributes of PCPs and the characteristics of the unheated cheese. Harvey et al. (1982) found that the flowability of heated processed Cheddar increased

markedly (from 0.5- to 2-fold) with age (from 3 to 6 months) of the Cheddar cheese used, the effect becoming more pronounced as the processing time of the PCP increased; no data on proteolysis were presented. Arnott etal. (1957) found no relationships between the levels of fat, moisture, pH or the level of proteolysis (measured by tyrosine content) in commercial Cheddar cheeses of different age (0-340 day) and the meltability (flowability) of the resultant PCPs. Variability in the flowability of the PCPs was attributed to the interactive effects of the different cheese characteristics. Surprisingly, Holsinger et al. (1987) reported that the melt index of processed Cheddar decreased as the proportion of mature (135-278 days) to young (90 days) Cheddar (stored at - 1 7 . 8 ~ increased from 100:0 through to 0:100. While few experimental details were given, the results of the latter study suggest an increased creaming reaction as the proportion of mature Cheddar increased. Lazaridis et al. (1981) investigated the effect of increasing the level of proteolysis in a pasteurized processed model chemically-acidified curd system by treating the processed curd (varying conditions: 40-55 ~ pH 5.5-9.0) with a proteinase from Aspergillus oryzae. In contrast to the studies cited earlier, there was a strong positive relationship ( r = 0.96) between the flowability and the extent of proteolysis (non-protein N). Excessive proteolysis was, however, associated with textural defects, including overshortness, faulty body and graininess. In a subsequent study (Mahoney et al., 1982), the same group found that optimal flowability of the processed chemicallyacidified curd was obtained when the proteolysis products were in the molecular mass range 10-25 kDa; smaller peptide sizes ( < 1 0 kDa) gave an excessively soft PCP which overflowed on cooking. In model experiments with processed Gouda, Ito et al. (1976) found an inverse relationship between the age (and hence level of proteolysis) and its emulsifying capacity (defined as ml of added oil absorbed per gram of cheese protein). A lower DE, due to greater proteolysis, would be expected to reduce the contribution of emulsified fat globules to structure building and the creaming effect, favour more oil-release during melting, and improve the flowability of the melted PCP (cf. Rudan and Barbano, 1998; Guinee et al., 2000b). Thus, in the studies of Lazaridis et al. (1981) and Mahoney et al. (1982), a decrease in the DE may explain the increase in flowability of the melted PCPs as the level of proteolysis in the raw cheese increased. Blend ingredients: rework Rework refers to a PCP which, for various reasons, is not packaged but instead is stored (refrigerated at a low

Pasteurized P r o c e s s e d C h e e s e and Substitute/Imitation C h e e s e Products

temperature or frozen) and reused (re-processed/ reworked) as a blend ingredient in later batches of PCP. It is obtained from left-overs in the cooking/filling machines, damaged packs and batches, which are overcreamed or are too viscous to pump. Meyer (1973) identified three types of rework: (A) that made from young cheese, quickly processed and long in structure; (B) that with a typical creamed character (i.e., processed cheese with texture characteristics considered normal for the product type) and (C) over-creamed product with a brittle structure. 'Hot melt', a North-American term, is a type of PC rework, which is the hot 'hardened' PCP that is removed from the packing pipelines following a plant breakdown, especially during continuous processing operations (Kal~ib et al., 1987). Microscopical examination of 'hot melt', which may be considered as an overcreamed rework, revealed the presence of dark areas (Fig. 15) that developed to a degree depending on the extent of heating and the melting salt used (Kal~ib et al., 1987). The dark areas represent regions where the protein absorbed an increased concentration of osmium during fixation. Klostermeyer and Buchheim (1988) reported that the protein matrix of processed cheese heated at 140 ~ contained areas of compacted protein as revealed by a freeze-fracturing technique followed by replication with platinum and carbon. It is probable that the areas of compacted protein observed in the study of Klostermeyer and Buchheim (1988) correspond to the osmiophilic dark areas reported by Kalab et al. (1987). Klostermeyer and Buchheim (1988) also observed that the dimensions of areas of relatively low protein concentration in the protein matrix of PCP decreased as the creaming effect increased: 1-2 b~m in diameter with no creaming (melting time, 4 min), - 0 . 5 p~m with mild creaming (melting time,

Scanning electron micrograph of a processed cheese food showing the presence of an electron-dense area (black area shown by arrow) that developed after holding the product for an extended time (5 h) at 82 ~ Bar corresponds to 0.2 i~m (adapted from Kalab et aL, 1987).

375

6 min) and completely absent at optimal creaming (melting time, 9 min), resulting in a uniform protein matrix. Rework that is free of crystals can sometimes be useful for initiating, or enhancing, the creaming effect in blends that are slow to thicken during processing. The recommended usage levels of rework types A, B and C are 1-2%, w/w, 2.0-30%, w/w and 0.0-1.0%, w/w (maximum), respectively (Meyer, 1973). Type A rework is particularly useful to impart creaming to PCS blends with a high proportion of mature (e.g., intact casein level, "--70% total) or very mature (e.g., low intact casein level, , 09

o o

800

600

09

> t-"

400

(1) Q.

200


2P were used as ES. The results of studies to date suggest that pH probably exerts its influence on the rheology and texture of PCPs and ACPs via its effects on protein-protein interactions and casein hydration, and on the calcium sequestering ability of the ES (Marchesseau et al., 1997; Cavalier-Salou, 1991; cf., 'The role of ES in the formation of a physico-

chemically stable product' and 'Characteristics of different ES in the manufacture of PCPs and ACPs'). However, further studies are required to elucidate the direct effect of pH.

Stabilizers (binding agents) and hydrocolloids Stabilizers, which include carob bean gum, guar gum, carageenan, sodium alginate, gum karaya, pectins and carboxy methylcellulose, are permitted in PCS at a maximum level of 0.8%, w/w (Code of Federal Regulations, 1986). These products stabilize by virtue of their waterbinding and gelation capacities (Phillips et al., 1985). In cheese processing, they are normally used at a level of 0.1-0.3%, w/w, to firm up the structure in instances of high water content or low creaming action (thin consistency) due to, for example, the use of over-ripe cheese or an unsuitable ES blend. More recently, they have found application in reducing firmness, and improving the spreadability and cooking properties (meltability and flowability) of reduced-fat PCPs (Brummel and Lee, 1990; Swenson et al., 2000). While it is difficult to determine the efficacy of the hydrocolloids in the latter studies due to the absence of low-fat controls, both firmness and flowability varied significantly with the type and the level used. Hydrocolloids (locust bean gum, guar gum, modified starch, xanthan gum, low methylated pectin) have recently been investigated as substitutes for sodium phosphate ES (Pluta etal., 2000); a mixture of locust bean gum (0.8%, w/w) and modified starch (2%, w/w) was claimed to give a stable ES-free product and was recommended as a substitute for sodium phosphate in the manufacture of PCPs. Various food-grade emulsifiers (e.g., lecithin, Tweens and Spans) have been used in PCPs, especially in reduced-fat products, to impart softness and improve flowability on melting (Drake et al., 1999). Lee et al. (1996) reported the effects of adding low molecular weight emulsifiers [(sodium dodecyl sulphate (SDS), Nacetyl-N,N,N-trimethylamonium bromide (CTAB), lecithin, mono- and diglycerides)] on the rheological properties of model PCPs. All emulsifiers led to finer dispersions compared to the controls, but their effect on the rheological properties was largely determined by protein-emulsifier interactions which depended on the emulsifier charge. The cationic CTAB increased hardness and elasticity while the anionic SDS gave a PCP which was softer and less elastic than the control; the neutral lecithins and glycerides had little effect.

A n a l o g u e cheese products (ACPs) Analogue cheese products may be classified as cheese substitutes or imitations, which partly or wholly substitute or imitate cheese and in which milk fat, milk protein or both are partially or wholly replaced by

380

Pasteurized Processed Cheese and Substitute/Imitation Cheese Products

non-milk-based components, principally of vegetable origin. However, their designations and labelling should, by law, clearly distinguish them from cheese or PCPs. The labelling requirement for imitation and substitute cheeses has been reviewed by McCarthy (1991). In the USA, an imitation cheese is defined as a product which is a substitute for, and resembles, another cheese but is nutritionally inferior, where nutritional inferiority implies a reduction in the content of an essential nutrient(s) present in a measurable amount but does not include a reduction in the caloric or fat content (Food and Drugs Administration Regulation 101.3, Identity Labelling of Food in Packaged Form (e)). A substitute cheese is defined as a product which is a substitute for, and resembles, another cheese and is not nutritionally inferior. Outside the USA, there is little specific legislation covering imitation or substitute cheeses. Few, if any, standards relating to permitted ingredients or manufacturing procedures

exist for imitation cheese products. For more pertinent information regarding designation and labelling, the reader is referred to IDF (1989), McCarthy (1991), current National Regulations and Codex Alimentarius. Other cheese-like products, which may be classified as imitation or substitute, are Tofu and Filled Cheeses; the latter products have been discussed briefly by Fox et al. (2000) and will not be reviewed here. The general aspects of ACPs have been reviewed recently (Ennis and Mulvihill, 1997; Fox et al., 2000; Guinee, 2002b). Analogue cheese products are cheeselike products manufactured by blending various edible oils/fats, proteins, other ingredients and water into a smooth homogeneous blend with the aid of heat, mechanical shear and ES. The array of ingredients used in ACPs and their functions are listed in Table 7. The effects of various ingredients, processing conditions and low temperature storage on the quality of imitation cheese products have been reported extensively

Ingredients used in the manufacture of cheese analoguesa,b,c, d

Ingredient

Main function~effect

Examples

Fat

Gives desired composition, texture and meltability characteristics; butter oil imparts dairy flavour Give desired composition, semi-hard texture with good shreddability, flow and stretch characteristics on heating Assist in the formation of physico-chemical stable product Gives required composition Low cost relative to casein Rarely, if ever, used commercially as sole protein owing to product defects; may be used at low levels (e.g., 2-3% w/w) Substitution for casein and cost reduction

Butter, anhydrous milk fat, native or partially hydrogenated soya bean oil, corn oil, palm kernel oil Casein, caseinates Whey protein

Assist in the formation of physico-chemically stable product; modify textural and functional properties Enhance product stability; modify texture and functional properties See Table 1 See Table 1 See Table 1 See Table 1 See Table 1 Improve nutritive value

Sodium phosphates and sodium citrates

Milk proteins

Vegetable proteins

Starches Stabilizers Emulsifying salts

Hydrocolloids Acidifying agents Flavours and flavour enhancers Sweetening agents Colours Preservatives Minerals and vitamin preparations

Soya bean protein Peanut protein, wheat gluten

Native and modified forms of maize, rice, potato starches

Hydrocolloids: guar gum, xanthan gum, carageenans See Table 1 See Table 1 See Table 1 See Table 1 See Table 1 Magnesium oxide, zinc oxide, iron, vitamin A palmitate, riboflavin, thiamine, folic acid

a Modified from Guinee (2002b). b The ingredients permitted are subject to the prevailing regulations in the region of manufacture. c Whey proteins mainly for products used in cooking applications where flow resistance is required. d See text for more details on effects of different ingredients (see 'Influence of various parameters on the consistency and cooking characteristics of PCPs and ACPs' and 'Formulation')

Pasteurized Processed Cheese and Substitute/Imitation Cheese Products

(Abou El-Ella, 1980; Lee and Marshall, 1981; Yang and Taranto, 1982; Marshall, 1990; Cavalier-Salou and Cheftel, 1991; Kiely et al., 1991; Suarez-Solis et al., 1995; Ennis and Mulvihill, 1997; Abou E1-Nour et al., 2001). Many of these have been discussed in Influence of various parameters on the consistency and cooking characteristics of PCPs and ACPs'. Similarities with PCPs include: 9 the use of many ingredients in common, including ES, stabilizers, non-cheese dairy ingredients, colours, flavours and flavour enhancers; 9 similar manufacturing technology, involving the application of heat and shear to the formulated blend, followed by hot filling, packing and cooling; 9 similar microstructures which may be generally described as an o/w emulsion, stabilized by hydrated (para) caseinate which occurs as a concentrated dispersion (e.g., high-moisture, low-protein ACPs) or as a weakly gelled (para) caseinate dispersion, depending on product composition and hardness (see 'The role of ES in the formation of a physico-chemically stable product' and 'Micro-structure of PCPs and ACPs'); 9 the absence of a ripening period (even though relatively minor changes can take place during cold storage of PCPs and ACPs (cf., Tamime et al., 1990; Guinee, 2002b) 9 the diverse range of textures, flavours, cooking properties and packaging formats; 9 the use of both as alternatives for natural cheese and in similar applications (cf., 'Cheese as an Ingredient', Volume 2). The major difference between ACPs and PCPs is in the permitted ingredients (as discussed in 'Formulation'), with most commercial analogues containing vegetable-derived fat, rather than milk fat, as in natural and processed cheeses. Analogue cheese products may be arbitrarily categorized as dairy, partial dairy or non-dairy depending on whether the fat and/or protein components are from dairy or vegetable sources (Shaw, 1984; Fox et al., 2000). Partial dairy analogues, in which the fat is mainly vegetable oil (e.g., soya oil, palm oil, rapeseed and their hydrogenated equivalents) and the protein is dairy-based (usually rennet casein and/or caseinate) are the most common. Non-dairy analogues, in which both fat and protein are vegetable-derived, have little or no commercial significance and, to the authors' knowledge, are not commercially available. Dairy analogues are not produced in large quantities because their cost is prohibitive. Partial dairy ACPs were introduced to the market in the USA in the early 1970s and constitute by far the largest group of imitation or substitute cheese products.

381

Since then, the commercial manufacture of analogues of a wide variety of natural cheeses (e.g., Cheddar, Monterey Jack, Mozzarella, Parmesan, Romano, Blue, Cream cheese) and PCPs have been reported in the trade literature (Dietz and Ziemba, 1972; Graf, 1981; Anonymous, 1982, 1986; Shaw, 1984; Morris, 1986). Based on feedback from the marketplace, current annual production of analogue cheese in the USA, the primary manufacturer, is - 3 0 0 000 tonnes (personal communication: Martin O'Donovan, BL Ingredients LLC, Chicago) with the major products being low-moisture Mozzarella, Cheddar and pasteurized processed Cheddar. These products have numerous applications: frozen pizza toppings, slices in beef burgers and ingredient in salads, sandwiches, cheese sauces, cheese dips and ready-prepared meals. Compared to the USA, European production is estimated to be relatively small (e.g., 20 000 tonnes/annum). This may be attributed to the lack of a common European effective legislation policy, the efforts of groups concerned with the protection of the designation of origin of milk and dairy products and/or the relatively low consumption of pizza and cheese as an ingredient in Europe (cf., Guinee, 2002c). Moreover, cheese flavour ingredients (e.g., EMCs) are still insufficiently developed to give analogue cheeses, which could be consumed as table cheeses (K.N. Kilcawley, personal communication), which is the major form of EU cheese consumption. The following have contributed to the success of (partial dairy) ACPs in the USA: (i) their lower cost relative to natural cheeses, coupled with the increase in overall cheese consumption; the low cost of analogues is due to the low cost of vegetable oils (compared to butterfat) and of price-subsidized casein imported from Europe, the absence of a maturation period, which for natural cheeses amounts to -US$1.6/tonne/day and the relatively low cost of manufacturing plant relative to that for natural cheese; (ii) the diversity they can offer by way of functionality (e.g., flowability, melt resistance, shreddability), made possible by tailor-making formulations, coupled with their relatively high functional stability during storage; (iii) the popularity of fast food and ready-prepared meals; (iv) their ability to meet special dietary needs and to act as a vehicle for health benefits/supplements, e.g., lactose-free, low in calories, low in saturated fat, vitaminenriched (Andreas, 1985; Anonymous, 1986; Morris, 1986; Keane and Glaeser, 1990); this is made possible by formulation changes. The following discussion relates to partial dairy analogues, especially analogue low-moisture Mozzarella cheese (LMMC), frequently referred to as analogue pizza cheese, APC.

382

Pasteurized Processed Cheese and Substitute/Imitation Cheese Products

APC" principles and manufacturing protocol

The principles of manufacture of APC from rennet casein are similar to those for PCPs involving: 9 the sequestration of Ca from the rennet casein by added ES at the high temperatures (typically ---80-84 ~ 9 upward pH adjustment of the blend by the added ES; 9 concomitant hydration of the casein by the ES, shear and heat; 9 dispersion of added fat by the shear and its emulsification by the hydrated para-caseinate; 9 structure formation during cooling. The manufacturing technology for ACPs is also very similar to that for PCPs (Ennis and Mulvihill, 1997; Fox et al., 2000; Guinee, 2002b), as described in 'Manufacturing protocol for PCPs'. While production methods vary, a typical manufacturing procedure (Fig. 17) involves the following sequence of events: simultaneous addition of required quantities of water and dry ingredients (e.g., casein, ES), addition of oil

I Formulation of blend i A

B

C

Cheese cooker Mix for -1-2 min

+

I

Process: heat to +85 ~ shear continuously

+ Homogeneous molten mass pH -8.5 Homogeneous molten mass I pH +6.0-6.4 I

+

i Mould and hot pack I

+

I Storeat4to-4~

and cooking to ---85 ~ (using direct steam injection) while continuously shearing until a uniform homogeneous molten mass is obtained (typically 5-8 min). Flavouring materials (e.g., EMC, starter distillate) and pH-regulator (e.g., citric acid) are then added and the mixture is blended for a further 1-2 min and hot-packed. Horizontal twin-screw cookers (e.g., Damrow, Blentech), operating at a typical screw speed of 40 rpm, are used in the manufacture of APC. This cooker design ensures adequate blending and a relatively low degree of mechanical shear (e.g., compared to the homogenizing effects of some processed cheese cookers). These process conditions, together with the correct formulation, promote a low degree of fat dispersion and hence a relatively large fat globule size (e.g., 5-25 I~m; Neville and Mulvihill, 1995; Ennis and Mulvihill, 1997; Neville, 1998; Guinee et al., 1999). The relatively large fat globule size ensures a sufficient degree of oiling-off from the APC topping when baked on pizza; this, in turn, limits dehydration of the cheese topping and is conducive to satisfactory flow and succulence characteristics (cf., Rudan and Barbano, 1998; Guinee et al., 2000b; 'Pasta-Filata Cheeses' and 'Cheese as an Ingredient', Volume 2). As for PCPs, there is generally an inverse relationship between the DE and the flowability of APCs (Neville, 1998; Mounsey, 2001). Addition of the acid at the end of manufacture, rather than at the beginning, ensures a high pH (--~8-9) in the blend during processing. This procedure is desirable in the manufacture of ACPs where insoluble rennet casein is the major protein ingredient. A high pH during processing leads to greater sequestration of calcium by the sodium phosphate ES, higher negative charge to the casein and higher degree of para-casein hydration. These changes enhance the conversion of the calcium para-casein to sodium para-caseinate, which binds water and emulsifies the vegetable oil (cf., 'The role of ES in the formation of a physico-chemically stable product' and 'Characteristics of different ES in the manufacture of PCPs and ACPs'). Thus, reducing the pH of the blend during processing increases the time required for the formation of the ACPs and probably affects its properties (e.g., firmness, meltability). The addition of flavouring ingredients, such as EMC, towards the end of processing minimizes the loss of flavour volatiles at the high temperature of processing.

I

Formulation Typical manufacturing procedures (A, B, C) for lowmoisture Mozzarella cheese analogue. The procedures differ with respect to the order in which the ingredients (1-5) are added, e.g., casein (1) followed by oil (4) and water (5) in procedure B.

A typical formulation (Table 8) shows that it differs from that for PCPs by the absence of cheese (though some cheese may be optionally introduced as a

Pasteurized Processed Cheese and Substitute/Imitation Cheese Products

Typical formulation of low-moisture analogue Mozzarella cheese a

Ingredient

Addition level (%, w/w)

Casein and caseinates Vegetable oil Starch Emulsifying salts Flavours and flavour enhancers Stabilizers Acidifying agent Colour Preservative Water and condensate

18-24 22-28 0.0-3 0.5-2 0.5-3 0.0-0.50 0.2-0.36 0.04 0.10 45-55

a Modified from Guinee (2002b).

flavouring agent) and the inclusion of vegetable oil and a relatively large level of casein(ate)s (cf., Table 1). The major protein source in dairy-based ACPs is caseinate or rennet casein (Nishiya et al., 1989; Ennis and Mulvihill, 1999), with the former being used mainly for spreadable products. Rennet casein is favoured for semi-hard block products and, especially, for APC where it generally imparts better stringiness and stretchability than acid casein or sodium or calcium caseinates. Rennet casein is formed by rennet coagulation of skim milk at normal pH, dehydration of the gel by cutting, stirring and heat treatment, washing of the curd to remove lactose, concentration of the curd by centrifugation and drying, grinding and separation of the dried casein into powders of different mean particle size (Mulvihill, 1992). At the micro-structural level, each powdered particle may be considered as a portion of dried skim milk cheese, with the casein in the form of an agglomerate of aggregates of paracasein. Similar to cheese, various types of attractions are expected to maintain the integrity of the paracasein aggregates (cf., Walstra and van Vliet, 1986), e.g., electrostatic bonds, hydrophobic bonds and calcium phosphate bridges. A further similarity between rennet casein and a young skim milk cheese (with a high level of intact casein) is insolubility in water (cf., Ennis et al., 1998; Fenelon and Guinee, 2000; Feeney et al., 2001). By choosing the appropriate blend of ES, the concentration of calcium cross-linking the paracasein molecules can be reduced to the desired level to give textural and cooking characteristics tailor-made to suit the envisaged application of the product (Fox et al., 2000). On cooking cheese, functional properties such as flow and stretch involve the partial displacement of contiguous layers of the para-casein on the application of stress (see 'Cheese as an Ingredient', Volume 2); a moderate displacement is desirable in cooked pizza cheese (Fox et al., 2000; 'Cheese as an

383

Ingredient', Volume 2). The level of displacement on cooking an ACP depends on the concentration of calcium cross-linking the casein molecules in the final product, which in turn is dependent on the type of casein ingredient used, its total calcium level, the colloidal calcium-to-casein ratio and the concentration and type of ES. For rennet casein which has a high calcium-to-casein ratio (--~36 mg/g casein), the degree of calcium sequestration and para-casein aggregation is easily controlled by using the correct blend of ES to give the desired degree of casein hydration/aggregation and fat emulsification in the ACP (Guinee, 2002b). This, in turn, gives the desired degree of flow and stretchability on cooking the APC. Compared to rennet casein, caseinates tend to over-hydrate, resulting in a degree of casein aggregation which yields good flowability but which is too low to achieve satisfactory stretchability. Owing to the relatively high cost of casein, much effort has been vested in its partial replacement by cheaper substitutes. Increasing the level of substitution of rennet casein by total milk protein, in the range 0-50%, resulted in a progressive increase in firmness and a decrease in the flowability of ACP (Abou-E1-Nour et al., 1996). In a subsequent study, Abou-E1-Nour et al. (2001) investigated the effects of replacing rennet casein by native phosphocasein (NPC) prepared by microfiltration and diafiltration with water (NPC-W) or ultrafiltered milk permeate (NPC-P) in block APC. At 20%, w/w, replacement, the addition of NPC resulted in an increased flowability of the melted APC, with the effect of the NPC-W being significantly greater than that of the NPC-P. In contrast, the NPC-W resulted in a slight decrease in firmness of the unheated ACP whereas the NCP-P gave a marked increase. A comparison between the NPC preparations and the MPC, prepared by UE indicated that the latter gave notably higher firmness in the unheated APC and lower flowability of the heated APC (Abou-E1-Nour et al., 2001). This trend concurs with that of previous studies showing that the addition of whey proteins to PCPs or ACPs, as a substitute for cheese or casein, impairs flowability and increases firmness (cf., 'Blend ingredients: cheese base (CB), ultrafiltered milk retentate (UFMR), cheeses from high heat-treated milks and whey proteins'). Hence, whey proteins are not used owing to the negative impact on flowability, except in applications where flow-resistant ACPs may be needed (e.g., cheese insets in burgers) when --~1-3%, w/w, whey protein is added. Studies have been undertaken on the effects of replacing casein in ACPs by various types of vegetable proteins, e.g., soybean (Lee and Marshall, 1981; Taranto and Yang, 198i; Yang and Taranto, 1982; Yang et al.,

384

Pasteurized Processed Cheese and Substitute/Imitation Cheese Products

1983; Kim etal., 1992; Ortega-Fleitas etal., 2001), peanut (Chen etal., 1979), pea protein (E1-Sayed, 1997) or wheat protein (Anonymous, 1981). These proteins gave varying results, depending on the ingredient preparation (e.g., soy flour or soy isolate, pH, fat content) and the type and level of other ingredients (e.g., hydrocolloids). However, the use of these protein substitutes, especially at a level > 10-20%, w/w, of the total protein, has, in general, been found to give ACPs which have a quality inferior to that made using casein only. Common defects include lack of elasticity, lower hardness, an adhesive/sticky body, impaired flow and stretchability and/or poor flavour. Hence, vegetable proteins are rarely used in the commercial manufacture of APCs. To date, starch has been the most effective low-cost casein substitute. Native maize starch appears to be the main type used commercially, with starches from other sources and with different types of modification (pre-gelatinized and/or chemically or enzymatically modified) being used less frequently (Ennis and Mulvihill, 1997). Native starches are used successfully commercially at a level of 2-4%, w/w, to replace ---10-15%, w/w, of total casein. At higher levels of substitution, product defects become noticeable- an increase in the firmness and brittleness of the unheated ACP and a decrease in the fluidity and flowability of the melted cheese, especially if the starch has a high amylose-to-amylopectin ratio (Mounsey and O'Riordan, 1999, 2001; Guinee, 2002b; Figs 18, 19). Moreover, on shredding, the unheated APC with added starch tends to fracture more easily to form curd fines and also tends to exude free moisture after a short period of cold storage, which often leads to sticking and bailing 70

during shredding operations. These defects, which occur to a degree dependent on the type and level of added starch (Mounsey and O'Riordan, 1999, 2001; Mounsey, 2001; Figs 18, 19), cooking temperature and time, degree of agitation and cooling rate, are probably related to storage-related retrogradation and gelation of the starch molecules (especially amylose). Starches (e.g., maize, ,#heat) with a high ratio of amylose to amylopectin tend to retrograde and undergo gelation more readily than those (e.g., waxy maize, rice, potato) with a lower level of amylose (cf., Miura et al., 1992) during storage of the ACP. The other factors above probably influence the degree of gelatinization of the starch during the manufacture of the ACPs and, thus, the concentration of free amylose molecules available for gelation. It is envisaged that a starch gel would impede the flow of the heated cheese when cooked on pizza. The adverse effects of starch may also be related to an increased degree of fat emulsification (Mounsey and O'Riordan, 2001), as a result of a higher apparent viscosity of the APC blend during manufacture when starch is added, especially at high levels. Composition and functionality

Analysis of commercial APC indicates large intra- and inter-factory variations in composition (Guinee et al., 2000c; Guinee, 2002), e.g., moisture, 40-52%, w/w; fat, 60

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Pasteurized Processed Cheese and Substitute/Imitation Cheese Products

22-30%, w/w; protein, 13-21%, w/w; 31-38 mg Ca/g protein. Such variations undoubtedly reflect differences in formulation, which suggest that formulation change is a key approach used by manufacturers in the production of APCs with customized nutritional, textural and/or functional (cooking) characteristics. Comparison with commercial Low Moisture Mozzarella Cheese (LMMC) shows that APC has a lower protein content, higher concentrations of moisture and fat, and higher ratios of Ca- and P-to-protein (Guinee et al., 2000c). The higher ratios of Ca- and P-to-protein reflect the use of rennet casein (which has higher concentrations of Ca and P on a protein basis than most natural cheeses) and the inclusion of sodium phosphate ES during formulation. Moreover, the mean value for the sum of moisture, fat, protein and ash in commercial APC is --96.5%, w/w, compared to ---99.5%, w/w, in the LMMC, indicating the addition of carbohydrate-based ingredients during formulation (Guinee et al., 2000c). The heat-induced functional properties of LMMC are discussed in detail in 'Pasta-Filata Cheeses', Volume 2. These generally change fairly markedly with storage time at ar ~ as reflected by reductions in apparent viscosity and an increase in the flowability of the heated cheese; depending on the cheese type, the stretchability of the melted cheese generally increases at first and decreases thereafter. The changes in these functional attributes are due to various factors including age-related physico-chemical changes in the cheese, including proteolysis, solubilization of casein-bound calcium and increases in para-casein hydration and in the level of non-globular fat (Kindstedt, 1995; Guinee 2002c). Similar to PCPs, the functionality of freshly manufactured APCs (e.g., after storage at 4~ for o

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0.7) that are generally of a magnitude which results in large deformation and fracture (i.e., breakdown into smaller pieces): portioning of cheese into retail sizes, shredding into thin narrow cylindrical pieces (e.g., 2.5 cm long and 0.4 cm diameter), dicing into very small cubes (0.4 cm) and comminution by forcing precut cheese through die plates with narrow apertures. Similarly, when eaten, cheese is subjected to a number of strains which reduce it to a paste capable of being swallowed; first, the cheese is bitten (cut by the incisors), compressed (by the molars) on chewing and sheared (between the palate and the tongue, and between the teeth). The behaviour of the cheese when exposed to the different size-reduction methods constitutes a group of important functional properties, which are summarized in Table 1. In general, apart from shreddability, there is little information in the scientific literature on the functional properties of unheated cheese or how they

Rheological properties of unheated cheese which affect its functionality as an ingredient

Types of properties

Description

Measurements 1

Elasticity and related properties (springiness, toughness)

Tendency of cheese to recover to original dimensions following removal of the applied stress (o-, force per unit surface area)

Fracturability

Tendency of cheese to fracture into pieces when a stress (o-) is applied, e.g., during compression or extension

Recovery of sample after compression, obtained using Texture Profile Analysis Fracture stress (o-f) - force to fracture Fracture strain (sf) -displacement at fracture

(and related terms)

- brittleness

-Iongness

- crumbliness Firmness (and related terms) - Firm - Soft Adhesiveness

Tendency to fracture into pieces at a low deformation or displacement (strain; s, i.e., after a low-percentage compression). Low deformation at fracture, i.e., low sf Tendency to fracture at a large deformation, i.e., high 8 f The tendency to break down easily into small, irregular shaped particles (e.g., by rubbing) Resistance of a cheese to be deformed (e.g., compressed) when subjected to a stress (o-) High resistance to deformation, i.e., high O m a x Low resistance to deformation, i.e., low O m a x Tendency to be sticky and resist separation from a material it contacts

Firmness (O-max)- - stress (o-) required to achieve a given compression/extension

Texture Profile Analysis

References used in compilation: van Vliet (1991), Visser (1991), Fox etaL (2000), Guinee (2002). See 'Rheology and Texture of Cheese', Volume 1 for details of rheology tests. 1 Measurements obtained from large strain deformation tests, as in compression testing or Texture Profile Analysis using a Texture Analyzer; see 'Rheology and Texture of Cheese', Volume 1 for details of tests.

400

Cheese as an Ingredient

may be related to its rheological properties, which determine:

over the pizza base, preparation of sandwiches and use in salad bars. Mature Camembert or Chaumes, which are soft, short and adhesive, are very unsuitable for 9 the magnitude of the stress required to fracture shredded/diced cheese applications because of their (fracture stress, of); tendency to stick to the shredding equipment and of 9 the degree of strain (e.g., change in dimensions) the shredded cheese to bailing and clumping. Howrequired to fracture (fracture strain, ~f); ever, the ability of these cheeses to undergo plastic 9 the level of force or stress required to achieve a fracture and flow under shear (i.e., spread) makes given deformation (O'max); them ideal for spreading on crackers and for blending 9 the type of fracture (i.e., clean or jagged); with other materials such as butter, milk or flour in 9 the degree to which a piece of cheese recovers (in the preparation of fondues and sauces. The brittleness size dimensions) after being strained (e.g., comand tendency of hard cheeses, such as Parmesan and pressed or sheared). Romano, with low levels of moisture and fat-in-dry The various rheological terms (o'f, el, O'max) matter, to undergo elastic fracture (clean fracture withdescribed above are easily measured from the force (or out flow) endows them with excellent gratability stress, o-)/displacement (or strain, e) curve obtained (when crushed between rollers) and suitability as a during compression of a cheese sample, as described free-flow condiment for sprinkling, e.g., onto pasta in 'Rheology and Texture of Cheese', Volume 1. On dishes. However, these properties render the latter consideration of the forces operative during deforma- cheeses unsuitable for food applications that require tion, and the structure and the biochemistry of cheese, slices (e.g., filled sandwiches, cheeseburgers) or shredit can be inferred that relationships do exist between ded cheese. The crumbliness of Feta and Stilton makes the functional and the rheological characteristics of them very desirable for use in tossed salads and Greek unheated cheese. Similarly, cheese texture, which is a salads as the irregularly shaped, curd-like particles crecomposite sensory attribute resulting from a combin- ate an image of 'real' cheese and are more visually ation of physical properties that are perceived by the appealing to the consumer than cheese shreds. senses of touch (including kinaesthesis and mouthfeel), sight and hearing, has been found to be related Factors influencing the rheological (functional) to rheological (stress-strain) characteristics of cheese properties of unheated cheese (Szczesniak, 1963; Sherman, 1969; Brennan, 1988). Cheese rheology and the factors that affect it have The relationships between some common functional been studied (Culioli and Sherman, 1976; Vernon properties and the rheological parameters of the raw Carter and Sherman, 1978; Chen et al., 1979; Creamer cheese, as described below, are given in Table 1. and Olson, 1982; Green et al., 1985; Luyten, 1988; The rheological characteristics of the raw cheese Visser, 1991; Fenelon and Guinee, 2000) and reviewed have a major impact on how it behaves during com- extensively (van Vliet, 1991; Visser, 1991; Rao, 1992; minution and its usability as an ingredient (Table 2). Prentice et al., 1993; Fox et al., 2000; 'Rheology and Thus, it is difficult to cleanly portion hard cheeses Texture of Cheese', Volume 1). The rheology of cheese which have a relatively a low fracture strain (Parmesan) is a function of the combined effects of various factors, or which fracture in a jagged fashion (e.g., an over- including its composition, micro-structure (i.e., the acid Cheddar or Cheshire) owing to their tendency to spatial arrangement of its components and the break at the edges. Similarly, these cheeses are unsuit- strength of attractions between the structural elements) able for applications where shredded cheese is and the physico-chemical state of its components (e.g., required (e.g., pizza) because of their susceptibility to degree of casein hydrolysis). Moreover, it is difficult to fracture/shattering and the resultant formation of a quantify the direct effects of any of the gross composhigh level of curd fines/dust on the surface of the itional components (fat, protein or moisture) separately, uncooked pizza, which is aesthetically unappealing. owing to the fact that these tend to vary simultanConversely, other hard cheeses, such as Cheddar, low- eously, especially where large changes in the concenmoisture Mozzarella (LMMC) and Gouda-type, are tration of a particular component (e.g., fat) occur and unsuitable for grating owing to their lack of brittleness in the absence of process interventions. However, and to their elasticity and relatively high o'f and el, for convenience, the effects of individual factors are which enables a relatively high degree of recovery to discussed separately below. their original shape and dimensions following crushing. However, the latter cheeses generally shred very Protein level well to give pieces of uniform size which are relatively The concentration and the type of protein have a non-adhesive, which makes them ideal for distribution major influence on the rheological properties, as

Cheese

confirmed by the positive correlation between the volume fraction of the casein matrix and cheese firmness (O'max) and the o-f; de Jong, 1977; Guinee et al., 2000a; (Fig. 3), and by the effects of gel fineness or coarseness on the rheological characteristics of the matrix (Green etal., 1983; Green, 1990b; Guinee etal., 1993b). Hence, reduced-fat Cheddar, which has a high volume fraction of para-casein matrix relative to full-fat Cheddar, is firmer, and has a higher o-f, than the latter (Fenelon and Guinee, 2000). The large influence of protein becomes apparent when the effects of an applied stress to cheese structure are considered; the protein matrix provides the first resistance to deformation. The stress-bearing capacity of the matrix is dependent on its volume fraction and homogeneity, which determine the number of stress-bearing strands per unit area. Considering a gel to which a relatively small stress (i.e., much less than the fracture stress) is applied in the direction x, the elastic shear modulus (G', i.e., ratio of shear stress to shear strain, ofT), which is an index of elasticity or strength of the gel, can be related to the number of strands per unit area according to the equation (Walstra and van Vliet, 1986): d2A G' = C N ~ dx 2

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firmness

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Ingredient

401

determining the geometry of the network; dA = change in elastic energy when the aggregates in the strands are moved apart by a distance, dx, on application of the stress. The number of strands per unit area of a gel are determined by: 9 the concentration of gel-forming protein; 9 the fineness or coarseness of the gel, with a fine gel network having a greater number of stress-bearing strands than a coarse gel. As the concentration of casein in the matrix increases, the intra- and the inter-strand linkages become more numerous, and the matrix more elastic (Ma et al., 1997) and more difficult to deform (de Jong, 1976, 1978a; Chen et al., 1979; Prentice et al., 1993). At low temperatures ( < 5 ~ milk fat is predominantly solid and adds to the elasticity of the casein matrix. The solid fat globules limit the deformation of the casein matrix, as deformation of the latter would also require deformation of the fat globules enmeshed within its porous structure. However, the contribution of fat to the elasticity of cheese decreases rapidly as the ratio of solid-to-liquid fat decreases with increasing temperature and is very low at 40 ~ where all the milk fat is liquid (Guinee and Law, 2002). High heat treatment (HHT) of milk and denatured whey proteins High heat treatment of milk increases the level of in-situ denaturation of whey proteins and their complexation with K-CN at the micelle surfaces. The denatured whey proteins form appendages which protrude from the micelle surfaces and render the Phe]05mMetl06 bond of K-CN less susceptible to hydrolysis by rennet (van Hooydonk et al., 1987; McMahon et al., 1993b). These changes coincide with a reduction in the degree of casein aggregation/fusion during rennet-induced gel formation and the remaining post gel-cutting cheesemaking operations and an increased level of denatured whey proteins incorporated into the gel matrix (Pearse et al., 1985; Green, 1990a,b). Consequently, rennetinduced milk gels from HHT milk have a relatively fine structure, low porosity and an increased waterholding capacity. Cheese prepared from HHT milk (e.g., 82 ~ for 15 s) has lower o-f and Crmax than cheese made from milk pasteurized at a normal temperature (e.g., 72 ~ for 15 s; E1-Koussy et al., 1977; Marshall, 1986; Green et al., 1990a,b; Guinee et al., 1998). These effects are attributable to the reduced degree of para-casein aggregation, the increased level of denatured whey proteins in the protein network and the generally higher moisture level. Owing to its effect on cheese rheology, high levels of denatured whey proteins in cheese milk may

402

Cheese as an Ingredient

be exploited as a means of improving the texture (reducing the firmness and elasticity) of low-fat cheeses which tend to be excessively firm and rubbery (Guinee et al., 1998). For similar reasons, the inclusion of whey protein-based fat mimetics (e.g., Simplesse | 100 and Dairy Lo TM) in reduced-fat Cheddar reduces of, ef and O'ma x (Lucey and Gorry, 1994; Fenelon and Guinee, 1997). The whey proteins in these preparations, at least in the case of Dairy Lo TM, appear to interact with the casein to form a complex- type gel during Cheddar manufacture. Various studies have examined the effects of adding denatured whey proteins, in the form of partially de-natured whey protein concentrates (PDWPC; prepared by the Centriwhey, Lactal or UF processes), to cheese milk for the manufacture of hard or semi-hard cheeses, primarily as a means of enhancing cheese yield. The addition of WPC increases the moisture content, actual yield and moisture-adjusted yield, with the extent of the increase being correlated positively with the degree of denaturation of the added WPC (van den Berg, 1979; Brown and Ernstrom, 1982; Banks and Muir, 1985; Baldwin et al., 1986; Punidadas et al., 1999; Meade and Roupas, 2001). However, the addition of PDWPC has, generally, been found to cause defective body (greasy, soft) and flavour (unclean, astringent) characteristics in Gouda and Cheddar cheeses (van den Berg, 1979), with the intensity of the defects becoming more pronounced with increasing level of the PDWPC added. It has been suggested that these defects may be due to the large size of whey protein particles (aggregates) which do not fit compactly within the pores of the para-casein matrix, and thereby impede its shrinkage and syneretic potential (van den Berg, 1979). Fat c o n t e n t

reduction in the concentration of intact casein. Moreover, liquid fat confers viscosity and also acts as a lubricant on fracture surfaces of the casein matrix and thereby reduces the stress required to fracture the matrix (Marshall, 1990; Prentice et al., 1993). Similarly, reducing the fat content (e.g., from 21-25%, w/w, to "--9-11%, w/w) of low- (47.7-51.8%, w/w) or high(52.2-57.4%, w/w) moisture Mozzarella cheeses resulted in significant increases in hardness and springiness at 1 and 6 weeks, with the magnitude of the effect being the most pronounced for hardness (Tunick et al., 1993). There was a significant effect of the interaction between scald temperature and fat content on hardness, with the effect of fat reduction on hardness being more pronounced as the scald temperature was raised from 32.4 to 45.9 ~ This suggests a higher degree of para-casein aggregation at the higher temperature, an occurrence that would be expected to impede the level of displacement of contiguous casein layers obtained for a given stress. Owing to its effect on the ratio of solid-to-liquid fat in the cheese, temperature has a marked influence on cheese rheology, with the elastic shear modulus (G'), E, o'f and O'max decreasing as the temperature increased (Guinee and Law, 2002; 'Rheology and Texture of Cheese', Volume 1). The effect of the solid-to-liquid fat ratio, as affected by temperature, on the rheological properties of cheese and its use as an ingredient is evident in many instances. Hence, in pizza manufacture, cheese is tempered to, and maintained at, a low temperature prior to shredding (e.g., - 2 ~ so as to maximize the elastic contribution of fat and reduce the tendency of the cheese to stick or clump, and thereby facilitate free flow and distribution onto the pizza surface. Similarly, cheeses are maintained at refrigeration temperatures prior to portioning and slicing to get clean cutting and reduce the risk of surface smearing and greasiness by 'sweated' fat.

Alteration of the fat content has a major effect on the rheological properties of cheese varieties, including Cheddar, LMMC and Cottage cheeses (see Guinee and Law, 2002). Such effects are expected because of the differences in the viscoelastic contributions of fat and casein, as discussed above. However, the overall effects of the changing fat content may be attributed in large part to the interactive effects of changes in the levels of fat, moisture and protein. This is because a reduction in fat content (especially if large, e.g., >4%, w/w) is generally paralleled by increases in moisture, protein, intact casein and Ca. At temperatures of ~--4-20 ~ increasing the level of fat in Cheddar cheese results in decreases in elasticity (E), of, el, Crmax, cohesiveness, springiness, chewiness and gumminess and an increase in adhesiveness. The latter trends are expected because of the concomitant

Homogenization of cheesemilk and degree of fat emulsification Homogenization of milk is practised in the manufacture of some cheese varieties where lipolysis is important for flavour development, e.g., Blue cheese, to increase the accessibility of the fat to mould lipases and thereby increase the formation of fatty acids and their derivatives (e.g., methyl ketones; Fox et al., 1996). Moreover, homogenization is an essential step in the manufacture of cheeses from recombined milks and some acid curd varieties with a high fat content (e.g., Cream cheese; see 'Acid- and Acid/Rennet-Curd Cheeses: Part A Quark, Cream Cheese and Related Varieties, Part B Cottage Cheese, Part C Acidheat Coagulated Cheeses', Volume 2). Homogenization reduces the mean fat globule size and increases the surface area of the fat by a factor of 5-6 (McPherson et al.,

C h e e s e as an Ingredient

1989). The newly formed fat globules are coated with a membrane consisting of casein micelles, sub-micelles, whey proteins and some of the original fat globule membrane (Walstra and Jenness, 1984; Keenan et al., 1988). The membrane enables the newly formed fat globules to behave as pseudo-protein particles which can interact with the casein micelles and become an integral part of the gel matrix formed during acid or rennet gelation of milk (van Vliet and Dentener-Kikkert, 1982; Green et al., 1983; Lelievre et al., 1990; Tunick et al., 1997; Michalski et al., 2002). Hence, the effective protein concentration of, and the overall level of protein-protein interactions in, the casein matrix are thereby increased. Homogenization of cheesemilk, e.g., at respective first and second stage pressures of 17.6 and 3.5 MPa, generally results in a higher moisture level and decreases in the magnitude of o-f and O'max o f reducedfat Cheddar (Emmons et al., 1980; Metzger and Mistry, 1994). Similarly, homogenization of milk for full-fat Mozzarella (---22%, w/w, fat) cheese, at combined first and second stage pressures of 250 or 500 kPa, resulted in significant decreases in hardness and springiness and an increase in cohesiveness; simultaneously, there were non-significant decreases in gumminess and chewiness (Jana and Upadhyay, 1991). The magnitude of these changes, which increased with homogenization pressure, coincided with a decrease in protein content and increases in the contents of moisture (i.e., - 5 % , w/w) and MNFS. In contrast, Tunick etal. (1993) reported that two-stage homogenization of milk at combined first and second stage pressures of 10.3 or 17.2 MPa resulted in a general increase in the hardness of low-fat ( - 9 % , w/w) or high-fat (~25%, w/w) Mozzarella cheese after storage for 1-6 weeks, the effect being more pronounced for low-fat cheese. Moreover, there was a significant effect of the interaction between homogenization pressure and scald temperature used in cheese manufacture, with the increase in hardness being more pronounced for the higher scald temperature cheeses. The higher hardness at the higher scald temperature probably reflects an increase in the degree of casein aggregation, an effect that would be enhanced as the effective casein concentration increases with homogenization of the milk. Rudan et al. (1998) reported that homogenization of cheesemilk or cream (first and second stage pressure, 13.8 and 3.45 MPa) did not significantly affect the hardness or springiness of reducedfat ( - 8 % , w/w) Mozzarella cheese at 30 days. The discrepancies between the latter two studies, in which the moisture content of the control and the homogenized milk cheeses were similar, may reflect differences in homogenization conditions, test conditions, age of cheese and fat content (see Fox et al., 2000; 'Rheology and Texture of Cheese', Volume 1).

403

Moisture content

Increasing the moisture content, while maintaining the ratios of the other compositional parameters relatively constant, reduces the concentration of protein and the volume fraction of the casein matrix (de Jong, 1978a). Hence, increasing the moisture content of Dutch-type Meshanger cheese from 40 to 60%, w/w, resulted in a marked reduction in O'max. Similarly, increasing the moisture content of 7.5-month-old Gouda cheese from --~32 to 46%, w/w, resulted in progressive decreases in E, crf and O'max (Luyten, 1988; Visser, 1991); the ef increased slightly with moisture content to an extent dependent on cheese pH and maturity. Similarly, Watkinson et al. (2002) reported that an increase in the moisture content of model Cheddar-like cheeses, from 40 to 48%, w/w, resulted in a large decrease in E and degree of cracking at fracture and large increases in ~f and adhesiveness (stickiness). Creamer and Olson (1982) reported a linear decrease in of as the moisture content of Cheddar was increased from 34.0 to 39.7%, w/w, with of at the lower moisture level being almost twice that at the higher moisture level. Salt (NaCI) content The effects of salt in the moisture phase (S/M) in the range 0.4-12%, w/w, on the rheology of model Goudatype cheeses, in which the levels of the other compositional parameters were relatively constant, were studied by Luyten (1988) and Visser (1991). The range of S/M investigated was inclusive of the values that span the spectrum of different varieties, e.g., from --~2.0%, w/w, in Emmental to --~12%, w/w, in Feta. Increasing the concentration of S/M in this range resulted in progressive increases in E, o-f (from --~28 kPa at 0.4%, w/w, S/M to --~83 kPa at 11.3%, w/w, S/M) and O-max (Visser, 1991). The fracture strain, el, increased slightly to a maximum at 4.5-5.0%, w/w, S/M, then decreased sharply to a value which was about half the maximum at 5.5%, w/w, S/M and thereafter remained relatively constant as the S/M was increased to 11.3%, w/w. The effects of salt are probably attributable to its effects on the degree of protein hydration. In low-concentration brines (i.e., -,o~ o ~

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96%, w/w, dry matter) Outline of production processfor cheese powder.Abbreviations:EMDIs, enzyme-modifieddairy ingredients;MSG, monosodium glutamate.

C h e e s e as an Ingredient

whey or skim milk solids, starches, maltodextrins and butter-fat. In addition to antioxidants, fat encapsulation technology, which reduces the level of free fat in the powder, may be used to reduce the susceptibility to oxidative rancidity. The type and the level of ingredients used in the formulation depend on powder type (e.g., natural or extended), wettability and solubility characteristics and application (Anonymous, 1991). Typical formulations of the slurries required for natural and extended cheese powders with different levels of cheese solids are given in Table 4. The flavour profile and intensity of the final cheese powder is determined by the type(s) of cheese used and the type(s) and level(s) of other flavouring agents (such as EMC, hydrolysed butter-fat, starter distillates) and flavour enhancers (e.g., sodium chloride, monosodium glutamate, autolysed yeast extract). Generally, mature cheese with an intense flavour is used so as to impart a strong flavour to the final product. Apart from their lack of flavour-imparting properties, young cheeses with a high level of intact casein are unsuitable as they result in very viscous slurries, which are difficult to atomize and dry efficiently. Filling materials in extended cheese powders are usually added to replace cheese solids and thereby reduce the formulation costs. However, they may influence the flavour, wettability and mouth-coating characteristics of the product in which the cheese powder is used. (b) Processing of the blend and slurry formation. Processing principles and technology are similar to those used for the manufacture of PCPs. Processing involves heating the blend (using direct steam injec-

417

tion) to a temperature of---75-85 ~ in a processed cheese-type cooker, or in large (e.g., 5000 L) 'dissolving tanks' (e.g., Limitech) with shearing blades, while continuously shearing (e.g., at 1500-3000 rpm). Maintaining the temperature