Cheese: Chemistry, Physics & Microbiology

Cheese Chemistry, Physics and Microbiology Volume 2

Major Cheese Groups

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Cheese Chemistry, Physics and Microbiology Volume 2

Major Cheese Groups

Third edition

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

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

This book is printed on acid-free paper First published 1987 by Elsevier Applied Science Second edition 1993 by Chapman & Hall Third edition 2004 Copyright © 2004, Elsevier Ltd. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the publisher. Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone: (44) 1865 843830, fax: (44) 1865 853333, e-mail: [email protected]. You may also complete your request on-line via the Elsevier homepage (http://www.elsevier.com), by selecting ‘Customer Support’ and then ‘Obtaining Permissions’. Elsevier Academic Press 84 Theobald’s Road, London WC1X 8RR, UK http:/ /www.elsevier.com Elsevier Academic Press 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http:/ /www.elsevier.com British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Library of Congress Control Number: 2004105783 Volume 1 ISBN 0-1226-3652-X Volume 2 ISBN 0-1226-3653-8 Set ISBN 0-1226-3651-1 Cover images: Bacteria (detail): reprinted with permission from Donald J. McMahon, Food Structure, 1993, Vol. 12, pp. 251–258, Fig. 3A. Sensory Circle: adapted with permission from Pierre Lavanchy et al., A Guide to the Sensory Evaluation of Texture of Hard and Semi-Hard Cheeses, 1994, Institut National de la Recherché Agronomique, Paris. Electropheresis gel: Published with kind permission of Vivek Upadhyay, University College Cork, Republic of Ireland.

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Contents

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

vii ix xiii xv xvii 1

General Aspects of Cheese Technology R.J. Bennett and K.A. Johnston

23

Extra-Hard Varieties M. Gobbetti

51

Cheddar Cheese and Related Dry-salted Cheese Varieties R.C. Lawrence, J. Gilles, L.K. Creamer, V.L. Crow, H.A. Heap, C.G. Honoré, K.A. Johnston and P.K. Samal

71

Gouda and Related Cheeses G. van den Berg, W.C. Meijer, E.-M. Düsterhöft and G. Smit

103

Cheeses with Propionic Acid Fermentation M.T. Fröhlich-Wyder and H.P. Bachmann

141

Surface Mould-ripened Cheeses H.-E. Spinnler and J.-C. Gripon

157

Blue Cheese M.D. Cantor, T. van den Tempel, T.K. Hansen and Y. Ardö

175

Bacterial Surface-ripened Cheeses N.M. Brennan, T.M. Cogan, M. Loessner and S. Scherer

199

Cheese Varieties Ripened in Brine M.H. Abd El-Salam and E. Alichanidis

227

Pasta-Filata Cheeses P. Kindstedt, M. Cari´c and S. Milanovi´c

251

Cheeses Made from Ewes’ and Goats’ Milk M. Medina and M. Nuñez

279

Acid- and Acid/Rennet-curd Cheeses Part A: Quark, Cream Cheese and Related Varieties D. Schulz-Collins and B. Senge

301

Acid- and Acid/Rennet-curd Cheeses Part B: Cottage Cheese N.Y. Farkye

329

Acid- and Acid/Rennet-curd Cheeses Part C: Acid-heat Coagulated Cheeses N.Y. Farkye

343

vi Contents

Pasteurized Processed Cheese and Substitute/Imitation Cheese Products T.P. Guinee, M. Caric´ and M. Kala´b

349

Cheese as an Ingredient T.P. Guinee and K.N. Kilcawley

395

Index

429

Foreword

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

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List of Contributors

Professor M.H. Abd El-Salam Dairy Department National Research Centre Dokki Cairo Egypt

Professor M. Caric´ University of Novi Sad Faculty of Technology Bulevar Cara Lazara 1 Novi Sad Serbia and Montenegro

Professor E. Alichanidis Laboratory of Dairy Technology School of Agriculture Aristotle University of Thessaloniki 541 24 Thessaloniki Greece

Professor T.M. Cogan Dairy Products Research Center Teagasc, Moorepark Fermoy Co. Cork Ireland

Professor Y. Ardö The Royal Veterinary and Agricultural University Department of Dairy and Food Science Rolighedsvej 30 1958 Frederiksberg C Denmark

Dr L.K. Creamer Fonterra Research Centre Private Bag 11 029 Dairy Farm Road Palmerston North New Zealand

Dr H.P. Bachmann Agroscope Liebefeld-Posieux Swiss Federal Institute for Animal Production and Dairy Products Schwarzenburgstrasse 161 CH-3003 Bern Switzerland

Dr V.L. Crow Fonterra Research Centre Private Bag 11 029 Dairy Farm Road Palmerston North New Zealand

Mr R.J. Bennett Institute of Food Nutrition and Human Health Massey University Palmerston North New Zealand

Dr E.-M. Düsterhöft NIZO Food Research PO Box 20 671O BA Ede The Netherlands

Dr N.M. Brennan Dairy Products Research Centre Teagasc, Moorepark Fermoy Co. Cork Ireland Dr M.D. Cantor Danisco A/S Innovation Langebrogade 1 1001 Copenhagen K Denmark

Professor N.Y. Farkye Dairy Produce Technology Center California Polytechnic State University San Luis Obispo CA 93407 USA Professor P.F. Fox Department of Food and Nutritional Sciences University College Cork Ireland

x

List of Contributors

Dr M.T. Fröhlich-Wyder Agroscope Liebefeld-Posieux Swiss Federal Institute for Animal Production and Dairy Products Schwarzenburgstrasse 161 CH-3003 Bern Switzerland Dr J. Gilles Deceased 19 January 2003 (Retired from the New Zealand Dairy Research Institute.) Professor M. Gobbetti Dipartimento di Protezione delle Piante e Microbiologia Applicata Università di Bari Via G. Amendola 165/a 70126 Bari Italy Dr J.-C. Gripon Unité de Biochimie et Structure des Protéines Instituto National de La Recherche Agronomique 78350 Jouy-en-Josas France Dr T.P. Guinee Dairy Products Research Centre Teagasc, Moorepark Fermoy Co. Cork Ireland Dr T.K. Hansen The Royal Veterinary and Agricultural University Department of Dairy and Food Science Rolighedsvej 30 1958 Frederiksberg C Denmark Dr H.A. Heap Fonterra Research Centre Private Bag 11 029 Dairy Farm Road Palmerston North New Zealand Dr C.G. Honoré Fonterra Research Centre Private Bag 11 029 Dairy Farm Road Palmerston North New Zealand Mr K.A. Johnston Fonterra Research Centre Private Bag 11 029 Dairy Farm Road Palmerston North New Zealand

Dr M. Kaláb Agriculture and Agri-Food Canada Food Research Program Guelph Ontario, K1A OC5 Canada Dr K.N. Kilcawley Dairy Products Research Centre Teagasc, Moorepark Fermoy Co. Cork Ireland Dr P. Kindstedt Department of Nutrition and Food Sciences University of Vermont Burlington VT 05405-0044 USA Dr R.C. Lawrence 23 Pahiatua Street Palmerston North New Zealand (Retired from the New Zealand Dairy Research Institute.) Dr M. Loessner Technical University of Munich 21EL, Abtilung Microbiologia Weihenstephan D-85354, Freising Germany Dr P.L.H. McSweeney Department of Food and Nutritional Sciences University College Cork Ireland Dr M. Medina Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) Crta. de la Corun~ a km. 7,5 28040 Madrid Spain Dr W.C. Meijer NIZO Food Research PO Box 20 6710 BA Ede The Netherlands Professor S. Milanovic´ University of Novi Sad Faculty of Technology Bulevar Cara Lazava 1 Novi Sad Serbia and Montenegro

List of Contributors xi

Dr M. Nuñez Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) Crta. de la Corun~ a, km. 7,5 28040 Madrid Spain Professor G. Ottogalli Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche Sezione di Microbiologia Agraria Alimentare Ecologica Via G. Celoria 2 20133, Milano Italy Dr P.K. Samal Britannia Industries Limited Britannia Gardens Airport Road Bangalore 560 017 India (Formerly of Fonterra Research Centre, Private Bag 11 029, Dairy Form Road, Palmerston North, New Zealand.) Professor S. Scherer Technical University of Munich 21EL, Abtilung Microbiologia Weihenstephan D-85354, Freising Germany Dr D. Schulz-Collins Arrabawn Co-Op. Nenagh Co. Tipperary Ireland

Dr B. Senge Technische Universität Berlin Faculty of Process Sciences Department of Food Rheology Königin-Luise-Str. 22 Sekr. KL-H1 D-14195 Berlin Germany Professor G. Smit NIZO Food Research PO Box 20 6710 BA Ede The Netherlands Dr H.-E. Spinnler Laboratoire de Génie et Microbiologie des Procédés Alimentaires Instituto National de La Recherche Agronomique 78850 Thiverval-Grignon France Dr G. van den Berg NIZO Food Research PO Box 20 6710 BA Ede The Netherlands Dr T. van den Tempel Chr. Hansen A/S Cheese Culture Technology Bøge Allé 10-12 2970 Hørsholm Denmark

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Preface to the First Edition

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

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Preface to the Second Edition

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

P.F. Fox

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Preface to the Third Edition

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

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Diversity of Cheese Varieties: An Overview P.L.H. McSweeney, Department of Food and Nutritional Sciences, University College, Cork, Ireland G. Ottogalli, Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Sezione di Microbiologia Agraria, Alimentare, Ecologica, Milano, Italy P.F. Fox, Department of Food and Nutritional Sciences, University College, Cork, Ireland

Introduction A great diversity of cheeses are produced from the same raw materials (usually bovine, ovine, caprine or buffalo milks, lactic acid bacteria (LAB), coagulant and NaCl); indeed it has been said that ‘there is a cheese for every taste preference and a taste preference for every cheese’ (Olson, 1990). Although cheesemaking is an ancient art (see ‘Cheese: An Overview’, Volume 1), modern cheese production relies on the application of much science and technology, including the use of industrial enzymes, complex fermentations, sophisticated engineering and a dynamic biochemistry during ripening. Indeed, if cheese was developed today, it would be hailed as a triumph of biotechnology! Cheese production has a long history (see ‘Cheese: An Overview’, Volume 1) which is reflected in the wide range of technologies used for their manufacture. The idea of protecting and preserving the traditional diversity of foods, including cheese, commenced at the Paris Convention of 1883 where the term Appellation d’Origine Contrôlee (AOC) was introduced to recognize the specific heritage of food products from particular regions, while guaranteeing product authenticity (Bertozzi and Panari, 1993). This concept became widespread in Europe and was replaced by the EU scheme, Protected Designations of Origin (PDO), which applies to foodstuffs which are produced, processed and prepared in a given geographical area using recognized technology. Foods with the designation ‘Protected Geographical Indication’ (PGI) have a geographical link with a particular region during at least one stage of production, processing or preparation while ‘Foods with Tradition Speciality Guaranteed’ (TSG) status have a traditional character, either in their composition or means of production. A number of cheeses have PDO status (e.g., Roquefort, Stilton, Manchego, Grana, Padano, Parmigiano Reggiano, Gruyère de Comté). Unlike commercial trademarks, PDO denomination reflects a collective heritage and

may be used by all producers of a particular variety in a defined geographical area. PDO cheeses are protected by the European Union under various international agreements (Bertozzi and Panari, 1993). A list of cheeses with PDO status is shown in Table 1. Other varieties may be produced outside the country or region of origin, e.g., Cheddar, Emmental, Gouda, Gruyère and Camembert, but the name of the producing country is often included. The FAO/WHO has published standards for several major cheese varieties in various editions of Code of Quality Standards for Cheese forming part of the Joint FAO/WHO Codex Alimentarius (see www.codexalimentarius.net). The concept of a Codex Alimentarius evolved from a meeting of European Governments at the Italian city of Stresa in 1951 but the idea for such a Codex Alimentarius dates from the end of the nineteenth century; in the Austro-Hungarian Empire between 1897 and 1911, a collection of standards and product descriptions for a wide variety of foods was developed as the Codex Alimentarius Austriacus.

Classification Schemes for Cheese A considerable international trade exists in the principal varieties of cheese, many of which are produced in several countries but which may not be identical. To assist international trade, to provide nutritional information and perhaps for other reasons, e.g., research, a number of attempts have been made to develop classification schemes for cheeses. There is no definitive list of cheese varieties. Sandine and Elliker (1970) suggest that there are more than 1000 varieties. Jim Path (University of Wisconsin) has compiled a list of 1400 varieties of cheese (available at ww.cdr.wisc.edu). Walter and Hargrove (1972) described more than 400 varieties and listed the names of a further 400 varieties, while Burkhalter (1981) classified 510 varieties (although some are listed more than once).

Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 2: Major Cheese Groups ISBN: 0-1226-3653-8 Set ISBN: 0-1226-3651-1

Copyright © 2004 Elsevier Ltd All rights reserved

2 Diversity of Cheese Varieties: An Overview

Table 1 Cheeses with protected designations of origin (PDO) or protected geographical indication (PGI) Country

Variety

PDO

Belgium

Fromage de herve

X

Denmark

Danablu Esrom

Germany

Allgäuer Bergkäse Allgäuer Emmentaler Altenburger Ziegenkäse Odenwälder Frühstückskäse

X X X X

Anevato Batzos Feta Formaella Arachovas Parnassou Galotyri Graviera Agrafon Graviera Kritis Kalathakai Limnou Kasseri Katiki Domokou Kefalograviera Kopanisti Ladotyri Mytilinis Manouri Metsovone Pichtogalo Chanion San Michali Sfela Xynomyzithra Kritis

X X X X X X X X X X X X X X X X X X X

Cabrales Idiazábal Mahón Picón Bejes-Tresviso Queso de Cantabria Queso de l’Alt Urgell y la Cerdanya Queso de La Serena Picón Bejes-Tresviso Queso de Murcia Queso de Murcia al vino Queso Majorero Queso Manchego Queso Palmero o Queso de la Palma Queso Tetilla Queso Zamorano Quesucos de Liébana Roncal Abondance Beaufort

X X X X X X X X X X X X X

Bleu d’Auvergne Bleu des Causses Bleu du Haut-jura,de Gex, de Septmoncel Bleu du Vercors Brie de Meaux Brie de Melun Brocciu Corse ou brocciu Cantal ou Forme de Cantal ou Cantalet Camembert de Normandie Chabichou du Poitou

X X X

Greece

Spain

France

PGI

Country

Variety

PDO

France

Chaource Comté Crottin de Chavignol ou Chavignol Emmental de Savoie Emmental français est-central Epoisses de Bourgogne Fourme d’Ambert ou fourme de montbrison Laguiole Langres Livarot Maroilles ou Marolles Mont d’or ou vacherin du Haut-Doubs Morbier Munster ou Munster-Géromé Neufchâtel Ossau-Iraty Pélardon Picodon de l’Ardèche ou Picodon de la Drôme Pont-l’Evêque Pouligny-Saint-Pierre Reblochon ou reblochon de Savoie Rocamadour Roquefort Saint-Nectaire Sainte-Maure de Touraine Salers Selles-sur-Cher Tomme de Savoie Tomme des Pyrénées

X X X

X X

X X X X X X

X X X X X X X

PGI

X X X X X X X X X X X X X X X

X X X X X X X X X X X

Ireland

Imokilly Regato

X

Italy

Asiago Bitto Bra Caciocavallo Silano Canestrato Pugliese Casciotta d’Urbino Castelmagno Fiore Sardo Fontina Formai de Mut Dell’alta Valle Brembana Gorgonzola Grana Padano Montasio Monte Veronese Mozzarella di Bufala Campana Murazzano Parmigiano Reggiano Pecorino Romano Pecorino Sardo Pecorino Siciliano Pecorino Toscano Prouolone Valpadana Quartirolo Lombardo Ragusano

X X X X X X X X X X X X X X X X X X X X X X X X

Diversity of Cheese Varieties: An Overview 3

Table 1 continued Country

Variety

Italy

Raschera Robiola di Roccaverano Taleggio Toma Piemontese Valle d’Aosta Fromadzo Valtellina Casera

PDO X X X X X X

The Netherlands Boeren-Leidse met sleutels Kanterkaas,Kanternagelkaas, Kanterkimijnekaas Noord-Hollandse Edammer Noord-Hollandse Gouda

X X

Austria

X X

Portugal

PGI

X X

Gailtaler Almkäse Tiroler Almkäse/ Tiroler Graukäse Tiroler Bergkäse Tiroler Graukäse Vorarlberger Alpkäse Vorarlberger Bergkäse

X X X X

Queijo de Azeitão Queijo de Cabra Transmontano Queijo de Évora Queijo de Nisa Queijo do Pico

X X X X X

Country

Variety

Portugal

Queijo Mestiço de Tolosa Queijo Rabaçal Queijo São Jorge Queijo Serpa Queijo Serra da Estrela Queijo Terrincho Queijos da Beira Baixa

Sweden

Svecia

United Kingdom

Beacon Fell Traditional Lancashire cheese Bonchester cheese Buxton Blue Dorset Blue cheese Dovedale cheese Exmoor Blue cheese Single Gloucester Swaledale cheese, Swaledale ewe’s cheese Teviotdale cheese West Country Farmhouse Cheddar cheese White Stilton cheese, Blue Stilton cheese

PDO

PGI X

X X X X X X X X X X X X X X X X X X

Source: http://europa.eu.int/comm/agriculture/qual/en/pgi_01en.html

However, many of these varieties are very similar and should be regarded as variants rather than varieties. Walter and Hargrove (1972) suggested that there are probably only about 18 distinct types of natural cheese, no two of which are made by the same method, i.e., they differ with respect to: setting the milk, cutting the coagulum, stirring, heating, draining, pressing and salting of the curds or ripening of the cheese. They listed the following varieties as typical examples of the 18 types: Brick, Camembert, Cheddar, Cottage, Cream, Edam, Gouda, Hand, Limburger, Neufchatel, Parmesan, Provolone, Romano, Roquefort, Sapsago, Swiss, Trappist and whey cheeses. The authors acknowledged the imperfection and incompleteness of such a classification scheme and indeed a cursory glance at the list of the examples highlights this, e.g., listing Edam and Gouda as clearly distinct families appears highly questionable while exclusion of Feta and Domiati and all heat-acid coagulated cheeses appears to be major omissions. Attempts to classify cheese varieties exploit a number of characteristics of the cheese: • texture, which is dependent mainly on moisture content; • method of coagulation as the primary criterion, coupled with other criteria; • ripening indices.

Classification schemes based on texture

The difficulties in classifying cheese varieties were discussed by Schulz (1952) who reviewed earlier attempts to do so. Schulz (1952) was critical of these earlier schemes because they relied excessively on knowledge of the manufacturing process. He proposed a modified scheme consisting primarily of five groups based essentially on moisture content (moisture in fat-free cheese, MFFC): dried (40% MFFC), grated (40–49.9% MFFC), hard (50–59.9% MFFC), soft (60–69.9% MFFC) and fresh (70–82% MFFC). The fresh, soft, hard and grated groups were each sub-divided into two sub-groups (i.e., eight sub-groups) based on whether or not the cheeses were cooked and/or pressed. An interesting development was the sub-division of each of the eight sub-groups into six sub-sets (a–g) on the basis of the concentration of calcium in the fat-free, NaCl-free solids, which reflects the rate and extent of acidification: 2.5%, 2.1–2.5%, 1.6–2.0%, 1.1–1.5%, 0.6–1.0% and 0.6%. Davis (1965) discussed the problems encountered in attempting to classify cheese and suggested a number of possible schemes. One scheme (Table 2) was based on the rheological properties, or, more precisely, on the moisture content of the cheese. In fact, most schemes include a similar criterion. In a second scheme (Davis, 1965), cheeses were classified primarily into hard,

4 Diversity of Cheese Varieties: An Overview

Table 2 Suggested classification of cheeses based on rheological propertiesa Type

Moisture, %b

pV

pM

pS

Very hard Hard Semi-hard Soft

25 25–36 36–40 40

9 8–9 7.4–8 7.4

6.3 5.8–6.3 5.8 5.8

2.3 2–2.3 1.8–2 1.8

pV, viscosity factor, logarithmic scale; pM, elasticity factor, logarithmic scale; pS springiness factor, logarithmic scale. a From Davis (1965). b Suggested moisture levels appear to be very low.

semi-hard and soft (Table 3); varieties were listed within each category according to type of milk, method of coagulation, cutting of the coagulum, scalding of the curds, drainage of whey and method of salting and moulding. Walter and Hargrove (1972) classified cheese into eight families (Table 4). However, this scheme has a number of inconsistencies, e.g., traditionally, Brick and Münster cheeses are smear-ripened varieties but are listed in category 3.1 and are thus separated from the other smear cheeses in category 3.2. Likewise, although Mysost and Primost are unripened (category 4.2), they are quite hard. The species from which the milk is obtained was not included. Burkhalter (1981) classified 510 varieties based essentially on three criteria (Table 5): species of dairy animal (cow, sheep, goat, buffalo), moisture content and characteristic ripening agent. Scott (1986) also classified cheeses primarily on the basis of moisture content, i.e., hard, semi-hard and soft, and sub-divided these groups on the basis of cooking (scalding) temperature and/or secondary microflora (Table 6). The mechanism of coagulation was not considered by Scott (1986) and rennet-, acid- or acid/heatcoagulated cheeses are included in some groups. An alternative classification scheme suggested by Prof P. Walstra is shown in Table VIII in Fox (1993). Innovations were the use of the water:protein ratio rather than moisture content as the primary criterion for classification and replacement of cooking temperature by starter type, i.e., mesophilic, thermophilic. Classification schemes based on method of coagulation

The fundamental event in cheese manufacture is the conversion of liquid milk to a visco-elastic gel (coagulum). In fact one of the three coagulating agents may be used: rennet, acid and acid/heat, which suggests a clear primary criterion for classification. Rather surprisingly, the mechanism of coagulation was not used as a

classification criterion until Fox (1993) suggested the classification of cheeses into super-families based on the coagulating agent: • Rennet cheeses: most major international varieties. • Acid cheeses: e.g., Cottage, Quarg, Queso Blanco, Cream cheese. • Heat/acid: e.g., Ricotta, Manouri, Sapsago, Ziger, Schottenziger, some forms of Queso Blanco. Rennet-coagulated cheeses represent ⬃75% of total cheese production and almost all ripened cheeses. Acid-curd cheeses (‘Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels’, Volume 1; ‘Acid- and Acid/Rennet-Curd Cheeses’, Volume 2) represent ⬃25% of total cheese production and are generally consumed fresh. Coagulation by a combination of heat and acid is used for a limited number of varieties, including Ricotta and Manouri. Traditionally, they were by-products produced from the whey obtained from rennet-coagulated cheeses although today they are also produced from mixtures of milk and whey or even milk alone (see ‘Acid- and Acid/Rennet-Curd Cheeses: Part A Quark, Cream Cheese and Related Varieties, Part B Cottage Cheese, Part C Acid-heat Coagulated Cheeses’, Volume 2). A minor group of cheeses are produced in Norway by the concentration of whey and crystallization of lactose, e.g., Mysost. Fox (1993) suggested that the classification schemes of Davis (1965), Walter and Hargrove (1972) and Burkhalter (1981) can be applied to rennet-coagulated cheeses, which form the most complex family, but are not really applicable to the other two super-families since most are high-moisture, soft cheeses and most, normally, are not ripened. The classification scheme of Fox (1993) was expanded and modified by Fox et al. (2000). Rennetcoagulated varieties were subdivided into relatively homogeneous groups based on the characteristic ripening agent(s) or manufacturing technology. The most diverse family of rennet-coagulated cheeses are the internal bacterially ripened varieties which include most hard and semi-hard cheeses. The term ‘internal bacterially ripened’ is somewhat misleading since indigenous milk enzymes and residual coagulant also play important roles in the ripening of these cheese varieties. This group may be subdivided based on moisture content (extra-hard, hard or semi-hard), the presence of eyes or a characteristic technology (e.g., cooking/stretching of pasta-filata varieties or ripening under brine). Many varieties in large-scale industrial production are included in this group. Grana-type cheeses (extra-hard), which are often used in grated form, are characterized by a high cooking temperature during their manufacture (‘Extra-Hard Varieties’,

Table 3 Features of manufacture – summary of fundamental cheese types (modified from Davis, 1965)

Cheddar Semi-hard

Port du Salut Brick

Pecorino Edam Gouda Caciocavallo Soft Surfacesmear

Cambridge Limburg

Surface mould

Camembert

Mouldripened (blue veined) Acid coagulated

Roquefort

Cottage Sapsago

Cream

Cream





   

 ()  ()















 











 







 







 















   

 

 



 









 



 

 





 







 

Pressure



Hand



Hoop



Brine



()



 

Shaping Cheese



Salting

Curd



 

Hoop



Vat



Medium

 

Low

 

None

 

Small



Large

Very hard Large gas holes No gas holes Fairly firm, mild flavour Fairly strong, sweetish flavour Sheep’s milk Fairly firm Mellow Full flavour, long keeping Unripened Strong flavour, bacterial ripening Strong flavour, surface mould ripening Peppery flavour, internal mould ripening Soft lactic flavour, flavoured by herbs Made from cream

Ladled

Parmesan Emmental

Rennet

Very Hard

Acid

Characteristics

Drainage

Ripened

Cheese variety

Scalding

Skimmed

Type

Cutting

High

Method of coagulation

Milk















 

5

6 Diversity of Cheese Varieties: An Overview

Table 4 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 Ripened by bacteria, without eyes: Cheddar, Granular, Caciocavallo 2.2 Ripened by bacteria, with eyes: Emmental, Gruyère 3. Semi-soft 3.1 Ripened principally by bacteria: Brick, Münster 3.2 Ripened by bacteria and surface micro-organisms: Limburger, Port du Salut, Trappist 3.3 Ripened principally 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

Table 5 Classification of cheese according to source of milk, moisture content, texture and ripening agents* 1. 1.1

Cow’s milk Hard (42% H2O)

1.1.1 Grating cheese (extra-hard) 1.1.2 Large round openings 1.1.3 Medium round openings 1.1.4 Small round openings 1.1.5 Irregular openings 1.1.6 No openings 2.

Sheep’s milk Hard; semi-hard; soft; blue-veined; fresh

3.

Goat’s milk

4.

Buffalo’s milk

1.2

Semi-hard/ semi-soft (43–55% H2O) 1.2.1 Small round openings 1.2.2 Irregular openings 1.2.3 No openings 1.2.4 Blue veined

1.3

Soft (55% H2O)

1.4 Fresh, rennet

1.5 Fresh, acid

1.3.1 Blue veined 1.3.2 White surface mould 1.3.3 Bacterial surface smear 1.3.4 No rind

* Modified from (Burkhalter, 1981); unless otherwise stated, the cheeses are internal bacterially ripened.

1.6 Fresh

Diversity of Cheese Varieties: An Overview 7

Table 6 Classification of cheese according to moisture content, cooking temperature and secondary microfloraa Hard cheese (moisture content 20–42%) Low-scald Ns

Medium-scald Ns

High-scald Ns or Pr

Plastic curds Ns or Pr

Edam (NL) Gouda (NL) Cantal (F) Fontina (I) Cheshire (UK)

Cheddar (UK) Glouchester (UK) Derby (UK) Leicester (UK) Svecia (S) Dunlop (UK) Turunmaa (SF)

Grana (Parmesan; I) Emmental (CH) Gruyère (CH) Beaufort (F) Herrgardsost (S) Asiago (I) Sbrinz (CH)

Scamorza (I) Provolone (I) Caciocavallo (I) Mozzarella (I) Kaaseri (Gr) Kashkaval (YU) Perenica (Cz)

Semi-hard cheese (moisture content 44–55%; low-scald) Ns

Bs

Bv

St Paulin (F) Caerphilly (UK) Lancashire (UK) Trappist (BiH) Providence (F)

Herve (B) Limburg (B) Romadur (G) Münster (F) Tilsit (G) Vacherin-Mont d’Or (S) Remoudou (B) Srainbuskerkase (G) Brick (USA)

Stilton (UK) Roquefort (F) Gorgonzola (I) Danablu (D) Mycella (D) Wensleydale (UK) Blue Vinny (UK) Gammelost (N) Adelost (S) Tiroler-Graukäse (D) Edelpitzkäse (A) Aura (Ice) Cabrales (E)

Soft cheeses (moisture content 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 (P. camemberti); Bv, blue-veined internal mould (P. 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. Gruyère 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 Diversity of Cheese Varieties: An Overview

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

Enzyme-Modified Cheese Acid-Coagulated Cottage, Cream, Quarg

Dried Cheeses

Heat/Acid Coagulation Ricotta

Cheese

Concentration/Crystallization Mysost Processed Cheese Most varieties of cheese may be processed

Rennet-Coagulated

Natural Cheese

Surface-ripened Havarti Limburger Münster Port du Salut Trappist Taleggio Tilsit

Mould-ripened

Internal bacterially ripened

Internal mould Surface mould (usually P. camemberti ) (P. roqueforti ) Brie Roquefort Camembert Danablu Stilton

Cheeses with eyes Extra-hard

Hard

Grana Padano Parmesan Asiago Sbrinz

Cheddar Cheshire Graviera Ras

Semi-hard Caerphilly Mahon Monterey Jack

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

High-salt varieties Domiati Feta

Pasta-filata varieties Mozzarella Kashkaval Provolone

Dutch-type (Eyes caused by citrate metabolism) Edam Gouda

Figure 1 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

Class B Fresh cheeses (unripened); Interior – soft, exterior – rindless, ripening – absent; IM  2–5; IL  1–2

Class A Fresh cheeses, rarely ripened

Class

Class C Short ripened cheeses; Interior – soft, exterior – usually rindless or thin rind; IM1  2–10, IL2  1–5

Formatica

Formatica

Lacticinia

Group

Table 7 Classification of cheeses according to Ottogalli (1998, 2000a,b, 2001)

Family

Description

Examples

1

Yoghurt-like product, but with loss of some whey

2

Milk coagulated by addition of organic acid

3

Acid addition and heating of whey (goat or ewe)

4

Acid addition and heating of whey (cow) Acid addition and heating of cream Acid addition and heating of buttermilk

Lebneh (Middle East); Fromage Blanc (Switzerland, France); Sauer-milchkäse, 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)

5 6

7

Acid addition and heating of colostrum or beestings

1

Acid-rennet coagulation

2

Rennet-acid coagulation

3

Goat or sheep

4

Fresh-kneaded or plastic or stretched cheeses

5

Coagulum cut into cubes and/or flakes cooked, drained, washed and water cooled

1

Rindless, very short ripening phase

2

Thin rind, short ripening (1 month)

3

Same as C1 or C2 but from goats’ or ewe’s milk

4

Kneaded curds

5

White-brined

Petit Suisse, Pates fraiches (France); Frischkase Quargel (Germany); Cream cheese (USA) Gervais™ (France); Jonchée, Caillebotte (France); Primo sale (Italy). Caprino (Italy); Goat cheese (UK); Cadiz, Soria, Villeria (Spain); Bruscion (Switzerland) Mozzarella di bufala, Fiordilatte (Italy); Oaxaca (Mexico); Pizza cheese (America) Cottage (UK, USA); Huttenkase (Germany); Farkost (Sweden)

Crescenza (Italy); Butterkase (Austria); Cremoso (Argentina) Caciotta, Italico, Bel Paese™ (Italy); St. Paulin, Port Salut™ (France); Tetilla (Spain); Passendale (Belgium); Caerphilly (UK); Richelieu (Canada) Burgos, Azeitao, Puzol, Villalon (Spain); Capricorn goat (UK); Robiola di Roccaverano (Italy) Scamorza (Italy); Cascaval (Romania); Ostiepok (Czech Republic) Feta (Greece); Telemes (Romania); Domiati (Egypt); Brinza (Israel); Peynaz peynir (Turkey); Surati panir (India); Halloumi (Cyprus); Lightvan (Iran) continued

10 Diversity of Cheese Varieties: An Overview

Class E Blue-veined cheeses; Interior – soft to semi-soft, blue veins, exterior – soft rind with felt or smear; IM  60–70, IL  10–15

Class D Soft, surface-ripened cheeses; Interior – soft, exterior – felt of mould or smear; IM  25–35, IL  10–15

Class

Class F Semi-hard cheeses; Interior – semi-hard, exterior – hard rind; IM  10–15 (or depending on family); IL  Depends on family

Formatica

Formatica

Formatica

Group

Table 7 continued

Family

Description

Examples

1

White-moulded rind

2

Smear surface

3

Same as D1 or D2 or D4 but goats’ or ewes’ milk

4

Mould-ripened (white or blue) and smeary surface

Camembert, Caprice de Dieux, Brie, Coulommiers, Chource, Carré de l’Est (France); Bouchester (UK); Tomme de Vadois (Switzerland); Casanova (Denmark); Scimudin (Italy) Romadour (Belgium); Brick, Liederkranz (USA); Havarti, Esrom (Denmark); Epoisses, Langres, Livarot, Maroilles, Münster (France); Kernhem (The Netherlands); Ridder (Norway); Vacherin Mont d’Or (Switzerland); Limburger (Germany) Crottin, Chabichou, Bouche de Chèvre, Pouligny, Saint Maure, Rocamadour (France); Altenburger (Germany); Capricorn goat (UK) Taleggio, Quartirolo, Robiola (Italy); Chaumes, Pont l’Eveque, Reblochon (France)

1

Cows’ milk

2

White moulded rind

3

Ewes’ or goats’

1

Untextured, usually semi-cooked and pressed

2

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

3

4

Kneaded curds (‘pasta filata’)

5

Propionic cheeses. Big round eyes

6

Textured (and dry salted) curd

7

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-Iraty (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

1Index 2Index

Class

Family

Description

Examples

Class G Hard and extra-hard cheeses; Interior – hard, exterior – hard rind, long ripened; IM  depends on family, IL  depends on family

1

Untextured, usually cooked and pressed

2 3

Washed curd, long ripened Same as G1 but goats’ or ewes’ milk

4

Kneaded curds (‘pasta filata’)

5

Cheeses with eyes

6

Textured (and dry salted) curd (‘Cheddaring’)

7

Smeared rind The microbial coat causes the development of strong aroma

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) Gruyère (Switzerland, France); Puzzone di Moena (Italy); Tete de Moine (Switzerland)

Class H Cheeses made using various technologies

Miscellanea

Formatica

Group

Table 7 continued

1

Melted

2 3 4

Smoked Grated or fractionated Mixed with other ingredients (fruit, vegetables, spices)

5

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

6

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

7

Processed cheese, Spread cheese, Sottilette™ Oak-smoked Cheddar (United Kingdom) ‘Grating cheeses’ Friesan Clove cheese (NL); Sage Derby (UK); Kummelkasë, Käse mit Champignons (Germany); Sapsago (Switzerland); Ciboulette (France) Devon Garland (United Kingdom); Bruss (Italy); Kopanisti (Greece); Tupi (Spain); Fromage fort (France) Philadelphia™ (USA); Belgioioso™ (Italy)

‘Imitation cheeses’, Filled cheeses

of maturation (IM)  soluble N  100/total N. 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  0.8). The organoleptic and microbiological characteristics of the families of cheeses in Table 7 are summarized in Table 8. According to this classification, cheeses and related products can be presented as in Table 7 or as in Fig. 2. An advantage of the system of Ottogalli (1998, 2000a,b, 2001) is that it allows the comparison of

cheeses from all over the world and the classification of products with similar characteristics. A disadvantage stems from the detailed and sharp sub-division of cheeses which necessitates exact knowledge of their technology. In addition, some products may move from one category to another during ripening (e.g., varieties which are consumed as semi-hard cheeses early in ripening but later become extra-hard varieties), and some varieties which are in fact quite different (e.g., white-mould cheeses and smear-ripened cheeses) are in the same class (D), although in different families. In addition, Quarg and Queso Blanco are placed in different families whether they are made with (B2) or without (A1) rennet. Finally, cheeses made from ultrafiltration retentate are grouped together in Family H6 although they may in fact be quite similar to cheeses in other families made using traditional technology. Classification based on ripening indices

Davis (1965) suggested the possibility of classifying cheese according to the extent of chemical breakdown during ripening and expressed the view that it might be possible within a few years (from 1965) to classify cheese on the basis of chemical fingerprints; nearly 40

Table 8 Organoleptic and microbiological characteristics of the families of cheese described in Table 7 (Ottogalli, 1998, 2000a,b, 2001). See Table 7 for descriptions of the classes and families of cheese Cheeses character Soft

Rind

Body Semi-hard and hard

Rind

Microflora Rindless Rind with white surface mould Rind with smeared surface Rind with mould and smeared surface

No openings Blue-veined Brushed and cleaned during ripening Absence of cleaning operations

Body

No openings

Small openings

Large openings

Penicillium camemberti Geotrichum candidum Red-orange bacteria Penicillium spp. (or other moulds) and red-orange bacteria Lactic acid bacteria Penicillium roqueforti Microflora usually irrelevant

Relevant microflora (mainly moulds) Relevant microflora (mainly bacteria) Homofermentative lactococci and lactobacilli Heterofermentative lactococci and lactobacilli Propionic acid bacteria

Family/class A, B D1, D3 D2, D3 D4

B–G E1, E2, E3 F1, F2, F3, F5, F6 G1, G2, G3, G5, G6 F4, G4 F7, G7 F1, F3, F4, F6 G1, G3, G4, G6 F2, G2

F5, G5

Diversity of Cheese Varieties: An Overview 13

FRESH CHEESES (soft)

C

SHORT RIPENED CHEESES (soft)

E

QUARK

A3

RICOTTA (goat or sheep)

Whey derived A4

RICOTTA (cow)

A5

Cream derived MASCARPONE

A6

Buttermilk derived SKYR

A7

(Not available)

"LACTICINIA" fresh or ripened (soft or hard)

B

D

Milk derived A2

B1

PETITE SUISSE (acid curd)

B2

C1

CRESCENZA

C2

D1

CAMEMBERT (white mould rind)

E1

STILTON (cow)

PRIMO SALE

ASIAGO PRESSATO

B3

CAPRINO (goat or sheep)

B4 MOZZARELLA (plastic or kneaded; cow or buffalo)

B5

COTTAGE (cubes or flakes)

C3

CACIOTTA (goat or sheep)

C4 SCAMORZA (plastic or kneaded curd)

C5

FETA (Ripened under brine)

D2 LIVAROT (smeared surface)

TALEGGIO D3 TRONCHETTO DI CAPRA D4 (goat or sheep; smeared or (smeared or mould rind) mould rind)

E2

E3

Colostrum derived KOLOSTRUM KAESE (Not available)

SURFACE RIPENED CHEESES (soft)

CAMBOZOLA (white rind)

FORMATICA

A

LEBNEH

LACTICINIA

A1

ROQUEFORT (goat or sheep)

BLUE VEINED CHEESES (SOFT)

F1

MONTASIO (semicooked)

F2

FONTAL (washed curd)

F3 CANESTRATO (goat or sheep; ripened)

F4 CACIOCAVALLO (kneaded curd; ripened)

F5 MAASDAMER (cheese with eyes; ripened)

F6 CANTAL (structured; ripened)

F7

FONTINA (smeared rind; ripened)

G1

GRANA (cooked; ripened)

G2

EDAM (washed; ripened)

G3 PECORINO (goat or sheep; ripened)

G4 PROVOLONE (kneaded curd; ripened)

G5 EMMENTAL (cheese with eyes; ripened)

G6 CHEDDAR (structured; ripened)

G7

APPENZELLER (smeared rind; ripened)

H3

H4

H5

H6 SPECIAL TECHNOLOGY H7 IMITATION CHEESES CHEESES (vegetable Substitutes)

F SEMIHARD CHEESES

HARD CHEESES

H1 PROCESSED CHEESES H2 (melted cheeses) H

SMOKED

GRATED

"MISCELLANEA"

MIXED (different ingredients)

FROMAGES FORT

(ripened under special conditions)

MISCELLANEA

G

Figure 2 Examples of cheese from the principal groups of Ottogalli (1998, 2000a,b, 2001); see Table 7 for further details. (See Colour plate 1.)

years later it is still not possible to do so reliably although some progress has been made in this area. An obvious problem encountered when attempting to fingerprint cheeses chemically arises from the fact that ripening cheese is a dynamic system and therefore the age at which the cheeses are fingerprinted creates a major problem of definition. Within any particular variety there is considerable variability with respect to any particular characteristic for several reasons, including the type (specificity) of the rennet, the activity and specificity of several enzymes from the primary starter, secondary starter or adventitious bacteria, the differences in composition, including zonal differences due to salt diffusion and/or the evaporation of water. At present, there is insufficient information, even on the major varieties, to permit such a chemical fingerprinting. However, it seems worthwhile to speculate on some possible methods and criteria that might be useful for the classification of cheese. The most effective analytical methods are:

• Urea–polyacrylamide gel electrophoresis (PAGE) for resolving and identifying the large, water (or pH 4.6)-insoluble peptides. Sodium dodecyl sulphate (SDS)–PAGE or capillary electrophoresis should also be effective but to date have been used much less widely than urea–PAGE. • Reverse-phase (RP)–HPLC for resolving and perhaps identifying small, water (pH 4.6)-soluble peptides. Interfacing RP–HPLC and mass spectrometry (MS) should greatly facilitate the identification of small peptides. However, LC/MS is rarely used, possibly owing to cost. • The free amino acid profile of cheese may be a useful criterion for classification. While there is a considerable amount of information on the concentration of amino acids in a number of cheeses (see Fox and Wallace, 1997), we are not aware of its use as a criterion for cheese classification. • Profile of volatile compounds as determined by GC or GC–MS; attempts to classify cheeses based on their volatile flavour compounds are discussed in more

14 Diversity of Cheese Varieties: An Overview

detail in ‘Cheese Flavor: Instrumental Techniques’, Volume 1. Since many cheese varieties contain the same volatile compounds and many of the same proteolytic products, albeit at different levels (i.e., different varieties do not possess unique compounds), multivariate statistical approaches to data handling seem the most-promising. • Most cheese classification schemes are based on, or include, an item for texture (and thus on moisture and fat content). Texture is usually assessed subjectively or indirectly by determination of moisture content. Classification schemes based on rheological measurements would be precise and sensitive. Davis (1965) recommended such a scheme (Table 2). A number of chemical or physico-chemical studies have been performed to compare different cheese varieties (e.g., Smith and Nakai, 1990; Martin-Hernandez et al., 1992; Fox, 1993; McGoldrick and Fox, 1995; Dewettinck et al., 1997; Dirinck and De Winne, 1999; Dufour et al., 2001; Manca et al., 2001) or to distinguish between cheeses of the same variety differing in age or quality attributes (e.g., Fritsch et al., 1992; Rohm, 1992; O’Shea et al., 1996; Garcia-Palmer et al., 1997; Frau et al., 1998; Contarini et al., 2001; Peres et al., 2002).

Brief Descriptions of the Principal Categories of Cheese The objectives of Volume 2 of this book are to discuss the chemistry, physics and microbiology of the manufacture and the ripening of the major groups of cheese. Discussion of different cheese varieties generally follows the modified classification scheme of Fox et al. (2000; Fig. 1). In addition chapters are included on general aspects of cheese technology, processed cheese products, cheeses made from sheep’s and/or goats’ milk and uses of cheese as a food ingredient, including a brief discussion of enzyme-modified cheese. The remainder of this chapter will serve as an introduction to this volume by providing brief outlines of the science and technology of major groups of cheese. Most of these groups were reviewed in chapters in the second edition of this book, usually by authors from the same institution. Some groups of cheeses reviewed in the second edition have been omitted, e.g., Iberian cheeses, Italian cheeses, North European varieties, varieties produced in the Balkans and former USSR and non-European cheeses. However, these changes are due more to rearrangements than to omissions; the principal varieties from the above regions are covered under other headings, hopefully in a more objective way.

Extra-hard varieties

Extra-hard cheeses (‘Extra-Hard Varieties’, Volume 2) include a number of varieties which are ripened for a long period (usually 6–24 months). They are characterized by a hard granular texture, an aromatic flavour which can range from delicate to strong, very suitable for grating and are usually used as condiments for other foods, like pasta, as a topping or as a seasoning. Granatype cheeses, which have a brittle, grainy texture when mature, are made from raw cows’ milk which is partially skimmed; the starters used are thermophilic lactobacilli (often as a whey culture) and the curds are scalded in the vat at 50–55 °C for 20–30 min. During the long ripening period (c. 2 years), the temperature must not exceed 20 °C (to avoid fat liquefaction or ‘sweating’ and a propionic acid fermentation) and the rind is brushed and oiled frequently. The best known extra-hard cheeses are the Italian ‘Grana’ types (Grana Padano, Granone Lodigiano, Parmigiano Reggiano), Asiago, Bagozzo, Bra, Formai de Mut; in addition, the ‘Pecorino’ cheeses (Pecorino Romano, Pecorino Sardo, Pecorino Siciliano, Pecorino Toscano, Pecorino Pepato, Fiore Sardo), which are made from ewes’ milk, are included in this group, as are the Swiss varieties, Tete de Moine, Sbrinz, Sapsago, the Spanish cheeses, Cebrero, Pedroches and Manchego, the Greek cheeses, Kefalotiri and Gravera and Reggianito from South America. It must be emphasized that many of these cheeses may be consumed as hard or semi-hard cheeses at an earlier stage of ripening. Cheddar and related varieties

Cheddar cheese originated in England and is one of the most important cheese varieties made worldwide (see ‘Cheddar Cheese and Related Dry-salted Cheese Varieties’, Volume 2). It is a hard cheese, usually made from pasteurized, standardized cows’ milk which is coagulated using calf rennet or a rennet substitute. A mesophilic starter (usually defined strains of Lactococcus) is used to acidify the milk, and the coagulum is cut and cooked to 37–39 °C. The drained curds are ‘cheddared’, which traditionally involves forming beds of drained curds along the sides of the vat, cutting the beds into blocks and inverting and piling the blocks of matted curds at regular intervals. The cheddaring process allows time for acidity to develop in the curds (pH decreases from c. 6.1 to 5.4) and places the curds under gentle pressure, which assists in whey drainage. The curd granules fuse during cheddaring and the texture of the curd mass becomes rubbery and pliable. When the pH has reached c. 5.4, the blocks of curd are milled into small chips and dry-salted. The salted curds are moulded and pressed overnight. Traditionally, Cheddar cheese was ripened in

Diversity of Cheese Varieties: An Overview 15

insulated rooms without temperature control. However, more recently, Cheddar is matured at 4–8 °C (although a higher temperature, up to 14 °C, is used occasionally) for a period ranging from ⬃3 months to 2 years, depending on the maturity desired. Although the traditional manufacturing procedure is still practised on a farmhouse level and in small factories, most Cheddar cheese is now manufactured in highly automated factories using multiple vats which provide a semi-continuous supply of cheese curd. Cheddaring is mechanized using a large tower in which the curds at the bottom are pressed gently by the weight of that above or using a belt system. Milling and salting are also mechanized. Pressing and moulding are done automatically using a ‘block former’ (a large tower in which the salted curds are compressed by their own weight and a close texture is ensured by applying a vacuum). Most Cheddar is now produced in block form, although traditional Cheddar cheeses were cylindrical, weighing 10–20 kg. Annatto or similar colorant may be added to the milk for Cheddar cheese; the resulting product is known as ‘Red’ Cheddar. The British Territorial varieties, Cheshire, Derby, Gloucester and Leicester, are dry-salted cheeses manufactured by a protocol similar to that for Cheddar cheese. Cheese with propionic acid fermentation

Cheeses with a propionic acid fermentation (see Volume 2) are characterized by the presence of many large (up to ⬃2 cm in diameter) round openings, called ‘eyes’, due to the metabolic activity of propionic acid bacteria which metabolize lactate, produced by LAB from lactose, to propionic acid, acetic acid, CO2 and H2O; they also contribute to the development of the typical mild, nutty flavour of these varieties. For proper eye development, at least three conditions are necessary: • ripening of the cheese at 20–24 °C for a period to permit the rapid growth of propionic acid bacteria and to soften the cheese for eye development; • a relatively low level of salt to which the propionic acid bacteria are very sensitive; • the physical properties of the curd, which must be sufficiently elastic and flexible to contain the gas and form the eyes. Emmental, which was first manufactured in the Emm valley in Switzerland, is traditionally made from raw milk acidified by thermophilic LAB but this cheese is now produced in Switzerland, France, Germany, USA, Finland and elsewhere. The curds are cooked at c. 54 °C, which denatures most of the rennet. Immediately after cooking, the curds are moulded and acidification

occurs mainly after whey drainage, leading to a high level of calcium in the cheese which, together with the low rennet activity caused by the high cooking temperature (which results in a low level of proteolysis), gives the cheese an elastic texture. Emmental cheese is ripened for at least 4 months, including ⬃3–6 weeks at c. 22 °C for eye formation. Semi-hard cheeses with a propionic acid fermentation include Maasdamer, Leerdamer and Jarlsberg. Some other cheeses, e.g., Gruyère, may have eyes but they are not essential. Gouda and related varieties

Gouda cheese originated in The Netherlands but it, and similar varieties, is now produced worldwide from pasteurized cows’ milk acidified by a mesophilic starter containing citrate-positive bacteria (see ‘Gouda and Related Cheeses’, Volume 2). The milk is coagulated using calf rennet or a rennet substitute and, after the coagulum has been cut, the curds and whey mixture is stirred for 20–30 min. Some (c. 30%) of the whey is then removed and replaced by hot water which has the effect of cooking the curds and removing some lactose (which helps to control the development of acidity after the curds are moulded). After cooking at 36–38 °C and whey drainage, the curds are pressed under whey before being moulded, pressed and brine-salted. Traditionally, Gouda is coated with yellow wax and matured for 2–3 months at c. 15 °C (although some are ripened much longer, e.g., up to 2 years). Gouda is an internal, bacterially ripened cheese, the ripening of which is also characterized by the catabolism of citrate to diacetyl, other volatile flavour compounds and CO2. The CO2 produced causes a few small eyes in the cheese. Edam is a Dutch variety similar to Gouda but is made from semi-skimmed milk (c. 2.5% fat). It has a characteristic spherical shape and, traditionally, is covered with red wax. Other Dutch-type varieties include Maribo and Danbo (Denmark), Colonia and Hollanda (Argentina), Norvegia (Norway) and Svecia (Sweden). Pasta-filata cheeses

Pasta-filata varieties (see Volume 2) are also known as ‘kneaded’ or ‘plastic curd’ cheeses, the curds for which are heated to c. 55–60 °C, kneaded and stretched. Pasta-filata cheeses are characterized by a unique texture which is malleable, smooth, fibrous and sliceable. These qualities arise mainly from the cooking/stretching step which is common to all these varieties, whether they are soft, semi-hard or hard. By far the most important pasta-filata cheese is Mozzarella which originated in southern Italy and was made originally from buffalo milk (Mozzarella di bufala).

16 Diversity of Cheese Varieties: An Overview

Mozzarella di bufala is still made on a small scale but most Mozzarella is made from pasteurized, partly skimmed cows’ milk and is often referred to as Pizza cheese or, in the United States, low-moisture, partskimmed Mozzarella. The milk for this cheese is coagulated with calf rennet (or suitable substitute), acidified using Streptococcus thermophilus and a thermophilic Lactobacillus as starter; the coagulum is cut and the curds/whey mixture cooked to c. 41 °C. The whey is drained off and the curds are held to allow acidification (and may be cheddared). When the curd pH reaches 5.1–5.3, the curds are heated, kneaded and stretched in hot water or dilute brine (c. 78 °C to a curd temperature of c. 58–60 °C) by hand, in the same fashion as a baker might knead dough, or mechanically, which is usually used in industry. Mozzarella cheese may be brine- or dry-salted and is usually consumed within a few weeks of manufacture; traditional Mozzarella is consumed as soon as possible after manufacture. Pasta-filata cheeses can be consumed fresh (often as a topping on pizzas) or ripened (semi-hard or hard) or smoked. Mozzarella is used mainly as a pizza topping for which its principal characteristics are its physicochemical functional properties, especially meltability and stretchability. The functionality of biologically acidified Mozzarella improves to a maximum after ripening for ⬃2 weeks at 6–8 °C and then deteriorates due to excessive proteolysis. Chemically acidified Mozzarella is very functional immediately after manufacture. In addition to the use of Mozzarella cheese as a pizza topping, pasta-filata varieties include Mozzarella di Bufala (buffalo milk), Mozzarella di vacca (cows’ milk; also called Fiordilatte, Scamorza or Provola), Caciocavallo, Cascaval, Kashkaval, Provolone, Kasseri and Kasar peyniri.

without cooking, into moulds to drain. When the curd is sufficiently cohesive, the moulds are removed and the cheese is cut into pieces and salted. The cheese pieces are then transferred to barrels or tin-plated cans, covered by a brine solution (c. 14% NaCl) and ripened at 14–16 °C for c. 7 days until the pH has decreased to c. pH 4.5. The cheese-containing cans are then transferred to rooms at 3–4 °C and stored for at least 2 months. Surface mould-ripened varieties

Surface mould-ripened varieties (e.g., Camembert and Brie) are soft cheeses characterized by the growth of Penicillium camemberti on the cheese surface. Mould spores may be added to the cheesemilk or sprayed onto the cheese after manufacture. Cheese milk is acidified using a mesophilic starter and coagulated using rennet extract. After the coagulum has formed, it is usually ladled directly, without cutting, into moulds, where drainage occurs. The cheeses are usually brine-salted and ripened at c. 12 °C for 10–12 days for mould development. As discussed in ‘Metabolism of Residual Lactose and of Lactate and Citrate’, Volume 1 and ‘Surface Mould-ripened Cheeses’, Volume 2, the ripening of white-mould cheese is characterized by the extensive catabolism of lactate at the surface of the cheese by the mould, causing an increase in the pH of the surface zone (and thus creating a pH gradient from the surface to the core of the cheese) and the migration of lactate from the core. Calcium phosphate precipitates at the elevated pH of the surface and soluble calcium phosphate migrates through the cheese towards the surface. These changes, together with proteolysis, cause considerable softening of the cheese and mature whitemould varieties may flow under their own weight. Blue cheese

Cheeses ripened under brine

Feta, Domiati and related cheeses (e.g., Brinza, Beli Sir, Telemes, Kareish, Beyaz Peiniri; see ‘Cheese Varieties Ripened in Brine’, Volume 2), evolved in the eastern Mediterranean and Balkan regions; they are also known as ‘pickled cheeses’, so-called because they are ripened under brine. Feta is a Greek cheese made from sheep’s milk with PDO status. However, similar whitebrined cheeses are also made from pasteurized cows’ milk on a large industrial scale outside Greece, often using ultrafiltration technology (see ‘Pasta-Filata Cheeses’, Volume 1). Milk for Feta cheese is coagulated using rennet (which may also have lipase activity) and acidified using a thermophilic or mesophilic lactic starter. The coagulum is cut into small cubes and scooped,

Blue cheese varieties (‘Blue Cheese’, Volume 2) are characterized by blue/green veins throughout the cheese caused by the growth of Penicillium roqueforti. The milk for these varieties is coagulated by rennet extract; the curds are acidified using a mesophilic lactic culture and are cooked at a low temperature before being transferred to moulds. Some varieties of blue cheese are salted by repeated surface application of dry NaCl while others are brine-salted. The salted cheeses are ripened at a temperature and relative humidity which favour mould growth. Since P. roqueforti requires O2 for growth, the texture of Blue cheese must be open to allow the fungal spores and hyphae to germinate and grow. This open texture is achieved by encouraging mechanical openings during manufacture (by not pressing the curds after moulding) and by piercing the

Diversity of Cheese Varieties: An Overview 17

cheeses with needles (by hand or a special machine). The ripening of Blue cheese is characterized by extensive lipolysis. Blue cheeses have a soft texture and a strong flavour dominated by n-methyl ketones which are produced by the mould from fatty acids. Blue cheese varieties include Bleu d’Auvergne, Cabrales, Gorgonzola, Danablu (Danish Blue) and Stilton, all of which are made from cows’ milk, and Roquefort which is made using sheep’s milk. Bacterial surface-ripened cheese

Bacterial surface-ripened (‘smear-ripened’) cheeses are a diverse group of varieties characterized by the growth of a complex Gram-positive bacterial flora on the surface during ripening (‘Bacterial Surface-ripened Cheeses’, Volume 2). Soft smear cheeses usually are acidified using a mesophilic culture, are not cooked to a high temperature and are brine-salted. These cheeses have a high moisture content and are typically moulded as small cylinders (⬃200 g), with a high surface area:volume ratio which allows the surface smear to have an important influence on the characteristics of the mature cheese. During manufacture, the surface of the cheeses is washed periodically with a brine solution, a process referred to as ‘smearing’. In many factories, old cheeses (which have fully developed surface microflora) are smeared first and the same smear liquid is used to smear young cheeses, which are thus inoculated. This practice, called ‘old-young’ smearing, assists in the development of the surface microflora, but has been criticized on the grounds of hygiene. Soon after manufacture, the surface microflora of smear cheeses is dominated by yeasts (e.g., Debaroymces hansenii) and Geotrichum candidum. Growth of these micro-organisms deacidifies the cheese surface and encourages the growth of coryneform bacteria (e.g., Corynebacterium, Arthrobacter, Brevibacterium), Micrococcacae and Staphylococcus. These bacteria gain access to the cheese from the milk (particularly for raw milk cheeses) or through post-pasteurization contamination. These cheeses are characterized by a strong aroma and high levels of proteolysis and lipolysis, mainly at their surface. Sheep’s and goats’ milk cheeses

Although sheep and goats are minor dairying species (each produces ⬃2% of total world milk production compared with ⬃11% and ⬃85% for buffalo and cattle, respectively), they are quite significant in Mediterranean and Balkan countries, where most of their milk is used for cheese production. Many famous and popular cheeses are produced from sheep’s milk, e.g., Roquefort, Feta, the various Pecorino varieties, Kashkaval and

Manchego. A more complete list of cheeses produced from sheep’s and goats’ milks is given by Kalantzopoulos (1993), and ‘Cheeses Made from Ewes’ and Goats Milk’, Volume 2 is devoted to goats’ and ewes’ milk cheeses. These cheeses are considered together because the conditions of management of these relatively small animals are very similar and in many countries they are farmed in mixed flocks. In many cases, due also to the limited lactation period, cheeses are prepared from the mixed milk of these two species. Goats’ and ewes’ milk cheeses are produced mainly around the Mediterranean basin and in the Balkans. The gross composition of ewes’ milk is markedly different from that of goats’ and cows’ milks, which have generally similar gross composition, although goats’ and cows’ milks differ in many respects, including their proteins and fatty acid profiles. These differences influence the characteristics of cheeses made from sheep’s or goats’ milk. Goats’ milk cheeses are usually consumed fresh or ripened for a short period of time. Sheep’s milk contains high levels of fat and protein, which are the main cheesemaking constituents. The coagulum is firm, the syneresis is rapid and the NaCl diffusion is slow due to the low moisture content of these cheeses. By modifying the cheesemaking technology, it is possible to obtain a range of cheeses (fresh, short-, medium- and long-ripened) from ewes’ milk. Acid-curd cheeses

Acid-coagulated cheeses are varieties for which milk or cream is coagulated on acidification to c. pH 4.6. Acidcurd cheeses were perhaps the first type of cheese produced since such products may arise from the souring of milk by the adventitious microflora. These cheese varieties are distinguished from yoghurt because their manufacture involves dehydration by removal of at least some whey. Acidification is usually achieved by the action of a mesophilic starter culture but direct acidification is also practised. A small amount of rennet may be used in certain varieties (e.g., Cottage or Quarg) to increase the firmness of the coagulum and to minimize casein loss in the whey but its use is not essential. The coagulum may or may not be cut or cooked during manufacture but it is not pressed. Acid-coagulated cheeses (e.g., Cream, Cottage, Quarg, some Queso Blanco) are characterized by a high moisture content and are usually consumed soon after manufacture. Acid-coagulated varieties represent ⬃25% of total cheese production (considerably higher in some countries, ‘Cheese: An Overview’, Volume 1). They are usually consumed when fresh although there are some minor varieties of ripened acid-curd cheese. The acid coagulation of milk is described in ‘Formation, Structural Properties and Rheology of Acid-coagulated

18 Diversity of Cheese Varieties: An Overview

Milk Gels’, Volume 1, and acid-coagulated cheeses are discussed in detail in ‘Acid- and Acid/Rennet-Curd Cheeses: Part A Quark, Cream Cheese and Related Varieties, Part B Cottage Cheese’ Part C Acid-heat Coagulated Cheeses’, Volume 2. Cheeses coagulated by a combination of heat and acid

A small group of cheeses are produced by a combination of heat and acid. The best-known and perhaps the most important member of this group is Ricotta, an Italian cheese variety (the name derives from ricottura, ‘reheating’) which is produced from rennet cheese whey, perhaps with some milk added, by heatinduced coagulation (85–90 °C) and some acidifying agent (e.g., lemon juice or vinegar). Ricotta curd is transferred to moulds surrounded by ice where drainage occurred. Mascarpone is made by a process similar to that for Ricotta except that it is made from cream and a slightly higher cooking temperature is used. The resulting cheese is creamier than Ricotta and is usually salted at a low level and whipped and formed into a cylindrical shape. Other heat-/acid-coagulated varieties and their country of origin include Ricotta Forte (Italy), Brocciu (Corsica), Cacio-ricotta (Italy, Malta), Mizthra and Manouri (Greece) and Ziger (former Yugoslavia). Some of these varieties were described by Kalantzopoulos (1993) and some aspects are discussed in ‘Acid- and Acid/Rennet-Curd Cheeses: Part A Quark, Cream Cheese and Related Varieties, Part B Cottage Cheese, Part C Acid-heat Coagulated Cheeses’, Volume 2. Processed cheese products

Processed cheeses differ from natural cheese by not being made directly from milk but from various ingredients such as natural cheese (usually), emulsifying salts, milk solids, butter oil, other dairy ingredients, vegetable oils or other ingredients (Fox et al., 2000; see ‘Pasteurized Processed Cheese and Substitute/Imitation Cheese Products’, Volume 2). Processed cheese is produced by blending shredded natural cheeses, varying in maturity, with emulsifying salts and often other ingredients, and heating the blend under vacuum with constant agitation until a homogeneous blend is obtained. Although connoisseurs of cheese often regard processed cheese as inferior to natural cheese, the former has a number of advantages, including stability and consistency and they provide an outlet for inferior quality cheese which might otherwise be difficult to sell. The nutritional value of processed cheese is generally similar to that of natural cheese; although it has usually a higher sodium

content than the latter, this can be reduced (see ‘Pasteurized Processed Cheese and Substitute/Imitation Cheese Products’, Volume 2). Reducing the fat content of processed cheese has less undesirable consequences than for natural cheese. Since processed cheese can be produced in a wide range of flavours, shapes and consistencies, it is particularly popular for ingredient applications. About 2  106 tonnes of processed cheese are produced annually, i.e., ⬃14% of natural cheese. Cheeses made for use as food ingredients

In addition to processed cheese and cheese analogues (see below), most of which are used in ingredient applications, an increasing proportion of natural cheeses produced worldwide is consumed as an ingredient in other food products (see Guinee, 2003; ‘Cheese as an Ingredient’, Volume 2). While many traditional varieties have flavour or functional properties amenable to their use as ingredients (e.g., low-moisture Mozzarella for use as a pizza topping), it is likely that new ‘varieties’ of natural cheese will evolve in the future to meet requirements for cheese with tailor-made functional properties. Enzyme-modified cheeses (EMCs) are products with concentrated cheese flavours formed by the enzyme-catalysed hydrolysis of cheese curd or other ingredients by the action of exogenous proteinase, peptidase and/or lipase preparations (see Kilcawley et al., 1998; Wilkinson and Kilcawley, 2003; ‘Cheese as an Ingredient’, Volume 2). The advantages of EMCs over other sources of cheese flavours are their flavour intensity, range of flavours available, reduced production costs and shelf-life. Because of their high flavour intensity, EMCs are typically added as flavourings to foods at a very low level (c. 0.1%, w/w). Enzyme-modified cheeses are relatively new products, especially on a commercial scale. It seems very likely that their production will increase, made possible by the availability of purer and more specific enzymes and selected cultures. At present, Cheddarlike EMCs are the principal products but it is likely that the production of other varieties will increase. At present, EMCs are used only as ingredients; however, with increased knowledge of the biochemistry of cheese ripening in general, and of EMCs in particular, and of the flavour impact compounds in various cheese varieties, it seems conceivable that EMCs may evolve into table cheeses. Norwegian whey ‘cheese’

A unique type of ‘cheese’ is produced in Norway by evaporation of water from whey by concentration to ⬃80% total solids and crystallization of the lactose. This

Diversity of Cheese Varieties: An Overview 19

type of cheese originated in Norway. Strictly speaking, it could be argued that such varieties are not cheeses in sensu stricto. These cheeses (‘Brunost’, brown cheese) are characterized by having a smooth but firm body, a sweet, caramel-like flavour and a long shelf-life. Sweet whey is the usual starting material although acid whey may be used for some brands. Sometimes, skim milk or cream is added to the whey to give a whiter, smoother product. Types of Brunost include Primost, Gjetost, Mysost, Niesost, Fløtemyost and Gudbrandsdalost. The manufacture of these cheeses involves concentration of whey (or whey/cream mixture) by evaporation to high total solids to form a plastic mass. The Maillard reaction is encouraged and is important for the final colour and

flavour of the product. The concentrate is then cooled, kneaded and packaged. Crystallization of lactose is controlled so as to avoid sandiness in the product. Non-European cheeses

The majority of commercially important cheese varieties originated in Europe, and Europe and North America remain the most important regions for cheese production (see ‘Cheese: An Overview’, Volume 1). However, numerous minor cheeses are produced in Asia, Africa and Latin America, some of which are listed in Table 9 and were discussed by Phelan et al. (1993).

Table 9 Some non-European cheese varieties (modified from Phelan et al., 1993). Varieties discussed elsewhere in this volume are not listed Country Asia Afghanistan Bangladesh Bhutan

China India

Indonesia Iran Iraq Jordan Lebanon

Nepal

Pakistan Philippines Qatar Saudi Arabia Syria

Cheese

Remarks

Karut Kimish Panier Chhana Ponir Chhana Churtsi Durukhowa

Very hard cheese made from skim milk Semi-hard unripened cheese obtained by acid coagulation Acid coagulation of boiled milk Semi-hard ripened cheese (as above) Hard cheese made from yak and chauri milk Hard, rubbery cheese made from yaks’ and chauris’ milk (known as Chugga or Chhurpi in Nepal) Similar to Edam Acid-curd cheese from inner Mongolia Sour milk cheese made from cows’ milk Soft heat-acid coagulated variety Fresh rennet cheese made from heated milk Sort cheese made from cows’ and buffaloes’ milk coagulated with vegetable rennet (bromelain) Variety ripened under brine similar to Feta Similar to Liqvan Hard brittle cheese with a sharp flavour Hard sun/air dried variety made from sheep’s or goats’ buttermilk Hard cheese made from sheep’s and goats’ milk Soft fresh cheese made from whole milk White cheese varieties

Long Giang Hurood Chhanna Paneer Panir Tahu Susu Atau Dadih Liqvan ‘White cheese’ Awshari Djamid Shankalish Akawieh Baladi/Baida/ Hamwi Chelal Karichee ‘Fresh cheese’ Umbris Chhana Chhurpi Shosim Langtrang Panir Peshawari Kesong Puti ‘White cheese’ Ekt Mesanarah Medaffarah Shankalish

Cheese in the form of strings or ropes Soft whey cheese Soft rennet-coagulated cheese Soft spreadable cheese made from raw goats’ milk (as above) Similar to Durkhowa Soft cheese made from yaks’ and chauris’ milk Semi-hard cheese made from yaks’ and chauris’ milk Soft cheese variety Semi-hard cheese made from whole or partly skimmed cows’ milk Soft fresh cheese made from carabao and cows’ milk (as above) Sun-dried cheese made from sheep’s buttermilk Sun-dried rennet-coagulated cheese made from sheep’s milk Pasta-filata variety made from sheep’s milk Rennet coagulated cheese made from partially skimmed milk continued

20 Diversity of Cheese Varieties: An Overview

Table 9 continued Country

Cheese

Remarks

Asia Turkey

Beyaz Peyneri

Yemen

Kasar Peyneri Mihalic Peyneri Tulum Peyneri Aomma Taizz

Semi-hard cheese ripened under brine made from sheep’s milk or mixtures of milks Hard, pasta-filata variety Hard cheese made from raw sheep’s milk and ripened under brine Hard cheese made from sheep’s milk or mixtures of milks Pasta-filata variety No details available

Africa Algeria

Benin

Chad Dem.Rep.Congo Egypt

Ethiopia Kenya Madagascar Mali Niger Nigeria

Sudan

Latin America Argentina Bolivia

Brazil

Chile Colombia Costa Rica Cuba Dominican Republic Ecuador Honduras

Takammart Aoules Takamart Woagachi/Wagashi Wagassirou/ Gassigue Pont Belie Mashanza Ras Karish/Kareish Daani Mish Ayib Mboreki Ya Iria ‘Fromage’ ‘Fromage blanc’ Wagashi Tchoukou Wara/Awara Chukumara Dakashi ‘Country cheese’ Karish Braided cheese Gibbna Mudafera ‘White cheese’ Goya Tafi Altiplano Quesillo Queso Benianco/ Quieso Chaqueno Queijo de Coalho Queiso de Manteiga Queijo Minas Queijo Prato Chanco ‘Queso Blanco’ Palmito Patagras Queso de Freir Queso Andino Quesillo de Honduras

Rennet-coagulated cheese made from goats’ milk and air/sun-dried Heat/acid coagulated cheese made from buttermilk and air/sun-dried Rennet-coagulated cheese made from goats’ milk and sun-dried Soft fresh cheese made from cows’ milk coagulated using the sap of Calotropis procera Fresh cheese made from goats’ or sheep’s milk Soft fresh cheese made from cows’ milk Hard, bacterially ripened variety Fresh, low salt acid-coagulated cheese variety Soft cheese made from sheep’s or sheep’s/goats’ milk Karish cheese ripened in Mish (pickling solution) Heat/acid coagulated variety made from buttermilk Fresh soft cheese made from cows’ or goats’ milk Semi-hard cheese made from cows’ milk Fresh soft cheese made from skimmed cows’ milk (see above) Hard sun-dried cheese made from various milks Soft unripened variety similar to Wagashi Tough-textured cheese Heat-coagulated colostrum Hard cheese variety (see above) Semi-hard, braided cheese variety Similar to Feta or Domiati Semi-hard cheese made from cows’ milk Soft white cheese ripened under brine Hard, ripened cheese made from cows’ milk Semi-hard ripened cheese made from raw whole milk Soft fresh cheese made from raw whole cows’ and sheep’s milk Fresh, unripened soft cheese. Also known as Banela in Mexico, Paraguay cheese in Paraguay and Queso Blanco in Nicaragua Semi-hard ripened cheese made from whole cows’ milk Semi-hard ripened cheese Processed cheese Semi-hard cheese made from raw cows’ milk Semi-hard ripened cheese Semi-hard ripened cheese made from whole cow’s milk Generic name for rennet- and acid-coagulated cheeses Pasta-filata cheese made from raw whole cows’ milk Semi-hard, ripened cheese Type of Queso Blanco consumed after frying Soft, ripened cheese Pasta-filata cheese made from cows’ milk

Diversity of Cheese Varieties: An Overview 21

Table 9 continued Country Latin America Mexico

Peru Uruguay Venezuela

Cheese

Remarks

Chihuahua Cotija Oaxaca Panela Queso Andino Requeson Colonia Yamandu De Mano Guayanes Llanero/Americano

Semi-hard, ripened cheese Hard, ripened cheese made from cows’ or goats’ milk Pasta-filata cheese variety Fresh, unripened cheese (see above) Heat/acid coagulated cheese similar to Ricotta Semi-hard, ripened cheese made from cows’ milk Semi-hard, ripened cheese made from cows’ milk Semi-hard unripened pasta-filata cheese Semi-hard unripened cheese Similar to Queso Blanco

Imitation and substitute cheese products

A wide range of imitation and substitute cheese products are produced worldwide, which may be classified into three broad categories: analogue cheese, filled cheeses and tofu-based products. Cheese analogues are cheese-like products produced by blending various oils/fats, proteins (usually rennet casein), flavours and other ingredients with water into a smooth homogeneous cheese-like blend with the aid of heat, shearing forces and emulsifying salts (Guinee, 2003). Analogues of low-moisture Mozzarella, Cheddar, Monterey Jack and processed (Cheddar) cheeses are produced and have the advantages of being cheaper and more easily manufactured than natural cheese; their functional properties may be tailor-made for specific applications. Developments in EMC technology should make it possible to improve and diversify the flavour of analogue cheese products. Filled cheeses differ from natural cheeses in that the milkfat is partially or totally replaced by vegetable oil which is dispersed using high-speed mixing and homogenization in skim milk or skim milk reconstituted from various dairy ingredients such as skim milk powder, whey and total milk protein dispersed in water. The filled milk is then used as the starting material for conventional in-vat cheesemaking (Fox et al., 2000). Tofu is a cheese-like product produced from soybeans which has been a staple food in the Orient for many centuries. Although the appearance resembles that of fresh cheese, and has similar culinary applications, its physico-chemical properties are clearly different from all the classes described in this chapter.

References Bertozzi, L. and Panari, G. (1993). Cheeses with Appellation d’Origine Contrôlée (AOC): factors that affect quality. Int. Dairy J. 3, 297–312.

Burkhalter, G. (1981). Catalogue of Cheese. Document 141, International Dairy Federation, Brussels, Belgium. Contarini, G., Povolo, M., Toppino, P.M., Radovic, B., Lipp, M. and Anklam, E. (2001). Comparison of three different techniques for the discrimination of cheese: application to the ewe’s cheese. Milchwissenshaft 56, 136–140. Davis, J.G. (1965). Cheese, Vol. 1, Basic Technology, Churchill Livingstone, London. Dewettinck, K., Dierckx, S., Eichwalder, P. and Huyghebaert, A. (1997). Comparison of SDS-PAGE profiles of four Belgian cheeses by multivariate statistics. Lait 77, 77–89. Dirinck, P. and De Winne, A. (1999). Flavour characterisation and classification of cheeses by gas chromatographic–mass spectrometric profiling. J. Chromatogr. 847, 203–208. Dufour, E., Devaux, M.F., Fortier, P. and Herbert, S. (2001). Delineation of the structure of soft cheeses at the molecular level by fluorescence spectroscopy – relationship with texture. Int. Dairy J. 11, 465–473. Fox, P.F. (1993). Cheese: an overview, in, Cheese: Chemistry, Physics and Microbiology, Vol. 1, 2nd edn, P.F. Fox, ed., Chapman & Hall, London. pp. 1–36. Fox, P.F. and Wallace, J.M. (1997). Formation of flavour compounds in cheese. Adv. Appl. Microbiol. 45, 17–85. Fox, P.F., Guinee, T.P., Cogan, T.M. and McSweeney, P.L.H. (2000). Fundamentals of Cheese Science, Aspen Publishers, Gaithersburg, MD. Frau, M., Simal, S., Femenia, A. and Rossello, C. (1998). Differentiation and grouping of chemical characteristics of Mahon cheese. Z. Lebensm. Unters. Forsch. 207, 164–169. Fritsch, R.J., Martens, F. and Belitz, H.D. (1992). Monitoring Cheddar cheese ripening by chemical indexes of proteolysis. 1. Determination of free glutamic acid soluble nitrogen and liberated amino groups. Z. Lebensm. Unters. Forsch. 194, 330–336. Garcia-Palmer, F.J., Serra, N., Palou, A. and Gianotti, M. (1997). Free amino acids as indices of Mahon cheese ripening. J. Dairy Sci. 80, 1908–1917. Guinee, T.P. (2003). Cheese as a food ingredient, in, Encyclopedia of Dairy Sciences, Vol. 1, H. Rogenski, J.W. Fuquay and P.F. Fox, eds, Academic Press, London. pp. 418–427. Kalantzopoulos, G.C. (1993). Cheese from ewes’ and goats’ milk, in, Cheese: Chemistry, Physics and Microbiology,

22 Diversity of Cheese Varieties: An Overview

Vol. 2, 2nd edn, P.F. Fox, ed., Chapman & Hall, London. pp. 507–553. Kilcawley, K.N., Wilkinson, M.G. and Fox, P.F. (1998). Enzyme-modified cheese. Int. Dairy J. 8, 1–10. Manca, G., Camin, F., Coloru, G.C., Del Caro, A., Depentori, D., Franco, M.A. and Versini, G. (2001). Characterization of the geographical origin of Pecorino Sardo cheese by casein stable isotope (C-13/C-12 and N-15/N-14) ratios and free amino acid ratios. J. Agric. Food Chem. 49, 1404–1409. Martin-Hernandez, C., Amigo, L., Martinalvarez, P.J. and Juarez, M. (1992). Differentiation of milks and cheeses according to species based on the mineral content. Z. Lebensm. Unters. Forsch. 194, 541–544. McGoldrick, M. and Fox, P.F. (1995). Intervarietal comparison of proteolysis in commercial cheese. Z. Lebensm. Unters. Forsch. 208, 90–99. Olson, N.F. (1990). The impact of lactic acid bacteria on cheese flavor. FEMS Microbiol. Lett. 87, 131–147. O’Shea, B.A., Uniacke Lowe, T. and Fox, P.F. (1996). Objective assessment of Cheddar cheese quality. Int. Dairy J. 6, 1135–1147. Ottogalli, G. (1998). A global comparative method for the classification of world cheeses (with special reference to microbiological criteria). Ann. Microbiol. Enzimol. 48, 31–58. Ottogalli, G. (2000a). A global comparative method for the classification of world cheeses (with special reference to microbiological criteria). Revised edition. Ann. Microbiol. 50, 151–155.

Ottogalli, G. (2000b). Proposta di aggiornamento nella classificazione dei formaggi con particolare riferimento agli aspetti microbiologici. Alimenta 8, 147–165. Ottogalli, G. (2001). Atlante dei Formaggi, Hoepli, Milan. Peres, C., Viallon, C. and Berdague, J.L. (2002). Curie point pyrolysis–mass spectrometry applied to rapid characterisation of cheeses. J. Anal. Appl. Pyrol. 62, 161–171. Phelan, J.A., Renaud, J. and Fox, P.F. (1993). Some nonEuropean cheese varieties, in, Cheese: Chemistry, Physics and Microbiology, Vol. 2, Major Cheese Groups, 2nd edn, P.F. Fox, ed., Chapman & Hall, London. pp. 421–466. Rohm, H. (1992). Regional classification of Swiss cheese based on its chemical composition. Z. Lebensm. Unters. Forsch. 194, 527–530. 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. Schulz, M.E. (1952). Klassifizierung von Kasë. Milchwissenshaft 7, 292–299. Scott, R. (1986). Cheesemaking Practice, Elsevier Applied Science Publishers, London. Smith, A.M. and Nakai, S. (1990). Classification of cheese varieties by multivariate analysis of HPLC profiles. Can. Inst. Food Sci. Technol. J. 23, 53–58. Walter, H.E. and Hargrove, R.C. (1972). Cheeses of the World, Dover Publications, Inc., New York. Wilkinson, M.G. and Kilcawley, K.N. (2003). Enzyme modified cheese, in, Encyclopedia of Dairy Science, Vol. 2, H. Roginski, J.W. Fuguay and P.F. Fox, eds, Academic Press, London. pp. 434–437.

General Aspects of Cheese Technology R.J. Bennett, Senior Lecturer in Dairy Technology, Institute of Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand K.A. Johnston, Principal Research Technologist, Fonterra Research Centre, Palmerston North, New Zealand

Introduction Cheesemaking involves the conversion of liquid milk (an unstable, bulky but highly nutritious raw material) into cheese (a stable, flavoursome, concentrated product that provides eating pleasure and has an extended shelf-life). Cheesemaking has been practised for many thousands of years, for most of the time as a cottage industry. Towards the end of the nineteenth century, as industrialisation progressed, cheese manufacture moved to the factory; since then, there has been a progressive development of the technology, especially equipment, to the situation today with large, highly automated, modern factories employing minimal staff. This move has been driven by several factors – scale, cost and availability of labour, increased hygiene and need for product uniformity and consistency. This development has been at the cost of some individuality and variety; therefore, in parallel with the increased mechanisation of manufacture, there has been a resurgence of many small boutique cheesemakers. The impact of computers and automation on the cheesemaking process has been dramatic, with many of the previously manual-controlling, programming, analysis and data-logging operations being replaced by computers. Thus, greater uniformity of production has been made possible. This chapter aims to introduce the steps involved in the cheesemaking process, explaining their purpose and then describing the equipment and the processes that have been developed to facilitate large-scale manufacture. Not all equipment types are included in detail but rather the major types to illustrate their purpose. Cheeses may be classified in various ways. The diversity of cheese types arises from composition (the manufacturing process) and from the cultures or microflora involved ( Johnson and Law, 1999). This chapter focuses on the manufacturing process. A useful primary classification from a manufacturing technology viewpoint is based on cheese firmness (effectively, moisture content) and the salting technology involved. This is illustrated in Table 1 and forms the basis of the discussion of the manufacturing processes for the major cheese varieties

outlined in Fig. 1. The initial focus is on common steps to the end of the vat stage of manufacture. This is followed by discussion of the technology used for hard, dry-salted varieties such as Cheddar, with that used for other types being discussed in later sections.

Cheese Manufacture in the Vat Milk preparation

The milk used for cheesemaking comes from cows, sheep, goats and buffaloes. As the key ingredient, its quality and preparation are of vital importance. As the equipment and processes used are standard dairy operations, they are not described in detail. Excellent explanations are provided by Bylund (1995a) and Muir and Tamime (2001). Hygienic milk harvesting, refrigeration and gentle handling are essential features of milk harvesting and transport to the factory. The absence of inhibitory substances such as antibiotics is also necessary for satisfactory cheese manufacture. Removal of foreign matter is a necessary first step in factory processing, and this is achieved by filtration through an appropriate mesh or by centrifugal clarification. Compositional adjustment of the milk is often required to achieve the desired final product specifications. This commonly involves centrifugal separation of part of the milk stream into skim milk and cream, followed by blending of the skim milk with the whole milk to achieve the desired fat content. For some products, a higher fat level may be necessary and this is achieved by incorporating additional cream. More recently, it has become feasible to also adjust the protein content of the milk. This is normally achieved through the use of ultrafiltration technology (discussed in ‘Application of Membrane Separation Technology to Cheese Production’, Volume 1). Skim milk is concentrated and then blended with other components to achieve the desired final composition. Advantages include a more uniform starting material, more profitable use of a lactose stream and greater throughput of milk solids through the cheese vat, as the milk is effectively partially concentrated.

Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 2: Major Cheese Groups ISBN: 0-1226-3653-8 Set ISBN: 0-1226-3651-1

Copyright © 2004 Elsevier Ltd All rights reserved

24 General Aspects of Cheese Technology

Table 1 Classification of cheese based on hardness and salting technology

Hardness

Salting technology

Examples

Hard/semi-hard Hard/semi-hard Soft/semi-soft

Dry Brine Brine

Cheddar, Cheshire Emmental, Gouda Camembert, Blue vein

Control of the microbiology of the cheese milk is a vital issue affecting the final product, and there is an ongoing vociferous debate on the merits of raw milk cheese versus cheese for which heat treatment, normally pasteurisation, has been used (Johnson and Law, 1999). Pasteurisation, through the use of a plate heat exchanger and holding tubes with typical time/temperature relations of 72 °C/15 s, is standard practice to

Milk preparation

VAT STAGE

Starter culture and coagulant addition

• Setting • Cutting • Cooking • Washing (some types)

POST-VAT STAGES Brine-salted types

Hard, drysalted types

Dewheying

Whey to further processing

Drying

Texturing (cheddaring) or stirring

Milling

Pasta-filata types

Cooking/stretching

Salting

Moulding

Pressing

Brining

Ripening

Despatch

Figure 1 Basic steps in cheese manufacture.

Hard/semihard types

Soft mouldripened types

Dewheying

Dewheying

Pre-pressing (some types)

Moulding

Pressing

Acid development

Brining

Brining

Ripening

Ripening

General Aspects of Cheese Technology 25

kill pathogenic organisms. If the raw milk is to be stored refrigerated for a long period before pasteurisation, thermisation (66 °C/15 s) is recommended to prevent the growth of psychrotrophic organisms and their associated production of lipases and proteinases. Alternative processes for the reduction of bacterial load include the use of specially designed centrifuges (bactofuges) and microfiltration (Maubois, 2002). These avoid some of the perceived detrimental effects of thermal processes and are especially useful for the removal of spores, such as Clostridia, that survive pasteurisation and can cause problems in the final product. Following thermal or other treatments, the milk enters the cheese vat, typically at 32 °C. Starter culture preparation and addition

The use of cultures of micro-organisms, including bacteria, yeasts and moulds, is an integral component of cheese manufacture. ‘Starter Cultures: Genetics’, ‘Starter Cultures: Bacteriophage’, ‘Secondary and Adjunct Cultures’, of Volume 1 are devoted to detailed discussion of these cultures. The micro-organisms have two primary roles – the reduction of the pH during manufacture due to the production of lactic acid from lactose, and the biochemical and physical changes during the curing or ripening phase after manufacture of the initial cheese curd. The cultures responsible for acid development are typically lactic acid bacteria and are commonly referred to as cheese starters, although they are also involved during ripening. Organisms, the primary role of which is post-initial manufacture, are known as starter adjuncts. Both groups of cultures are commonly incorporated into the milk in the cheese vat, although for some varieties, such as smear-ripened cheeses, the formed cheese may be inoculated with the culture. Cheese starters used for hard varieties such as Cheddar are commonly composed of Lactococcus lactis subsp. cremoris. The quantity of culture required for controlled, rapid acid development in the vat means that a substantial inoculation is necessary. This may be provided in a variety of ways, such as the direct addition of a powdered concentrated culture provided by a culture manufacturer. This may be frozen or freezedried and may need to be reconstituted before addition to the vat. The very successful system used in New Zealand, described by Heap (1998), depends on the use of frozen single-strain cultures, which are then grown in a heat-treated reconstituted skim milk in a pH-controlled environment, to produce a concentrated culture that is then metered into the milk at a level such as 0.3%, v/v, as the vat is being filled with milk. A simple fermentation vessel is used for bulk culture

production. The greatest hazard to the production of starter and satisfactory acid development in the vat is the presence of bacteriophage. Multiple vat filling throughout the day in large plants increases the potential for phage build-up. Stringent hygiene precautions, the use of several carefully selected phage-unrelated strains and the use of a phage-inhibitory growth medium for starter preparation are the techniques designed to minimise this risk. Starter adjunct cultures can be added directly to the vat, usually from ‘pottles’ or suspensions of culture especially prepared by a culture manufacturer. As they are adjuncts, the quantities required are much smaller than the quantity of acid-producing starter. Coagulant addition

The most fundamental step in the cheesemaking process involves the conversion of the liquid milk into a semi-solid gel. Subsequent syneresis, or shrinkage and loss of whey from this gel, results in the formation of cheese curd. Detailed discussion of coagulants and syneresis is provided in ‘Rennets: General and Molecular Aspects’, ‘Rennet-induced Coagulation of Milk’, ‘The Syneresis of Rennet-coagulated Curd’, ‘Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels’, Volume 1. Coagulation involves the aggregation of the casein and is normally achieved by the addition of a coagulant to the milk in the vat stage of manufacture, although it can also be accomplished by pH reduction through acidification for some varieties, such as Cottage cheese. Traditionally, the coagulant of choice has been rennet, derived from the abomasum of young milk-fed calves, in which the principal active ingredient is chymosin. For reasons of supply, economy and ethics, alternatives are now also used frequently, derived from fungal sources such as Rhizomucor meihei or a natureidentical chymosin produced by genetic engineering technology. The coagulants are normally supplied by the manufacturer as stable liquid concentrates, which can be metered directly into the cheese vat at the appropriate stage via a distribution system. As the coagulant is a highly concentrated enzyme system, the quantity required is much lower than that of cheese starter, typically 0.01%, v/v, for calf rennet. As the enzymes are also involved in the ripening process, the level of addition and the enzyme characteristics are of vital importance to the cheese being produced. Vat stage

The cheese vat or cheese tank is the part of the cheesemaking equipment in which milk is converted from

26 General Aspects of Cheese Technology

a standardised liquid to a semi-solid gel. This part of the process concentrates the casein and the fat of the milk by removing moisture (whey). The first part of the process involves the addition of the coagulant to the milk, this being known as setting the vat. The coagulant is added and mixed in, as already described, and the vat contents are then left undisturbed. Determination of the appropriate coagulum strength for the next stage can be made by an experienced operator observing the curd or by using instruments such as the Stoelting Optiset® probe and others, discussed by Law (2001). Once a satisfactory coagulum has been formed, usually after about 40 min, the gel is cut into cubes of 6–10 mm size, to encourage moisture expulsion (syneresis). In most cheesemaking processes, the curds/whey mixture is then cooked to a higher temperature while lactose is fermented by the starter bacteria and acid is produced. Acid development is an important step in most cheesemaking processes and controls the rate and extent of syneresis, the composition, the final cheese pH and, perhaps of most importance, the degree of mineral solubilisation that occurs during the process. The cooking process has a fundamental role in controlling syneresis by influencing curd shrinkage and acid development. Following cooking, the curds/whey mixture is stirred until the drain pH target is reached and curds/whey separation (draining) or dewheying is initiated. For some varieties, a reduction in the lactose content of the curd and whey in the vat may be accomplished by partial removal of the whey followed by addition of water, which may be heated to also assist with cooking. This operation can be described as washing. Historically, cheese curd was produced in large, open, jacketed, square-ended, stainless steel vats. The cutting and stirring mechanisms were mounted above the vat and often both curd processing (e.g., cutting, cooking and stirring) and curd conditioning (e.g., cheddaring) were carried out in the vat. Labour costs were high and quality was often variable. Although this system is still used successfully in some small plants, more exacting hygiene standards, coupled with the demand for higher throughputs at reduced cost, resulted in the introduction of enclosed vat systems in the late 1960s. Since then, enclosed vat systems have been further refined to meet the needs of an increasingly mechanised and automated industry, an industry that in some countries is also having to deal with processing increasingly larger milk volumes because of extensive and rapid amalgamation of a number of smaller plants. This vat stage of cheese production is a batch process, and, for continuous throughput, a factory must have a number of vats, usually at least 6–8, to enable production to be sequenced to ensure a continuous output.

The majority of the enclosed vat systems available contain: • one or two revolving knife panels of various designs, which are used for both cutting and stirring operations, depending on their direction of rotation; • a fully or partially surrounding (steam or hot water) heating jacket; • whey removal systems for predraw and in-vat washing; • automated rennet addition, cleaning-in-place (CIP) and computer-controlled options for cutting/stirring speeds and cooking recipes (later models only). The choice of equipment for the vat stage of the cheesemaking process depends on many external factors, including the type of cheese to be made, downstream curd processing, flexibility, cost and throughput. Internal vat factors are also important. For example, the configuration of the vat and its cutting and stirring mechanisms, how the vat is heated and emptied, rennet addition and CIP configurations are also important. How the coagulum is cut is of particular significance. The cutting operation, together with the speed of stirring following cutting, influences how large the particles will be at draining and how much of the milk components (fat and casein) are lost to the whey. Johnston et al. (1991) showed that the speed and the duration of cutting in Damrow vats determined the curd particle size at draining and hence the moisture content in the final cheese, and that whey fat losses could be minimised depending on the cutting programme used. They also proposed a model for cutting that explains how variation in cutting speed and duration of cutting, followed by a constant stirring speed, determines the curd particle size distribution in a Damrow cheese vat. A similar study ( Johnston et al., 1998) using Ost vats (30 000 l) gave similar trends. However, the study on Ost vats also showed that, although similar, the trends were sufficiently different from those for Damrow vats, to warrant characterisation of each vat type as to the effect of the speed and the duration of cutting on cheesemaking efficiency,before implementing a specific cutting regime. A number of vat types are available, including OST, Damrow, Scherping and APV CurdMaster. These are discussed in turn. There is a similar discussion of vats and their design in Law (2001). The OST vat

One of the first and the most popular choices of enclosed cheesemaking vat was the Tetra Tebel OST (Ost Sanitary Tank) vat. To date, five models have been produced (OST I, II, III, IV and V) and there are two versions for each model – with or without predraw capability.

General Aspects of Cheese Technology 27

Both the OST I and the OST II vats were upright, single silo-shaped tanks with one (OST I) or two or more (OST II) vertically mounted knife panels. The tank volume ranged from 2000 to 20 000 L and these two models were first made in 1969. Manual, semi-automatic and fully automatic versions were available; however, in all cases, an operator was still required to add the coagulant. The last delivery of these models was made in 1977. The OST III vat was the first horizontally mounted vat of the OST series and its design was driven by a need to process larger (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

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

5

2

6

4

3

1

Figure 2 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

Manway with safety grid and switch

Inspection lamp Special design CIP heads to ensure efficient cleaning

Air vent

Solid shaft with heavy duty agitator

Fully cleanable bottom bearing

Control panel – manual or fully programmable design Legs with adjustable ball feet

Agitators designed for effective stirring and cutting with minimum fat and fine losses Dual bottom outlet for rapid and efficient emptying (Double-O DB only)

All stainless steel construction. lnsulated as standard to minimise energy costs and ensure stable process temperatures

Figure 3 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 l. 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: ⬇16 000 l, ⬇18 000 l and ⬇30 000 l. 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 l. 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

"L"

"W" WHEY STRAINER

"H"

35"

Figure 4 Damrow DOH horizontal cheese vat. Courtesy of Damrow Inc., USA.

The APV 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: • for variable degrees of filling from 40 to 100%; • all shaft seals to be located above product level;

Figure 5 Scherping horizontal cheese vat. Courtesy of Scherping Systems, USA.

30 General Aspects of Cheese Technology

Tight and hygienic shaft sealing

Silent and reliable drive system

Efficient and hygienic air vent

Water sprinkle system for hot or cold water

Open, easily CIPable shaft bearings

Movable bottom support

Steam inlet

Air vent

Condensate outlet

Figure 6 APV CurdMaster cheese vat. Courtesy of Invensys APV, UK.

• efficient horizontal and vertical mixing; • 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 l. 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 ultrafiltration 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 ultrafiltration stage ( Jameson, 1987); however, the Sirocurd equipment is not now in operation.

General Aspects of Cheese Technology 31

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. Dewheying

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

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

Figure 7 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

1

3 4 2 5

7 6

Figure 8 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

Figure 9 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

Figure 10 APV Cheddarmaster belt system. Courtesy of NZMP Whareroa, New Zealand. (See Colour plate 2.)

34 General Aspects of Cheese Technology

Vertical agitators

Draining belt

Draining screen

Curd and whey inlets

Matting and/or stirring belts with optional washing Peg stirrer with parking position clear of curd on belt Curd mill Figure 11 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 process – 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/salting 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, multi-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-salt 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

Agitator traverse rack

Agitator assembly Curd agitator arms

Salt distributor

Curd forking paddle Curd unloading door Curd unloading paddle

Whey collection tank

Curd rake

Air conveying valve (omitted with vacuum curd unloading)

Whey drain trench Figure 13 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

Figure 14 Laude block mould. Courtesy of Laude bv, 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 multi-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 °C) 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 °C 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 °C for 1 month. If a slower rate is required, a temperature of 2 °C 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. Figure 15 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. 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 °C 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 °C. 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

Initial cooling of dry-salted cheese.

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 39

Operation diagram Vacuum distributor valves

Discharge sequences Cyclone

Stage 1

Stage 2

Stage 3

Stage 4

Curd feed valve

Milled salted curd

Vacuum pump

Interceptor vessel Drain Curd feed Guillotine

Stage 5 Bag

Ejector Whey drain valve

Elevator platform

Bag Conveyor loader

Figure 16 Blockformer operating principles. Courtesy of Tetra Pak, Sweden.

Figure 17 Blockformers with bag presenters. Courtesy of NZMP Edendale, New Zealand. (See Colour plate 6.)

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

Figure 18 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-salted 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

Figure 19 Robot stacking of cheese blocks. Courtesy of NZMP Hautapu, New Zealand. (See Colour plate 8.)

General Aspects of Cheese Technology 41

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

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

3

2a

2 1

2 4

Figure 20 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.

1

2 3

4

3

6

7

8

5

9

Figure 21 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

Brining

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 NaCl. It should also contain an appropriate level of CaCl2, 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 °C. 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

Figure 22 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

Figure 23 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 °C, followed by 6–7 weeks at 22–25 °C for eye development, and storage/curing at 8 °C for several months. For Gouda, conditions may be several weeks at 10–12 °C, followed

Figure 24 APV tray brining system. Courtesy of Invensys APV, UK. (See Colour plate 11.)

General Aspects of Cheese Technology 45

Figure 25 Deep brining system. Courtesy of NZMP Lichfield, New Zealand. (See Colour plate 12.)

by 3–4 weeks at 12–18 °C, followed by several months at 10–12 °C (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 l 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) 10B 3B

1 10A

7 8A 8B

6

9

2 3A

4

5

Figure 26 Process line for soft cheese with Contifiller. Courtesy of Invensys APV, UK.

The development of various systems, such as the Guerin process, is described in more detail by Bertrand (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 pressure – just gravity is used. For cheese such as Blue where 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 47

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 impingertype 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.

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 °C, with the cooked curd temperature varying between 55 and 65 °C. 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. Almac s.r.l.

Almac s.r.l. 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.l. 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.l. 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

Figure 27 Almac cooker/stretcher. Courtesy of Almac, Italy. (See Colour plate 13.)

Stainless Steel Fabricating, Inc.

Stainless Steel Fabricating (SSF) also manufactures 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 113 kg/h (250 lbs/h) to 9080 kg/h (20 000 lbs/h) make up SSF’s SUPREME cooker/stretcher range. An example is shown in Fig. 28. In contrast to the Almac design, SSF cooker/stretcher models use inclined twin augers to cook and condition the Pasta-filata curd. At the base of the incline, curd is cooked in circulating hot water. Curd conditioning takes place at the top of the incline, where the cooking water is encouraged to drain back to the base of the incline. Stainless Steel Fabricating also manufactures a range of moulders with capacities up to 1816 kg/h (4000 lbs/h) depending on the size of the mould. In addition, SSF produces an extruder for String cheese production. String cheese, which is essentially a thin stick of Mozzarella, is a popular snack food in the US and is used by some pizza makers, such as Pizza Hut, to fill the crusts of their ‘stuffed-crust’ pizzas. Each stick

is characterised by the fibrous nature of its texture and more specifically the lengthwise alignment of the protein fibres. Using the SSF extruder, a series of continuous ropes of Mozzarella is formed. The length of stick can be varied by adjusting the location of the string cutter, which is activated by an electronic sensing device. Other models of the automatic string cutters can produce ropes up to 3 m in length. Construzioni Meccaniche E Technologia S.p.A. (CMT)

Construzioni Meccaniche E Technologia S.p.A., like Almac, is an Italian-based company and also produces a range of Mozzarella equipment including cooker/stretchers for customers similar to those supplied by Almac. As with SSF, CMT also produces equipment to make String cheese (Fig. 29). However, in the CMT machine, the String cheese is moulded rather than extruded. Construzioni Meccaniche E Technologia S.p.A. claims certain advantages, including the same fibrous structures as those obtained by extrusion but also more consistent weight and dimension control.

Conclusions Cheesemaking is a centuries-old process that has developed from an art to a science as the demand for the product and the scale of production have increased. Conversion from a cottage industry to the

General Aspects of Cheese Technology 49

Figure 28 SSF cooker/stretcher. Courtesy of Stainless Steel Fabricating, Inc., USA. (See Colour plate 14.)

highly complex automated factories in use today has demanded major developments in technology. There have been many ingenious approaches to the technology requirements and the consumer has benefited from having very consistent, safe, nutritious and palat-

able products. Further technological developments will occur as our understanding of cheese increases and our ability to fractionate milk to its various components and reassemble them into desired products increases.

Figure 29 CMT String cheese moulder. Courtesy of Construzioni Meccaniche E Technologia, Italy. (See Colour plate 15.)

50 General Aspects of Cheese Technology

Acknowledgements The authors are grateful to Tetra Pak AB, Sweden, for permission to use illustrations from the Dairy Processing Handbook and other sales literature. The following are also gratefully thanked for the supply and the use of technical sales information: Almac s.r.l., Italy; Alpma GmbH, Germany; Construzioni Meccaniche E Technologia S.p.A., Italy; Damrow Inc., USA; Hivolt Services Ltd, New Zealand; Invensys APV Ltd, Denmark, UK and New Zealand; Laude bv, The Netherlands; Scherping Systems, USA; Stainless Steel Fabricating, Inc., USA; Stoelting Inc., USA; Tetra Pak (New Zealand) Ltd; Tetra Pak Tebel bv, The Netherlands. Permission to use photographs from numerous sites of NZMP Ltd, New Zealand, is gratefully acknowledged, as is the assistance given by P. Jeffery from Massey University, Palmeston North, New Zealand, with the preparation of the figures.

References Bertrand, F. (1987). The main steps in manufacture, in, Cheesemaking, Science and Technology, 2nd edn, Eck, A., ed., Technique et Documentation–Lavoisier, France. pp. 413–443. Bylund, G. (1995a). Collection and reception of milk, in, Dairy Processing Handbook, Tetra Pak Processing Systems, Sweden. pp. 65–71. Bylund, G. (1995b). Cheese, in, Dairy Processing Handbook, Tetra Pak Processing Systems, Sweden. pp. 287–329. del Prato, O.S. (2001). Pasta Filata cheeses, in, Mechanisation and Automation in Dairy Technology, Tamime, A.Y. and Law, B.A., eds, Sheffield Academic Press, Sheffield. pp. 266–295. Heap, H.A. (1998). Optimising starter culture performance in New Zealand cheese plants. Aust. J. Dairy Technol. 53, 74–78. Jameson, G.W. (1987). Manufacture of Cheddar cheese from milk concentrated by ultrafiltration: the develop-

ment and evaluation of a process. Food Technol. Aust. 39, 560–564. Johnson, M. and Law, B.A. (1999). The origins, development and basic operations of cheesemaking technology, in, Technology of Cheesemaking, Law, B.A., ed., Sheffield Academic Press, Sheffield. pp. 1–32. Johnston, K.A., Dunlop, F.P. and Lawson, M.F. (1991). Effects of speed and duration of cutting in mechanized Cheddar cheesemaking on curd particle size and yield. J. Dairy Res. 58, 345–354. Johnston, K.A., Luckman, M.S., Lilley, H.G. and Smale, B.M. (1998). Effect of various cutting and stirring conditions on curd particle size and losses of fat to the whey during Cheddar cheese manufacture in Ost vats. Int. Dairy J. 8, 281–288. Kosikowski, F.V. and Mistry, V.V. (1997). Cheese with eyes, in, Cheese and Fermented Milk Foods, 3rd edn, F.V. Kosikowski, LLC, Westport, CT. pp. 226–251. Kristensen, J.M.B. (1999). Salting of the cheese, in, Cheese Technology – A Northern European Approach, International Dairy Books, Aarhus, Denmark. pp. 137–139. Law, B.A. (2001). Cheddar cheese production, in, Mechanisation and Automation in Dairy Technology, Tamime, A.Y. and Law, B.A., eds, Sheffield Academic Press, Sheffield. pp. 204–224. Martley, F.G. and Crow, V.L. (1996). Open texture in cheese: the contributions of gas production by microorganisms and cheese manufacturing practices. J. Dairy Res. 63, 489–507. Maubois, J.L. (2002). Membrane microfiltration: a tool for a new approach in dairy technology. Aust. J. Dairy Technol. 57, 92–96. McLeavey, L.J. (1995). Setting and Cutting of Curd in Scherping Cheese Vats. Diploma in Dairy Science Technology Thesis, Massey University, Palmerston North, New Zealand. Muir, D.D. and Tamime, A.Y. (2001). Liquid milk, in, Mechanisation and Automation in Dairy Technology, Tamime, A.Y. and Law, B.A., eds, Sheffield Academic Press, Sheffield. pp. 53–93. Pointurier, H. and Law, B.A. (2001). Soft fresh cheese and soft ripened cheese, in, Mechanisation and Automation in Dairy Technology, Tamime, A.Y. and Law, B.A., eds, Sheffield Academic Press, Sheffield. pp. 250–265.

Extra-Hard Varieties M. Gobbetti, Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università di Bari, Bari, Italy

Introduction The various schemes proposed for the classification of cheeses (see ‘Diversity of Cheese Varieties: an Overview’, Volume 2) indicate that the description of extra-hard varieties is not always unequivocal. The FAO/WHO Codex Alimentarius defines as hard and extra-hard, those cheeses having values of moisture on fat-free basis (MFFB) and fat in dry matter (FDM) lower than 56% and higher than 45%, respectively. Davis (1965) proposed a classification of cheeses based primarily on moisture content and assigned values of 25–36% and 25% to hard and very-hard cheeses, respectively. Burkhalter (1981) used the same primary criterion but did not separate hard and extra-hard varieties, and characterised as hard cheeses those with a moisture content lower than 42%, further dividing them in subgroups based on the source of milk (e.g., cows’, sheep’s or goats’ milk), texture and ripening agents. The temperature of cooking, low to high scald, the type of secondary microflora and the extent of chemical breakdown during ripening are other standards which have been used to differentiate cheeses within the hard and the extra-hard group (Walter and Hargrove, 1972). Davis (1965) proposed values for the classification of cheeses as hard, semi-hard and soft, based on viscosity, elasticity and springiness. A few preliminary considerations may, therefore, emerge: (i) the moisture content is probably the primary criterion by which extra-hard cheeses are differentiated; (ii) although based on the use of several standards, the distinction between hard and extra-hard varieties is not always well defined; (iii) extra-hard varieties are manufactured from cows’, sheep’s or goats’ milk or their mixtures; and (iv) different names for the same or very similar cheeses are used in countries which are large producers of these types of cheese. The selection of extra-hard varieties is further complicated since a particular cheese may be consumed as an extra-hard variety but also after a shorter period of ripening, when the cheese is soft. This chapter will describe cheeses which, although sometimes consumed as a different category, are manufactured mainly as extra-hard varieties.

Most of the extra-hard varieties are produced in Italy. Some of them, like Parmigiano Reggiano, Grana Padano and Pecorino Romano, rank amongst the most famous international cheeses and have maintained their traditional features over time in spite of great changes in cheesemaking technology. Parmigiano Reggiano, Grana Padano, Asiago and Pecorino Romano are used traditionally as grated cheeses as flavouring for Italian ‘pasta’. Swiss, Spanish, Russian, Balkan and nonEuropean extra-hard cheese varieties are also well known. Most of the European extra-hard varieties are produced under Protected Denominations of Origin (PDO). For instance, of the 979 060 tonnes of cheese produced in Italy in 2001, 441 360 tonnes were of cheeses which are legally designed by a PDO. Of the latter, 343 838 tonnes (c. 78%) were hard or extra-hard cheeses (Industria Lattiero-Casearia Italiana, 2002). Table 1 shows the production of the more important hard and extra-hard Italian cheeses. Except for Fossa (pit) cheese, all other extra-hard Italian cheeses have PDO status. All these cheeses, with the exception of Grana Padano, Parmigiano Reggiano, Asiago, Montasio, Provolone and Ragusano, are, or may be, produced from ewes’ milk alone or mixed with cows’ milk. Most of the Italian cheeses made from ewes’ milk are identified by the name ‘Pecorino’.

Main Chemical and Technological Features The main chemical and technological features of the more representative extra-hard cheeses are shown in Tables 2 and 3, respectively. Nevertheless, the characterisation of some varieties is very poor and the related technological features are incomplete. Long-ripened pasta-filata cheeses, like Provolone and Ragusano, are described in ‘Pasta-Filata Cheeses’, Volume 2. The use of raw milk and natural thermophilic starters, cooking of the curd to a high temperature, long ripening, a very low moisture content and, generally, an ancient tradition are features common to most of the extrahard cheeses. Some of the main relevant technological traits of the more famous extra-hard varieties are described below.

Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 2: Major Cheese Groups ISBN: 0-1226-3653-8 Set ISBN: 0-1226-3651-1

Copyright © 2004 Elsevier Ltd All rights reserved

52 Extra-Hard Varieties

Table 1 Production of the principal extra-hard Italian cheeses, 2001 Cheese production, tonnes

Cheese yield, kg milk/kg cheese

Cheese variety

Animal species

Milk quantity, tonnes

Grana Padano Parmigiano Reggiano Asiago Montasio Pecorino Romano Pecorino Siciliano Pecorino Sardo Fiore Sardo Canestrato Pugliese Castelmagno

Cow Cow

2 057 054 1 554 793

138 080 108 425

14.90 14.34

2 16 051 8 66 865 2 22 458 38 340 3 708 5 040 1 001 690

22 611 91 578 35 310 7 100 600 700 180 65

9.56 9.47 6.30 5.40 6.18 7.20 5.56 10.55

2 23 000 90 975

21 400 10 150

10.42 9.0 6.50

Provolone Valpadana Ragusano Fossa

Cow Cow Sheep Sheep Sheep Sheep Sheep Cow/sheep and Goat Cow Cow Sheep/cow

Source: Industria Lattiero-Casearia Italiana 2002.

Italian cheeses Parmigiano Reggiano

Parmigiano Reggiano, also internationally known as Parmesan, is, together with Grana Padano, a ‘Grana’ cheese due to the granular texture of the ripened cheese. In addition to these, there are Grana Bagozzo and Grana Lodigiano which, because of their limited production, have practically disappeared from the market. Parmigiano Reggiano cheese is produced according

to a traditional and well-defined technology in a restricted area of the Pianura Padana. For the manufacture of Parmigiano Reggiano, feeding of the cows is regulated carefully: (i) the ratio between forage and other feeds must be 1 to limit the dry matter (DM) derived from feeds which are rich in starch and proteins; (ii) 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

Table 2 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 Castelmagno Fossa Sbrinz Mahón 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.

Table 3 Main ripening characteristics of the principal extra-hard cheese varieties Type of rennet

Cooking,°C*

Salting

Natural whey culture (thermophilic, rod-shaped lactic acid bacteria) Natural whey culture (thermophilic lactic acid bacteria) None or natural culture in whey or milk Natural whey culture

Calf, powder

53–54

22–24 days in saturated brine

Calf, powder

54–55

Calf, powder or liquid

40–46

Calf, powder or liquid

48–50

Natural culture in ‘scotta’

Lamb, paste

45–46

None or natural culture in whey or milk Natural whey culture

Lamb, paste

40–45

Calf, paste

40–45

Natural whey culture

Lamb or goat, paste

No heat treatment

None or natural culture in whey or milk

Calf, powder or liquid

No heat treatment or 45

Cheese

Type of milk

Starter

Grana Padano

Cow, raw, partly skimmed

Parmigiano Reggiano

Cow, raw, partly skimmed

Asiago

Cow, raw, partly skimmed

Montasio

Cow, raw, partly skimmed

Pecorino Romano

Sheep, raw or thermised, whole Sheep, raw or thermised, whole Sheep, raw or thermised, whole Sheep, raw, whole

Pecorino Siciliano Pecorino Sardo Fiore Sardo

Canestrato Pugliese

Sheep, raw

Ripening, months

Proteolysis

Lipolysis

pH

12–16, at 18–20 °C

Deep, slow

Weak

5.5–5.6

20–23 days in saturated brine

18–24, at 18–20 °C

Deep, slow

Weak

5.4–5.5

Dry-salting for 10–12 days Dry-salting for 10–12 days or 4–7 days in saturated brine Dry-salting for 30–60 days

12–24, at 15–16 °C

Fairly deep

Weak

5.5

12, at 15–18 °C

Fairly deep

Moderate

5.0–5.5

8–12, at 10–14 °C

Deep

Strong

5.3–5.4

Dry-salting for 10–20 days

6–8, at 12–15 °C

Fairly deep

Strong

5.2–5.5

Saturated brine and/or dry salting Saturated brine and/or dry salting Dry-salting for 4–6 days

2–12

Fairly deep

Strong

5.2–5.5

3–6, at 12–16 °C

Fairly deep

Strong

5.1–5.3

4–12, at 11–14 °C

Fairly deep

Fairly strong

5.0–5.2

continued

53

54 Table 3 continued Type of rennet

Cooking, °C*

Salting

None or natural culture in whey or milk

Calf, powder or liquid

No heat treatment

Dry-salting for 4–6 days

None or natural culture in whey or milk

Calf, powder

No heat treatment

Saturated brine, dry-salting for 1 day

Cow, raw

Natural culture in whey

Calf, powder or liquid

54–56 °C

Cow, mix of cow and sheep, raw Sheep, raw or pasteurised

None or natural culture in milk

Calf, powder or liquid

No heat treatment

18–22 days in saturated brine Saturated brine for 2 days

Natural culture in milk

No heat treatment

Sheep, raw or pasteurised Sheep, raw

None or natural culture in milk

Calf, powder or liquid, or microbial rennet Calf, powder or liquid

No heat treatment

None or natural culture in milk Natural culture in milk

Calf, powder or liquid Calf, powder or liquid

No heat treatment No heat treatment

Cheese

Type of milk

Starter

Castelmagno

Cow, mix of cow, sheep and goat, raw, partly skimmed Sheep, mix of cow and sheep, raw

Sbrinz

Mahón

Fossa

Manchego

Roncal

Idiazabal Kefalotyri

Sheep, goat, mix of sheep and goat

* The duration of cooking is variable, for details see ‘Main Chemical and Technological Features’.

Saturated brine for 2 days, dry-salting for 1 day or both Saturated brine or dry salting for 1 day Saturated brine for 2 days Dry-salting

Ripening, months

Proteolysis

Lipolysis

pH

6–12, at 10–12 °C

Fairly deep

Fairly strong

5.0–5.3

6–8, at 12–14 °C and 17–25 °C 6–12, at 16–20 °C

Deep

Moderate

5.0–5.2

Deep, slow

Moderate

5.0–5.5

10–12, at 10–12 °C

Deep

Moderate

5.0–5.2

10–12, at 10–12 °C

Fairly deep

Moderate

5.1–5.3

10–12, at 10–12 °C

Fairly deep

Fairly strong

5.0–5.3

6–8, at 12–14 °C 6–8, at 10–12 °C

Deep

Moderate

5.0–5.2

Fairly deep

Moderate

5.0–5.5

Extra-Hard Varieties 55

within the district where Parmigiano Reggiano is legally produced; 25% of the DM of the forage used may be produced in territories adjacent to the district; (iii) the feeding of silage as fodder is not allowed to minimise the number of spore-forming, gas-producing, bacteria in the milk; also, the storage of silage on the same farm is prohibited. The use of additives, other than rennet and NaCl, for cheesemaking is prohibited. A mixture of milk from two consecutive milkings is used; evening milk is partially skimmed after overnight creaming at c. 20 °C in special tanks, ‘bacinelle’ (capacity, 10–50 hl), which contain a shallow body of milk. A slight microbial acidification occurs during creaming. After that, the partially skimmed milk is mixed in a ratio 1:1 with the whole milk from the following morning’s milking. The fat content of the milk for Parmigiano Reggiano is c. 2.4–2.5%. The natural whey cultures used as starters for Parmigiano Reggiano and Grana Padano are prepared from the whey from the previous cheesemaking, which is held in a temperature gradient (from c. 50 to c. 35 °C) for 24 h. The microbial composition of the natural starter is very complex, subject to environmental factors and dominated by thermophilic lactic acid bacteria (c. 109 cfu/ml) such as Lactobacillus helveticus, Lb. delbrueckii subsp. lactis, Lb. delbrueckii subsp. bulgaricus and Lb. fermentum. The ratio of obligately homofermentative to heterofermentative species is c. 10:1 or higher. A large amount of the natural whey culture, c. 3% (v/v), is added to the milk. The calf rennet used for Parmigiano Reggiano contains less than 3–4% pepsin, based on clotting activity. The curd cooking temperature ranges between 53 and 55 °C, and the time from rennet addition at 32–34 °C to the end of cooking is 22–23 min. The vats used for the manufacture of Parmigiano Reggiano and Grana Padano cheeses have a capacity of 10–12 hl and, traditionally, have the shape of an inverted bell. From each vat, two cheeses, each weighing 35–37 kg after ripening, are produced. Parmigiano Reggiano is ripened for 20–24 months at c. 18 °C and an environmental humidity of c. 85%. Parmigiano Reggiano and Grana Padano have a cylindrical shape with a diameter of 33–45 cm and a height of 18–25 cm. The cheeses have a very low moisture content (c. 30%), a typical compact texture, with or without many very small eyes, and melt in the mouth with a sweet flavour, which is the result of very slow ripening, during which proteolysis is the main biochemical event (Bottazzi, 1962; Consorzio del Formaggio Parmigiano Reggiano, 1989; Gobbetti and Di Cagno, 2002). Grana Padano

Grana Padano cheese is manufactured in several provinces of the Pianura Padana. Several major features distinguish it from Parmigiano Reggiano. For

Grana Padano, the feeding of high-quality silage fodder is allowed, and the cheese is produced from two consecutive milkings which are stored at 8 °C on the farm. The milk is skimmed by creaming in ‘bacinelle’ or very large tanks (300–500 hl) for c. 12 h at 12–15 °C. The total microbial count of the milk after holding in the ‘bacinelle’ is low, c. 103–104 cfu/ml compared to c. 106 cfu/ml for milk for Parmigiano Reggiano, also due to the lower temperature of creaming (Bottazzi, 1979). The fat content of the milk for Grana Padano is c. 2.1–2.2% and ripening lasts 14–16 months (Bottazzi, 1962; Consorzio per la Tutela del Formaggio Grana Padano, 1990; Gobbetti and Di Cagno, 2002). Asiago

Several types of Asiago cheese are manufactured which differ mainly in the duration of ripening. Asiago d’Allevo is a hard or extra-hard cheese variety, ripened for c. 12 months, and typically produced in the Veneto region. Previously, the cheese was manufactured from ewes’ milk, but only cows’ milk is used now. Raw milk from one or two consecutive milkings is partly skimmed by a creaming protocol similar to that described for Parmigiano Reggiano cheese. The natural whey culture used as starter is dominated by thermophilic lactic acid bacteria. The cooking of the curds is generally for 20–30 min and is divided into two steps. After cutting, the curds are heated to 40–42 °C and held for 5–7 min; then, the temperature is increased to 46 °C and held for 15–25 min. After moulding, the curds are pressed for c. 12 h. Generally, the cheeses are ripened for 1 year, exceptionally for 2 years. The cheeses are cylindrical in shape, 9–12 cm high and 30–35 cm in diameter and weigh 8–12 kg. The texture is rather compact and the flavour is slightly sweet (Battistotti et al., 1983; Ottogalli, 2001; Gobbetti and Di Cagno, 2002; Innocente et al., 2000). Montasio

The cheese derives its name from the homonymous place located in the Julian Alps. Currently, its manufacture has been extended to the Friuli region and to several provinces of the Veneto region. A mixture of cows’ milk from two consecutive milkings is used; the milk from the evening milking is partially skimmed after overnight creaming. The natural whey culture used as starter is added to the milk at 31–35 °C and coagulation by calf rennet takes place in 30–40 min. After cutting to the dimensions of rice grains, the curds are cooked at 48–50 °C for several minutes, pressed for 24 h and dry salted or immersed in saturated brine. Ripening of extra-hard Montasio cheese lasts 12 months at c. 18 °C and an environmental

56 Extra-Hard Varieties

humidity of c. 80%. Cheeses have a cylindrical shape with a diameter of 30–40 cm, height of 8–10 cm and weigh 5–9 kg. The mature cheese has a brown rind, a granular texture with very small eyes and a pronounced and moderately piquant flavour (Battistotti et al., 1983; Ottogalli, 2001). Pecorino Romano

Pecorino Romano cheese is manufactured in the regions around Rome and in Sardinia. It is the best-known extrahard ewes’ milk cheese. Pecorino Romano is usually made from raw or thermised milk which is inoculated with a natural culture, ‘scotta fermento’, which is produced by acidifying the ‘scotta’, the whey obtained from the manufacture of Ricotta. Thermophilic lactic acid bacteria, such as Streptococcus thermophilus, Lb. delbrueckii subsp. lactis and Lb. helveticus, dominate the microflora of this natural starter. The milk is coagulated at 37–39 °C using lamb rennet paste and after cutting, the curds are cooked at 45–46 °C. After removal from the vat, the curds are placed in moulds, pressed manually and pierced with the fingers or a stick to increase whey drainage. The cheese is ripened for 8–12 months at 10–14 °C to develop the characteristic flavour. The cheese is cylindrical in shape, 25–32 cm high and 25–30 cm in diameter and weighs 22–32 kg. The sensory characteristics of Pecorino Romano cheese depend mainly on lipolysis by enzymes (pre-gastric esterase) in the lamb rennet paste, and flavour intensity is related to the content of free butanoic, hexanoic and octanoic acids. Proteolysis may show wide variations but the soluble nitrogen is always less than 30% of the total nitrogen (Battistotti et al., 1983; Ottogalli, 2001; Gobbetti and Di Cagno, 2002). Pecorino Siciliano

This variety of Pecorino cheese is manufactured only in Sicily, between October and June, when whole ewes’ milk is available. Only natural whey cultures, containing mainly thermophilic lactic acid bacteria, are used as starter. Lamb paste rennet is used for coagulation, which occurs within 40–60 min. The coagulum is broken into pieces the dimensions of a pea using a traditional wooden tool, known as a ‘rotella’. The curds are partially cooked at 40 °C for c. 10 min by adding hot water (c. 70–80 °C) and moulded in a circular vessel, traditionally called ‘canestro’, where the curds are pressed slightly. The cheeses are ripened for at least 6–8 months to develop the moderate piquant flavour. The cheese has a cylindrical shape, 12–18 cm high and 35 cm in diameter, and weighs 4–15 kg. Pepato (peppery) is a variant of Pecorino Siciliano cheese which differs by the addition of black pepper to the curds during moulding (Battistotti et al., 1983; Ottogalli, 2001; Gobbetti and Di Cagno, 2002).

Pecorino Sardo

This is a variety of Pecorino cheese, the manufacture of which is limited to several provinces of Sardinia. Raw or thermised whole ewes’ milk, natural whey or milk cultures and calf rennet paste are used in cheesemaking, which does not differ substantially from that of Pecorino Siciliano cheese. The ripening of this hard variety may last for 12 months. The shape of the cheeses is cylindrical, 10–13 cm high, 15–20 cm in diameter and weigh 1.7–4 kg. The straw-yellow rind is smooth and springy initially, but later it becomes darker and harder. The mature cheese has a pleasant pungent flavour and a firm, hard, fairly granular texture (Battistotti et al., 1983; Ottogalli, 2001; Gobbetti and Di Cagno, 2002). Fiore Sardo

The production of Fiore Sardo cheese is strictly limited to some provinces of Sardinia. Traditionally, it was produced by the shepherds in their cottages. Raw whole ewes’ milk from a single milking is used. A large part of the milk is produced by an indigenous breed of sheep. Starters are not deliberately added and lamb rennet paste is used to coagulate the milk. The curds are not cooked and are pressed slightly during moulding. Treatment of the curds with hot water is necessary to make the rind thick and resistant. The cheeses are ripened for c. 6 months or more, and during the early phase of ripening, they may be smoked slightly by exposing them to smoke from the wood of Mediterranean scrub trees. During ripening, the cheeses are often rubbed with a mixture of olive oil and sheep fat. The cheeses have a cylindrical or wheel shape with curved sides, are 13–15 cm high and weigh 1.5–5 kg. The flavour of Fiore Sardo is pronounced, aromatic, moderately spicy and the rind varies from deep gold to dark brown with a sour smell (Battistotti et al., 1983; Ottogalli, 2001; Gobbetti and Di Cagno, 2002). Canestrato Pugliese

Canestrato Pugliese is manufactured only in the Apulia region. The cheese derived its name and traditional shape from the rush basket, ‘canestro’, in which the curds are ripened. Raw, whole ewes’ milk of one or two daily milkings is generally used, but thermised or pasteurised milk may be processed also. A natural whey culture, composed mainly of thermophilic lactic acid bacteria, may be added, and liquid or powdered calf rennet, or, exceptionally, lamb paste rennet, is used. After cutting, the curds–whey mixture is heated to 45 °C and held for 5–10 min. This treatment is generally not considered as ‘cooking’. The cheeses are drysalted for c. 2 days and, during ripening (4–12 months) in the ‘canestro’, are turned regularly and rubbed with a mixture of oil and vinegar. Ripening in the ‘canestro’

Extra-Hard Varieties 57

is limited to a few days for industrial production. Colonisation of the surface by moulds from the environment frequently becomes evident during ripening, which are removed by brushing after few months. The cheeses have a cylindrical shape, 10–14 cm high, 25–34 cm in diameter and weigh 7–14 kg. The rind is brown to pale yellow, and the interior is compact with small eyes. The flavour is pronounced and tends to be moderately piquant (Battistotti et al., 1983; Ottogalli, 2001; Gobbetti and Di Cagno, 2002). Castelmagno

This cheese has been manufactured in the Grana valley, near Cuneo (Piedmont), since the twelfth century. Raw cows’ milk is partly skimmed according to a protocol similar to that described for Parmigiano Reggiano cheese. Rarely, a mixture of cows’, ewes’ and goats’ milks is used. The traditional technology does not involve the use of a natural starter, and acidification is due to the indigenous lactic acid bacteria. Liquid or powdered calf rennet, which may be combined with a small amount of lamb rennet paste, is used for coagulation. After cutting the coagulum and removal of most of the whey, the curds are traditionally harvested in cloth bags which are hung for 10–12 h at room temperature, allowing the removal of further whey. The cheese is ripened in natural caves at 10–12 °C and 85–90% relative humidity for more than 6 months. The cheeses have a cylindrical shape, are c. 20 cm high, 20–25 cm in diameter and weigh 4–6 kg. Penicillium spp. from the environment colonise the cheese surface, and occasionally the interior of the cheese. Castelmagno cheese may be considered as a hard Blue cheese variety with a compact but friable texture and a moderately piquant flavour (Battistotti et al., 1983; Ottogalli, 2001; Gobbetti and Di Cagno, 2002). Fossa

The tradition of Fossa (pit) cheese originated in the Emilia-Romagna region (north of Italy) in the Middle Ages. The typical feature of this cheese involves ripening in flask-shaped pits which are dug in the tufa ground of this region. Cheesemaking is typically from raw ewes’ milk but in some cases, mixed ovine-bovine milk is used. Traditionally, the cheese is produced only during a defined period of the year. Natural thermophilic starters in milk, comprised of indigenous lactic acid bacteria, are added to the cheese milk. The curds are not cooked and after moulding are held at c. 28 °C for 4–8 h. Curds are generally ready for ripening in pits after a period (up to about 3 months) of maturation in rooms, which is necessary to achieve a certain degree of consistency and to eliminate the risk of whey losses when the cheeses are pressed into the pits.

Before they are placed and pressed in the pits, the cheeses are wrapped individually in cloths. The sides of the flask-shaped pits are covered with straw which is fixed by canes, horizontally linked by wooden rings. The pits are open during August and when completely filled with cheeses, they are hermetically sealed. The humidity inside the pits is close to saturation and the temperature ranges from 17 to 25 °C. Traditionally, the pits are opened on 28th November; at this time, the cheeses have been ripened for at least 6 months, including maturation in rooms. Due to the pressure inside the pits, the shape of the cheeses varies from cylindrical to very irregular and the weight ranges from 1.0 to 1.5 kg. The flavour is generally full, sharp, balanced and moderately piquant (Gobbetti and Di Cagno, 2002). Extra-hard Swiss cheeses

Most of the Swiss cheeses classified as hard or extrahard varieties are discussed in ‘Cheese With Propionic Acid Fermentation’, Volume 2, which deals with cheeses with the propionic acid fermentation. A few others are described below. Sbrinz

The cheese derived its name from the locality of Brienz in Switzerland but now its manufacture has been extended to France, Germany and Italy. Raw, whole cows’ milk of one day’s milking is used. The milk is warmed to 34–38 °C, and a natural whey culture, containing mainly thermophilic lactic acid bacteria, is added. Liquid or powdered calf rennet is used to give coagulation in 15–20 min. During heating and mixing at 45–48 °C, the coagulum is cut to the dimensions of wheat grains. Cooking is at 54–56 °C for a few minutes. After harvesting, the curds are pressed for 24 h, salted in brine for 18–22 days and ripened for 6–12 months at c. 18 °C and an environmental humidity of c. 80%. The cheeses have a cylindrical shape, are 10–14 cm high, 40–50 cm in diameter and weigh 20–25 kg. The rind is yellow to brown at the end of ripening, and the interior is compact with a Grana-like texture. The moisture content is less than 35% and the flavour is pronounced (Battistotti et al., 1983; Fessler et al., 1999). Saanenkäse

This cheese is made from cows’ milk of two consecutive milkings which is coagulated at 32 °C by addition of calf rennet. After cutting, the curds are cooked at 50–52 °C and pressed. Ripening lasts from 2 to 5 years and the moisture content is c. 25%. The cheeses have a cylindrical shape, are 10–14 cm high, 40–60 cm in diameter and weigh 20–40 kg. The interior and taste

58 Extra-Hard Varieties

are similar to those of Parmigiano Reggiano and Sbrinz cheeses (Battistotti et al., 1983).

compact with small eyes. The extra-hard variety has a moisture content of c. 35% and its flavour is pronounced (Marcos and Esteban, 1993).

Spanish extra-hard cheeses

All the Spanish cheeses listed below have a PDO status as established by national and European rules. Mahón

The cheese takes its name from the capital of Minorca (Balearic Islands), where it is produced. Raw or pasteurised cows’ milk, containing 5% of indigenous ewes’ milk, is used for cheesemaking. Natural whey cultures may be used as starters; the milk is coagulated at 30 °C and, after cutting, the curds are pressed and salted in brine. Several variants are produced, including an extrahard cheese which is ripened for at least 10 months. The cheeses have a parallelepipedal shape, weigh 2–4 kg, the rind is white to yellow, oily due to treatment with olive oil, and the interior is compact with small eyes. The extra-hard variant has a moisture content less than 32% and its flavour is pronounced (Alcalá et al., 1982; Esteban et al., 1982; Marcos et al., 1983). Manchego

Manchego takes its name from the La Mancha region where the cheese was traditionally made from raw sheep’s milk by shepherds. Because of increasing market popularity, its manufacture has spread throughout Spain. Cheesemaking at an industrial level uses ewes’ milk collected over two consecutive days from herds in a demarcated area. The milk is pasteurised and a mesophilic starter culture (Lactococcus lactis subsp. lactis and Lc. lactis subsp. cremoris, mainly) and calf rennet or microbial rennet from Rhizomucor miehei are added. After c. 35 min at 30 °C, the coagulum is cut into pea-sized grains. The curd particles are heated to 37 °C for 20 min and then stirred for another 30 min. After removal of the whey, the grains are transferred to a curd strainer and the beds of drained curd are cut into cube-shaped blocks, each of which is placed in a cylindrical PVC hoop, lined with a smooth cloth, in which the curds are moulded and pressed pneumatically at 0.3 MPa for 5 h. The cloths are removed and the curd pressed again at the same pressure for 17 h, after which it is immersed in a circulating brine bath at 14 °C for 36 h. The blocks of curd are then placed in a drying room at c. 14 °C and 85% environmental humidity where they are stored, with periodic turning, for 10 days, after which they are transferred to a curing chamber at c. 9 °C and 95% environmental humidity. After 12 months, the cured cheeses are brushed and, in some cases, coated with a polyvinyl acetate emulsion containing an anti-fungal agent. The cheeses have a cylindrical shape, c. 20 cm in diameter and weigh 2.5–3 kg. The rind is green to black and the interior is

Roncal

This cheese takes the name from the Navarrese valley where it is produced from December to July. It is manufactured from ewes’ milk and the main technological traits are similar to those for Manchego cheese, except for the smaller dimensions of the ripened cheeses. A typical microflora composed of deliberately added mesophilic starter lactococci and adventitious lactobacilli persists during ripening (Ordóñez et al., 1980; Marcos and Esteban, 1993). Idiazabal

This is another extra-hard variety made from whole ewes’ milk in mountain caves of the Basque country. Raw milk from the ‘latxa’ breed of sheep is coagulated at 25–35 °C in 30–45 min by addition of lamb rennet. The coagulum is cut to rice-sized grains, heated to and maintained at 40–45 °C for several minutes, before being placed in moulds where the curds may be seasoned before pressing. Salting is performed by rubbing the rind with dry salt or by immersion of the cheese in brine for 24 h. The cheese is ripened for several months until a moisture content of c. 33% is reached. The cheeses have a cylindrical shape and weigh 1–2 kg. The rind of artisanal cheeses is engraved with drawings or symbols characteristic of the Basque culture. An optional smoking may be performed at the end of ripening by using smoke of wood from beech, birch, cherry or white pine trees. The taste is strong and pronounced, slightly acidic and piquant with a characteristic sheep milk flavour (Marcos and Esteban, 1993; Arizcun et al., 1997a,b). Other extra-hard cheeses Russian cheeses

Sovetskiı~ , Altaiskiı~ and Briskiı~ are hard or extra-hard varieties made from cows’ milk and are similar to Swiss-type extra-hard cheeses. The use of a mixed starter culture composed of Sc. thermophilus and Lb. helveticus, cooking of the curds at 50–55 °C, pressing for 4–8 h and ripening for at least 6 months are the main technological traits. Generally, cheeses have a moisture content of 32–36%, weigh 10–18 kg and have a rectangular shape (Gudkov, 1993). Balkan cheeses

Kefalotyri is an extra-hard, salty Greek cheese, made exclusively from ewes’ or goats’ milk with the use of thermophilic and propionic starters. After coagulation by calf rennet at 35 °C, the coagulum is broken to dimensions of c. 6 mm and pressed for 5–10 h. Salting

Extra-Hard Varieties 59

is performed by rubbing the rind with dry salt and the cheeses are ripened for more than 3 months. The cheeses have a flat cylindrical shape, are c. 30 cm in diameter and weigh 5–10 kg. The flavour is strong, piquant and salty (Pejic´, 1956; Scott, 1981). Manura is a Greek traditional farmhouse hard cheese variety manufactured on Sifnos island in the Aegean sea from raw ewes’ milk or from a mixture of raw ewes’ and goats’ milks of local herds. Typically, after 3–4 months of ripening in straw beds, the cheeses are held for several days in red wine to soften them and then for a few days in wine residues. Cheeses are small and weigh c. 600 g. Pas´ki cheese means cheese from Pag, which is the name of the Adriatic island (Greece) where it is produced. It is a very-hard cheese made from ewes’ milk which is ripened for at least 6 months. The cheeses have a high DM content, a firm compact texture, with no holes, and the flavour tends to be strong and piquant (Pejic´, 1956). Other extra-hard cheeses

Very-hard and hard varieties are produced in several non-European countries. Most of them are manufactured from ewes’ and/or goats’ milk, a starter culture is not always used, pressing of the curds is a very common feature and the cheeses are ripened for at least 6 months. Typical examples are Djamid from Jordan (Phelan et al., 1993), Ras from Egypt (Hofi et al., 1970) and Paphitico and Graviera from Cyprus (Phelan et al., 1993).

Ripening Although most of the extra-hard varieties considered above have high market popularity and are of great economic relevance, only a few of them have been characterised extensively. In addition, since the same cheese may be produced in a number of hard or extra-hard variants which differ with respect to the type of milk, season of milking, technology and ripening, the results available on cheese characterisation may differ markedly. Owing to the large size and the prolonged brine and/or dry salting, most extra-hard cheeses are commonly characterised by a decreasing NaCl gradient from the surface to the centre and by an opposite moisture gradient, which is reflected in the water activity (aw) values. These gradients persist for a very considerable period after salting, and consequently, ripening in these cheeses shows variations which depend on the cheese zone. Changes in microflora during ripening

The lack of fermentable carbohydrates, low pH, aw (mainly due to NaCl) and temperature, and the presence of bacteriocins make the environmental conditions in extra-hard cheeses very hostile during ripening.

Generally, this favours a sharp decline of the number of thermophilic starter bacteria which are gradually replaced by mesophilic bacteria. For the extra-hard varieties, mesophilic bacteria are derived mainly from the raw milk used but environmental contamination is not excluded, as well as survival of bacteria following sub-pasteurisation or thermisation for those cheeses for which heat-treated milk is used. The composition of this population may vary but non-starter lactic acid bacteria (NSLAB) constitute the major part. Pediococcus spp., Lb. casei, Lb. casei subsp. pseudoplantarum and Lb. rhamnosus are the predominant bacteria in Parmigiano Reggiano and Grana Padano cheeses (Bottazzi, 1979, 1993; Gobbetti and Di Cagno, 2002). Pediococci seem to be fundamental for maintaining the equilibrium within the cheese-related microbial community, probably also interfering negatively with the growth of clostridia, while Lb. casei, as the major part of the NSLAB, is very important for its peptidase activity (Gobbetti et al., 1999a,b). In Parimigiano Reggiano cheese, NSLAB decrease from c. 108 cfu/g at 5 months to 104 cfu/g after 24 months of ripening (Coppola et al., 1997). Lb. plantarum, Lb. casei and Enterococcus faecium prevail in Manchego cheese after 1 month of ripening (Núñez et al., 1989). Lb. plantarum and Lb. curvatus were the species isolated most frequently from Fossa cheese, with fewer numbers of Lb. paracasei subsp. paracasei (Gobbetti et al., 1999c). A more heterogeneous microflora, consisting of Lb. plantarum, Lb. pentosus, Lb. curvatus, Lb. brevis, Lb. paracasei subsp. paracasei and Leuconostoc spp., was found in Canestrato Pugliese cheese (Albenzio et al., 2001; Corbo et al., 2001). Lb. curvatus and Lb. paracasei subsp. paracasei were also found in Fiore Sardo at the end of ripening (Mannu et al., 2000). Together with components of the NSLAB microflora such as Lb. curvatus, Lb. plantarum and Lb. fermentum, and with a heterogeneous population of enterococci, the thermophilic Lb. dekbrueckii subsp. lactis was found occasionally in ripened Pecorino Romano (Di Cagno et al., 2003). Lb. casei, Lb. plantarum and Ln. mesenteroides subsp. mesenteorides and Ln. mesenteroides subsp. dextranicum were the bacteria found in aged Roncal and Idiazabal cheeses (Arizcun et al., 1997a). In most extrahard and hard cheeses, NSLAB reach c. 107–108 cfu/g after few months, which is generally maintained for a long time during ripening (McSweeney et al., 1993; Gobbetti et al., 1999c; Mannu et al., 2000; Albenzio et al., 2001). All these differences and changes in the microbial population are considered relevant factors which affect the cheese during subsequent ripening, especially regarding the extensive secondary proteolysis, which leads to an elevated concentration of small peptides

60 Extra-Hard Varieties

and amino acids, which is undoubtedly related to the peptidase activity of mesophilic bacteria.

6.5

Sugar concentration (mg/g)

16

10

5

2

3

4

5

6

7

8

9

10 11 12

Time (hours) Figure 1 Changes in the concentrations of lactose (䊉), glucose (䊏) and galactose (䉱) in the external (- -) and internal (—) zones of Parmigiano Reggiano cheese (Mora et al., 1984).

10 5.5

pH

Lactic acid (mg/g)

The lactic acid fermentation has been studied extensively in Parmigiano Reggiano cheese during the first 48 h after manufacture (Mora et al., 1984) (Figs 1 and 2). There are no comparable data for other extra-hard cheeses but the fermentation is generally similar in those varieties which are cooked to a high temperature and have a rather large size. The growth of the starter thermophilic lactic acid bacteria and the hydrolysis of lactose depend mainly on the rate at which the curds cool after removal from the cheese vat. Depending on the weight of the cheese, the temperature at the centre of the curd remains relatively high, e.g., 50 °C for 12–16 h for Parmigiano Reggiano, while the exterior of the cheese cools rather suddenly (c. 2 h) to c. 42 °C. Consequently, bacterial growth starts earlier and is more intense in the external zone. While the residual lactose is consumed throughout the cheese within 8–10 h, bacterial numbers, pH and lactic acid concentration do not attain equal values in the centre and exterior of the cheese for a longer time. The concentration of lactic acid also may vary during ripening. For Manchego cheese, the concentration of lactic acid was c. 1.2 and 1.0% in the exterior and interior of the cheese during 2 weeks, then decreased to 1.0 and 0.8% after 3 weeks, but then increased again to c. 1.2% throughout the cheese, probably due to loss of moisture (Marcos and Esteban, 1993).

1

6.0

15

Lactose metabolism

5 5.0 2

4

6

8

10

12

24

48

Time (hours) Figure 2 Changes in pH (䊉) and lactic acid concentration (䊏) in the external (- -) and internal (—) parts of Parmigiano Reggiano cheese (Mora et al., 1984).

Lipolysis

The ripening of most cheeses is accompanied by a low level of lipolysis but extensive lipolysis occurs in several extra-hard varieties. The length of ripening strongly influences lipolysis and since ripening varies markedly within the same variety, cheeses ready for the market may differ greatly. Lipolysis may be due to the action of the indigenous lipase in cheese made from raw milk, to the action of microbial lipases, even though the lactic acid bacteria in starter cultures have only weak lipolytic activity, or to the action of the lipases present in rennet paste used in cheesemaking for certain varieties. Several extra-hard Italian cheeses are probably unique in that rennet paste is used commonly. The desirable flavour which characterises the Pecorino cheeses (Romano, Siciliano and Sardo) and Fiore Sardo is due mainly to the action of pre-gastric esterase (PGE) in rennet paste, which is used as the source of both coagulant and lipolytic agents in cheese manufacture. Rennet pastes are prepared by grinding the engorged stomachs, including curdled milk, of young calves, kid goats or lambs which are slaughtered immediately after suckling or pail-feeding. The stomachs and contents are generally held for c. 60 days prior to grinding. Pregastric esterase, the physiological role of which is to aid in the digestion of fat by the young animals which have limited pancreatic lipase activity, is secreted during suckling and is carried into the stomach with ingested milk. The strong, balanced piquant flavour which characterises Pecorino cheeses and Fiore Sardo is due primarily to the relatively high levels of short-chain free fatty acids (FFAs), especially butanoic, hexanoic and octanoic acids. Although there are some inter-species differences, lamb,

Extra-Hard Varieties 61

calf and kid PGEs preferentially hydrolyse fatty acids esterified at the sn-3 position of glycerol (Woo and Lindsay, 1984), which explains the relatively high rate of release of butanoic acid from milk fat, in which 90% of the butanoic acid is attached at the sn-3 position. Calf PGE does not hydrolyse monobutyrin and hydrolyses dibutyrin very slowly compared to tributyrin (Richardson and Nelson, 1967). The moderate accumulation of short-chain FFAs characterises the ripening of Parmigiano Reggiano, Canestrato Pugliese and Fossa cheeses, for which rennet paste is not used (Woo and Lindsay, 1984; Carboni et al., 1988; Gobbetti et al., 1999c; Albenzio et al., 2001). Table 4 shows the FFA profile of some extra-hard cheeses. The average values which are reported refer to ripened cheeses, with a high popularity on the market, but in general there is no standard flavour for such extra-hard Italian cheeses which is acceptable to all segments of the population. For Pecorino Romano cheese, there is a direct relationship between the flavour intensity and the concentration of butanoic acid (Richardson and Nelson, 1967) but the relationship between flavour desirability and butanoic acid concentration is more variable. Flavour desirability is influenced mainly by the relative proportions of the various FFAs. A strong, balanced, piquant Pecorino Romano cheese may be characterised by c. 10 500 mg/kg total FFAs, principally butanoic (C4:0), together with hexanoic (C6:0), tetradecanoic (C14:0), hexadecanoic (C16:0) and octadecenoic (C18:1) acids (Table 4). It was shown that among these compounds, butanoic and hexanoic acids are the most important components of the aroma quality of Pecorino Romano cheese. The total FFA content of Parmigiano Reggiano approaches 20% of that generally found in Pecorino

cheeses, with variations in the proportions of FFAs. Congeners of C18 fatty acids dominate the FFA profile at the end of ripening (Carboni et al., 1988). The crude vacuum distillate of Grana Padano cheese contains large amounts of butanoic and hexanoic acids, which represent 50 and 35% of the total FFAs, respectively. These two FFAs may be important for the background aroma of Grana Padano cheese. A small change in the relative proportions of butanoic and hexanoic acids was found between 12 and 24 months of ripening (Moio and Addeo, 1998). Canestrato Pugliese and Fossa cheeses show very similar FFA profiles, although the former has a higher total concentration of FFAs (Gobbetti et al., 1999c; Albenzio et al., 2001). Butanoic acid, which occurs at the highest concentration, hexanoic, decanoic (C10:0), hexadecanoic and congeners of C18 acids dominate. Probably due to the lipolytic activity of moulds which colonise the cheese surface during the early period of ripening, Canestrato Pugliese also shows a rather high proportion of octadecenoic (C18:1) and octadecadienoic (C18:2) acids. A qualitative and semi-quantitative comparison of the FFA profiles of other extra-hard varieties produced from ewes’ milk showed that butanoic, hexanoic, octanoic (C8:0) and decanoic acids were the dominant FFAs in Roncal, Pecorino Sardo and Fiore Sardo; levels were highest in the last cheese (Larráyoz et al., 2001; Di Cagno et al., 2003). Of 14 samples of Manchego cheese analysed, all contained high levels of short-chain FFAs, butanoic acid being the most abundant (Villaseñor et al., 2000). Extra-hard cheeses produced without the use of rennet paste may vary greatly in the concentration of FFAs depending on whether raw or pasteurised milk is used. Several studies have shown a higher level of

Table 4 Concentration of individual and total free fatty acids (mg/kg cheese) in Parmigiano Reggiano, Pecorino Romano, Canestrato Pugliese and Fossa cheeses

Fatty acid

Parmigiano Reggiano

Pecorino Romano

Canestrato Pugliese

Fossa

Butanoic (C4:0) Hexanoic (C6:0) Octanoic (C8:0) Decanoic (C10:0) Dodecanoic (C12:0) Tetradecanoic (C14:0) Hexadecanoic (C16:0) C18 congeners Total free fatty acids

172 48 44 107 107 225 565 1033 2301

3 043 1 428 429 1 009 690 778 1 306 1 843 10 526

425 178 42 98 46 85 172 322 1368

247 123 55 84 35 62 137 251 994

C18 congeners refer to octadecanoic (C18:0), octadecenoic (C18:1), octadecdienoic (C18:2) and octadectrienoic (C18:3) acids. The values indicated represent the average of several determinations made by different authors on cheeses which had a slightly different ripening time: Parmigiano Reggiano, 16–18 months; Pecorino Romano, 10–12 months; Canestrato Pugliese, 6–10 months; Fossa, 6–8 months. Source: Woo and Lindsay (1984); Carboni et al. (1988); Gobbetti et al. (1999c); Albenzio et al. (2001).

62 Extra-Hard Varieties

FFAs in cheese made from raw milk than in that made from pasteurised or thermised milk. Such differences are attributed mainly to heat-induced changes to the indigenous lipoprotein lipase of milk and to the lipase and esterase activities of the milk microflora, especially NSLAB, and become greater as the time of ripening increases. Studies on NSLAB (Gobbetti et al., 1996, 1997) showed that Lb. plantarum contains lipase and esterase which show a substrate specificity comparable to PGE and pancreatic lipase and since there is a very large population of NSLAB in cheese during ripening, their contribution to lipolysis has been suggested. Proteolysis

Proteolysis in extra-hard varieties does not differ substantially from that in other hard/semi-hard internal bacterially ripened cheeses. The low moisture and high salt content, which cause the persistence of gradients of moisture and NaCl in the cheese, and the absence of a fungal microflora, which is evident only on the surface of Canestrato Pugliese and Castelmagno, are all factors which influence proteolysis during ripening. The principal proteolytic agents in the curd are the coagulant, depending on the intensity of the cooking treatment, starter and NSLAB proteinases and peptidases and indigenous milk proteinases (particularly plasmin) (Fox et al., 1996). Proteolysis in Parmigiano Reggiano and Grana Padano cheeses has been studied using many different analytical methods. Polyacrylamide gel electrophoresis and isolectric focusing in a polyacrylamide gel (Addeo et al., 1988) showed the rapid hydrolysis of s1-casein (CN) to the primary degradation product, s1-CN (f24–199) and the formation of -CNs from -CN

during the first month of ripening. The latter, together with the very low levels of -CN f1–192 and -CN f1–189, the primary products of -CN hydrolysis by chymosin, indicates considerable plasmin activity. Hydrolysis of -CN by chymosin during ripening is inhibited by 5% NaCl and, in general, during curd cooking most of the chymosin activity is destroyed. The same pattern for -CN hydrolysis was found in Pecorino Romano cheese. Overall, -CN is rapidly and almost totally hydrolysed during the ripening of Parmigiano Reggiano, Grana Padano and Pecorino Romano cheeses, while s1-CN undergoes relatively less proteolysis (Fig. 3). One-year-old cheeses generally do not contain -CN, while at the end of ripening Parmigiano Reggiano cheese still contains unhydrolysed s1-CN. These findings confirm that chymosin, which is the primary proteolytic agent acting on s1-CN, is not very active in these cheeses. Addeo et al. (1988) proposed the ratio -CNs/-CN as an index of proteolysis in Parmigiano Reggiano during ripening. During the first year, the -CNs represent c. 20% of the oligopeptides, 1-CN being c. 30% of the total -CNs. After this period, the percentage of 1-CN decreased, while that of 2- and 3-CNs increased due to hydrolysis of the former by plasmin. SDS-PAGE and a specific anti--CN monoclonal antibody identified

1- and 2-CNs in Grana Padano cheese during ripening which showed a correlation with the extent of ripening (Gaiaschi et al., 2001). Nevertheless, the same authors found that the area of cheesemaking, season of production, length of ripening and type of dairy are all factors which may influence proteolysis. The urea-PAGE profiles of pH 4.6-insoluble fraction of ewes’ milk Fossa cheeses vary (Fig. 4). Nevertheless,

αS1 PL3 αS1PL2 αS1 αS1 PL1 β γ1 γ3 γ2

13

13

12

15 15 15

16

17 17 19 20 20

7

5

4

8

6

C

Figure 3 Urea-polyacrylamide gel electrophoretograms of pH 4.6-insoluble nitrogen fraction of Parmigiano Reggiano cheeses at different times of ripening. C, whole casein (Addeo et al., 1988). PL, refers to s1-casein fragments with different electrophoretic mobility.

Extra-Hard Varieties 63

So

1

2

3

4

5

6

7

β-CN

αS1-CN

Figure 4 Urea-polyacrylamide gel electrophoretograms of pH 4.6-insoluble nitrogen fraction of Fossa cheeses. Lanes: Sb, bovine Na-caseinate; So, ovine Na-caseinate standard; 1–7 Fossa cheeses (Gobbetti et al., 1999c).

the profiles are commonly characterised by the complete degradation of s1-CN after 6 months of ripening, while much of the -CN persists unhydrolysed (Gobbetti et al., 1999c). Fossa cheese is produced without cooking the curd and chymosin may remain active during ripening. The same was found for Manchego (Ordóñez et al., 1978) and Canestrato Pugliese (Albenzio et al., 2001; Corbo et al., 2001) cheeses. In the last case, since the cheese may be produced from raw, thermised or pasteurised ewes’ milk, RP-FPLC analysis of the water-soluble fraction showed a more complex peptide pattern in raw milk cheese which was positively linked to more intense proteolysis. The hydrolysis of the caseins leads to an increased proportion of water-soluble N which has been used as a ripening index for Parmigiano Reggiano (Panari et al., 1988). Fig. 5 shows the changes in the percentage ratio, soluble N/total N for Parmigiano Reggiano during ripening. The increase is very fast during the first 8–10 months, after which hydrolysis proceeds very slowly. At the end of ripening, the water-soluble N is c. 34% of the total N. Similar values (c. 32%) are found in Grana Padano cheese (Addeo and Chianese, 1990; Toppino et al., 1990). Since the pH of many extra-hard cheeses is in the range 5.0–5.5, the values of water-soluble and pH 4.6-soluble N do not differ significantly. Values of pH 4.6-soluble N/total N ranging from 19 to 29% were found in Romano-type cheese which

coincided approximately with those for the 12% TCAsoluble N (Guinee and Fox, 1984; Guinee, 1985). Since pH 4.6-soluble N is produced principally by rennet, while starter and non-starter bacterial enzymes are principally responsible for the formation of 12% TCAsoluble N, these data support the view that rennet is not very active in this cheese and that once it produces soluble peptides, bacterial peptidases hydrolyse them relatively rapidly. Contradictory results were reported for proteolysis in Pecorino Romano which varied with the zones of the cheese. At the beginning of ripening, some authors found greater proteolysis in the interior of the cheese, which from 40 days onward was more extensive in the surface zone due to the inward diffusion of NaCl. Other authors (Guinee and Fox, 1984; Guinee, 1985) did not find differences in the level of water- and pH 4.6-soluble N at various locations in the Romano-type cheese throughout ripening. The level of pH 4.6-soluble N is very high also in Fossa cheese, ranging from 30 to 39% of the total N. The water-soluble N may range from 13 to 30% of the total N in Canestrato Pugliese cheese, depending on several factors including NSLAB activity. The extrahard Spanish varieties may be divided into two groups: Mahón and Idiazabal, with a content of soluble N of c. 30%, and Manchego and Roncal with slightly lower values of c. 25% of total N (Ordóñez et al., 1980; Marcos and Esteban, 1993). Variations in the concentration of free amino acids during ripening may be considered as another index by which some extra-hard varieties can be compared (Table 5). Free amino acids accumulate in

40

30 NS/NT %

Sb

20

10

0 0

6

12

18

24

Months Figure 5 Increase (%) in the level of water-soluble nitrogen (SN)/total nitrogen (TN) in Parmigiano Reggiano cheese during ripening. Open circles are the average of several cheeses, of the same age, at the end of ripening (Panari et al., 1988).

Table 5 Concentration individual free amino acids (mg/g cheese) in Parmigiano Reggiano, Canestrato Pugliese and Fossa cheeses

Amino acids Histidine Arginine Serine Aspartic acid  asparagine Glutamic acid  glutamine Threonine Glycine Alanine Tyrosine Proline Methionine Valine Phenylalanine Isoleucine Leucine Cysteine Ornithine Lysine Tryptophan Total free amino acids

Parmigiano Reggiano

Canestrato Pugliese

Fossa

8.20 2.50 13.60

3.82 5.01 8.85

2.44 0.25 3.09

18.60

2.99

4.09

45.50 12.30 6.40 6.90 6.30 n.d. 7.20 18.40 13.20 15.90 22.20 n.d. 3.80 30.80 n.d. 231.80

15.34 3.23 2.55 2.87 1.66 8.65 3.25 8.33 5.88 6.54 10.99 1.57 n.d. 13.31 0.03 104.87

19.19 2.07 1.8 5.83 2.02 5.6 3.97 9.56 5.42 6.24 13.83 5.00 n.d. 13.09 n.d. 103.49

The values indicated represent the average of several determinations made by different authors in cheeses which had a slightly different ripening time: Parmigiano Reggiano, 16–18 months; Canestrato Pugliese, 6–10 months; Fossa, 6–8 months. n.d., not determined. Source: Resmini et al. (1988); Gobbetti et al. (1999c); Albenzio et al. (2001); Corbo et al. (2001).

Parmigiano Reggiano until 15 months of ripening, after which their concentration remains relatively constant (Fig. 6) (Resmini et al., 1988). At the end of ripening, the average concentration of total free amino acids is c. 230 mg/g protein, which corresponds to c. 23% of the total protein content; therefore, Parmigiano Reggiano is one of the richest cheese in free amino acids. The same trend, with similar values, was found for Grana Padano, showing that the extension of ripening to more than 18 months did not produce a significant increase in free amino acids (Resmini et al., 1990). Nevertheless, large variability was found for the amino acid profile of cheeses of the same age. This variability is reduced by expressing the amino acid content as a relative percentage. A chemometric model was proposed to estimate the age and the organoleptic quality of Parmigiano Reggiano based on the level of serine, glutamine, arginine and ornitine which were used as markers (Resmini et al., 1988).

Free AA / Tot Prot %

64 Extra-Hard Varieties

25 20 15 10 r2 = 0.922 DSt = 1.231

5 0 0

2

4

6

8 10 12 14 16 18 20 22 24 26 Months

Figure 6 Changes in the total concentration of free amino acids in Parmigiano Reggiano cheese during ripening (Resmini et al., 1988).

Long-ripened Mahón cheese may be differentiated from young cheese by the content of glutamic acid, glycine, serine and threonine, while cheese produced from raw or pasteurised milk can be differentiated by the concentration of asparagine and glutamine (Frau et al., 1997). The total concentration of free amino acids in Fossa cheese varies greatly between samples (Gobbetti et al., 1999c), which is relatively high compared to Cheddar cheese (Lynch et al., 1996) and even to internally mould-ripened cheeses such as Gorgonzola (Gobbetti et al., 1998). Canestrato Pugilese cheese manufactured from raw ewes’ milk also has a high level of free amino acids (Albenzio et al., 2001). Apart from the high concentrations of threonine, isoleucine and phenylalanine in Parmigiano Reggiano cheese, glutamic acid, proline, valine, leucine and lysine are the amino acids commonly present at high concentrations in Parmigiano Reggiano, Pecorino Romano, Canestrato Pugliese, Fossa, Mahón and Manchego cheeses (Ordóñez et al., 1980; Resmini et al., 1988; Frau et al., 1997; Gobbetti et al., 1999c; Albenzio et al., 2001; Di Cagno et al., 2003). Volatile compounds

Cheese flavour is the result of several non-enzymatic and many enzymatic reactions. Decarboxylation, deamination, transamination, desulphuration and cleavage of side chains convert amino acid to aldehydes, alcohols and acids which together with other compounds, derived by other routes (e.g., lipolysis and catabolism of fatty acids), compose the volatile profile of extra-hard cheeses. Based on High Resolution Gas Chromatography (HRGC)–Mass Spectrometry (MS) and different methods of extraction, volatile compounds in some longripened cheeses have been characterised (Moio and Addeo, 1998; Izco and Torre, 2000; Villaseñor et al., 2000; Larráyoz et al., 2001; Di Cagno et al., 2003) (Table 6). Overall, large variations for the same cheese

Extra-Hard Varieties 65

Table 6 Several volatile compounds ( g/kg) found in Grana Padano, Canestrato Pugliese, Fiore Sardo, Pecorino Romano and Manchego cheeses

Compounds

Grana Padano

Canestrato Pugliese

Fiore Sardo

Pecorino Romano

Manchego

Esters Methyl butanoate Ethyl butanoate Methyl hexanoate Ethyl hexanoate 3-Methylbutyl butanoate Methyl octanoate Ethyl octanoate Ethyl decanoate

2 223 4.5 737 8 5 229 24

n.d. n.d. n.d. 4.17 n.d. n.d. 3.53 3.47

n.d. n.d. n.d. 3.68 n.d. n.d. 3.30 3.28

n.d. n.d. n.d. 3.69 n.d. n.d. 3.29 3.12

41 289 37 115 21 4 64 n.d.

Ketones 2-Pentanone 3-Hydroxy-2-butanone 2-Hexanone 2-Heptanone 8-Nonen-2-one 2-Nonanone 2-Undecanone 2-Tridecanone

143 70 4 320 20 172 38 3

3.64 7.49 1.65 4.95 0.54 4.31 3.45 3.68

3.58 8.88 2.31 5.67 1.93 4.46 3.54 3.87

3.69 6.02 1.55 4.63 0.0 4.17 3.61 3.79

737 n.d. n.d. 368 n.d. 44 n.d. n.d.

Alcohols 1-Butanol 2-Pentanol 3-Methyl-3-buten-1-ol 3-Methyl-1-butanol 3-Methyl-2-buten-1-ol 1-Hexanol 2-Heptanol 1-Octanol 2-Nonanol 1-Decanol Furanmethanol Phenethyl alcohol

37 47 430 290 8.6 20 94 25 6 n.d. n.d. n.d.

7.88 3.83 2.55 3.09 2.67 4.80 3.58 3.08 2.92 2.34 2.55 1.82

2.17 3.36 2.27 2.49 2.08 2.19 2.74 1.87 1.57 1.62 2.24 2.03

4.12 2.24 2.34 3.07 2.24 2.87 2.37 2.03 2.23 1.81 2.38 1.98

167 307 n.d. 180 n.d. n.d. 25 n.d. n.d. n.d. n.d. n.d.

Aldehydes 3-Methyl-butanal 3-Methyl-thiopropanal Benzaldehyde Nonanal Hexadecanal Octadecanal 2-Furancarboxaldehyde Benzaldehyde

8 3 7 n.d. n.d. n.d. n.d. n.d.

n.d. n.d. n.d. 3.43 3.43 3.15 2.17 1.65

n.d. n.d. n.d. 2.90 3.32 3.13 2.27 1.71

n.d. n.d. n.d. 2.83 3.62 3.10 2.03 1.85

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

Lactones

-Hexanolactone

-Octanolactone

-Decalactone

-Decalactone

-Dodecalactone

-Dodecanolactone

-Dodecenolactone

-Tetradecanolactone

-Hexadecanolactone

n.d. n.d. 2 5 n.d. n.d. n.d. n.d. n.d.

1.99 2.11 2.70 4.15 3.95 4.02 2.99 3.20 2.29

2.78 1.94 2.44 4.07 3.63 4.02 2.62 3.10 2.21

2.39 3.74 4.43 5.53 4.15 3.12 3.30 2.24

n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. continued

66 Extra-Hard Varieties

Table 6 continued

Compounds

Grana Padano

Canestrato Pugliese

Fiore Sardo

Pecorino Romano

Manchego

Miscellaneous Phytene A Phytene B Phytadiene Phytanol Phytol 4-Methyl phenol 3-Methyl phenol Dimethyl disulphide Dimethyl trisulphide Methional Limonene

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

2.87 3.22 1.43 1.96 3.00 2.37 2.10 1.55 1.67 4.17 1.08

4.16 2.99 3.24 2.34 2.33 1.62 0.61 1.51 1.70 4.19 0.55

2.89 3.19 1.89 2.09 1.99 1.48 1.44 1.57 1.71 3.76 1.38

n.d. n.d. n.d. n.d. n.d. n.d. n.d. 4 n.d. n.d. 11

The values indicated represent the average of several determinations made by different authors in cheeses of different manufacture. n.d., not determined. Source: Moio and Addeo (1998); Villaseñor et al. (2000); Di Cagno et al. (2003).

variety were found to be related to cheesemaking practices, season of manufacture, duration of ripening and type of secondary microflora. Esters were the main neutral constituents in the aqueous distillate of Grana Padano cheese, constituting c. 41% of the total neutral volatiles (Moio and Addeo, 1998). Esters with a few carbon atoms have a perception threshold 10-fold lower than their alcohol precursors. Ethyl esters of butanoic, hexanoic, octanoic and decanoic acids represent c. 95% of the total esters. Ethyl hexanoate, with a distinct aroma of unripe apples, is present in the greatest quantity, c. 60% of the total esters. In 12-month-old Grana Padano cheese, this odorant is 10-fold the level found in fresh bovine milk (Moio et al., 1993). Ethyl butanoate is the second most important ester. Esters are the main volatile components of Canestrato Pugliese cheese (Di Cagno et al., 2003) and ethyl esters are the predominant esters in Manchego, Roncal, Mahón, Fiore Sardo and Pecorino Romano cheeses (Martinez-Castro et al., 1991; Moio et al., 1993; Izco and Torre 2000; Villaseñor et al., 2000; Larráyoz et al., 2001; Di Cagno et al., 2003). Ketones represent the second largest class of volatile compounds in Grana Padano cheese, accounting for c. 33% of neutral volatiles, similar to the amount found in Parmigiano Reggiano cheese, where they are the most abundant volatiles, representing c. 26% of total headspace chromatographic area (Barbieri et al., 1994; Moio and Addeo, 1998). The total concentration of methyl ketones in Parmigiano Reggiano (0.075 mol/g fat) is quite low compared to Blue cheese (5.18 mol/g fat for Roquefort) (Arnold et al., 1975; Piergiovanni and Volonterio, 1977; Gallois and Langlois, 1990). Ketones were also found to be the dominant volatile flavour compounds in Fiore Sardo (Di Cagno et al., 2003) and the second most abundant group of volatiles

in Roncal cheese (Izco and Torre, 2000) and were considered to be one of the major classes of volatiles which varied in Mahón cheese during ripening (Mulet et al., 1999). The major representatives of the 2-alkanones with odd numbers of carbon atoms in Grana Padano cheese were 2-pentanone, 2-heptanone, 2-nonanone and 2-undecanone. 2-Pentanone and 2-heptanone are the most abundant methyl ketones in aged Manchego cheese (Villaseñor et al., 2000), while 2-heptanone and 2-nonanone were the two methyl ketones found at the highest level in Canestrato Pugliese, Fiore Sardo and Pecorino Romano cheeses (Di Cagno et al., 2003). All the methyl ketones with an odd number of carbons (C39C9) were detected in Roncal, Pecorino Sardo, Manchego and Fiore Sardo cheeses at higher levels than those with an even number of carbons (C49C12) (Izco and Torre, 2000; Villaseñor et al., 2000; Larráyoz et al., 2001; Di Cagno et al., 2003). In Pecorino Sardo cheese, the concentration of methyl ketones generally increases during ripening. It was also presumed that the FFAs liberated through lipolysis are catabolised to methyl ketones by microbial activity (Izco and Torre, 2000). Alcohols represent the third class of volatiles in Grana Padano cheese, accounting for c. 23% of the total neutral volatiles. Those present in greatest quantity are 2-pentanol, 3-methyl-3-buten-1-ol, 3-methyl-1-butanol and 2-heptanol. 1-Octen-3-ol is a key aroma compound of mushrooms and has long been recognised as an important flavour compound produced by Penicillium roqueforti in Blue cheeses (Shimp and Kinsella, 1977). Alcohols are the predominant group of volatile compounds in Roncal and Pecorino Romano cheeses (Izco and Torre, 2000; Di Cagno et al., 2003). Butan-2-ol and propan-1-ol have been detected in the largest quantities

Extra-Hard Varieties 67

in the Spanish cheese, while 1-butanol and 1-hexanol characterised the Italian variety. Parmigiano Reggiano cheese contains at least 16 different chiral alcohols, the most abundant secondary alcohols found being 2butanol, 2-pentanol, 2-heptanol, 2-nonanol and 1octen-3-ol (Mariaca et al., 2001). Aldehydes and lactones contribute c. 0.6 and 0.1%, respectively, of the total neutral volatiles of Grana Padano cheese (Moio and Addeo, 1998). Low levels of aldehydes indicated a normal maturation; at higher levels, they were found to cause off-flavour. Lactones are the second largest class of volatiles in several Italian ewes’ milk cheeses like Canestrato Pugliese, Fiore Sardo and Pecorino Romano, -dodecalactone and -dodecanolactone being found at the highest levels (Di Cagno et al., 2003). Eleven lactones were detected in the Parmigiano Reggiano cheese; -decalactone and -dodecalactone were found most commonly (Mariaca et al., 2001).

Nutrition Some of the most famous Italian extra-hard varieties have also been characterised from the point of view of nutrition (Table 7). Compared to other varieties, Parmigiano Reggiano and Grana Padano are described as those cheeses with the highest content of protein and a low content of lipids and cholesterol (Turchetto, 1988; Berra and Ottina, 1990; Califfi and Mazzali, 2000). These cheeses have an energy value of c. 374–384 kcal/100 g which is similar to that of Pecorino Romano. One hundred grams of Parmigiano Reggiano cheese add c. 33 g

of protein to the diet. The protein content of these Italian varieties is of high quality since a great part is already digested to peptides of various size and amino acids which either facilitate digestion or stimulate the gastric secretions. A large part of the total free amino acids are essential amino acids (e.g., leucine, lysine, isoleucine and valine) and also the level of non-essential amino acids is very high which effectively reduces the metabolic energy expended on biosynthetic reactions. Except for cysteine  methionine, c. 50 g of Parmigiano Reggiano and Grana Padano cheeses are enough to meet the daily requirements of the other essential amino acids. Parmigiano Reggiano and Grana Padano cheeses contain c. 28% lipids, triacylglycerols being the main component. Cholesterol is present at a concentration less than 80–85 mg/100 g of cheese (Marchetti, 1988). The high content of calcium, c. 1.2%, and, especially, the optimum calcium/phosphorus ratio, is another important nutritional feature of these cheeses. Besides, the ratio of calcium/lipids is very high compared to other cheeses, which means that ingestion of an optimum intake of calcium (e.g., 800 mg/day) is not negatively correlated with an energetic surplus due to an elevated intake of lipids. Parmigiano Reggiano and Grana Padano cheeses are also very rich in other mineral constituents, e.g., 63 mg of potassium and 18 mg of iodine per 50 g of cheese which represent c. 20% of the human daily requirement (Ferri, 1990; Marchetti, 1990). Parmigiano Reggiano and Grana Padano cheeses have considerable levels of fat-soluble vitamins, A and D, and especially are highly appreciated for the elevated amount of vitamin B12 (Marchetti, 1990).

Table 7 Average value of nutritional compounds for 100 g of Grana Padano cheese

Acknowledgements

Compound

Concentration

Mazco Gobbetti wishes to thank Prof. Bruno Battistotti for the friendly and skilled revision of this chapter.

Moisture Protein Soluble peptides Free amino acids Lipids Carbohydrates Calcium Phosphorus Ratio calcium/phosphorus NaCl K, Mg2, Zn2, Fe2, Cu2, Se2, I Vitamin A, D3 and E Vitamin B1, B2, B6 Vitamin B12 Pantothenic acid Choline Biotin

32 g 33 g 1.5 g 6g 28 g absent 1165 mg 692 mg 1.7 1.4 g 881.5 g 227.5 g 494 g 3 g 246 g 20 g 6 g

The energy value of 100 g of Grana Padano cheese is 384 kcal (252 kcal from lipids and 132 kcal from proteins). Source: Califfi and Mazzali (2000).

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Cheddar Cheese and Related Dry-salted Cheese Varieties R.C. Lawrence, J. Gilles,* L.K. Creamer, V.L. Crow, H.A. Heap, C.G. Honoré, K.A. Johnston and P.K. Samal, Fonterra Research Centre, Palmerston North, New Zealand

Introduction In the warm climates in which cheesemaking was first practised, cheeses would have tended to have a low pH as a result of the acid-producing activity of the lactic acid bacteria and coliforms in the raw milk. In colder climates, it would have been logical either to add warm water to the curds and whey to encourage acid production (the prototype of Gouda-type cheeses) or to drain off the whey and pile the curds into heaps to prevent the temperature falling. In the latter case, the piles became known as ‘Cheddars’, after the village in Somerset, England, where the technique is said to have been first used about the middle of the nineteenth century. The concept of cheddaring was quickly adopted elsewhere. The first Cheddar cheese factory, as opposed to farmhouse cheesemaking, was in operation in the United States (NY State) in 1861, followed by Canada (Ontario) in 1864 and by New Zealand and England in 1871. Development of cheddaring

Cheddar cheese was apparently made originally by a stirred curd process without matting, but poor sanitary conditions led to many gassy cheeses with unclean flavours (Kosikowski and Mistry, 1997). Cheddaring was found to improve the quality of the cheese, presumably as a result of the faster and greater extent of acid production. As the pH fell below about 5.4, the growth of undesirable, gas-forming organisms, such as coliforms, would have been increasingly inhibited. The piling and repiling of blocks of warm curd in the cheese vat for about 2 h also squeezed out any pockets of gas that formed during manufacture. Cheesemakers came to believe that the characteristic texture of Cheddar cheese was a direct result of the cheddaring process. It is now clear that recently developed methods of manufacturing Cheddar cheese do not involve a traditional cheddaring step but the cheese obtained has a texture identical to that of traditionally made Cheddar.

* Deceased 19 January 2003.

The development of the fibrous structure in the curd of traditionally made Cheddar does not commence until the curd has reached a pH of 5.8 or less (Czulak, 1959). The changes that occur are a consequence of the development of acid in the curd and the consequent loss of calcium and phosphate from the protein matrix. Therefore, it is important to recognize that ‘cheddaring’ is not confined only to Cheddar cheese. All cheeses are ‘cheddared’ in the sense that all go through this same process of chemical change. The only difference is one of degree, i.e., the extent of flow varies due to differences in calcium level, pH and moisture (Lawrence et al., 1983, 1984). In addition, with brine-salted cheeses, flow is normally restricted at an early stage in manufacture by placing the curd in a hoop. However, if Gouda curd is removed from a hoop, it flows in the same way as Cheddar curd. Similarly, the stretching induced in Mozzarella by kneading in hot water is best viewed as a very exaggerated form of ‘cheddaring’. All young cheese, regardless of the presence of salt, can be stretched in the same way as Mozzarella, provided that the calcium content and pH are within the required range (Lawrence et al., 1993). Development of dry-salting

In the early days of cheesemaking, the surface of the curd mass was presumably covered with dry salt in an attempt to preserve the cheese curd for a longer period. In localities where the salt was obtained by the evaporation of seawater, it would have been a rational step to consider using the concentrated brine rather than wait for all the liquid to evaporate. The technique of dry-salting, i.e., salting relatively small pieces of curd before pressing, appears to have evolved in England, probably in the county of Cheshire, where rock salt is abundant. Cheshire has been manufactured for at least 1000 years and is thus a more ancient cheese than Cheddar. Variants of Cheshire and Cheddar were developed in specific localities of Britain and have come to be known as British Territorial cheeses. Blueveined cheeses such as Stilton, Wensleydale and Dorset are also dry-salted.

Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 2: Major Cheese Groups ISBN: 0-1226-3653-8 Set ISBN: 0-1226-3651-1

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72 Cheddar Cheese and Related Dry-salted Cheese Varieties

Dry-salting overcomes the major disadvantage of brine-salting, i.e., the ‘blowing’ of the cheese due to the growth of such bacteria as coliforms and clostridia, but introduces new difficulties because the starter organisms and lactic acid formation are also inhibited by the salt. This inhibition is not a problem when the pH of the curd granules is allowed to reach a relatively low value prior to the application of salt, as in Cheshire and Stilton manufacture. However, the manufacture of a dry-salted cheese in the medium pH range (5.0–5.4), such as Cheddar, is more difficult than that of the Gouda-type cheeses in which the pH is controlled by limiting the lactose content of the curd by the addition of water to the curds/whey mixture in the vat. At the time of salt addition, a relatively large amount of lactose is still present in Cheddar curd (Turner and Thomas, 1980). However, this is not detrimental to the quality of the cheese provided that the salt-in-moisture (S/M) level is greater than 4.5% and the cheese is allowed to cool after pressing (Fryer, 1982). Differences obviously exist in the procedures used for the manufacture of dry- and brine-salted cheeses but these have relatively little effect on the finished cheeses; the production of dry-salted cheeses is similar in principle to that of brine-salted cheeses. Clearly, the rate of solubilization of the casein micelles and the activity of the residual rennet and plasmin in the curd will be affected more rapidly by dry-salting than by brining but only during the first few weeks of ripening. There is no evidence to suggest that the mechanisms by which the protein is degraded are affected by the changes in salt concentration as the salt diffuses into the curd. Any differences between dry- and brinesalted cheeses of the same overall chemical composition will therefore decrease as the cheeses age. Traditional Cheddar cheese is visually different from the common brine-salted cheeses such as the Goudaand Swiss-type cheeses, which are more plastic in texture and have ‘eyes’. However, both these characteristics are a result of the relatively high pH and moisture of these cheeses and not of brine-salting itself. The texture of a brine-salted cheese is less open than that of traditionally-made Cheddar cheese because the curd is pressed under the whey to remove pockets of air before brining. As a close texture is a pre-requisite for the formation of ‘eyes’, it has come to be generally believed that ‘eyes’ can be obtained only in brine-salted cheese. The technique of vacuum pressing allows the removal of air from between the particles of dry-salted curd. This can result in a closeness of texture similar to that of Gouda-type cheeses. Therefore, it is now possible to manufacture dry-salted cheese with ‘eyes’ provided that the chemical composition is similar to that of tradi-

tional brine-salted cheeses and if the starter contains gas-producing strains (Lawrence et al., 1993). Present and future role of Cheddar-like cheeses

Traditionally, Cheddar was a so-called ‘table cheese’ and was purchased by the consumer shortly before consumption. In line with the global changes in the dairy and food industries (Creamer et al., 2002), cheese, Cheddar in particular, is commonly purchased from the manufacturer, repackaged, often in vacuum packs, and sold on to supermarkets or food wholesalers. It is also used as the base material for a range of processed cheeses (‘Pasteurized Processed Cheese and Substitute/ Imitation Cheese Products’, Volume 2) and ‘cheesefood’ products (‘Cheese as an Ingredient’, Volume 2). Because of our understanding of the factors controlling the development of Cheddar cheese flavour and texture during maturation, it is possible to produce cheeses with a range of pre-determined characteristics using semi-automated mechanized manufacture (‘General Aspects of Cheese Technology’, Volume 2). Cheese, as a major ingredient in a food, needs to fulfil certain requirements, such as retention of the flavour and textural characteristics it confers on the food over a substantial storage period. This is coupled with strict composition and price criteria. A good example of meeting this challenge is outlined in detail by Chen and Johnson (2001) in producing a dry-salted cheese using a mesophilic starter suitable for hot-melt products, such as Pizza pies, without using the pastafilata (Mozzarella) process.

Manufacture of Cheddar Cheese During the latter half of the twentieth century, there were a number of significant changes to the way in which Cheddar cheese is manufactured. The single most important factor supporting those changes has been the availability of reliable starter cultures. The successful development of continuous mechanized systems for Cheddar manufacture has depended upon the ability of the cheesemaker to control precisely both the expulsion of moisture and the increase in acidity required in a given time. This in turn has led to the recognition that the quality of cheese, now being made on a very large scale in modern cheese plants, can be guaranteed only if its chemical composition falls within pre-determined ranges. Nevertheless, Cheddar cheese is still a relatively difficult variety to manufacture because the long ripening period necessary for the development of the required mature flavour can also be conducive to the formation of off-flavours. In addition, its texture can vary considerably. The intermediate position of

Cheddar Cheese and Related Dry-salted Cheese Varieties 73

5.4

Stage of lactation

Colby Swiss

5.2

Gouda 5.0

Milk composition Cheddar

4.8

Standardization

Cheshire

Production of acid

Heat

Fat in curd

Stilton

4.6

Curd acidity/pH 700

600

500

400

Dimensions of curd

Salt

mmoles calcium/kg solids-not-fat

Moisture expulsion

Figure 1 Classification of traditionally manufactured cheese varieties by their characteristic ranges of the ratio of calcium to solids-not-fat and pH.

Cheddar cheese in the total cheese spectrum (Lawrence et al., 1984) (Fig. 1) is particularly exemplified by its textural properties, which lie between the crumbly nature of Cheshire and the plastic texture of Gouda. The traditional manufacture of Cheddar cheese consists of: (a) coagulating milk, containing a starter culture, with rennet; (b) cutting the resulting coagulum into small cubes; (c) heating and stirring the cubes with the concomitant production of a required amount of acid; (d) whey removal; (e) fusing the cubes of curd into slabs by cheddaring; (f) cutting (milling) the cheddared curd; (g) salting; (h) pressing; (i) packaging and ripening (Fig. 2). Although it is impossible to separate the combined effects of some of these operations on the final quality of the cheese, they will, as far as possible, be considered individually. Effect of milk composition and starter culture

(Whitehead and Harkness, 1954; Lawrence et al., 1983; Johnston et al., 1991). In order to achieve uniform cheese quality in large commercial plants, the manufacturing procedures must be as consistent as possible. The first requirement is uniformity of the raw milk. This is achieved by bulking the milk in a silo to even out differences in milk composition from the various districts supplying milk to the cheese plant. Preferably, the milk should be bulked before use so that its fat content can be standardized accurately. For Cheddar cheese varieties, the milk is normally standardized to a casein/fat ratio between 0.67 and 0.72. The more fat present in the cheese milk, and therefore in the rennet coagulum, the more difficult it is to remove moisture under the same manufacturing conditions because the presence of fat interferes mechanically with the syneresis process. Standardization has traditionally involved manipulation of the fat content of the cheese milk to give a specific casein/fat ratio. This is usually achieved either by partially removing the fat from the whole milk stream or by removing all the fat from the whole milk and adding back a portion to the skim milk stream. However, recent developments in membrane

Dr y S

Cheddaring 30–40 min

Mellowing

Pressing

2–5 min 20–40 min 30 min

35–40 min 2 h 20 min–2 h 45 min

Mill

tir cur

d

ey off Stir off Run W h

off Heat

Cut Stir on Heat on

Set

Cheesemaking basically involves the removal of moisture from a rennet-induced coagulum (Fig. 3). The four major factors involved are the proportion of fat in the curd, the curd particle size, the cooking (scalding) temperature and the rate and extent of acid production

Figure 3 The main factors in the expulsion of moisture from a rennet-induced milk coagulum.

Hoop

800

Salt

pH at day 1

Breed

Feed

1 h 50 min–2 h

Figure 2 A typical manufacturing schedule for Cheddar cheese.

1h

≈16 h

74 Cheddar Cheese and Related Dry-salted Cheese Varieties

processing technologies have meant that the protein component of the whole milk can now be standardized also. There are a number of options by which the protein content of cheese milk can be standardized. An example is concentrating the level of protein in a skim milk stream by ultrafiltration and adding the retentate back to the whole milk stream to boost the protein concentration in the whole milk to the target level, which is typically between 3.5 and 4%. The manufacture of Cheddar cheese is more dependent on uniform starter activity than that of washed curd cheeses, such as Gouda. The proper rate of acid development, particularly before the whey is drained from the curd, is essential if the required chemical composition of the cheese is to be obtained (Whitehead and Harkness, 1954; Lawrence et al., 1984). However, the curd is ‘cooked’ to expel moisture at a temperature that normally adversely affects the starter bacteria. The cheesemaker must therefore exert judgement to ensure that the desired acid development in the curd is reached at about the same time as the required moisture content. The starter system used in New Zealand cheese plants is based on the continuous use of a single triplet starter comprising three defined strains of Lactococcus lactis subsp. cremoris selected primarily on the basis of their acid production, phage resistance and flavour development (Heap, 1998). Defined starter systems are now widely used in the United States (Richardson et al., 1981), Ireland (Timmons et al., 1988), Scotland and Australia (Heap and Lawrence, 1988; Limsowtin et al., 1996) and have replaced the undefined commercial mixed-strain cultures of the type still used exclusively for the manufacture of Gouda-type cheeses in The Netherlands (Stadhouders and Leenders, 1984). If the cooking temperature is kept constant (for instance at 38 °C) throughout the cheesemaking year and standardized milk is used, by far the most important factor in producing Cheddar cheese of uniform quality is the extent of acid production in the vats. In New Zealand, this is managed successfully in two ways: (a) the use of reconstituted skim milk or suppliers’ milk of good quality for the preparation of bulk cheese starter; (b) the ability of the cheese industry to produce neutralized bulk cheese starter and to control the ratios of the starter strains added to the cheese milk (Heap, 1998). To compensate for seasonal changes in milk composition, it is normally necessary only to vary the percentage inoculum of starter to achieve the required acidity at draining. Effect of coagulant

The amount of rennet added should be the minimum necessary to give a firm coagulum in the set-to-cut time

(time between rennet addition and cutting) required. In Cheddar cheese manufacture, the set-to-cut time is usually in the range 35–45 min. There is a range of animal, microbial and recombinant rennets to choose from and their advantages and disadvantages are discussed in ‘Rennets: General and Molecular Aspects’, Volume 1. Calf rennet, high in chymosin, has been used traditionally for Cheddar cheese production. The advantage of using a high chymosin content calf rennet is that the flavour and the texture of aged Cheddar are more predictable, with less bitterness. The same could be said for the recombinant chymosins. However, some customers have strong aversions to the use of genetically engineered ingredients in cheese. Some cheese manufacturers are now investigating the use of microbial rennets, which provide the added advantage of being suitable for Kosher, Halal and some vegetarian products. In addition, use of microbial rennets in Cheddar cheese production opens up the options for downstream whey products (whey protein concentrates, milk protein concentrates, etc.). Changes in the volume of rennet added, an increase or decrease in the setting temperature, addition of calcium chloride and/or pH adjustment may be required to avoid any seasonal changes in milk composition and functionality. The rennet-induced coagulum consists of a continuous network of protein that entraps both water and fat globules. The protein network is composed of small units of protein held together by various forces. Several reports (Eino et al., 1976; Green et al., 1981, 1983) have concluded that the microstructure of the coagulum produced by different types of milk coagulant is a major factor determining the structure and texture of Cheddar cheese. It has been suggested (Green et al., 1981) that ‘the structure of the protein network is laid down during the initial curd-forming process and is not fundamentally altered during the later stages of cheesemaking and that the fibrous and more open framework of curd formed by bovine and porcine pepsins might be a reason for the softer curd associated with their use’ (Eino et al., 1976). This implies that different milk coagulants significantly affect the initial arrangement of the network of protein structural units. However, it is more likely that the proportion of minerals lost from the coagulum, as a result of the change in pH throughout the entire process, largely determines the texture of a cheese. As one would expect, the type of rennet used and the amount retained in the cheese curd affect the degree of proteolysis as the cheese ripens (Stanley and Emmons, 1977; Creamer et al., 1985) (cf. ‘Rennetinduced Coagulation of Milk’, ‘Biochemistry of Cheese Ripening: Introduction and Overview’, ‘Metabolism of Residual Lactose and of Lactate and Citrate’, ‘Lipolysis

Cheddar Cheese and Related Dry-salted Cheese Varieties 75

and Catabolism of Fatty Acids in Cheese’, ‘Proteolysis in Cheese during Ripening’, and ‘Catabolism of Amino Acids in Cheese during Ripening’, Volume 1). The early stages of Cheddar cheese manufacture, specifically gel assembly and curd syneresis, have been reviewed (Fox, 1984; Green, 1984) (‘Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels’, Volume 1). Electron microscopy studies (Kimber et al., 1974; Kalab, 1977; Stanley and Emmons, 1977) have shown that the casein micelles, which are separate initially, aggregate, coalesce and finally form a multi-branched casein network. The fat globules, also separate at first, are gradually forced together as a result of shrinkage of the casein network. After the coagulum is cut, the surface fat globules are exposed and washed away as the curd is stirred. This leaves a thin layer depleted of fat at the curd granule surface. During matting, the layers of adjacent curd granules fuse, leading to the formation of fat-depleted junctions (Lowrie et al., 1982). Starter bacteria are trapped in the casein network near the fat–casein interface, which has been shown to be the region of highest water content in the mature cheese (Kimber et al., 1974). In all cheese varieties, the outline of the original particles of curd formed when the rennet-induced coagulum is cut can be readily distinguished by scanning electron microscopy (Kalab et al., 1982). In addition, in traditionally-made Cheddar cheese, the boundaries of the milled curd pieces can be seen (Lowrie et al., 1982). These curd granules and milled curd junctions in Cheddar cheese are permanent features, which can still be distinguished in aged cheese. Effect of cutting

The objective of cutting the coagulum, and indeed the objective of the heating and stirring stages that follow cutting, is to facilitate syneresis (‘The Syneresis of Rennet-coagulated Curd’, Volume 1). However, the cutting operation, together with the speed of stirring following cutting, also influence how large the particles will be at draining and how much of the original milk components (fat and protein) are lost to the whey. The size distribution of the particles at draining is one of the key factors for controlling the moisture content of cheese. The larger the particles, the more moisture that is retained (Whitehead and Harkness, 1954). Maximizing moisture (or moisture in the nonfat substance (MNFS)) and minimizing losses (fat and cheese fines) to the whey will ensure the highest possible yield and profitability (Lawrence and Johnston, 1993). Therefore, cutting is a key operation in cheesemaking and influences not only the composition but also the yield of the finished cheese.

Johnston et al. (1991) showed that the speed and duration of cutting in 20 000 l Damrow cheese vats during commercial Cheddar cheese production determines the curd particle size distribution at draining and hence the moisture content of the final cheese. The whey fat losses could be minimized by the choice of the cutting protocol used. They concluded that, as cutting proceeded, the particle size distribution increasingly favoured smaller particles and that there were two different effects (Fig. 4). In region I, where the cutting cycle is too short, large curd particles remaining after cutting will be reduced in size by smashing during the subsequent stirring phase. Smashing results in small curd particles and fines at draining and high whey fat losses. Between regions I and II, the curd particle size following cutting is small enough to avoid smashing during subsequent stirring and therefore the curd particle size is at a maximum and whey fat losses are at a minimum. In region II, continued cutting gives rise to a greater proportion of smaller curd particles and, in the absence of smashing, whey fat losses remain low. Based on this explanation, Johnston et al. (1991) proposed a model (Fig. 5) for cutting that explains how variations in cutting speed and duration of cutting, followed by a constant stirring speed, determine the curd particle size distribution in a Damrow cheese vat. Each of the five curves (Fig. 5) represents the variations in curd particle size distribution with the duration of cutting, for a constant speed of cutting. Each curve depends on the duration of cutting and is characterized by a specific duration of cutting at which the curd particle size is at a maximum. As the cutting speed is reduced and the duration of cutting is increased to avoid shattering during stirring, the maximum curd particle size increases. Cutting beyond a certain duration, irrespective of the speed of cutting, does not further reduce the curd particle size. A similar study (Johnston et al., 1998) using Ost vats (30 000 l) showed similar trends. However, the Ost vat study also showed that, although similar, the trends were sufficiently different to warrant the characterization of each vat type as to the effect of the speed and duration of cutting, before implementing a specific cutting regime. Effect of heating (cooking) the curd

During cooking, the curds are heated to facilitate syneresis and aid in the control of acid development. The moisture content of the curds is normally reduced from approximately 87% in the initial gel to below 39% in the finished Cheddar cheese. The expulsion of whey is aided by the continued action of rennet as

76 Cheddar Cheese and Related Dry-salted Cheese Varieties

80 I

II

0.50 60

Curd size 0.40

50

Fat content

0

20

40 Total revolutions of knife

60

Fat content (%)

Proportion of curd <7.5 mm (%)

70

0.30

80

Figure 4 Effect of the speed and duration of cutting on the proportion of curd particles 7.5 mm at draining and the fat content of the whey at running.

well as the combined influence of heat and acid. The temperature should be raised to 38–39 °C over a period of about 35 min. The curds shrink in size and become firmer during cooking.

Proportion of curd <7.5 mm (%)

80

Acid production at the vat stage

70

60

50

40

0

5

10 15 20 Duration of cutting (min)

25

Figure 5 Curves showing the effect of the speed and duration of cutting on the proportion of particles 7.5 mm. Cutting speeds were: (䊊), 2 rev/min; (䉱), 4 rev/min; (䉭), 5 rev/min; (䊐), 6 rev/min; (䊏), 8 rev/min. The continuous lines show the data of Johnston et al. (1991) and the dotted lines are the anticipated trends.

The single most important factor in the control of Cheddar cheese quality is the extent of acid production in the vat (Fig. 6) because this largely determines its final pH (Lawrence and Gilles, 1982; Creamer et al., 1988) and the basic structure of the cheese (Lawrence et al., 1983). As the pH of the curds decreases, there is a concomitant loss of colloidal calcium phosphate from the casein sub-micelles and, below about pH 5.5, the sub-micelles dissociate into smaller aggregates (Roefs et al., 1985). As the amount of rennet added and the temperature profile are normally constant in the manufacture of Cheddar cheese, the pH change in the curd becomes the important factor in regulating the rate of whey expulsion (Van Slyke and Price, 1952; Lawrence et al., 1984). In mechanized cheesemaking systems, the cheese is usually made to a fixed time schedule. In New Zealand, the time between ‘setting’ (addition of milk coagulant) and ‘running’ (draining of whey from the curds; also called ‘pump out’) is normally 2 h 40 min  10 min. The percentage of starter added determines the increase in titratable acidity or

Cheddar Cheese and Related Dry-salted Cheese Varieties 77

Acid production in vat

Decrease in pH of curd

Loss of calcium and phosphate

pH and mineral content of curd at draining

Basic structure of cheese

Breakdown of casein network during ripening

Cheddar texture and flavour Figure 6 Relationship between the extent of acid production up to the draining stage and the production of Cheddar cheese flavour and texture.

decrease in pH between ‘cutting’ and ‘running’. The extent of acidity increase at this stage is particularly important because it also controls the increase in acidity from ‘drying’ (when most of the whey has been removed) onwards (Dolby, 1941). The actual increase in acidity may need to be adjusted at intervals throughout the year to achieve the required pH in the cheese at 1 day. This depends upon changes in the chemical composition of the milk, which, in turn, are determined by both the feed of the cow and the lactational cycle. The pH at draining also determines the proportions of residual chymosin (calf rennet) and plasmin in the cheese (Holmes et al., 1977; Lawrence et al., 1984; Creamer et al., 1985). Chymosin plays a major role in the degradation of the caseins during ripening and in the consequent development of characteristic cheese flavour and texture. While curds remain in the whey, there is a continual transfer of lactose to the curds. The whey thus provides a reserve of lactose which prevents any great decrease in lactose concentration in the curd. After the whey has been removed, this reserve is no longer available and the lactose content of the curd falls rapidly as the fermentation proceeds. Curd that has been left in contact with the whey for a longer period has a

higher lactose content than curd of the same pH value from which the whey has been removed earlier (Dolby, 1941; Czulak et al., 1969). Acid production can be under complete control only if defined starter systems, such as the single triplet starter, where the individual strains have been selected based on their sensitivity to the manufacturing temperature profile, are used (Lawrence and Heap, 1986; Heap, 1998). Use of this culture has allowed New Zealand cheesemakers to reduce the time from ‘set’ to ‘salt’ to about 4 h 30 min. Even shorter times are potentially possible but these are limited by the rate at which moisture can be expelled from the curds in the traditional Cheddar process. Experience has shown that it is preferable to produce lactic acid relatively slowly during the early stages of curd formation and cooking, followed by an increasing rate after draining the whey from the curds. This procedure retains more of the calcium and phosphate in the curd. A recent trend in Europe has been to include a thermophilic strain in starter blends comprising mesophilic strains used for making pressed cheeses (Beresford and Cogan, 1997), as well as soft-ripened cheeses. The rationale for the inclusion of this strain would appear to be in terms of providing a relatively slow rate of acid production at a low temperature of manufacture (high-pH, white-mould cheeses). However, phage attack of the mesophilic strains in these starter blends has led to variable rates of acid production in the cheese vats and problems controlling the final moisture content of the cheese (Heap, personal observation). Effect of cheddaring

The series of operations consisting of packing, turning, piling and re-piling the slabs of matted curd is known as cheddaring. The curd granules fuse under gravity into solid blocks. Under the combined effect of heat and acid, matting of the curd particles proceeds rapidly. The original rubber-like texture gradually changes into a close-knit texture, with the matted curd particles becoming fibrous. The importance attached to flow in the past varied markedly from country to country. In Britain, it was common for each Cheddar block to be made to spread into a thin, hide-like sheet covering an area of about a square metre, whereas in New Zealand only moderate flow was induced, the final Cheddar block being little different in dimensions from when first cut. Czulak and his colleagues (Czulak and Hammond, 1956; Czulak, 1958, 1959; King and Czulak, 1958) initially concluded that extensive deformation and flow were essential in Cheddar cheesemaking. However, further research in Australia

78 Cheddar Cheese and Related Dry-salted Cheese Varieties

(Czulak, 1962), New Zealand (Harkness et al., 1968) and Canada (Lowrie et al., 1982) slowly led to the view that ‘cheddaring’ is not an essential step and serves no purpose other than to provide a holding period during which the necessary acidity develops and further whey can be released from the curds. This loss of whey is controlled by the acidity and temperature of the curd. The temperature is important, both directly and indirectly, because the rate of acid development is also influenced by temperature. In general, a higher temperature during cheddaring increases the expulsion of whey from the curds. In the traditional process, manipulations of the curd, i.e., the cutting of the matted curd into different sized blocks, the height of piling and the frequency of turning the curd blocks, also aid in moisture control. Mechanical forces – pressure and flow – have been shown (Czulak and Hammond, 1956; Czulak, 1959) to be an important factor in the development of the fibrous structure in the curd. This is clearly seen in the arrangement of the fibres, which follow the direction of the flow. However, a fibrous structure cannot be brought about by pressure and deformation unless the curds have reached a pH of 5.8 or less (Czulak, 1959). This suggested that pressure and flow serve to knit, join, stretch and orientate the network of casein fibres already partly formed in response to rising acidity. The readiness to flow, the type of fibres and the density of their network are also influenced by temperature and moisture. The warmer the curd and the higher its moisture content, the more readily it flows and the finer, longer and denser are the fibres. Czulak (1959) also concluded that it is possible to influence curd structure by manipulating pH, pressure and temperature and that a direct relationship exists between the structure and the water-holding capacity of the curd. This was confirmed by Olson and Price (1970), who showed that extension and rapid flow of curd during cheddaring produced a higher moisture content in the resulting cheese. Fluorescence microscopy has demonstrated the change of the casein from spherical granular particles to a fibrous network (King and Czulak, 1958). Whereas some granular structure was evident in curd grains, the conversion to the fibrous form was complete in cheddared curd. The fibrous shreds of cheddared curd consist of flattened, elongated curd particles that overlap each other, forming a networktype structure with the protein as a continuous phase. The exact mechanism responsible for these observed changes in cheddared curd is not known with certainty but the loss of minerals from the casein micelles in the curd is likely to be the major factor. The loss of calcium phosphate will destabilize the casein micelles,

resulting in a change in the conformation of the caseins. The concomitant loss of moisture from the casein micelles may also possibly contribute to the conformational change. Czulak (1962) concluded that the characteristic close texture of Cheddar cheese could be obtained without cheddaring. However, he suggested that in mechanizing the cheesemaking process it was probably most convenient, while holding the curd for acidity to develop, to allow the particles to mat together ‘but to apply no labour or equipment for its fusing beyond that necessary for ready handling’. Almost all modern mechanized Cheddar cheesemaking systems are based upon these conclusions and involve little or no flow of the curd mass. This development was supported by the success achieved in the manufacture of cheese of normal Cheddar characteristics, particularly in the United States, by ‘the stirred curd’ process. This strongly indicates that flow and the cheddaring process itself are of little or no significance in the Cheddar cheesemaking process. Similar conclusions were also reached by research workers (Harkness et al., 1968) in New Zealand. Effect of milling

The milling operation consists of mechanically cutting the cheddared curd into small pieces in order to: (a) increase the surface area of the curd and so enable more uniform salt distribution into the curd; (b) encourage whey drainage from the curd; (c) assemble the curd in a convenient form for hooping or block-forming. There is a practical upper limit to the cross-section of milled curd before salting for two reasons: (a) there is inadequate whey drainage after salting with large particles; (b) the larger the curd particles, the smaller is the surface/volume ratio. With larger particles, a higher salting rate is therefore required to achieve a given final level of S/M in the cheese. This increases the chance of seaminess (Conochie and Sutherland, 1965a) and gives higher salt losses in the whey (Gilles, 1976). The longer time required for salt penetration allows a greater development of acid in the centre of large curd particles than in smaller particles and this may result in a ‘mottled’ appearance of the final cheese. Gilbert (1979) pointed out that ideally the curd should be cut into spheres to obtain a uniform mass/surface area profile. However, the best that can be achieved (Breene et al., 1965; Gilbert, 1979) is to use a curd mill that produces a shredded curd, flakes of curd or finger-like pieces of curd. The curd mill speed can be increased or decreased to change the curd particle size and shape, which in turn affect the

Cheddar Cheese and Related Dry-salted Cheese Varieties 79

Salt (and more specifically S/M) plays a number of roles in the quality of Cheddar cheese by controlling: (a) the final pH of the cheese (Thomas and Pearce, 1981; Lawrence and Gilles, 1982), (b) the growth of microorganisms, specifically starter bacteria and undesirable species such as coliforms, staphylococci and clostridia, and (c) the overall flavour and texture of the cheese. The S/M level controls the rate of proteolysis of the caseins by the rennet, plasmin and bacterial proteases. Proteolysis, and thus the incidence of bitterness and other off-flavours, decreases with an increase in salt concentration (Thomas and Pearce, 1981; Pearce, 1982). At S/M levels 5.0%, bitter flavours are rarely encountered (Lawrence and Gilles, 1969); below this level there is more or less an inverse linear relationship between S/M and the incidence of bitterness. General aspects of salt in cheese are considered in ‘Salt in Cheese: Physical, Chemical and Biological Aspects’, Volume 1; some specific aspects in relation to Cheddar are considered below.

thereby released to dissolve more salt. The proportion of moisture in the curd and the amount of salt added both affect the rate of solution of the salt. The high salt content of the surface of the milled curd particles reduces the tendency of the particles to fuse together. The difference between dry-salting and brine-salting is, in effect, the availability of water at the surface of the curd. With brine-salting, salt absorption begins immediately; release of whey occurs, as in dry-salting, but is not a pre-requisite for salt absorption. In modern cheese plants, it is essential that the curd particles prior to salting are consistent from day to day with respect to moisture content, particle size and shape, acidity level and temperature, and that the application of salt is uniform. This gives the cheesemaker control over both the mean salt content and, equally important, variations (standard deviation) within a day’s manufacture. Cheese specifications normally require both moisture and S/M to be within specified ranges. This means that in practice variations in the moisture content of the curd prior to salting must not be greater than 1%. It has been suggested (Sutherland, 1974; Gilbert, 1979) that the size of the salt crystals used is important for both salt uptake and moisture control. In practice, however, the major requirement in mechanized cheese plants is that the size range of the salt crystals should be narrow. If the range is variable, the delivery of salt from the equipment is erratic. The presence of large amounts of very fine crystals also results in excessive salt dust within the plant environment. Although salt promotes syneresis, it should not be used in mechanized Cheddar cheesemaking as a means of making a significant adjustment to the moisture content of the curd. However, in practice, because of variations in milk buffering capacity, starter activity and plant breakdowns, day-to-day variations in the curd pH and moisture are not uncommon. Therefore, slight adjustments are made to the quantity of added salt to attain consistency in the salt and moisture contents in the curd. The salting techniques commonly used in mechanized cheesemaking are: boom-salting, in one or two stages, and trommel salting. The former uses salt addition to the curd on a mass/volume ratio whereas the latter uses a mass/mass ratio. More details on salting systems are included in ‘General Aspects of Cheese Technology’, Volume 2.

Salting of milled curd

Mellowing after salting

The salt crystals dissolve on the moist surfaces of the milled curd particles and form a brine. This diffuses into the curd matrix through the aqueous phase, causing the curd to shrink in volume, and more whey is

Sufficient time must be allowed after salting (the mellowing time) to ensure the required absorption of salt on the curd surface and continued free drainage of whey. It was suggested earlier that the curd could be

cheese S/M ratio (Samal, personal observation). The more uniform the ratio of surface area to curd mass after milling, the more uniform will be the rate of salt diffusion into the milled curd particles and more consistent will be the amount of salt retained. It is worth noting that these conditions are more closely satisfied if the curd is not cheddared but is kept in the granular state prior to salting. Milling has little role in granular curd cheesemaking; see ‘Stirred curd or granular cheese’. Mellowing prior to salting

In the traditional procedure for Cheddar cheese manufacture, the milled ‘chips’ were left until the newly cut surfaces glistened as a mixture of whey and fat exuded from them. The mellowing period provided time to produce sufficient surface moisture to dissolve the salt crystals when they were applied and gave rise to better salt retention. The purpose of the traditional mellowing period (‘dwell time’) was to allow for further moisture release and acidity increase. In modern mechanized Cheddar cheese plants, salt is added to the curd pieces immediately after milling, and continuous agitation of the milled particles is used to encourage whey flow and salt absorption. Effect of salting

80 Cheddar Cheese and Related Dry-salted Cheese Varieties

hooped as soon as it had been salted. However, this led to problems in cheese made by these shorter processes (Czulak, 1963), specifically to the entrapment of whey and consequently to excessive moisture and uneven colour in the cheese. As a result, a number of investigations have been carried out to determine the factors that influence the amount of salt absorbed and the speed of its absorption (Breene et al., 1965; Sutherland, 1974; Gilles, 1976; Gilbert, 1979). The amount of salt absorbed by the curd and the rate of subsequent whey drainage are related to the availability of dissolved salt on the curd particle surfaces, and to the physical characteristics of the curd, e.g., fat-free curd allows faster diffusion (Sutherland, 1977). Even when a mellowing time of more than 30 min is maintained and the level of salt addition is uniform, large variations may still occur in the salt content of cheeses because other conditions that affect salt absorption are not controlled. For instance, the curd temperature, the depth of curd, the extent of stirring after salt addition and the degree of structure development in the curd are also significant factors in the control of salt absorption and subsequent whey drainage (Sutherland, 1974; Gilles, 1976). Therefore, it is not surprising that there have been conflicting reports as to how long the mellowing time after salting should be. It is clear that holding for at least 15 min is necessary to minimize the loss of salt during pressing (Breene et al., 1965). Other reports suggest that the pressing of the salted curd should be delayed for at least 30 min (Gilles, 1976) and preferably for 45–60 min (Breene et al., 1965). Some loss of salt occurs even when the mellowing time is extended to 60 min. However, an increase in the mellowing time substantially reduces the proportion of whey expelled during pressing and greatly improves the degree of salt absorption (Sutherland, 1974). Mechanized cheese plants nowadays have a mellowing time of 20–40 min, which is usually adequate for satisfactory salt uptake and whey removal (C.G. Honoré and P.K. Samal, unpublished results). The irregular effect of curd temperature on the extent of salt absorption was thought (Breene et al., 1965) to be caused by a protective layer of fat exuding from the surface of curd particles. Less fat was present on curd surfaces at 26 °C than at 32 °C. Above 38 °C, such fat was melted and dispersed in the brine solution that was present on the surface. In general, however, a decrease in the curd temperature at salting increases the S/M of the final cheese (Sutherland, 1974). Curd salted at a high pH retains more salt (Dolby, 1941) and is more plastic than curd salted at a low pH. Similarly, for a given salting level, the S/M is high when the titratable acidity is low (Gilles, 1976).

Salting the curd under the most favourable conditions for salt absorption reduces the proportion of salt required and reduces salt losses (Gilles, 1976) and also helps to overcome the defect of seaminess (Czulak, 1963; Czulak et al., 1964). Equilibration of salt within a cheese

The rate of penetration of salt into cheese curd is very slow (McDowall and Dolby, 1936; Guerts et al., 1974; Sutherland, 1977; Morris et al., 1985) and a mean diffusion rate of 0.126 cm2/day for salt in the water of Cheddar cheese has been reported (Sutherland, 1977). This corresponds well with salt migration values for Gouda cheese of the same moisture content (Guerts et al., 1974), suggesting that the matrix structures of the two cheese types are similar. Despite the low rate of salt diffusion, it was nevertheless believed that the S/M concentration in Cheddar cheese was essentially uniform within a few days (McDowall and Dolby, 1936). Reports (Morris, 1961; Fox, 1974; Sutherland, 1977; Thomas and Pearce, 1981) suggested that wide variations occurred in salt content between blocks from the same vat and even within a block. Also, there was appreciable variation in the salt and moisture contents of small plug samples taken from different cheeses from the same vat (Sutherland, 1977; Thomas and Pearce, 1981). With increased plant mechanization and automation, better designed cheese vats and improved salting devices, the S/M variation within a block of cheese and between blocks of cheese from the same vat is reasonably well controlled. As the consistency of cheese flavour is directly related to the extent of variability in S/M, the need to produce a curd mass consisting of particles of uniform cross-section at the time of salting cannot be over-emphasized (Fig. 7).

Acidity development Curd particle Curd acidity/pH cross-section at salting at salting

Moisture content of curd

Salt uptake

Salting rate

Salt-in-moisture Figure 7 The main factors that affect the salt uptake and S/M level in Cheddar cheese.

Cheddar Cheese and Related Dry-salted Cheese Varieties 81

Seaminess and fusion

When curd particles are dry-salted, discrete boundaries are set up between the individual particles, in contrast to brine-salted cheeses where there is only one boundary, i.e., the cheese rind or exterior. The addition of dry salt causes shrinkage of the curd and a rapid rate of release of whey containing calcium and phosphate, particularly in the first few minutes of pressing. It has been suggested that the salted surface of the curd particle acts as a selective permeable membrane, thereby concentrating calcium and phosphate at the surface of the curd particle (McDowall and Dolby, 1936). It is possible that this calcium gradient is also accentuated, under some circumstances, by the variations in pH between the surface and the interior of the salted curd particle owing to inhibition of starter activity by the high salt concentration at each curd boundary. The establishment of a pH gradient leads, in turn, to a shallow calcium gradient (Le Graet et al., 1983), the magnitude of which will depend on the size of the curd particle and the proportion of salt added. In its most extreme form, the deposition of calcium phosphate crystals results in the phenomenon of seaminess in Cheddar cheese (Czulak, 1963; Conochie and Sutherland, 1965a; Al-Dahhan and Crawford, 1982), a condition in which the junctions of the milled curd particles are visible after pressing. Seaminess is more frequent and more marked with cheese of low moisture and high salt content and in some cases persists after the cheese has matured (Czulak et al., 1964). The binding between curd particles is usually weak, due to incomplete fusion. This often leads to crumbling when the cheese is sliced or cut into small blocks for packing. Photomicrographs show that, in both seamy and non-seamy Cheddar cheese, crystals of calcium orthophosphate dihydrate are dispersed throughout the cheese mass (Conochie and Sutherland, 1965a), but in seamy cheese they are concentrated in the vicinity of the surfaces of the milled curd particles to which salt was applied. To a depth of about 20 m below these surfaces, the protein appears to be denser than elsewhere, suggesting that severe dehydration of the surface occurs on contact with dry salt. The observation (Van Slyke and Price, 1952; Czulak, 1963) that seaminess is reduced by washing the curd after milling and before salting can be explained by the removal of calcium and/or phosphate from the surface layer. In addition, the provision of more water will lessen the dehydrating and contracting effect of salt on the surface layer. Seaminess and poor bonding between the curd particles occur together and treatment with warm water corrects both defects. Poor fusion of the curd as a con-

sequence of heavy salting results from changes in the protein at the surface, from poor contact between the hardened surfaces, from the physical separation brought about by the presence of salt crystals and, when these have disappeared, from the growth of the calcium ortho-phosphate crystals (Conochie and Sutherland, 1965a). Fusion of the particles is improved by an increase in the pH, temperature or moisture content of the curd. Effect of pressing

Traditionally, Cheddar cheese was pressed overnight using a batch method. The development of the ‘blockformer’ system (Wegner, 1979; Brockwell, 1981; Tamime and Law, 2001; ‘General Aspects of Cheese Technology’, Volume 2) offered two major advantages for modern cheesemaking plants: firstly it is a continuous process and secondly the residence time is reduced to about 30–45 min. The curd is fed continuously into an extended hoop (tower) under a partial vacuum, and mechanical pressure is applied at the base of the tower for a very short period, usually for about 1 min. In traditionally made Cheddar, the two common types of textural defect are mechanical- and slitopenness. Mechanical-openness (occurrence of irregularly shaped holes) is evident in very young cheese but decreases markedly during the first or second week after manufacture and changes little thereafter (Czulak et al., 1962; Irvine and Burnett, 1962; Price et al., 1963). However, in Cheddar cheese blocks from block-formers, mechanical-openness is barely visible immediately after manufacture and becomes prominent at about 4 weeks after manufacture (Samal, personal observation). Slitopenness is usually absent in freshly made cheese (Robertson, 1965a) but develops during maturation (Hoglund et al., 1972a). The extreme expression of this defect, known as fractured texture, is found only in mature cheese. A comprehensive survey of commercial Cheddar cheese on the United Kingdom market carried out during 1958–1961 showed that mechanically open cheese was usually almost free of fractures and conversely that badly fractured cheese usually had few mechanical openings (Robertson, 1965b). The term ‘fracture’ is normally used to describe long slits, i.e., slits longer than about 3.5 cm. As a result of the growth of the cheese pre-packaging trade, the importance attached to fractures in cheese has greatly increased because fractures can result in the break-up of cheese during prepackaging. The basic mechanisms for the formation of openness in Cheddar cheese depend firstly on mechanical-openness, i.e., microscopic nuclei or larger air spaces in the cheese structure, and secondly on gas production by microorganisms (Martley and Crow, 1996).

82 Cheddar Cheese and Related Dry-salted Cheese Varieties

From the observation (Walter et al., 1953) that cheese hooped under whey had a completely close texture and from their own studies of curd behaviour, Czulak and Hammond (1956) concluded that air entrapped during compression of curd was responsible for mechanically open texture. They considered that during compression of the salted, granular curd, the spaces between the granules diminish until they form a complex of narrow channels filled with air. Under further pressure, some of the air is forced out, the escape of the remainder being blocked by closure of the channels at various points and the high surface tension developed by traces of whey in the remaining narrowed outlets. The isolated pockets of trapped air form numerous small irregular holes in the cheese. Conventionally-made Cheddar cheese has a significantly closer texture than Granular cheese. The effect of cheddaring on texture appears to be due to the presence of milled strips of curd (fingers) compared with the relatively small granules of uncheddared curd present in Granular cheese. The larger the fingers of curd, the fewer are the pockets of trapped air and the closer is the texture of the cheese. As mentioned previously, however, there is a practical limit to the size of milled curd because large curd fingers may result in inadequate whey drainage after salting. During the last 50 years, there has been a marked reduction in the incidence of both texture defects by: (a) the use of higher pressures during curd fusion (Whitehead and Jones, 1946); (b) the change from the manufacture of 36 kg rinded cheese to smaller 20 kg rindless cheese; (c) the introduction of vacuum pressing; (d) the use of defined single-strain cultures from which gas-producing strains have been omitted. The beneficial effects of these modifications are undoubtedly associated with a reduction in the gas content of the cheese. The production of carbon dioxide during ripening by non-starter bacteria has been associated with the development of slit-openness (Hoglund et al., 1972a) but gas production is considered to be of secondary importance compared with manufacturing conditions (Hoglund et al., 1972b). It is the relatively insoluble and biologically inactive nitrogen in the entrapped air that contributes to the ultimate openness of the cheese because the oxygen is rapidly metabolized during ripening. Vacuum pressing

It was a logical step to prevent the entrapment of air between the curd particles by pressing the curd under vacuum, a procedure first patented in Canada by Smith et al. (1959). A moderately high vacuum, approximately 33 kPa pressure, is required. Vacuumtreated cheese is free, or almost so, of mechanical-

openness when 2 weeks old and remains free throughout maturation (Robertson, 1965a). There was some disagreement among the various research groups as to the optimum conditions for vacuum pressing (Robertson, 1965b). Initially, the cheddared and salted curd was pre-pressed under vacuum for 30 min before dressing – or trimming, followed by normal pressing (Czulak et al., 1962). Later work suggested that pressures greater than 180 kPa appeared to be required during and after vacuum pressing to achieve a close texture (Robertson, 1965b). An important development in Australia was the hooping of granular, salted curd and pressing under vacuum (Czulak, 1962). It was found that the use of vacuum pressing ensured the characteristic close texture of Cheddar cheese and thus eliminated the need for cheddaring. This observation was particularly significant for the complete mechanization of Cheddar cheese manufacture. Trials in New Zealand (Robertson, 1965a) quickly confirmed the Australian conclusions. Maximum reduction in openness was achieved with the combined use of vacuum pressing of granular curd and a homofermentative starter (Hoglund et al., 1972a). Presumably, air can be removed more readily by vacuum from granular curd than from the closer textured cheddared curd. The technique used in the ‘block-former’ system of filling hoops under a partial vacuum is particularly effective in achieving a close texture (Brockwell, 1981; Tamime and Law, 2001). Mechanical-openness is rarely found in cheese made by the ‘block-former’ system, if the recommended operating procedures are practised in the blockformer, e.g., filling time, residence time, pressing time and appropriate vacuum. However, slit-openness does develop if gas-producing organisms are present. A factor that formerly restricted the size of Cheddar cheese blocks was the tendency for large cheeses to show severe mechanical-openness. With the aid of vacuum pressing, it has been found quite practicable to form curd into very large blocks (Robertson, 1967) which by extrusion into cutting equipment can be subdivided into 20 kg blocks. Rapid cooling

An unwelcome side effect of large blocks is that a temperature gradient is set up within the block because of the relatively slow cooling of the block interior. This, and the demands of the marketplace for evenly ripened blocks of cheese, has necessitated the introduction of rapid cooling of blocks by placing them in open stacks in well-ventilated areas of the cool room or in especially designed cooling devices. Generally, the core temperature of each block is cooled to below 18 °C in 24 h to keep the growth of the non-starter

Cheddar Cheese and Related Dry-salted Cheese Varieties 83

lactic acid bacteria to a minimum (Fryer, 1982). Further details on the rapid cooling process and equipment are provided in ‘General Aspects of Cheese Technology’, Volume 2.

Chemical Composition and Cheddar Cheese Quality Developments in cheese marketing, coupled with increasing consumer standards, have resulted in a demand for cheese of greater uniformity of composition than in the past. Such uniformity is best achieved by a grading system based on compositional analysis, because the relationship between the composition and the quality of Cheddar cheese is now well established (Robertson, 1966; Lyall, 1968; O’Connor, 1971; Gilles and Lawrence, 1973; Fox, 1975; Pearce and Gilles, 1979). Lyall (1968) briefly reported on a procedure for evaluating chemical analyses of cheese, points being assigned on the basis of composition. However, the only scheme in commercial use for assessing Cheddar cheese quality by compositional analysis appears to be that proposed by Gilles and Lawrence (1973). Suggested ranges of MNFS, S/M, fat-in-dry matter (FDM) and pH for both first and second grade cheeses are given in Fig. 8. All New Zealand export Cheddar cheese is subject to compositional grading to ensure that it meets the appropriate specification. In addition, a sensory flavour assessment is carried out to ensure that the cheese is free from flavour defects (Lawrence and Gilles, 1980). Burton (1989) concluded that grad-

MNFS 50–56

S/M 4.0–6.0

MNFS 52–54

S/M 4.7–5.7

ing on the basis of composition may be a satisfactory method for deciding which cheese should be allowed to mature for the British market and which should be sold more quickly. Any grading system based on compositional analysis will be relatively complex because a further factor, the rate and extent of acid production at the vat stage, must also be considered (Lawrence et al., 1984; Lawrence and Gilles, 1986). The point in the process at which the curd is drained from the whey is the key stage in the manufacture of Cheddar cheese because it controls to a large extent its mineral content, the amount of residual chymosin in the cheese, the final pH and the moisture/casein ratio (Lawrence et al., 1984). All these factors influence the rate of proteolysis in the cheese. A relationship has also been found between the calcium content of the cheese, the concentration of residual chymosin and protein breakdown during ripening (Lawrence et al., 1983) and between the rate of acid development in the early stages of manufacture and proteolysis in the cheese (O’Keeffe et al., 1975). The calcium level is therefore an index of the extent of acid production up to the draining stage and also offers a rough indication of the rate of proteolysis that is likely to occur during ripening. Significant differences in the calcium content of Cheddar cheese would suggest differences in the proportions of residual chymosin in the cheese and thus differences in the rate of proteolysis and the development of flavour. However, variations in calcium content have a much smaller effect on Cheddar cheese quality than MNFS, S/M and pH. It is important to recognize that these three parameters are interrelated (Lawrence and Gilles, 1986) and must be controlled as a group to ensure first-grade cheese. Nevertheless, the effect of each of these factors will, as far as possible, be examined separately. Effect of MNFS

First grade

FDM 52–56

FDM 50–57

pH 5.1–5.3

pH 5.0–5.4

Figure 8 Suggested ranges of salt-in-moisture (S/M), moisture in the non-fat substance (MNFS), fat-in-dry matter (FDM) and pH for first grade (shaded) and second grade Cheddar cheese. Analyses 14 days after manufacture.

There is considerable circumstantial evidence that the main factor in the production of the characteristic flavour of hard and semi-hard cheese varieties is the breakdown of casein. This is supported by the finding that the ratios of moisture to casein and of salt to moisture are critical factors in cheese quality (Gilles and Lawrence, 1973; Lawrence and Gilles, 1986) because both parameters affect the rate of proteolysis in cheese (Thomas and Pearce, 1981). Traditionally, cheesemakers describe cheese in terms of its absolute moisture content but the ratio of moisture to casein is much more important because it is the relative hydration of the casein in the cheese that influences the

84 Cheddar Cheese and Related Dry-salted Cheese Varieties

course of the ripening process (Lawrence and Gilles, 1980). However, it is difficult to measure the casein content of cheese accurately and most commercial plants analyse for only fat and moisture. Therefore, a practical compromise is to determine the ratio of moisture to non-fat substance rather than measure the moisture/casein ratio. The non-fat substance is not the same as the casein in the cheese but is equal to the moisture plus the solids-not-fat. Approximately 85% of the solids-not-fat consist of casein. Therefore, there is a relationship between the moisture/casein ratio and the MNFS. The level of MNFS in cheese gives a much better indication of potential cheese quality than the moisture content of the cheese in the same way as the S/M ratio is a more reliable guide to potential cheese quality than is the salt content of the cheese per se (Lawrence and Gilles, 1980). In large mechanized cheese plants, a significant relationship exists between the FDM and MNFS values for a cheese (Lawrence and Gilles, 1986), probably as a result of the relative inflexibility of the procedures available for the control of moisture. This is of commercial interest because changing the FDM is an effective way of controlling the MNFS in the cheese as the composition of the milk changes throughout the season. The actual MNFS percentage for which a cheesemaker should aim depends on the storage temperature used and when the cheese is required to reach optimum quality. Experience has shown that if Cheddar cheese is to be stored at 10 °C and the cheese is to be consumed after 6–7 months, then the MNFS of the cheese should be about 53%. The higher the MNFS percentage, the faster is the rate of breakdown. Thus, if one anticipates that the cheese will be consumed after 3–4 months, the MNFS percentage can be increased to about 56%. However, the higher the MNFS, the more rapidly Cheddar cheese will deteriorate in quality after reaching its optimum. The same is true for a Cheddar cheese with a relatively low S/M, i.e., less than 4%, or with a high acid content. Such cheeses tend to develop gas and sulphide-type off-flavours after they have reached maturity.

a key factor in determining the pH of dry-salted cheese (Fig. 9). However, the salting pH/titratable acidity is to a large extent controlled, in turn, by the pH/titratable acidity developed at draining (Lawrence and Gilles, 1982). The potential for a further decrease in pH after salting depends upon the residual lactose in the curd and its buffering capacity. The residual lactose will be determined by the rate at which an inhibitory level of NaCl is absorbed by the cheese curd and the salt tolerance of the starter strains used. The buffering capacity is largely determined by the concentrations of protein and phosphate present, and to a much lesser extent by ions such as calcium. The concentrations of phosphate and calcium retained in the cheese are influenced mainly by the rate of acidification prior to the separation of the whey from the curd. The buffering capacity is also influenced by seasonal, regional and lactational factors. Given reliable starter activity at the vat stage, the actual pH reached in dry-salted cheeses is determined by the S/M value because this controls the extent of starter activity after salting, the rate of lactose utilization in the salted curd and thus the pH reached. An S/M concentration of 6% will inhibit the activity of all Lactococcus lactis subsp. cremoris strains, the starter organisms of choice for Cheddar manufacture (Lawrence and Heap, 1986). The proportion of residual lactose that remains unmetabolized in such cheese will be high even after 2 months (Turner and Thomas, 1980). However, in a cheese with S/M of 4.5%, the starter will not be inhibited completely and the lactose will be metabolized rapidly. This explains why the pH of 1-day Cheddar cheese may range from 5.3 (which is about the pH of the curd at salting) to pH 4.9. In general, the higher the pH, the greater

Milk composition

Starter percentage

Casein and mineral content of curd

Time between cutting and draining

Curd acidity/pH at draining

Effect of pH

Every cheese variety has a characteristic pH range (Lawrence et al., 1984), within which the quality of the cheese is dependent upon both its composition and the way in which it is manufactured (Lawrence et al., 1983). The pH value is important in that it provides an indication of the extent of acid production throughout the cheesemaking process. In normal manufacture, the curd pH/titratable acidity at salting is

Curd acidity/pH at salting

Buffering capacity

pH

Starter activity

Residual lactose in salted curd

Figure 9 The main factors that determine the pH of Cheddar cheese.

Cheddar Cheese and Related Dry-salted Cheese Varieties 85

is the amount of lactose left unmetabolized. Under normal circumstances, this residual lactose does not affect the quality of Cheddar cheese at maturity (Gilles and Lawrence, unpublished results). The importance of measuring the pH at day 1 has been generally overlooked in the past, probably because it is relatively difficult to measure the pH of cheese accurately. This has led to a lack of appreciation of the significance of relatively small changes in pH. In addition, a pH value per se is sometimes difficult to interpret unless considered in conjunction with the calcium level in the cheese (Lawrence and Gilles, 1980), as well as the pH/titratable acidity at draining. Effect of S/M

The main factors that determine the S/M percentage of Cheddar cheese are summarized in Fig. 7. In young Cheddar cheese, the S/M ratio is the major influence controlling water activity. This in turn determines the rate of bacterial growth and enzyme activity in the cheese, specifically the proteolytic activity of chymosin (Fox and Walley, 1971; Pearce, 1982; Fox, 1987), plasmin (Richardson and Pearce, 1981) and starter proteinases (Martley and Lawrence, 1972). If the S/M value is low (4.5%), the starter numbers will reach a high level in the cheese and the chance of off-flavours due to the starter bacteria is greatly increased (Lowrie and Lawrence, 1972; Breheny et al., 1975). For this reason, cheesemakers normally aim for an S/M value in Cheddar cheese between 4.5 and 5.5% (Lawrence and Gilles, 1980, 1982). Within this S/M range, the rate of metabolism of the lactose is controlled by a second factor, the temperature of the cheese during the first few days of ripening, because this controls the rate of growth of non-starter bacteria such as lactobacilli and pediococci (Fryer, 1982). Although nonstarter bacteria grow on energy sources other than lactose in cheese, undoubtedly the presence of lactose encourages their rapid growth. This tends to result in a more heterolactic metabolism of lactose, usually with the production of acetate, ethanol and carbon dioxide, and may lead to flavour and textural defects. Clearly, the initial number of non-starter bacteria in the salted curd should be controlled by hygiene during manufacture. Thereafter, their rate of growth, particularly after the first few days of ripening, should be kept to a minimum and this is largely controlled by the temperature of the cheese (Fryer, 1982). For this reason, large mechanized Cheddar cheese plants in New Zealand and Australia have incorporated rapid cooling systems, which reduce the core temperature of the 20 kg cheese blocks to less than 18 °C within 24 h of manufacture.

Compositional ranges were introduced into the grading system to reduce variability within a day’s manufacture, especially with respect to S/M. This measure has helped to reduce the previous variability. The rate of ripening will differ but all of the cheese is likely to be acceptable as long as its composition is within the required compositional range. For instance, variations in the moisture content and acidity of the curd before salting, in the accuracy of salt delivery by salting equipment and in the dimensions and structure of the milled curd will all result in considerable variation in salt uptake (Lawrence and Gilles, 1982). Despite improved understanding of salt diffusion (Baldwin and Wiles, 1996; Wiles and Baldwin, 1996) in large mechanized Cheddar cheese plants, as well as new improved ideas and equipment for salting cheese curds, inherent variation of S/M still exists. The effect of manufacturing Cheddar cheese with reduced sodium and S/M levels, on cheese quality, is discussed in ‘Salt in Cheese: Physical, Chemical and Biological Aspects’, Volume 1. Effect of FDM

The FDM in Cheddar cheese is less important than MNFS, S/M or pH, in that it normally influences cheese quality only indirectly through its effect on MNFS (Whitehead, 1948). Nevertheless, the FDM has more relevance to the cheesemaker than the fat content per se because moisture is volatile and legal limits for fat are usually specified in terms of FDM. Use of FDM has the further advantage that it can be controlled directly by altering the casein/fat ratio of the milk.

Texture of Cheddar Cheese Most consumers of Cheddar cheese consider texture and flavour to be its most important attributes (McEwan et al., 1989; Jack et al., 1993). On the other hand, Cheddar purchased for repackaging also needs to withstand cutting, slicing and moulding. Hence the rheological properties are important (Gunasekaran and Ak, 2003). Cheddar destined to be used as a functional ingredient has the rather different requirements to give particular textural (and flavour) characteristics to the final product (‘Rheology and Texture of Cheese’, Volume 1). The desirable textural characteristics of a Cheddar cheese are different for different consumers, and this usually involves personal assessments of breakdown in the mouth, evenness of dissolution (melting), amount of chewing required, gritty remnants, etc. Traditionally, the textural properties of cheese sold for immediate

86 Cheddar Cheese and Related Dry-salted Cheese Varieties

human consumption are determined by trained graders. For texture research, such assessments are often made by consumer panels, and there have been many studies using either rheological parameters or the results from specific texture-measuring devices. Such studies have shown that perceived texture correlates moderately well with the indices investigated and developed by Szczesniak (1968, 1987) and discussed by Fox et al. (2000) and Gunasekaran and Ak (2003), and less well with the traditional rheological measures (Breuil and Meullenet, 2001). Nevertheless, there is still no clear method for discerning instrumentally which blocks of Cheddar cheese have acceptable textural properties. The complex interrelationships between the parameters that affect cheese texture make it almost impossible to design simple experiments in which the effect of a single parameter, such as fat content, can be examined in isolation. The wide-ranging experiments carried out by the New Zealand group in the 1970s and 1980s laid the basis for a good understanding of the factors underlying the production of Cheddar cheese of appropriate texture and flavour throughout the maturation cycle (Lawrence et al., 1993). Nevertheless, some recent studies demonstrate that fat content (Fenelon and Guinee, 2000) and pH (Pastorino et al., 2003) can affect the rheological properties of Cheddar cheese. However, by using modern statistical approaches, it is now possible to segregate the effects of several parameters, although each experiment needs to be very large and use many cheese samples (C.J. Coker, T.M. Dodds, S.P. Gregory, K.A. Johnston and L.K. Creamer, unpublished results, 2000). Cheddar cheese has a texture that is intermediate (Fig. 10) between those of the relatively high pH cheeses, which flow readily when a force is applied, and the low pH cheeses which tend to deform, by shattering, only at their yield point. Scanning electron

microscopy has established that cheese consists of a continuous protein matrix but that this matrix is clearly different in the various cheese types (Hall and Creamer, 1972). The structural units in the protein matrix of Gouda are essentially in the same globular form (10–15 nm in diameter) as in the original milk. In contrast, the protein aggregates in Cheshire are much smaller (3–4 nm) and are apparently in the form of strands or chains, i.e., the original sub-micellar protein aggregates appear to have lost much of their identity. Cheddar is intermediate between Gouda and Cheshire, i.e., much of the protein in Cheddar is in the form of smaller particles than in Gouda (Fig. 10). As the pH decreases towards that of the isoelectric point of para-casein (approximately 4.5), the protein assumes an increasingly compact conformation and the cheese becomes shorter in texture and fractures at a smaller deformation (Creamer and Olson, 1982; Walstra and van Vliet, 1982). The texture of Cheddar cheese has a wider range of consumer acceptability than the texture of other varieties as a consequence of the intermediate position of Cheddar in the cheese spectrum. The high moisture and relatively high pH (5.2) of American Cheddar resulted traditionally in a more cohesive and waxy texture (Kosikowski and Mistry, 1997) than that of traditional English and New Zealand Cheddar. In North America, a relatively low level of acid was developed in the curd up to the salting stage (less than 0.65% titratable acidity). In contrast, English cheesemakers strove for a high salting acidity (about 0.85%) with consequently a low final pH (about 4.9). New Zealand cheesemakers aimed for a final pH of 5.0 and a moisture content of about 35%, in contrast to the 38–39% moisture level found in both American and English Cheddar. In recent times, however, most Cheddar-producing countries have tended

Swelling of renneted micelles 15

pH Texture (14 days) Typical cheeses

5.5

5.4

10 nm 10

5.3

5.2

Springy

5 nm

5.1

5.0

4.9

4.8

4.7

4.6

Short Mealy

Cheddar

Size of sub-micelles

3 nm

Cheddary

Plastic Gouda

5

Non-cohesive Cheshire Stilton

Figure 10 Diagrammatic representation of the effect of the pH on the microstructure and texture of cheese.

Cheddar Cheese and Related Dry-salted Cheese Varieties 87

towards the American style of ‘sweet’ Cheddar cheese with a final pH between 5.1 and 5.3 now being common. Effect of pH, calcium and salt

Although the mineral content plays an important role in establishing the characteristic structure (Lawrence et al., 1983, 1984), the texture of Cheddar cheese appears to be more dependent upon pH than on any other factor (Lawrence et al., 1987). For the same calcium content, the texture at 35 days can vary from curdy (pH 5.3) to waxy (pH 5.3–5.1) to mealy (pH 5.1). Trials in New Zealand have shown that, for any given pH value, the concentration of calcium in Cheddar can vary over a range of 15 mmoles/kg with only a slight effect on the texture (Lawrence et al., 1993), although there is a general tendency for the cheese to become less firm as the calcium content decreases. However, the dominant effect of pH on texture can be modified by other compositional factors, particularly the levels of moisture, salt and calcium. Between pH 5.5 and 5.1, much of the colloidal calcium phosphate and a considerable part of the casein are dissociated from the sub-micelles (Roefs et al., 1985). These changes in the size and characteristics of the sub-micelles significantly increase their ability to absorb water (Tarodo de la Fuente and Alais, 1975; Snoeren et al., 1984; Creamer, 1985; Roefs et al., 1985), casein hydration reaching a maximum at about pH 5.35. More relevantly, Creamer (1985) found that casein hydration in renneted milk increased greatly in the presence of NaCl between pH 5.0 and 5.4. Furthermore, at any given pH, the extent of solubilization of the micelles by the NaCl decreased as the calcium concentration in the solution increased. This finding is in agreement with the effects of calcium in brine on the solubilization of the rind of Gouda-type cheese (Guerts et al., 1972). It also explains the observations that a higher Ca2/Na ratio results in a firmer cheese (Walstra and van Vliet, 1982), and that Cheddar cheese made from milk to which calcium has been added has a reduced protein breakdown and is of poorer quality (Babel, 1948; Ernstrom et al., 1958). The high level of calcium in buffalo milk (Rajput et al., 1983) may also account for the difficulty in manufacturing Cheddar cheese from buffalo milk. The extent of proteolysis is low (Neogi and Jude, 1978), presumably because the degree of solubilization of the casein micelles by the NaCl is reduced. As a result, Cheddar cheese needs to be stored for a long period before its characteristic texture and flavour develop.

Therefore, it is not surprising that the texture of Cheddar cheese changes markedly as the pH varies between 5.4 and 4.9. A wide range of casein aggregates is present and differences in the sodium and calcium ion concentrations, as well as the proportion of water to casein, markedly affect the extent of swelling of the sub-micelles (Fig. 10). Salt also has a more direct effect on the texture of Cheddar cheese; excessive salting (i.e., an S/M  艐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  艐4%) results in a pasty cheese with abnormal ripening and flavour characteristics. Such factors as enzyme activity and the conformation of S1- and -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 NaCl 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 S1-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 S1- and -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 et al., 1983; C.J. Coker, T.M. Dodds, S.P. Gregory, K.A. Johnston and L.K. Creamer, unpublished results, 2000).

Flavour of Cheddar Cheese 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 et al., 1997). Cheddars made from raw milk were more intensely flavoured and had atypical flavours, with farmhouse cheeses 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 S1-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 -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 89

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

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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. Effect of proteolysis

Acceptable Cheddar flavour

Off-flavours

Figure 11 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). The free amino acids are also precursor substrates 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 H2S and CH3SH, compounds that are extremely susceptible to oxidation, decreased as the

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). Role of starter

The absence of any Cheddar flavour in glucono lactoneacidified cheese and the development of typical, balanced Cheddar flavour in starter-only cheese (Reiter et al., 1966) established that Cheddar starter, normally Lactococcus lactis subsp. cremoris as discussed in ‘Effect of milk composition and starter culture’, has a role in the development of cheese flavour. However, the exact role has been much more difficult to determine. An important indirect role of the starter is considered to be providing a suitable environment that allows the development of characteristic cheese flavour (Lowrie et al., 1974). Starter activity results in the required redox potential, pH and moisture content in the cheese that allows enzyme activity to proceed favourably. In addition, the temperature during manufacture and the S/M must be controlled to ensure that the net metabolic activity of the starter organisms is low (Lawrence et al., 1972; Lowrie et al., 1974) but nevertheless adequate to allow the required pH at day 1 to be reached. Should the starter reach too high a population or survive too long, flavour defects such as 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 1% by 3 months (Martley and Lawrence, 1972). The decrease in starter viability is generally an indication of starter autolysis, which is considered to have important consequences for the control of bitterness, as discussed in ‘Effect of proteolysis’, and for other ripening developments in Cheddar (Wilkinson et al., 1994a,b; Crow et al., 1995). Starter autolysis in Cheddar is influenced by the choice of starter strains (Martley and Lawrence, 1972), and the extent and the rapidity of autolysis are modified by manufacturing conditions, particularly

Cheddar Cheese and Related Dry-salted Cheese Varieties 91

cook temperature, pH and salting, and the composition of the final cheese. A balance between intact and autolysed starter cells is important for good quality Cheddar flavour. Sufficient intact cells are needed for lactose removal and potentially for other physiological reactions such as oxygen removal (Crow et al., 1995). There is indirect evidence that starter autolysis results in higher concentrations of small peptides and free amino acids (Wilkinson et al., 1994a,b). The intracellular peptidases are released by autolysis before they can be very active in cheese as the peptide transport systems are probably inactive in the stressed starter cells (Crow et al., 1993). Role of starter enzymes

It is likely that a number of starter enzymes have a direct role in flavour development (Lawrence and Thomas, 1979; Law and Wigmore, 1983; Farkye et al., 1990). The starter proteinase is a cell-associated endopeptidase (lactocepin), which has been studied extensively, biochemically and genetically (Reid and Coolbear, 1998) and makes important contributions to proteolysis in cheese ripening (see reviews, Pritchard and Coolbear, 1993 and Kunji et al., 1996). Proteinasenegative starters have been used in different ratios with normal starter strains to demonstrate that a lower level of this enzyme during Cheddar ripening can reduce the development of bitter flavours (Mills and Thomas, 1980). Using proteinase-negative starters, Lane and Fox (1997) have shown that the absence of starter proteinase during cheese ripening gives rise to decreased levels of small peptides and amino acids, and a poorer quality Cheddar. Therefore, a balance of proteinase activity is often important to Cheddar flavour. There are also different starter proteinases (mainly types I and III), and their specificity differs (Broadbent et al., 1998) and/or their stability in cheese differs (Reid and Coolbear, 1999), factors that contribute to the starters’ influence on proteolysis and bitterness. The other proteolytic activities of starters that are important for proteolysis in Cheddar are the peptidases (see reviews, Pritchard and Coolbear, 1993 and Kunji et al., 1996). The early appearance of free amino acids in Cheddar is due mainly to the starter peptidases (O’Keeffe et al., 1976). The mesophilic starters have about 15 peptidases with different specificities (McSweeney and Sousa, 2000); a number have been shown to be intracellularly located and the activities vary between strains (Crow et al., 1994). Although the collective importance of the peptidases in Cheddar proteolysis is reasonably well established, the individual roles of the peptidases are not clear. Genetic modi-

fication of the peptidase expression in the starter may clarify their roles (Kok and Venema, 1995). For example, Cheddar flavour was not accelerated using a starter that overproduced the general aminopeptidase, PepN (McGarry et al., 1994). This may not be surprising if the suggestion by Fox and Wallace (1997) is correct, that production of amino acids in cheese ripening is not rate limiting. To date, there is no strong evidence to suggest that the lactococcal starters have a true lipase that is capable of hydrolysing the milk triglycerides. However, they have esterase activity that acts on milk monoand di-glycerides, with the short chain fatty acids being released preferentially (Holland et al., 2002). There is evidence that the starter esterase can also produce short-chain fatty acid esters (Nardi et al., 2002). Starter strains have a range of esterase activity with a significant proportion located on the cell surface (Crow et al., 1994). Both the hydrolysis and ester synthesis reactions of the starter esterase probably play a role in Cheddar flavour but more investigations are needed to define the extent to which this impacts on Cheddar flavour. Role of other starter activities

In the young Cheddar curd, starters ferment the remaining lactose to lactic acid and possibly to other minor fermentation products. Some starters will also ferment citric acid to products including diacetyl, acetate and carbon dioxide. These products can contribute to flavour (Lawrence and Thomas, 1979) and the fermentations are dependent on intact viable cells (Crow et al., 1993). Other ripening reactions by starter that may be important in cheese are the modifications of amino acids and fatty acids (McSweeney and Sousa, 2000). It has been suggested that the enzymatic or chemical modification of amino acids is a rate-limiting factor in cheese ripening (Fox and McSweeney, 1996). Starters have a wide range of abilities to metabolize amino acids. This includes converting arginine to ornithine (Crow and Thomas, 1982), leucine, methionine and phenylalanine to their corresponding aldehydes (MacLeod and Morgan, 1958) and a range of amino acids to their corresponding organic acids (Nakae and Elliot, 1965). Many of the amino acid conversions by starters and other dairy bacteria rely on transamination reactions that are believed to be rate limited by the availability of the -ketoglutarate (Tanous et al., 2002). Enhancement of amino acid metabolism increased the aroma (Banks et al., 2001) and the flavour maturity (Shakeel-Ur-Rehman and Fox, 2002) in Cheddar made with added -ketoglutarate. Despite all this information, it is still not clear what constitutes a proper balance of amino acid transformations

92 Cheddar Cheese and Related Dry-salted Cheese Varieties

by starter with respect to a balanced Cheddar flavour. In mature commercial Cheddar, the availability of amino acids increases with time when the adventitious microflora are likely to contribute. In addition, at this later stage of ripening in good quality Cheddar, there is normally significant starter autolysis, which could affect the ability and the way the starters metabolize amino acids. It is clear that starters, because of the initial high biomass in young curd and associated ripening enzymes and fermentative abilities, will contribute to Cheddar flavour. In the past and currently, the choice of starter has been dictated more by its importance to commercial curd manufacture (particularly reliable acid production and associated phage resistance) than by its ripening properties, which have been focused mainly on minimizing flavour defects, such as bitterness. With the increasing understanding of Cheddar ripening and the role of the starter, it will be possible to use more lactococcal strains with specialized ripening attributes. Such strains can be used either as starters or as flavour adjuncts if their starter properties are compromised. Role of non-starter lactic acid bacteria

Cheddar contains a heterogeneous adventitious microflora originating from the milk and/or the manufacturing environment (Peterson and Marshall, 1990; Martley and Crow, 1993). In Cheddar, the main microflora identified are mesophilic lactobacilli and occasionally pediococci (Jordan and Cogan, 1993; Crow et al., 2001), commonly referred to as NSLAB. The most common species are Lactobacillus paracasei, Lb. casei, Lb. rhamnosus, Lb. plantarum and Lb. curvatus. Strains of heterofermentative lactobacilli (Lb. brevis and Lb. fermentum) are identified occasionally. The common species vary between countries (Fox et al., 1998) and the species and strains can vary between factories, within a factory and within a block of cheese during ripening (Crow et al., 2002). Cheddar cheese made under controlled bacteriological conditions and containing only starter streptococci develops balanced, typical flavour (Reiter et al., 1966) but it is intriguing that cheeses made in open vats develop such flavour more rapidly (Reiter et al., 1967; Law et al., 1976, 1979). This suggests that NSLAB present as a result of post-pasteurization contamination are beneficial. Nevertheless, there have been reports that conclude that NSLAB have little effect on normal Cheddar cheese flavour development (Law and Sharpe, 1977, 1978). The role of NSLAB in contributing positively to Cheddar cheese flavour has yet to be elucidated (Peterson and Marshall, 1990; Martley and Crow,

1993). In general terms, the numbers and types of dominant NSLAB are important (Crow et al., 2002). These factors are influenced by milk quality, factory hygiene, the rate of cooling of the cheese, the ripening temperature and the cheese composition (Lane et al., 1997; Fox et al., 1998). The inherent variability of the initial NSLAB strains makes consistent control of ripening by NSLAB a challenge. The rate of cooling of the cheese, after pressing the curd, appears to be a significant factor in controlling the cheese flora (Fryer, 1982) and appears to offer the easiest method of controlling cheese flavour (Miah et al., 1974). Recent evidence suggests that selected NSLAB can be used as adjunct cultures to provide an important additional tool in controlling Cheddar flavour (see ‘Role of adjuncts’). The NSLAB have a diversity of metabolic and enzyme activities. Different NSLAB strains have a wide range of proteinase (Broome et al., 1991a), peptidase (Broome et al., 1991b) and esterase (Williams and Banks, 1997) activities and types, can catabolise a range of amino acids (Christensen et al., 1999) and can produce esters (Liu et al., 1998). Different NSLAB are likely to grow on different energy sources in cheese (Fox et al., 1998) and influence the redox potential in different ways (Thomas et al., 1985). The balance of all these activities is probably important to good quality Cheddar ripening. The prolonged presence of high numbers of some NSLAB species in Cheddar has been associated with ripening defects such as off-flavours, slits and crystals (Crow et al., 2001). Off-flavours can be produced by Lb. brevis and Lb. plantarum (Puchades et al., 1989). Slits have been attributed to heterofermentative lactobacilli (Laleye et al., 1987) and the formation of white spots of calcium lactate pentahydrate crystals has been associated with the racemizing activity of certain NSLAB (Thomas and Crow, 1983; Johnson et al., 1986, 1989; Dybing et al., 1988; Bhowmik et al., 1990). In Cheddar, the total lactate (usually the L() isomer) is at a concentration close to crystallizing out. Crystallization of calcium lactate on the surface of Cheddar cheese is a common and troublesome defect (Pearce et al., 1973). A number of lactobacilli isolates and all pediococci isolates can convert the L() isomer of lactate to the D() isomer in Cheddar such that an equilibrium is eventually reached where there is an equal mixture (a racemic mixture) of both isomers. As the racemic mixture of lactate is more insoluble than the separate isomers, there is a higher possibility of lactate crystallization in Cheddar containing a racemic mixture of lactate. Role of adjuncts

Cheddar-type varieties traditionally have starter cultures as the only dairy microorganisms deliberately added to the milk. Adjuncts, cultures that are added deliberately

Cheddar Cheese and Related Dry-salted Cheese Varieties 93

for features other than for acid production, and used in some other cheese types (e.g., propionibacteria in Swisstype cheese), have generally not been used for Cheddar. There has been an increasing interest in the use of adjuncts for Cheddar, usually for flavour acceleration but also for flavour consistency or for contributing to unique flavour profiles (Fox et al., 1998). A number of cultures with putative health attributes are also being investigated as adjuncts to produce probiotic cheese, including Cheddar (Ross et al., 2002). Much of the published work has concentrated on the use of NSLAB as adjuncts. Other adjuncts studied include attenuated thermophilic lactobacilli to accelerate ripening (Wilkinson, 1993). Addition of non-attenuated thermophilic lactobacilli, particularly Lb. helveticus, has been shown to accelerate ripening, reduce bitterness and provide a different flavour profile (Fox et al., 1998). Some other adjuncts studied in less detail include smear bacteria, Enterococcus, Pseudomonas and yeast (Crow et al., 2002). There is some commercial use of NSLAB adjuncts, but time and economics will determine if their use is sustained (Crow et al., 2002). Earlier work (Lane and Hammer, 1935; Reiter et al., 1967) has been followed by a recent increased effort in studying their use (Puchades et al., 1989; Broome et al., 1990; McSweeney et al., 1994; Lynch et al., 1996; Muir et al., 1996; Crow et al., 2001). In these studies, the NSLAB adjuncts often improved the flavour intensity. Although the desirable ripening mechanisms for suitable adjuncts are not known, some analysis shows that flavour improvement is associated with an increase in the concentration of amino acids and small peptides and that the volatiles are produced in a different ratio (Fox et al., 1998). Some strains tested produced flavour defects (e.g., Lee et al., 1990). The dynamics of the interactions between adventitious and adjunct NSLAB growing in Cheddar are not fully understood (Fox et al., 1998). For use in New Zealand Cheddar, successful NSLAB adjuncts are carefully selected from good quality cheese; to achieve competition against the range of adventitious NSLAB and to provide a balance of cheese ripening attributes, an adjunct is made up from more than one strain (Crow et al., 2002). Provided that the milk quality and the factory hygiene are high (i.e., a low level of adventitious NSLAB), the adjunct strains can overgrow the adventitious NSLAB and be the main population of NSLAB throughout ripening, thus providing consistency to mature Cheddar flavour development.

Grading of Cheddar Cheese There is no one standard for measuring cheese quality. Young Cheddar cheese is judged on the basis of whether it has properties characteristic of its variety.

Compositional analysis provides an objective method for detecting atypical cheese and is to be preferred to subjective grading methods. In the case of mature cheese, quality assessment is largely a matter of specific market preference, with consumers in different countries differing considerably in their requirements with respect to cheese flavour. Cheddar cheese flavour requirements is specific to country, ethnicity and endapplication. Whereas sensory profiling of cheese provides a powerful tool for quality assurance and new product development, grading is a highly efficient method of identifying out-of-specification cheese early in the ripening period (Muir, 2002). Grading of cheese encompasses sensory evaluation and functionality tests on the finished product, and is carried out in tandem with chemical and microbiological analyses as part of the manufacturer’s quality assurance programme. Grading is carried out as a series of checks during ripening to determine whether or not the manufacturer has achieved what he initially set out to achieve (S.P. Gregory, personal communication). Although the assignment of a grade to a consignment of cheese may be improperly influenced by the sample because differences may exist between blocks of cheese made from the same vat of milk, it has thus far been the most practical way of grading. Flavour defects, such as fruitiness and sulphide off-flavours, have sometimes been located in particular areas within a cheese (Gilles and Lawrence, unpublished results). Such lack of uniform flavour usually results from variations in S/M (Lawrence and Gilles, 1982). Differences between cheeses have also been attributed to an uneven cooling of cheese blocks stacked closely on pallets while the cheese is still warm (Conochie and Sutherland, 1965b). It is therefore possible that a grade score is highly biased if the assessment of a whole vat depends on a single randomly drawn sample (Sutherland, 1977). The texture of Cheddar cheese changes dramatically during the first few days of ripening. The simplest explanation for this observation is that the cheese microstructure consists of an extensive network of S1-casein and that cleavage by chymosin (or rennet substitute) of just a few peptide bonds of S1-casein greatly weakens this network (Creamer and Olson, 1982). This results in a relatively large change in the force necessary for deformation. It is differences in this force that a grader attempts to assess when he rubs down a plug of cheese between his thumb and forefinger. From this assessment of the texture, after the cheese has been allowed to ripen for about 30 days, the grader proceeds to predict what the quality of the cheese will be after it has matured (Lawrence et al., 1983).

94 Cheddar Cheese and Related Dry-salted Cheese Varieties

Based on the grade or quality attributes, the cheese is identified as one with potential for long-hold (mature Cheddar), medium-hold or short-hold (mild Cheddar). Bitterness is the most common flavour defect detected in Cheddar cheese. Therefore, the sensory method of prediction traditionally used by graders has some validity because the rate of change in cheese texture during the first few days of ripening is determined by the same factors, i.e., the pH at day 1, the salt/moisture ratio and the moisture/casein ratio, that also influence the quality of the cheese at maturity (Fig. 12). Experience has long shown that a Cheddar cheese with an atypical texture seldom, if ever, develops a characteristic flavour but unfortunately the reverse is not true. A good-textured cheese does not always develop an acceptable flavour, because off-flavours can still be produced if unsuitable manufacturing and ripening procedures are used (Lawrence et al., 1983). Detection of atypical cheese can be achieved more directly and objectively by compositional analysis.

Varieties of Cheddar Cheese Low-fat Cheddar cheese

Although a relatively minor product, low-fat Cheddar cheese is important in today’s health-conscious society, where consumers have become more concerned about the amount of fat in their diet. However, it has been difficult to produce a low-fat Cheddar cheese with the same flavour and texture characteristics as

Acid production at draining

those of a full-fat cheese (Guinee and Law, 2002). The flavour and acceptability at 3 months decrease with decreasing fat content (Banks et al., 1989). Cheddar cheeses containing only 15–30% fat are noticeably more firm and less smooth, when young, than full-fat cheese. The differences in texture, although marked in the early stages of ripening, apparently narrow after the cheese has matured for 1 or 2 months (Olson, 1984a). There has been some success at improving the quality of low-fat Cheddar cheese (Guinee and Law, 2002), but there is still a poor consumer perception of lower fat cheeses, judging by their relatively low consumption. The approaches taken to improve both the flavour and the texture have been reviewed (Guinee and Law, 2002). A novel idea for improving the body and texture of low-fat Cheddar cheese was through the use of sweet ultrafiltered buttermilk (Mistry et al., 1996). However, the differences in texture, seen at 4 weeks, between cheeses made with and without buttermilk, were smaller after 24 weeks. By using a combination of manufacturing changes and novel starter and starter adjuncts, Johnson et al. (1998) claim to have achieved a cheese with more acceptable texture, but with a flavour, although improved, that is not identical to that of Cheddar. Guinee et al. (1999) claim to have developed a halffat Cheddar cheese with improved sensory acceptability by modification of the cheesemaking procedure, including increasing the pasteurization temperature, and using selected starter cultures and bacterial culture adjuncts. The addition of fat mimetics to the milk is

pH change and mineral loss

Basic structure

Proteinase activity (salt-in-moisture; moisture-in-casein)

Quality at maturity

Sensory evaluation

Texture and flavour at grading

Figure 12 An explanation for the general validity of traditional sensory testing of Cheddar cheese.

Cheddar Cheese and Related Dry-salted Cheese Varieties 95

also a method that is claimed to improve the quality of reduced fat cheese (Guinee and Law, 2002).

style and other washed-curd cheese types using a method similar to that outlined for traditional Colby and Monterey.

Stirred curd or granular cheese

As discussed earlier, Granular cheese preceded, historically, the manufacture of traditional Cheddar cheese. It is made as for normal Cheddar cheese except that the curd fusion or cheddaring step is omitted. Therefore, more acid has to be developed at the vat stage to compensate for the shorter total manufacturing time. Starter systems are available that allow very acceptable Granular cheese to be made. Maintaining curd in the granular form, without the need for milling prior to salting, has obvious attractions. However, there is a tendency for the curd to mat after drying unless it is agitated, and continued stirring may lead to higher fat losses. Moisture expulsion is also faster than during cheddaring. The salted curd particles take some time to fuse together, the rate of bonding depending largely upon the pH of the curds at salting. However, there are advantages in mechanized cheesemaking systems in having the curds in a granular form. The salt readily mixes with the curd, and the salted granules flow and can be hooped easily. Stirred curd cheesemaking is now widely used in the manufacture of ‘barrel’ (bulk pressed) cheese, although variations in moisture level may occur as a consequence of different temperatures within the block (Olson, 1984b; Reinbold and Ernstrom, 1988). The pressing of granular curd gives rise to opentextured cheese as a result of air being entrapped within the cheese (Czulak and Hammond, 1956). However, this has been overcome by the development and widespread use of methods of pressing the curd under vacuum (Brockwell, 1981; Tamime and Law, 2001). Granular cheese resembles Cheddar cheese in composition but it matures somewhat differently because of the relatively low acidity at which the curds are salted. Curds hooped in the granular form give a texture at 14 days which, although completely close, is just perceptibly different from that of normal Cheddar cheese (Czulak, 1962). However, this difference in texture diminishes as the cheese matures. Washed curd varieties

There has been a substantial increase in recent years in the consumption of washed-curd varieties of Cheddar cheese (Olson, 1981). Varieties such as Colby and Monterey are milder in flavour and have a more plastic texture than Cheddar. They are relatively high-moisture cheeses (39–40%) and ripen rapidly. It has also become popular in New Zealand and Australia to manufacture dry-salted Gouda-style, Edam-

Colby and Monterey

The recent improvements in the production of granular Cheddar for processing are also indirectly responsible for the production of Colby and Monterey because these varieties are in fact washed-curd, granular cheeses. Traditionally, whey is drained off until the curds on the bottom of the vat are just breaking the surface and cold water is added to reduce the temperature of the curds/whey to about 27 °C (Fig. 13). The moisture content of the cheese can be controlled by the temperature of the curds/whey/water mix. The moisture content decreases as the temperature is increased between 26 and 34 °C. The pH of the cheese is determined by the proportions of both whey removed and water added. The length of time the water is in contact with the curd is also important because this determines the level of residual lactose. As salt penetration into the interior of the granular curd particles is rapid, no pH gradient occurs and seaminess is not a problem.

Manufacture as for Cheddar up to running stage

Proportion of whey removed and water added

Contact time Temperature of curd/whey/water mixture Residual lactose

pH

Moisture content

Typical texture of Colby

Figure 13 The main factors that determine the characteristic texture of Colby cheese.

96 Cheddar Cheese and Related Dry-salted Cheese Varieties

The calcium content of Colby tends to be slightly lower than that of Cheddar because of the higher percentage of starter used and a further small loss of calcium at the washing stage. However, as discussed previously, it is the pH that determines to a large extent the texture of a cheese. The addition of water results in a small increase (about 0.1–0.2 units) in the pH of the finished cheese but this is sufficient to give the cheese a more plastic texture than Cheddar. Recent trials (Creamer et al., 1988) have shown that the calcium content of Colby cheese can vary between 120 and 180 mmoles/kg cheese without influencing significantly the texture of the cheese as long as the pH is greater than about 5.2. The characteristic texture of Colby cheese is thus influenced almost entirely by its pH and moisture level (Fig. 13). Traditionally, Colby had a mechanically open texture but the use of shorttime pressing systems (Wegner, 1979; Brockwell, 1981; Tamime and Law, 2001), in which the curd is transported to be pressed under a partial vacuum, results in a texture that is as close as that of Goudatype cheese varieties. Monterey cheese has many similarities to Colby but is usually softer (Kosikowski and Mistry, 1997).

Acknowledgements The editorial assistance of Ms Claire Woodhall and the advice of Mr Philip Watkinson are gratefully acknowledged.

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Gouda and Related Cheeses G. van den Berg, W.C. Meijer, E.-M. Düsterhöft and G. Smit, NIZO Food Research, Ede, The Netherlands

Origin and Characteristics Gouda and related cheeses are the main representatives of the class of semi-hard cheeses and can be characterised by: (a) the use of fresh pasteurised cow’s milk, the milk normally being partly skimmed (generally leading to at least 40% fat in the dry matter of the cheese); (b) milk clotting by means of rennet (usually extracted from calves’ stomachs); (c) the use of, preferably, mixed-strain starters consisting of mesophilic lactococci and usually leuconostocs, that generally produce CO2; (d) a water content in the fat-free cheese below 63% (ratio of water to solids-not-fat 1.70); (e) pressing the cheese to obtain a closed rind; (f) acidification mainly in the curd block after separation of the whey during pressing, holding and the first hours of brining; (g) salting after pressing, usually in brine; (h) absence of an essential surface flora; (i) being at least somewhat matured (for 4 weeks) and thus having undergone significant proteolysis. Consequently, the cheese normally has a semi-hard consistency and a smooth texture, usually with small holes; the flavour intensity varies widely. After prolonged natural ripening, the consistency will be hard and short and the formation of amino acid crystals is common. Variation within cheese type and ripening time is considerable: (a) loaf size may be between 0.2 and 20 kg; (b) loaf shape may be a sphere (Edam), a flat cylinder with bulging sides (Gouda), a block, like a loaf of bread, etc.; (c) fat content in the dry matter ranges from 40 to over 50%; (d) water content in the fat-free cheese ranges from 53 to 63%; (e) salt content in the cheese water ranges from 2 to 7%; (f) pH may be anywhere from 4.9 to 5.6 (brittle Edam or old Gouda); (g) maturation may take from 2 weeks to 2 years under drying conditions, although foil-ripening of

block-form cheeses at a rather low temperature is applied now in large volumes. Recently, several companies have begun to produce cheese with a reduced fat content. In order to avoid too tough a consistency and too flat a flavour, modified technology is used. The introduction of adjunct starters, often of thermophilic nature, to enhance flavour development, has also given more opportunities to achieve the desired texture. Besides calf rennet, the use of fermentation-produced chymosin and some microbial rennets is also practised nowadays in various countries. Generally, a larger loaf is likely to have a lower water content (initially) and is matured for a longer time than a smaller one. To this end, Baby Gouda (0.2–1.0 kg) is manufactured with a higher water content than the larger normal Gouda cheese. Smaller loaves, with a relatively large surface through which more moisture evaporates, are used for shorter ripening under natural conditions. Herbs or spices are sometimes added, particularly cumin (i.e., the seeds of Cuminum cyminum). Traditionally, two main types of cheese were made in The Netherlands: Gouda and Edam. Gouda cheese was made in fairly large loaves of flat cylindrical shape (mostly 4–14 kg) from fresh whole milk and was matured for a variable period (6–60 weeks) under natural conditions; it is still made on some farms from raw milk in much the same way (‘Goudse boerenkaas’). Edam, a sphere of 1 or 2 kg, was made from a mixture of skimmed evening milk and fresh morning milk, leading to about 40% fat-in-dry-matter (FDM); the cheese had a somewhat shorter texture than Gouda and was usually matured for 6 months or more. Mimolette (‘Commissiekaas’), a high-coloured sphere of 4 kg, is also related to this type. Later, a greater range of cheeses, differing in shape, body and taste, evolved from these types. Most modern types have a somewhat higher pH and moisture content than the cheeses had previously; one reason for this change was to obtain better sliceability of the matured cheese. It is good to be aware of the industrial scale on which production occurs. The past 45 years especially have witnessed drastic changes in the cheese industry. Refrigeration of the milk at the farm (⬃4 °C) and collection of this milk every second or third day have

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104 Gouda and Related Cheeses

become the accepted system in many countries. Rigorous control of the hygienic quality of the milk leads to far smaller variation in composition, thus facilitating the introduction of systems for process control. Cheese factories have been modernised and merged into plants with high capacity. Plants with an annual production of 30 000 tonnes of cheese, manufactured in a 6-day working week, are more or less standard now for Gouda-type cheesemaking. These plants are highly mechanised, automated and computerised, producing cheese of the desired quality at relatively low labour costs but with very costly equipment. Individual plants are often specialised in the manufacture of a single cheese variety. They process about 50 000 l milk/h divided among three cheese vats (batches). Improved insights into cheese technology gained by much research made these developments possible.

Manufacture Process principles

The milk for cheesemaking is stored and prepared by standardisation to produce a product of the desired composition. After pasteurisation, the milk is cooled to renneting temperature and starter, rennet and possibly other ingredients are added. The milk coagulates and forms a gel that is cut rather soon in order to be able to divide the curd without too much stress on the still-fragile particles. The increased surface of the curd particles and gradually stirring more vigorously facilitates the expulsion of whey (syneresis). After some time, sufficient whey has been liberated and a significant part of this first (non-diluted whey) whey is removed to enhance the forces on the curd grains and to make room for the addition of curd-washing water. This water serves to dilute the whey to reduce the lactose content of the curd. Moreover, this water is heated to increase the temperature of the curds and whey mixture, which enhances syneresis to further reduce the moisture content of the curd. To this end, stirring is also intensified somewhat. This step takes approximately 30 min (90% of lactose diffusion is reached) before the curd particles are allowed to settle. After ‘matting’, the whey is drained off at a limited speed, especially after the whey surface reaches the top of the curd mass. Particularly in this stage, inclusion of air between curd particles must be avoided carefully because it impairs later eye formation in the cheese. When the drainage is nearly complete, the curd particles have started to fuse and the bed is compacted, it is possible to cut the mass into blocks. These blocks are transferred to cheese moulds, with a liner, in which the mass still flows for a while to fill the mould entirely.

Then, the moulds with curd are transported to the press, where all cheeses from one batch are pressed at the same time. In the meantime, whey leaks from the blocks of curd, until pressing closes the surface. During pressing, pressure on the cheese surface is increased gradually to approximately 25 kPa in three or four steps. The cheese acquires its desired shape and a thin rind is formed, facilitated by the liner, to protect the loaf against the forces during subsequent process steps and greatly inhibits moisture loss until salting. After demoulding, the loaves are still held for further acidification upside down in a mould, without a liner when possible, for some time, depending on the cheese type. Then, the cheeses are salted, usually by immersion in brine. After brining, the cheeses are dried somewhat and coated, and curing starts. During ripening, the loaves are kept in an air-conditioned room to protect the integrity of the rind, control water evaporation from the cheese and prevent visible microbiological growth on the cheese surface. These essential process steps largely determine cheese composition and the efficiency of production. Important aims are to obtain maximum yield, to control cheese composition (and hence quality), and to keep the process as short as possible, while following a fixed time schedule. The main points to be considered in relation to cheese composition are: final fat and water content, pH of the cheese, quantity of calcium phosphate remaining in the cheese, and the quantity of rennet retained in the curd. To regulate the process, the rate of syneresis is of paramount importance. Several process parameters are important in this respect and have their impact on the water content of the cheese, its MNFS (moisture in the non-fat substances) in particular. An increase in the fat content of the milk, the heat load by pasteurisation and thermisation (see ‘Milk quality’), of the curd cube size, the amount of curd wash water, and a decrease in scalding temperature, will diminish syneresis. Syneresis is enhanced by adding CaCl2, adding more starter or using a more active starter, which causes a faster pH drop in the curd during the process. It is also enhanced by increasing the (scalding) temperature, the stirring intensity and time, the amount of whey removed before curd drainage and the time from filling the moulds until pressing. Increasing the moisture content by such process parameters also increases the inclusion of lactose, resulting in a lower pH of the cheese, unless there is a concomitant increase in the amount of curd wash water. Pre-acidification of cheese milk (which is not advisable), increasing the quantity of starter added, the rate of growth of starter bacteria and the time needed until the curd is separated from the whey, all reduce the pH of the curd before the rind is closed during pressing and,

Gouda and Related Cheeses 105

consequently, reduce the concentrations of calcium and inorganic phosphate retained in the cheese. Lower concentrations of these give a somewhat lower cheese yield, a lower buffering capacity and consequently a slightly lower pH, and may affect cheese texture somewhat, the consistency becoming slightly softer and shorter. During normal cheesemaking, acidification by lactose fermentation hardly occurs before filling the cheese moulds. Thereafter, pH should decrease considerably and acidification will be complete after approximately six hours in brine. A flow sheet for the production of Gouda cheese is shown in Fig. 1 to give an example of the steps in a practical process. The manufacturing process is discussed in more detail in the following sections. If more detailed information about the process equipment is desired, the reader is referred to van den Berg (2001). Milk treatment

The aim of treatment of the milk is to improve or maintain the quality of the milk for cheesemaking, with respect to cheese quality and composition, yield and ease of manufacture. Milk quality

Milk quality may be defined so as to include composition. The fat and the casein contents of the milk naturally affect cheese yield and fat content; lactose content affects cheese acidity (see ‘Control of pH and water content’). Off-flavours, particularly if associated with the fat, may be carried over into the cheese. Although in a well-ripened Gouda-type cheese very limited lipolysis occurs, it is never enhanced to avoid any soapy off-flavour. Physical dirt should be absent as it shows up in the cheese. It can be removed easily by filtering or centrifugation. Such issues are among the official quality properties in the payment scheme for farm milk in The Netherlands, as mentioned in a survey on this subject by IDF (1995). The bacteriological quality of cheese milk is of great importance. Pathogenic organisms may survive in cheese, which may be a problem in raw milk cheese. Pathogenic Enterobacteriaceae and staphylococci might grow in cheese. However, proper manufacturing procedures, e.g., the use of an active lactic starter, ensure that throughout the cheese, either the sugar is fully and rapidly converted into acid by the starter organisms (van Schouwenburg-van Foeken et al., 1979), or that the salt content is already high enough to prevent the growth of pathogens. This is an important reason why there are no incidents with pathogens in well-made Gouda cheese. For more details on pathogens in cheese, the reader is referred to ‘Growth and Survival of Microbial Pathogens in Cheese’, Volume 1. Several bacteria present can cause

defects in the cheese: coliforms, Lactobacillus spp., Streptococcus thermophilus, faecal streptococci, propionic acid bacteria, Clostridium tyrobutyricum (see ‘Butyric acid fermentation’). The growth of psychrotrophic bacteria in the raw milk may lead to the production of sufficient thermostable lipolytic enzymes to cause undesired lipolysis in the cheese (Driessen, 1983); bacterial proteinases do not seem to cause undesirable effects but may break down casein prematurely and reduce cheese yield. Nowadays, milk is stored at the farm for some days so that the growth of psychrotrophs may have started already. Temperature control and good cleaning procedures are necessary and stimulated by quality demands on the total microbiological count of farm milk (IDF, 1995). However, such milk is usually thermised (e.g., 10 s at 66 °C) upon reception at the cheese factory, sufficient to kill several types of bacteria, including most psychrotrophs, but not to greatly alter the milk otherwise (Stadhouders, 1982; van den Berg, 1984). Pasteurisation

The milk is pasteurised, usually high temperature-short time (HTST) e.g., 15 s at 73–74 °C, just before cheesemaking. However, it is noteworthy that such an indication of pasteurisation intensity is not sufficient to indicate the total heat load to which the milk is exposed. Therefore, it is necessary to be aware of temperature changes with time when passing through the heat exchanger, including a large regeneration section (de Jong, 1996). Figure 2 gives time–temperature relations for effects that are important for cheesemaking; it should be realised that these are only examples since there is considerable variation in the thermolability of micro-organisms, etc. Pasteurisation serves the following functions: (a) Killing of pathogens, including Listeria monocytogenes, which means that heating may be slightly more intensive than that needed to inactivate alkaline phosphatase only. (b) Killing of spoilage organisms. Spores of Clostridium tyrobutyricum survive but Enterobacteriaceae, propionic acid bacteria and most lactic acid bacteria are killed. Some species of Lactobacillus and Streptococcus may survive, but they are seldom present at high numbers in the milk. However, Streptococcus thermophilus may grow on the metal surface downstream of the large regeneration section of a modern heat exchanger (Bouman et al., 1982) and thus attain high numbers if pasteurisation is continued for a long time (e.g., 10 h). This may lead to undesirable flavour and texture in the cheese (see ‘Thermo-resistant streptococci’). (c) Inactivation of milk enzymes. This is probably important only with regard to lipoprotein lipase (EC 3.1.1.34), but even the usefulness of this is variable.

106 Gouda and Related Cheeses

Milk

Thermisation 15 s 66 °C

Thermisation 15 s 66 °C

Unripened cheese

Separation Standardisation e.g., 3.5% fat

Pasteurisation 10 min 95 °C

Coating with latex

Skim milk Storage 4–6 °C

Drying

Cream

Pasteurisation 15 s 73 °C

Sludge

Sterilisation

Bactofugation

Mixing

Whey cream

Brining 5 days

Separated whey

Separation

Holding 1h

Demoulding and turning

Starter 0.6% Setting 20 min 31 °C

Curd fines

Pressing 80 min

CaCl2 0.020% Cutting 20 min

Sieve or hydrocyclone

Draining and moulding

Draining 45%

Whey

Buffer tank 34.5–33.5 °C

Scalding 34.5 °C

Stirring 20 min

Partial draining

Rennet 0.020%

Hot water 25%

NaNO3 0.0025%

Figure 1 Example of a modern flow sheet for the manufacture of 12 kg Gouda cheese (until curing). Time from start of cutting to start of moulding – 60 min. A curd-filling machine with vertical columns and plastic moulds with a nylon gauze lining is used. NaCl content of brine – 18%, brine temperature – 14 °C, brine pH – 4.5. If bactofugation is omitted, about 0.015% NaNO3 is added.

Gouda and Related Cheeses 107

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Several lipases and proteinases produced by psychrotrophic bacteria are not inactivated (Driessen, 1983). (d) Because the milk should be pasteurised shortly before renneting, it also serves to undo, more or less, the adverse effects of cold storage on the casein micelles and on the salt equilibrium (which in turn affects renneting properties; ‘Rennet-induced Coagulation of Milk’, Volume 1), to melt the fat in the globules and to bring the milk to renneting temperature. Heat treatment can also have undesirable effects, particularly if its intensity is distinctly higher than that needed to inactivate alkaline phosphatase: (a) Considerable denaturation of serum proteins by more intense pasteurisation leads to slow renneting, a weak curd and poor syneresis (‘Rennet-induced Coagulation of Milk’, ‘The Syneresis of Rennet-coagulated Curd’, Volume 1). It may also cause a cheese of poorer quality, particularly the development of bitterness and sulphide flavour (van den Berg et al., 1996b). The first defect is probably caused by stronger binding of chymosin and the second by breakdown of denatured (more heat-sensitive) sulphur-containing whey proteins. A small amount of denatured whey proteins, which is mainly precipitated on caseins, has little adverse effect on the renneting process or cheese quality, e.g., when denaturation occurs during sterilisation of small vol-

umes, such as bactofugate or starter milk which are then blended into a large volume of milk. Since heat denaturation also causes a profitable increase in cheese yield, fairly rigorous control is exerted in some countries, e.g., via the nitrogen content of the whey (which, for example, should be at least 95% of that of the whey made from raw milk). As sampling of the original raw milk has become increasingly difficult when (sometimes days later) the whey from the cheese vat is sampled for this goal, a new and more sensitive method has been introduced in The Netherlands. This is based on the different kinetics of denaturation of the individual whey proteins, e.g., bovine serum albumin and immunoglobulin G are denatured more rapidly between 70 and 100 °C than -lactoglobulin. A small increase in pasteurisation temperature causes a distinct change in the relation between the contents of these native heat-sensitive proteins and that of native -lactoglobulin in the whey (van den Bedem and Leenheer, 1988). (b) Useful milk enzymes may be inactivated, especially xanthine oxidase (EC 1.2.3.2). This enzyme is needed to slowly convert (added) NO3 to NO2, which is essential for the desired action of nitrate against clostridia (see ‘Butyric acid fermentation’). Although pasteurisation of cheese milk is widespread and has certainly helped to considerably improve average cheese quality, it is often held to be responsible for a certain lack of flavour, especially in well-matured varieties. This may be due to inactivation of lipoprotein lipase and killing of bacteria that may contribute to raw milk cheese flavour, which usually is more variable than the flavour of cheese made from pasteurised milk. Less-severe heat treatment may improve flavour. An old-fashioned way of improving milk quality is to let the fresh milk cream at a low temperature (5–10 °C); in this way, most bacteria accompany the cream because of agglutination (Stadhouders and Hup, 1970). By heat-treating the cream, but not the skimmilk, most bacteria are killed without greatly affecting milk enzymes. Bactofugation

Another way to enhance bacteriological quality is bactofugation. This may be applied when cheese is made from raw milk. However, the main purpose is the removal of spores of Clostridium tyrobutyricum. As said before, these spores survive pasteurisation and will pass into the cheese, where they may cause butyric acid fermentation (BAF). This defect is often called ‘late-blowing’ and is a well-known risk for Gouda and related cheeses (see ‘Butyric acid fermentation’). The addition of nitrate is effective against this defect but not desired in various markets. The use of a bactofuge, in particular the

108 Gouda and Related Cheeses

self-desludging type, is a successful answer to this question. The bactofuge is a kind of centrifuge for milk with special properties to remove the heavy sludge via nozzles mounted at the outside of the separator bowl. Bacterial spores have a rather high density in comparison with the bacteria themselves. The original idea of using a separation technique to improve the bacteriological quality of milk, in that case for liquid milk, came from Simonart and Debeer (1953). The bactofuge is mounted in line with the milk pasteuriser or the thermiser and is fed from the regeneration section because the best efficiency of the process is obtained at approximately 60 °C. This treatment reduces the number of spores drastically, even to about 2–3% of the number in raw milk (van den Berg et al., 1980, 1988). The sediment obtained contains the spores but also more casein than the original milk and its removal would cause a significant reduction in cheese yield (about 6%). Consequently, the sediment is commonly ultrahigh temperature (UHT) heated to kill the spores and added back to the milk; the concomitant denaturation of serum proteins is acceptable (see ‘Pasteurisation’). Double bactofugation increases the efficacy of spore removal, but is more costly. Nevertheless, this treatment is necessary when no nitrate addition is allowed. In such a modern line, the continuously discharged heavy phase from the second separator is fed back into the milk supply to the first bactofuge because there is not much difference in the spore load of the two liquids. On the other hand, the heavy phase from the first bactofuge, mixed with the intermittent discharges (by the self-desludging mechanism) from both machines, is the total volume to be sterilised. For further details of this technique, the reader is referred to van den Berg (2001). The technique of microfiltration of the skimmed milk fraction is an alternative to bactofugation. The effect on the removal of spores might be similar to double bactofugation but the investments are higher. Moreover, the cream and the milk retentate must be sterilised, which is a considerably larger volume than in the case of bactofugation. Standardisation and additives

In most cases, after thermisation, the milk is standardised so as to yield the desired FDM of the cheese (see ‘Standardisation’). This generally implies some skimming of the milk. Usually, part of the milk is passed through a separator (which also removes dirt particles) and sufficient cream is removed. Skimmed milk, and often some sterilised whey cream, is added to whole milk shortly after reception. This standardised milk is stored until further processing. However, nowadays automatic in-line standardisation systems are receiving some interest in

large cheese factories because of possibilities to minimise storage tank volume. Substances added to the milk after pasteurisation and before renneting may include: (a) CaCl2 to speed up, and particularly to reduce variability in, renneting and syneresis. (b) Nitrate, to prevent early blowing by coliforms and the growth of C. tyrobutyricum. Nowadays, nitrate is often added later, i.e., to the curds–whey mixture after the first whey has been removed. This serves both to save on nitrate and to avoid producing large quantities of whey that contains nitrate. (c) Colouring, either -carotene or annatto (an extract of the fruits of Bixa orellana), for obvious reasons. Its use appears to be waning and is often omitted, although some types are highly coloured, e.g., Mimolette. Spices, if any, are commonly added to the curds and whey mixture before draining, e.g., in the buffer tank. Cheesemaking Curdmaking

Renneting is usually done at 30–31 °C and cutting starts 20–25 min afterwards. About 20 ml of rennet with a specific activity of 150 IMCU (International Milk Clotting Units) and 10–20 g CaCl2 are usually added to 100 l of cheese milk. The pH at renneting is somewhat reduced by the addition of CaCl2 and starter; 6.50–6.55 is the usual value. The aim is to produce a coagulum (gel) that can be cut easily and stirred without excessive loss of ‘fines’ in the whey and which shows rapid syneresis. If the milk is drawn from a very large quantity, and moreover if the calving pattern of the cows is fairly evenly spread throughout the year, milk composition is generally sufficiently constant to give good results with fixed quantities of rennet and CaCl2 always. The coagulum is cut, usually into pieces of any given form, some 8–15 mm size, initially. When the coagulum pieces already have been cut sufficiently but still some time is required for syneresis, the mass is stirred (by turning the knives in the opposite direction) until the desired amount of whey can be drained off. This stirring should be carried out very gently to minimise loss of fines and fat. Curd fines are usually defined as the fraction 1 mm size and can easily be lost in the separated whey. After a while, the curds are allowed to settle and part of the whey is removed so that stirring becomes more effective, i.e., the forces acting on the curd grains are higher and thus promote syneresis. After removing the first whey, stirring is immediately restarted to avoid too much curd fusion, as curd lumps may persist during

Gouda and Related Cheeses 109

the rest of the process. The temperature is increased (scalding), also to speed up syneresis, but not to a temperature high enough to harm the (mesophilic) starter organisms, which usually implies keeping below 38 °C. The curd wash water must be carefully spread over the cheese vat in order to prevent local overheating. During scalding, agitation should be more vigorous, again to avoid local curd fusion. Inside such curd lumps, whey expelled by individual curd particles cannot be removed and the washing of lactose is inhibited, resulting in acid, soft and ‘nesty’ spots (clusters of small irregular openings) in the cheese. Scalding can be done by indirect heating or by adding heated whey or hot water. The latter practice is the most common, since water usually has to be added anyway to regulate pH (see ‘Control of the pH’). Initially, an increase in the size of the cheese vats became possible with the separation of the process steps of curdmaking, separation of curds and whey (curd drainage) and moulding. The time schedule for successive batches (vats) is programmed in such a way that (nearly) finished curds–whey mixtures can be pumped separately at constant time intervals into a buffer tank before being introduced into the continuously working curd drainage and moulding machine. The open cheese vats of the past have been developed into large enclosed tanks with integral cutting and stirring devices, built-in filling, emptying and dosing (of rennet and starter) pipework and metering devices (for sucking whey) and control and automation modules. Recently, the increased need for process control has also led to the successful introduction of a laser optical fibre device built into each cheese vat to control the renneting process. It indicates the beginning of aggregation, the increasing firmness of the coagulum and may warn when the desired gel firmness has been reached (ten Grotenhuis, 1999). All milk takes part in the renneting process and will contribute to cheese yield. However, during curdmaking and successive processing steps some loss of curd fines and fat is inevitable because of mechanical stress by the processing equipment. The cheesemaker has to perform the optimal process in relation to the equipment used and the curd properties desired at final drainage (curd size, water content, temperature, etc.). The losses in the whey of fat and, in particular, curd fines, should be minimal. Most of the fat in whey is already liberated when cutting the coagulum because cutting initiates the loss of the fat globules from the newly created surfaces. The amount of whey expelled during the first cuts has a very high fat content that decreases during syneresis and expulsion of whey at the end of cutting to 9% of the fat percentage of the cheese milk. Some further curd damage by mechanical stress during the process creates

new curd surfaces and also results in losses of fat and curd fines. This is particularly at risk when curd is allowed to settle before sucking the first whey and starting stirring and adding the washing water. Then, extra force is needed to avoid curd lumps during the next stage of curdmaking. The design of the cheese tank (vat) is important with respect to such losses and most types of vat have been thoroughly tested, as demonstrated by de Vries and van Ginkel (1984). The difference between the fat content of the whey at the end of curdmaking and after dilution with the wash water (calculated from the first whey) should be no more than 0.02. Curd fines, expressed as dry curd solids, in the first whey should be 150 mg/l whey. This level may increase somewhat in the whey outside the curd particles during further processing, including the holding time in the buffer tank before drainage. However, it must be realised that during drainage, especially in the vertical draining cylinders, most of these fines are still entrapped between the larger curd particles. Draining and moulding

In the past, after the curds had lost enough moisture (the water content being ⬃68% in the case of Gouda cheese and the pH 6.45), stirring was stopped and the curd grains were allowed to settle. Partial fusion of deforming curd grains now occurs, whey is separated, and a continuous mass of curd is formed that can be cut into blocks. Considerable loss of whey from the curds occurs during these stages (Walstra et al., 1985). This was often promoted by applying some pressure (e.g., 400 Pa) by placing perforated metal plates on top of the bed of curd or later by the curd layer itself being deep enough; pressure also promotes curd fusion. The lower the fat content, the less easy curd fusion becomes, because of the lower scalding temperature and reduced ability of the curd grains to deform. As a consequence, more whey may be left between the curd grains after drainage, finally resulting in a more open texture. If a very low water content is desired, the drained curds may be stirred or worked; this causes considerable additional syneresis, but also loss of fines and fat and a cheese with an open texture (many irregular, small holes). The blocks of curd are transferred to cheese moulds. The traditional square block of curd is allowed to spread and fill the round mould after some time. Between the corners, it flows out and at the corners it may even be compressed, causing in the cheese locally a higher and a lower water content, respectively (Arentzen, 1972). So certain flow properties of the curd block are necessary. At these stages until brining, the temperature of the curd blocks should be maintained high enough for good curd flow, to continue acidification and achieve good rind formation during pressing.

110 Gouda and Related Cheeses

To this end, moulds are still warm after cleaning and the room temperature should be approximately 20 °C and often the presses are practically closed. Forcing the flow of the curd block, e.g., by the premature start of pressing, should be avoided because it may locally destroy curd fusion and in such places more whey collects between the curd particles. This turns into ‘nesty’, soft and acid spots in the final cheese because this whey (with lactose) is resorbed, while the distance between these stronger acidifying curd grains remains too large and these spaces are filled later with gas. Essentially, the mechanism is similar to what may happen in the case of curd lumps (see ‘Curdmaking’). The introduction of specific curd-sedimentation or pre-pressing vats, with a moving, perforated belt, was the first step in mechanisation of the Gouda cheesemaking process. At one end of this vat, the settled and drained curd layer is mechanically cut into pieces of the desired dimensions, which are transferred to moulds to be pressed. Such curd blocks are still square and require time to deform and fill the round Gouda cheese moulds. Furthermore, greater variation of water content in the cheese will occur. The water content of the curd blocks also deviates within a batch due to differences in drainage time before the transfer to the moulds. Although this system is still applied with improved equipment, in many cases it has been superseded by the following technique. The curds–whey mixture is fed from the buffer tank into continuously working machines that separate the whey from the curds, shape the curd blocks and fill them into moulds. The most common machine is the Casomatic®, which operates with a downward curd stream. It has draining columns for cylindrical or rectangular cheeses. Technologically, these machines have the advantage that the weight of the loaves can be controlled accurately, the relative standard deviation being 0.5–1.5%; this is especially important for small loaves, e.g., Edam cheese. These machines were less flexible than the pre-pressing vats for the production of cheeses of different shapes and sizes. However, modern models have been improved in this regard. During the mould-filling process, syneresis of the curd proceeds and would cause the water content of cheese to decrease if no precautions were taken. Therefore, syneresis during the moulding operation is slowed down by stirring the curds–whey mixture in the buffer tank only gently, but sufficiently to keep the mixture homogeneous, and by gradually reducing the temperature by, e.g., 1 °C when the time between batches is more than 15–20 min. Because the first curd blocks of a batch contain more moisture and will loose more moisture before pressing than the others, the height of the curd block can be adapted during drainage of the batch. In this way, the moisture

content and the weight of the individual final cheeses can be controlled fairly accurately in combination with the time between filling the mould and start of pressing (de Vries and Staal, 1974). Nowadays, two buffer tanks are often used, alternately filled with the next batch, in order to let any fine air bubbles entrapped during stirring and pumping to escape. These could later disturb the eye pattern in the cheese, as they are entrapped during drainage. Consequently, the stirring time in the cheese vat is shortened. Figure 3 shows a section through a draining column of the Casomatic®. The process in the draining columns was investigated thoroughly by Akkerman et al. (1996). During ‘free’ drainage, there is still considerable ongoing syneresis because whey is separated and the hydrostatic pressure has disappeared. At the same time, compaction, deformation of the curd particles and curd fusion take place, blocking the pores between the curd particles, and drainage decreases (Akkerman et al., 1994). This also slows down the compaction rate. In the draining column, the liquid pressure gradient between the centre and the wall of the drainage equipment is the important driving force. The column is divided into three sections in which this gradient increases in each section downwards in parallel with the compaction rate. To this end, the control of the outflow of the whey through the perforated wall has always been a very important feature of this machine. It is managed in relation to variables like the geometry of the drainage column, the softness (moisture content) of the curd grains and their size, especially the amount of curd fines. At the outlet, below the draining column, curd blocks are cut off (for round moulds, circular blocks), often pre-pressed slightly (without deformation of the block) to prevent loosening of curd during successive handling and transferred to the moulds. The filled moulds are moved to the press and on their way, a cover is put on top. The whey obtained at various stages may be collected separately, because it differs in pH, added water and added nitrate. The whey is usually separated and the cream obtained is pasteurised, e.g., 30 min at 95 °C, to destroy fully any bacteriophage that may be present. The whey cream is used to adjust the fat content of the next lot of cheese milk. Curd fines are usually separated from the whey by means of hydrocyclones or filters, but are not recycled to the curd because of the danger of contamination (bacteria and phage). They are pressed and often used for processed cheese. Pressing

The blocks of curd are pressed in moulds to obtain the typical shape of the cheese and a closed rind. The latter is necessary to avoid further loss of moisture until

Gouda and Related Cheeses 111

Figure 3 Cut-away view of the Casomatic CSC, the single column continuous whey drainage and portioning machine (reproduced with permission of Tetrapak Tebel B.V., Leeuwarden, The Netherlands). 1, Curds and whey supply; 2A–C, Whey drainage sections with pressure indicators; 3, Whey collection tube; 4, Whey collection vessel; 5, Pneumatically operated cylinder for opening and closing the curd knife; 6, Pneumatically operated cylinder for removing the curd block to fill the cheese mould.

112 Gouda and Related Cheeses

also ‘Draining and moulding’ and ‘Control of water content’). In traditional cheesemaking, the freshly pressed loaves were turned in the moulds with a flat cover and left there until the next day for ‘shaping’ (Dutch: omlopen), i.e., to attain a symmetrical shape. After removing the cloths and before turning, the pressing rind created between the cover and the mould was trimmed off, which caused some risk of tearing open during brining. The main change occurring was, however, the complete conversion of lactose to lactic acid. This is important because during brining, fermentation by the starter bacteria is slowed down and even effectively stopped in the rind, owing to the combined effects of low temperature and high salt content. Nowadays, a somewhat greater quantity of a fastergrowing starter is usually added, which implies that much more lactic acid has already been produced a few hours after adding starter. The course of acidification in the core of the cheese is illustrated in Fig. 4. The short holding between pressing and brining is facilitated by a better design of the modern cheese moulds so that ‘trimming’ and ‘shaping’ are not really necessary any more. The shape of the curd block also approaches that of the mould better and the time necessary for curd flow before pressing diminishes. So the modern mould has made it possible to speed up the process to a great extent, thanks to improved starter technology. Instead of pressing cheese loaves in stacks, it has become common to press them in a single layer by individual pressing cylinders or one cushion for a great number of cheeses. Pressing a pile of cheeses in a column

6.2 pH

6

5.8 4 Lactose

5.4

2

5.0 Lactic acid

0

4.6

g lactose or g lactic acid 100 g–1 H2O

6.6

pH

brining, to achieve an even salt penetration through the cheese surface, to withstand the tension during brining and to serve as a barrier for micro-organisms during brining and ripening. Originally, wooden moulds were used, and the blocks of curd were wrapped in cloth; a pressure of 50–100 kPa was applied for several hours and the cheese developed a very distinct, firm rind. Nowadays, cheese loaves are formed in finely perforated metal or plastic moulds, or plastic moulds lined with a gauze or some kind of cloth to promote drainage and rind formation. The pressure is usually much lower and is applied for a shorter time. Consequently, only a weak rind is formed; although the rind should be fully ‘closed’, i.e., free of visible openings, it is not a fully effective barrier against the high numbers of micro-organisms during brining (Wilbrink et al., 1981). In order to have an undisturbed process, sticking of the cheese to the liner of the mould must be avoided. This is achieved by the correct combination of a gradual increase of pressure over time and the firmness of the curds. Closing of the rind is due to complete fusion of the outermost layer of curd grains, promoted by local deformation of the surface around threads of the liner or corners of the perforations of the mould (Mulder et al., 1966) and good drainage of the liberated whey. Essentially, it is a locally intensified syneresis process with a rearrangement of casein strands. Pressing, as it is applied nowadays, is generally insufficient to cause complete fusion throughout the mass of the freshly pressed loaf. This implies that some moisture can still move fairly freely via small interstitial openings left between the curd particles through the cheese mass, possibly leading to uneven moisture distribution. During brining, the temperature decreases and the remaining whey is resorbed, while the interstitial openings practically disappear. Within 1–2 days, curd fusion is nearly complete and the cheese has a closed texture. The cheese looses considerable moisture during pressing, but moisture loss is slight once a closed rind has formed (Straatsma and Heijnekamp, 1988). This implies that starting the pressing earlier and applying a higher pressure lead to less moisture loss, and hence to a cheese with a higher water content. By varying the time between mould-filling and the start of pressing, water content can thus be regulated to some extent, especially to ensure the same water content in cheese loaves of the same batch. During stirring in the whey, syneresis proceeds. Consequently, the blocks from a batch formed last in a continuously operating draining and moulding machine have the lowest water content, and pressure should be applied to those loaves soon to ‘keep in the moisture’, while the blocks formed earlier should be left for a longer time before pressing (see

10 14 4 6 8 12 Time after adding starter (h) Pressing Wash water In brine 0

2

Figure 4 Acid production during Gouda cheese manufacture (conditions comparable to those in Fig. 1). Quantity of lactose and lactic acid, and the pH of curd and cheese (core) as a function of the time after starter addition to the cheese milk.

Gouda and Related Cheeses 113

under one pressing head, as in the past, started pressing of the first, more moist cheeses of the batch earlier instead of later (see ‘Draining and moulding’). Nowadays, presses are filled and emptied continuously. Continuous pressing, however, is not applied because differences in water content still exist between loaves of a batch at the beginning and the end of moulding, due to differences in the extent of syneresis of the curd. When time between batches is not too long, say 20 min, the whole batch is pressed for the same length of time and pressuring programme. With respect to demoulding, mechanical systems have been designed which prevent damage to the relatively weak cheese rind. The lid is lifted perpendicularly, the mould is emptied after rolling on the side or the cheese is blown out of the linerless rectangular mould. When held for further acidification, the cheeses are kept in normal moulds. Then, the cheeses are weighed on their way to the brine as part of the system to control composition and yield. Brining

Nowadays, the cheese is put into brine within 1 h after pressing when some residual lactose is still present in the curd. (Incidentally, this means that even in a 2-week-old cheese, the outermost layer may contain about 0.2% lactose in the cheese moisture and also that the brine contains lactose.) The use of pasteurised milk, strict hygiene

and adequate rind treatment are needed to prevent the growth of undesirable micro-organisms, which might profit from the presence of lactose. Another requirement is a sufficient decrease in pH; after pressing, ⬃5.9 and, when it occurs, after 1.5 h holding, 5.4–5.5. Moreover, the modern curing room provides better drying conditions for the rind of the cheese. Otherwise, such cheeses will easily develop a slimy surface which is hard to repair. Brining is done primarily to provide the cheese with the necessary salt. Moreover, it serves to cool the loaves rapidly to below 15 °C (to stop further syneresis, and prevent or slow down the growth of undesirable bacteria), and to give them a certain rigidity (due to the high salt content in the rind) during the necessary handling shortly after brining. Brining causes a considerable loss of water (2–3 times the quantity of salt taken up) and loss of a little soluble matter (0.2% of the cheese mass). Factors affecting these processes are discussed in ‘Salt in Cheese: Physical, Chemical and Biological Aspects’, Volume 1. Figure 5 indicates the effect of different salt concentrations in the brine on salt uptake and water loss during the process of manufacturing normal Gouda cheese. The brining process of Gouda and related cheeses was investigated extensively by Geurts et al. (1980). An increased fat content in the cheese hinders the process. Conversely, an increased water content before brining enhances salt uptake, relatively more than the

Salt content of dry matter (SDM)

Water content (W)

(%)

(%)

3.5 46 3.0

SDM

SDM

2.5 45 2.0 44

1.5

1.0 43 W

0

1

2

3

0.5

W

4

5

6

Days

Figure 5 Changes (idealised) in water content (W) and salt content of dry matter (SDM) of Gouda cheese during brining for a normal process and for a longer brining time at a lower water content and weaker brine (dotted lines), according to van den Berg et al. (1975).

114 Gouda and Related Cheeses

water loss. The shape and the size of the cheese have considerable impact on local diffusion processes. It may be expected that increased temperature and brine concentration speed up the process. However, in practice, strong dewatering of the rind zone hinders salt uptake and the net effect hardly increases above 20% salt or 18 °C. These phenomena are better described by the Maxwell-Stefan equation than by Fick’s law, which Geurts et al. (1980) applied originally, as discussed by Payne and Morison (1999). The average salt content of the young Gouda cheese is generally 4–5 g/100 g water in the cheese; of course, it takes considerable time (up to 2 months in a large loaf) for the salt to become more or less evenly distributed throughout the cheese. The brine usually contains 17–18% NaCl, but weaker brines might sometimes be used to allow brining the loaves for exactly 1 week. This poses the risk of growth of salt-tolerant lactobacilli in the brine that may even penetrate the cheese to some extent and cause flavour defects (Stadhouders et al., 1974; Wilbrink et al., 1981). Regular cleaning of the equipment and basin walls around and above the brine level is desirable (Stadhouders et al., 1985). Chemical disinfection of brine has negative effects on cheese flavour and, certainly in case of chlorine, forms toxic compounds. For similar reasons, irradiation of brine was not successful. Filtration techniques may be reasonably effective but are still too complicated to find wide application. The brine should contain enough Ca2(0.2% at 17% NaCl) and acid (pH 4.5) to prevent dissolution of cheese protein, which would cause a slimy rind (Geurts et al., 1972). A weak brine has a similar effect but may contain more Ca2 (van den Berg et al., 1975). Brine is used for years, with daily removal of the surplus brine and adjusting the salt concentration. A certain balance exists in the non-salt components in the brine and the moisture of the cheese. These solids also serve to buffer the pH so that only a limited amount of HCl is needed to control it. The lactose concentration in brine is higher when the cheese is salted at a higher pH, e.g., in case of rectangular blocks designated for foil-ripening. A consequence of brining is the loss of moisture, resulting in the dimensions of the cheese shrinking by a comparable percentage. Loaves with corners and edges shrink more at these places than elsewhere. The cheeses are transported by floating in the brine that is circulated continuously through the whole brining bath. The density of the cheese is approximately 1.07 while that of the brine is 1.10 g ml1, depending on salt concentration. A bypass flow is used for cooling (the brine temperature is maintained at 13 °C)

and for the uptake of salt from a silo. The daily amount of salt necessary to maintain a constant salt concentration is calculated from salt uptake by the cheese plus the increase of the brine volume by moisture from the cheese liberated by pseudo-osmosis. Two brining systems are in use. One uses stainless steel racks with horizontally tightened nylon nets to form several horizontal compartments; the racks can be moved up and down in a deep brine basin (2–3 m). Filling and, after brining, emptying of successive compartments is realised by opening gates, consisting of a frame with bars, at the front or the rear that do not hinder brine flow through the basin when closed. This circulation also serves to maintain an even salt concentration around all cheeses in order to control salt uptake by the cheese. Stronger agitation is, however, not necessary because salt uptake is, in effect, controlled by (slow) diffusion in the cheese when brine concentration is maintained. Moreover, increasing agitation by air injection through pipes with small openings at the bottom of the basin is not advised because precipitated calcium phosphates may block the openings. The air bubbles become smaller and yeast growth is enhanced (van den Berg et al., 1975). A high yeast count in the brine must be avoided because high counts on the cheese surface will inactivate natamycin (see ‘Rind treatment and curing’). In the other brining system, the loaves are salted while floating in a shallow layer of circulating brine (⬃0.4 m); brine sprinkling devices or rollers that periodically immerse the loaves take care of salting of the top of the loaves. The latter system is nowadays rarely installed in new factories because of the very large ground surface needed. The brine volume increases by about 2.5% of the cheese mass that is brined. The resulting surplus is often simply mixed with the effluent of the whole cheese plant. However, demands on the quality of the effluent and the (still threatening or already existing) financial consequences of discharging will make this solution less acceptable in many areas. Several techniques have been studied; concentration by evaporation under vacuum seems to be the most attractive one (Zoon et al., 1991). Rind treatment and curing

For Gouda and related cheese varieties, development of micro-organisms on the cheese surface is undesirable, because they may negatively affect cheese quality. In particular, the growth of moulds must be prevented, since they adversely affect the appearance of the cheese and some species may produce mycotoxins, e.g., sterigmatocystin by Aspergillus versicolor (Veringa et al., 1989). In former days, cheese was pressed in such a way as to obtain a thick and very tough rind; mould development

Gouda and Related Cheeses 115

was reduced by regular rubbing of the cheese rind with a dry cloth and unboiled linseed oil. After hardening, the coat formed also reduced water evaporation from the cheese rind, thus permitting it to remain relatively supple and springy. This practice was abandoned after the introduction of special plastic dispersions, which offer superior protection and permit the production of cheese with a weaker rind. This is one of the improvements that allowed mechanisation and a marked speeding up of cheesemaking. These plastic dispersions form, on drying, a coherent plastic film of hydrophilic nature that offers a protective coating against mechanical damage. When compared with oil treatment, it reduces the evaporation of water by ⬃50%. After the first week, water evaporation slows down steadily because diffusion inside the cheese is the limiting factor, while the relative humidity of the air is constant (Bouman, 1977). The film mechanically hinders mould growth, but it may also contain fungicides, e.g., natamycin (pimaricin), an antibiotic produced by Streptomyces natalensis, which is active only against moulds and yeasts, or calcium or sodium sorbate. In many countries, like The Netherlands, only natamycin is allowed while in some other countries only sorbates are permitted. Compared to sorbates, natamycin has the advantages that its migration into the cheese is generally limited to the outer 1 mm of the rind, it inhibits most of the microorganisms found on the surface of the cheese and it does not adversely affect the appearance, taste and flavour of the cheese (de Ruig and van den Berg, 1985). Moreover, natamycin is much more effective than sorbates; for comparable protection from mould growth, the amount of sorbate needed is about 200 times that of natamycin. Recently, growth of Penicillium discolor, which appeared to be less sensitive to natamycin, caused some trouble with Gouda cheese but adequate hygiene measures can prevent this when using this fungicide (van Rijn et al., 1997). With respect to public health, an acceptable daily intake of 0.3 mg natamycin/kg body weight has been established. The EU cheese regulations limit the quantity to 1 mg/dm2 of cheese surface when the cheese is sold. Recently, alternatives for plastic coatings, based on milk proteins, have been introduced. Generally, the cheese is cured at 12–15 °C and 85–88% RH. The conditions must allow the coating to dry rather quickly, otherwise undesirable micro-organisms like yeasts, and successively coryneform bacteria or moulds, may develop and cause off-flavours and a sticky and dirty surface. However, if the coating layer dries too quickly, cracks may form in the plastic film, allowing mould growth in the cheese rind because natamycin penetrates into the cheese only to a very limited extent. Some residual moisture is necessary to keep this film sufficiently flexible. Particularly at the beginning of ripen-

ing, the cheese inevitably expels a little moisture, causing a high humidity between the loaf and the shelf, which favours bacterial growth. To prevent this and to allow the cheese to retain a good shape, loaves are turned frequently during this period. Upon prolonged ripening, this frequency is reduced. Other measures to maintain shape are sufficient evaporation of moisture to replace, to some extent, the ‘framework’ created during salting that wanes by salt diffusing to the core, and to maintain not too high a temperature. In this respect, 17 °C is a practical hurdle. Above this temperature, most milk fat is liquid and the cheese exudes fat (‘sweating’), while the consistency of the cheese may become greasy. If a higher temperature is desired to accelerate ripening, then a paper banderole is sometimes used around the cheese. A higher temperature increases the risk of ‘late blowing’ and more attention to the cheese surface is required to keep it clean. After brining, the cheeses are often dried slightly at room temperature, firstly removing the excess of brine. It is preferable to do this by means of a suction mouth just above the surface of the cheese. At this stage, the rind is under stress because of the brining process, and forced drying by heated air must be avoided. Cheese, just after removal from the brine and after application of the first coating, exudes extra moisture that will remain between the cheese and the shelf. The cheese will stick to the shelf, which hampers the following treatments of the cheese and destroys the plastic coating. (Even by pressing a finger on such quickly dried cheese, some moisture will appear immediately.) When the cheese is free of visible liquid, the first coating layer is applied on the top and most of the sides. Then, the cheese is conveyed in the same position to the store and automatically fed on to shelves to dry quietly for 1 or 2 days. The plastic coating is applied two or three times during the first 2 weeks and, during longer curing, repeated with gradually diminishing frequency. Care is taken that the cheese surface is sufficiently dry before each treatment. The cheese is turned and a coating machine with turning flexible flaps spreads the plastic dispersion evenly over the cheese. This treatment is combined with cleaning the upper surface of the shelf, and the cheeses are positioned on their dry and still clean other side. The drying of the cheese surface (and the shelves) is controlled by the climatic conditions of the curing room and, consequently, the decrease of the water content of the cheese or its weight loss. Treatments in curing rooms also have been highly mechanised: transport, coating and turning of cheese, cleaning of shelves, etc. Much progress has been made in controlling the temperature, relative humidity and velocity of air, in order to approximate the ideal situation in which each loaf is stored under

116 Gouda and Related Cheeses

identical conditions. Good insulation of the curing rooms is very important to prevent the condensation of water on the walls at the desired high humidity of the air, resulting in mould growth. Wooden shelves are normally used because they have the advantage of absorbing some moisture from the (young) cheese. However, they require special attention from a hygienic point of view. A strict maintenance programme of cleaning and drying combined with the treatment of the cheeses may guarantee sufficient safety. New wooden shelves need more attention because considerable quantities of fermentable sugars are still present in the wood. Thorough leaching in water, cleaning, disinfection and drying are necessary. Also, suitable glues must be used for the construction of these shelves (see ‘Natural ripening’). Smaller cheeses, like baby types and Edam, are sometimes ripened hanging in coarse plastic nets or placed in perforated holes of a stainless steel plate where they can dry on all sides. The relative humidity of the air should be slightly higher to prevent excessive evaporation. Because such cheeses are coated on all sides, it is difficult to avoid the pattern of the net being imprinted on the surface. However, these cheeses are usually waxed before distribution. Just before they are put on the market, cheeses may be treated with paraffin (cheese wax), generally after they have been treated with latex. Before waxing, the loaves must have had, during the whole ripening procedure, a clean, dry surface, since increased pH by previous bacterial growth on the cheese and a high humidity between the cheese and the wax layer favours bacterial growth, causing off-flavours and gas formation. In such cases, washing and drying of the cheese before waxing does not prevent these defects. Consequently, wax is applied predominantly to a matured cheese, mainly to prevent weight loss during transport. In some cases, especially for Baby Gouda or Baby Edam, often red wax is applied when the cheese has dried briefly after brining (so-called peel-off wax). Wax layers must be closed and cracks and pinholes must be avoided. Such cheese is stored under sufficiently cool conditions to maintain its shape in the cartons on pallets, consequently avoiding cracks due to bulging. Some cheeses are made in rectangular or square loaves for curing while wrapped in plastic foil. Treatment of the cheese, as mentioned above, is unnecessary. This type of cheese is particularly suitable for the processed cheese industry. However, it is also of increasing interest for customers who sell this type of cheese in prepacked portions or slices. Prolonged curing at the usual temperature, e.g., 14 °C, however, tends to produce cheese of poor flavour and consistency. Therefore, the cheese is kept at a low temperature (8 °C); the resulting flavour is rather flat (see

also ‘Foil-ripening’). A starter with low CO2-producing capacity is used to prevent loosening of the wrapping and too open a texture.

Important Topics in the Manufacture of Gouda Cheese Milk standardisation and cheese yield

Cheese yield is usually defined as the mass of cheese obtained from a certain quantity of milk. When making cheese, individual milk components, including water, are converted to varying extents into the final cheese. During the process, salt is added. The conversion factors of the milk components are related to the process carried out and may differ slightly among cheese factories. The final cheese must have a certain composition because of legal and quality requirements. This primarily concerns water content and FDM but the actual parameter is the ratio of protein (para-casein) to water in the cheese. During the process, the conversion of protein from milk to cheese is of primary importance. This is why it has been advocated to calculate yield/kg para-casein in the milk (van den Berg et al., 1996a). This approach has also a close relation to the common practice of filling a cheese vat with such an amount of milk that a constant amount of cheese is obtained and that down-stream equipment is in full use. The cheese milk composition should be standardised to obtain the desired cheese composition. To this end, the fat content is adjusted in a ratio to the protein content, as determined by infrared analysis at the plant. However, this protein content serves as a parameter for the para-casein content that is converted to cheese and may be obtained by the difference in protein content between milk and (undiluted) whey. It is important to calculate precisely the desired fat content of the cheese milk. The yield of cheese should also be predictable. Comparison with the ultimate analytical results may enhance understanding of the cheesemaking process. Usually, all cheese made from one vat of milk is weighed before brining, and again later. If this is always performed in much the same way it may be a valuable help, since it gives a first indication of cheese composition when milk composition and conversion factors of the main components are known. Standardisation

Under practical cheesemaking conditions, establishing the correct fat content of the milk causes specific problems. Firstly, the cheese mass is always inhomogeneous, causing difficulties in determining its real FDM. For this reason, borer samples may give considerable bias and therefore the whole loaf, a sector from it, or a quarter from a square-shaped cheese, is ground.

Gouda and Related Cheeses 117

Secondly, one has to take into account that, generally, the fat content of different loaves from one batch is not identical, the standard deviation often amounting to about 0.5% FDM. Moreover, FDM decreases slightly during ripening, since proteolysis involves ‘conversion’ of some water into dry matter. For these reasons, a safety margin is taken into account, i.e., the initial fat content is adjusted to a somewhat higher level than is required. The plus sign in notations on Dutch cheese, like 40 or 60, refers to this margin. However, the ratio of the price of fat to that of protein is a factor that affects this margin in practice. In the Dutch regulations, the FDM must be within a margin of 4% on top of the indicated value, i.e., Edam cheese has an FDM of 40–44%, Gouda 48–52%, etc. It will be obvious that when fat is cheaper than protein, one leans to a higher level and vice versa. Such adjustments will affect the ratio of water to protein in the cheese because, according to Dutch regulations, the water content of the cheese should be constant; pH is affected as well (see ‘Control of pH’). Difficulties in standardisation are also caused by the multiplicity of variables affecting the ratio of fat to dry matter in the finished product: (1) The composition of the milk changes with season and shows short-term fluctuations. Moreover, changes may occur during prolonged cold storage. The fat content of the whole milk is not a reliable basis for standardisation because the fat/protein ratio is not constant. A much greater certainty is obtained if the protein content of the milk is estimated, or still better, its para-casein content, as noted above. Almost fixed proportions of the fat and the calcium para-casein–calcium phosphate complex are carried over from the milk into the cheese. So a good ratio between fat and protein can be found for standardisation purposes, as calculated by Lolkema (1991). (2) The method of making the cheese. Important aspects are: (a) Pasteurisation of the milk – denatured serum proteins are incorporated into the curd, increasing its solids-non-fat (SnF) content. (b) Cutting of the coagulum, which affects fat losses into the whey and the amount of curd fines. (The latter fraction has a lower fat content than the curd itself.) (c) The quantity of wash water used, which affects the SnF in the moisture of the cheese. (d) The amount of acid produced in the curd, and thereby the loss of calcium phosphate into the whey. (e) The quantity of salt absorbed by the cheese.

(3) Maturation of the cheese; the quantity of fat hardly changes but the SnF does, since water is ‘converted into dry matter’ during the hydrolytic processes. To standardise the cheese milk, the ratio between its fat content (v) and (crude) protein content (p) may simply be used as a basis. Suppose that F is the fraction of the fat that is transferred from the milk to the cheese, and that K kg fat-free dry cheese, including added salt, originated from 1 kg of milk protein, then Fv/Kp represents the ratio of fat to SnF in the cheese. As far as the making of Dutch-type cheese is concerned, both F and K approximate 0.9. Hence, the ratio of fat to protein in the milk may be adjusted to the ratio that is desired between fat and SnF in the cheese. In the Dutch cheesemaking industry, more detailed formulae (Posthumus et al., 1967) are in use, e.g.: v  rp  q

(1)

Under normal conditions of Gouda and Edam cheesemaking, r depends primarily on the desired FDM content of the cheese. For 40% FDM, r  0.67, for 48% FDM, r  0.91. The last factor, q, refers to the fat lost in the first and the second wheys. The loss increases more than proportionally with v. For cheese with 20% FDM, q  0.05, for 40% FDM, q  0.14, for 48% FDM, q  0.20, for 60% FDM, q  0.40. Some cheesemakers use more elaborate calculations to determine v. On the other hand, the conversion of SnF (of which para-casein is the main component) into cheese is important. For standard Dutch Gouda cheese, the average protein content of the NaCl-free SnF of the cheese was found by van den Berg et al. (1996a) to be 86.2%. In fact, this content is crucial for standardisation of cheese milk and was used indirectly by Lolkema (1991) for his standardisation system. Yield

The yield of cheese is often defined as kg of product (fat  protein  other solids  water) obtained from 100 kg of cheese milk. Most factors that affect the ratio of fat to dry matter also influence cheese yield. An important variable is undoubtedly the water content of the cheese and, hence, the loss of whey (syneresis) during curdmaking and pressing. Many factors affect the water content (see ‘Control of pH and water content’). Its standard deviation between loaves of one batch often amounts to 0.5–1% (Straatsma et al., 1984). It may be calculated that yield increases by nearly 0.2 kg if the average water content increases by 1%, i.e., from 41 to 42%. If we consider the water content as fixed, milk

118 Gouda and Related Cheeses

composition and conversion of various components can be affected in various ways, determining yield. Milk protein content and composition are of primary importance because the conversion of many other SnF components is more or less related to para-casein. There are different factors that may affect protein composition, in particular para-casein content of the milk (Walstra, 2000). Natural variations in milk composition and processing affect the amount of protein converted to cheese but there is also the risk of premature proteolysis resulting in the loss of peptides in the whey. In practice, modern infrared techniques are used successfully for milk analysis in the cheese plant. Season. Under Dutch conditions, yield is relatively high in autumn and low in spring, the discrepancy amounting to over 10%. This is due to the variation of the protein content, more precisely the para-casein content, because the ratio of casein/total (or true) protein is also not constant. The variation in non-protein-nitrogen (NPN) content greatly contributes to this phenomenon. The different types of milk protein and their analysis, para-casein in particular, in relation to cheesemaking, are discussed by van den Berg et al. (1996a). However, other SnF components that are converted into cheese (calcium, phosphate, citrate) also do not have a constant level in milk; even their ratio to casein is variable. Genetic variants of milk proteins. These may affect

cheese yield (van den Berg et al., 1992; Jakob and Puhan, 1995). The presence of the B variant of -lactoglobulin correlates with a higher ratio of casein to total protein (TP) than the A variant. The B variant of -casein is correlated with a higher (-)casein content and better coagulation properties than the A variant. This results in a somewhat better protein recovery in spite of the loss of more caseinomacropeptide. Bulk milk does not have the ideal composition as regards these genetic variants, but improvement will cost much time and much logistic effort. Mastitis. Severe mastitis leads to the production of

milk with a reduced casein content and a reduced casein/total N ratio (Barbano, 2000). Actually, since large quantities of bulk milk are used, real problems are seldom encountered but control of somatic cell count (SCC) is still important with respect to cheese yield. Cold storage of the milk. The indigeneous milk

enzyme, plasmin, is continuously active, even at low storage temperature. It slowly decomposes -casein into -caseins and proteose-peptones (PP) and also s2-casein is hydrolysed; some of the PPs are lost into the whey. van den Berg et al. (1998) found in good quality milk (low SCC) during 3 days storage at 4 °C an increase of TP in whey by 0.018%. This increase

holds for raw as well as for thermised milk and the rate of proteolysis is constant with time. Such loss may increase when SCCs or the psychrotrophic counts are considerably increased. Pasteurisation of the milk. Increased intensity of

pasteurisation will increase yield (see ‘Pasteurisation’). Rennet type. Proteolysis, other than the hydrolysis of

-casein, during cheesemaking will reduce yield, but the loss is usually negligible, unless some older types of microbial rennets are used. Starter. A change in the amount of starter added introduces several other changes. Firstly, the incorporation into the curd of denatured serum proteins will increase with the quantity of starter. Banks and Muir (1985) reported such an effect for Cheddar cheese and such could be confirmed by the authors with Gouda cheese. The casein from the acid starter liquid behaves during renneting like normal milk casein but a very small amount of casein is decomposed during propagation of the starter bacteria. This amount is, however, practically negligible in comparison with the amount of denatured whey proteins. So, the higher yield is caused by the increased the retention of serum proteins from the starter, since it is prepared from severely-heated milk. Secondly, when more starter is used to increase the acidification it inevitably requires, in Gouda-type cheese manufacture, more curd wash water (see ‘Control of pH and water control’), which increasingly dilutes the whey and hence reduces the yield. The net result of both factors may be almost nil. Increasing the rate of acidification, by more active starter in particular, reduces the pH of milk and curd, dissolving more calcium and inorganic phosphate. Probably, the subsequent loss in y is small, say 30 g for 10 kg cheese produced if the pH at the separation of curds from whey is as low as 6.25 instead of 6.5. Moreover, somewhat more PP will leave the micelles at a lower pH and will be lost in the whey. A lower yield (a higher protein content in the whey) might be expected but the findings of van den Berg and de Vries (1975) do not point to such an effect in normal cheesemaking. At an extremely high acidification rate, a slight increase in the protein content of the whey during pressing was observed. Bactofugation. The severe heat treatment of the sludge

that is added back to the milk (see ‘Bactofugation’) means an increase in yield because of the denatured whey proteins. Practically all whey proteins are denatured, just as is the case with the starter milk. For the amounts used in practice, it does not harm cheese quality. By increasing the pasteurisation temperature of the cheese milk by only 4–8 °C, a similar increase in yield may be obtained. However, in that case the more

Gouda and Related Cheeses 119

heat-sensitive whey proteins are primarily involved and, as mentioned in ‘Pasteurisation’, they may even cause deterioration of cheese flavour (van den Berg et al., 1996b). increase of colloidal calcium phosphate in the micelles (Walstra and Jenness, 1984). When 1 mmol/l CaCl2 is added to enhance the clotting, it will presumably increase cheese yield by ⬃30 g/100 kg of milk. Inclusion of native whey proteins. An appreciable increase in cheese yield may be obtained by accumulation of serum proteins in the curd when ultrafiltration and renneting of the milk is followed by no, or very short, curdmaking and quick pressing (Buijsse, 1999). However, this technique is hardly practised in Gouda cheesemaking. In standard Gouda cheese, no more than approximately 1% of the protein in the cheese consists of native whey proteins (de Koning et al., 1981). In view of the results of van Boekel and Walstra (1989), on steric exclusion of serum proteins with respect to (para)-casein micelles, it is likely that Goudatype (and many other types of) cheese produced in the usual manner (no ultrafiltration) contains insignificant quantities of native serum proteins. Curd washing. The dilution of whey with water at

scalding affects y. Increasing the quantity of added water, e.g., from 30 to 40% (expressed per mass of curd and whey after the first whey has been sucked off) reduces cheese yield by approximately 0.5%. The effect is illustrated in Fig. 6. In the calculation of cheese yield, the efficiency of reducing the concentration in the curd particles by the washing was taken to be 90% for lactose and other low molecular weight substances, and 50% for the serum proteins. Lolkema (1991) found a similar relation between wash water and cheese yield at a higher yield level. Salting. Absorption of NaCl obviously causes a gain in weight of the cheese. Against this profit there is usually a greater loss of moisture (see ‘Brining’), and hence a net loss of weight. This moisture also contains dissolved components from the cheese. The quantity of salt absorbed varies, e.g., from 1 to 3%, and the net weight loss may vary from, say, 0.02–0.06 kg/kg cheese produced. The loss of solids-not-salt ranges from 1 to 3 g/kg; this may include losses caused by mechanical damage during salting (such as slight amounts of curd pressed between the cover and the mould). Mechanical losses. During curdmaking and handling there are risks of mechanical damage to the curd that causes losses of fat and fines, as discussed in ‘Curd-

10.5 kg cheese /100 kg milk

CaCl2 . Addition of CaCl2 to the milk causes some

10.6

10.4

10.3

10.2

10.1 0

20

40

60

80

Water added to curds + whey (kg/100 kg) Figure 6 Yield of Gouda cheese (12 day-old, 41% water) as a function of the quantity of ‘curd wash’ water used. Water content and pH of the cheese are assumed to remain constant (recalculated from Posthumus et al., 1963; from Cheese: Chemistry, Physics and Microbiology, 2nd edn, Fox, P.F., ed., Volume 2, Chapter 2, Chapman & Hall, London, with kind permission from Kluwer Academic Publishers).

making’. Moreover, during cutting of the drained and fused curd mass to be moulded, pieces of curd may be lost, but with modern draining and moulding machines such losses are normally rather limited. Strictly speaking, yield refers to the ultimate product, excluding curd remnants, fines and any rind trimmings which have to be discarded; y includes the (dried) latex coating. Clearly, yield cannot be predicted very precisely, the cheesemaking process being too complex. Even the random variation in water content makes exact prediction difficult. To predict the yield of cheese from a given vat of standardised milk one obviously will proceed on its protein content. When taking into account a standard process and the usual conversion factors, the weight of the cheese before brining already gives an indication of the water content at an early stage. Calculations of the conversion of many milk components into the final cheese are given by van den Berg et al. (1996a), including various practical complications of analytical methods and methods to set up a moisture control system. Control of pH and water content

Very few quantitative data have been published on this subject. Process control has made considerable progress during recent decades because most variables have been identified and can be controlled (Straatsma and Heijnekamp, 1988). Besides the control of the curdmaking

process, a very important factor is the degree of acidification of the curd and the freshly made cheese. The control of starter activity is a key factor and will be discussed separately in ‘Starters: composition and handling’. However, certain minimum variations will still exist because of practical analytical uncertainties and imperfections. For example, the use of a continuous draining and moulding machine makes the demarcation of batches somewhat arbitrary. When weighing batches of cheese to get an idea of the expected water content of the final cheese, one will only decide about adjustment of process parameters after a number of batches have passed into the brine. It is not easy to adjust the water content and pH of cheese independently of each other. In ‘Interrelations’, interrelationships under varying conditions are considered. On the other hand, nowadays modelling of such a complex process is desired in order to be able to adjust the process immediately, but reliably. This has been done for Gouda cheesemaking and maturation by de Jong et al. (2002). In addition, the need to have an in-line measuring method for the moisture content of the individual cheeses before brining still exists and the recent approach of this problem by Frankhuizen et al. (2002) is interesting. Control of water content

The basic information with respect to this subject is outlined in ‘The Syneresis of Rennet-coagulated Curd’, Volume l. In fact, we have to deal here with MNFS, rather than with the absolute water content of the cheese, which, within one cheese type, decreases fairly proportionally with increasing fat content. Besides FDM, MNFS is characteristic of the type of cheese and affects textural properties. As a matter of fact, numerous factors affect the water content, as discussed in ‘Cheesemaking’. How they turn out under practical conditions was investigated by Straatsma and Heijnekamp (1988). The effects of some process parameters are illustrated in Fig. 7. Under normal manufacturing conditions, however, the number of process parameters available in practice that can actually adjust MNFS turns out to be restricted. Important are: (a) Cutting of the coagulum. The smaller the grains, the higher the syneresis rate, causing a lower water content. Cutting the coagulum very finely, however, seems to increase MNFS, and it causes a greater loss of fines and fat into the whey. An inhomogeneous cheese mass may result if the initial size of the grains differs widely. (b) Stirring of the curds–whey mixture. This concerns the intensity of stirring, which increases with the stirring rate and on any removal of part of the whey, the

Change in water content (% units)

120 Gouda and Related Cheeses

+2 0 –2 60

70

80

Past. temp. (°C)

5.5

6.0

6.5 33

pH after 4 h

35

37

Scalding temp. (°C)

+1 0 –1 40

60

80

Time (min)

5

6

0

2

4

Grain size (mm) CaCl2 (mmolar)

Figure 7 The effect of some variables in the treatment of milk and in curdmaking 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; from Cheese: Chemistry, Physics and Microbiology, 2nd edn, Fox, P.F., ed., Volume 1, Chapter 5, Chapman & Hall, London, with kind permission from Kluwer Academic Publishers).

duration of stirring and (scalding) temperature which is, in practice, the first factor used to adjust water content. Extended stirring causes a lower ultimate water content. If the temperature of the curd mass is kept constant after separation of the whey, then the time of separation should affect the final water content only slightly. This indicates that in practical cheesemaking, lowering of the temperature after whey drainage rapidly restrains syneresis. (c) The above process steps also play a part in the acidification rate, since the temperature affects the activity of the starter bacteria, and since stirring can be stopped earlier at a higher syneresis rate, hence at a higher pH. Other variables occurring at renneting, pressing, shaping and salting have effects also but they are less suitable process parameters through which to adjust the water content from one batch to another. However, it is necessary for optimising the process that all effects of different steps, in particular when starting a new production line, are analysed. In this respect, it is important to study the variability of the water content within the cheese, between the cheeses of one batch, between batches and between production days. This gives insight into possible systematic deviations caused in different stages of the process, so that related process parameters can be found that must be adjusted (Straatsma et al., 1984). To these measures also belongs the cooling programme in the buffer tank to slow down further syneresis (see ‘Draining and moulding’). This provides a means to control moisture content during

Gouda and Related Cheeses 121

final drainage of the curd and filling the successive moulds of the batch by inhibiting syneresis until closing the rind during pressing. This should be done in relation to batch time, and time between the mould filling and the start of pressing (see ‘Pressing’). Differences in water content, size and shape of the cheese types made on the same line have to be considered also because of differences in moisture loss before brining (Geurts, 1978). In this way, the most profitable cheesemaking process, with low losses, a good yield and a good quality cheese of the right composition, can be found. If the process has been established in this way, then the cheesemaker will adjust the water content primarily with small variations in the scalding temperature. To give an idea of the increase in the dry matter content (DM) at different stages of a modern process for normal Gouda cheese, the following values may be mentioned: milk, 12.8%; end of curd preparation, 32%; at mould filling, 42%; after pressing, 53%; after brining, 57%. Control of the pH

Here, we deal with the pH at one or a few days after making the cheese; after this, the pH gradually but slowly increases as a result of citrate fermentation, loss of CO2 and proteolysis. The course of pH and lactose content in the core of normal Gouda cheese is illustrated in Fig. 4. During brining, the pH of the outer rind zone will decrease further because of the low pH of the brine. During ripening under well-controlled conditions, the pH of the outer rind soon increases to a level slightly higher than in the core. Any (undesired) visible microbial growth on the surface will increase the rind pH considerably. The lactose in the rind after brining (see ‘Brining’) will disappear within a few weeks. The pH of the cheese results mainly from the amount of lactic acid, on the one hand, and that of the buffering compounds on the other. The acid is produced by the starter bacteria metabolising the available lactose. The main buffering substance in curd and cheese is the calcium para-casein–calcium phosphate complex, of which calcium phosphate contributes to roughly one-third of the buffering capacity. Lactic acid itself is a buffer in cheese at low pH (pK  3.9). The other salts in the curd moisture presumably play a minor part; after about 2 weeks, citrate to a large extent has been fermented by starter organisms (DL-type). During cheesemaking, acidification should be under control. If Gouda cheese is brined shortly after pressing, some lactose is usually left in the outer rind portion, but it disappears under normal ripening conditions within a few weeks. In the core of Gouda cheese, all lactose is metabolised within 12 h, mainly to lactic acid. If we presume that the water content of the MNFS is adjusted to its desired value, important compositional characteristics of the milk in relation to the final pH are:

(a) The lactose content of the milk serum (rather than the lactose to casein ratio in the milk). (b) The quantity (and composition) of calcium phosphate in the casein micelles; a changed buffering capacity of the curd is due predominantly to a different calcium phosphate concentration. As soon as the cheese milk has been collected and bulked, these variables (a and b) are fixed. To make the desired type of cheese from this milk, the important process parameters involved are: (a) Factors affecting the water content of the cheese. The higher the water content, the more lactose, or its equivalent as lactic acid, that is present in the cheese, and the lower the pH will be. In other words, from the moment the cheese loaf is formed, the ratio between incorporated lactose and buffering substances controls the pH. It has been observed that, other things being equal, increasing the water content of Gouda cheese by 1% decreases the pH by 0.1 unit. (b) The decrease in pH during curdmaking, and the ensuing loss of calcium and phosphate into the whey, may play a role too. These phenomena depend on the buffering capacity of the curd, on the amount of added calcium chloride (which reduces the pH slightly), and on the degree of acid production, which is, in turn, affected by the amount and type of starter added, the temperature, any pre-acidification, infection with bacteriophage and the presence of inhibitory components – antibiotics and disinfectants, agglutinins (active in milk but not in curd and cheese) and the peroxidase–H2O2–thiocyanate system. These factors should be under control. Stronger acidification will reduce the pH by only ⬃0.2 units at moulding; this causes dissolution of little calcium phosphate and hence it would have only a minor effect on the final pH, but it may affect texture. On the other hand, a decrease in pH increases the syneresis rate, which affects the water content and hence the pH (via point (a)). If the water content is kept constant by other means, a small effect still remains, since now a slightly smaller quantity of the buffering calcium phosphate is incorporated into the curd, causing a lower final pH and a lower cheese yield (see ‘Yield’). So a well-controlled process is necessary to maintain a constant buffering capacity in the cheese. (c) If this is under control, then the best process parameter to adjust the pH is washing. After the addition of water to the curds–whey mixture, lactose diffuses from the grains into the whey to equalise the lactose concentrations inside and outside the particles, although equilibrium is rarely reached. When the size distribution of the particles is normal and the contact time with the wash water is 25 min, the efficiency of reducing the lactose concentration in

122 Gouda and Related Cheeses

the curd is at least 90% (van den Berg and de Vries, 1974). More water causes a lower yield (see ‘Yield’), as well as a less-valuable whey. Traditionally, curd washing is a one-stage process but recently it has been proposed to reduce the total amount of washing water by a two-stage process. After removal of the first whey, a small amount of water is added and ⬃20 min later some whey is removed, followed by the addition of another small amount of water. This might reduce water usage by 10–20%, while still giving the same washing effect (Verschueren et al., 2002).

Water to curd + whey (kg/100 kg)

Figure 8 illustrates the quantities of water to be added under conditions related to normal Gouda cheese manufacture. The apparent lactose content in the fat-free dry matter represents the ratio between lac-

35 A 484

30

495 25

506

20 4.3

4.4

4.5

4.6

4.7

Lactose in milk (%) 40 Water to curd + whey (kg/100 kg)

B 30 460 20 440 10

tose and buffering substances (see point (a)). The adjective ‘apparent’ means the lactose still present plus the lactose already fermented into lactic acid before salting; these values are combined in order to be able to make calculations. Note that increasing the water content of the cheese before salting from 45 to 47% increases the amount of water to be added by 15%. A 0.2% higher lactose content of the milk necessitates 5% more added water. Interrelations

If a cheese with a high water content is desired, e.g., 47% after salting, and a normal pH of 5.15, then in addition to gentle cutting and stirring, much water at a relatively low temperature must be added. To obtain a normal content and a high pH, whey drainage and water addition may be repeated. If the water content is to be normal and the pH low (4.9–5.0), addition of water should be omitted. Then, scalding can preferably be achieved by means of hot water in the vat jacket, or by adding heated whey (which formerly was a common practice in Edam cheese manufacture). If an extremely low pH is desired, giving a typical short consistency, the milk may be preacidified. When making a very brittle type of Edam cheese, even the addition of lactose to the milk has been practised. To obtain a low water content and a normal pH, the coagulum should be cut rather finely and, after removal of part of the whey, it should be stirred vigorously at a rather high scalding temperature. Heating should, again, be indirect and slow to prevent the formation of curd particles with a ‘skin’. It will be obvious that such a process approaches that for hard cheese and it explains why in Emmental and Parmesan cheesemaking, the curd is never washed. Normally, the water content of the cheese is considered but concerning the control of pH, MNFS is primarily involved. Sometimes, FDM may be changed (see ‘Standarisation’), which means that MNFS is changed also. It has been calculated for Gouda cheese that increasing FDM by 2% at a constant water content requires an increase of washing water by 7%. Starters: composition and handling

0 42

44

46

Water in cheese (%) Figure 8 Amount of curd wash water to be used in relation to the lactose content of the milk (A) and the water content of the cheese before brining (B). Figures near the curves indicate (apparent) lactose (%) in the fat-free dry matter of the finished cheese (A) and lactose (%) in the milk (B). In A, the water content of the cheese is 46%; in B, the (apparent) lactose content in the fat-free dry cheese is 4.85% (from van den Berg and de Vries, 1976).

Industrial cheese starters are usually composed of acidforming lactococci, Lactococcus lactis subsp. lactis and Lc. lactis subsp. cremoris, possibly in combination with citrate-positive strains of Lc. lactis subsp. lactis and/or Leuconostoc spp. Cheese starters are categorised into O, L and D type of starters. The L refers to the presence of Leuconostoc spp. in the starter, the D to citrate-positive strains in the starter. DL-Starters contain both types of citrate-utilising organisms, while O-starters lack both

Gouda and Related Cheeses 123

types of organisms. Selection of the type of starter is strongly dependent on the suitable properties of the starter, such as flavour formation, phage resistance and eye formation. Detailed information on these organisms, for example on their taxonomy, physiology, biochemical characteristics, phages and phage resistance, and on the composition of starters and their propagation can be found in ‘Starter Cultures: General Aspects’, Volume 1. Specific information on Dutch starters for cheesemaking was published by Stadhouders (1974) and Stadhouders and Leenders (1984). Industrial cheese starters can be divided into two groups, undefined and defined starters. Artisanal starter cultures, derived from raw milk production practice, are traditional undefined mixtures of strains. These spontaneously developed starters are still used in traditional, small-scale factories, located in various parts of the world, e.g., in Southern Europe. Their composition is complex, relatively variable and often poorly defined. The artisanal starters are phage-carrying and partly phage-resistant. Therefore, these starters can be used in cheese farms/factories without any controlled protection against air-borne bacteriophages. They do not suffer complete failure of acid production when they become contaminated with disturbing phages, but the strain composition of the starter is greatly affected and the rate of acidification may vary considerably (Cogan, 1996; Limsowtin et al., 1996; Mäyrä-Makinen and Bigret, 1998). Modern large-scale cheese factories require the use of starters with consistent activity. Acid production in cheese must proceed fairly quickly and at a constant rate, the latter being essential for the control of syneresis and the water content of the cheese. Therefore, mixedstrain starters, originally derived from artisanal production practice, are propagated under controlled conditions. This ensures a more uniform bacterial composition of starters and controls their rate of acidification when they are propagated under complete protection from phage. In The Netherlands, the mixed-strain starters currently used were selected originally from artisanal practice, according to their taste and flavour formation properties, rate of acidification, capability to induce eye formation and phage resistance. They are kept as inoculated milk in a frozen condition. Sub-culturing is minimised, which preserves their functional properties, population and phage resistance. These so-called mother starters serve for the production of concentrates for bulk starter preparation. The concentrates are distributed to the cheese factories in a frozen state (Stadhouders and Leenders, 1984). The most common procedure for the manufacture of bulk starter is as follows. Bulk starter milk is pasteurised, e.g., for 30 min at 95 °C or 1 min at 110 °C.

The intensity of the heat treatment is aimed at the destruction of thermo-resistant phages in the milk. Specially designed bulk starter equipment offers an effective barrier against air-borne phages. Generally, the room above the milk in these tanks is provided with an over-pressure of phage-free air made up by passing a (high efficiency particulate air) HEPA filter. Moreover, a special device is mounted on top, enabling decontamination of the outer side of boxes of starter concentrate with hypochlorite solution before the starter is introduced into the tank (Stadhouders et al., 1976; Lankveld, 1984). Additional precautions should be taken to avoid accumulation of disturbing phages in the factory, which especially could affect the rate of acidification of the curd in the vat. These measures include: the manufacture of bulk starters in separate rooms, use of closed equipment, cheese vats in particular, frequent cleaning and disinfection of all installations. Cheese whey is a specially dangerous source of phage contamination, and its processing equipment, the self-desludging separators in particular, is also separated from the cheesemaking room. Starters are propagated for 18–24 h at 20 °C. In almost all modern factories, the starter is automatically metered and added to the cheese vat. Starters may be kept for a limited time (e.g., 24 h) below 5 °C without loss of activity. The activity of the bulk starter should be the same on successive days of manufacture. Activity is usually assessed by an IDF-standardised activity test (Stadhouders and Hassing, 1980) performed with a standard, pasteurised, reconstituted, high-quality skim-milk powder, and also with the pasteurised cheese milk, which ought to be skimmed. The activity of the starter in either of these milks should be constant. Any change in activity can be an indication of (i) a contamination of the starter with disturbing bacteriophages, (ii) a reduced activity of the starter (e.g., if it had been kept too long at a low temperature), (iii) the presence of antibiotics and/or disinfecting agents in the cheese milk or (iv) variations in the composition of the milk. To a certain extent, variations in activity may be corrected by adjusting the quantity of starter added to the cheese milk, or by adjusting other conditions during curdmaking, e.g., the scalding temperature. It must be remarked that results of the activity test and the acidification rate of cheese are not always precisely related because of different conditions in milk and fresh cheese, notably phage concentration, but this should be under control. According to practical standards for the Dutch cheese industry, the pH of cheese should be 5.8–5.9 after 4 h from the start of manufacture, and 5.4–5.5 after 5.5 h (Northolt and Stadhouders, 1985), as indicated in Fig. 4.

124 Gouda and Related Cheeses

Nowadays, most commercial suppliers market undefined mixed-strain starters for direct vat inoculation (DVI) as well. This requires a much higher concentration of the micro-organisms in the deep-frozen concentrate to obtain similar acidification rates compared with bulk starters. The technology of DVI eliminates unnecessary sub-culturing within the factory and reduces many difficulties associated with it (Sandine, 1996). Defined-strain starters are blends of two or more strains. They are frequently used nowadays instead of the former undefined mixed-strain starters. Since the risk of phage attack is greater here than with the use of undefined mixed-strain cultures, cultures with different phage-sensitivity profiles are used in rotation. Definedstrain starters are less common for Gouda-type cheese than, for example, for Cheddar cheese production (Pearce, 1969; Heap and Lawrence, 1976; Limsowtin et al., 1977; Heap, 1998). However, the use of single strains as an adjunct starter in combination with a DVI (undefined or defined) starter is increasingly popular in semi-hard cheeses, such as Gouda-type. The single-strain adjunct starters are highly flexible in generating various cheese features, such as eye size and cheese flavour.

the cheese is unequal, primarily concerning salt and water, as shown in Fig. 9. The more concentrated the brine and/or the greater the weight loss by evaporation in the curing room, the higher the water content before brining should be to obtain an equal water content after 2 weeks when cheese composition is officially checked in The Netherlands. This results in a higher water content in the core after brining and this will affect consistency and ripening processes (de Vries, 1978). The water content in the core of the cheese during the first weeks of ripening has practically already been established during pressing. As far as salt has diffused during brining, the water content has decreased by pseudo-osmosis and this reduction will continue during ripening. At the surface of the cheese, the salt concentration in the cheese moisture (Sw) is similar to that of the brine, and the stronger the brine, the lower the water content is in the outer rind zone. This is a totally different condition compared to cheese made from dry-salted curd, like Cheddar cheese. Further, the calcium and the Salt concentration in the cheese moisture (Sw) (%)

Water content (W) (%)

Maturation

48

18 W

Maturation is the result of numerous changes occurring in the cheese. The structure and composition and organoleptic properties of cheese alter greatly. Development of cheese properties is due particularly to the conversion of lactose, protein, fat and, in Gouda-type cheeses, of citric acid. Cheese technology and composition greatly affect cheese consistency and performance, both directly and indirectly, as discussed by Lawrence and Gilles (1986). Cheese composition during ripening

During ripening, enzymes such as residual chymosin or other clotting enzymes, residual plasmin and proteolytic enzymes from the starter bacteria are still active (see ‘Proteolysis in Cheese during Ripening’, Volume 1). Environmental factors in the cheese, e.g., available water (aw), salt concentration, lactate concentration, pH and lack of oxygen, as well as curing room conditions, are important for their activity. The degree of lysis of the starter bacteria, liberating their enzymes, is also affected by these conditions. In this respect, the composition of the cheese and the method of producing it are key factors for ripening, as discussed by van den Berg and Exterkate (1993). Composition after brining

After brining, when ripening has started, the cheese contains all components but the composition within

16

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12

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8

36

6

34

4

32

2 Sw

Sw 0

1

2

3

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6

Distance from surface (cm) Figure 9 Gradients (idealised) of water content (W) and salt concentration in cheese moisture (Sw) in a zone of 6 cm below the rind of Gouda cheese, just after brining, for a normal process (4 days) and for a longer brining time (7 days) at a lower water content and weaker brine (dotted lines), according to van den Berg et al. (1975).

Gouda and Related Cheeses 125

phosphate contents have also been established before brining, mainly by the acidification rate. Control of the pH has been discussed (see ‘Control of the pH’). During normal Gouda cheesemaking, ⬃7% of the chymosin added to the cheese milk is retained in the curd (Zoon et al., 1994). It is mainly adsorbed by the protein and not inactivated during processing because the scalding temperature is mild, in contrast to the high cooking temperature in hard cheesemaking. The amount of residual active chymosin will be increased by (for a–d see Stadhouders and Hup, 1975): (a) (b) (c) (d)

using more of this coagulant; lower pH at curd drainage; probably by low scalding temperature; probably by increased moisture content of the cheese (at least enhances activity); (e) more intensive milk pasteurisation (van den Berg et al., 1996b). Some plasmin is present in the fresh Gouda cheese but not as much as can be expected in hard cheese from high-cooked curd (Fox and Stepaniak, 1993). Another essential variable is the bacterial population in the fresh cheese as it greatly affects ripening and the possible development of defects (see ‘Possible microbial defects’). This concerns starter bacteria and contaminating organisms, respectively. Concerning the latter, strictly enforced hygienic measures must be taken to prevent the growth of, e.g., mesophilic lactobacilli (Stadhouders et al., 1983c). The viable count of lactic starter bacteria (LAB) in the standard process is practically constant, at 1  109/g cheese (Stadhouders, 1974). This number has been reached after 3–4 generations at normal inoculation; the pH is then 5.7, which is the case shortly after pressing. This count will soon start to decrease due to cell death, usually followed by lysis during ripening.

Cheddar cheese. So Gouda cheese is ready for consumption at 4 weeks after manufacture, when it has a mild flavour. However, salt diffusion into the core continues during further curing and in the meantime the water content decreases. The water content in the rind zone of 0.5 cm decreases to 30% within 3 months. It has been found that proteolysis has practically stopped in that area, as shown in Fig. 10. This drying and lack of proteolysis gives the rind of a mature cheese its tough and hard consistency and a horny appearance. It is clear that the lack of proteolysis in the cheese rind reduces the values for N fractions, e.g., SN, pH 4.6-SN, PTA-SN (or AN) of the usual representative cheese samples. In the outer rind zone, another phenomenon is worth mentioning, although all its consequences are not well understood. Inside the cheese, the redox potential (Eh) is approximately –140 mV, caused by lactic acid fermentation, which means that the core of the cheese is anaerobic. This implies that in the rind zone a certain redox potential gradient must be present because outside the cheese, normal oxygen pressure exists. This explains why slowly growing microaerophilic moulds can grow under the plastic coating of cheese contaminated by long-time contact with contaminated shelves (Stadhouders and Labots, 1962). Slight lipolytic activity from moulds and yeasts on the TN(%) 50

40

+

+

30 +

Natural ripening

During ripening, normal Gouda cheese loses water by evaporation. The decrease in the overall water content of the cheese during the first 10 days is ⬃1.5% but the rate decreases steadily with time. Within a year, the water content will have decreased by ⬃10%. The air humidity is the driving factor to maintain a water gradient in the cheese, and water diffusion within the cheese is the limiting factor in weight loss, except during the first 10 days, when increasing air velocity also enhances evaporation (Bouman, 1977). Certainly during the first month, ripening in the core of the cheese is practically not hindered by the increasing salt concentration or the decreasing water content. This and the higher curing temperature of at least 12 °C favours the ripening process in comparison with, for example,

20 +

10

+ +

0 0

2

4

6

8

10

12

Age of cheese (months) Figure 10 Course of proteolysis in the core (sector sample minus 15 mm rind) and in the rind (outer 5 mm) of Gouda cheese during 1 year maturation (van den Berg and de Vries, unpublished results); SN and AN as percentage of TN.  – SN (core);   AN (core); –䊐– – SN (rind).

126 Gouda and Related Cheeses

surface has been found in the rind zone of cheese (Stadhouders and Mulder, 1959). It may be expected that their growth, although practically invisible, cannot be fully prevented and the rind zone will acquire a slightly higher fat acidity (see also ‘Proteolysis and lipolysis’). Further investigations are desirable on whether more micro-organisms can grow under such conditions. Moreover, it would not be surprising if other chemical or enzymatic reactions in this zone produce relevant flavour compounds (see also ‘Foil-ripening’). It will be obvious that because of the direct contact between the cheese and the shelf, adequate hygienic measures must also be part of the treatment scheme for the shelves, but transfer of off-flavours of, e.g., chlorophenolic compounds into the cheese must be avoided also. In this respect, glues with phenolic components used for the construction of the shelves are dangerous, because disinfection by hypochlorite will easily result in the formation of these compounds. Foil-ripening

Nowadays, a considerable amount of Gouda or Edam cheese is packed after brining in a plastic foil with very low permeability for water and gases to avoid weight loss, mould growth and all the effort needed to keep the naturally ripening cheese in the curing room (see ‘Rind treatment and curing’). Such cheeses are made in a rectangular shape and piled in palletised cases to keep their shape. After ripening, they can easily be cut into consumer-size packages without losses. However, there are clear differences when foil-ripening cheese is compared with naturally ripened cheese (see ‘Yield’): (a) A constant average water content during curing. (b) Before brining, the water content is lower by 2–3%; otherwise the cheese will be too soft after the usual (rather short) ripening time. (c) The ripening temperature is lower, namely 5–6 °C. (d) There is no oxygen available at the surface of the cheese. (e) Brining time is longer because of the lower water content. (f) The cheeses are brined immediately after pressing, which means that the lactose content in the outer rind zone is still higher during the first weeks. At the start of brining, the pH is still so high that the final growth of the starter bacteria might be hindered by cooling in the brine. (g) Diffusion of salt and water after brining is still slow due to the lower curing temperature, the lower water content and the weaker water gradient in the cheese, because no water evaporation occurs. (h) Eventually, the physical rind zone disappears and is gradually subjected to normal proteolysis.

The majority of foil-ripened cheese is ripened for a short period of 1 month. After a longer time, when the proteolytic ripening parameters like the soluble nitrogen (SN) and the amino acid nitrogen fraction of total nitrogen (AN) are comparable with those of naturally ripened cheese, flavour formation is, however, generally poorer and consistency is softer and tends to be sticky. The differences in consistency may be caused to a large extent by the water content, at that time being higher than that of naturally ripened cheese. The influence of the absence of oxygen at the surface of the cheese is not well understood and warrants further investigation. An advantage seems to be that the lowripening temperature prevents the cheese from ‘lateblowing’. However, if, after ripening, the temperature is not controlled during transport and storage, this risk still exists. Fermentation of lactose and citric acid

The formation of lactic acid by the starter bacteria is paramount for the preservation of cheese. By their action they: (a) ferment lactose quickly and almost completely; consequently, the cheese soon lacks available carbohydrate. (b) produce lactic (and a little acetic) acid and reduce the pH of the cheese to 5.1–5.2. At the end of fermentation (after about 10 h), the lactic acid concentration in the cheese moisture is about 3%. Part (usually 4–7%) of the lactic acid is present in its undissociated (i.e., bacteriostatic) form, the more so if the pH is lower. (c) reduce the redox potential of the cheese to about 140 to 150 mV at approximately pH 5.2, as measured with a normal hydrogen electrode (Langeveld and Galesloot, 1971; Northolt and Stadhouders, 1985). All these changes aid in inhibiting the growth of undesired micro-organisms; salt uptake by the cheese, the presence of a protective cheese rind and the adequate treatment of this rind also contribute (see ‘Rind treatment and curing’). Microbial defects should always be prevented (see ‘Possible microbial defects’). Individual strains in a mixed-strain starter may differ greatly as to growth rate, the maximum number to which they grow in cheese and the rate at which they lose viability and subsequently lyse during cheese ripening. Cheese milk is commonly inoculated at a level of 5  106–107 starter bacteria/ml of milk. Mechanical inclusion in the curd leads to 5  107–108 cfu/g of curd, where the bacteria grow to, at most, ⬃109 cfu/g; this implies that starter bacteria generate (divide) only a few times in the fresh cheese. After growth, fermentation

Gouda and Related Cheeses 127

is far from complete (pH of cheese ⬃5.7), and during further conversion of lactose, growth and fermentation are uncoupled. Fermentation of citric acid is of particular importance to eye formation in Dutch-type cheese. The DLand L-starters used in the manufacture of Dutch-type varieties ferment citric acid, but DL-starters do so more rapidly and produce more CO2; they are, therefore, used if more extensive eye formation is desired. The rate of decrease of the citric acid content in the young cheese may be used as an indication of the capability to induce eye formation (Northolt and Stadhouders, 1985); the rate of citric acid fermentation is, however, not the only factor involved in eye formation (see ‘Texture’). Proteolysis and lipolysis Proteolysis

Protein breakdown in Gouda-type cheese is due mainly to the remaining action of coagulating enzymes, enzymes of starter bacteria and, to a much lesser extent, milk proteinases. Basic information about these proteolytic systems is given in ‘Proteolysis in Cheese during Ripening’, Volume 1. The separate and combined action of these systems in Gouda cheese has been studied intensively by making use of aseptic milking and cheesemaking techniques (Kleter and de Vries, 1974; Kleter, 1975, 1976, 1977; Visser and de Groot-Mostert, 1977; Visser, 1977a–d). Effectively, the action of calf rennet is determined predominantly by the amount remaining in the curd. Gouda-type cheese contains approximately 0.2 ml rennet (strength 150 IMCU)/kg of cheese (see ‘Composition after brining’). The action of the coagulant enzymes, predominantly chymosin, is characterised by the rapid degradation of s1-casein at the onset of maturation, about 70–80% being hydrolysed within 2 months in standard cheese. -Casein is degraded far more slowly, about 40–50% remaining even after 6 months (van den Berg and de Koning, 1990). When using more calf rennet and a lower scalding temperature, the degradation of s1-casein is even more rapid (Visser, 1977d). Rapid breakdown of s1-casein is particularly favoured by the pH of the cheese being near to the optimum (about 5) for rennet action, and a still low NaCl content in the cheese moisture of the core (see ‘Composition after brining’). -Casein degradation is slowed down considerably, even at this low NaCl content (Noomen, 1978b). Calf rennet appears to be responsible for the formation of most of the SN and the liberation of high and low molecular weight (MW) peptides, but only very low amounts of amino acids. After the primary proteolysis of the casein, the caseinderived peptides are hydrolysed to small peptides and

amino acids in the secondary proteolysis due to the action of the complex proteolytic system of the starter bacteria. The proteolytic system of dairy LAB has been studied extensively (Pritchard and Coolbear, 1993; Poolman et al., 1995; Christensen et al., 1999). The proteolytic system can be divided roughly into three main components with specific modes of action, as follows: (a) The first step in the degradation of the caseinderived peptides is catalysed by an extracellular proteinase, which is a member of the serine proteases of the subtilisin family. The proteinase is anchored to the cell membrane and is located extracellularly. The proteinase hydrolyses the casein-derived peptides to oligopeptides (Hugenholtz et al., 1984; Exterkate et al., 1993; Kok, 1993). (b) Subsequent degradation of the oligopeptides is catalysed by intracellularly localised peptidases. Therefore, peptide transport systems are crucial. Various transport systems with different specificities have been characterised. Amino acid transport systems, two ditripeptide carriers and an oligopeptide transport system have been characterised in dairy lactococcal cells (Kunji et al., 1995). All these transport systems are active, energy-driven processes. (c) The oligopeptides transported into the cells by the transport systems are subsequently hydrolysed by various peptidases into small peptides and amino acids. Approximately 13 different peptidases have been characterised with different specificities (Kok and de Vos, 1994). During cheese manufacture, the carbon source is depleted, which de-energises the microbial starter. Active transport of the oligopeptides into the cell is, for this reason, reduced. Starvation of the starter during cheese ripening and, subsequently, permeabilisation of the starter cells is essential for the ongoing amino acid production in cheese. Starters may vary greatly in their sensitivity to lysis (W.C. Meijer, F. Kingma, A. van Boven and J. Hugenholtz, unpublished results). Bacterial lysis is of great importance for the final cheese flavour, since amino acids are the main substrate for the final cheese flavour (Wilkinson et al., 1994; Crow et al., 1995; Morgan et al., 1995). When acting alone in cheese, milk proteinases may hydrolyse s1-, s2- and -caseins to some extent during prolonged ripening, due to which small amounts of low-MW peptides and amino acids are liberated (Visser, 1977c). The pH and the NaCl content sof mature Gouda-type varieties are not very favourable for the activity of most enzymes; in particular, plasmin activity is reduced greatly (Noomen, 1978a). In normal cheeses, where all enzyme systems act together, no clear mutual stimulation or inhibition of

128 Gouda and Related Cheeses

the systems in the formation of soluble N components is observed. The action of rennet clearly stimulates the starter bacteria to produce amino acids and low-MW peptides, which is most likely due to the progressive degradation by starter peptidases of the higher-MW products of rennet action. Contents of soluble N compounds (Visser, 1977c) reflect the ‘width’ of ripening. The ‘depth’ of ripening is defined as the ratio between the amount of degradation products of low MW, e.g., amino acids or peptides with MW 1400, and the total amount of soluble breakdown products. In that sense, the ‘width’ of ripening of Gouda-type cheese is predominantly determined by rennet action, and the ‘depth’ by the action of starter bacteria. Serum proteins seem to be hardly degraded in cheese; an exemption might be made for denatured, heat-sensitive whey proteins (see ‘Pasteurisation’). Many selections of starter bacteria have been made on the basis of proteolytic activity and/or lysis sensitivity. Use of these bacterial starters has demonstrated the huge impact of these parameters on flavour intensity and flavour diversification. However, shortening the ripening time of Gouda cheese with organoleptic characteristics exactly corresponding to those of the normal cheese is still a challenge. The application of specific, highly proteolytic, thermophilic starter bacteria has resulted in cheese which ripens more quickly and develops a typical, so-called thermophilic cheese flavour. An example of this is the Proosdij-type cheese, which has been successfully introduced in The Netherlands under various brand names such as Parrano®. In addition to mesophilic starter bacteria, a mixture of thermophilic lactobacilli and streptococci is used in its production, which otherwise follows the normal process for Gouda cheesemaking (van den Berg and Exterkate, 1993). These kind of adjunct starters are also used for low-fat cheeses (see ‘Origin and characteristics’). Lipolysis

In Gouda-type varieties, some lipolysis usually occurs and is even desirable, but it should be limited, otherwise the cheese has a soapy flavour. Factors that affect lipolysis have been studied intensively by Stadhouders and Mulder (1960) and Stadhouders and Veringa (1973). Cheese made from raw milk shows the distinct action of milk lipase; if made from aseptically drawn milk containing a negligible number of lipolytic bacteria, fat acidity increases gradually (Stadhouders and Mulder, 1957). The HTST-treatment of milk, e.g., 15 s at 72 °C, largely, but not completely, inactivates milk lipase. Cheese made from aseptically drawn, low-temperature pasteurised milk still shows an increase in fat acidity during maturation, although this increase is slight or even

scarcely noticeable (Kleter, 1976, 1977). One may question to what extent variations in the earlier results were caused by differences in susceptibility of the milk to lipolysis, e.g., due to mechanical damage of the fat globules. Under well-controlled conditions during the making and the curing of cheese made from pasteurised milk, lipolysis in cheese will result predominantly from the action of starter bacteria, residual milk lipase and, possibly, heat-stable lipases of psychrotrophic organisms. Such conditions, in particular, imply a good bacteriological quality of the milk prior to thermisation or pasteurisation (especially, psychrotrophs should be virtually absent), and the absence of visible microbial growth on the cheese surface during maturation. Enzymes of the starter bacteria have very low activity on triglycerides, but are able to produce free fatty acids (FFAs) from mono- and di-glycerides formed by milk lipase and/or other microbial lipases (Stadhouders and Veringa, 1973). The latter might originate from NSLAB. The activity of milk lipase is reduced by NaCl in the cheese. The action of milk lipase is also affected by the pH of cheese. Although this action has been found to decrease markedly with decreasing pH when assayed on substrates greatly different from cheese, the acidity of cheese fat has been reported to increase faster in cheese of low pH (Raadsveld and Mulder, 1949a). The explanation is unclear, though it should be noted that at a lower pH a higher proportion of any short-chain fatty acids present will be in the fat phase (Veringa et al., 1976). Lipase activity in cheese increases markedly with temperature (Raadsveld and Mulder, 1949b). In cheese made from milk containing high numbers of psychrotrophic bacteria (or their heat-stable lipases), lipolysis may be increased to undesirable levels. Also, growth of organisms on the cheese surface, e.g., moulds, coryneform bacteria and yeasts, may contribute to increased acidity of the fat. Growth of such organisms, however, is usually minimised but cannot be fully prevented; consequently, the rind portion of the cheese generally acquires a somewhat higher fat acidity. Homogenisation of the cheese milk greatly enhances lipolysis in cheese, but is seldom practised. The FFA of cheese milk normally amounts to, say, ⬃0.5 mmol/100 g of isolated fat. In a 6-week-old Gouda cheese made from HTST-pasteurised milk, this value averages about 1.3 mmol, which increases to about 1.6 after 6 months and to about 1.8 after 1 year. Cheese made from raw milk usually shows a higher FFA level. Flavour development

During the last decade, it became clear that flavour development in cheese results from a series of

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(bio)chemical processes for which the starter cultures provide the enzymes. The balance between proteolysis and peptidolysis is very important in order to prevent a bitter off-flavour and also results in the release of FAAs. In particular, the enzymes which catabolise these FAAs play a major role. Various enzymatic and chemical reactions have been identified in the conversion of amino acids to volatile flavour compounds, and a good balance between all the flavour components is essential for the desired sensory quality of Gouda or related cheeses. Apart from flavour components derived from the caseins, in Gouda-type cheeses some flavour components derived from carbohydrate metabolism should also be mentioned. The main conversion of lactose obviously leads to the formation of lactate by LAB, which affects the flavour, giving a mild fresh acid taste. However, a fraction of the intermediate pyruvate can alternatively be converted to various flavour compounds such as small amounts of diacetyl, acetoin, acetaldehyde or acetic acid, some of which contribute to typical yoghurt flavours. Moreover, citrate serves as the main source for diacetyl due to the presence of citrate-fermenting strains in the starters used for Gouda preparation. In young cheese, citrate-fermenting strains in the starters used for Gouda cheese manufacture. In young cheese, diacetyl flavour can be easily distinguished but after 3 months this flavour has disappeared to a great extent because diacetyl is converted to acetoin during further ripening. Nevertheless, the conversion of caseins is undoubtedly the most important biochemical pathway for flavour formation in Gouda and related cheeses (van Kranenburg et al., 2002). Degradation of caseins by the activities of rennet enzymes and the cell-envelope proteinase and peptidases from LAB yields small peptides and FAAs. The balance between the formation of peptides and their subsequent degradation to FAAs is very important, since the accumulation of peptides might lead to a bitter off-flavour in cheese (Visser et al., 1983; Stadhouders et al., 1983b; Smit et al., 1996, 1998). Various bitter-tasting peptides have been identified in Gouda cheese and especially these peptides should be degraded rapidly in order to prevent bitterness (Visser et al., 1983; Stadhouders et al., 1983b; Smit et al., 1998). Specific cultures have been selected with good ability to degrade bitter-tasting peptides (Smit et al., 1998) and such cultures are nowadays used frequently in the preparation of various varieties of Gouda cheese. For specific flavour development, further conversion of amino acids is required to produce various alcohols, aldehydes, acids, esters and sulphur compounds involved in the flavour perception of Gouda cheese. Amino acids can be converted in many different ways by enzymes such as deaminases, decarboxylases, transamin-

ases (aminotransferases) and lyases. Transamination of amino acids results in the formation of -keto acids that can be converted to aldehydes by decarboxylation and, subsequently, to alcohols or carboxylic acids by dehydrogenation. Many of these components are odouractive and contribute to the overall flavour of the cheese (Fig. 11). Using biochemical and genetic tools, the various flavour-forming routes from amino acids and the enzymes involved have recently been identified, mostly in Lc. lactis (Alting et al., 1995; Gao et al., 1997; Yvon et al., 1997, 1998, 2000; Engels et al., 2000; Smit et al., 2000; Yvon and Rijnen, 2001; van Kranenburg et al., 2002). Aromatic amino acids, branched-chain amino acids and methionine are the most relevant substrates for flavour development in Gouda cheese. Volatile sulphur compounds derived from methionine, such as methanethiol, dimethyl sulphide and dimethyl trisulphide, are regarded as essential components in Gouda cheese varieties (Urbach, 1995). In fact, a Gouda cheeselike flavour can be generated by incubation of methionine with cell-free extracts of Lc. lactis (Engels and Visser, 1996). Conversion of methionine can occur via an aminotransferase-initiated pathway by branched-chain or aromatic aminotransferases, or via an , - elimination of methionine by the lyase activities of cystathionine lyase (CBL), cystathionine -lyase (CGL) or methionine

-lyase (MGL) (Alting et al., 1995; Bruinenberg et al., 1997; Yvon et al., 1997; Gao and Steele, 1998; Rijnen et al., 1999; Engels et al., 2000; Fernández et al., 2000, 2002; van Kranenburg et al., 2002). It has already been mentioned that various LAB strains differ in their amino acid-converting abilities and that these activities are in fact linked to the ability to synthesise amino acids. Ayad et al. (1999, 2000) focused on the ability of Lactococcus strains isolated from various natural sources, the so-called ‘wild lactococci’. These strains originated from dairy and nondairy environments and had unique flavour-forming properties, when compared to commercially available starter strains. Using combinations of these strains makes it possible to develop tailor-made starter cultures for Gouda and related cheeses, a development with strong application possibilities. Texture Structure

Cheese consistency is discussed in ‘Rheology and Texture of Cheese’, Volume 1 of this book. For Gouda and Edam cheeses, a detailed study has been carried out by Luyten (1988). The main factors affecting the consistency in the core of these cheese varieties are moisture content, extent of proteolysis, pH, NaCl and fat level,

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any inhomogeneity of these variables throughout the cheese mass and, of course, temperature. Levels of calcium and phosphate are fairly constant under normal cheesemaking conditions but they are somewhat lower than in Emmental and higher than in other cheese types (e.g., Cheddar and Camembert). The influence of most of these parameters in Gouda cheese has been discussed by Visser (1991). During natural ripening, several changes occur that may be important to texture: (a) Structure and composition become more uniform, particularly during the early stages, due to further fusion of curd grains and reduction of salt and pH gradients. The moisture gradient persists for a long time. (b) The cheese looses water by evaporation and ongoing syneresis, especially near the rind. (c) Maturation primarily implies breakdown of the para-caseinate network, s1-casein quickly, followed by -casein more slowly (remaining after 6 months, ⬃20% and 40–50% respectively, as reported by Visser (1977b), van den Berg and de Koning (1990)); it also causes a slight increase in pH (formation of alkaline groups by proteolysis, degradation of lactic acid). (d) Gas is formed. The common result is that during maturation, the apparent elastic modulus of the cheese increases, the deformation at which fracture occurs decreases and the fracture stress at first decreases and subsequently increases again (Luyten, 1988; Zoon, 1993). This is shown in Fig. 12. During maturation, proteolysis dimin-

ishes the strain, and the stress increases mainly because of the lower water content and to a lesser extent by the increased salt concentration (salt/water). The modulus seems, for the same pH and NaCl content, to depend on the water content in the fat-free matter only. The only rheological parameter that appears to correlate well with the degree of maturation of the cheese is the deformation at fracture, as was, for instance, found in a study of several, widely different cheeses ranging in age from 4 to 20 weeks (H. Oortwijn, unpublished results). The relative deformation (Hencky strain) at fracture is, say, 1.6 for unsyneresed curd, higher than 1 shortly after salting and about 0.5 after 3 months of maturation (Luyten, 1988). The complete fusion of the curd within few days, the still low salt concentration and the rapid start of casein degradation in the core soon give the cheese a smooth texture, while its ‘longness’ disappears gradually with ripening and tan increases. These changing viscoelastic properties of curd and cheese are very important. During cheese formation, the curd deforms during pressing and fusion but, after a relatively short time, it remains deformed because casein bonds are broken and new ones are formed, within the curd particles as well as between caseins of neighbouring curd particles. After complete curd fusion, the cheese should still be able to flow slowly during the first month, which is a prerequisite for the creation of spherical holes. A cylindrical sample (obtained with a trier) of young Gouda cheese can be bent extensively before it breaks; during maturation of the cheese this flexibility is increasingly smaller. The rind zone of Gouda cheese shows a different development from the interior of the cheese. After

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Figure 12 Stress–strain curves obtained for Gouda cheese in a compression test at 2 (—), 6 (......), 13 (-----) and 26 () weeks (Zoon, 1993).

132 Gouda and Related Cheeses

brining, this zone is white (‘salted out’), short and brittle. Some weeks later, when the salt concentration declines below 7.5%, these properties have disappeared. In this zone, casein degradation is hindered by the high salt concentration and later even inhibited by the low moisture content. Then, the rind becomes gradually more tough and springy and is still distinguishable as a somewhat darker and translucent zone in the older cheese. This zone slowly increases in thickness; 3–4 mm after 1 year, depending on the degree of water evaporation (see also ‘Natural ripening’). This is not the case with foil-ripening (see ‘Foil-ripening’) because no water evaporates. In the end, the high water content and proteolysis throughout the whole cheese give a soft and sticky consistent body without a rind zone. Eye formation

In Gouda and related cheeses, the number, the size and the shape of holes are considered an important texture characteristic. Figure 13 shows a section of a typical Gouda cheese. Conditions allowing hole formation have been studied in some detail by Akkerman et al. (1989). Holes can be formed if gas pressure exceeds saturation and if sufficient nuclei are present. The gas is commonly N2 already present in the milk because this is saturated with air at 4 °C when received at the cheese plant. During cheese manufacture, any O2 present is consumed by the starter organisms and, during the next few weeks, CO2 is produced by starter organisms. This CO2, and possibly H2 produced by undesirable bacteria (see ‘Possible microbial defects’), can also con-

Figure 13 Section through a normal Gouda cheese of 12 kg.

tribute to the formation of the holes. The supersaturation needed for hole formation (by approximately 0.3 bar) can be achieved when the rate of CO2 production is relatively fast (which depends on temperature, type and number of bacteria and citrate content), its rate of diffusion (D  3  1010 m2 s1) out of the cheese is slow (mainly depending on loaf size and shape) and if the partial pressure of N2 is high (usually ⬃0.9 bar). Quantitative relations have been given by Akkerman et al. (1989). Nuclei are usually small air bubbles, either incorporated as such between the curd particles when the curd mass is ‘worked’ after draining off all whey, or already present in the milk and incorporated within the curd particles. The latter nuclei presumably exist as tiny air bubbles adhering to dirt particles and very small granules or partially coalesced fat globules; they can remain only if the milk is (almost) saturated with air. After normal pasteurisation of, usually, cold-stored milk, the time in the cheese vat before renneting allows sufficient deaeration by gentle stirring; otherwise, many pinholes will be formed in the cheese. Incomplete fusion of the curd, local inclusion of whey (curd lumps and disturbance of the curd block – see ‘Curdmaking’ and ‘Draining and moulding’) and inclusion of air at drainage may also serve as nuclei or may even disturb regular eye formation (‘nesty’ spots). Nucleation predominantly determines the number of holes, and their shape depends on cheese consistency, while both characteristics also depend on the rate of gas production. If the latter is not too fast and the cheese consistency allows for viscous flow of the cheese body, eyes (i.e., spherical holes) develop. If the consistency is

Gouda and Related Cheeses 133

short, or, more precisely, if the fracture stress of the material at slow deformation is low, slits may develop because the cheese mass fractures in the vicinity of the holes. Such may be the case for a cheese of low pH, low calcium phosphate content and considerable proteolysis at the time of gas production, but quantitative relations cannot be given yet. The problem is that most variables causing the fracture stress to be low (implying easy slit formation) also cause the elongational viscosity of the cheese to be low (implying a low overpressure in the hole and thus less possibility of fracture; see Akkerman et al., 1989). Possible microbial defects Butyric acid fermentation

Butyric acid fermentation (BAF) is characterised by the catabolism of lactic acid principally to butyric acid, CO2 and H2: 2CH3CHOHCOOH : CH3CH2CH2COOH  2CO2  2H2 Consequently, the growth of anaerobic, spore-forming, lactate-fermenting butyric acid bacteria (BAB), especially of Clostridium tyrobutyricum, may cause the ‘late blowing’ of cheese due to excessive production of CO2 and H2, and a very bad off-flavour. Grass silage, used as a feed in winter, but sometimes also as a part of the ration in summer, represents the main source of contamination of the milk with spores of BAB, especially when it is insufficiently preserved. Such silage contains large numbers of BAB spores, which survive passage through the digestive tract of the cow and are concentrated in dung. The degree of contamination of the milk with spores therefore strongly depends on hygienic conditions during milking (de Vries and Stadhouders, 1977), but even with modern methods of milking, slight contamination with faeces present on the udder cannot be prevented. This problem is more serious since the spores fully survive the HTST-treatment normally applied to cheese milk. The spores do not germinate in milk but in the cheese, where they can grow at 7 °C under anaerobic conditions with lactate as C-source. The redox potential will then be reduced from ⬃130 mV to 200 mV, which may be used as an indicator for BAF. Gouda cheese, as a brined cheese, is especially vulnerable to BAF, 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

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 microfiltration 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 NaCl 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 °C (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 BAF. 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 °C, 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 NaCl 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 BAF, 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 BAF. 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/l 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/l need 2.5 g nitrate/100 l milk and ⬃10 000 spores/l need 15 g nitrate/100 l milk. The latter number of spores may occur in the winter season. If then, only 2.5 g nitrate/100 l 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 combination with single bactofugation, this 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 °C. 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% NaCl. 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 a weak brine (e.g., 14% NaCl) is kept sufficiently acid (pH 4.6) and cold (13 °C), growth of the organisms usually does not occur and they die gradually. However, increased numbers in brine originate from their growth in deposits, which are often present on the walls of basins just above the brine level, on racks and other equipment, and so contaminate the brine. Growth conditions for the lactobacilli are more favourable in these deposits as a result of the action of salt-tolerant yeasts increasing the pH, a lower NaCl concentration (due to absorption of water) and a somewhat higher temperature than that of the brine. Measures to keep the number of lactobacilli low in brine include good hygiene in the brining room with removal of deposits, and adjustment of the NaCl content of the brine to at least 16% and of its pH to 4.5 (Stadhouders et al., 1985). Thermo-resistant streptococci

These bacteria are normally present in raw milk. In particular, strains of Sc. thermophilus may be responsible for cheese of inferior quality. In contrast to the mesophilic streptococci, they can grow at 45 °C and survive thermisation (e.g., 10 s at 66 °C) and, to some extent, pasteurisation (e.g., 15 s at 72 °C) of milk. During such heat treatments, after some time a few organisms may become attached to the walls of the

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regeneration section of the heat exchanger and may then start to multiply very rapidly (minimum generation time, 15 min); this may depend on their initial number in the milk. Continuous use of heat exchangers for too long a period without cleaning may cause heavy contamination of the cheese milk (about 106/ml). Cleaning of the thermiser and the pasteuriser within 8 h is normally necessary. As a result of their high number in curd and growth during the early stage of cheesemaking, their number may increase to more than 108/g of cheese. They render the flavour of cheese ‘unclean’ and ‘yeasty’. Moreover, CO2 production by these bacteria may yield cheese with an excessively open texture after approximately 5 weeks, especially if a starter with high CO2-producing capacity is used for cheesemaking (Hup et al., 1979; Bouman et al., 1982). Propionic acid bacteria

Very considerable growth of these organisms in cheese results in the development of a sweet taste and a very open texture, due to excessive gas formation. Propionic acid bacteria can convert lactate into propionic acid, acetic acid, CO2 and H2O according to: 3CH3CHOHCOOH : 2CH3CH2COOH  CH3COOH  CO2  H2O Consequently, the pH of the cheese does not change significantly. Because the bacteria develop very slowly in cheese at the commonly applied ripening temperature and salt content, any serious defects occur only after prolonged ripening. Several conditions determine their growth in cheese. The pH is decisive, significant growth starting only from 5.1 and increasing at higher values. Increasing the concentration of NaCl retards their growth. So higher salt concentrations near the rind of the cheese may be inhibitory. A higher storage temperature favours the growth of propionic acid bacteria. Nitrate hinders their growth. When conditions allow growth of these bacteria in cheese, the development of BAB (if present) may also be expected, provided that growth of the latter is not otherwise prevented. Propionic acid bacteria are killed by normal pasteurisation of milk, e.g., 15 s at 73 °C. Therefore, they are predominantly of interest in the manufacture of cheese made from raw milk, farm-made cheese in particular. However, in factories also making Maasdam cheese, cross-contamination must be prevented. Yeasts and coryneform bacteria

Abundant growth of yeasts and coryneform bacteria (and in extreme cases of B. linens) on the cheese surface may lead to a somewhat slimy rind and various

discolourations or pink appearance. Growth of these organisms is favoured by insufficient acidification of the cheese, leading to a significant lactose content in the rind, salting of cheese in brine with a low NaCl content and a high pH, inadequate drying of the cheese rind after brining (this is the main factor in practice) and the use of insufficiently cleaned shelves. Inadequate drying is the main factor in practice because when the other items are under control, it still may happen. Consequences for cheeses waxed after maturation have been discussed (see ‘Rind treatment and curing’). Growth of moulds causes discolouration and may under extreme conditions pose a health hazard because of mycotoxin formation. To prevent their development, special attention must be paid to the treatment of the cheese rind, the drying of the cheese surface and the hygienic conditions in curing rooms (see ‘Rind treatment and curing’).

Acknowledgements As this text is an adapted version of the chapter on Dutch-type varieties by Walstra, Noomen and Geurts in the previous edition, the authors are grateful for their kind permission.

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van den Bedem, J.W. and Leenheer, J. (1988). Heat treatment classification of low heat and extra low heat skimmilk powder by HPLC. Neth. Milk Dairy J. 43, 311–326. van den Berg, M.G. (1984). The Thermization of Milk. Bulletin 182, International Dairy Federation, Brussels. pp. 3–11. van den Berg, G. (2001). Semi-hard cheeses, in, Mechanisation and Automation in Dairy Technology, Tamime, A.Y. and Law, B.A. eds, Sheffield Academic Press Ltd., Sheffield. pp. 225–249. van den Berg, G. and de Koning, P.J. (1990). Gouda cheesemaking with purified calf chymosin and microbially produced chymosin. Neth. Milk Dairy J. 44, 189–205. van den Berg, G. and de Vries, E. (1974). Der Zusammenhang zwischen den Faktoren, die den pH van Käse beeinflussen (relation between factors influencing the pH of cheese). Milchwissenschaft 29, 214–218. van den Berg, G. and de Vries, E. (1975). Whey composition during the course of cheese manufacture, as affected by the amount of starter and curd washing water. Neth. Milk Dairy J. 29, 181–197. van den Berg, G. and de Vries, E. (1976). Het gebruik van wrongelwaswater (the use of curd washing water). Zuivelzicht 68, 878–879, 924–926. van den Berg, G. and Exterkate, F.A. (1993). Technological parameters involved in cheese ripening. Int. Dairy J. 3, 485–507. van den Berg, G., Stadhouders, J., Smale, E.J.W.L. and de Vries, E. (1975). Voor- en nadelen van “slappe” kaaspekel (merits and demerits of lower brine concentrations). Zuivelzicht 67, 984–988. van den Berg, G., Hup, G., Stadhouders, J. and de Vries, E. (1980). Application of the “Bactotherm” process (selfdesludging bactofuge, type MRPX 314 SGV, in combination with bactofugate sterilizer) in the manufacture of Gouda cheese. Technological effects on manufacture of Gouda cheese. NIZO-Report R112, Ede, The Netherlands. van den Berg, G., Daamen, C.B.G., de Vries, E., van Ginkel, W. and Stadhouders, J. (1988). Test of the bacteriaremoving separators, manufactured by Westfalia Separator AG, for the manufacture of Gouda cheese. NIZO-Report R127, Ede, The Netherlands. van den Berg, G., Escher, J.T.M., de Koning, P.J. and Bovenhuis, H. (1992). Genetic polymorphism of -casein and -lactoglobulin in relation to milk composition and processing properties. Neth. Milk Dairy J. 46, 145–168. van den Berg, M.G., van den Berg, G. and van Boekel, M.J.S. (1996a). Mass transfer processes involved in Gouda cheese manufacture, in relation to casein and yield. Neth. Milk Dairy J. 50, 501–540. van den Berg, G., Neeter, R., Allersma, D. and de Jong, C. (1996b). Ripening of Cheese Made from Milk with Different Heat Treatments. Bulletin 317, International Dairy Federation, Brussels. p. 40. van den Berg, G., Boer, F. and Allersma, D. (1998). Koel bewaren van melk van invloed op kaasopbrengst (consequences of coldstorage of milk for cheese yield). Voedingsmiddelentechnologie 31(4), 101–104. van Kranenburg, R., Kleerebezem, M., Van Hylckama Vlieg, J.E.T., Ursing, B.M., Boekhorst, J., Smit, B.A., Ayad, E.H.E., Smit, G. and Siezen, R.J. (2002). Flavour forma-

tion from amino acids: predictions from genome sequence analysis. Int. Dairy J. 12, 111–121. van Rijn, F.T.J., Hoekstra, E.S., van der Horst, M.I., Samson, R.A. and Stark, J. (1997). Penicillium discolor in de Nederlandse kaasindustrie. Aanwezigheid leidt meestal niet tot problemen. Voedingsmiddelentechnologie 30(20), 19–23. van Schouwenburg-van Foeken, A.W.J., Stadhouders, J. and Witsenburg, W.W. (1979). The number of enterotoxigenic Staphylococcus aureus reached in Gouda cheese made under normal acidification conditions and the amount of enterotoxin produced. Neth. Milk Dairy J. 33, 49–59. Veringa, H.A., van den Berg, G. and Stadhouders, J. (1976). An alternative method for the production of cultured butter. Milchwissenschaft 31, 658–662. Veringa, H.A., van den Berg, G. and Daamen, C.B.G. (1989). Factors affecting the growth of Aspergillus versicolor and the production of sterigmatocystin on cheese. Neth. Milk Dairy J. 43, 311–326. Verschueren, M., van den Hoven, G.A. and de Jong, P. (2002). Enhancement of curd washing efficiency: a twostage extraction process. Aust. J. Dairy Technol. 57, 144. Visser, F.M.W. (1977a). Contribution of enzymes from rennet, starter bacteria and milk to proteolysis and flavour development in Gouda cheese. 1. Description of cheese and aseptic cheesemaking techniques. Neth. Milk Dairy J. 31, 120–133. Visser, F.M.W. (1977b). Contribution of enzymes from rennet, starter bacteria and milk to proteolysis and flavour development in Gouda cheese. 2. Development of bitterness and cheese flavour. Neth. Milk Dairy J. 31, 188–209. Visser, F.M.W. (1977c). Contribution of enzymes from rennet, starter bacteria and milk to proteolysis and flavour development in Gouda cheese. 4. Protein breakdown: a gel electrophoretical study. Neth. Milk Dairy J. 31, 247–264. Visser, F.M.W. (1977d). Contribution of enzymes from rennet, starter bacteria and milk to proteolysis and flavour development in Gouda cheese. 5. Some observations on bitter extracts from aseptically made cheese. Neth. Milk Dairy J. 31, 265–276. Visser, J. (1991). Factors affecting the rheological and fracture properties of hard and semi-hard cheese, in, Rheological and Fracture Properties of Cheese. Bulletin 268, International Dairy Federation, Brussels. pp. 49–61. Visser, F.M.W. and de Groot-Mostert, A.E.A. (1977). Contribution of enzymes from rennet, starter bacteria and milk to proteolysis and flavour development in Gouda cheese. 3. Protein breakdown: analysis of the soluble nitrogen and amino acid nitrogen fractions. Neth. Milk Dairy J. 31, 210–239. Visser, S., Slangen, C.J., Hup, G. and Stadhouders, J. (1983). Bitter flavour in cheese. 3. Comparative gel-chromatographic analysis of hydrophobic peptide fractions from twelve Gouda-type cheeses and identification of bitter peptides isolated from a cheese made with Streptococcus cremoris HP. Neth. Milk Dairy J. 37, 181–192. Walstra, P. (2000). General principles, in, Practical Guide for Control of Cheese Yield, Special Issue 0001, International Dairy Federation, Brussels. pp. 6–13.

140 Gouda and Related Cheeses

Walstra, P. and Jenness, R. (1984). Dairy Chemistry and Physics, John Wiley, New York. Walstra, P., van Dijk, H.J.M. and Geurts, T.J. (1985). The syneresis of curd. 1. General considerations and literature review. Neth. Milk Dairy J. 39, 209–246. Wilbrink, A., Spoelstra, T. and Strampel, J. (1981). Scheurvorming in kaas bij gebruik van slappe kaaspekel. Zuivelzicht 73, 16–19. Wilkinson, M.G., Guinee, T.P., O’Callaghan, D.M. and Fox, P.F. (1994). Autolysis and proteolysis in different strains of starter bacteria during cheese ripening. J. Dairy Res. 61, 249–262. 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 flavour compounds. Appl. Environ. Microbiol. 63, 414–419. Yvon, M., Berthelot, S. and Gripon, J.C. (1998). Adding -ketoglutarate to semi-hard cheese curd highly enhances

the conversion of amino acids to aroma compounds. Int. Dairy J. 8, 889–898. Yvon, M., Chambellon, E., Bolotin, A. and Roudot-Algaron, F. (2000). Characterization and role of the branchedchain aminotransferase (BcaT) isolated from Lactococcus lactis subsp. cremoris NCDO 763. Appl. Environ. Microbiol. 66, 571–577. Zoon, P. (1993). Physical properties of cheese, in, Proceedings of the Cheese Research & Technology Conference, April 13–14, 1993, Center of Dairy Research, Madison, WI. pp. 55–61. Zoon, P., Straatsma, J. and Allersma, D. (1991). Indampen van kaaspekel en de gevolgen voor de kaaskwaliteit (Concentration of cheese brine by evaporation and its effect on cheese quality). Voedingsmiddelentechnologie 24(11), 13–16. Zoon, P., Ansems, C. and Faber, E.J. (1994). Measurement procedure for the concentration of active rennet in cheese. Neth. Milk Dairy J. 48, 141–150.

Cheeses with Propionic Acid Fermentation M.T. Fröhlich-Wyder and H.P. Bachmann, Agroscope Liebefeld-Posieux, Swiss Federal Institute for Animal Production and Dairy Products, Switzerland

Introduction The propionic acid fermentation leads to characteristic eyes and nutty flavour and can either occur spontaneously or can be achieved by a culture of selected propionibacteria. A spontaneous fermentation leads to irregular eye formation, because strain diversity of the natural propionibacterial flora is great. The number and size of eyes vary markedly, and cracks or splits are quite common. Comté and Beaufort are typical examples of cheese varieties with a spontaneous propionic acid fermentation. The application of a culture of selected propionibacteria allows a more regular eye formation as a result of a propionic acid fermentation which is under control. Such cheese varieties are often called Swiss-type cheeses. The body and texture correspond to those of hard or semi-hard cheeses. They were manufactured originally in the Emmental (Emmen valley) in Switzerland. Emmental is probably the best-known Swiss-type cheese and is frequently referred to simply as ‘Swiss cheese’. There is no internationally recognised definition of Swiss-type cheeses that differentiates them from other varieties. Swiss-type cheeses have round regular eyes which vary in size from medium to large. The characteristics of Swiss Emmental are: • • • •

cylindrical shape; firm dry rind; weight – 60–130 kg; 1000–2000 round eyes per loaf of diameter 1–4 cm, caused by propionic acid fermentation; • flavour – mild, slightly sweet, becoming more aromatic with increasing age; • cheese body – ivory to light-yellow, slightly elastic. Today, Emmental-type cheese (Fig. 1) is produced in many countries and a great variety of other Swisstype cheeses is also available on the market, including Jarlsberg, Maasdamer, Leerdamer and generally many other products denoted as Swiss cheese. Their body and texture correspond to those of hard and semi-hard cheeses. They are manufactured by methods differing

from traditional Swiss procedures. Thus, the treatment of milk, the extent of mechanisation, the starters used, the weight, shape, ripening time and shelf life are often different from the original. Descriptions and analytical values presented in this chapter focus on Swiss Emmental cheese but most of the information is applicable to other cheese varieties with a propionic acid fermentation.

Fermentations Lactic acid fermentation

Especially in the production of hard Swiss cheese varieties, mainly thermophilic lactic acid bacteria are used as starters, often as mixed cultures of lactobacilli (Lactobacillus helveticus, Lb. delbrueckii subsp. lactis) and streptococci (Streptococcus salivarius subsp. thermophilus). They guarantee the homofermentative catabolism of lactose to 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.

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142 Cheeses with Propionic Acid Fermentation

Figure 1 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 l of milk). Propionic acid fermentation begins about 30 days after the start of manufacture at about 20–24 °C for roughly 7 weeks and then continues at a slower rate at 10–13 °C. 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 °C, but growth occurs also at 14 °C. 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; Fröhlich-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 of aspartate, the fermentation of lactate is coupled 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

Figure 2 Scanning electron micrograph of a culture of Propionibacterium freudenreichii (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 CO2 + 1mol ATP

(B) Formation of succinate during propionic acid fermentation by CO2-fixation: 3 mol lactate

(2 – x) mol propionate + 1 mol acetate + (1 – x) mol CO2 + 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 CO2 + 6 mol succinate + 6 mol NH3 + 3 mol ATP

Figure 3 Metabolic pathways for the utilisation of lactate by propionic acid bacteria according to Crow and Turner (1986) and Sebastiani and Tschager (1993).

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 (Fröhlich-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

Succinate (mmol/kg)

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 (Fröhlich-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 800 nmol 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

10

5

0 0

1

2

3

4

5

6

7

Asp + Asn (mmol/kg) Figure 4 Linear regression, with a 95% confidence interval, of succinate and sum of aspartate (Asp) and asparagine (Asn) in 6-month-old Emmental cheese (䉲, propionibacteria with high aspartase activity; 䉱, propionibacteria with weak aspartase activity) (Wyder et al., 2001).

144 Cheeses with Propionic Acid Fermentation

Table 1 Mean values of metabolites, proteolytic parameters and propionibacterial counts in Emmental cheese (6 and 12 months) made with propionibacteria with weak or strong aspartase activity (Wyder et al., 2001) Emmental cheeses at 6 months

Emmental cheeses at 12 months

Parameter

Weak (N  10)

Strong (N  8)

t-test

Weak (N  10)

Strong (N  8)

t-test

Lactatea L()-Lactatea pH Free SCAa Acetatea Propionatea n-Butyratea n-Caproatea Succinatea CO2a Propionibacteriab Total nitrogenc WSNa TCASNa Free amino acidsa Aspartatea Asparaginea

57.4  10.5 31.1  9.3 5.75  0.02 114.4  5.2 48.4  1.3 60.1  4.4 1.1  0.2 0.4  0.1 4.0  0.6 27.6  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

45.3  17.4 17.0  8.9 5.79  0.02 126.0  5.2 53.1  5.1 67.1  10.2 1.2  0.1 0.5  0.1 11.9  1.7 33.6  2.0 nd 3.20  0.07 720.4  26.1 470.4  40.1 165.58  30.20 00 0.125  0.237

ns ** ** ns * ns ns * *** ***

47.0  8.5 25.4  8.1 5.63  0.06 117.4  5.9 47.6  0.6 63.2  4.2 1.7  0.9 0.5  0.1 5.1  2.8 nd 6.7  0.9 3.16  0.08 901.0  28.3 682.6  50.3 266.92  34.51 4.834  0.585 1.886  0.494

11.3  6.7 2.9  2.4 5.73  0.02 148.1  5.0 58.7  1.7 83.6  3.6 1.7  0.1 0.7  0.1 17.7  2.5 nd 8.4  0.3 3.21  0.06 926.3  28.7 687.3  46.8 246.86  22.95 0.588  0.097 0.054  0.154

*** *** *** *** *** *** ns ** ***

ns ns ns ns *** ***

*** ns ns ns ns *** ***

a mmol/kg. b log CFU/g. c mol/kg. SCA, Short Chain Acids; WSN, Water-soluble N; TCASN, 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 increased number of propionibacteria. 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.

25

26

27

Figure 5 Emmental cheese (12 months old) made with propionibacteria with strong (no. 26–27) or weak (no. 25) aspartase activity (Wyder et al., 2001).

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

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

Table 2 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 6 months

Emmental cheeses of 12 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  0.6 4.7  0.6 4.9  0.3 5.4  0.4 4.9  0.4 4.4  0.8 4.3  0.5 3.0  0.3 3.1  0.2 2.3  0.2 1.9  0.3 2.0  0.2 1.8  0.4 19.1  1.5

4.6  0.6 5.3  0.4 5.8  0.6 5.5  0.3 4.6  0.5 5.3  0.6 4.7  0.3 3.3  0.3 3.5  0.2 2.2  0.1 2.3  0.2 2.2  0.2 1.7  0.4 21.3  1.7

* * ** ns ns * ns ns *** ns ** ns ns *

4.6  0.6 4.4  0.6 4.5  0.5 5.3  0.7 4.6  0.6 6.5  0.5 4.5  0.6 3.6  0.3 3.7  0.4 2.5  0.3 2.3  0.2 2.6  0.3 1.8  0.4 18.1  1.8

4.6  0.8 5.4  0.3 5.8  0.6 5.0  0.6 4.7  0.4 6.8  0.4 4.4  0.5 3.5  0.3 3.8  0.3 2.4  0.2 2.5  0.3 2.8  0.4 1.9  0.2 20.6  1.0

ns *** *** ns ns ns ns ns ns ns ns ns ns **

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 (Fröhlich-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

Figure 6 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 Fröhlich-Wyder et al., 2002).

146 Cheeses with Propionic Acid Fermentation

Table 3 Water, lactate, pH and proteolytic parameters for Emmental cheese (Fröhlich-Wyder et al., 2002) Water (g/kg) Factor Feeding Grass Hay Propionibacteria Weak Strong Lb. casei Added Not added Lb. helveticus Added Not added

Lactate (mmol/kg)

pH

TN (g/kg)

WSN (% TN)

NPN (% WSN)

Free AA (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

* – – –

*** – – –

– *** *** –

*** – *** **

*** – – –

*** ** * –

*** – – ***

** ** – *

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.

Table 4 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) (Fröhlich-Wyder et al., 2002) Factor Feeding Grass Hay Propionibacteria Weak Strong Lb. casei Added Not added Lb. helveticus Added Not added ANOVA Feeding Propionibacteria Lb. casei Lb. helveticus

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

* * *** –

*** *** – –

*** *** *** –

*** – – –

** ** – –

*** *** *** –

– *** *** –

*** – *** *

– – *** –

– *** – –

–, not significant. *p  0.05. **p  0.01. ***p  0.001. C1, formate; C2, acetate; C3, propionate; C4, butyrate; C6, caproate.

Cheeses with Propionic Acid Fermentation 147

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,

100 Propionate (mmol.kg–1 )

Wyder, 2000). Jimeno et al. (1995) found growth inhibition of propionibacteria in cheese of up to 80% compared to the control without facultatively 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 facultatively 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 facultatively 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.

90 80 70

Prop96 Prop90

60 no

yes

50 Addition of Lb. casei Figure 7 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) (Fröhlich-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. delbrueckii 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 105 cfu/ml1. The same authors presented evidence for an inhibitory substance in milk, which is heat-stable and has a low molecular mass (Piveteau et al., 2000). It is removed by Lb. helveticus strains as a result of proteolysis, but not by Lb. delbrueckii or Lb. lactis strains. Consequently, the activation of propionibacterial growth may be the result of stimulation by the proteolytic activity of lactobacilli-liberating peptides and

148 Cheeses with Propionic Acid Fermentation

free amino acids and/or the removal of an inhibitory substance by the action of Lb. helveticus (Kerjean et al., 2000).

Technology The milk used for cheese manufacture should contain as few bacteria as possible so that the added starter cultures can have an optimum effect. If raw milk is used, the bacteriological requirements are particularly stringent. The microbial and hygienic state of farm milk, of course, also depends on the duration and temperature of storage, and secondary contamination before or after processing must be avoided. In Switzerland, Emmental cheese must be manufactured from raw milk from cows receiving no silage feeds. The whole technological sequence of operations is geared to creating optimum conditions for the propionic acid fermentation. A fundamental step is the addition of water (12–18%) to the milk or to the curd. This leads to a relatively high pH after the lactic fermentation (5.20–5.30), which consequently accelerates the propionic acid fermentation. Furthermore, this step leads to a soft and elastic texture and is also the explanation for the high calcium content of the cheese. A soft and elastic texture is crucial for regular eye formation. To avoid undesired fermentations, no residual sugar (galactose) should remain after the lactic fermentation. Most Swiss-type cheese varieties need a high cooking or scalding temperature. Emmental curd is heated to 52–54 °C after cutting. During pressing, the temperature remains at around 50 °C for many hours (Fig. 8).

At this temperature, the curd dries and most of the undesirable micro-organisms are eliminated. This is not only important for the hygienic safety, but also for avoiding too intensive proteolysis in depth (peptidolysis), which leads to a ‘shorter’, i.e., more crumbly texture of the cheese, and increases the risk of splits and cracks during ripening. The high cooking temperature also causes the complete inactivation of the chymosin. A temperature above 54 °C impairs the propionibacteria too strongly. Swiss-type cheeses are often manufactured in copper vats (Fig. 9) and pressed in cylindrical moulds (Fig. 10). The copper content should not be too high, because copper inhibits the formation of lactic and propionic acids. On the other hand, copper forms complexes with sulphur compounds originating from the catabolism of amino acids, and thus has a positive impact on the flavour and aroma of the cheese. A copper content between 120 and 200 mol/kg is optimal; it comes mainly from the vat surface. Because propionibacteria are sensitive to salt, brining is less intensive than for other cheese varieties. The average salt content in Swiss Emmental cheese is between 3 and 5 g/kg. Brining leads to a firm and dry rind, which reduces the loss of CO2 during the propionic acid fermentation and thus supports eye formation. The rind is also responsible for the sturdy shape of the cheeses during ripening. Today, Swiss-type cheeses are manufactured in many countries by technologies differing from the traditional Swiss procedure. Considering the technological aspects, Swiss-type cheeses are always cooked cheeses. On the

50 centre

48

outer zone 46 44 °C

42 40 38 36 34 32 30 1

2

3

4

5

6

7

8

9

10 11 12 13 14 15 16 17 18 19 20

Pressing hours Figure 8 Temperature profile of Emmental cheese during pressing (Steffen and Schnider, 1978).

Cheeses with Propionic Acid Fermentation 149

Ripening General aspects

Figure 9 Cutting of the curd. (See Colour plate 17.)

other hand, the treatment of milk, the extent of mechanisation, the weight and shape, the average composition (hard or semi-hard varieties both with different fat contents), ripening time and shelf life are frequently very different. Quite often, Swiss-type cheeses are manufactured in the form of blocks and ripened in plastic bags for large-scale production.

To initiate the typical propionic acid fermentation, the ripening temperature for the cheese must be raised to approximately 20–24 °C for a certain period of time. As soon as the development of sufficient eyes has been accomplished, the propionic acid fermentation is retarded by storing the cheese at a lower temperature (10–13 °C). In the case of Swiss Emmental cheese, the period of the eye formation is between 40 and 60 days. By tapping the cheeses, the cheesemaker determines the right time for the transfer from the warm to the ripening room. The relative humidity in the ripening room is rather low (70–80%). This, and also the brining as mentioned above, leads to a firm and dry rind, which reduces the loss of CO2 and leads to the sturdy shape of the cheeses. Furthermore, the low humidity accelerates the sweating of the cheeses (secretion of fat) and reduces the growth of moulds on the surface of the cheeses and, therefore, the time needed for manual cleaning. The ripening period varies widely; some cheese varieties are sold directly after the propionic acid fermentation while others are ripened for more than 1 year. Lately in Switzerland, ripening under humid conditions (in rock caves) has gained more importance. By the action of specific moulds, the cheese surface becomes black and proteolysis is further affected, although the proteolytic enzymes of the surface flora do not penetrate into the cheese mass. An indirect, but strong proteolytic, effect arises from the accelerated increase in pH of the outer zones of the cheeses due to deacidification of the surface by the moulds. Moreover, soluble substances produced by the surface flora diffuse into the cheese mass. Flavour formation

Figure 10 Filling of the curd into the cheese moulds. (See Colour plate 18.)

Flavour development in cheese is very strongly dependent of the microbial flora of milk. Whilst the indigenous flora of milk is generally composed of unwanted microorganisms, which can influence the flavour directly by their fermentative activities or indirectly by other enzymatic reactions, the desired lactic acid bacteria must be added to the cheese milk as starter or adjunct cultures. The addition of rennet and the different operations involved in cheesemaking and cheese ripening influence flavour development. In Switzerland, Emmental cheese is made from raw milk. Certain flavour compounds in milk are in fact lost and others are produced when it is subjected to thermisation or pasteurisation before processing. The high temperatures applied during the early stages of manufacture and pressing of Emmental cheese are essential for flavour development. Other important factors are the fermentation and ripening processes.

150 Cheeses with Propionic Acid Fermentation

During ripening for 3–12 months, the intensity of odour, aroma, saltiness and sourness increase (Table 5). Sweetness and bitterness decrease slightly. Due to the propionic acid fermentation, the sweet taste is about 1–1.5 units higher than in other hard cheese varieties without the propionic acid fermentation. Warmke et al. (1996) evaluated the following substances as potent taste compounds: acetic, propionic, lactic, succinic and glutamic acids, each in free form and/or as ammonium, sodium, potassium, magnesium or calcium salts, as well as the corresponding chlorides and phosphates. Magnesium and calcium propionate mainly caused the sweetish note in the taste profile of Emmental. Although bitter-tasting amino acids and peptides occurred in the cheese, they were not detected in the taste profile. For analytical reasons, the flavour components are generally divided into two major groups: volatile and non-volatile compounds. The volatile compounds derive from glycolysis, proteolysis and lipolysis during ripening and include volatile short chain acids (Table 4), primary and secondary alcohols, methyl ketones, aldehydes, esters, lactones, alkanes, aromatic hydrocarbons and different sulphur- and nitrogen-containing compounds (Table 6). Methional and acetic and propionic acids are the most important volatile compounds for typical Emmental flavour. Ethyl butanoate, ethyl 3-methylbutanoate and ethyl hexanoate contribute to the fruity odour note. The two furanones are responsible for the caramel-like flavour in Emmental cheese. Proteolysis is considered to be essential to the ripening process, contributing to flavour formation by the liberation of free amino acids. Recent results, however, showed that an increase of free amino acids in cheese could not be correlated with flavour formation. It is concluded that the rate-limiting factor in the formation of flavour is the degradation of amino acids by the micro-organisms present in cheese rather than their release (Yvon and Rijnen, 2001). Lactic acid bacteria are the main contributors, but propionibacteria Table 5 Flavour description of Emmental cheese (Mean values  standard deviation for n  10) (taken from the work by Wyder et al., 2001) Cheese age (months) Parameter

Scale

3

6

12

Odour intensity Aroma intensity Sweetness Saltiness Sourness Bitterness

0–7 0–7 0–7 0–7 0–7 0–7

3.0  0.3 2.5  0.3 2.5  0.1 1.9  0.2 2.1  0.3 2.0  0.3

3.1  0.2 3.1  0.2 2.3  0.2 1.9  0.3 2.0  0.2 1.8  0.4

3.6  0.3 3.7  0.4 2.5  0.3 2.3  0.2 2.6  0.3 1.8  0.4

Table 6 Concentration of odorants ( g/kg dry matter, except ammonia) in Emmental cheese (Mean values  standard deviation for n  4) (unpublished results from the work by Wyder et al., 2001) Cheese age (months) Odorant

3

6

12

2,3-Butandione 2-Methylbutanal 3-Methylbutanal Ethylbutanoate Ethyl 3-methylbutanoate 2-Heptanone Dimethyltrisulfide Methional Ethylhexanoate 1-Octen-3-one 4-Hydroxy-2, 5-dimethyl3(2H)-furanone 5-Ethyl-4-hydroxy2-methyl-3(2H)furanone 2-sec-Butyl-3methoxy-pyrazine Skatole

-Decalactone 3-Methylbutyric acid Ammonia, mg/kg

431  147 181  40 145  22 27  17 0.40  0.16

605  354 251  43 167  16 73  23 0.78  0.21

522  60 0.11  0.10 67  33 51  21 0.06  0.02 1186  276

770  57 2783  1517 0.16  0.08 0.21  0.16 68  22 50  6 164  63 351  156 0.05  0.02 0.06  0.02 658  297 1002  387

531  470 372  88 152  15 148  72 2.48  1.25

253  72

255  86

547  232

0.07  0.03

0.05  0.03

0.04  0.01

47  15 34  6 3751  1216 1680  97 20  10 30  10 560  150

720  160

37  10 1171  132 30  10 970  190

also have a high ability to convert branched-chain amino acids to acids. Isoleucine/Leucine is converted mainly to isovaleric acid (Thierry and Maillard, 2002). The cause of lipolysis in Emmental cheese can be bacterial lipase and the indigenous lipoprotein lipase in milk which is, however, thermolabile and therefore its activity is reduced by cooking to a temperature over 50 °C. Lactic acid bacteria have only limited lipolytic activity, with Sc. thermophilus having the highest (Gobbetti et al., 1996). Propionibacteria, in contrast, have high strain-dependent lipolytic activity, 10–100 times more than lactic acid bacteria (Dupuis, 1994). Lipolysis in Emmental is caused mainly by propionibacteria and is generally recognised as necessary to produce typical Emmental cheese flavour. The amount of free fatty acids present varies from 2 to 7 g/kg (Isolini et al., 2001). Nevertheless, higher contents give flavour defects (Bachmann, 1998b). The release of free fatty acids starts in the warm room simultaneously with the growth of propionibacteria (Chamba and Perreard, 2002). Long-chain free fatty acids, which have a minor influence on cheese flavour, accumulate during ripening, whereas short- and medium-chain free fatty acids are most probably transformed by -oxidation and esterification to volatile flavour compounds (Bosset

Cheeses with Propionic Acid Fermentation 151

et al., 1995; Chamba and Perreard, 2002). Since propionibacteria have very strain-dependent lipolytic activity and also are the main lipolytic agents in Emmental, it is important to include the potential for lipolysis in the selection of new propionibacterial cultures. The non-volatile flavour compounds include peptides, free amino acids, amines (Table 1), free fatty acids, salt and minerals (Table 7). The peptides and free amino acids contribute to the background flavour. Free glutamic acid is mainly responsible for the umami taste. Salt (NaCl) and other minerals directly influence the saltiness and indirectly the total aroma intensity. Cheese off-flavour depends quite often on the properties of the cheese milk. Certain plants and feeds such as bulbous plants, leeks, vegetable wastes, herb mixtures and different mineral salt mixtures fed to dairy cows can influence the taste of milk and produce offflavours. Certain milk enzymes also can induce offflavours, e.g., rancidity induced by lipase. Today, Swiss-type cheese can be found on the market over a wide range of maturity from very young, elastic cheese with the typical sour lactic aroma and sweet taste up to cheese ripened for a very long period in humid caves (Fig. 11) with a more intensive flavour and a nutty and spicy note. Texture formation

Cheese body and texture are very important qualities for both traders and consumers. Variations from what is considered normal in body and texture within the same cheese variety are not tolerated because there is a close relationship between the body and texture and other qualities, such as eye formation, taste and shelf life. Texture denotes the structure and the consistency of the cheese body. A soft and elastic texture is crucial for regular eye formation. The high calcium content, which is the result of the high pH after the lactic fermentation (5.20–5.30), is very important for a ‘long’, elastic texture. Table 8 shows the development of the Table 7 Concentration of non-volatile components (g/kg dry matter) in Emmental cheese (mean values  standard deviation for n  4) (unpublished results from the work by Wyder et al., 2001) Cheese age (months) Taste component

3

6

12

Glutamic acid Sodiuma Potassiuma Magnesiuma Calciuma Phosphatea

5.4  0.6 5.2  0.6 1.3  0.1 0.7  0.1 6.6  0.8 10.6  1.2

8.1  0.3 4.5  0.2 1.0  0.1 0.6  0.1 6.5  0.6 13.9  1.5

11.6 4.5 1.2 1.0 10.6 13.2

a Concentration in the aqueous extract.

Figure 11 Emmental, about 12-months-old, matured in a cave with a high relative humidity.

rheological parameters and penetrometry data during ripening from 3 to 12 months. The texture of Swiss Emmental changes during ripening from elastic and relatively soft to less elastic, more friable and more firm. Figure 12 shows the typical texture profile of 6-month-old Emmental cheese. Because of the low water content, Swiss-type cheese melts at a relatively high temperature. The average softening point measured with an automatic dropping point apparatus is 74 °C. The sum of free amino acids measured 1 day after manufacture by the Cadmium-ninhydrin method and the trichloroacetic acid-soluble nitrogen after 20 days allow an early prediction of flavour and texture development in Emmental cheese (Bachmann et al., 1999). Emmental cheese is characterised by a higher ratio of s1-:-casein than in cheeses which are not cooked at 50–55 °C. This is due to the specificity of primary proteolysis: s1-casein is hydrolysed at a slower rate and -casein at a faster rate. The slower proteolysis of s1-casein is due to inactivation of the coagulant, either chymosin or microbial enzymes, during cooking. The faster rate of primary proteolysis of -casein is due to the activity of plasmin. The high ratio of s1-:-casein Table 8 Results of penetrometry and uniaxial compression test on Emmental cheese during ripening (mean values  standard deviation for n  10) (adapted from Bachmann et al., 1999) Cheese age (months)

     

1.2 0.7 0.1 0.1 0.7 0.6

Parameter

Unit

3

6

12

Penetrometry Strain at fracture Stress at fracture Stress at 33% deformation

mm 3.7  1.0 2.5  0.3 4.6  0.6 % 68.9  3.2 63.7  2.5 46.5  5.4 kN/m2 614  121 437  58 319  48 kN/m2 147  16 157  20 244  30

152 Cheeses with Propionic Acid Fermentation

Firmness 5

Humidity

4 3

Friability

2 1

Elasticity

Solubility

Adhesiveness Figure 12 Texture profile of 6-month-old Emmental cheese (adapted from Lavanchy and Bütikofer, 1999).

is jointly responsible for the soft and elastic texture (Kerjean et al., 2001). The proteolytic activity of propionibacteria is insignificant. The course of proteolysis in Swiss Emmental cheese is shown in Table 9. Insufficient proteolysis may cause different cheese defects. If the level of protein breakdown is too low, the taste is flat and the body consistency too ‘long’, i.e., rubbery. Sometimes, uneven openings also appear. Excessive proteolysis results in an overripe and sharp taste and a shorter body. Frequently, the extent of proteolysis in a cheese loaf varies from one zone to the other, a phenomenon that is due to a changing temperature profile in the cheese loaf during the lactic acid fermentation. Since the outer zone cools faster, it often develops a bacterial flora which is proteolytically more active than the flora of the centre of the loaf. This usually leads to cheese defects such as a short and firm body, or a sharp taste, or the development of white colour under the rind.

diversity of the indigenous propionibacterial flora is great. The number and size of eyes vary markedly, and cracks or splits are quite common. The application of a culture of selected propionibacteria allows a more regular eye formation as a result of a propionic acid fermentation which is under control. Small quantities of CO2 are already produced during the lactic acid fermentation and the degradation of citrate. The fermentation of citrate leads to a higher number of eyes in the initial stage of the propionic acid fermentation and to a lower number of eyes in the mature cheese (Fig. 13). To initiate the typical propionic acid fermentation, the ripening temperature for the cheese must be raised to approximately 20–24 °C. As soon as the development of sufficient eyes is accomplished, the propionic acid fermentation is retarded by storing the cheese at a lower temperature (10–13 °C). The different stages of CO2 production and eye formation may be summarised schematically as follows: CO2 production and CO2 diffusion (starts at the beginning of the lactic acid fermentation) b Accumulation of CO2 in the cheese body (degradation of citrate and propionic acid fermentation) b Over-saturation at the centres of future eye formation (propionic acid fermentation) b Onset of eye formation at these centres (after approximately 20–30 days)

Eye Formation The propionic acid fermentation leads to the formation of characteristic eyes and a nutty flavour and can occur either spontaneously or be achieved by a culture of selected propionibacteria. A spontaneous fermentation leads to irregular eye formation, because strain

b Increase in the number of eyes and their enlargement (propionic acid fermentation  decarboxylation of amino acids)

Table 9 Proteolysis in Swiss Emmental cheese (mean values  standard deviation for n  10) (adapted from Bachmann et al., 1999) Cheese age (months) Parameter

Unit

2/3

3

6

12

Water-soluble nitrogen 12% trichloroacetic acid-soluble nitrogen Sum of free amino acids

mmol/kg mmol/kg

218  17 90  9

610  31 386  39

693  33 469  47

901  28 683  60

mmol/kg

16  4

120  32

169  24

267  35

Cheeses with Propionic Acid Fermentation 153

Number of eyes

150

100

with Lb. casei

50

without Lb. casei

0 40 days

180 days Stage of ripening

Figure 13 Influence of the citrate metabolism on the number of eyes in Emmental cheese (adapted from the work by FröhlichWyder et al., 2002).

The development of eye formation depends mainly on: • • • •

time, quantity and intensity of CO2 production; number and size of the areas of future eye formation; CO2 pressure and diffusion rate; body texture and temperature.

At the beginning of eye formation, i.e., about 30 days after manufacture, only a few eyes appear; thereafter, the number of new holes increases progressively. The maximum rate is attained after about 50 days, which is also the time of rapid eye enlargement. The appearance of new eyes declines with decreasing CO2 production and simultaneous hardening of the cheese body. The quantity and distribution of the eyes also depend on other factors such as those mentioned above. The number of eyes is increased by the nonhomogeneity of the curd, physical openness and the content of gas. Centrifugation and heat treatment of the milk or application of vacuum after filling of the curds and during pressing of the cheese are performed in order to obtain a lower number of eyes. In cheeses produced from microfiltered milk, the number of eyes is generally much lower, even when the retentate is added. This indicates that air bubbles are important areas of future eye formation. As regards eye formation, proper dip filling of the moulds is imperative since air inclusions can lead to undesirable openness. Figure 14 shows the deformation of the granules around eyes. In a cheese loaf of approximately 80 kg, total CO2 production is about 120 l before the cheese is sufficiently aged for consumption. About 60 l remain dissolved in the cheese body, ⬃20 l are found in the eyes and ⬃40 l diffuse out of the loaf (Flückiger et al., 1978).

The development of CO2 pressure shows two major phases (Fig. 15). The first covers the period of proper eye formation in the ripening room. During this period, the CO2 pressure remains relatively low, between 1500 and 2500 Pa, because of the low resistance of the soft cheese mass to gas compression at 22–24 °C. During storage, i.e., the second stage, the CO2 pressure increases to 4000–8000 Pa. The difference in pressure between various loaves is higher in the second stage than in the first. The pressure increase in the second stage is explained by the higher resistance to gas compression of the cheese mass, which is due to the decrease in temperature from 22 to 12 °C, and by continued gas production. During the first stage there is a marked pressure increase within the eyes. These observations go back to a period where it was common to use propionibacteria

Figure 14 Deformation of curd granules around eyes (L) in Emmentaler cheese (Rüegg and Moor, 1987).

154 Cheeses with Propionic Acid Fermentation

5

40

4

3 20 2

Eye volume (ml)

Overpressure (kPa)

30

10 1

0

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Figure 15 Eye volume (ⵧ) and CO2 overpressure (䉬) in Emmental cheese (adapted from Flückiger, 1980).

with high aspartase activity (Flückiger, 1980). More recent results are not available. Excessive proteolysis becomes particularly evident when a large amount of casein is degraded to lowmolecular compounds (high non-protein nitrogen level). Additional CO2 production by decarboxylation of amino acids clearly reduces the keeping quality of the cheese and leads to oversized eye formation. In addition, the propionibacteria are stimulated which increases the production of CO2 (aspartate metabolism). The cheese body often cannot withstand the pressure of the gas and cracks or splits appear. This defect is referred to as late or secondary fermentation.

The decrease of the number of pathogens can be explained by the so-called hurdle technology, which implies the synergistic effects of a high milk quality, short milk storage (effect of active antimicrobial enzyme systems of fresh raw milk), antagonistic starter culture flora, rapid acidification, antimicrobial effect of lactic acid, high curd cooking temperature, brining, and ripening at an elevated temperature for at least 4 months. All these factors are also important determinants of flavour and texture quality.

8

Hygienic Safety In Switzerland, Emmental cheese must be manufactured from raw milk. Despite intense hygienic efforts, contamination of raw milk by pathogenic microorganisms cannot be completely excluded. Infectious diseases in dairy cows or contamination of milk during milking, storage, transport or processing present potential hazards. This has led to discussions on their hygienic safety of raw-milk cheeses. Bachmann and Spahr (1995) and Spahr and Schafroth (2001) examined the ability of potentially pathogenic bacteria to grow and to survive during the manufacture and ripening of Swiss Emmental cheese made from raw milk (Figs 16 and 17). They concluded that the hygienic safety of Swiss Emmental cheese is comparable to cheese produced from pasteurised milk.

Log cfu/ml, g

7 6 5 4 3 2 1 0

Milk

Curd Cheese Cheese Cheese after 1d 7d 30 d cooking

Figure 16 Behaviour of Aeromonas hydrophila (䉫), Campylobacter jejuni (䉬), Escherichia coli (), Listeria monocytogenes (䉱), Pseudomonas aeruginosa (䊊), Salmonella typhimurium (I), Staphylococcus aureus (ⵧ) and Yersinia enterocolitica (䊏) during manufacture and ripening of Swiss Emmental cheese made from raw milk (only data for batches with longest survival are shown); detection limit, (Bachmann and Spahr, 1995).

Cheeses with Propionic Acid Fermentation 155

6

log10 cfu/g

5

R2 = 0.923

4 3 2 1 0 0

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Ripening days Figure 17 Inactivation curves for Mycobacterium avium subsp. paratuberculosis in Swiss Emmental cheese during 120 days of ripening (Spahr and Schafroth, 2001).

References Bachmann, H.P. (1998a). Die Vergärung von Aspartat durch Propionsäurebakterien steigert das Risiko von Nachgärung beim Emmentaler Käse, Agrarforschung 5, 161–164. Bachmann, H.P. (1998b). Lipolyse im Käse: nicht zu viel, nicht zu wenig. Agrarforschung 5, 293–295. Bachmann, H.P. and Spahr, U. (1995). The fate of potentially pathogenic bacteria in Swiss hard and semihard cheeses made from raw milk. J. Dairy Sci. 78, 476–483. Bachmann, H.P., Bütikofer, U. and Meyer, J. (1999). Prediction of flavour and texture development in Swiss-type cheeses. Food Sci. Technol. -Lebensm. -Wiss. Technol. 32, 284–289. Baer, A. (1995). Influence of casein proteolysis by starter bacteria, rennet and plasmin on the growth of propionibacteria in Swiss-type cheese. Lait 75, 391–400. Baer, A. and Ryba, I. (1999). Interactions between propionic acid bacteria and thermophilic lactic acid bacteria. Lait 79, 79–92. Bosset, J.O., Gauch, R. and Mariaca, R. (1995). Comparison of various sample treatments for the analysis of volatile compounds by GC-MS. Application to the Swiss Emmental cheese. Mit. Gebiete Lebensm. Hyg. 86, 672–698. Brendehaug, J. and Langsrud, T. (1985). Amino acid metabolism in propionibacteria: resting cell experiments with four strains. J. Dairy Sci. 68, 281–289. Chamba, J.F. (2000). Emmental cheese: a complex microbial ecosystem. Consequences on selection and use of starters. Sci. Alim. 20, 37–54. Chamba, J.F. and Perreard, E. (2002). Contributrion of propionic acid bacteria to lipolysis of Emmental cheese. Lait 82, 33–44. Crow, V.L. (1986a). Utilization of lactate isomers by Propionibacterium freudenreichii subsp. shermanii: regulatory role for intracellular pyruvate. Appl. Environ. Microbiol. 52, 352–358. Crow, V.L. (1986b). Metabolism of aspartate by Propionibacterium freudenreichii subsp. shermanii: effect on lactate fermentation. Appl. Environ. Microbiol. 52, 359–365.

Crow, V.L. and Turner, K.W. (1986). The effect of succinate production on other fermentation products in Swiss-type cheese. NZ J. Dairy Sci. Technol. 21, 217–227. Crow, V.L., Martley, F.G. and Delacroix, A. (1988). Isolation and properties of aspartase-deficient variants of Propionibacterium freudenreichii subsp. shermanii and their use in the manufacture of Swiss-type cheese. NZ J. Dairy Sci. Technol. 23, 75–85. Dupuis, C. (1994). Activités protéolytiques et lipolytiques des bactéries propioniques laitières. Thèse ENSA, Rennes. Fessler, D. (1997). Characterisation of Propionibacteria in Swiss Raw Milk by Biochemical and Molecular-biological Methods, Thesis No. 12328, ETH Zürich. Flückiger, E. (1980). CO2- und Lochbildung im Emmentalerkäse. Schweiz. Milchzeitung 106, 473–480. Flückiger, E., Montagne, D.H. and Steffen, C. (1978). Beitrag zur Kenntnis der CO2-Bildung im Emmentalerkäse vor Beginn der Propionsäuregärung. Schweiz. Milchwirt. Forsch. 7, 73–78. Fröhlich-Wyder, M.T., Bachmann, H.P. and Casey, M.G. (2002). Interaction between propionibacteria and starter/ non-starter lactic acid bacteria in Swiss-type cheeses. Lait 82, 1–15. Gobbetti, M., Fox, P.F. and Stepaniak, L. (1996). Esterolytic and lipolytic activities of mesophilic and thermophylic lactobacilli. Ital. J. Food Sci. 2, 127–135. Isolini, D., Casey, M.G., Fröhlich-Wyder, M.T., Meyer, J., Sollberger, H. and Collomb, M. (2001). Propionsäurebakterien und ihre lipolytische Aktivität: eine mögliche Screening-Methode. FAM Intern. Bericht 35, 1–9. Jimeno, J. (1997). Lactobacillus casei et Lactobacillus rhamnosus citrate () et citrate () des MK 3007 et 3008: Croissance et antagonisme dans l’emmental modèle. FAM Intern. Ber. Biochem. 14, 1–18. Jimeno, J., Lazaro, M.J. and Sollberger, H. (1995). Antagonistic interactions between propionic acid bacteria and nonstarter lactic acid bacteria. Lait 75, 401–413. Kerjean, J.R., Condon, S., Lodi, R., Kalantzopoulos, G., Chamba, J.F., Suomalainen, T., Cogan, T. and Moreau, D. (2000). Improving the quality of European hard-cheeses by controlling of interactions between lactic acid bacteria and propionibacteria. Food Res. Int. 33, 281–287. Kerjean, J.R., Bachmann, H.P. and Cogan, T. (2001). Technical note: Cooking temperature of whey and curd during Emmental cheesemaking. Milchwissenschaft 56, 556–556. Lavanchy, P. and Bütikofer, U. (1999). Caractérisation sensorielle de fromages à pâte dure ou mi-dure fabriqués en Suisse. Mitt. Lebensm. Hyg. 90, 670–683. Perez Chaia, A., Pesce de Ruiz Holgado, A. and Oliver, G. (1987). Interaction between Lactobacillus helveticus and Propionibacterium freudenreichii subsp. shermanii. Microbiol. Aliment. Nutr. 5, 325–331. Piveteau, P.G., Condon, S. and Cogan, T.M. (1995). Interactions between lactic and propionic acid bacteria. Lait 75, 331–343. Piveteau, P., Condon, S. and Cogan, T.M. (2000). Inability of dairy propionibacteria to grow in milk from low inocula. J. Dairy Res. 67, 65–71.

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Richoux, R. and Kerjean, J.R. (1995). Technological properties of pure propionibacteria strains: test in small scale Swiss-type cheese. Lait 75, 45–59. Rüegg, M. and Moor, U. (1987). The size distribution and shape of curd granules in traditional Swiss hard and semi-hard cheeses. Food Microstruct. 6, 35–46. Sebastiani, H. and Tschager, E. (1993). Succinatbildung durch Propionsäurebakterien – Eine Ursache der Nachgärung von Emmentaler? Dt. Molk.-Ztg. 114, 76–80. Sieber, R., Collomb, M., Lavanchy, P. and Steiger, G. (1988). Beitrag zur Kenntnis der Zusammensetzung schweizerischer konsumreifer Emmentaler, Greyerzer, Sbrinz, Appenzeller und Tilsiter. Schweiz. Milchwirt. Forsch. 17, 9–16. Sollberger, H. and Wyder, M.T. (2000). Propionsäurebakterien und fakultativ heterofermentative Laktobazillen. Schweiz. Milchzeitung 126, 5. Spahr, U. and Schafroth, K. (2001). Fate of Mycobacterium avium subsp. paratuberculosis in Swiss hard and semihard

cheese manufactured from raw milk. Appl. Environ. Microbiol. 67, 4199–4205. Steffen, C. and Schnider, J. (1978). Erhebungen über den Temperaturverlauf im Emmentalerkäse. Schweiz. Milchzeitung 104, 383. Thierry, A. and Maillard, M.-B. (2002). Production of cheese flavour compounds derived from amino acid catabolism by Propionibacterium freudenreichii. Lait 82, 17–32. Warmke, R., Belitz, H.D. and Grosch, W. (1996). Evaluation of taste compounds of Swiss cheese (Emmentaler). Z. Lebensm. Untersuch.-Forsch. 203, 230–235. Wyder, M.T., Bosset, J.O., Casey, M.G., Isolini, D. and Sollberger, H. (2001). Influence of two different propionibacterial cultures on the characteristics of Swisstype cheese with regard to aspartate metabolism. Milk Sci. Int. 56, 78–81. Yvon, M. and Rijnen, L. (2001). Cheese flavour formation by amino acid catabolism. Int. Dairy J. 11, 185–201.

Surface Mould-ripened Cheeses H.-E. Spinnler, Laboratoire de Génie et Microbiologie des Procédés Alimentaires, INA-PG, Thiverval-Grignon, France J.-C. Gripon, Unité de Biochimie et Structure des Protéines, INRA, Jouy-en-Josas, France

Introduction Surface mould-ripened soft cheeses are characterised by the presence of a felt-like coating of white mycelia due to the growth of Penicillium camemberti on the surface. The presence of this mould gives these cheeses a characteristic appearance, as well as a typical aroma and taste. It also leads to more complex ripening patterns than in other varieties of cheese with more simple microflora. These cheeses are becoming increasingly popular with consumers, and the demand for them increases. In the present chapter, after reviewing briefly the different methods of production and technologies of these cheeses, the microflora, the various biochemical changes that occur during their ripening, their aroma and textural properties and the control of their ripening will be considered.

Diversity of Surface Mould-Ripened Cheeses A typical example of surface mould-ripened cheeses is Camembert, which is a cheese with a soft consistency and a flat cylindrical form, approximately 11 cm in diameter and 2.5 cm thick. Camembert originated in the Normandy region of France. It is believed by some to date from about 1790 and has been attributed to a farmer’s wife, called Marie Harel, from the small village of Camembert. Though first made on farms, Camembert has been made by industrial companies since the beginning of the twentieth century. The manufacture of surface mould-ripened cheeses became progressively widespread in France, followed by other European countries. Camembert manufactured in Normandy and meeting certain manufacturing norms benefits from a protected designation of origin (PDO; Camembert de Normandie). Conversely, the name ‘Camembert’ is not protected and is used for cheeses manufactured elsewhere in France or in other countries. The other principal cheeses with a surface mould are Brie, Coulommier and Carré de l’Est (a mild fermented cheese). Products also exist which are marketed with a trade name. Brie from Meaux or from Melun are characterised by their large diameter (35 and 27 cm, respectively) and also

possess a PDO. France produced aproximately 300 000 tonnes of Camembert, Brie, Coulommier and Carré de l’Est in 2000 (CNIEL, 2002). Total production of surface mould-ripened cheeses is a little greater, as products marketed under trade names should be included. Germany produces 18 000 and 2000 tonnes of Camembert and Brie, respectively (again, these figures are under-estimates as they do not include products manufactured under other names). Denmark also produces significant quantities of surface mould-ripened cheeses. Many other countries around the world, including USA, Australia, Argentina, New Zealand and several European countries produce cheeses with a surface mould, but in limited amounts. In the absence of precise figures, it can reasonably be estimated that in 1999, surface mould-ripened cheeses represented 7–8% of the total production of cheese in the 15 countries of the European Union and 2–3% of the world production. (Reprinted from Encyclopedia of Dairy Sciences, Gripon, J.-C., pp. 401–403, 2 paragraphs only, with permission from Elsevier.)

Technology Traditional Camembert is made from raw milk with the addition of a mesophilic starter. The pH at renneting is approximately 6.4 and the coagulation time is 30–45 min. Transfer of the coagulum to the moulds is by means of a ladle (5 ladles/mould), either manually or using an automated system. Draining takes place spontaneously through the perforated sides of the moulds during the first hours at 26–28 °C, then at a progressive reduction in temperature approaching approximately 20 °C by the end of draining. A curd with a low mineral content is thus obtained, with a pH of 4.6–4.7 at the end of draining. The cheese is drysalted and maturated for a minimum period of 21 days in cellars at 11–13 °C and 90% relative humidity. Camembert, without designation of origin, is manufactured from raw or pasteurised milk. Coagulation generally takes place continuously in an Alpma-type production system. The coagulum is cut into cubes of 2–2.5 cm/side and the curds are moulded (manually or automated, in multi-moulds) 30–50 min after cutting.

Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 2: Major Cheese Groups ISBN: 0-1226-3653-8 Set ISBN: 0-1226-3651-1

Copyright © 2004 Elsevier Ltd All rights reserved

158 Surface Mould-ripened Cheeses

Microbial flora The composition and the evolution of the flora of surface mould-ripened cheeses are complex, particularly when raw milk is used. Traditional Camembert is a good example of this complexity. In changing different parameters (salt, water activity, pH), cheese technologists hinder most of the microbial growth but the fungal and the bacterial flora of mould-ripened cheeses remain very diverse. In raw milk cheese, the physico-chemical treatments select the ‘technological’ flora but in pasteurised milk cheese, most of the flora are nowadays added to the milk as starters. These micro-organisms (yeast, Geotrichum candidum, coryneform bacteria) generate different compounds responsible for different functions; change in texture, taste, flavour, colour, antimicrobial activity and organoleptic qualities are tailormade by exploiting the properties of these different micro-organisms. The succession of micro-organisms is determined by the changes in the chemical environment. First, the lactic acid bacteria (mainly Lactococcus lactis subsp. lactis and Lc. lactis subsp. cremoris), by reducing the pH, will select acidophilic micro-organisms such as yeasts and filamentous fungi. On growing on the surface, stimulated by the oxygen, they will permit the development of ripening bacteria (mainly coryneform bacteria) which are adapted to the curd composition and ripening environment (Fig. 1). One can consider that the pH 5.8 is a barrier pH (Fig. 2) below which ripening bacteria cannot grow. Fungi

Very soon after manufacture, yeasts grow on the surface (Leclercq-Perlat et al., 1999) forming a dense layer

14 °C RH = 85% Viable cell concentration (log10 CFU/gDM)

Curds are salted in brine. Distribution requirements, as well as market demands, have led to modifications in existing technology and have created new types of surface mould-ripened cheeses. The latter, called solubilised or stabilised cheeses, are often sold under trade names. Pasteurised milk is renneted after a very short maturation period. The coagulum is cut into cubes, 0.7–1 cm in size, and the curds are stirred and washed. Part of the whey is drawn off before moulding. The starter used consists of thermophilic streptococci or a mixture of streptococci and lactococci. The curd obtained is much less acidic than that of Camembert. These cheeses rapidly acquire a soft texture, giving them a mature appearance; their taste is milder than that of traditional Camembert, and their storage properties are improved. (Reprinted from Encyclopedia of Dairy Sciences, Gripon, J.-C., pp. 401–403, 2 paragraphs only, with permission from Elsevier.)

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Figure 1 Changes in the number of D. hansenii (䊊), G. candidum (䉭) and B. linens (䊐) in Camembert ripening (Leclercq-Perlat et al., 2003).

about 200 m thick; Kluyveromyces lactis, Saccharomyces cerevisiae and Debaryomyces hansenii are the most common yeast species (Baroiller and Schmidt, 1990). The mould, Geotrichum candidum, appears at the same time as the yeasts but its growth is limited by salting. These fungi and Penicillium camemberti, by consuming lactate for their growth (Fig. 3), raise the pH (Fig. 2) and permit the growth of bacteria adapted to the water activity of the cheese such as staphylococci or coryneform bacteria. Debaryomyces hansenii and Kluyveromyces marxianus are usually added because their substrate consumption profiles are quite different. Indeed, Kluyveromyces consumes first lactose and only then lactate, but D. hansenii is able to consume both at the same time. In the past, Geotrichum candidum caused concern to cheese technologists because of its proteolytic activity and by causing a ‘toad skin’-like surface on the cheese. But by its enzymatic properties, it plays a major role in taste and flavour formation. Now, the selection of strains which do not cause ‘toad skin’, and better control of their use, has led to the widespread use of this species. Nowadays, in order to improve the organoleptic quality of Camembert made from pasteurised milk, selected strains of Geotrichum candidum, yeast and coryneform bacteria are generally added to the cheese milk, giving a product closer to traditional Camembert, and closer to the expectations of most consumers. Geotrichum is very sensitive to salt and therefore drysalting may stop its growth for a while. These yeasts, in starting to hydrolyse proteins and fat, will prepare the curd and help the growth of Penicillium. After 6 or 7 days of ripening, the growth

Surface Mould-ripened Cheeses 159

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Figure 2 Changes in pH at the surface (䉱) and in the core (䉭) in Camembert ripening (Leclercq-Perlat et al., 2003).

of P. camemberti is observed and a white felt covers the entire surface of the cheese. The growth of P. camemberti is extremely fast compared to that of the other members of the ripening flora. In 2 or 3 days, its growth is completed and changes surface pH, exhausts lactate at the surface and produces a large amount of CO2 which may change the gaseous environment of the ripening cellar. It is clear that P. camemberti plays a major role and imparts its characteristics to the cheese. However, the secondary flora plays an essential complementary role in the development of the organoleptic quality of traditional products.

Initially, two species of Penicillum were distinguished, P. caseicolum and P. camemberti. P. caseicolum is now considered to be a white mutant of P. camemberti. Different forms of P. camemberti can be distinguished (Moreau, 1979): • a form with a fluffy mycelium, white at first becoming grey-green; • a form with ‘short hair’, rapid growth, white, closenapped mycelium; • a form with ‘long hair’, rapid growth, white, loose, tall mycelium;

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Time (Days) Figure 3 Changes in lactate (䊊䊉) and in residual lactose (䉭䉱) at the surface (closed symbols) and at the core (open symbols) during the ripening of Camembert cheese (Leclercq-Perlat et al., 2003).

160 Surface Mould-ripened Cheeses

• ‘Neuchatel form’ – vigorous, rapid growth, giving a thick white-yellow mycelium. Only the white forms are used for cheesemaking. Commercial strains differ mainly in the rapidity of their growth on cheese and the density of their mycelium. Penicillium spores are produced by specialised companies after culturing in a fermentor or in ‘Roux flasks’. They can be added to the cheese milk, added to the surface in the form of a powder after curdmaking or mixed with the salt (when dry-salting is used). Bacteria

After 15–20 days, when the Penicillium has catabolised the lactic acid and deacidified the cheese, an aerophilic acid-sensitive bacterial flora becomes established on the surface. When the pH increases above 5.8, many bacteria grow again at the surface. Until recent work on taxonomy, most of the species involved in the ripening of white-mould cheese were described as belonging to Micrococcaceae and coryneform bacteria. Recent phylogenetic analysis of DNA changed the taxonomy of these groups (Irlinger et al., 1997; Stackebrandt et al., 1997; Irlinger and Bergère, 1999). Most of them belong to the huge coryneform bacteria group and others to Staphylococcus group and coliforms (e.g., Hafnia alvei). The most commonly found of these bacteria is Brevibacterium linens but a large diversity of coryneform bacteria, such as Arthrobacter, Micrococcus, Corynebacterium and Brachybacterium, is also present on these cheeses. They play a major role in flavour generation and on the appearance of the cheese. Interactions between micro-organisms

Microbiologists are not at ease with complex ecosystems like that of cheese, but it is clear that the ripening flora should be considered as a whole. Many strains of the bacteria mentioned above are able to produce a specific flavour when alone in a medium (even a cheese curd medium), but when associated to the other microorganisms, the results are completely different and most of the time the interesting flavour detected in pure culture is, for several reasons, lost when the organism grows in the cheese ecosystem. The first reason is that bacteria may not grow because of competition or inhibition but it is also possible that the metabolic pathways are not expressed because of chemical changes to the environment. Inside the cheese, lactococci are clearly predominant; the yeast population remains much lower than on the surface (about 106 cells g1 instead of 108 cells g1; Leclercq-Perlat et al., 2003). In the production of cheeses from pasteurised milk, the microflora

is less diverse, containing mostly micro-organisms added as starters, e.g., Lactococcus and P. camemberti. The populations of other micro-organisms are reduced and the cheese obtained has a more neutral aroma.

Glycolysis The lactic starters used to make Camembert are homofermentative mesophilic lactococci, and lactose breakdown leads essentially to the production of lactic acid by the hexose diphosphate pathway. For traditional Camembert, rennet is added to the milk after ripening when the pH is about 6.4. Intense acidification occurs mainly during draining, and the pH of the curd when taken from the mould is about 4.6. After the end of curdmaking, the surface fungal flora (i.e., yeast, Geotrichum and Penicillium in particular) use lactic acid for their growth. There is, as a result, a marked increase in the external pH and an internal migration of lactate towards the surface of the cheese. The surface pH increases steadily to about 7.0 at the end of maturation; the increase is slower in the interior, where the final pH is about 6.0 (Fig. 2). This neutralisation in cheese plays at least four different roles in the ripening process: 1. As previously mentioned, acid-sensitive bacteria, including micrococci and coryneform bacteria become established on the surface of mouldripened soft cheese and contribute to their traditional flavour qualities. 2. Neutralisation also favours the activity of ripening enzymes, the pH optimum of which is often close to neutrality. 3. Neutralisation also causes migration of minerals in the curd. Le Graet et al. (1983) showed considerable migration of calcium and phosphate towards the exterior of Camembert during mould growth on the surface. The rind of surface mould-ripened cheese attains high concentrations of calcium and inorganic phosphorus (17 and 9 g kg1, respectively) while the concentrations of these decrease at the centre. Le Graet et al. (1983) observed that the high pH of the surface causes the formation of insoluble calcium phosphate, immobilising this salt at the rind. Electron microscopic studies of the rind showed the presence of crystals which were identified tentatively as calcium phosphate. This is of nutritional interest as far as the mineral supply in surface mould-ripened cheese is concerned, depending on whether the rind is eaten or not, since at the end of ripening, the rind contains about 80% of the calcium and 55% of the phosphorus of the cheese.

Surface Mould-ripened Cheeses 161

4. As discussed below, the increase in pH markedly modifies the rheological properties and gives rise to a softer curd.

Proteolysis Although less important than in Blue cheeses, proteolysis in surface mould-ripened cheeses is quite significant. In the outer part of a ripe raw-milk Camembert, pH 4.6-soluble nitrogen represents about 35% of total nitrogen; within the cheese, there is less breakdown and only 25% of the nitrogen is soluble at pH 4.6. The soluble nitrogen fraction contains mainly small peptides (nitrogen soluble in 12% trichloracetic acid is about 20% of total nitrogen). In ripe traditional Camembert cheese, ammonia, resulting from the deamination of amino acids, is also present. Electrophoretic studies reveal strong degradation of s1-casein in the whole cheese while -casein is highly degraded in the outer part but clearly less in the centre. This high level of proteolysis is due to the presence of three agents: rennet, plasmin and microbial proteinases, among which enzymes synthesised by P. camemberti are dominant. Camembert retains more rennet in the curd than other cheese varieties because acidification occurs during draining. It has been observed that about 50% of the rennet added remains in the curd while about 15% is retained in pressed cheese (Vassal and Gripon, 1984). A degradation product of s1-casein by rennet, s1-I casein ( s1-CN f24-199), is detected by electrophoresis in Camembert after 6 h of draining and the concentration of this peptide increases during ripening. However, the pH of the outer part of Camembert increases quickly, reaching 6 or more after 2 weeks and can reach 7.0 after 3–4 weeks (Fig. 2). Under these conditions, one may suppose that the action of rennet (the pH optimum of which on caseins is about 5.5) decreases at the end of ripening when the pH has increased. An increase in the level of the -caseins, resulting from the degradation of -casein by plasmin, is observed at the end of ripening. This increased activity is not surprising since the pH of the outer region of Camembert at the end of ripening is not far from the optimum for plasmin (about 8.5). At the end of ripening, in the outer part of Camembert, this enzyme is probably more active than in semi-hard cheeses where the pH remains at about 5.2. Studies on aseptic curds (Desmazeaud et al., 1976), in which P. camemberti developed alone with no other micro-organism, have shown an extensive production of high and low molecular weight peptides, as well as of free amino acids. Thus, this mould has a high proteolytic potential due to the production of extracel-

lular endo- and exo-peptidases. It synthesises appreciable quantities of a metalloproteinase and an aspartate proteinase (Lenoir, 1984), which are optimally active at pH 5.5–6.0 and 4.0, respectively. Strains of P. camemberti have very similar enzyme profiles, with a variability of about 2-fold. The evolution of the proteolytic activity in curd has been studied in Camembert during ripening (Lenoir, 1970). At the centre of the cheese, this activity is very low. However, in the outer region it increases abruptly after 6–7 days of ripening, i.e., when the Penicillium begins to grow. Aspartyl proteinase and metalloproteinase are both synthesised in cheese and their concentrations are maximal after about 15 days and then decrease slowly. These two enzymes are thus fairly stable in cheese. Lenoir (1970) noted that the difference in the degree of proteolysis between the centre and the surface of Camembert was proportionally lower than the difference in proteolytic activity and suggested that the peptides migrate towards the centre of the cheese. Scanning electron microscopic studies of Camembert cheese show some lysis of the mycelium. However, electrophoregrams of cheese do not show the appearance of new hydrolytic products, indicating that intracellular proteinases play a much more limited role than the extracellular proteinases. P. camemberti produces large amounts of amino acids in cheese (Desmazeaud et al., 1976) due to the synthesis of extracellular exopeptidases. Ahiko et al. (1981) described an acid carboxypeptidase produced by P. camemberti, which is a serine enzyme with an optimum pH of 3.5, able to reduce the bitterness of a casein hydrolysate by releasing hydrophobic amino acids. An alkaline aminopeptidase, with a pH optimum of 8.5, has also been characterised (Matsuoka et al., 1991). Geotrichum candidum synthesises intra- and extracellular proteinases (pH optima near 6.0; Gueguen and Lenoir, 1976), but enzyme-production varies significantly from one strain to another (Gueguen and Lenoir, 1975). It is considered that its proteolytic action in cheese is clearly lower than that of P. camemberti since the proteolytic activity of the outer region of Camembert does not increase during the growth of Geotrichum but only during that of Penicillium (Lenoir, 1984). Also, Geotrichum alone seeded on the surface of the curds causes less proteolysis than P. camemberti alone (Vassal, personal communication). The proteolytic role of yeast is considered to be low. Schmidt (1982) observed an intracellular caseinolytic activity with an optimum pH of about 6.0 in 165 strains isolated from Camembert cheese. B. linens secretes extracellular proteolytic enzymes; several proteinases have been demonstrated (Foissy, 1974; Hayashi et al., 1990) and Rattray et al. (1995,

162 Surface Mould-ripened Cheeses

1996) purified and characterised an extracellular proteinase with pH and temperature optima of 8.5 and 50 °C, respectively. Extra- and intra-cellular aminopeptidases have been isolated and characterised (Foissy, 1978; Hayashi and Law, 1989; Rattray and Fox, 1997). These enzymes could participate in proteolysis of Camemberttype cheeses during late ripening but probably to a low extent. Rattray and Fox (1999) reviewed in detail the properties of the proteolytic system of B. linens. The presence and the action of lactic acid bacteria should not be forgotten. The cell-wall proteinase and the various peptidases contribute, as in other type of cheeses, to the hydrolysis of peptides produced by rennet, plasmin and microbial proteinases. The increase of pH in the outer part of Camembert could favour the action of the various peptidases since their optimum pH is generally near neutrality. Most of these enzymes have been isolated and characterised in the case of Lc. lactis, and their genes have been sequenced (see Christensen et al., 1999; Bolotin et al., 2001).

Lipolysis The intense degradation of fat is a common characteristic of mould-ripened cheeses. Moulds and yeasts are able to secrete a large diversity of lipases. These enzymes are active at the interface between fat globules and the continuous serum phase. Lipases (EC 3.1.1.3) hydrolyse triglycerides to form diglycerides, monoglycerides and free fatty acids. They are not very specific but can hydrolyse triglycerides more or less rapidly according to their molecular weight, with preferential liberation for fatty acids at positions Sn1 and Sn3. However, stricter specificities depending on the nature of the fatty acid can be observed. G. candidum synthesises two lipases, one of which preferentially liberates oleic acid and the other unsaturated C18 fatty acids at the Sn2 position of triglycerides (Veeraragavan et al., 1990; Bertolini et al., 1995). P. camemberti produces large quantities of an extracellular alkaline lipase (pH optimum – 9.0). At pH 6.0, this enzyme retains 50% of its maximal activity and remains very active at the temperature of ripening (Lamberet and Lenoir, 1976). It is the main lipolytic agent in Camembert cheese. It is more active on triglycerides composed of low molecular weight fatty acids. Other acids, such as C1–C4 or branched-chain C4 and C5 acids result from the action of micro-organisms on amino acids. The degradation of lactose by the same micro-organisms leads to acetic acid and propionic acid. For Camembert cheese, Kuzdzal-Savoie and Kuzdzal (1966) estimated that 5% of total free acids are not produced by lipolysis. From the organoleptic point of view, fat in cheese plays three main functions:

1. it is a component involved in the determination of the texture; 2. it is a solvent of the flavours molecules; 3. it constitutes a major precursor for the development of flavours. The role of fat in texture is as a lubricant, which gives the cheese a soft sensation in the mouth. The monoglycerides are very efficient emulsifiers and may reduce the size of fat globules in the cheese which may help to get a smoother mouth-feel and may also change flavour release (Wendin et al., 1999; Miettinen et al., 2002). Lipolysis is not homogeneous throughout the cheese and occurs mainly under the rind. Hassouna and Guizani (1995) reported that lipolysis is twice as intense just under the rind than in the interior of the cheese. The association of this phenomenon with proteolysis, and particularly the relatively high pH, gives the characteristic texture of the soft part of the cheese under a Camembert rind after a long ripening. As most of the flavour compounds are hydrophobic, most are more soluble in fat than in the serum. It is common to get ten times more flavour in the fat than in the water phase for flavour compounds with more than six carbon atoms (Overbosch et al., 1991). Free fatty acids are linear and have an even number of carbons. These compounds have quite high olfactive thresholds from a few mg kg1 to several hundreds mg kg1. But usually in this type of cheese the level of lipolysis is high. Blue cheeses undergo more intense lipolysis, reaching up to 50 meq/100 g of fat in a Danish Blue cheese while it is much less in surface mouldripened cheeses. In Camembert, lipolysis is usually less than 25 meq/100 g of fat and as low as 11 meq/100 g of fat in Brie (Vanbelle et al., 1978).

Flavours Flavours provided by fat catabolism Fatty acids

Long-chain free fatty acids (more than 12 carbon atoms) play a minor role in flavour, given their high perception thresholds. Short- and intermediate-chain even-numbered fatty acids (4–12 carbons) have much lower perception thresholds and each has a characteristic note (Molimard and Spinnler, 1996). Butyric acid has a rancid, cheesy odour. Octanoic, 4-methyloctanoic and, especially, 4-ethyloctanoic acids have odorous notes like that of goats. In goat cheeses, branched-chain fatty acids have much lower thresholds than the linear fatty acids. 4-Ethyl octanoic acid, has an olfactive threshold about 500 times lower than that of decanoic acid, which is linear with the same number of carbons. These fatty acids play a major role in the ‘goaty’ characteristics of these

Surface Mould-ripened Cheeses 163

cheeses. The young unlipolysed cheeses are much less goaty than the more ripened one. These branchedchain fatty acids are also present in ewes’ milk cheeses (Ha and Lindsay, 1991) but not in cows’ milk cheeses. According to their concentration and perception thresholds, volatile fatty acids can contribute to the aroma of the cheese or even, for some, give a rancidity defect. It is, in fact, the undissociated form of these acids which is aromatic. This form is found in the fat phase of the cheese, while the aqueous phase contains both forms, undissociated and ionised. Low pH reduces ionisation and increases volatility of the acids. Compounds produced by partial ␤-oxidation

A homologous series of methyl ketones with an odd number of carbon atoms, from C3 to C15, is one of the most important aroma compounds in the aroma of Blue and surface mould-ripened cheeses (Gallois and Langlois, 1990). The longer chain ketones, probably, are much less important than the intermediate ones because of their lipophilicity, which probably limit their volatility in a fatty matrix, like cheese. Several studies have established the pathway for the formation of these products in cheese (Dartey and Kinsella, 1973; Okumura and Kinsella, 1985). In Camembert, methyl ketones are by far the most abundant volatile flavour compounds, in the order of 25–60 mmol/100 g of fat, the two major compounds being nonan-2-one and heptan-2-one. All methyl ketones found in Camembert are also present in Blue cheese (Gallois and Langlois, 1990). Concentrations of heptan2-one and nonan-2-one in white-mould cheeses are very high and out of proportion considering the quantity of octanoic and decanoic acids present in milk fat, where the main fatty acid is palmitic acid (C16:0). We can therefore suppose that the corresponding fatty acids are not the only precursors of methyl ketones. Thus, the study of the oxidation of 14C-labelled palmitic and lauric acids by P. roqueforti spores has permitted elucidation of the formation of methyl ketones from long-chain fatty acids by successive cycles of -oxidation. Furthermore, addition of oleic acid (C18) in a milk-based medium causes an increase in the production of heptan-2-one and nonan-2one by P. camemberti, but the addition of lauric acid (C12) does not increase the production of undecan-2-one. Dumont et al. (1974a,b) isolated the aroma compounds of 11 samples of Norman Camembert by vacuum distillation and found 11 methyl ketones, all alkan-2-ones from C4 to C13, as well as octan-3-one (trace). The authors also identified 3-methylpentan-2one, 4-methylpentan-2-one, methylhexan-2-one (trace), nonan-2-one and undecan-2-one in larger amounts. The amounts of nonan-2-one, heptan-2-one and undecan-2one increased steadily during ripening.

In Camembert and Brie, most of the methyl ketones are present from the eighth day of ripening onwards but Moinas et al. (1973) identified butan-2-one and pentan2-one in young Camembert only (1–5% of the methyl ketones). These methyl ketones seemed to disappear during ripening. On the other hand, they observed an increase in the concentration of nonan-2-one during cheese ripening (1–5% of the methyl ketones in young Camembert compared with 20–40% of the methyl ketones in ripe Camembert) while the amount of heptan2-one remained constant (1–5% of the methyl ketones). From their odour notes typical of Camembert cheeses and from the amounts present in these cheeses, we are able to understand the important role played by ketones and methyl ketones in the aroma of these products. These volatile compounds are not only found in Camembert-type cheese, they are abundant in blue-veined cheeses in which heptan-2-one is the methyl ketone present in the largest quantity. Nonan-2-one, decan-2-one, undecan-2-one and tridecan-2-one are described as having fruity, floral and musty notes while heptan-2-one has a Blue Cheese note (Rothe et al., 1982). Oct-1-en-3-one has a mushroom note in aqueous media and a metallic note in lipid-rich media (Teranishi et al., 1981). Octa-1,5-dien-3-one is described as having a soil-like odour, octan-3-one a mushroom note and damascenone a woody note (Karahadian et al., 1985a). The principal agents in the formation of methyl ketones in mould-ripened cheeses are moulds, and the precursors are fatty acids. Methyl ketones are formed in a metabolic pathway which is connected to the -oxidation pathway. P. camemberti, P. roqueforti and Geotrichum candidum possess an enzymatic system which permits a diversion from the normal -oxidation pathway. The free fatty acid is oxidised to -ketoacylCoA. The action of a thiolase yields a -ketoacid which is rapidly decarboxylated by a -keto-acyl-decarboxylase to give a methyl ketone with one less carbon than the initial fatty acid; this metabolism has been extensively studied in yeasts (Fig. 4). For the micro-organism, this metabolic pathway represents a method for detoxifying fatty acids in the media. It needs only one molecule of coenzyme A, while complete degradation needs two. This mechanism allows faster recycling of the co-factor (Kinsella and Hwang, 1976). At low concentrations, fatty acids are oxidised completely to CO2 and H2O, and very low amounts of methyl ketones are formed (Margalith, 1981). -Oxidation is a particularly important metabolic pathway since 60% of the carbonyl compounds produced by P. camemberti on a milk-based medium are methyl ketones (Okumura and Kinsella, 1985). The mycelium of P. camemberti

164 Surface Mould-ripened Cheeses

O

R

C OH

ATP, CoASH AMP, ppi, H2O

Acyl CoA synthetase O R C SCoA

FAD

Acyl CoA oxidase

FADH2 O

R C

SCoA

H2O

Δ-Enoyl CoA hydratase R

O

C

Intra-chain oxidation of unsaturated fatty acids

SCoA

OH NAD

L-3-Hydroxy acyl CoA dehydrogenase

NADH2 O R

C SCoA

O

Thiolase HSCoA O R

C OH O Decarboxylase

CO2

work on the production of methyl ketones by moulds, especially P. roqueforti (Creuly et al., 1992). The production of heptan-2-one continues to attract attention because it is preponderant in Blue-type cheeses. New flavourings with high aromatic power appeared on the market to flavour sauces, crackers, etc. Secondary alcohols found in mould-ripened cheeses are mainly heptan-2-ol and nonan-2-ol, which represent, together with the methyl ketones from which they are derived, 10–20% and 5–10%, respectively, of all aroma compounds found in Camembert (Moinas et al., 1973). Dumont et al. (1974b) also isolated significant quantities of pentan-2-ol from ripe Camembert. However, Moinas et al. (1973) did not report this alcohol in mature Camembert and have identified this alcohol in young samples only.

R O

Figure 4 Fatty acid catabolism to methyl ketones by yeasts (from Ratledge, 1984).

is more sensitive to inhibition by fatty acids than that of P. roqueforti in spite of the fact that it uses fatty acids more rapidly. Mycelium and spores, but not germinating spores of Penicillium, are able to metabolise fatty acids to methyl ketones. The latter seem to be more sensitive to the inhibitory effect of fatty acids (Fan et al., 1976). G. candidum produces methyl ketones, including pentan-2-one, heptan-2one, nonan-2-one and undecan-2-one. It also produces pentan-3-one, which was found for the first time by Jollivet et al. (1994) in cultures of eight strains of G. candidum. The increasing demands for Blue cheese aroma compounds from the food industry gave rise to much

Linoleic and linolenic acid are precursors of 8C aroma compounds, particularly oct-1-en-3-ol, oct-2-en-1-ol, octa-1,5-dien-3-ol and octa-1,5-dien-1-ol and ketones such as octan-2-one, oct-1-en-3-one and octa-1,5dien-3-one. Oct-1-en-3-ol is well known for its raw mushroom odour. Considering its low perception threshold (0.01 mg kg1), it gives Camembert cheese aroma a characteristic note. This compound is, without a doubt, one of the key compounds in the overall aroma of Camembert. Flavouring attempts by Moinas et al. (1975) on a neutral cheese base have shown that it is possible to mask partially the blue note of methyl ketones by oct-1-ene-3-ol. At concentrations of 5–10 mg kg1 in the cheese base, these authors obtained a flavour close to mature Camembert. If it is present in too large amounts, it is responsible for an aroma defect. Oct-1-en-3-ol represents 5–10% of the volatile compounds in Camembert. On the other hand, it is present in only very small amounts in young Camembert. Furthermore, its production is bound to P. camemberti metabolism, appears only late in cheese ripening and is a result of the secondary metabolism (Spinnler et al., 1992). Even-chain methyl ketones, except for butan-2-one, are probably produced by intra-chain oxidation. They are never present in large amounts, except in very ripe cheese. Camembert made from raw milk contains more even-chain methyl ketones and branched-chain methyl ketones. It was suspected by Karahadian et al. (1985a,b) that P. camemberti is capable of intra-chain oxidation of linoleic and linolenic acids, as are basidiomycetes (Chen and Wu, 1984). The principal enzymes believed to be involved are a lipoxygenase and a hydroperoxide-lyase, which are commonly present in moulds. Recently, Perraud and Kermasha (2000)

Surface Mould-ripened Cheeses 165

demonstrated that P. camemberti has lipoxygenase activity able to produce 9-, 10- and 13-hydroperoxy acids from polyunsaturated fatty acids. Perraud et al. (1999) also demonstrated lipoxygenase activity in G. candidum. Some aldehydes found in Camembert cheese, such as hexanal, heptanal and nonanal, are due to fat oxidation. Hexanal and (E)-hex-2-enal are known to give the green note of immature fruit. Their perception threshold in water is 9 and 24 g kg1, respectively (Ahmed et al., 1978). Octanal, nonanal, decanal and dodecanal are described by these authors as having an aromatic note, resembling orange. Their perception threshold in water is 1.4, 2.5, 2 and 0.5 g kg1, respectively. Lactones

Lactones found in Camembert are -decalactone,

-decalactone, -dodecalactone and -dodecalactone. These compounds have also been identified in Blue cheeses (Gallois and Langlois, 1990). From an organoleptic point of view, lactones are generally characterised by very pronounced fruity notes (peach, apricot, coconut). -Lactones have a generally higher detection threshold than those of

-lactones. These thresholds are relatively low for

-octalactone, -decalactone and -dodecalactone (7–11 g kg1 in water) and are lower for shorterchain lactones (Dufossé et al., 1994). Lactone precursors are hydroxylated fatty acids. The intra-molecular esterification happens under the action of pH and/or micro-organisms. The action of micro-organisms in the production of lactones has never been clearly demonstrated in cheese. Hydroxyacids, which are direct precursors of lactones, are present in triglycerides in milk. Lipases can liberate them and they are then cyclised to form lactones. Nevertheless, hydroxylated fatty acids can come from the normal catabolism of fatty acids and can be generated from unsaturated fatty acids by the action of lipoxygenases or hydratases. P. roqueforti spores can form 12-carbon lactones from long-chain saturated fatty acids (C18:1, C18:2). Chalier and Crouzet (1992) performed the bioconversion with spores of P. roqueforti using soya and copra oils as substrates. Flavour compounds produced from amino acid catabolism

The most common pathway, used by micro-organisms for amino acid breakdown, is Erhlich’s pathway which leads to the production of branched-chain aldehydes, branched-chain alcohols or branched-chain acids from the branched-chain amino acids. Primary and secondary alcohols, along with ketones, are considered to be very important compounds in the

aroma of soft, mould-ripened cheeses. Regarding primary alcohols, 3-methylbutan-1-ol is present in relatively large quantities in Camembert and has an alcoholic, floral note. Phenyl-2-ethanol, with a perception threshold in a cheese base of 9 mg kg1 and a characteristic rose floral note (Roger et al., 1988) and its ester, phenylethylacetate, play an important role in raw-milk Camembert where they are always present in important amounts (Dumont et al., 1974b). This alcohol is one of the major compounds in Camembert after 7 days of ripening, at a concentration of 1.15 mg kg1. Its concentration stabilises at approximately 1 mg kg1 at the end of ripening. It is lower than the detection threshold (9 mg kg1) but is close to the detection threshold of the most sensitive panelist of the panel used in the study of Roger et al. (1988). In fact, these authors thought that phenylethanol and its esters have cumulative effects to give the perceptible floral note in certain Camembert cheeses. This alcohol is produced mainly during the first week of ripening, because it is, mainly, a metabolic product of yeasts (Lee and Richard, 1984). Ethanol, propan-2-ol, butan-2-ol, octan-2-ol and nonan-2-ol are also encountered in most soft cheeses. Eleven alcohols have been identified and quantified in two types of Brie. Ethanol and short-chain linear alcohols only have a limited aromatic role in cheese but are the precursors of several esters. By way of oxidative deamination or transamination, amino acids can be transformed to -ketoacids which can then be decarboxylated to aldehydes. The aldehydes can then be reduced to the corresponding primary alcohols or oxidised to acids. It has been shown in many models that the deamination/transamination step is very often a rate-limiting step in amino acid catabolism. Products arising from the reduction of aldehydes include 2-methylpropanol, 3-methylbutanol, 2-methylbutanol, 3-methylpropanol and phenylethanol. Production of phenylethanol from phenylalanine seems to be mainly performed by yeasts (Lee and Richard, 1984). In the same way, P. camemberti catabolises valine to 2-methylpropanol and leucine to 3-methylbutanol. The eight strains of G. candidum studied by Jollivet et al. (1994) produced isobutanol. Aldehydes

The main aldehydes found in Camembert are 2methylbutanal, 3-methylbutanal and benzaldehyde. These compounds, mostly at trace levels, are present as early as the first week of ripening in surface mouldripened cheeses like Brie and Camembert. Benzaldehyde is described as having an aromatic note reminicent bitter almond. Its detection threshold in water is 350 g kg1 (Buttery et al., 1988). With

166 Surface Mould-ripened Cheeses

detection thresholds in malt culture media of 0.1, 0.13 and 0.06 mg kg1, respectively, 2-methylpropanal, 2-methylbutanal and 3-methylbutanal are also encountered in cheeses, including mould-ripened cheeses (Margalith, 1981). These compounds, can be oxidised to isobutyric, 2-methyl butyric and isovaleric acids. These acids are described as having a mild odour, reminicent of sweat. Aldehydes originate from amino acids either by transamination, leading to an -ketoacid which can be decarboxylated, or by chemical degradation. This last reaction is simple and can occur without enzymatic catalysis during ripening. Aldehydes are transitory compounds in cheese since they are transformed rapidly to alcohols or corresponding acids. Yeasts can contribute to the production of ethanal when alcohol dehydrogenase is less active than pyruvate decarboxylase. The biosynthesis pathway for benzaldehyde was determined recently by Nierop-Groot and de Bont (1999). It was shown that a chemical breakdown of phenylpyruvic acid is catalysed by divalent cations such as Mn2. This pathway seems also to be used for other amino acids, such as methionine, producing 2-methyl thioethanal which has a green-apple flavour (Yvon et al., 2001). Amines

For some micro-organisms, the breakdown of amino acids starts by decarboxylation, with the production of amines. Numerous volatile amines have been identified in Camembert cheese, including methylamine, ethylamine, n-propylamine, isopropylamine, n-butylamine, 1-methylpropylamine, n-amylamine, iso-amylamine, anteiso-amylamine, n-hexylamine, ethanolamine, dimethylamine, diethylamine, dipropylamine and dibutylamine (Adda and Dumont, 1974). Dimethylamine has been detected in Camembert and in Blue cheeses at 0.811–1.623 mg kg1. Nitrosamine have also been described in Camembert at a level of 25 nmol/10 g but has not been identified in Blue cheeses. We should keep in mind that ammonia, derived from amino acid deamination, is also an important element of Camembert aroma. P. camemberti, G. candidum and B. linens play major roles in ammonia production by deamination of amino acids (Karahadian and Lindsay, 1987). Many volatile amines are described as having fruity, alcoholic or varnish-like aroma notes. Ethylamine and butylamine have perception thresholds in water from 0.83 to 3.63 mg kg1 and 0.24 to 13.9 mg kg1 of free base, respectively (Laivg et al., 1978). Methylamine, dimethylamine and propylamine have perception thresholds in water of 182, 34.4 and 62.4 mg kg1 of free base, respectively. Tertiary amines have much lower perception thresholds. Triethylamine,

with a fishy odour, is perceived at a concentration of 0.47 g kg1 of free base in water. Some people have a specific anosmia for this amine which is a widespread pheromone in mammalian species. Amine biosynthesis

Decarboxylation of amino acids leads to the production of CO2 and amines. This reaction needs the presence of pyridoxal-phosphate and co-enzyme. Decarboxylation of leucine gives isobutylamine, phenylalanine gives phenylethylamine and tyrosine gives tyramine. A low oxygen pressure favours these reactions. Amines are not final products but are subjected to oxidative deamination to form aldehydes. They can also be the starting point of compounds like N-isobutylacetamide encountered in Camembert, presumably by reaction with acetic acid. Catabolism of amino acid side chains Indole ring

Degradation of the side chains of tyrosine and of tryptophan by tyrosine-phenollyase and by tryptophan-indole lyase, respectively, leads to the formation of phenol and indole. Parliment et al. (1982) considered that phenol found in Limburger results from degradation of tyrosine by B. linens. The catabolism of tryptophan by B. linens has been recently studied by Ummadi and Weimer (2001). In model media, tryptophan was broken down to anthranilic acid at a high rate. However, the physicochemical environment of ripening cheese is quite far from optimal conditions and these authors concluded that it is unlikely that B. linens could be responsible for faecal, putrid or meaty-brothy defects in Cheddar cheese. Sulfur compounds

During their work on the identification of minor components present in aromatic extracts of Camembert, Dumont et al. (1976a,b) isolated four sulfur compounds from a fraction with a garlic flavour note: 2,4-dithiapentane, diethyldisulfide, 2,4,5-trithiahexane and 3-methylthio-2,4-dithiapentane. These authors also identified traces of a sulfur-containing alcohol, 3-methylthiopropanol (or methionol), and ethyldisulfide. Other sulfur compounds are also found in Camembert cheese. Disulfides are generally absent from young cheeses. In these cheeses, a low level of proteolysis yields only a low level of sulfur amino acids, precursors of disulfides. In late ripening, sulfur compounds are quantitatively reduced and even disappear in some products. This can be explained by their high volatility. Nevertheless, in Brie cheese, Karahadian et al. (1985a) found sulfur compounds (dimethyldisulfide, dimethyltrisulfide and methionol) only in aged Camembert cheeses with a growth of

Surface Mould-ripened Cheeses 167

Brevibacterium linens and other coryneform bacteria. Sulfur compounds found in cheeses are described as having a strong garlic or ‘very ripe cheese’ odour. Furthermore, these compounds have a very low detection threshold in water, from 0.02 g kg1 for methanethiol to 0.3 g kg1 for dimethylsulfide (Shankaranarayana et al., 1974). Sulfur compounds originate principally from methionine degradation, resulting from a carbon–sulfur bond cleavage by a methionine- -demethiolase. This amino acid is a precursor of methanethiol which is itself the starting point for some other compounds, including dimethyldisulfide and dimethyltrisulfide. Many microorganisms are able to produce methanethiol from methionine. Among the ripening fungi many have this potential, such as P. camemberti, G. candidum and Y. lipolytica (Bonnarme et al., 2001; Spinnler et al., 2001). Molimard et al. (1997) have shown that some strains of G. candidum, although its growth was quite early in the ripening process, were able to change the characteristics of a Camembert cheese irrespective of which of the four strains of P. camemberti were used (Fig. 5). One strain of G. candidum caused the development of cabbage and cowshed notes. It was then shown that G. candidum growing in a curd medium enriched with methionine was able to accumulate a large variety of sulfur compounds, including various thioesters such as methylthioacetate, methylthiopropionate, methylthiobutyrate, methylthioisobutyrate, methylthioisovalerate (MTIV) and methylthiohexanoate (MTH; Berger et al., 1999a). These thioesters have various flavour notes, from cheesy (MTIV) to fruity (MTH; Berger et al., 1999b). Recently, the metabolism of G. candidum was explored and it was shown that this species, unlike B. linens, was able to accumulate 2-keto-4-methylthiobutanoic acid as an intermediate in catabolism

(Bonnarme et al., 2001). The origins of different sulfurflavour compounds are summarised in Fig. 6. Smear bacteria have also been studied but mainly B. linens or Arthrobacter spp. (Bonnarme et al., 2000). Among these micro-organisms, coryneform bacteria, especially B. linens, are considered as the key agents in the production of sulfur compounds in cheeses in which they grow. The production of sulfur compounds by pure cultures of B. linens has been studied by Tokita and Hosono (1968) and Law and Sharpe (1978). Jollivet et al. (1994) studied the production of dimethyldisulfide by six out of eight strains of G. candidum in a cheese-based model system. It was recently shown that dimethylsulfide is produced by G. candidum using a separate pathway than methanethiol production from methionine (Demarigny et al., 2000). Styrene

Styrene has a very strong plastic-like odour. Its perception threshold in cream is 5 g kg1. This compound has been described as a trace component in several cheeses, including Camembert (Dumont et al., 1974c). Adda et al. (1989) found an abnormally high quantity of styrene (5 mg kg1) in Camembert with a pronounced celluloid taste. These authors demonstrated the role played by P. camemberti in the production of this hydrocarbon. Spinnler et al. (1992) observed a correlation between the production of styrene and oct-1-en-3-ol in a minimal medium. These two compounds were produced after 15 days of culture, when there is no more glucose in the growth medium. Oct-1-en-3-ol is produced 2–3 days before styrene. 13C-styrene is produced from 13C-phenylalanine (Spinnler, unpublished data) suggesting that this amino acid is the precursor of styrene.

PCA 2 (26.7%)

Miscellaneous compounds Esters

5 4 3 2 1 0 –1 –2 –3 –4

Cabbage, cow-shed

P2G2

Intensity, Blue Cheese

P3G2 Milky, creamy

P4G3 P3G3 P4G1 –6

–4

P4G2 P2G3 P4 P1G3 P3G1

–2

P2G1 P1G2

P2

P1G1 P3

0 2 PCA 1 (45.8%)

Cardboard,

P1 styrene 4

6

8

Figure 5 Changes in the flavour profile of Camembert cheese made with a pure culture of P. camemberti (strains P1–P4) or in association with G. candidum (strains G1–G3). Results where obtained with a trained panel (Molimard et al., 1997).

There is a great diversity of esters in cheese. Esters have been identified to the corresponding acids and alcohols present in Camembert. 2-Phenylethylacetate and 2-phenylethylpropanoate are qualitatively important in the flavour of Camembert cheese. On the seventh day of ripening, 2-phenylethylacetate is the principal compound in the aromatic profile, at a concentration of 4.6 mg kg1. This concentration then decreases and stabilises around 1 mg kg1 (Roger et al., 1988). Methylcinnamate, identified in Camembert by Moinas et al. (1975), seems to be particularly important in the aroma of this cheese. When varying the concentration of this compound in a neutral cheese base, to which heptan-2-one, heptan-2-ol, oct-1-en-3-ol,

168 Surface Mould-ripened Cheeses

L-Methionine (CH3S–CH2–CH2–CH(NH2)–COOH)

L-Methionine

γ-lyase

Aminotransferase (+ acceptor)

α-KG GDH

Methylthioacetaldehyde

NH3 α-KB

Reduction α-HB

(CH3–CH2–CO–CO2H) + Acyl-CoA + METHANETHIOL

Glu KMBA

Reduction HMBA

(CH3S–CH2–CH2–CO–CO2H)

KMBAdecarboxylase

KMBAdemethiolase

Methional (CH3S–CH2–CH2–CHO) α-KB +

METHANETHIOL (CH3SH)

Thioesters (CH3S–CO–R)

Methionol and methionol acetate

Auto-oxidation

AA, FFA, Sugars DMDS, DMTS

Other volatile sulfur compounds

Figure 6 Pathways for the catabolism of methionine by B. linens (left), in G. candidum the pathway using a transaminase and demethiolation of the KMBA (right) has been demonstrated but the existence of a methionine- -lyase (left) cannot be excluded at the moment. -KG, -ketoglutarate, KMBA, -keto- -methyl thio butyric acid; HMBA, 2-hydroxy-4-methylthio-butanoic acid; -KB, -keto butyrate; AA, amino acids; FFA, free fatty acids; DMDS, dimethyl disulphide; DMTS, dimethyl trisulphide; GDH, glutamate dehydrogenase.

nonan-2-ol, phenol and butyric acid had been added, these authors developed a characteristic Camembert note. Most of the esters found in cheeses are described as having fruity, floral notes. The most-cited aromatic notes of these compounds are pineapple, banana, apricot, pear, floral, rose, honey and wine. Some of these esters have a very low perception threshold, e.g., isoamylacetate which is detectable in water to a concentration of 2 g kg1 (Piendl and Geiger, 1980). Low carbon number esters have a perception threshold approximately ten times lower than the corresponding alcohol. Esterification reactions occur between alcohols derived from lactose fermentation (ethanol) or from amino acid catabolism, and short- to medium-chain carboxylic acids. For example, acetates come from transesterification of alcohols with acetyl-CoA. These reactions are well-known detoxification reactions in media, enabling the elimination of toxic alcohols and carboxylic acids. A wide variety of enzymes are involved in esterification reactions, including carboxylesterases, which have a very wide range of substrates, and arylesterases, present in most of the micro-organisms which con-

tribute to cheese ripening. Ester formation has been studied widely in fermented beverages in which they play an important aromatic role. Their production is due to yeast activity. In all cheeses, micro-organisms involved in ester production seem to be mainly yeasts. Production of esters occurs early during ripening. G. candidum is capable of producing numerous esters, some of which have a very pronounced melon odour. However, Latrasse et al. (1987) observed ester production by only one strain of G. candidum. Terpenes

Terpene alcohols such as 2-methylisoborneol (2-MIB) are produced by P. camemberti. 2-Methylisoborneol having a musty flavour but a very low detection threshold (0.1 g kg1) is the reason for its role in the soft and the mould-ripened cheeses (Karahadian et al., 1985a,b). Pyrazines

Dimethylpyrazine and trimethylpyrazine were identified in Camembert by Dumont et al. (1976a,b). 2,5Dimethylpyrazine, which has a ‘toasted hazel nut note’, can be produced from threonine. 2-Methoxy-3-iso-

Surface Mould-ripened Cheeses 169

propylpyrazine comes from the degradation of L-valine, as demonstrated in Pseudomonas taetrolens (Gallois, 1984). This pyrazine is responsible for an aroma defect in Camembert, in which it causes a rotten soil, raw potato note. Its perception threshold is very low (0.002 g kg1 in a milk medium) and therefore it is important when present, even in very low amounts. Volatile contaminants

Many chlorinated compounds, present at trace levels, have been found in Camembert including chloroform, carbon tetrachloride, dichloroethane, trichloroethane, tetrachloroethylene, dichlorobenzene and trichlorobenzene. For those compounds, an external origin is likely: pesticide, cleaning agents, pollution or artefacts due to solvent extraction during analysis. Likewise, benzene and its derivatives have been identified, e.g., ethylbenzene, dimethylbenzene, trimethylbenzene, and the one derivative of toluene. Most of the studies demonstrating the presence of traces of these compounds have been done on extracts obtained by the use of solvents, where the origin of these compounds is likely to be impurities in the solvent used. But the recent intensive use of dynamic headspace confirms the occurrence of these compounds in cheeses, the fat of which is a good trap for these volatiles (Spinnler, 2003). Among the large number of compounds present in the volatile fraction of Camembert, methyl ketones and alcohols (oct-1-en-3-ol, 2-phenylethanol, etc.), as well as 2-phenylethanol acetate, are quantitatively the most important. These products, along with sulfur compounds, play an important role in the aromatic note of this cheese. On the other hand, we have very little knowledge at this time on the aromatic importance of most of the molecules often present at trace levels in this cheese. At present, it is not possible to say which compounds determine the organoleptic quality of these cheeses. The aroma of soft and mouldripened cheeses is, in fact, the result of a subtle and fragile equilibrium between all the numerous volatile compounds they contain.

Texture The outer part of Camembert undergoes considerable modification of texture, and the curd which is firm and brittle at the beginning of ripening, later becomes soft. Softening is visible in a cross-section of the cheese and gradually extends towards the centre. The water content of Camembert is about 55% and, if it is too high, the outer part tends to flow when the ripe cheese is cut. These changes were previously attributed to the high level of proteolysis created by P. camemberti.

However, the diffusion of fungal proteases is limited and can affect only the outer few millimetres. The most important change caused by P. camemberti and the surface flora is the establishment of a pH gradient from the surface to the centre due to the consumption of lactic acid and the production of ammonia (Fig. 7). This pH gradient can be simulated by incubating young Camembert (3 days of ripening without inoculation with Penicillium) in an ammoniacal atmosphere. The ammonia dissolves in the curd and, after equilibration, the pH gradient established is expressed by cheese softening; this process is more evident near the surface where the pH is highest (Vassal et al., 1984). Increasing the pH, therefore, plays an important role causing the cheese to soften. This may be explained by the fact that the increase in pH augments the net charge on caseins and modifies protein–protein and protein–water interactions. It also changes the water absorption capacity and the solubility of the caseins. According to Noomen (1983), the physico-chemical conditions (water content and pH) in Camembert alone cannot explain softening, which could also be related to rennet action. Indeed, experimental cheeses, containing no rennet and incubated in an ammoniacal atmosphere, do not soften but become hard and springy, while cheese with rennet activity softens. The softening of Camembert could thus be explained by three processes: breakdown of s1-casein by rennet, increase in pH caused by the surface flora and to the outward migration of Ca2 in response to the pH gradient (see Fig. 7).

Control of Ripening The choice of the P. camemberti strain is important in the production of soft surface mould-ripened cheeses. However, the proteolytic activity of the different strains does not vary as much as their lipolytic and -oxidative activities (Lenoir and Choisy, 1970; Lamberet et al., 1982). The choice of a P. camemberti strain is also guided by the growth rate, colour, density and height of the mycelium, which contribute to the appearance and attractiveness of surface mould cheeses. Salting has a selective effect on the mould in soft cheeses. Too much salting limits the growth of G. candidum, while the growth of P. camemberti is much less affected. In whey culture, the growth of P. camemberti is slowed down when 10–15% salt is present. Conversely, too little salt, combined with insufficient draining, causes excessive growth of G. candidum and hinders the implantation of Penicillium, giving defective cheese; this defect is called ‘toad skin’. Under-salting may also favour the surface implantation of Rhizomucor, altering

170 Surface Mould-ripened Cheeses

Inner cheese mass

Inner cheese mass = 8–14 mm; sub-rind = 0–6 mm and cheese rind = 1–3 mm

(higher)

Lactate concentration gradient

(lower)

(higher)

Soluble Ca/PO4 concentration gradient

(lower)

(higher)

H+ concentration gradient

(lower)

Water gradient

(lower)

(lower)

NH4 concentration gradient

(higher)

(lower)

ASN and NPN concentration gradients

(higher)

(higher) +

Lactate metabolised

Ca3(PO4)2

NH3 produced

Cross-sectional view Cheese surface Surface microflora enzymes Figure 7 Gradients in Camembert cheese which may lead to the biological deacidification of the cheese centre through the migration of lactate. (ASN) Acid-soluble nitrogen, (NPN) Non-protein nitrogen (Leclercq-Perlat et al., 1999).

the appearance of the cheese; this defect is called ‘cat hair’. Reducing the water activity by higher salting and using a Penicillium strain that implants quickly helps to correct this defect (Choisy et al., 1984). Salting also influences the activity of Penicillium enzymes, and at 4% it reduces the degree of proteolysis in Camembert (25% of pH 4.6-soluble nitrogen versus 40% in an unsalted control). The effects of humidity and temperature in the ripening room on the growth of P. camemberti and the quality of Camembert-type cheese have been described by von Weissenfluh and Puhan (1987). The production of soft surface mould-ripened cheeses using milk highly contaminated with psychotrophic bacteria leads to organoleptic defects. The lipolytic activity of these bacteria is expressed by increased lipolysis and a rancid taste; bitterness has also been reported (Dumont et al., 1977). Listeria monocytogenes is able to survive the Camembert cheesemaking process and grow during ripening of the cheese. Control of L. monocytogenes (not detectable in 25 g of cheese) is obtained by the selection of good quality milk, adequate heat treatment and avoiding contamination during cheesemaking through good hygienic practices (good equipment design and appropriate cleaning and disinfection). Bacteriocin-producing lactic acid bacteria can also be used for cheesemaking. The number of L. monocytogenes in curd can be reduced very much by using strains of Lc. lactis that produce nisin (Maisnier-Patin et al., 1992). As mentioned above, uncontrolled development of G. candidum causes defects in the appearance and the taste of cheese, even though this mould probably contributes

significantly to the taste qualities of Camembert. Some strains of G. candidum clearly improve the taste and the aroma of Camembert cheese made from pasteurised milk. Their controlled growth results in a more typical Camembert flavour, close to that of traditional Camembert (Molimard et al., 1997). As in all cheeses, acidification of surface-mould cheese plays an essential role by controlling syneresis and the degree of mineralisation. When acidification is too high, the Camembert curd is too dry and brittle and enzyme activities are limited; insufficient acidification results in a cheese, the moisture content of which is too high at the end of ripening. The last 20 years have witnessed an increasing interest in ‘stabilised’ cheeses. Washing the curd permits a higher pH to be obtained at the end of draining. This gives a less demineralised cheese that seems more ripe than a traditional cheese of the same age. These cheeses made from pasteurised milk have a milder taste and keep better than Camembert made using the traditional technology. This could be due to more limited activity of P. camemberti, perhaps because of the lower levels of available lactose and lactate. Due to their higher pH, these products are more sensitive to coliform bacteria. An investigation in France by Pelissier et al. (1974) showed that mould-ripened cheeses are more sensitive to bitterness than other varieties and the intensity of this defect may cause considerable damage to cheese quality. More recently, Molimard et al. (1994) have shown that G. candidum is able to ameliorate this defect, possibly due to its very efficient peptidase system compared to P. camemberti, which is more proteolytic than peptidolytic. P. camem-

Surface Mould-ripened Cheeses 171

berti plays a crucial role in the appearance of bitterness in Camembert. Excessive growth of the mycelium can lead to the defect; if Penicillium growth is limited by the presence of G. candidum or by incubating the cheese in an ammoniacal atmosphere, proteolysis is reduced and the defect does not occur. Therefore, this defect could occur when there is too much proteolysis by Penicillium proteases (Vassal and Gripon, 1984). The level of rennet used and its augmentation does not seem to cause bitterness, perhaps because the pH of Camembert does not favour the action of rennet proteinases at the end of ripening. Lactic acid bacteria and their proteinases have also been reported to affect the occurrence of bitterness. The defect appears when a high population of lactic acid bacteria is present in the curd; on the other hand, if these populations are reduced (for example, by infection with bacteriophage), bitterness does not occur (Martley, 1975). This seems to be related to the degree of curd acidification, since the probability of bitterness is increased if the pH is low at the end of draining (Vassal, personal communication). Bitterness might not result directly from high amounts of lactic acid bacteria but could be related to Penicillium, the growth of which, and protease production, might be higher in very acid curds. In Camembert cheese, another very important point is mass transfer in the curd, from the core of the cheese to the surface or in reverse from the surface to the core. Due to different parameters such as relative humidity, lactate concentration gradient, moisture gradient, pH gradient and microbial activity, lactate migrates from the core to the surface leading to deacidification from the surface towards the core (Fig. 7). This deacidification changes the texture under the rind and even inside the curd. The faster the migration, the quicker is the ripening of Camembert. The stimulation of the microflora activity may lead to an exhaustion of their usual substrates, lactose and lactate. In that case, proteins and lipids are broken down, and is one of the possible reasons for the production, by P. camemberti, of styrene or 1-octen-3-ol. This risk increases when the ripening temperature is abnormally high or when the curds have been washed (stabilised curd technology; Spinnler et al., 1992).

Conclusion The particular characteristics of P. camemberti are expressed in surface mould-ripened cheeses, giving the cheeses their characteristic appearance and contributing to the development of the rheological and sensory qualities. However, the secondary micro-flora contribute to the attainment of the traditional quality

of this variety. Great progress has been made during the last 20 years in our knowledge of the mechanisms of ripening in surface mould-ripened cheeses. However, the processes are very complex and no close relationship can yet be seen between the composition and the quality of mould-ripened cheese. While studies on traditional mould-ripened cheeses should not be abandoned, it should be remembered that more cheeses are now being produced in large, automated factories. The good quality of these products must be maintained, taking into account consumer taste, which often favours rather mild products. Improving the storage life of surface mould-ripened soft cheese should also make it easier to distribute and to expand its production.

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174 Surface Mould-ripened Cheeses

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Blue Cheese M.D. Cantor, Danisco A/S, Innovation, Denmark T. van den Tempel, Chr. Hansen A/S, Cheese Culture Technology, Denmark T.K. Hansen Leo Pharma A/S, Microbiological Research Laboratory, Denmark Y. Ardö, The Royal Veterinary and Agricultural University, Department of Dairy and Food Science, Frederiksberg, Denmark

Introduction

Microenvironment in Blue Cheese

Blue cheeses are characterised by the growth of the mould Penicillium roqueforti, giving them their typical appearance and flavour. Many countries have developed their own types of Blue cheese, each with different characteristics (Table 1) and involving different manufacturing methods (Fig. 1). The bestknown varieties today, worldwide, are considered to be Gorgonzola, Roquefort, Stilton and Danablu, all of which have been granted the status of Protected Designation of Origin/Protected Geographical Indication (PDO/PGI), together with a number of other European Blue cheeses. Blue cheeses have probably been produced for a long time, either deliberately or by accident, before they were described in writing. Gorgonzola was the first Blue-veined cheese to be mentioned in the literature, in 879, while Roquefort was described in customs papers in 1070; however, already in the eighth century chronicles from monasteries mention the transport of Roquefort across the Alps (Kloster, 1980). Stilton was not mentioned until the seventeenth century. In Denmark, the production of Danablu and Mycella, Blue cheeses from cow’s milk, started in the 1870s. In 1916, a method for homogenising the cream was developed and used for the production of Danablu, making the cheese as white as the traditional Roquefort made from sheep’s milk. Additionally, homogenisation influences ripening by accelerating lipolysis. As Blue cheeses are becoming more and more popular, there has been increased interest in the scientific characterisation of the various types. This chapter aims to review the present knowledge of different aspects of Blue cheese ripening, emphasising changes in the microenvironment, micro-organisms that contribute to ripening and various biochemical changes, i.e., lipolysis, proteolysis and aroma formation. Finally, recommendations for the selection of appropriate starter and mould cultures, as well as new, possible adjunct cultures, will be discussed.

The microenvironment in Blue cheese is, in general, heterogeneous with pronounced gradients of pH, salt, water activity (aw), etc. The ripening temperature is typically 8–15 °C, depending on the variety. Furthermore, there are considerable structural differences within these cheeses, which influence the level and distribution of O2 and CO2. These parameters and their changes during the course of ripening have a great impact on the growth and biochemical activity of the various micro-organisms present in the cheese and thereby the quality of the final product. Therefore, knowledge of the levels encountered at different ripening times is important in order to construct realistic model systems. The minimum pH of Blue cheese ranges from approximately 4.6–4.7 in Danablu (Hansen, 2001), Mycella (Hansen et al., 2001) and Stilton (Madkor et al., 1987a) to 5.15–5.30 in Gorgonzola (Gobbetti et al., 1997), Picón Bejes-Tresviso (Prieto et al., 1999, 2000) and Cabrales (Alonso et al., 1987). The conversion of lactose to lactic acid by the lactic acid bacteria (LAB) of the primary starter culture is facilitated by the manufacturing procedure; the curd is very moist when placed in hoops and no pressure is applied during whey drainage (2–3 days), giving the LAB access to large amounts of lactose. The amount of residual lactose decreases very quickly. In 15-day-old Gamonedo Blue cheese, the lactose content was only 0.15% of total solids (Gonzàlez de Llano et al., 1992) whereas 0.9% lactose was found in 1-day-old Picón Bejes-Tresviso cheese, after which it was no longer detectable (Prieto et al., 2000). During ripening, the pH of Mycella increases to 6.5 in the core and to 5.9 in the surface layer (Hansen et al., 2001). Similar values were found in Danablu, as depicted in the partial least squares (PLS) plot in Fig. 2 (Hansen, 2001), and for other varieties of Blue cheeses (Zarmpoutis et al., 1997). However, higher pH values have been reported as well (Gonzàlez de Llano et al., 1992; Zarmpoutis et al., 1996, 1997; Gobbetti et al., 1997). The pH of the interior rises more rapidly than

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176 Blue Cheese

Table 1 A few examples of Blue cheeses and the range in reported gross composition

Name

Origin

Cabralesx Chetwynd Danablux Gamonedoa

Spain Ireland Denmark Spain

Gorgonzolax Kopanistix

Italy Greece

Kvibille Ädel Picón Bejes-Tresvisox

Sweden Spain

Roquefortx Stiltonx

France Great Britain

Type of milk used for production Raw cows’ milk Pasteurised cows’ milk Thermised cows’ milk Raw cows’, goats’ and ewes’ milk Pasteurised cows’ milk Raw cows’, goats’ or ewes’ milk or a mixture of these Pasteurised cows’ milk Raw cows’ milk or raw cows’ and raw ewes’ milk Raw ewes’ milk Pasteurised cows’ milk

% moisture

% fat

% protein

% NaCl

Reference

35.4–41.6 49.2–50.2 42.7–47.3 33–40.4

33.8–38.2 26 29–31 29.2–32.3

20.4–23.6 19.3–20.8 18.5–23.9 23.3–27.5

1.8–3.4 3.2–3.8 3–3.9 3.1–4.9

2 4 1,4,9,11 3

42.2–49.6 44.6–69.4

29.6–31 13–30

19–22.9 14.2–27

1.6–2.9 1–4.7

4,5,9 12

43 36.9–41.5 40.4–45.1 42–44 37–41.6

29 36.7–40.4 30.6–34.1 29 32–35.2

21 20.3–23.1 20.8–23.8 20 21–28.7

3–4 1.8–2.1 3.2–4.4 4.1 2.2–2.7

6 7,8 10,11 1,4,9

x Cheeses with PDO/PGI. a Gamonedo cheeses are smoked for 3–4 weeks. 1: Madkor et al. (1987a); 2: Marcos et al. (1983); 3: Gonzàlez de Llano et al. (1992); 4: Zarmpoutis et al. (1997); 5: Gobbetti et al. (1997); 6: Palmquist and Brelin (1993); 7: Prieto et al. (1999); 8: Prieto et al. (2000); 9: Muir et al. (1995); 10: Matsui and Yamada (1996); 11: de Boer and Kuik (1987); 12: Kaminarides (1986).

that of the surface (Gobbetti et al., 1997; Hansen et al., 2001), as the level of NaCl is lower, and therefore allows a faster and earlier growth of the mould cultures. The increase in pH is due to the metabolism of lactic acid to CO2 by yeasts and moulds and the increased proteolysis, leading to production of NH3 from amino acids (Godinho and Fox, 1982; Zarmpoutis et al., 1996, 1997). Salting, done by immersing the cheeses in brine or applying dry salt to the cheese surface, is an important step in the manufacture of most Blue cheeses. Both methods create a NaCl gradient from the surface of the cheese to the core, which equilibrates slowly during ripening (Fig. 2) (Godinho and Fox, 1981b; Gobbetti et al., 1997; Hansen et al., 2001). The overall NaCl content in ripe Blue cheese ranges from 2 to 5% (Madkor et al., 1987a; Zarmpoutis et al., 1996, 1997; Gobbetti et al., 1997; Prieto et al., 2000). The high salt content is due to a fairly long salting period for these cheeses (e.g., 2 days brine-salting for Danablu), the high moisture content and the loose structure of the cheese matrix. The diffusion of NaCl into the cheese core is faster in the piercing channels and in areas with fissures creating an even more uneven salt distribution. The NaCl concentration measured in Danablu cheeses after 8 weeks of maturation was approximately 2.0% (w/w) in the core and 4.0% (w/w) in the surface layer, corresponding to a NaCl in moisture of 7.5 and 10.0%, respectively (Hansen, 2001). The concentration of NaCl, lipolysis and proteolysis, especially the increase in low molecular weight peptides, influence the water activity, aw, significantly in Blue

cheeses (Marcos, 1993). Furthermore, the fat content influences cheese structure and thereby the diffusion coefficient of NaCl and the equilibration of aw throughout the cheese (cf. ‘Salt in Cheese: Physical, Chemical and Biological Aspects’, Volume 1). In Danablu, the highest aw, c. 0.98, is found in the interior after 1 week of ripening, while the value for the exterior region ranges from 0.85 to 0.90 (Fig. 2). After 5 weeks, aw for both the interior and the exterior regions of Danablu is usually in the range 0.91–0.94 (Fig. 2). Similar values have been found for Mycella (Hansen et al., 2001) and Picón Bejes-Tresviso cheese (Prieto et al., 1999, 2000). It is well known that the growth of fungi is affected by the gaseous composition in cheese, i.e., the concentrations of O2 and CO2. The level of O2 has been shown to decrease rapidly throughout the cheese; in Danablu, after 1 week of ripening, a 50% decrease was found 4 mm under the rind, whereas after 13 weeks, O2 was completely absent, except in the outer 0.25 mm (van den Tempel et al., 2002). This anaerobic environment was evident already after 3 weeks of ripening, except from small areas in the cheese, probably in fissures. The results are in accordance with observations in white-mould cheese (Boddy and Wimpenny, 1992), but are lower than values found in Roquefort (Thom and Currie, 1913), where oxygen was measured in the gas phase of the cheese. P. roqueforti is well adapted to growth inside Blue cheese where a low level of O2 is combined with a high level of CO2 (20–40%), as this does not significantly affect its growth (van den Tempel and Nielsen, 2000; Taniwaki et al., 2001).

Blue Cheese 177

Ax

Cows’, goats’ or ewes’ milk or a mixture of 2 or 3 milk types ↓

B

Pasteurised cows’ milk (c. 73 °C × 40 s) ↓

Raw milk, thermisation (c. 62 °C × 15 s) or pasteurisation

Inoculation with starter (mesophilic + thermophilic starter

(c. 72 °C × 15 s)

culture, c. 106 cfu/ml of cheese milk)

↓

↓

Addition of rennet

Inoculation with Penicillium roqueforti

Addition of starter (optional)

↓

Addition of Penicillium roqueforti (optional)

Addition of liquid calf rennet

↓

↓

Coagulation, cutting and stirring

Coagulation at 30–34 °C

↓

↓

Moulding

Cutting of the coagulum (size of curd: c. 2.0–2.5 cm)

↓

↓

Whey drainage for c. 10–48 h. No pressure applied, but

Stirring

moulds are inverted frequently

↓

↓

Moulding

Brine salting or dry salting for 24–48 h

↓

↓

Whey drainage, no pressure applied, held at 18 °C for 10 h

Piercing of the cheeses (optional)

with 4 turns

↓

↓

Ripening (in general at c. 10 °C, c. 85–95% relative humidity, for some varieties in caves)

Salting (c. 200 g of salt spread over each cheese), 22 °C for 40 h ↓ Ripening at 4–6 °C for 83 days, 85–90% relative humidity ↓

x

Times, temperatures, etc. depend on the variety being produced.

Cheeses are pierced after 12 and 20 days

Figure 1 An outline of general steps in the manufacture of different varieties of Blue cheese (A) and the steps involved in the production of Gorgonzola (B).

Micro-organisms that Contribute to Ripening of Blue Cheese Several micro-organisms make up the complex microbiota of Blue cheeses, contributing at different levels to ripening. The primary and secondary starter cultures, LAB and P. roqueforti, respectively, are the most import-

ant, but yeast and non-starter lactic acid bacteria (NSLAB), even though they are not added deliberately to the cheese milk, most probably also influence ripening. It should also be noted that some varieties of Blue cheeses are ripened naturally, i.e., cultures are not

(0,1)

(1,1) (1,0)

(0,0)

1.0

pH, 1 week

1.0

pH, 5 weeks 5.0 5.2

4.40

5.4 5.6

4.45 5.8

y

y 4.50

6.0

4.55

6.2

4.60 6.4 1.0

0.0

0.0

1.0 x

x 1.0

NaCl, 1 week

1.0

NaCl, 5 weeks

4.5

6.0 5.0

4.0

4.0

y

3.5

y 3.0 3.0 1.0

2.0 2.5 2.0 1.0

0.0

1.0

0.0

x 1.0

x

aw, 1 week

1.0

aw, 5 weeks

0.87 0.89

0.92

0.91 0.93

0.93

y

y

0.95 0.97

0.94

0.99

0.0

1.0 x

0.0

1.0 x

Figure 2 Partial least squares (PLS) contour plots of pH, NaCl and aw in Danablu 50 after 1 and 5 weeks of ripening. The contour plots show the gradients from the core to the surface of the cheese, corresponding to the grey area on the cheese depicted. (Data were visualised by PLS regression using SIMCA-P, ver. 3.01 (UMETRI AB, Sweden)).

178

Blue Cheese 179

added during manufacturing. However, the abovementioned groups of micro-organisms are present in both naturally ripened Blue cheeses and Blue cheeses with added cultures, and these groups, and their characteristics, will be described in the following sections. Lactic acid bacteria

Mesophilic and thermophilic LAB are used as the primary starter culture for the production of different varieties of Blue cheese. A mesophilic, undefined mixed culture will typically contain lactic acid-producing Lactococcus lactis (Lc. lactis subsp. lactis and Lc. lactis subsp. cremoris) and sometimes also citrate-positive strains of Lc. lactis subsp. lactis and Leuconostoc species, which produce CO2 and open up the structure to facilitate the penetration of air and development of the mould. The thermophilic starters used in Blue cheese usually contain Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. The most important role of the LAB starter culture is to acidify the milk by metabolising lactose to lactate. In general, the numbers of LAB (lactococci and lactobacilli) in the core decrease slowly from about 109 cfu/g after salting to 107–108 cfu/g at the end of maturation. The number on the surface after brining is 108–1010 cfu/g and remains almost stable to the end of maturation (Devoyod et al., 1968; Nuñez, 1978; Ordonez et al., 1980; Gonzàlez de Llano et al., 1992; Gobbetti et al., 1997; Hansen et al., 2001). Investigations of the LAB in the core and on the surface of Danablu, for which

only a mesophilic starter is used, immediately after brining to 4 weeks of ripening indicated that the number of lactococci decrease markedly in the surface layer during the first weeks of ripening. At the same time, an increase of a new microbiota dominated by Lactobacillus spp. was observed. For the whole period, the population in the core was dominated by lactococci (Hansen, unpublished results). Penicillium roqueforti

Penicillium roqueforti has previously been known under other names, but several species, including P. stilton, P. italicum, P. gorgonzola, P. glaucum, P. bioruge, P. suavolens and P. aromaticum, were found to belong to the same species and collected under the taxon P. roqueforti (Pitt, 1979; Stolk et al., 1990). Taxonomically, P. roqueforti is classified under the genus Penicillium Link, the subgenus Penicillium and the species roqueforti Thom (Pitt and Hocking, 1997). The taxonomy of the fungi is based on phenotypic analysis though genotypic methods are becoming more and more common. Different methods used to determine the taxonomy of P. roqueforti are shown in Table 2. Conidia of P. roqueforti may be added directly to the cheese milk, sprayed on the curd or colonise the cheeses naturally. The addition of conidia is crucial for the quality of Blue cheese varieties made from pasteurised milk. P. roqueforti can assimilate all the main carbohydrates that occur in cheese, i.e., lactose, glucose and galactose, utilise lactate and citrate and grow without

Table 2 Methods used for the taxonomical classification of Penicillium roqueforti Methods

Analysis

Reference

Classical methods based on phenotypic classification

Micro- and macromorphology, growth rate on specific media, assimilation of carbohydrates and acids, growth on different nitrogen sources, resistance to preservatives and chemicals Production of secondary metabolites under specific and controlled conditions (assayed by TLC, HPLC and GC) Production of aroma compounds and their specific profile (assayed by GC and MS) RAPD ITS-PCR rDNA-RFLP AFLP Based on the same criteria as the classical methods, but instead of visual analysis of the macromorphology, digital image analysis and multivariate data analyses are used

Samson et al. (1977, 1995) Pitt (1979) Pitt and Hocking (1997)

Profiles of secondary metabolites Aroma profiles PCR-based methods

Image analysis

Frisvad (1982) Lund et al. (1995) Boysen et al. (1996) Larsen and Frisvad (1995a,b) Geisen et al. (2001) Boysen et al. (1996, 2000) Boysen (1999) Dörge et al. (2000)

TLC, Thin layer chromatography; HPLC, High performance liquid chromatography; GC, Gas chromatography; MS, Mass spectroscopy; RAPD, Random amplified polymorphic DNA; ITS-PCR, Internal transcribed spacer-Polymerase chain reaction; RFLP, Restriction fragment length polymorphism; AFLP, Amplified fragment length polymorphism.

180 Blue Cheese

difficulty at the pH and temperatures encountered during ripening of Blue cheese (Cerning et al., 1987; Vivier et al., 1992). P. roqueforti is the Penicillium species with the highest tolerance to low levels of O2 (Pitt and Hocking, 1997). It has been demonstrated that the rate of growth of P. roqueforti is not significantly affected in the range 4–21% O2 (Thom and Currie, 1913; van den Tempel and Nielsen, 2000; Taniwaki et al., 2001), but growth seems to be affected by interactions between the levels of O2 and CO2. P. roqueforti grows in the presence of 25% CO2 (van den Tempel and Nielsen, 2000) and O2 in the range 0.3–21%. Taniwaki (1995) found that growth and sporulation of P. roqueforti occur at 20% CO2 in an atmosphere with 0.5% O2. P. roqueforti grows in fissures and piercing channels in the cheese. The colour of the mould varies from white through several shades of green to brownish, depending on the strain and its age. The growth rate of P. roqueforti is strongly affected by increasing concentration of NaCl. The influence of aw on growth, sporulation and germination of four strains of P. roqueforti was investigated in laboratory media containing added NaCl at concentrations corresponding to aw in the range 0.99–0.92 (0–13%, w/w, NaCl). The growth of most strains was stimulated by 3.5% NaCl, corresponding to aw 0.98 (Hansen and Jakobsen, 2003). Similar results have been reported by other authors (Godinho and Fox, 1981a; López-Díaz et al., 1996b; Valik et al., 1998). Higher concentrations of NaCl cause a decrease in the growth rate, e.g., a 92% reduction at aw 0.92 compared to the optimum growth rate at aw 0.98 (Hansen and Jakobsen, 2003). Concerning sporulation, an optimum was observed at aw 0.98 for three of the four strains of P. roqueforti examined; the fourth strain showed an optimum at aw 0.96. Sporulation was strongly inhibited at aw 0.94 for the three salt-sensitive strains whereas the NaCl-tolerant strain still showed a pronounced sporulation at aw 0.94, but not at aw 0.92 (Hansen and Jakobsen, 2003). Germination of P. roqueforti conidia is stimulated by 1–3% NaCl for most strains, but differences in NaCl tolerance have been observed (Godinho and Fox, 1981a; López-Díaz et al., 1996b). Below aw 0.96, the rate of germination decreases with decreasing aw (Hansen and Jakobsen, 2003) and it was observed that NaCl inhibits the rate of swelling of the conidia as well as the further development of the germ tube. Germination rate was also influenced by the microenvironment in which the conidia were produced, i.e., conidia produced and harvested at aw 0.92 germinated faster at aw 0.99 than conidia produced at a higher aw (Hansen and Jakobsen, 2003).

The aw in the core of Blue cheeses after brining is optimal for germination and growth, and the concentration of NaCl is in the range where P. roqueforti is stimulated (Godinho and Fox, 1981a). During the first 3 weeks of ripening, the NaCl concentration in the core increases to a level that induces sporulation and reduces the germination rate and growth of mycelia. These changes influence the appearance of the cheese as the blue-green colour is due to the conidia and also prevents the growth of a thick mycelium in fissures and piercing channels. A thick mycelium feels like rubber in the mouth and is therefore undesirable in Blue cheese. Due to the NaCl gradient, the development of P. roqueforti occurs from the interior to the exterior part of the cheese. The conidia in the exterior part of the cheese will germinate with a significantly prolonged lag-phase and a slow development of hyphae, compared to conidia in the interior. This difference in the rate of germination will persist only until the concentration of NaCl in the exterior part is close to the concentration in the interior. Concerning the further growth of P. roqueforti, the aw values determined in the surface layer of, e.g., Danablu and Mycella, indicate that mycelial growth will not occur in the surface layer, which might be of importance with regard to the possible differences in enzymatic activity of conidia and mycelium. Yeast

It is not widely appreciated that yeasts can be an important component of the microbiota of many cheese varieties. However, yeasts form a substantial part of the microbiota in surface-ripened cheeses (Eliskases-Lechner and Ginzinger, 1995; Bockelmann and Hoppe-Seyler, 2001), white-mould cheeses (Schmidt and Lenoir, 1980a,b) and Blue cheeses (de Boer and Kuik, 1987; Gonzàlez de Llano et al., 1992; Roostita and Fleet, 1996a; Gobbetti et al., 1997; van den Tempel and Jakobsen, 1998). Yeasts occur spontaneously in almost all types of cheese, and it is not unusual to find yeast counts as high as 107–108 cfu/g (Fleet, 1990; Viljoen and Greyling, 1995; Tzanetakis et al., 1998). Yeasts seem to originate from the raw milk and, for brine-salted cheeses, from the brine. Investigations have shown that yeasts can be found only at low numbers (10 cfu/g) in Danablu cheese before brine-salting (van den Tempel, 2000). Changes in the sensory properties of cheese do not become apparent until the yeasts have grown to a population of 105–106 cfu/g (Fleet, 1992), but despite the frequent occurrence of yeasts in Blue cheeses, they seem

Blue Cheese 181

generally not to cause defects except brown spots (Weichhold et al., 1988; Nichol and Harden, 1993). Origin of yeasts in Blue cheese Raw milk. The predominant yeasts found in raw milk

from four Danablu dairies in Denmark included Debaryomyces hansenii (Candida famata), C. catenulata, C. lipolytica, C. krusei and Trichosporon cutaneum. Yeast populations exceeded 101–104 cfu/ml and a total of 37 isolates were identified (van den Tempel and Jakobsen, 1998). Other authors have also described the presence of D. hansenii (C. famata) in raw milk in Australia (Fleet and Mian, 1987; Fleet, 1990) and a German investigator (Engel, 1986) demonstrated the occurrence of other yeast species, including C. curvata and Saccharomyces spp. Pasteurisation (72 °C  15 s) or thermisation (61 °C  15 s) generally does not kill yeasts (Vadillo et al., 1987). Fleet and Mian (1987) reported 103 cfu/ml of pasteurised milk, with C. famata as the predominant species, followed by Kluyveromyces marxianus. Brine and the dairy environment. Salting, especially

brine-salting, is a source of yeasts (Devoyod and Sponem, 1970; Kaminarides and Laskos, 1992; Eliskases-Lechner and Ginzinger, 1995; van den Tempel and Jakobsen, 1998). The composition and environmental conditions of the brine vary from country to country and from dairy to dairy. In France, for example, the brines used for Blue cheese production are typically 19–20% NaCl (w/v), pH 4–6 and 13–16 °C (Seiler and Busse, 1990), whereas brines used in Denmark have a higher NaCl content (22–23%), a higher temperature (19 °C) and a pH of 4.5 (van den Tempel and Jakobsen, 1998). The environmental conditions in cheese brines select for salt-tolerant yeast species originating mainly from the dairy environment, the brine and the cheeses (Tudor and Board, 1993). Brines used for Danablu production may have a yeast population ranging from 104 to 106 cfu/ml, depending on the dairy (van den Tempel and Jakobsen, 1998). In spite of distinct differences in the composition of the yeast flora among the dairies, D. hansenii (C. famata) was the predominant species in the brines, except from one dairy, where C. globosa predominated. Several brines used for the production of soft surfaceripened cheeses have shown the occurrence of 104–105 cfu/ml, with C. famata as the predominant yeast species (Seiler and Busse, 1990). The frequent occurrence of C. famata in brines used for cheesemaking is explained by its high tolerance to salt (Devoyod and Sponem, 1970; Kaminarides and Laskos, 1992; Eliskases-Lechner and Ginzinger, 1995). Other species

found were C. catenulata, C. lipolytica, Zygosaccharomyces spp., T. cutaneum and Cryptococcus laurentii. Occurrence and growth of yeasts in Blue cheeses

Yeasts develop spontaneously during the manufacturing, ripening and storage of Blue cheeses. Their occurrence is not unexpected because of their tolerance to low pH, elevated salt concentrations and low storage temperatures (Fleet, 1990). Furthermore, high concentrations of lactate, residual unfermented carbohydrates and small amounts of citric and acetic acids will assist the growth and prevalence of particular species of yeast. Blue cheese like Roquefort, made traditionally from raw milk, may reach a population of 107–108 and 105–106 cfu/g on the surface and in the interior, respectively, before brine-salting (Besancon et al., 1992). The same investigators showed that the yeast population on the surface decreases significantly (99%) after brinesalting, causing changes in the yeast population towards asporogenous yeast forms, C. famata in particular. These results confirm earlier investigations, which also showed a 90% reduction in the yeast flora and changes in yeast population, selecting for very salt-tolerant species, especially Candida spp. (Devoyod and Sponem, 1970; Galzin et al., 1970). The yeast flora in the interior of the cheese remains unaffected by salting during the early period of ripening, due to the slow diffusion of the salt from the surface to the interior of the cheese (Galzin et al., 1970; Hansen et al., 2001). Yeasts start to multiply on the surface of the cheese after a short adaptation period. There is an almost parallel development of the yeast population in the interior of the cheese, but with numbers 100-fold lower (Hansen et al., 2001). This can be explained by the low level of available oxygen and the high level of CO2, which reduce the growth of yeasts (van den Tempel and Nielsen, 2000). All investigators seem to show the predominance of D. hansenii (C. famata) in Blue cheese, except the Greek variety, Kopanisti, in which T. cutaneum seems to dominate over D. hansenii (C. famata) (Kaminarides and Anifantakis, 1989). A survey of the literature on yeasts isolated from Blue cheeses demonstrates the great diversity of the yeast flora (Table 3). In Blue cheeses such as Danablu, the yeast flora develops from a heterogeneous population towards a more homogeneous population as ripening progresses. On day 1 after salting, Danablu contains different species of yeasts, including C. famata, C. lipolytica, Zygosaccharomyces spp., C. rugosa and C. sphaerica. After 28 days of ripening at 10 °C, C. famata was the predominant yeast, reaching 6.2  106 and 1.4  108 cfu/g in the

182 Blue Cheese

Table 3 Species of yeast isolated from Blue cheeses Isolated species

Type of Blue cheese

Debaryomyces hansenii (Candida famata)

Roquefort1, 3, 4, Cabrales2, Gorgonzola3, 9, Danablu3, 10, Bleu d’Auvergne3, Bleu de Bresse3, Gamonedo5, Kopanisti6, Valdeón7, Australian Blue8, unknown brand of Blue cheese8 Roquefort1, 3, 4, Cabrales2, Gorgonzola3, Danablu3, 10, Bleu d’Auvergne3, Bleu de Bresse3, Kopanisti6, Valdeón7, unknown brand of Blue cheese8 Roquefort3, Gorgonzola3, Danablu3, 10, Bleu d’Auvergne3, Bleu de Bresse3, Valdeón7, Australian Blue8, unknown brand of Blue cheese8 Roquefort1, Cabrales2, Gorgonzola9 Danablu10, Gamonedo5, Valdeón7 Kopanisti6, Valdeón7 Australian Blue8, unknown brand of Blue cheese8, Danablu10 Valdeón7, unknown brand of Blue cheese8 Roquefort3, Gorgonzola3, Danablu3, 10, Bleu d’Auvergne3, Bleu de Bresse3 Cabrales2, Danablu10 Roquefort3, Gorgonzola3, Danablu3, Bleu d’Auvergne3, Bleu de Bresse3, Valdeón7 Roquefort3, Gorgonzola3, Danablu3, Bleu d’Auvergne3, Bleu de Bresse3, Valdeón7 Roquefort1, unknown brand of Blue cheese8 Roquefort3, Gorgonzola3, Danablu3, Bleu d’Auvergne3, Bleu de Bresse3, Kopanisti6 Kopanisti6, Danablu10 Australian Blue8, unknown brand of Blue cheese8 Unknown brand of Blue cheese8 Danablu10 Valdeón7 Unknown brand of Blue cheese8 Danablu10

Kluyveromyces marxianus (Candida sphaerica) Yarrowia lipolytica (Candida lipolytica) Pichia spp. Cryptococcus laurentii Rhodotorula spp. Candida catenulata Candida colliculosa Candida lambica Candida rugosa Candida zeylonoides Geotrichum candidum Kluyveromyces marxianus (C. kefyr) Saccharomyces cerevisiae Trichosporon cutaneum Cryptococcus albidus Candida intermedia Candida norvegensis Candida parapsilosis Candida tropicalis Zygosaccharomyces spp.

1: Devoyod and Sponem (1970); 2: Nuñez et al. (1981); 3: de Boer and Kuik (1987); 4: Besancon et al. (1992); 5: Gonzàlez de Llano et al. (1992); 6: Kaminaride and Anifantakis (1989); 7: López-Díaz et al. (1995); 8: Roostita and Fleet (1996a); 9: Gobbetti et al. (1997); 10: van den Tempel and Jakobsen (1998).

interior and on the surface of the cheese, respectively (van den Tempel and Jakobsen, 1998). Examination of Blue cheeses of different origin and age (12 weeks) showed that D. hansenii, or its asporogenous form C. famata, dominated in all cheeses examined (Table 4). Strong growth in the presence of salt, growth at a low temperature and the ability to utilise lactate and citrate are likely the key determinants that encourage the predominance of D. hansenii (C. famata) in cheeses (van den Tempel and Jakobsen, 2000). Another yeast frequently found in Blue cheese produced from raw milk is Kluyveromyces marxianus (C. sphaerica). It assimilates and ferments lactose and, due to gas production, could play an important role in the formation of the characteristic open texture of Blue cheese (Lenoir, 1984; Fox and Law, 1991; Roostita and Fleet, 1996a). Furthermore, strains of K. marxianus (C. sphaerica) are able to assimilate lactic and citric acids, and have weak proteolytic and lipolytic properties (Lenoir, 1984; Fleet and Mian, 1987; Besancon et al., 1992; Roostita and Fleet, 1996b). They have, however, been shown to exhibit a pronounced inhibitory effect

on the growth of P. roqueforti (Kaminarides et al., 1992; Hansen and Jakobsen, 1998). A yeast species less frequently found in Blue cheese is Yarrowia lipolytica (C. lipolytica) which is characterised by the inability to ferment carbohydrates or assimilate nitrate and has strong lipolytic and proteolytic properties (Roostita and Fleet, 1996a; Freitas et al., 1999; van den Tempel and Jakobsen, 2000). Non-starter lactic acid bacteria

Non-starter lactic acid bacteria (NSLAB) are found in several cheese varieties during ripening, including Blue cheeses. As in many other cheeses, they are commonly facultatively heterofermentative strains of Lactobacillus, i.e., mainly of the Lb. paracasei/casei complex and Lb. plantarum. Other NSLAB found in Blue cheese are Lb. fermentum, Lb. brevis, Pediococcus spp. and Leuconostoc spp. (Gonzàlez de Llano et al., 1992; Martley and Crow, 1993; Fox et al., 1996; Gobbetti et al., 1997; López-Díaz et al., 2000). Nonstarter lactic acid bacteria grow in Blue cheese from

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Table 4 Occurrence of Debaryomyces hansenii in selected Blue cheeses

Cheese

Surface, yeast/g cheese

Interior, yeast/g cheese

D. hansenii (%)

Roquefort Petite Fourme Bleu d’Auvergne Cambozola Saint Agur Gorgonzola Fourme d’Ambert

1.7  107 2.1  107 1.7  107 2.7  105 2.0  107 3.6  106 2.1  107

4.4  106 2.0  105 5.5  106 2.6  104 1.9  105 7.3  103 1.4  106

74 66 64 70 75 62 70

Modified from van den Tempel (2000).

10–100 cfu/g after brining to about 107 cfu/g at the end of maturation and are assumed to originate from the raw milk and the dairy plant environment (Nuñez and Medina, 1979; Gonzàlez de Llano et al., 1992; Gobbetti et al., 1997). In Blue cheeses made from raw milk, a high number of Enterococcus spp. have been isolated (Devoyod and Desmazeaud, 1971; Gonzàlez de Llano et al., 1992; López-Díaz et al., 2000). However, it is not known how normal variations in the composition of the NSLAB flora influence ripening and flavour of Blue cheese. Contaminants

Micro-organisms other than P. roqueforti and LAB starter cultures can colonise and grow well on Blue cheeses, especially on the surface. Spoilage of cheese due to fungal growth is caused by the formation of off-flavours (Sensidoni et al., 1994), mycotoxins and possible discoloration of the cheese (Lund et al., 1998). The most important spoilage fungi of semi-soft cheeses are Penicillium spp., including P. commune and P. nalgiovense (Lund et al., 1995). Species of less importance are P. verrucosum, P. solitum and P. discolor, which were also found in Valdeón, an artisanal Spanish Blue cheese made from raw milk (López-Díaz et al., 1995; Lund et al., 1995; Filtenborg et al., 1996). Of special interest for Blue cheese is the newly discovered species, Penicillium caseifulvum, which is frequently found on Blue cheeses (Lund et al., 1998). P. caseifulvum has to date been found in various Blue cheese dairies in Denmark and France (Lund et al., 1998), where it was isolated from cheese curd (102 conidia/g), brine (101–5  102 conidia/g) and from the surface of Danablu (102–103 conidia/g). P. caseifulvum is sensitive to CO2 (van den Tempel and Nielsen, 2000), and therefore grows only on the surface of the cheese, where it can cause discoloration in the form of brown spots.

Another contaminant frequently found in Blue cheese is Geotrichum candidum (de Boer and Kuik, 1987; LópezDíaz et al., 1995), which can cause considerable inhibition of the growth of Penicillium spp. (Nielsen et al., 1998; van den Tempel and Nielsen, 2000). G. candidum has been isolated from Danablu at levels of 102–103 cfu/g, mainly from the interior of the cheese, as G. candidum is sensitive to salt at concentrations above 1% (Philip, 1985; Lecocq and Gueguen, 1994; van den Tempel and Nielsen, 2000).

Microbial Interactions During the production and ripening of Blue cheese, interactions between the primary starter culture, P. roqueforti, and the mould and yeast contaminants determine the maturation time, aroma, texture and appearance of the final cheese. The mechanisms behind these interactions can be broadly divided into two groups: Antagonism, representing negative interactions caused by antimicrobial metabolites, competition for nutrients or unfavourable changes to the microenvironment. Synergism, representing positive interactions as mutual use and production of nutrients, changes to a more favourable microenvironment and atmosphere composition, degradation of antimicrobial compounds, physical attachments between microorganisms and changes in microstructure. Microbial interactions have been deduced from examinations of Blue cheeses (Kaminarides et al., 1992; Hansen and Jakobsen, 1997; van den Tempel and Jakobsen, 2000; van den Tempel and Nielsen, 2000; Hansen et al., 2001), but there have been only a few detailed studies in this area. In the following, microbial interactions involving P. roqueforti will be described. Penicillium roqueforti and lactic acid bacteria

Positive and negative interactions between LAB and Penicillium spp. have been described (Salvadori et al., 1974; Gripon et al., 1977; Suzuki et al., 1991; Gourama and Bullerman, 1995; Roy et al., 1996; Gourama, 1997; Hansen and Jakobsen, 1997; Hansen, 2001). However, only a limited number of investigations have been carried out to examine how several strains of LAB, both starter and non-starter cultures, affect the growth and sporulation of different strains of P. roqueforti in model cheese systems. The results shown in Table 5 are based on screening of 20 strains of P. roqueforti and 15 strains of LAB in a model cheese system and a

184 Blue Cheese

laboratory substrate (Hansen and Jakobsen, 1997). As shown in Table 5, both negative and positive interactions were demonstrated. Furthermore, the type of interactions observed was related to the composition of the medium used. Positive interactions were stronger and found more frequently in the cheese agar compared to the laboratory medium. The interactions were found to be strain-specific for P. roqueforti as well as for the LAB. The positive interactions were seen as faster growth and more pronounced sporulation of P. roqueforti. Further, P. roqueforti developed a thicker and more velvet-like mycelium. Negative interactions were seen as a reduction in, or the absence of, the growth of P. roqueforti. A synergistic effect on casein breakdown between P. roqueforti and LAB has also been observed, indicated by the production of significantly higher amounts of non-protein nitrogen and phosphotungstic acid-soluble nitrogen when the enzymes of the two types of organisms were incubated together compared to the amounts produced by only one of them (Ottogalli et al., 1974). The benefits obtainable by selecting the right combination of cultures are emphasised by results showing that stimulation of the growth and sporulation of P. roqueforti was stronger at higher levels of NaCl. The levels of NaCl investigated corresponded to the concentration found in the surface layer of Blue cheese where the growth of P. roqueforti can be limited or even absent. The stimulation of mycelial growth and the sporulation of P. roqueforti obviously enhance maturation, because both the conidia and the mycelium contribute to proteolysis and lipolysis in the cheese during ripening. Penicillium roqueforti and yeasts

As for LAB and P. roqueforti, selecting the right combinations of yeast and P. roqueforti cultures may stimulate the growth and sporulation of P. roqueforti and thereby enhance ripening and improve the appearance of the cheese in general.

Table 5 Positive and negative interactions regarding the growth of Penicillium roqueforti in laboratory medium and cheese agar for 300 combinations of strains of P. roqueforti and lactic acid bacteria

Positive interaction Negative interaction No sign of interaction

Cheese agar

Laboratory media

136 49 115

22 195 83

Modified from Hansen and Jakobsen (1997).

Interaction experiments carried out under environmental conditions similar to those in Blue cheese production have demonstrated radial growth of P. roqueforti to be stimulated by selected strains of D. hansenii (van den Tempel, 2000). The mechanism behind the observed positive interactions might be explained by the stimulation of P. roqueforti by D. hansenii caused by the release of nutrients on autolysis due to low survival rates of yeasts at high levels of CO2 (Lumsden et al., 1986; Ison and Gutteridge, 1987; Dixon and Kell, 1989; van den Tempel and Nielsen, 2000). Positive interactions between a strain of Saccharomyces cerevisiae (FB7) and strains of P. roqueforti have also been demonstrated (Hansen and Jakobsen, 2001; Hansen et al., 2001). Measurement of radial growth and visual observations of sporulation showed that whole cell inocula of S. cerevisiae promoted faster growth, thicker and more velvet-like mycelia and a more intense blue colour of the conidia. No interactions were seen when the supernatant or the disrupted cells of S. cerevisiae were examined. The mechanism behind the positive fungal–yeast interaction was found to be correlated to a synergistic effect in the breakdown of casein, as shown by capillary electrophoresis. S. cerevisiae FB7 degraded casein, and co-culturing with P. roqueforti resulted in a higher number and different patterns of peptides. These findings have been confirmed in a large-scale production of Mycella cheese, showing that addition of S. cerevisiae FB7 to Mycella gave rise to faster growth and sporulation of P. roqueforti, a softer cheese texture and a significantly higher relative concentration of aroma compounds (Hansen et al., 2001). Inhibition of P. roqueforti by Y. lipolytica has been observed in laboratory trials (van den Tempel and Jakobsen, 2000). The interactions were strain-specific for Y. lipolytica as well as for P. roqueforti. However, all strains of Y. lipolytica investigated were inhibitory to mycelial growth and sporulation of P. roqueforti (van den Tempel, 2000). Competition for nutrients seems to be the most frequently occurring mechanism of interaction between yeasts and moulds in laboratory systems, but this does not exclude co-existence in more natural situations (Boddy and Wimpenny, 1992). The belief that competition for nutrients is the main interspecies mechanism between yeasts (e.g., Y. lipolytica and D. hansenii) and P. roqueforti is based mainly on investigations showing: (i) no inhibition of P. roqueforti when using culture supernatant or disrupted cells, (ii) the quick colonisation of yeasts, e.g., Y. lipolytica and D. hansenii, on cheese agar, (iii) the quantitative relationship between the numbers of yeasts and inhibition of P. roqueforti, (iv) the inhibitory effect of Y. lipolytica and D. hansenii being

Blue Cheese 185

absent or reduced by addition of nutrients (van den Tempel, 2000; van den Tempel and Jakobsen, 2000).

Table 6 Total concentration of fatty acids (FA) in different cheese varieties

Penicillium roqueforti and contaminants

Variety

FA, mg/kg

Variety

FA, mg/kg

Geotrichum candidum has shown a growth potential similar to P. roqueforti in the absence of salt, indicating a possible overlap between the two species in the interior of the cheese during the initial ripening stage. Contamination of Blue cheese by G. candidum can cause inhibition of growth and sporulation of P. roqueforti resulting in ‘blind spots’, which affect the quality of the cheese significantly. This emphasises the importance of good manufacturing practice in the production of Blue cheese to prevent contamination by G. candidum. Studies by Tariq and Campell (1991) showed that G. candidum might compete by antibiosis, as volatile metabolites from arthrospore suspensions of G. candidum were found to inhibit conidial germination and reduce the rate of hyphal extension in different species of fungi, including P. roqueforti. Recent studies by Dieuleveux et al. (1998) demonstrated that G. candidum produces and excretes 2-hydroxy-3phenylpropanoic acid with a broad-spectrum antibacterial effect. Colonisation of the mould contaminant, P. caseifulvum, can occur on the surface of Blue cheese without major inhibition by any of the species investigated, thus causing colour defects on the cheese (Lund et al., 1998). The occurrence of P. caseifulvum is unlikely to affect the growth and sporulation of P. roqueforti due to their different growth niches in Blue cheese.

Gamonedoc Blue (US) Cabralesb Danablua Roquefort Parmesan

75685 35230 33153 32639 32453 4993

Provolone Gruyere Brie Cheddar Camembert Mozzarella

2118 1481 1314 1028 681 363

Ripening of Blue Cheese Lipolysis

Lipolysis in Blue cheeses, like proteolysis, is very intense compared to other cheeses. As seen from Table 6, high amounts of free fatty acids are found during the ripening of various kinds of Blue cheeses. In other varieties, this extensive lipolysis would cause a rancid taste, but in Blue cheeses, the free fatty acids are neutralised when the pH increases. In general, the total level of free fatty acids increases with ripening time, especially after the mould has sporulated (Alonso et al., 1987; Madkor et al., 1987b; Contarini and Toppino, 1995; Gobbetti et al., 1997), but a decrease at the end of ripening has also been observed (Prieto et al., 2000). This decrease could be caused by conversion of the fatty acids to methyl ketones. Due to the higher NaCl concentration in the rind, which inhibits mould growth and thereby lipase production, a lower level of free fatty acids has been observed in the outer part

Adapted from Woo et al. (1984) except: a Unpublished results (Cantor). b Alonso et al. (1987). c Gonzàlez de Llano et al. (1992).

of the cheese compared to the core (Godinho and Fox, 1981c; Gobbetti et al., 1997). This effect can be altered to some degree by selecting more NaCl-tolerant strains of P. roqueforti. Generally, the levels of both saturated and unsaturated long-chained fatty acids (C12:09C18:3) in Blue cheese is higher than the levels of shortchained fatty acids (C4:09C10:0), which correspond to results obtained for P. roqueforti grown in butterfat emulsions (Larsen and Jensen, 1999). However, considerable differences in the levels of individual free fatty acids can be found between various types of Blue cheeses (Alonso et al., 1987; Madkor et al., 1987b; Prieto et al., 2000). Degradation of lipids in Blue cheeses is caused mainly by enzymes from P. roqueforti (Kinsella and Hwang, 1976; Coghill, 1979; Gobbetti et al., 1997). The lipolytic activity of commercial strains of P. roqueforti differs significantly, resulting in the release of different amounts of free fatty acids (Farahat et al., 1990; Larsen and Jensen, 1999) and thereby different flavour profiles of the cheeses produced (Farahat et al., 1990; Gallois and Langlois, 1990). P. roqueforti produces two extracellular lipases, an acidic and an alkaline lipase (Menassa and Lamberet, 1982; Lamberet and Menassa, 1983b; Mase et al., 1995). Intracellular lipase activity has also been reported (Niki et al., 1966; Stepaniak et al., 1980), but further research in this area is required. The acidic lipase has a pH optimum at 6.0 and a lesspronounced optimum at 2.8, with maximum stability between 3.7 and 6.0 (Lamberet and Menassa, 1983b). The optimum temperature is 35–40 °C, but it retains 37% of maximum activity at 5 °C. Optimum pH for the alkaline lipase is 8.8–9.0 at 30 °C and 9.0–10.0 at 20 °C, but activity is retained between 4.5 and 11.0 (Lamberet and Menassa, 1983b), e.g., 15 and 20% of maximum activity is retained at pH 4.5 and 6.0, respectively. The relative importance of the acidic and the alkaline

186 Blue Cheese

lipases in cheese has not been determined fully. However, Lamberet and Menassa (1983a) investigated the lipolytic activity at pH 5.5 on tricaproin (acid lipase) and at pH 8.0 on tributyrin (alkaline lipase) in suspensions of seven French Blue cheeses. Activity at pH 5.5 dominated and only two samples showed measurable activity at pH 8.0. Even though the pH of Blue cheeses in general favours activity of the acid lipase, it should be noted that the alkaline lipase has the higher activity on milk fat (Eitenmiller et al., 1970; Lamberet and Menassa, 1983a,b). P. roqueforti dominates the overall lipid degradation in Blue cheeses, but other lipolytic agents are also present. The native milk lipoprotein lipase contributes at the beginning of the ripening period, most significantly in Blue cheeses produced from homogenised milk, like Danablu and Stilton (Gripon, 1993). As lipoprotein lipase is almost completely inactivated by pasteurisation, its effect will be the most pronounced in cheeses produced from raw or thermised milk. The LAB, whether they are part of the starter culture or the non-starter microbiota, have very low lipolytic activity and are not likely to influence lipolysis in Blue cheese (El Soda et al., 1986; Meyers et al., 1996). Yeasts probably affect lipolysis, which could be positive (Jakobsen and Narvhus, 1996), but this is very dependent upon the yeast species present. Almost all yeasts present in cheese (e.g., D. hansenii, K. lactis, S. cerevisiae, Y. lipolytica, C. catenulata and Galactomyces geotrichum) have at least esterase activity, being able to hydrolyse short-chained fatty acids from triglycerides (Fleet and Mian, 1987; Roostita and Fleet, 1996a; Hansen and Jakobsen, 1998; van den Tempel and Jakobsen, 1998). Lipolysis of long-chained fatty acids has been demonstrated for Y. lipolytica, C. catenulata and G. geotrichum and the activity seems to be at the same level for these three yeasts (Roostita and Fleet, 1996b; van den Tempel, 2000). Regarding the release of free fatty acids, an increase has been observed when strains of S. cerevisiae grow in co-culture with P. roqueforti, whereas no effect was observed with strains of D. hansenii (Cantor, unpublished results). Strains of Y. lipolytica have strong lipolytic activity, which could be desirable in Blue cheese, but they also, in general, affect the growth of P. roqueforti negatively. However, these interactions are very strain-specific (Hansen and Jakobsen, 1998; van den Tempel and Nielsen, 2000). Recently, preliminary results indicated a positive effect of lipases from Y. lipolytica on the development of free fatty acids when interacting with P. roqueforti, both in Danablu and in milk (Cantor, unpublished results). This could be used to enhance the quality of Blue cheeses made from pasteurised milk, where lipolysis

and aroma formation are delayed and often weaker, unless the ripening period is prolonged. Proteolysis and amino acid catabolism

Several studies have revealed extensive proteolysis in Blue cheese compared to other cheeses (Marcos et al., 1979; Gonzàlez de Llano et al., 1995; Zarmpoutis et al., 1997). Casein is hydrolysed at more sites and at a considerably higher rate, and there are no intact caseins or primary breakdown products left in the ripened cheese (Marcos et al., 1979; Trieu-Cuot and Gripon, 1983; Fernandez-Salguero et al., 1989; Gonzàlez de Llano et al., 1992; Zarmpoutis et al., 1997). A larger number of different peptides are produced than in semi-hard cheeses (Fig. 3), and a high concentration of amino acids are released as a result of the peptidases, especially from mould and LAB working in concert (Ismail and Hansen, 1972; Gripon et al., 1977; Coghill, 1979; Zarmpoutis et al., 1997). The enzymes contributing to the complicated proteolysis in Blue cheese originate from the milk, rennet, starter and non-starter bacteria, moulds and yeasts, with the main contribution from the mould culture, P. roqueforti (Coghill, 1979). A significant increase in proteolysis has been observed when the mould has become visible in the cheese, typically after 2–5 weeks of maturation, depending on the cheese variety (Trieu-Cuot and Gripon, 1983; Zarmpoutis et al., 1996). While P. roqueforti is growing out, breakdown of the caseins is performed mainly by rennet (Hewedi and Fox, 1984). The main activity of rennet in cheese is on s1-casein to produce s1-CN (f24–199) and the peptide s1-CN (f1–23) (Table 7) whereas the milk protease, plasmin, hydrolyses -casein to -caseins and proteose-peptones mainly during the first day. The cell envelope-associated proteinase of the Lactococcus or Lactobacillus starter culture hydrolyses the peptides produced from casein by rennet and plasmin. A limited release of amino acids by the starter aminopeptidases occurs during these first weeks of ripening. After a couple of weeks, P. roqueforti dominates proteolysis, liberating both peptides and amino acids using a variety of enzymes (Table 7) (Madkor et al., 1987a; Zarmpoutis et al., 1996, 1997). P. roqueforti expresses two extracellular proteases: a metalloprotease and an aspartic protease. The activity of these enzymes is maximal in Blue cheese at the stage when P. roqueforti has grown out and begins to sporulate. Both proteases are rather stable in cheese. The metalloprotease is active at pH 4.5–8.5 and it has an optimum for casein hydrolysis at 5.5, which corresponds to the pH often found in Blue cheese during ripening. The metalloprotease has a broad specificity

Blue Cheese 187

1.0

αs1-CN (f1–13)

0.9

Proteose-peptones

Absorbance at 210 nm

0.8 phe

0.7

β-LA

trp

0.6

α-LA

B

0.5

tyr trp

0.4

phe

0.3 0.2

tyr

β-LA

α-LA

A

0.1 0.0 15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Retention time (min) Figure 3 Peptide profiles analysed by RP-HPLC of A: Danablu and B: Semi-hard yellow cheese of similar age (about 3 months). -LA: -lactalbumin; -LG: -lactoglobulin; proteose-peptones: breakdown products from plasmin activity on -casein; s1-CN (f1–13): breakdown product from Lactococcus protease activity on the rennet-derived peptide s1-CN (f1–23).

and hydrolyses both s1- and -caseins. Hydrolysis of s1-casein leads to eight peptides with molecular weights ranging from 7000 to 21 000 Da (Trieu-Cuot et al., 1982b). Hydrolysis of -casein in buffer gives nine peptides with molecular weights between 13 100 and 21 100 Da (Trieu-Cuot et al., 1982b), of which one has been shown to accumulate in Blue cheese (Le

Bars and Gripon, 1981; Trieu-Cuot and Gripon, 1983). Of special interest is that the metalloprotease cleaves -casein at Pro909Glu91, which is not often hydrolysed by proteases because of the proline residue and that, like plasmin, it cleaves a bond close to Lys289 Lys29 (Le Bars and Gripon, 1981; Trieu-Cuot et al., 1982b; Trieu-Cuot and Gripon, 1983).

Table 7 Main enzymes involved in proteolysis and amino acid release during ripening of Blue cheese with scrubbed surfaces to prevent the development of slime microflora (Gripon, 1993; Ardö, 2001) Enzyme

Specificity in cheese

Plasmin

-CN and s2-CN after basic amino acids (Lys, Arg) Hydrolyses -CN to -CN and proteose-peptones; preferred cleavage sites: -CN (28–29, 105–106, 107–108) Hydrolyses s1-CN to s1-CN (f24–199) and s1-CN (f1–23)

Chymosin and other coagulants Lactococcal lactocepin Lactococcal peptidases NSLAB, peptidases P. roqueforti aspartic protease P. roqueforti metalloprotease P. roqueforti serine carboxypeptidase (extracellular, acid) P. roqueforti metalloaminopeptidase (extracellular, alkaline) Yeast CN, casein.

Hydrolyses peptides produced from casein by the action of plasmin, rennet or P. roqueforti Produces different peptides from s1-CN (f1–23) depending on the lactococcal strain Releases amino acids from smaller peptides Broad-specificity aminopeptidase (e.g., PepN), PepC, PepX and dipeptidase specificity Contribute to the release of amino acid Hydrolyses -CN preferentially to produce -CN (98–209, 30–209, 1–29, 100–209, 1–97/99) Hydrolyses s1-CN Broad specificity Releases acidic, basic and hydrophobic amino acids Releases apolar amino acids (not next to Gly) Large variation between strains from no to excessive proteolytic activity

188 Blue Cheese

The aspartic protease is stable at pH 3.5–6.0 and has two optimal pH values for hydrolysis of casein, 3.5 and 5.5, which may be explained by conformation changes in the substrate. Casein is hydrolysed into mainly high molecular weight peptides and it does not hydrolyse di- or tri-peptides (Modler et al., 1974; Le Bars and Gripon, 1981). The first peptide released by the aspartic protease from s1-casein in solution corresponds in isoelectric point and molecular weight to s1-CN (f24–199), indicating specificity similar to chymosin (Trieu-Cuot et al., 1982a; Larsen et al., 1998). Later, this peptide is further hydrolysed to 4–5 new peptides. The aspartic protease hydrolyses -casein into five peptides; initially the peptides -CN (f98–209), (f30–209) and (f1–29) are released and then the peptides -CN (f100–209) and (f1–97/99) (Le Bars and Gripon, 1981; Trieu-Cuot et al., 1982a). In Blue cheese, -CN (f98–209) has been shown to accumulate (Houmard and Raymond, 1979; Le Bars and Gripon, 1981; Trieu-Cuot and Gripon, 1983). Le Bars and Gripon (1981), who compared electrophotograms of commercial Blue cheeses with those of sterile curds inoculated with either P. roqueforti or purified aspartic protease, found that the patterns were very similar, indicating the great importance of P. roqueforti, and especially of its aspartic protease, for proteolysis in Blue cheese during ripening. A relationship has been established between the development of P. roqueforti, the activity of the aspartic protease and the release of bitter peptides (Gripon, 1993). To break down these bitter peptides, as well as other peptides, P. roqueforti possesses several exopeptidases. An extracellular acid carboxypeptidase, with a broad specificity, releases acidic, basic and hydrophobic amino acids and may be important in the debittering process. It is a serine enzyme with a pH optimum for hydrolysing an artificial substrate at pH 3.5, and it is stable at pH 5.0–5.5. P. roqueforti also produces an extracellular alkaline metalloaminopeptidase with a pH optimum of 8.0. It is specific for hydrophobic amino acids, and consequently, the debittering activity of P. roqueforti may increase with pH in Blue cheese. Several intracellular peptidases have also been detected, among them are alkaline carboxy- and amino-peptidases (Gripon, 1993), but their contribution to ripening is not known. The proteolytic activity, as well as the level of proteases and peptidases produced by P. roqueforti, varies greatly between strains (Larsen et al., 1998). The growth of mould within a pierced Blue cheese leads to an increase in pH that stimulates other proteolytic activities in the cheese, such as the LAB cell wall protease (lactocepin) and the milk protease, plasmin. At this later stage of ripening, hardly any -casein

remains in Blue cheese and plasmin activity is limited to any remaining peptides containing its specific cleavage sites. Salt inhibits the development P. roqueforti and therefore also its proteolytic activity, which explains the hard and rather tasteless zone close to the rind of Blue cheese (Godinho and Fox, 1982). Yeasts in Blue cheeses belong mainly to the genus Candida (Table 3). Proteolytic activity, mainly intracellular, has been detected for a few Candida strains, but this property is poorly documented for larger number of strains within the same species (Pereira-Dias et al., 2000; Klein et al., 2002). Activity on casein at 10 °C was not shown by any of six tested strains of D. hansenii originating from Blue cheese (van den Tempel and Jakobsen, 2000). However, in the same experiment, five of six strains of Y. lipolytica isolated from Blue cheese digested all the caseins at 10 °C, showing that yeast may contribute to proteolysis, e.g., a specific strain of S. cerevisiae used as an adjunct culture in Mycella was shown to enhance proteolysis and texture in the cheese (Hansen et al., 2001). However, an excessive contribution to proteolysis may cause a detrimental effect on the cheese. Aminopeptidase activity on branched-chain amino acids was shown for all strains of both D. hansenii and Y. lipolytica, and because yeasts grow to large numbers in many Blue cheeses, this activity may contribute to the release of amino acids during ripening (Klein et al., 2002). Non-starter lactic acid bacteria have been isolated from Blue cheese and could, as in other cheeses, be expected to take advantage of the large amount of small peptides produced by the other micro-organisms present and produce mainly similar aroma compounds from amino acids as the starter bacteria. Amino acids, which are released at high amounts in Blue cheeses (Zarmpoutis et al., 1997), contribute to a background flavour, but further catabolism is needed to produce several aroma compounds characteristic of cheese (Hemme et al., 1982; Yvon and Rijnen, 2001). However, the specific characteristic flavours of Blue cheese originate not from amino acids, but from lipolysis and a significant production of methyl ketones. Cheese flavour compounds are produced by LAB and moulds through amino acid catabolism (Hemme et al., 1982; Yvon and Rijnen, 2001). The free amino acids found most commonly in Blue cheese are glutamic acid, leucine, valine and lysine (Madkor et al., 1987b; Zarmpoutis et al., 1996, 1997). The metabolic pathways of LAB, starting with aminotransferase activity, dominate in hard and semi-hard cheeses, all of which have a low redox potential (Ardö et al., 2002). These activities are not very well studied in Blue cheese, but they are present. Oxidative deamination of amino acids may be performed by P. roqueforti within

Blue Cheese 189

the cheese and microbial flora on the cheese surfaces (cf. ‘Bacterial Surface-Ripened Cheese’, Volume 2). This activity produces ammonia in amounts that contributes to the flavour of Blue cheeses. Compounds resulting from different pathways of amino acid catabolism have been found in Blue cheese (as reviewed by Gripon, 1993). Glutamic acid is decarboxylated to -aminobutyric acid (GABA) and CO2, and other amino acids are decarboxylated to amines and CO2 by P. roqueforti as well as by adventitious micro-organisms in and on Blue cheese. The concentrations of amines vary greatly and tyramine is usually observed in higher amounts than tryptamine and histamine (de Boer and Kuik, 1987). Catabolism of arginine to ornithine and citrulline has been shown in Blue cheese. The complex amino acid catabolism in Blue cheese varieties still needs much research to be understood fully. Formation of aroma compounds

A wide range of volatile and non-volatile aroma compounds are produced in Blue cheese during ripening, primarily by P. roqueforti, influencing both the taste and the aroma of the final product. The varying proportions of these compounds determine the specific flavour profiles of the different Blue cheeses (Gallois and Langlois, 1990). A general overview of the different aroma compounds produced, their concentrations and characteristics will be given here. For more detailed information, excellent reviews have been published in recent years (Molimard and Spinnler, 1996; Sablé and Cottenceau, 1999; McSweeney and Sousa, 2000). The characteristic flavour and taste of Blue cheeses stems mainly from lipid degradation. Free fatty acids contribute both to the taste and the aroma, but even more important are the compounds produced from

them, e.g., the methyl ketones, which are essential for the sensory quality of Blue cheeses (Kinsella and Hwang, 1976; Rothe et al., 1994; Moio et al., 2000). As mentioned previously, the lipolytic activity of commercial strains of P. roqueforti differs significantly, resulting in different amounts of free fatty acids produced (López-Díaz et al., 1996b; Larsen and Jensen, 1999) and thus leading to the different flavour profiles of the cheeses (Table 8) (Farahat et al., 1990; Gallois and Langlois, 1990). Most volatile fatty acids (C4:0–C12:0) have fairly low threshold values, rather pungent or rancid flavour notes, and are usually present at fairly high concentrations in Blue cheeses, but because of the high pH of Blue cheese, these acids are neutralised and hence contribute to the aroma of the cheese and not to a rancid defect (Molimard and Spinnler, 1996). Hexanoic and octanoic acids are especially important flavour compounds (Rothe et al., 1994; Molimard and Spinnler, 1996; Sablé and Cottenceau, 1999). Methyl ketones are the major aroma compounds in Blue cheeses (Table 8) (Day and Anderson, 1965; Dartey and Kinsella, 1971; Ney and Wirotama, 1972; Gallois and Langlois, 1990; Gonzàlez de Llano et al., 1990; de Frutos et al., 1991; Moio et al., 2000). They have been reported to constitute 50–75% of the total volatile flavour compounds in Blue cheeses (Madkor et al., 1987b; Gallois and Langlois, 1990; de Frutos et al., 1991; Moio et al., 2000; Hansen et al., 2001), and their concentration in the cheese can be correlated to the intensity of a ‘Blue cheese’ note (Rothe et al., 1982, 1986, 1994). The methyl ketones found at the highest concentrations are 2-heptanone and 2-nonanone, but 2-pentanone and 2-undecanone are also important (Madkor et al., 1987b; Gallois and Langlois, 1990; Gonzàlez de Llano et al., 1990; de Frutos et al., 1991; Contarini and Toppino, 1995). The total concentration

Table 8 Total concentration ( g/kg cheese) of major groups of aroma compounds in Blue cheeses produced with different strains of P. roqueforti Aroma compounds

Roquefort strain PF a

Roquefort strain PO b

Roquefort strain PG c

Bleu de Caussesd strain PG c

Bleu d’Auvergne e unknown strain

Ketones Alcohols Esters Lactones Aldehydes

11 095 4 025 1 390 50 5

14 350 3 305 2 985 255 10

34 940 7 670 3 835 325 15

9345 3795 3155 425 0

9780 8110 2950 2230 250

Modified from Gallois and Langlois (1990). a Low proteolytic/low lipolytic activity; 210 days ripening. b High proteolytic/high lipolytic activity; 210 days ripening. c Medium proteolytic/medium lipolytic activity; 210 days ripening. d 100 days ripening. e Approximately 45 days ripening.

190 Blue Cheese

of methyl ketones in Blue cheese depends on manufacturing procedure, ripening time and the strain of P. roqueforti used (Table 8), whereas the proportions of the individual methyl ketones in the cheese depend mainly on the strain of P. roqueforti used (Gallois and Langlois, 1990). The odour impressions of the methyl ketones are, in general, fruity, floral and musty and, specifically for 2-heptanone, spicy and ‘Blue cheese’ (Molimard and Spinnler, 1996; Sablé and Cottenceau, 1999). As increasing concentrations of free fatty acids have been shown to inhibit the growth of P. roqueforti and thereby retard lipolysis, the formation of methyl ketones from free fatty acids has been proposed to be a detoxifying mechanism (Kinsella and Hwang, 1976). Methyl ketones with one less carbon atom are produced via part of the -oxidation pathway from the corresponding fatty acids. The first intermediate is a -keto acyl-CoA which is converted to a -keto acid by a thiohydrolase and then decarboxylated to a methyl ketone and CO2. However, at low concentrations, the fatty acids enter the Kreb’s cycle and are completely oxidised to CO2 (Molimard and Spinnler, 1996). Both conidia and mycelia are capable of producing methyl ketones (Fan et al., 1976). The majority of methyl ketones are derived directly from their fatty acid precursor, but the concentration of certain methyl ketones, e.g., 2-heptanone and 2-nonanone, is generally very high compared to the concentration of their precursors, C8:0 and C10:0 (Dartey and Kinsella, 1973a; Madkor et al., 1987b). Dartey and Kinsella (1973a,b) showed that these methyl ketones can also be produced from longer-chain fatty acids. Alcohols have been reported to represent between 15 and 30% of the total volatile flavour compounds in Blue cheese (Table 8) (Gallois and Langlois, 1990; Moio et al., 2000). Methyl ketones can be reduced to secondary alcohols, under anaerobic conditions, and these alcohols are generally more abundant in Blue cheese than primary alcohols. The main secondary alcohols are 2-heptanol, 2-nonanol and 2-pentanol, depending on the cheese type and strain of P. roqueforti used (Gallois and Langlois, 1990; Gonzàlez de Llano et al., 1990). Their flavour is more or less similar to the corresponding methyl ketones, but at higher concentrations they can give a musty or mouldy impression (Kinsella and Hwang, 1976). Primary alcohols are also present, with 3-methyl-1-butanol being the most abundant (Gallois and Langlois, 1990; Moio et al., 2000). Other aroma compounds with fruity and floral notes are esters and lactones; the latter are usually found at low concetrations (Table 8) (Gallois and Langlois, 1990; Gonzàlez de Llano et al., 1990). The higher

amount of lactones detected in Bleu d’Auvergne (Table 8) could be due to the use of pasteurised milk, as pasteurisation of milk has been shown to increase the level of lactones. Esters are believed to be formed in the cheese by microbial esterification of free fatty acids with alcohols. Apart from influencing the overall aroma profile of Blue cheese, the main contribution of esters is possibly by minimising the sharpness and bitterness arising from fatty acids and amines (Anderson and Day, 1966). Peptides and amino acids from proteolysis yield compounds which are important for the background flavour of the cheese and furthermore contribute with their own flavour, e.g., sweet, bitter, brothy (Nishimura and Kato, 1988; Yvon and Rijnen, 2001). Aldehydes are found at low levels (Anderson and Day, 1966; Ney and Wirotama, 1972; Gallois and Langlois, 1990; Table 8), but their impact on flavour is not known. The same is true for the volatile and non-volatile amines (Ney and Wirotama, 1972; Adda and Dumont, 1974), but it is believed that amines have an influence on the overall flavour sensation. The impact of sulphurcontaining compounds on Blue cheese flavour has not been investigated in detail, but they are presumed to make an important contribution (Kinsella and Hwang, 1976; Gallois and Langlois, 1990). Apart from P. roqueforti, yeasts also might contribute to the formation of aroma compounds, either directly, by producing the compounds, or indirectly, by influencing, e.g., growth, and thereby enzyme production of P. roqueforti. The use of S. cerevisiae FB7 as an adjunct culture in Mycella cheese leads to an increase in the concentration of aroma compounds in the experimental cheeses compared to the reference cheeses without added yeast (Hansen et al., 2001). Positive effects on aroma production with certain strains of Y. lipolytica growing together with P. roqueforti in a model cheese system has also been indicated, but this effect is very strain-specific regarding both the yeast and the mould (Cantor, unpublished results). Production and occurrence of mycotoxins

Penicillium roqueforti produces a range of secondary metabolites, or mycotoxins, like PR-toxin (Penicillium roqueforti-toxin) and its precursors, eremofortin A, B and C, the alkaloids roquefortine A, B and C and the isofumigaclavines, the marcfortines and mycophenolic acid (Scott, 1981; see ‘Toxins in Cheese’, Volume 1, for further discussion of mycotoxins). The occurrence of these metabolites in Blue cheese is shown in Table 9. The sesquiterpene PR-toxin is the most toxic of the secondary metabolites, inhibiting nucleic acid and protein syntheses, being cytotoxic in human and

Blue Cheese 191

Table 9 Mycotoxins, produced by P. roqueforti, detected in commercial Blue cheeses

Mycotoxin PR-toxin

PR-imine Roquefortine

Isofumigaclavine A Isofumigaclavine B Mycophenolic acid

No. of cheeses examined

No. of positive samples

13 60 30 60 10 16 12 13 30 16 16 32 100 10

0 0 0 50 1 16 12 13 30 13 6 4 38 0

Concentration ranges (mg/kg)

0.019–0.042 nra 0.05–6.8 0.16–0.65 0.2–2.29 0.05–1.47 traces–4.7 traces 0.25–5 nra–14.3

Detection limit reported (mg/kg)

Reference

0.2 0.0015 nra 0.001 0.03 0.03–0.05 0.016 0.05 nra 0.015–0.025 nra 0.075 0.02 0.02

1 2 9 2 3 4 5 6 9 4 4 7 8 3

a Not reported. 1: Engel and Prokopek (1979); 2: Siemens and Zawistowski (1993); 3: López-Díaz et al. (1996a); 4: Scott and Kennedy (1976); 5: Ware et al. (1980); 6: Schoch et al. (1984); 7: Engel et al. (1982); 8: Lafont et al. (1979); 9: Finoli et al. (2001).

porcine cell lines and in rat liver, in addition to being mutagenic (cited by Scott, 1981). Many strains of P. roqueforti used commercially as starter cultures or isolated from Blue cheeses have the ability to produce PR-toxin (Orth, 1976; Wei and Liu, 1978; Engel and Prokopek, 1979; Medina et al., 1985; Chang et al., 1991; Boysen et al., 1996; Geisen et al., 2001) or one or more of its precursors, eremofortin A, B or C (Moreau et al., 1980; Chang et al., 1991; Geisen et al., 2001) in synthetic media. Fortunately, PR-toxin is unstable in the cheese environment and is converted to the less-toxic PR-imine, which is also unstable, and PR-amide in the presence of basic and neutral amino acids (Scott and Kanhere, 1979; Chang et al., 1993). Furthermore, the optimum conditions for PR-toxin production, a high sugar content in the medium, a pH close to 4.5 and aeration, are far from the conditions prevailing in Blue cheese. PR-toxin has never been detected in commercial Blue cheeses or in experimental Blue cheeses made with known toxin-producing strains (Engel and Prokopek, 1979; Scott and Kanhere, 1979). Roquefortine C is a typical metabolite of P. roqueforti and P. carneum (Medina et al., 1985; Boysen et al., 1996; López-Díaz et al., 1996a), found very often in Blue cheese. Isofumigaclavine A and its stereoisomer, fumigaclavine A, is also a characteristic secondary metabolite for P. roqueforti (Scott et al., 1976; Boysen et al., 1996; Geisen et al., 2001). Data on the biological activity of these alkaloids are scarce; the only reported toxicity values are a LD50-value of 169–189 mg roquefortine/kg body weight and 340 mg isofumigaclavine A/kg body weight after intraperitoneal administration to mice (Ohmomo et al., 1975; Arnold et al., 1978).

Mycophenolic acid is not always produced by strains of P. roqueforti. Boysen et al. (1996) and Geisen et al. (2001) found that c. 50% of the strains investigated produced this metabolite, Engel et al. (1982) found it for 25% of the strains and López-Díaz et al. (1996a) detected mycophenolic acid from only one strain out of nine. In contrast, Lafont et al. (1979) reported that all 16 strains of P. roqueforti investigated produced mycophenolic acid. The LD50-values determined for mycophenolic acid is high, 2500 and 700 mg/kg for mouse and rat, respectively, but subacute toxic effects have been observed for monkeys and rats (Carter et al., 1969; Scott, 1981). However, taking into account the very low levels and the relatively low toxicity of the various mycotoxins present in the cheese, even large consumption of Blue cheese does not pose a risk to the health of the consumer.

Selection of Cultures Blue cheese is a very complex food ecosystem, with marked pH and NaCl gradients and variable, but generally, low levels of O2 and CO2. This heterogeneous microenvironment creates different habitats on the surface and in the core of the cheese, which select for specific micropopulations. The technological characteristics of the LAB of the starter culture and the P. roqueforti culture have significant influences on the quality of the cheese. The primary LAB culture must be able to reduce the pH and survive phage attack, as acidification of the cheese milk is essential for the renneting of the milk and syneresis of the curd, and thereby the fundamental part of the

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cheesemaking process. The secondary culture, P. roqueforti, is often chosen with regard to its proteolytic and lipolytic activities, depending on the type of Blue cheese, the targeted market and the desired shelf-life. The proteolytic activity of the strain of P. roqueforti used is extremely important for texture development, while the lipolytic activity determines the flavour profile. Next, the culture is chosen for its tolerance to NaCl, its growth rate and sporulation capacity. The right combination of these activities and characteristics is crucial for the development of a high quality product. It could, however, also be beneficial to include the interaction with the LAB as a factor and it should be considered whether to make use of the possible synergies in practice and to avoid antagonistic effects. Yeasts should also be considered as potential adjunct cultures as they are present in the cheese and have interesting technological characteristics. There are two major objectives in using yeasts as adjunct cultures in the production of Blue cheese: i) to secure the microenvironment by assimilating residual carbohydrates and organic acids, thereby promoting the growth of desired cultures and inhibiting the growth of spoilage and pathogenic micro-organisms, ii) to contribute directly to the desired cheese quality by their enzymatic activity and by stimulating P. roqueforti. But very careful selection is crucial to avoid undesirable antagonistic interactions between the different cultures and to avoid the production of pigments, undesirable aroma compounds and uncontrolled enzymatic activity. A few selected yeasts will be described as potential adjunct cultures for Blue cheese. However, it is important to remember that several of their technological characteristics are strain-specific and cannot be seen as a general characteristic of the yeast species. Although D. hansenii is the yeast species most frequently isolated from Blue cheese (Tables 3 and 4), it is rarely used as an adjunct culture and only a few applications have been reported, e.g., as a surface culture in the production of Roquefort (Besancon et al., 1992). D. hansenii is very weakly proteolytic and has low lipolytic activity. The strains do not enhance proteolysis, but they might alter the aroma profile slightly without changing it significantly. The potential use of D. hansenii as an adjunct culture seems to be linked with its osmo-tolerance and good growth in Blue cheese. Furthermore, it can create a stable microenvironment which protects against undesired microbial growth by assimilation of residual carbohydrates and organic acids (van den Tempel and Jakobsen, 2000; van den Tempel and Nielsen, 2000). The potential of Y. lipolytica as a ripening culture in cheese has been evaluated by Guerzoni et al.

(1998). It was demonstrated that Y. lipolytica possesses some of the essential properties for use as an adjunct culture: i) ability to grow and compete with other naturally occurring yeasts, such as D. hansenii and S. cerevisiae, even though it assimilates only galactose and lactate, ii) compatibility with and possible stimulation of LAB when co-inoculated, and iii) its remarkable lipolytic and proteolytic activities. Y. lipolytica is relatively salt-tolerant and its potential role as an adjunct culture in Blue cheese is linked mainly with early lipolysis at a time when the lipases from P. roqueforti are not present in significant amounts, but it may also contribute to proteolysis (van den Tempel and Jakobsen, 2000). Y. lipolytica could be a potential adjunct culture, but should be controlled very carefully because of its strong enzymatic activity, its inhibitory effect towards P. roqueforti and its ability to discolour the cheese (Weichhold et al., 1988; Nichol and Harden, 1993). However, unpublished results (Cantor) indicate that the addition of lipase from Y. lipolytica to the cheese milk could aid lipolysis in Blue cheeses made from pasteurised milk by assuming the role of the indigenous milk lipase. Strains of S. cerevisiae can stimulate the release of fatty acids by P. roqueforti, and a synergistic effect between P. roqueforti and S. cerevisiae has been demonstrated in the degradation of casein and the formation of aroma compounds (Hansen and Jakobsen, 2001; Hansen et al., 2001). S. cerevisiae can assimilate residual glucose, galactose and lactate. It has a relatively low tolerance to NaCl and would be a suitable yeast culture only for the production of Blue cheese with a low level of NaCl, such as Gorgonzola or Mycella. The purpose of using S. cerevisiae as an adjunct culture would be to make a controlled contribution to aroma formation and proteolysis, as well as creating a stable microenvironment. For all cultures, whether they are already in use or under consideration as adjunct cultures, a thorough screening of their technological characteristics is extremely important. Only a few selected characteristics have been investigated and described to date, but with the new knowledge, especially on microbial interactions, new possibilities for applying and combining cultures in different ways have become available.

Conclusion A number of aspects of Blue cheese ripening have been discussed in this chapter, but with the space available not all issues could be addressed. The importance of P. roqueforti for the quality of Blue cheese is indisputable, but newly gained knowledge on the microbial interactions and the importance of the adventitious

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microbiota point at new possibilities for improving existing Blue cheeses or developing new varieties. Adding yeast as adjunct cultures would be an obvious opportunity for diversifying the product range, but there is a strong need for further study of interactions with the other cultures present, not only concerning growth, but also on enzymatic activity. Progress has also been made in understanding the complex mechanisms of ripening, but further research is required, especially concerning proteolysis, e.g., on patterns of casein breakdown caused by P. roqueforti proteases in vitro and in different Blue cheeses. Furthermore, the amino acid metabolism in Blue cheeses still needs research to be fully understood. This could be useful for clarifying the effect of individual cultures on proteolysis and thereby on structure development and taste. Research on texture development and its relationship with the proteolytic activity of P. roqueforti together with texture analysis of Blue cheese is very limited. It would, however, be a valuable tool for further characterisation of the cheese itself and the effect thereon of the microbiota, especially the strain of P. roqueforti.

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Bacterial Surface-ripened Cheeses N.M. Brennan and T.M. Cogan, Teagasc, Moorepark, Fermoy, Co. Cork, Ireland M. Loessner and S. Scherer, Abteilung Mikrobiologic, ZIEL, Technical University of Munich, Germany

Introduction Many cheeses are characterized by the development of microbial growth on their surfaces during ripening. These are called surface-ripened cheeses and are subdivided into mould-ripened and bacterial-ripened cheeses, depending on the major micro-organisms involved. Mould surface-ripened cheeses include the well-known varieties, Brie and Camembert. Bacterial surface-ripened cheeses are less well-known and include Beaufort, Brick, Butterkäse, Comté, Epoisse, Esrom, Gruyère, Havarti, Italico, Limburger, Livarot, Mont d’Or, Münster, Pont l’Evêque, Port du Salut, Reblochon, Serra da Estrêla, Taleggio, Tetilla, Tilsit and Trappist. Bacterial surface-ripened cheeses are also called smear-ripened cheeses, because of the glistening appearance of the cheese surface, washed-rind cheeses, because their rind is washed several times with brine during ripening or red-smear cheeses, because of the red colour which characteristically develops on the surface of these cheeses. Bacterial-ripened cheeses are produced extensively in Austria, Belgium, Germany and France, but they are much less important in English-speaking countries. While these cheeses are made using lactic acid bacteria (LAB) as starters, their flavour is determined primarily by the growth of the surface microflora. The biochemical activity of these microflora results in the development of a cabbagy, garlicky or putrid flavour during ripening, due mainly to the production of sulphur compounds from methionine, particularly methanthiol (Adda et al., 1978; Hemme et al., 1982; Ferchichi et al., 1985; Manning and Nursten, 1985). Sulphur compounds have been identified in many cheese varieties and their importance in smear-ripened cheeses appears to be accentuated by their high concentration at the surface; interactions between the sulphur compounds generate the typical cheese flavour (Ferchichi et al., 1985; Gripon et al., 1991). The microbial composition of the smear of these cheeses is dominated by salt-tolerant yeast and Gram-positive bacteria, particularly coryneforms and staphylococci. Bacterial counts during ripening can exceed 109/cm2 while those of yeasts are generally ⬃107/cm2. The pH of the surface also increases during ripening due to the catabolism of

lactate and the production of NH3 through deamination of amino acids by the surface micro-organisms. The last review of these cheeses was that of Reps (1993) who evaluated their chemistry, biochemistry and microbiology. Since then, there has been little additional information on the first two aspects, except for the studies of Leclercq-Perlat et al. (2000a,b) (see ‘Brevibacterium linens’). The microbiology of the surface microflora of these cheeses is poorly understood and in this chapter we will review what is known about it, how it develops and is controlled during ripening, and the potential of the cheese surface to promote the growth of pathogens.

Factors that Affect Ripening of Smear Cheeses Manufacture

Typically, mesophilic mixed-strain cultures are used as starters for smear-ripened cheese, and the curds are cooked to a low temperature (35 °C), are lightly pressed and are usually brine-salted after moulding. Consequently, these cheeses have high moisture contents and are either soft, e.g., Reblochon and Limburger, or semi-hard, e.g., Tilsit and Pont l’Evêque. Beaufort, Comté and Gruyère cheeses are exceptions to this general rule; they are made with thermophilic cultures, are heated to a high temperature, are pressed at a high pressure and, consequently, have low moisture contents. Smear cheeses are normally salted by brining for 4–18 h, depending on the size of the cheese, with smaller cheeses being brined for shorter periods, after which the cheeses are drained for some hours to remove excess brine. Again, Beaufort, Comté and Gruyère cheeses are an exception to this rule as their surfaces are rubbed with dry salt several times throughout ripening. After brining, smear cheeses are either deliberately inoculated with commercial preparations containing different combinations of Brevibacterium linens, Debaryomyces hansenii and/or Geotrichum candidum (Busse, 1989; Hahn and Hammer, 1990) or, in some countries, particularly Germany, young cheeses are smeared or washed with smears from older cheeses. This so-called

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200 Bacterial Surface-ripened Cheeses

‘old-young’ smearing ensures that all the organisms required for surface ripening are transferred to the young cheeses (Kammerlehner, 1995). The main disadvantage of this approach is that undesirable contaminants, such as Listeria, if they are present on the surface of the old cheeses, will contaminate all the young cheeses (Hahn and Hammer, 1990, 1993). In Austria, B. linens is the only micro-organism deliberately inoculated on the surface of the cheese; all other organisms on the surface of Austrian smear cheeses are adventitious contaminants. The cheeses are ripened at a high relative humidity (RH) and temperature in the range 10–15 °C for a period ranging from 14 to 63 days (Table 1) and are washed frequently with a brine solution during the early stages of ripening. Sometimes, the cheese surface is inoculated a second time with the desired culture(s). Environmental factors

Environmental parameters like RH, ripening temperature, ripening time, microflora of the cheesemaking equipment, brine, etc. and the frequency of washing of the cheese all influence the development of the cheese microflora and dictate the surface characteristics of the final cheese. It is also likely that interactions occur between these parameters but this has not been studied. The high RH prevents the surface of the cheese from drying out while the relatively high temperature and the duration of ripening promote the growth of micro-organisms present while the washing ensures a uniform distribution of micro-organisms on the cheese surface. Generally, one can see visible growth on the cheese surface within a few days of the beginning of ripening. The range of ripening temperature is relatively narrow and is part of the tradition used to make each cheese. A higher ripening temperature is used for

Gruyère and Comté, to promote the growth of propionic acid bacteria (PAB) in these cheeses. There does not appear to be an obvious correlation between the time, the temperature of ripening and the salt and moisture contents of the different cheeses (Table 1). It has been suggested that the microflora of the brine influence the microflora of the immature cheese (Siewert, 1986) but Eliskases-Lechner and Ginzinger (1995b) could not confirm this. Distribution of the smear is vital, as rapid spreading of the micro-organisms from cheese to cheese ensures uniform ripening and reduces the risk of unwanted contaminants, like moulds, colonizing the cheese surface. Washing the cheese disrupts the micro-habitat and the moulds are brought into direct competition with other microorganisms and are out-competed due to their slow growth rates. As a result, a uniform bacterial smear develops. After 2–3 weeks, the desired microflora has developed, and soft and semi-soft cheeses are then wrapped or transferred to another ripening room at a lower temperature for further maturation. The development of the surface microflora is also influenced by the presence or absence of oxygen. If the shelves on which the cheese is ripened are solid, the cheeses must be turned frequently; infrequent turning will limit the amount of oxygen that reaches the surface of the cheese in contact with the shelf, thereby limiting microbial activity to the upper surface and the perimeter of the cheese. Physical and chemical characteristics of the cheese

pH, salt and moisture also affect the composition of the surface microflora. Variations in these factors, together with different treatments of the milk (pasteurized or raw), type of starter (indigeneous microflora or

Table 1 Composition of, and ripening conditions for, some bacterial smear-ripened cheeses Composition Cheese

Moisture%

Münster Port du Salut Reblochon Taleggio Pont l’Evêque Limburger

56 56 55 48–50 45–50 45–48 (50% max) 45–55 40–42 (44% max) 38–40 31

Tilsit Brick Beaufort Gruyère

From Robinson (1995).

Ripening conditions Salt%

Typical aw

%RH

Temperature (°C)

Time (days)

⬃90 ⬃90 ⬃90

18–20 12–18 15 3–4 12–13 10–15 4–10 15 8–12

14–21 42–56 35–42 42–56 28–42 14–21 42–56 30 14 56–89 120 14–21 21–90

1.1–1.3 2.0 1.8–3.0

0.98 0.98 0.97

80–85 ⬃90

2.5 1.8–2.5

0.96 0.98

90 ⬃90

1.1–1.3

⬃0.97

⬃92

8–12 10 15–18

Bacterial Surface-ripened Cheeses 201

commercial preparations), degree of cooking, pressing and salting of the curd before moulding and frequency of washing during ripening, ripening temperature and RH, and the length of the ripening period, have led to the development of the many different varieties of smear cheeses, few of which have been studied in detail. The pH of a young cheese, after acidification of the cheese curd by the LAB, is about 5.0. Yeasts and moulds can develop at this pH but it is generally felt that the salt-tolerant bacterial flora cannot grow at pH values less than 5.6, or even 6.0. Thus, the yeasts grow during the initial stages of ripening, de-acidifying the cheese surface. The consequent increase in pH allows the subsequent development of the salt-tolerant bacterial flora (see ‘Staphylococci and micrococci’ and ‘Coryneforms’). However, recent data show that many bacterial isolates from the cheese surface can grow at pH 4.9, in the presence of 8% salt (Brennan et al., 2002). Generally, smear cheeses are brine-salted and the salt diffuses into the cheese relatively slowly, resulting in a salt gradient with the highest concentration on the surface (see ‘Salt in Cheese: Physical, Chemical and Biological Aspects’, Volume 1). The surfaces of some cheeses are dry-salted, e.g., Comté. The salt level, in turn, will reflect the method and the length of brining. The longer the cheese is salted, the higher will be the level of salt on the surface. Both the salt and the moisture levels directly affect the water activity (aw). The moisture content of surface-ripened cheeses varies from 38 to 56 g/100 g and the salt level from 1.1 to 2.5 g/100 g (Table 1). The contribution of other solutes besides salt (e.g., other ions, like Ca, phosphate, etc., and amino acids) to aw is very low, so the aw of cheese calculated from the salt and the moisture contents should be an accurate indicator of how quickly the surface flora will grow. The calculated aw values, based on salt and moisture levels (Table 1), vary from 0.95 for Comté, which is a hard cheese, containing 40% moisture, to 0.98 for Münster, a soft cheese containing 56% moisture. Smear cheeses generally have a high surface area:volume (SA:V) ratio. The smaller the cheese, the higher will be the SA:V ratio and the larger will be the effect of biochemical changes produced by microorganisms present in the smear. The SA:V ratio also affects the salt level, as smaller cheeses will have more rapid salt diffusion.

content on the cheese surface. Little information is available on the initial number of yeast on the cheese surface. In an Austrian study (Eliskases-Lechner and Ginzinger, 1995b), where deliberate inoculation of the cheese surface with yeast is not practised, the number of yeast in cheese from 14 Tilsit cheese plants, 3 days after manufacture, ranged from 100 to 3  106/cm2, with an average of 104/cm2. There is some evidence that the initial number of yeast also determines the final level. For example, cheese with initial levels of 10 or 1000 yeast/cm2 reached final levels of 105 and 8  107/cm2 after 2–3 weeks of ripening, respectively (Eliskases-Lechner and Ginzinger, 1995b). In contrast, in another plant in which an unspecified soft cheese was made, the maximum number of yeast was 107–108/cm2 despite considerable variation in their initial number. The species of yeast found in several different redsmear cheeses are summarized in Table 2. Generally, D. hansenii and G. candidum are the most important species, with Trichosporon beigelii and Yarrowia lipolytica being important in some cheeses. Considerable variation occurred in the species present on Romadour, and to some extent on Limburger, produced in different factories. The significance of this is unclear. A succession of different species has also been noted at different stages of ripening (Wyder and Puhan, 1999). G. candidium has characteristics of both a yeast and a mould. In the past, it was often called the dairy mould but for the purpose of this review it is considered to be a yeast. During the past 50 years, it has gone through several name changes. In the older literature it is sometimes called Öospora lactis or Oidium lactis. Those strains isolated from soft cheeses grow more rapidly and have higher proteolytic activity than those isolated from hard cheeses. Therefore, two main biotypes can be distinguished based on growth rate and proteolytic activity: the first biotype has a rapid growth rate and a strong proteolytic activity, while the second biotype grows weakly and is only slightly proteolytic (Lenoir, 1984). There is considerable genetic diversity in G. candidum isolates from different French mould- and bacterial-ripened cheeses, including Chevre, Camembert, Livarot, Mont d’Or, Reblochon, Tomme de Savoie, Epoisses, St Nectaire, Pont l’Eveque, Brie, Morbier and some unknown cheeses (Marcellino et al., 2001; Gente et al., 2002). De-acidification

Micro-Organisms in the Smear Yeast

The growth of yeast on the surface of cheese is not surprising because of the low pH, relatively low moisture content, low temperature of storage and high salt

The yeasts on the surface of smear-ripened cheese have two major functions: de-acidification and production of compounds which stimulate the growth of the smear bacteria. They metabolize the lactic acid produced by the starter bacteria to CO2 and H2O, and deaminate amino acids, producing NH3, both of which

202 Bacterial Surface-ripened Cheeses

Table 2 Species of yeast found on the surface of different smear-ripened cheesesa

Teleomorph Candida catenulata Candida intermedia Candida rugosa Candida zeylanoides Cryptococcus laurentii Debaryomyces hansenii Galactomyces geotrichum Saccharomyces dairenensis Torulaspora delbrueckii Trichosporon beigelii Trichosporon ovoides Willopsis california Yarrowia lipolytica Reference

Anamorph

Weinkase

Romadour

Limburger

German

German

German

Factory

Factory

Factory

A

C

A

B 2 2

Candida famata Geotrichum candidum

86 4

95 1

D

A

3

C

Swiss

12

Tilsit

Reblochon

Austrian

French

2

10

69 6

3

55 21

64 17

85 2

22 52

79 5

2 59 6 28 6

Candida colliculosa

24 3

22

Candida lipolytica

3

87 1

1

1

1

19 1

1

1

1 4 20

7

2

3

4

1: Valdés-Stauber et al., 1997; 2: Wyder and Puhan, 1999; 3: Eliskases-Lechner et al., 1995b; 4: Bärtschi et al., 1994. a Results from references 1 and 2 are given as a percentage of the surface yeast microflora; other results are given as a percentage of the number of strains isolated and/or identified.

result in an increase of the pH of the cheese surface from its initial value of ⬃5.0 to 6.5. Thus, a pH gradient is set up with the core being more acid than the surface. In turn, this increase in pH permits the growth of the salt-tolerant bacterial flora. De-acidification not only enables the desirable bacteria to grow but also enhances the action of enzymes, important in ripening, the optimum pH of which is often close to neutrality (Gripon, 1997). The increase in pH modifies the rheological properties of the cheese, resulting in a soft body, which is typical of this type of cheese. Eliskases-Lechner and Ginzinger (1995b) examined the de-acidification properties of 305 strains of yeast in a medium containing 1% yeast-nitrogen-base solution and 3% lactate at pH 4.8. The strains differed in the final pH value attained and in the rate of pH increase: 23% of the strains neutralized (pH 7.0) the medium within 72 h, 33% within 94 h and 38% within 120 h. The inter-strain differences indicated that an unsuitable yeast flora can cause a delay in de-acidification. In such cases, the addition of rapidly de-acidifying yeast may be necessary. Differences in the rate of de-acidification by isolates of different origins were observed, especially in isolates from the brine, which showed very low de-acidification activity. This suggests that brine is not a major source of yeast. These workers also reported no correlation between the change in pH and the composition of the yeast flora on the cheese surface. In spite of low numbers of yeast, de-acidification on the cheese surface was

not delayed, indicating that de-acidification depends not only on the number of yeast present but also on the strain. Production of stimulatory compounds

Yeasts on the surface of these cheeses also produce stimulatory growth substances, which appear to be necessary for the growth of B. linens. A study by Purko et al. (1951a) on the associative action between some yeast and B. linens showed that the latter did not grow on a vitamin-free agar medium but when the same medium was inoculated with yeast, B. linens grew around the yeast colonies. These workers also showed that strains of yeast isolated from the surface of Limburger cheese produced significant amounts of pantothenic acid, niacin and riboflavin. Purko et al. (1951b) demonstrated that B. linens grown on a semi-synthetic medium containing either pantothenic acid or p-aminobenzoic acid did not require biotin for growth and that the rate and the amount of growth of B. linens increased greatly in the presence of pantothenic or p-aminobenzoic acid. Lubert and Frazier (1955) demonstrated that the autolysates of yeast isolated from the smear of Brick cheese contained substances that stimulated the growth of Micrococcus caseolyticus, Mc. freudenreichii and Mc. varians. Two of these species, Mc. caseolyticus and Mc. varians have subsequently been reclassified as Staphylococcus caseolyticus (Schleifer et al., 1982), which has been reclassified again as Mc. caseolyticus (Kloos et al., 1998).

Bacterial Surface-ripened Cheeses 203

Yeasts also contribute to the ripening process through their proteolytic and lipolytic activities which, although slight, would be active during several weeks of ripening. In addition, they prevent the surface of the cheese from drying out and influence flavour formation by producing volatile acids and carbonyl compounds (Siewert, 1986).

while Staphylococcus spp. have a low GC content and are included in the clostridial branch. Although they appear similar (clusters of cocci) under the microscope, they are easily separated from each other. Micrococci are resistant to furazolidone and lysostaphin and, when they produce acid from glucose, do so only under aerobic conditions, while staphylococci are sensitive to furazolidone and lysostaphin and produce acid from glucose anaerobically. Phylogenetic and chemotaxonomic analyses have shown that the genus Micrococcus is very heterogeneous and more closely related to Arthrobacter than to Staphylococcus. Recently, it has been separated into five genera, Micrococcus sensu stricto, Kocuria, Nesterenkonia, Kytococcus and Dermacoccus, and these genera, together with others such as Arthrobacter and Stomatococcus, belong to the family Micrococcaceae (Stackebrandt et al., 1995, 1997). The species of Staphylococcus and Micrococcus found on the surface of several cheeses are summarized in Table 3. Whether Casar de Cáceres should be considered a smear cheese is not clear. It is included because

Staphylococci and micrococci

Although coryneform bacteria play the most important role in the ripening of smear- cheese, recent studies have indicated that large numbers of micrococci and staphylococci are also found on the surface of these cheeses (Cácares et al., 1997; Irlinger et al., 1997; Valdés-Stauber et al., 1997; Carnio et al., 1999; Irlinger and Bergère, 1999; Bockelmann and Hoppe-Seyler, 2001; Brennan et al., 2002). Micrococcus and Staphylococcus have been placed in the same family, but they are not related phylogenetically. Micrococcus spp. have a high GC content and are included in the actinomycetes branch of the eubacteria,

Table 3 Species of Staphylococcus and Micrococcus (number of isolates) isolated from different smear-ripened cheese

Pont l’Eveque Staphylococci S. aureus S. capitis S. caprae S. caseolyticus S. cohnii S. epidermidis S. equorum S. gallinarum S. hominis S. intermedius S. lentus S. saprophyticus S. sciuri S. vitulis S. xylosus Unidentified Total number of strains Micrococci Micrococcus luteus Kocuria varians K. roseus K. kristinae Unidentified Total number of strains Raw milk Reference

2

2

Livarot

9 1

2 3

Munster

3

Maroilles

1

1

Several French cheesesa

Casar de Caceresb

1

6 1 1 36 5

89 3

2

1

1 1 1 6

2

9 3 18

1 6 23

1 3 15

5 2 8

7 7 3 20 16 1 145

3 115 183

22

3 3

7

/ 1

5 5 / 1

1 1 / 1

1: Michaux (1983); 2: Irlinger et al. (1997); 3: Cácares et al. (1997). a Mostly from the surface. b 1 cm below the surface. c Brachybacterium species.

1 1 8

3c / 1

10 2

 3

204 Bacterial Surface-ripened Cheeses

dry salt is spread on the surface a few times during ripening which may help it to develop a surface flora. Staphylococci are more important than micrococci with Staph. equorum, Staph. saprophyticus, Staph. caseolyticus and Staph. xylosus being the most prominent species. Staph. caseolyticus has been reclassified as Macrococcus caseolyticus (Kloos et al., 1998). Staph. equorum was more or less the dominant bacterium on the surface of different French and German cheeses in the studies of Carnio et al. (1999) and Bockelmann and Hoppe-Seyler (2001). Recently, two new species of staphylococci, Staph. fleuretti and Staph. succinus subsp. casei, have also been isolated from smear-ripened cheeses (VernozyRozand et al., 2000; Place et al., 2002). Recent data show that a progression of bacteria occurs in the smear; staphylococci are the major organisms found early in ripening and are replaced by coryneform bacteria some days later (Brennan et al., 2002). In addition, low numbers of coagulase-positive pathogenic species, e.g., Staph. aureus, Staph. intermedius and Staph. hyicus have been isolated from a few cheeses. The cheeses from which they were isolated are all raw-milk cheeses which may imply that they originated in the raw milk. The dominant micrococci appear to be Kocuria varians and Mc. luteus. Most strains of staphylococci and micrococci can grow in the presence of 10% salt, which means that a general-purpose medium containing sufficient salt to inhibit the starter bacteria would be a very good selective medium. Staphylococci are also inhibited by furazolidone, implying that furazolidone-containing media should be selective for micrococci. Plate Count Agar containing 5% NaCl was used by Eliskases-Lechner and Ginzinger (1995a) and Brennan et al. (2002) to isolate the bacteria from the smear of Tilsit and Gubbeen cheeses, respectively. At each stage of ripening (4, 16, 23 and 37 days) of Gubbeen, the predominant organisms were coryneforms, and very few staphylococci and no micrococci were found (the staphylococci were found mainly on day 4 of ripening). Significant numbers of non-pigmented micrococci (whether they were staphylococci or micrococci was not determined) have been found on the surface of Comté and Beaufort cheeses during ripening, and the flora was dominated by coryneforms (Piton-Malleret and Gorrieri, 1992). Coryneforms

Coryneform bacteria (Arthrobacter, Brachybacterium, Brevibacterium, Corynebacterium, Microbacterium and Rhodococcus spp.) and related taxa occur almost everywhere on living and non-living matter in the environment. They are especially important on the surface of smeared cheeses, and numerous species have been

found in different cheeses, particularly Tilsit (Table 4). The classification of coryneform bacteria is very confusing. The methods used in recent years include chemotaxonomic techniques, e.g., polyamine patterns (Altenburger et al., 1997; Busse und Schumann, 1999), fatty acids (Kämpfer und Kroppenstedt, 1996), numerical taxonomic analysis (Kämpfer et al., 1993) and assessment of the heterogeneity of partial 16S rRNA sequences by temperature-gradient gel electrophoresis (TGGE; Felske et al., 1999). Other methods, which place more emphasis on the identification of coryneform genera, have also been reported, e.g., analysis of physiological characteristics by the BIOLOG Identification System (Lindenmann et al., 1995) or the API (RAPID) Coryne database (Funke et al., 1997) or the RapID CB Plus system (Funke et al., 1998), comparative 16S rDNA sequence analysis (Bockelmann et al., 1997b), the use of genus-specific oligonucleotide probes (Kollöffel et al., 1997), fluorescence in-situ hybridization (FISH) and colony hybridization (Kollöffel et al., 1999), as well as Fourier-transform infrared spectroscopy (Oberreuter et al., 2002). The latter is receiving more attention, since an extensive database for the identification of bacteria from the two suborders Micrococcineae and Corynebacterineae, is now available (Oberreuter et al., 2002). Such studies have shown that many of the coryneform bacteria isolated from cheese have been misclassified. For example, Microbacterium flavum, which was isolated from an unspecified variety of cheese, has been redesignated as Corynebacterium flavescens (Barksdale et al., 1979), Caseobacter polymorphus, originally isolated from Limburger and Meshanger cheese, as C. variabilis (Collins et al., 1989), B. ammoniagenes as C. ammoniagenes (Collins, 1987) and methanethiol-producing coryneforms, isolated from Cheddar cheese and milk, as B. casei (Collins et al., 1983). In addition, the genus Aureobacterium has been amalgamated with Microbacterium (Takeuchi and Hatano, 1998). Such data suggest that many of the bacteria in Table 4 may be misidentified. New results from molecular bacterial taxonomy revealed that there is no taxon ‘coryneform bacteria’ (Stackebrandt et al., 1997). Today, the family names Micrococcaceae, Brevibacteriaceae and Corynebacteriaceae should be used with the corresponding genera (Table 5). For practical reasons, the expression, coryneform bacteria, is still widely used to group Arthrobacter, Brevibacterium, Corynebacterium and Microbacterium spp., and will be used in this paper, too. Brevibacterium linens

B. linens is a major micro-organism in the smear of surface-ripened cheeses. Its enzymes, especially its proteolytic and lipolytic ones, and biochemical characteristics

Bacterial Surface-ripened Cheeses 205

Table 4 Different species (as number of isolates) of coryneforms found in smear-ripened cheeses Limburger Species Arthrobacter citreus A. globiformis A. nicotianae A. variabilis Arthrobacter spp. Brachybacterium alimentarium Br. tyrofermentans Brevibacterium fermentans B. fuscum B. helvolum B. linens B. oxydans Brevibacterium sp. Corynebacterium ammoniagenes C. casei C. mooreparkense C. variabilis C. flavescens Corynebacterium sp. Curtobacterium insidiosum Curto. poinsettiae Curto. betae Microbacterium imperiale Microbacterium gubbeenense Total number Reference

a

b

Romadour

Weinkase

Harzer

1 5

1

5

2

5

1

2

7

2

Taleggio

Tilsit

 

19 102 10 14 43

Gubbeen

Gruyère

Beaufort

 

 

5

5

1

3

4

5

2

8

3

3

16

2



1

2 1 77 3 4 53 115 44

1

1 

4

3

1

2

6

5 25 8 12 4 8 33

27

11

13

33

13

1

1

1

1

1

385 2

3

4

1: Valdés-Stauber et al. (1997); 2: Piantanida et al. (1996); 3: Eliskases-Lechner and Ginzinger (1995a); 4: Brennan et al. (2002); 5: Schubert et al. (1996). a Includes Weinkase. b Includes Romadour cheese.

influence the ripening and final characteristics of smear surface-ripened cheeses (for review, see Rattray and Fox, 1999). Because of its ubiquitous presence on the surface of a variety of cheeses, such as Limburger, Münster, Brick, Tilsiter and Appenzeller, it has long been recognized as an important dairy micro-organism. B. linens is strictly aerobic, halotolerant, with a rod–coccus growth cycle, temperature growth optimum of 20–30 °C, and optimum growth at pH 6.5–8.5. The growth of B. linens on the cheese surface is stimulated by vitamins produced by the yeasts (Purko et al., 1951). Extracellular, cell wall-associated and intracellular proteinases have been reported for B. linens, as well as

the ability to produce different bacteriocins and pigments for colour development on the cheeses (see ‘Cheese pigmentation and colour development’). The early literature suggests that the only organism in the smear was Bacterium linens (Wolff, 1910), which was later reclassified as Brevibacterium linens (Breed, 1953). More recently, B. linens has been reported to represent only 30% of the bacteria on the surface of smear-ripened cheese (Brandl, 1980; Busse, 1989), 22% of the total microflora of Limburger cheese (El-Erian, 1969) and 15% of the total microflora of Tilsit cheese (Bockelmann, 1997). Recent studies have indicated that several other coryneform bacteria, besides B. linens, including

206 Bacterial Surface-ripened Cheeses

Table 5 Current hierarchic classification of bacterial families and genera with relevance for smear cheeses Actinobacteria Micrococcineae Micrococcaceae Arthrobacter, Micrococcus, Kocuria, Renibacterium Brevibacteriaceae Brevibacterium Microbacteriaceae Microbacterium/Aureobacterium, Clavibacter, Curtobacterium Dermabacteriaceae Brachybacterium, Dermabacter Corynebacterinea Corynebacteriaceae Corynebacterium, Turicella Nocardiaceae Rhodococcus, Nocardia Firmicutes with low GC content of DNA Staphylococcus, Enterococcus, Listeria, Bacillus From Bockelmann and Hoppe-Seyler (2001).

Arthrobacter citreus, A. globiformis, A. nicotianae, B. imperiale, B. fuscum, B. oxidans, B. helvolum, Brachybacterium alimentarium, Br. tyro-fermentans, Corynebacterium ammoniagenes, C. betae, C. casei, C. mooreparkense, C. insidiosum, C. variabilis, Curtobacterium pointsettiae, Microbacterium imperiale and Mc. gubbeenense, are also found on the surface of various cheeses (Table 4; Seiler, 1986; Busse, 1989; Eliskases-Lechner and Ginzinger, 1995a; Schubert et al., 1996; Irlinger et al., 1997; ValdésStauber et al., 1997; Carnio et al., 1999; Irlinger and Bergère, 1999; Bockelmann and Hoppe-Seyler, 2001; Brennan et al., 2002). The importance of each of the species listed in Tables 3 and 4, in the development of the flavour of smear-ripened cheese, has not been determined. However, Leclercq-Perlat et al. (2000a,b) carried out an extensive study of the growth of D. hansenii and B. linens, the utilization of lactate and the production of acid-soluble nitrogen and NPN, in aseptically made, model soft-cheese during ripening. D. hansenii grew rapidly during the first 2 days and then slowed down but it continued to grow exponentially (generation time ⬃70 h) until day 10 (pH of rind ⬃6), after which growth stopped. In contrast, B. linens did not begin to grow until about day 15 when the pH was ⬃7 but it then grew exponentially until day 70 (generation time 70 h). Total cell numbers of each micro-organism were 10–100-fold higher than their respective viable counts. The pH of the rind increased linearly during the first 23 days at a rate of 0.12 pH units/day while the lactate level in the rind decreased linearly at a rate of 12.5 mmol/kg of dry matter per day up to day 25, then more slowly until day 35 when it was essentially zero. The lactate level in the inner mass of the cheese also

decreased linearly until day 41. Acid-soluble nitrogen increased exponentially throughout ripening, and the rate was faster when the cheese was stored at 4 °C (day 43–day 76) than when the cheese was held at 13 °C from day 2 to day 43. The most rapid increase in NPN was between day 22 and day 42 when the cheese was held at 13 °C, although it also increased during storage at 4 °C. The NH3 concentration in the rind remained low during the first 24 days of ripening after which it increased linearly to a level of 2 g/kg DM on day 76. The changes in NPN correlated positively with the growth of B. linens. Corynebacterium

Corynebacterium spp. are facultatively anaerobic, Grampositive chemo-organotrophs, which are widely distributed in soil, plants and waste water (Crombach, 1972) and may occur in various foods. Some species of the genus Corynebacterium are major components of the smear microflora of bacterial smear surface-ripened cheeses, e.g., C. ammoniagenes (Table 4). Many Corynebacterium and Arthrobacter isolates in Table 4 have not been identified to soecies level and recently two new species, C. casei and C. mooreparkense, were found in large numbers, especially at the later stages of ripening on the surface of an Irish smear-ripened cheese (Brennan et al., 2001a). Methanethiol is considered to be an important compound in flavour determination of these cheeses and all ten isolates of C. mooreparkense and three of ten isolates of C. casei were able to produce it. In addition, C. mooreparkense and C. casei had esterase and leucine arylamidase activitities, and C. mooreparkense had lipase activity which probably play some role in cheese ripening (Brennan et al., 2001a). Other new species have also been isolated including Brachybacterium tyrofermentans and Br. alimentarium from the surface of Gruyére and Beaufort cheese (Schubert et al., 1996) and Microbacterium gubbeenense from Gubbeen cheese (Brennan et al., 2001b). It is probable that the surface of red-smear cheeses will be a source of further new species. Arthrobacteria

Arthrobacter spp. are Gram-positive, chemo-organotrophs which have a respiratory metabolism, never fermentative, are widely distributed among the bacterial population of soil, and are major components of the smear microflora of some surface-ripened cheeses (Mulder et al., 1966; ElErian, 1969; Eliskases-Lechner and Ginzinger, 1995a; Valdés-Stauber et al., 1997, Table 4). Mulder et al. (1966) isolated 22 strains of Arthrobacter, which formed grey-white colonies, from the surface of many types of cheese. In the presence of NaCl, they could develop at pH 5.5, i.e., considerably earlier than other coryneforms. El-Erian (1969) isolated 173 strains

Bacterial Surface-ripened Cheeses 207

of Arthrobacter from Limburger cheese and classified them into four groups according to the colour of the colonies formed during growth. The studies of Seiler (1986) indicated the importance of yellow-pigmented Arthrobacter species for ripening of Tilsit cheeses. Coryneform bacteria on 21 brick cheeses were isolated and identified by Valdés-Stauber et al. (1997). Of 148 isolates differentiated in microtiter plates by comparison to reference strains, 21 Arthrobacter sp. and 14 A. nicotianae strains could be identified; A. citreus appeared only sporadically. Bockelmann et al. (1997a) found that the development of red colour could be promoted by mixed-cultures of yellow Arthrobacter strains and B. linens. The importance of yellowpigmented A. nicotianeae for the development of the typical reddish-brown colour of semi-hard cheese has been confirmed (Bockelmann et al., 1997a). Despite their presence at high cell numbers on the surface of several cheeses, only a few studies on the enzymology of dairy Arthrobacter spp. have been reported (see ‘Flavour development’). Some Arthrobacter strains isolated from the surface of red-smear cheeses are known to produce bacteriocins or bacteriocin-like substances. Hug-Michel et al. (1989) isolated 80 bacterial strains from the surface of Vacherin Mont d`Or cheese, including seven A. protophormiae and one A. uratoxydans strains. A wide taxonomical distribution of the lin gene, responsible for production of Linocin M18 within coryneform bacteria, has been demonstrated by Valdés-Stauber and Scherer (1996); five out of six Arthrobacter strains tested possessed the gene necessary for the production of Linocin M18. From a total of 2613 individual colonies, eleven Arthrobacter strains were shown to produce clear zones of inhibition on solid media against six or more strains of Listeria monocytogenes (Carnio et al., 1999). The secondary flora Enterococci

Enterococci are ubiquitous bacteria which frequently occur in large numbers in dairy and other food products. Although they share a number of biotechnological traits (e.g., bacteriocin production, probiotic characteristics, usefulness in dairy technology) there is no consensus on whether enterococci pose a threat as food-borne pathogens (Giraffa et al., 1997). The presence of enterococci in dairy products has long been considered as an indication of poor hygiene during the production and processing of milk. On the contrary, many authors suggest that enterococci may have a potential desirable role in some cheeses because they occur in large numbers (up to 107–108 cfu/g) in the indigeneous microflora of many cheeses. For example, enterococci were found to be present in more than 96% of 48 Italian fresh, soft and semi-

hard cheeses examined, ranging from 101 to 106 cfu/g. In hard and semi-hard cheeses, the counts of Enterococcus spp. were greater, and the organisms persisted longer, than the other microflora (Gatti et al., 1993). Numerous strains of enterococci associated with food systems are capable of producing a variety of antibacterial proteins (enterococcins) with activity against food-borne pathogens, such as Listeria monocytogenes, pathogenic clostridia or Staphylococcus aureus (for review, see Giraffa, 1995). An overview of the potential of enterococcal bacteriocins in model cheese systems is given in ‘Enterococcal bacteriocins’. Non-starter lactic acid bacteria

Non-starter lactic acid bacteria (NSLAB) are mesophilic lactobacilli and pediococci, which form a significant portion of the microbial flora of most cheese varieties during ripening (Beresford et al., 2001). They are not part of the normal starter flora and, generally, they do not grow well in milk (Cogan et al., 1997). Lactobacilli are either (i) obligatory homofermentative, (ii) facultatively heterofermentative or (iii) obligatory heterofermentative (Kandler and Weiss, 1986). Many species of mesophilic lactobacilli have been isolated from surface-ripened cheese, but the most frequently encountered are Lactobacillus casei/Lb. paracasei, Lb. plantarum, Lb. rhamnosus and Lb. curvatus (Ennahar et al., 1996; Coppola et al., 1997). As NSLAB are found in cheeses made from raw or pasteurized milk, the major source of these microorganisms may be the raw milk itself, or in case of cheese made from pasteurized milk, either post-process contamination or failure of pasteurization to fully inactivate them (Beresford et al., 2001). Non-starter lactic acid bacteria may have some effect on the maturation of cheeses. Pediococci, which develop in Comté cheeses, have proteolytic, lipolytic and esterolytic activities (Bhowmik and Marth, 1990) and therefore could play a beneficial role during cheese ripening. In the same way, facultatively heterofermentative lactobacilli are potentially highly proteolytic (Khalid and Marth, 1990). Bouton et al. (1998) suggested that the high number of lactobacilli present at the end of ripening indicated that these bacteria could have an effect on flavour development. Only a few authors report significant numbers of NSLAB in red-smear cheeses, most of them in lowmoisture hard cheeses, whereas in other extensive studies (Carnio et al., 1999; Brennan et al., 2002), no information about the incidence of NSLAB is reported. Coppola et al. (1997) isolated lactobacilli (mainly Lb. paracasei subsp. paracasei and Lb. rhamnosus) and pediococci (Pediococcus acidilactici) from MRS agar during the later stages of Parmiggiano Reggiano cheesemaking. In the study of Bouton et al. (1998), the number of propionibacteria, facultative heterofermentative lactobacilli

208 Bacterial Surface-ripened Cheeses

and enterococci increased during the ripening of Comté cheese. On the other hand, Lb. plantarum WHE 92 was found at high levels (108 cfu/g cheese) among 1962 bacterial isolates from the surface of a soft smear cheese (Münster) and had anti-listerial activity (Ennahar et al., 1996; see ‘Bacteriocins of lactic acid bacteria’). Propionic acid bacteria

Propionic acid bacteria (PAB) grow in many cheese varieties and are the characteristic microflora associated with surface-ripened Swiss-type cheeses such as Gruyère, Appenzeller, or Comté. In cheese manufactured from raw milk, sufficient ‘wild’ PAB are present. However, with the advent of pasteurization, PAB are now added to the cheese milk at the beginning of manufacture to ensure that they are present in sufficiently high numbers (Vorobjeva, 1999). The cheeses undergo a propionic acid fermentation, and the propionic and acetic acids produced contribute to the development of characteristic flavours of the cheese. Flavour development Sulphur amino acids

Sulphur compounds are particularly important in the flavour of probably all cheeses, particularly smearripened varieties. Metabolism of sulphur amino acids by bacteria associated with cheese has been reviewed by Weimer et al. (1999) but was treated mainly from the involvement of lactococci and lactobacilli in Cheddar cheese. It was originally thought that methanethiol (CH3SH) was the major sulphur compound produced and that B. linens was the main organism responsible. Recent evidence suggests that other micro-organisms on the cheese surface, including Mc. luteus, S. equorum, B. acetobutylicum (but not B. flavum), C. glutamicum, Arthrobacter spp., several unidentified coryneforms, G. candidum and the starters, Lc. lactis and Lb. helveticus, also produce CH3SH (Bloes-Breton and Bergère, 1997; Dias and Weimer, 1998a; Berger et al., 1999; Bonnarme et al., 2000). These studies have also shown that other sulphur compounds, including dimethyl sulphide (DMS), dimethyl disulphide (DMDS), dimethyltrisulphide (DMTS), thiols (2-propanethiol, 2methylpentanethiol), thioesters (methylthioacetate, methylthiobutanoate, methyl thiopentanoate), are also involved. These are termed the volatile sulphur compounds (VSC) and quantitatively are produced in very small amounts but as they have very low flavour thresholds, small amounts ( g/kg) are important in flavour perception. CH3SH is produced enzymatically from methionine and the other compounds, particularly DMS, DMDS and DMTS are formed from it by chemical (non-enzymatic) means (see ‘Catabolism of Amino Acids in Cheese during Ripening’, Volume 1).

The most studied VSC is CH3SH, which is produced by two pathways. One involves its direct production from methionine by L-methionine- -lyase in an elimination reaction in which NH3 and -ketobutarate are also produced (Fig. 1). The second mechanism involves a two-step reaction involving an amino transferase or transaminase, which produces -keto- -methyl-thiobutyric acid (KMBA) and glutamate from methionine and -ketoglutarate, respectively. -Keto- -methyl-thiobutyric acid is subsequently transformed to CH3SH. Both pathways appear to be present in many of the organisms examined but which is more important has not been examined. The enzyme activities are very low but so are the flavour thresholds of these compounds. In addition, these enzymes must be active at the relatively high salt (5–10%) concentrations in these cheeses. The L-methionine- -lyase (EC 4.4.4.11) from B. linens has been purified (Dias and Weimer, 1998b). It consists of four identical 43-kDa sub-units. Its optimum temperature is 25 °C and its optimum pH 7.5. The enzyme is inhibited by carbonyl reagents and NaCl but not by metal-chelating agents; 5% NaCl reduces the activity to 10% implying that the enzyme may not be active in the cheese matrix. Dias and Weimer (1998a) found significant production of CH3SH by six of the seven strains of B. linens; however, a commercial strain, B. linens BLI, produced only insignificant amounts. B. acetylicum produced almost as much as the best strain of B. linens, and B. flavum (seven strains) produced none. Micrococci (four strains) were poor producers, but Lc. cremoris S1 and Lb. helveticus CNRZ 32 produced significant amounts. Production of CH3SH from methionine and KMBA by growing cells by five strains of bacteria, commonly found on the surface of cheese, was compared by Bonnarme et al. (2000). Very small amounts of CH3SH were produced, but significant amounts of DMDS and DMTS were produced by B. linens ATCC 9175, Mc. luteus 790 and C. glutamicum D13. Resting cells of B. linens 9175 were also efficient, with Mc. luteus 790 and an Arthrobacter sp. producing 50% of that produced by B. linens, while Staph. equorum 1265 and C. glutamicum D13 only produced 35 and 27%, respectively. Cell-free extracts produced very little CH3SH from KMBA except for C. glutamicum, while B. linens, C. glutamicum and the Arthrobacter sp. produced significant amounts of it from methionine; production by Staph. equorum and Mc. luteus was poor. Four of the ten strains of G. candidum have also been shown to produce significant amounts of CH3SH, DMS DMTS, S-methyl thioacetate, S-methyl thiopropionate, S-methyl thioisobutanoate, S-methyl thioisovalerate and S-methyl thiohexanate (Berger et al., 1999). Except for CH3SH, the exact pathways involved in the production

Bacterial Surface-ripened Cheeses 209

L-Methionine CH3–S–CH2–CH2–CH(NH2)–CO2H

(2)

(1) α-KG

Met ATase

L-Glu

L-Methionine

B. linens (++) M. luteus (+) Arthrobacter sp. (–) C. glutamicum (+) S. equorum (–)

KMBA CH3–S–CH2–CH2–CO–CO2H

γ-lyase

B. linens (++) M. luteus (+) Arthrobacter (+) C. glutamicum (+/–) S. equorum (+/–)

NM4+

MTL CH3SH

KMBA-demethiolase ( ≥ 90%) Chemical conversion ( ≤ 10%)

Chemical conversion

+

B. linens (++) M. luteus (+) Arthrobacter sp. (+/–) C. glutamicum (+/–) S. equorum (+/–)

DMDS CH3S–SCH3 and DMTS CH3S–S–SCH3

α-Ketobutyrate CH3–CH2–CO–CO2H Figure 1 Two pathways for the production of methanthiol. Reprinted from Bonnarme et al. (2000) with permission. Met ATase, methionine aminotransferase; KMBA, -keto- -methyl thio butyric acid; DMDS, dimethyl disulphide; DMTS, dimethyl trisulphide; -KG, -ketoglutarate; MTL, methanethiol.

of these compounds have not been determined but the redox potential (Eh) is thought to be important. Proteinases and lipases

The smear bacteria also contribute to the ripening process through their proteolytic and lipolytic activities. Hosono and Tokita (1970) studied the lipolytic properties of C. mycoderma and D. kloekeri isolated from the surface of Limburger cheese. Both yeasts produced extracellular lipases with a pH optimum of 4.5, and optimum temperatures of 35 and 30 °C for the lipases of C. mycoderma and D. kloeckeri, respectively. These workers also reported that the lipase of C. mycoderma released considerably more lauric, myristic and palmitic acids and less stearic acid from milk fat than the lipases of D. kloekeri. Three esterases, which were active on aliphatic and nitrophenyl esters, were isolated from Brevibacterium strain R312 (Lambrechts et al., 1995); they had an optimum pH of 6–8, 7.6 and 8, and optimum temperatures of 43, 36 and 30 °C, respectively. Two of them appeared to be sulphydryl enzymes. An extracellular proteinese and an intracellular aminopeptidase have been purified from B. linens ATCC 9174 (Rattray et al., 1995; Rattray and Fox,

1997). The proteinase is a dimer of molecular mass 126 kDa. Its optimum pH is 8.5, its optimum temperature 50 °C and it is activated by Mg2 and Ca2; Hg2, Fe2 and Zn2 strongly inhibited the enzyme. NaCl also stimulated the enzyme which appeared to be a serine proteinase. The aminopeptidase was a thiol enzyme with a mass of 59 kDa, had an optimum pH of 8.5, an optimum temperature of 35 °C and was inhibited by Co2 and Zn2. Some of the putative extracellular enzymes from A. nicontianae, which may be important in cheese ripening, including two serine proteinases, a proline iminopeptidase and an esterase, have been purified by Smacchi et al. (1999a,b, 2000). The proteinases had molecular masses of 54 and 71 kDa and pH optima at 9–9.5; they also had considerable activity at pH 6 on s1- and -caseins. The proline iminopeptidase hydrolysed proline-containing peptides under the pH, temperature and salt concentration of surface-ripened cheese. An extracellular proline iminopeptidase has also been purified from C. variabilis (Gobbetti et al., 2001). It is a serine enzyme with a pH optimum of 8.5; however, significant activity also occurred at pH 6 and in the presence of 7.5% NaCl, implying that it would also be active on the cheese surface during ripening.

210 Bacterial Surface-ripened Cheeses

G. candidum produces several intracellular and extracellular proteinases (Lenoir, 1984; Hannan and Gueguen, 1985) and lipases (Alifax, 1979; Sidebottom et al., 1991). Cheese pigmentation and colour development

The smear bacteria are also thought to be responsible for the development of the surface pigmentation characteristic of red-smear cheese. Considering the low percentage of B. linens in the smear reported by El-Erian (1969) and more recently by Bockelmann (1997), it is unlikely that this organism is solely responsible for the colour of the smear. Seiler (1986) demonstrated the importance of yellow-pigmented Arthrobacter species in the ripening of Tilsiter cheese, and Bockelmann et al. (1997a) reported that the development of the redorange colour in the cheese could be promoted by a combination of a yellow Arthrobacter spp. and B. linens. A more intense colour was observed on addition of casein hydrolysate to the growth medium, indicating that the strong proteolytic activity of B. linens may promote colour development. Lee et al. (1985) reported that the metabolism of phenylalanine and tyrosine is essential for the development of colour by coryneform bacteria isolated from cheese, while Ferchichi et al. (1986) reported that methionine is essential for the development of orange pigments by B. linens. These findings were confirmed by Bockelmann et al. (1997a), who observed that the development of the red-orange pigmentation was not achieved on addition of single amino acids to the growth medium. Tyrosine was reported to be the only amino acid which influenced colour, but the characteristic red-orange colour of smear-ripened cheese was not produced. Addition of combinations of tyrosine, phenylalanine and methionine, which have been reported to be responsible for colour development by coryneform bacteria, did not result in the development of pigmentation, indicating that the development of red-orange pigmentation is more complex. Piantanida et al. (1996) showed that the smear micro-organisms influence the surface colour of Taleggio cheese. A white surface indicated the presence of G. candidum, a yellow colour C. flavescens, yellow-green patches the presence of A. globiformis and A. citreus, while a uniform pink-red surface indicated the presence of B. linens.

Ripening and Spoilage of Red-Smear Cheese Origin of the surface microflora

Bacterial smear-ripened cheeses have a long tradition. Without the knowledge of the bacterial nature of the surface flora, a large variety of smear cheeses was pro-

duced long before 1900 (Fox, 1993). Numerous species of yeast and bacteria have been isolated from the surface of smear cheese (Tables 2, 4 and 5). This raises the question ‘where do these micro-organisms originate from?’ Deliberate inoculation of the cheese surface is generally confined to commercially available cultures of D. hansenii, G. candidum and/or B. linens. Many cheese producers do not rely solely on these cultures (see ‘Secondary and Adjunct Cultures’, Volume 1). Traditionally, the surface flora of mature cheeses is used as the source of micro-organisms for smearing young cheeses; the necessary micro-organisms are transferred from the mature cheese to the brine before smearing of the young cheeses is begun. However, commercially available surface starters do not reflect the microbial composition of the cheese surface. Thus, cheese milk, brine, air, utensils and shelves are all possible sources of the surface microflora. Since the introduction of pasteurization, which has considerably improved food safety, the cheese milk flora has less influence on the surface microflora of cheeses (Holsinger et al., 1997), the dependance on an intact ‘house microflora’ and starter cultures has increased considerably. For cheeses made from raw milk, the evolution of the microflora of Comté cheeses made in duplicates from different sources was followed during ripening (Bouton et al., 1998). Comparison of the cheeses revealed no significant difference in the development of the microflora. Most of the micro-organisms found on the cheese surface are very salt tolerant, so brines could also be a source (Bockelmann and Hoppe-Seyler, 2001). However, Eliskases-Lechner and Ginzinger (1995b) found pure cultures of D. hansenii on the surface of Tilsiter cheese at all stages of ripening, even when other species, e.g., Kluyveromyces marxianus and Trichosporon beigelii, were the dominant organisms in the brine. In another study (Eliskases-Lechner and Ginzinger, 1995a), Austrian bacterial surface-ripened cheeses were examined for changes in microbial composition of the smear. The bacterial flora was a mixed population. B. linens, which had been deliberately smeared on the cheese surface, comprised 30% of the total bacterial count, and A. globiformis and C. ammoniagenes were also dominant, although they were not added to the smear. All other species found occurred only sporadically or were found in the smear from a single plant. Thus, one can conclude that all bacteria which grow on the surface and which have not been added deliberately are probably adventitious contaminants, which grow well in the presence of the high salt concentrations and relatively high pH of the cheese surface. It is likely that the major sources are the brine and the wooden shelves on which the cheese rests during ripening (Beresford et al., 2001).

Bacterial Surface-ripened Cheeses 211

Galli et al. (1996) investigated several Taleggio cheese samples at the end of ripening. The bacteria on the surface layer (109–1010 cfu/g smear) were either Gram-positive cocci (Mc. sedenterius, Staph. xylosus, Staph. sciuri) or were Gram-positive irregular asporogeneous rods (Microbacterium lacticum, B. linens, B. casei). Mb. lacticum and B. linens, together with some Micrococcus and Staphylococcus isolates were found to be mainly responsible for the typical orange pigmentation of the ripe cheeses. Bockelmann and Hoppe-Seyler (2001) analysed the surface flora of semi-hard Tilsit, soft Chaumes and semihard goat’s milk cheese. D. hansenii was the predominant yeast at all stages of ripening, and 75–95% of the bacteria were coryneforms. Yellow-pigmented coryneform isolates (1–30%) were A. nicotianae, whereas B. linens was only 0–15%. Non-pathogenic staphylococci, mainly Staph. equorum, comprised 5–15% of the total flora. An extensive study of Danbo, a smear-ripened Danish cheese, showed that D. hansenii was the dominant yeast (Petersen et al., 2002). Using restriction analysis of mitochondrial DNA, these workers showed that none of the four strains in the brine were detected on the cheese surface when it was removed from the brine. Furthermore, there was a progression of strains, since another dominant strain of D. hansenii, which was different from those detected on the cheese early in ripening, developed later in ripening. This strain was able to grow better than the other strains at the pH and salt levels on the cheese surface. Recently, another extensive study was conducted (Brennan et al., 2002) of the bacteria on the surface of a farmhouse smear-ripened cheese at different stages of ripening. Of 400 isolates, 390 were lactate-utilizing coryneforms and 10 were coagulase-negative staphylococci. The cheeses were dominated by single clones of novel species of C. casei (50.2%), C. mooreparkense (26%) and Mb. gubbeenense (12.8%). B. linens BL2 was not found in the surface flora of the cheese, even though it had been deliberately inoculated on to the cheese surface; in fact, it was inhibited by all the Staphylococcus and many of the coryneform isolates. The source of C. casei, C. mooreparkense and Mb. gubbeenense on the cheese surface was not determined. Defined cultures

Deliberate inoculation of the cheese surface is generally confined to commercially available cultures of D. hansenii, G. candidum and/or B. linens. However, these micro-organisms do not reflect the microbial composition of the cheese surface. So far, there is little knowledge about the influence of different strains on colour development of red-smear cheese. Because B. linens produces orange (or red)-coloured colonies, several

groups of workers have distinguished between the colour of the colonies in the smear cheese, e.g., orange, yellow or non-pigmented (Seiler, 1986; Piton-Malleret and Gorrieri, 1992; Eliskases-Lechner and Ginzinger, 1995a; Hoppe-Seyler et al., 2000). The orange-coloured colonies are considered to be B. linens and the yellow-coloured ones, Arthrobacter spp. (Hoppe-Seyler et al., 2000). However, in the study of Piton-Malleret and Gorrieri (1992), the orange- and yellow-coloured colonies comprised 82 and 68% Micrococcaceae (whether they were micrococci or staphylococci was not determined), respectively; the remainder were coryneforms (genus also not designated). In the study of Eliskases-Lechner and Ginzinger (1995a), the cream- and yellow-coloured isolates were predominantly A. globiformis and C. ammoniagenes, respectively. Understanding the microbial ecology of the cheese surface is a prerequisite for the development of defined surface cultures for control of surface ripening (Bockelmann, 1997). Because of the numerous organisms found in smear cheeses, the development of defined-strain smear cultures is being investigated with promising results. Carminati et al. (1999) screened surface-smear organisms isolated from Taleggio cheese for their ability to inhibit different pathogens. Most of the isolates showing anti-listerial activity (19% of the total) were coryneform bacteria, mainly Mb. lacticum. Two bacterial mixtures (containing orange-pigmented strains and strains that inhibited Listeria) were studied as smear cultures for Taleggio cheese. However, the cheese did not undergo normal ripening, due to a delay in the growth of the smear bacteria. The successful use of a defined five-strain culture (D. hansenii, B. linens, A. nicotianae, C. ammoniagenes and Staph. sciuri) for Tilsit cheese was demonstrated by Bockelmann and Hoppe-Seyler (2001) on a pilot scale. No problems with yeast growth were observed, but ripening appeared to be slightly slower compared to another batch in which a commercial smear culture had been used. Nevertheless, after 6 weeks of ripening, cheese quality was good. However, commercially available surface starters do not reflect the diversity of the microbial composition of the cheese surface. Too much emphasis is put on B. linens because of the orange pigmentation. New results from Bockelmann and Hoppe-Seyler (2001) showed that the red-brown and orange pigments are most likely due to the growth of yellow-pigmented Arthrobacter spp. The mechanisms of developing the different shades of red are not yet understood. The presence of a rich and complex microflora on the surface of Taleggio cheese might help to control Listeria contamination. The role of the surface microflora, which develops during ripening, could exert a

212 Bacterial Surface-ripened Cheeses

‘self-protecting activity’ on the hygienic quality of the product (Carminati et al., 1999). Pathogens

Some cheeses, especially red-smear cheeses, may be risk products for bacterial contamination since they constitute a suitable medium for the growth of pathogens like Listeria monocytogenes (Terplan et al., 1986) and some other food-borne pathogens. This is partly due to the traditional ‘old-young’ smearing method, where unripe cheeses are inoculated with a complex, undefined microflora washed from the surface of ripened cheeses. A disadvantage of this approach is that undesirable contaminants will be transferred to the smear bath and will be spread throughout the factory. Smearing machines were found to be an important source of Listeria contamination (Hahn and Hammer, 1993). Listeria monocytogenes

Listeria monocytogenes is a food-borne pathogen, the control of which in food is made difficult by its ubiquity in the environment, its ability to grow at refrigeration temperature and its tolerance to low pH (pH 5.0) and NaCl levels up to 10% (Farber and Peterkin, 1991). Redsmear cheeses are especially at risk from a listeriosis point of view since they constitute a suitable medium for the growth of L. monocytogenes (Terplan et al., 1986). The increase in the pH of the surface layer during the ripening of bacterial surface-ripened cheeses, creates a more favourable environment for growth of microorganisms, including contaminating L. monocytogenes, the causative agent of food-borne human listeriosis. Concerning the so-called ‘YOPI’ group (young, old, pregnant and immuno-compromised), human listeriosis is often severe as mortality rates may approach 50% (Farber and Peterkin, 1991; Low and Donachie, 1997), and infection during pregnancy may lead to abortion or still-birth (Rocourt, 1994). Although L. monocytogenes is known to cause illness in those who may be predisposed to other illnesses, some authors (Farber and Peterkin, 1991; Aureli et al., 2000) have recently indicated that otherwise healthy individuals may also acquire listeriosis. Characteristically, contamination with Listeria is localized almost exclusively on the cheese rind (Farber and Peterkin, 1991). Since the rind of such cheeses is usually considered edible, the accidental presence of L. monocytogenes on surface-ripened cheeses can pose a potential health risk for certain consumers. As a consequence of some outbreaks of listeriosis, in which cheese was the major vehicle, several investigations into the occurrence of L. monocytogenes in different surface-ripened cheeses have been conducted (Terplan et al., 1986; Beckers et al., 1987; Breer and

Schopfer, 1988; Pini and Gilbert, 1988; Weber et al., 1988; Eppert et al., 1995; Loncarevic et al., 1995, 1998; Rudolf and Scherer, 2000, 2001). While Listeria was the emerging food-borne pathogen of the 1980s, it has faded somewhat from public attention. Recent systematic investigations on the hygienic status of red-smear cheese are rarely available. It is therefore unknown whether the lack of outbreaks in recent years caused by red-smear cheese is due to an improved hygienic status of these cheeses. Investigations of Terplan et al. (1986), Comi et al. (1990), and Rudolf and Scherer (2001) have shown that L. monocytogenes was isolated from none of the 99 Italian hard cheeses (0%), 2 of the 48 (4.2%) German hard cheeses and 2 of the 45 (4.4%) other European hard cheeses. These results generally agree with those from other surveys in which L. monocytogenes was found more frequently in high-moisture than in lowmoisture cheese (Ryser, 1999). As a consequence of recontamination, L. monocytogenes and other species of Listeria may still appear frequently in red-smear cheese, even when pasteurized milk has been used for cheesemaking. Beckers et al. (1987) reported that 9 of the 14 mould-ripened cheeses made from raw milk, but none of the 36 soft cheeses made from pasteurized milk, were positive for L. monocytogenes. Eight years later, Eppert et al. (1995) and Loncarevic et al. (1995) also found a higher incidence of L. monocytogenes in soft and semi-soft cheese made from raw milk (33 and 42%, respectively) than in cheese made from pasteurized milk (9 and 2%, respectively). In contrast, Breer and Schopfer (1988) found that the incidence of L. monocytogenes in cheeses made from raw or pasteurized milk was similar (13.9 and 12.2%, respectively). However, more recently Rudolf and Scherer (2001) reported that more pasteurized milk cheeses (8%) were positive for L. monocytogenes than those made from raw milk (4.8%). Surface counts of Listeria seem to increase throughout cold storage because, generally, higher numbers were found close to the end of the shelf-life of the cheese (Rudolf and Scherer, 2001). Contamination with and multiplication of organisms may also occur in the retail chain and in the domestic refrigerator (Greenwood et al., 1991). Therefore, the consumer can be exposed to a potentially large number of pathogens even if the initial level of contamination is low. The ability of L. monocytogenes to grow at low temperatures, the length of time needed for maturation of ripened cheeses and the prolonged or unspecified shelf-life assigned to many products make the complete exclusion of L. monocytogenes of great importance.

Bacterial Surface-ripened Cheeses 213

The incidence of L. monocytogenes in soft and semisoft cheese in different investigations ranges from 1.1 to 22%, whereas other species of Listeria have been found in 0.5–24% of the cheese samples examined. The surveys published throughout the last 14 years (Table 6) involved quite different sample sizes and detection methods, as well as different cheese types. Therefore, a direct comparison of the incidence of L. monocytogenes in these studies is not possible. However, there is no indication that the incidence of L. monocytogenes in European red-smear cheeses has decreased sufficiently during this time period and, therefore, it still presents a considerable public health risk. Other species besides L. monocytogenes, predominantly L. innocua, are frequently found on cheese, sometimes in combination with the pathogen. Obviously, similar occurrences in the environment and the identical conditions of growth of both species may therefore be considered as an argument for considering L. innocua as a marker organism for contaminated dairy plants (Hahn and Hammer, 1990). In any case, the presence of non-pathogenic Listeria in a product indicates an unsatisfactory process, at risk of contamination with L. monocytogenes. It is therefore recommended that food samples in general should be tested for the occurrence of the genus Listeria. Staphylococcus aureus

Many of the food poisoning outbreaks of bacterial origin and of known aetiology are caused by the ingestion of foods containing Staph. aureus enterotoxins. Rawmilk cheeses have been repeatedly involved in staphylococcal outbreaks (Table 7; De Buyser et al.,

1985) and enterotoxin A (SEA) is commonly implicated (De Buyser et al., 1996). The incidence and growth of Staph. aureus and, in some cases, the production of enterotoxins during manufacture and ripening of red-smear cheeses, have been studied by several researchers (Gomez-Lucia et al., 1992; Otero et al., 1993; Bachmann and Spahr, 1995; De Luca et al., 1997). In Italy, De Luca et al. (1997) found 16.3% of all cheese samples made from pasteurized milk to be positive for S. aureus, with cell numbers ranging from 10 to 315 000 cfu/g. E. coli O157:H7

Since its identification as a human pathogen, E. coli O157:H7 has become a major concern for the food and dairy industries because of its ability to cause severe illness. Many outbreaks of E. coli O157:H7 have been linked with the consumption of contaminated meat and other foodstuffs, such as water, lettuce, alfalfa sprouts and apple juice (Buchanan and Doyle, 1997). The consumption of unpasteurized milk and dairy products manufactured from raw milk has also been associated with the transmission of E. coli O157:H7. In 1999, over 11% of the total number of reported cases of infection caused by E.coli O157:H7 in England and Wales were due to dairy products (CDSC, 2000). So far, little information is available about the incidence and behaviour of E. coli O157:H7 in surfaceripened cheeses (Table 8). Manufacturing cheese on a laboratory scale from milk inoculated with E. coli O157:H7, Maher et al. (2001) demonstrated that the manufacturing procedure promoted substantial

Table 6 Incidence of Listeria spp. in red-smear cheeses from different countries since 1986 Origin of cheese

Type of cheese

No. of samples

L. monocytogenes (%)

Other Listeria spp.(%)

Authors/Year

D, other countries F A, DK, FIN, F, D, GR, NL, I, N, P, S, CH n.d. F F, UK, I, CY, D, DK, RL D, other countries I Other countries F, D A, DK, UK, F, D, GR, I, NL, N, RO, E, S A, DK, F, D, I, CH

Soft, semi-soft, hard Soft

420 69

3.3 14.5

3.3 n.d.

Terplan et al. (1986) Beckers et al. (1987)

Soft and semi-soft Red-smear cheese Soft and semi-soft Soft Soft, semi-soft, hard Soft Soft Soft

187 343 619 222 509 121 90 91

1.1 9.6 2.1 10.4 5.7 1.6 11.0 22.0

0.5 9.6 1.5 8.6 6.1 1.6 n.d. 24.2

Farber et al. (1987) Breer and Schopfer (1988) Gledel (1988) Pini and Gilbert (1988) Weber et al. (1988) Massa et al. (1990) Rørvik and Yndestad (1991) Eppert et al. (1995)

Soft and semi-soft Soft, semi-soft, hard

333 374

6.0 6.4

n.d. 11.8

Loncarevic et al. (1995) Rudolf and Scherer (2001)

A, Austria; CH, Switzerland; CY, Cyprus; D, Germany; DK, Denmark; E, Spain; F, France; FIN, Finland; GR, Greece; I, Italy; N, Norway; NL, The Netherlands; P, Portugal; RL, Lebanon; RO, Romania; S, Sweden; UK, England.

214 Bacterial Surface-ripened Cheeses

Table 7 Examples of S. aureus outbreaks in cheese in different countries Country

Year

No. of cases

Food implicated

Type of milk

Reference

Canada USA France England Scotland England Brazil

1980 1981 1983 1983 1984 1988 1994

62 16 20 2 27 155 7

Cheese curd Cheese Farm ewe cheese Cheese Ewe cheese Stilton cheese Cheese

Unspecified Pasteurized Raw Pasteurized Raw Unpasteurized Unspecified

Todd et al. (1981) Altekruse et al. (1998) De Buyser et al. (1985) Barrett (1986) Bone et al. (1989) Maguire et al. (1991) Pereira et al. (1996)

growth of E. coli O157:H7 to levels that permitted survival during ripening and extended storage. The authors concluded that the presence of low numbers of E.coli O157:H7 in milk, destined for manufacture of raw-milk cheese, could constitute a threat to the consumer.

of bacteriocin-producing cultures as biocontrol microorganisms, as starter cultures for fermented foods, as bacteriocin-containing microbial fermentates or as partially purified bacteriocins added directly to foods (Muriana, 1996). Bacteriocins of lactic acid bacteria

Bacteriocins

The most likely biological way to control pathogens in cheese is probably with bacteriocins. These are peptides, generally of low molecular mass, which are produced by many bacteria and inhibit the growth of other, generally closely related, species. They vary in their spectrum of activity, mode of action, molecular weight, genetic origin and biochemical properties. Klaenhammer (1993) defined four distinct classes of bacteriocins: class I, antibiotics; class II, small (10 kDa), relatively heat-stable, non-lanthionine-containing membrane-active peptides, subdivided into Listeria-active peptides with the N-terminal consensus sequence, -Tyr-Gly-Asn-GlyVal-Xaa-Cys- (class IIa), poration complexes requiring two different peptides for activity (class IIb) and thiolactivated peptides requiring reduced cysteine residues for activity (class IIc); class III, large (30 kDa), heatlabile proteins and class IV, complex bacteriocins that contain essential lipid or carbohydrate moieties in addition to protein. Bacteriocin-producing strains can be used in numerous applications as protective cultures, including the use

A large number of bacteriocins produced by LAB have been characterized in recent years, most of them class II bacteriocins. While most bacteriocin producers synthesize only one bacteriocin, several LAB produce multiple bacteriocins (Quadri et al., 1994; Bhugaloo-Vial et al., 1996; Casaus et al., 1997). The classic example of a commercially successful naturally produced inhibitory agent is nisin. Known since the work of Rogers (1928), it is produced by some strains of Lc. lactis and has been structurally characterized as a lanthionine-containing peptide by Gross and Morell (1971); nisin and nisinproducing strains have had a long history of application in food preservation, especially of dairy products (Molitor and Sahl, 1991). Lactic acid bacteria have been examined extensively for bacteriocin production because of their widespread use as food starter cultures (Nettles and Barefoot, 1993). Bacteriocins produced by LAB, however, are active mainly against Gram-positive bacteria and, therefore, many exploratory applications have been directed towards Listeria monocytogenes, which is likely to appear in red-smear cheese varieties.

Table 8 Pathogenic E. coli outbreaks in cheese in different countries Country

Year

No. of cases

Food implicated

USA

1983

170

Denmark

1983

The Netherlands

1983

69

Sweden

1983

66

Scotland

1994

22

Brie and Camembert cheeses Brie from the same plant as for USA, 1983 Brie from the same plant as for USA, 1983 Brie from the same plant as for USA, 1983 Farm cheese

Type of milk

Reference

Pasteurized

MacDonald et al. (1985)

Pasteurized

Nooitgetagt and Hartog (1988)

Pasteurized

Nooitgetagt and Hartog (1988)

Pasteurized Raw

Nooitgetagt and Hartog (1988) Rampling (1996); Ammon (1997)

Bacterial Surface-ripened Cheeses 215

Sulzer and Busse (1991) examined 14 bacteriocinogenic strains of Enterococcus faecalis, Lb. paracasei and Lc. lactis isolated from cheese or raw milk for their ability to control L. monocytogenes during the manufacture of Camembert cheese. Complete inhibition occurred when the inhibitory strain was used as a starter culture and there was a low level of contamination with Listeria sp. during the early stage of ripening. Very little inhibition occurred if the inhibitory strain was added together with the starter culture. Of 1962 bacterial isolates from a bacterial surfaceripened soft cheese (Münster) screened for activity against L. monocytogenes, six produced anti-listerial compounds other than organic acids (Ennahar et al., 1996). The strain which displayed the strongest antilisterial effect was Lb. plantarum WHE 92, which produced pediocin AcH, which is normally produced by Pediococcus acidilactici (Henderson et al., 1992). The activity spectrum included all ten Listeria strains tested, Enterococcus, Micrococcus, Staphylococcus and Bacillus spp. Gram-negative bacteria tested were not affected. The anti-listerial effect of the strain was investigated in Münster cheese (Ennahar et al., 1998). A cell suspension of Lb. plantarum WHE 92, sprayed on the cheese surface at the beginning of the ripening period, prevented the growth of L. monocytogenes. Moreover, the addition of Lb. plantarum WHE 92 did not adversely affect the ripening process. The anti-listerial potential of Lb. plantarum WHE 92, which is commercially available as ALC 01 (anti-listerial culture, Danisco, Germany), was investigated in more detail by Loessner et al. (2003). Growth of L. monocytogenes WSLC 1364, which was isolated from a cheeseborne outbreak of listeriosis, on red-smear cheese was examined in the presence and the absence of the pediocin AcH-producing Lb. plantarum ALC01. An initial level of 102 cfu of L. monocytogenes/ml of brine was nearly completely inhibited (Fig. 2A), while a pediocin-resistant mutant of L. monocytogenes grew to high cell numbers on the cheese surface (Fig. 2B). The inhibition was due to the production of pediocin AcH by Lb. plantarum during growth before it was added to the brine solution, rather than bacteriocin production in situ on the cheese. Pediocin resistance developed in vitro at different frequencies in all L. monocytogenes strains investigated, and a resistant mutant remained stable and multiplied easily in smear cheese over a 4-month production period in the absence of selection pressure. Therefore, it was concluded that the addition of a pediocin AcH-producing Lb. plantarum culture is a potent measure for combating Listeria in a contaminated production line but, due to easy development of resistance, its use should be restricted to emergency situations.

Bacteriocins of coryneform bacteria

Smear cheeses are characterized by the development of a relatively high pH (6.5) at the surface during ripening. This, together with a relatively high ripening temperature, allows the growth of salt-tolerant pathogenic micro-organisms on the surface of the cheese, particularly L. monocytogenes. The undefined microflora from the surface of ripe cheeses which are used for the ripening of commercial red-smear cheeses were shown to have a strong impact on the growth of Listeria spp. (Eppert et al., 1997). Therefore, several workers (HugMichel et al., 1989; Valdés-Stauber et al., 1991; Ryser et al., 1994; Martin et al., 1995; Carnio et al., 1999, 2001; Motta and Brandelli, 2002) have evaluated smear bacteria for their ability to prevent the growth of Listeria and other pathogens. Hug-Michel et al. (1989) tested 80 strains of microorganisms which had been isolated from the rind of Vacherin Mont d‘Or cheese for antagonistic activity against Listeria; 9 of the 80 strains (identified as Arthrobacter protophormiae, A. uratoxydans or Serratia marcescens) showed an inhibitory effect against all serotypes of Listeria monocytogenes tested; the identity of the inhibitor was not identified. Valdés-Stauber et al. (1991) showed that small numbers of B. linens (16 strains), A. nicotianae (4 strains) and A. nucleogenes (3 strains) inhibited 26–87% of the 91 strains of Listeria tested; B. linens M18 was particularly effective. The strain produces a substance (named linocin M18) that inhibits the growth of Listeria spp. and several coryneforms and other Gram-positive bacteria (Valdés-Stauber and Scherer, 1994). The structural gene for linocin M18 has been detected in several strains of B. linens, five Arthrobacter spp. and four Corynebacterium spp. (Valdés-Stauber and Scherer, 1996), suggesting that the ability to produce this bacteriocin is widely distributed in coryneform bacteria. Ryser et al. (1994) found that less than 0.1% of 125 000 isolates from 105 French smear cheeses showed visible zones of inhibition against L. monocytogenes. Isolates possessed anti-listerial activity against various strains of Enterococcus faecalis, Staph. xylosus, Staph. warneri and coryneform bacteria; one strain, B. linens OC2, which was isolated from Comté cheese, was particularly effective. These data suggest that the number of smear isolates which can inhibit Listeria is low. In crude extracts of both organisms, the inhibitory properties were associated with molecules of high molecular mass but the purified bacteriocins, linenscin OC2 and linocin M18, have molecular masses of ⬃1.2 and 31 kDa, respectively, and had broad spectra of activity inhibiting Staph. aureus, several Listeria, Bacillus, Arthrobacter, Brevibacterium and Corynebacterium spp. and LAB. Neither bacteriocin showed activity against

216 Bacterial Surface-ripened Cheeses

1.0E + 06 A 1.0E + 05

cfu/cm2

1.0E + 04 1.0E + 03 1.0E + 02 L. plantarum ALC 01 1.0E + 01 L. plantarum ATCC 14917 1.0E + 00 1

3

5

7

9

11

13

15 17 19 21 23 Days of ripening

25

27

29

31

33

35

37

1.0E + 08 B 1.0E + 07 1.0E + 06

cfu/cm2

1.0E + 05 1.0E + 04 WSLC 1364 WSLC 1364R WSLC 1364 WSLC 1364R

1.0E + 03 1.0E + 02 1.0E + 01 1.0E + 00 1

3

5

7

9

11

13

15 17 19 21 Days of ripening

23

25

27

29

31

33

35

Figure 2 (A) Inhibition of growth of L. monocytogenes WSLC 1364 by Lb. plantarum ALC 01. Control cheeses were ripened with the non-pediocin AcH-producing type strain Lb. plantarum ATCC 14917. The cheeses were contaminated with 2  102 cfu/ml Listeria on day 1. (B) Inhibition of growth of L. monocytogenes WSLC 1364 by Lb. plantarum ALC 01 (open symbols). The contamination levels were 102 cfu/ml (squares) or 103 cfu/ml brine solution (circles). Control cheeses were contaminated with a bacteriocinresistant mutant of L. monocytogenes WSLC 1364R (closed symbols). Ripening experiments were performed on soft cheese using a commercial, undefined multi-species microbial consortium (From Loessner et al., 2003).

Gram-negative bacteria. Linenscin OC2 lost no activity on heating at 100 °C for 10 min, while linocin M18 was inactivated on heating to 80 °C for 5 min but withstood heating to 50 °C for 30 min (Valdés-Stauber and Scherer, 1994; Maisnier-Patin and Richard, 1995). The ability of linocin M18-producing strains of B. linens and undefined microbial consortia from the surface of smear cheeses to inhibit L. monocytogenes on deliberately inoculated cheese, was compared by Eppert et al. (1997). The results showed that the linocin-producing strains reduced the number of L. monocytogenes by only 1–2 log

cycles, while some of the microbial consortia caused complete inhibition of the Listeria. This finding implies that linocin M18 only partly inhibits the development of L. monocytogenes in smear cheeses and that other factors are also important. Martin et al. (1995) isolated three strains of B. linens from the brine used for a red-smear cheese that produced anti-listerial activity. The anti-listerial compound was stable between pH 4 and 9 and remained active after heating (80 °C, 30 min) at acid pH. No data of its use in cheese ripening were given.

Bacterial Surface-ripened Cheeses 217

The anti-listerial potential of 19 consortia of bacteria isolated from different French smear-cheese was analysed by Carnio et al. (1999). Forty-eight of 2613 isolates caused clear inhibition of one or more strains of L. monocytogenes. In a study of 299 strains isolated from German dairy products, 30 strains inhibited at least one strain of L. monocytogenes. Bacteria with antilisterial potential were members of the genera Arthrobacter, Brevibacterium, Corynebacterium, Enterococcus, Micrococcus, Microbacterium and Staphylococcus. The highly inhibitory Staph. equorum WS 2733 was isolated from the surface of a French Raclette cheese (Carnio et al., 2000). This strain was shown to produce the antibiotic, micrococcin P1, which exhibited a bacteriostatic effect on a variety of Gram-positive bacteria. The anti-listerial potential of this strain as a protective starter culture was evaluated in in situ application in cheese-ripening experiments. A remarkable growth reduction of L. monocytogenes was achieved compared to control cheese ripened with a non-bacteriocin producing strain (Fig. 3). Carminati et al. (1999) screened surface-smear organisms isolated from Taleggio cheese for their ability to inhibit L. monocytogenes, Staph. aureus, E. coli and Hafnia alvei. Most of the isolates which showed anti-listerial activity (19% of the total) were coryneform bacteria, mainly Mb. lacticum. Two bacterial mixtures (containing orange-pigmented strains and strains inhibiting Listeria) were examined as surface-smear starter on commercial Taleggio cheese production; they partially but not com-

pletely inhibited Listeria monocytogenes Ohio. However, the cheese did not ripen normally, due to a delay in growth of the surface-smear bacteria. Recently, Motta and Brandelli (2002) identified a bacteriocin produced by the red-smear cheese bacterium, B. linens ATCC 9175. A crude bacteriocin obtained from the culture supernatant fluid was inhibitory to some indicator strains, including L. monocytogenes, but was inactive against the Gram-negative bacteria and yeasts tested. Enterococcal bacteriocins

Numerous strains of enterococci associated with food systems are capable of producing a variety of antibacterial proteins, called enterococcins, with activity against food-borne pathogens, such as L. monocytogenes, pathogenic clostridia or Staph. aureus (see Giraffa, 1995, for a review). In addition, several enterococcins have been assessed for their ability to inhibit Listeria spp. in dairy systems, where enterococci are often isolated as desirable microflora. Among nonlactic species, bacteriocins from Enterococcus spp., especially Ec. faecium and Ec. faecalis, are the most widely produced and characterized. Enterococcins share a number of common characteristics: (i) generally high heat stability; (ii) stability over a wide range of pH, but most often under acidic conditions; and (iii) broad spectra of activity on Gram-positive pathogenic bacteria, including L. monocytogenes. Although enterococci are generally considered to be harmless,

1.0E + 08 1.0E + 07 1.0E + 06

cfu/cm 2

1.0E + 05 1.0E + 04 1.0E + 03 DSMZ 20674 (bac–) WS 2733 (bac+)

1.0E + 02

DSMZ 20674 (bac–) 1.0E + 01

WS 2733 (bac+)

1.0E + 00 1

3

5

7

9

11 13 15 17 19 21 23 25 27 29 31 33 35 Days of ripening

Figure 3 Inhibition of growth of Listeria monocytogenes WSLC 1364 during ripening of cheese smeared with a bacteriocin-producing strain of S. equorum WS 2733 (open symbols) and a non-bacteriocin producing strain, DSMZ 20674 (closed symbols) after contamination at day 1 with 102 (circles) and 104 (squares) Listeria monocytogenes/ml brine (from Carnio et al., 2000).

218 Bacterial Surface-ripened Cheeses

the absence of haemolytic activity by enterococcinproducing strains should always be demonstrated. Only a few enterococcins have been assessed for their ability to prevent the growth of pathogens in model cheese systems. In preliminary experiments on Taleggio cheese, it was shown that Ec. faecium 7C5 bacteriocin was produced regularly in cheese during whey drainage and its activity was maintained at detectable levels until the end of ripening (Giraffa et al., 1995); an anti-listerial effect could also be observed on the surface of cheeses contaminated with 102 cfu/cm2 L. monocyto~ et al. genes Ohio (Giraffa and Carminati, 1997). Nu nez (1997) investigated the inhibitory effect of enterocin 4, a bacteriocin produced by Ec. faecalis INIA 4, on L. monocytogenes Ohio and Scott A during the manufacture and ripening of Manchego cheese. Counts of L. monocytogenes Ohio decreased significantly (3–6 log units during the first 7 days of ripening) in cheese made from milk inoculated with Ec. faecalis INIA 4, whereas L. monocytogenes Scott A was inhibited only slightly, when Ec. faecalis INIA 4 was used in combination with a commercial starter. In two different studies (Ennahar et al., 1998; Ennahar and Deschamps, 2000), the isolation of two bacteriocinproducing Ec. faecium strains from the cheese surface was reported. Both enterocin 81, produced by Ec. faecium WHE 81, and enterocin A, produced by Ec. faecium EFM01, displayed a narrow spectrum of activity, which is directed mainly against Listeria sp. The activity of both bacteriocins was shown to be equally active at pH values ranging from 4.0 to 8.0, which is of considerable interest with regard to possible use in fermented foods. Some preliminary experiments on soft cheese have shown that enterocin 81 offers good protection against L. monocytogenes during cheese ripening (Ennahar et al., 1998).

Limitations of application of bacteriocins

The potential of bacteriocins as food preservatives is well-demonstrated. They are not only effective, but are also safe for use in food (Cleveland et al., 2001). Application studies have, however, shown that there are limitations to the usefulness of bacteriocins as antimicrobial agents. For example, nisin is unstable at pH  5, and although bacteriocins of the pediocin family are able to extend the shelf-life of food products, full suppression of the spoilage microflora is rarely achieved. Moreover, one of the major concerns regarding the use of bacteriocins is the development of highly tolerant and/or resistant strains (Rasch and Knochel, 1998). It has been observed that Listeria develop tolerance to nisin and pediocin-like bacteriocins at a relatively high frequency in both the laboratory media and the model food systems (Ming and Daeschel, 1993; Rekhif et al., 1994; Gravesen et al., 2002; Loessner et al., 2003). Carnio et al. (1999) observed that the sensitivity of L. monocytogenes strains to the inhibitory activity of coryneform bacteria was quite different. On average, isolates from red-smear cheese were less sensitive to the inhibitory effects of red-smear bacteria than to animal or other food isolates. This observation indicates that selective pressure on the cheese surface might have resulted in the development of resistance mechanisms by micro-evolutionary adaption. From an applied point of view, the combined use of different bacteriocins is likely to be better than using one bacteriocin alone to prevent the growth of pathogenic bacteria. However, although bacteriocins in foods generally exhibit moderate anti-microbial activity, they are not suitable for use as a primary means of food preservation. However, they can be integrated into appropriate multi-hurdle preservation systems.

Anti-Listeria compounds of Geotrichum and Penicillium

Effects of complex smear cheese ripening consortia

As already outlined, yeasts contribute to the proper development and ripening of surface-ripened cheeses. It would be desirable if these organisms would also contribute to the inhibition of pathogenic micro-organisms during ripening. Only a few studies have discussed the possibility of using Penicillium spp. to inhibit the growth of undesirable micro-organisms in cheese (Larsen and Knøchel, 1997). In that study, ten foodrelated strains of P. camemberti inhibited pathogens like L. monocytogenes. The inhibition was due to the production of acetaldehyde, benzaldehyde, 3-methylbutanal and 1-octen-3-ol. Dieuleveux and Gueguen (1998) showed that G. candidum produced compounds that inhibited L. monocytogenes, which were identified as D-3-phenyllactic acid and D-3-indollactic acid.

The undefined microbial flora derived from the surface of ripe red-smear cheeses show a strong impact on the growth of Listeria spp. This antagonistic behaviour is a stable feature of these microbial consortia, since the inhibitory effects could be reproduced with the smear of cheese produced over a period of several months to 1 year (Eppert et al., 1997). Ripening of cheeses with different undefined starters led to similar developments of pH and cell counts of yeast and bacteria, whereas development of deliberately inoculated Listeria on the cheese surface was dependent on the culture used for ripening (Eppert et al., 1997; Rudolf, 2001). In some cases, the microbial consortia inhibit Listeria almost completely (Fig. 4; Eppert et al., 1997; Maoz et al., 2002).

Bacterial Surface-ripened Cheeses 219

1.0E + 08 1.0E + 07 1.0E + 06 cfu/cm2

1.0E + 05 1.0E + 04 1.0E + 03 MB KS WE Raclette

1.0E + 02 1.0E + 01 1.0E + 00 1

4

7

10

13

16

19 22 25 28 Days of ripening

31

34

37

40

43

Figure 4 Growth inhibition of Listeria monocytogenes by a complex consortium of soft red-smear micro-organisms from cheeses. Cheeses were artificially contaminated with 2–3  102 cfu Listeria monocytogenes WSLC 1364/ml brine solution at day 1 of ripening. Cheeses were obtained from German soft (MB, KS, WE) and semi-soft French (Raclette) cheese. The graph shows Listeria cells per cm2 of cheese surface. Ripening experiments were repeated twice over a period of several months. Note that different cheese microbial consortia display very different inhibitory activity against Listeria monocytogenes (from Loessner et al., 2003).

Nevertheless, the molecular basis of these effects is unknown. Bacteriocin production contributes to the inhibition of Listeria during the ripening of red-smear cheese, but the striking inhibitory effects observed with the industrial wash-off flora are not explained completely by bacteriocin production. Additional factors must be responsible for the inhibition of Listeria, e.g., the production of other inhibitory substances (Walstead et al., 1974) or unknown ecological interactions within the complex smear flora, such as competition and symbiotic relationships. A project partly funded by the EU is currently underway with two major objectives, viz., to obtain a clearer understanding of the surface microflora of five different cheeses, Limburger, Reblochon, Livarot, Tilsit and Gubbeeen, and to identify strains of yeast which have anti-listerial activity. Preliminary results show that such yeasts exist (http://www.teagasc.ie/ research/dprc/smearcheese.htm).

Conclusions The surface of a smear-ripened cheese is a very complex microbial ecosystem and this review has discussed the ripening of such cheeses in terms of the microflora, paying particular attention to Gram-positive, catalase-positive, salt-tolerant bacteria and the problems associated with the development of pathogenic bacteria, particularly L. monocytogenes. It is likely that on all bacterial smear-ripened cheese, yeasts dominate during the early stages of

ripening, where they metabolize the lactic acid produced by the starter bacteria and produce an increase in the pH of the cheese surface. The most common yeast is D. hansenii, followed by Kluyveromyces lactis, Geotrichum candidum and Yarrowia lipolytica. The bacteria on the surface of smear-ripened cheeses are Gram-positive, catalase-positive, salt-tolerant bacteria, which can be divided into two categories, coryneforms and staphylococci. The major difficulty in identifying these micro-organisms is that coryneform bacteria, as a whole, are not well defined taxonomically. Only after resolving their taxonomy can in-depth studies on the bacteria present in the smear and how these organisms interact with each other and their contribution to ripening be undertaken.

Acknowledgement Financial support from the EU under contract QLK1CT-2001-02228 is gratefully acknowledged.

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Cheese Varieties Ripened in Brine M.H. Abd El-Salam, Dairy Department, National Research Centre, Dokki, Cairo, Egypt E. Alichanidis, Laboratory of Dairy Technology, School of Agriculture, Aristotle University of Thessaloniki, Greece

Introduction Cheeses ripened in brine are the oldest known group of cheeses (Scott, 1986). Traditionally, the manufacture of these cheeses was limited to the Mediterranean basin and the Balkans. However, their production has been extended to several parts of the world as a result of their popularity and increased demand in the international market (Mann, 1999). The manufacture of cheeses ripened in brine was carried out for centuries on a small scale, which is difficult to standardize. However, the last decades of the twentieth century have witnessed the development of large-scale, mechanized and standardized production of cheeses ripened in brine. Advanced technologies have been adopted for their production, including ultrafiltration (UF) techniques. It has been estimated that UF-Feta cheese represented ⬇56% of the total UF cheeses produced throughout the world ( Jensen et al., 1988). These developments improved and better defined the characteristics of cheeses ripened in brine. Cheeses ripened in brine can be defined as those preserved in brine (pickle) from manufacture until they reach the consumer. They are characterized by the following: • Rindless, manufactured in various shapes and sizes but usually in pieces of less than 1 kg. • Clean, acid and salty taste when fresh; the ripened cheese has a sharp piquant flavour. • The cheese and brine have a high salt content which bestows good keeping quality in hot climates. • White colour arising from the use of sheep, goat or buffalo milk in their manufacture. When cows’ milk is used to make cheese ripened in brine, methods are used to decolourize cows’ milk fat in order to obtain the desired white colour. • Changes in the composition and properties of cheese during ripening and those of the brine used are interrelated. • Most varieties in this group are stored in sealed containers but some are stored in gas-permeable containers, which affect the biochemical changes which occur during ripening.

In this chapter, we will describe different cheeses ripened in brine, but special emphasis will be placed on Domiati and Feta cheeses, which are the most important members of this group and are recognized in international markets. Besides, more information about Domiati and Feta cheeses is available in the literature than other varieties ripened in brine.

Classification and Nomenclature Most cheeses ripened in brine are not well defined, which usually create problems in their classification. In many cases, cheese is described as ‘white pickled cheese’, a generic name that can apply to all cheeses ripened in brine. In addition, wide variations are found in the composition and texture of cheeses ripened in brine. Therefore, classification of these cheeses is necessary. The general systems used to classify cheese can be adapted for the classification of cheeses ripened in brine, as follows (country of origin is given in parentheses): 1. Soft cheeses (moisture content, 55–65%) 1.1. Acid-coagulated: Mish (Egypt). 1.2. Rennet-coagulated: 1.2.1. Salting of cheese curd (Feta type): Feta (Greece), Teleme (Romania), Brinza (Russia, Israel), Bli-sir-U-kriskama (Serbia), Bjalo or Belo Samureno sirene (Bulgaria), Chanakh (Russia), Beyaz peynir (Turkey), Akawi (Syria), Baida (Lebanon), Iranian white cheese (Iran). 1.2.2. Salting of cheese milk (Domiati type): Domiati (Egypt), Dani (Egypt; a variant of Domiati cheese made from sheep’s milk), Gibna bayda (Sudan). 2. Semi-hard cheeses (moisture content, 45–55%): Halloumi (Cyprus), Braided Meddafara and Magdula (Syria, Sudan), Nabulsi (Jordan).

Domiati Cheese Domiati cheese can be considered as the most important cheese ripened in brine in the Middle East in terms of the quantity of cheese produced or available

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228 Cheese Varieties Ripened in Brine

information. It is unique among cheeses ripened in brine in the addition of a large quantity of NaCl (8–15%) to the milk before renneting. This results in: • Partial solubilization of the colloidal calcium phosphate (Puri and Parkash, 1965). Addition of NaCl up to 6% to cows’ milk causes a significant increase in the level of soluble calcium (P  0.001) (Elzeny, 1991). • An increase in the acidity of milk (Abd El-Hamid et al., 1981) and a decrease in its pH (Elzeny, 1991). This has been attributed to a cation exchange reaction of Na for H. • The rennet coagulation time of cows’, buffaloes’, goats’ and sheep’s milk increases with the amount of NaCl added up to 7.5–10% but decreases slightly at a higher level of added salt (Abd El-Hamid et al., 1981). Aggregation of casein micelles is enhanced at a high NaCl concentration, being dependent on the size of the micelles and the time of exposure to salt (Elzeny, 1991). • Addition of NaCl (up to 1 M) to milk or casein micelles of variable sizes dispersed in simulated milk ultrafiltrate (SMUF) (Saito and Hirose, 1972; Abd El-Salam et al., 1978) reduces the turbidity of the system due to a decrease in the average micellar size and increased non-preferential solubilization of casein fractions. • Casein micelles in salted milk have an irregular shape rather than the spherical shape characteristic of normal micelles (Elzeny, 1991). • The extent of hydrolysis of -casein by rennet decreases by 43 and 61% on addition of 5 and 10% NaCl to cows’ milk, respectively (Elzeny, 1991). • Addition of NaCl reduces the action of the coagulant; chymosin is affected less than Rhizomucor meihei protease (Ibrahim et al., 1973). The overall effect of adding NaCl to milk before renneting is the need for more rennet and a longer coagulation time for Domiati cheese compared to other cheeses. In addition, the coagulum formed is usually weak and therefore is ladled into moulds without cutting, and whey is permitted to drain for a long time (24 h). The cheese is consumed either fresh or after ripening in sealed tins under salted whey from the same cheese. Ripening usually occurs at room temperature (20  5 °C). Details of the manufacture of Domiati cheese have been described (Fahmi and Sharara, 1950; Abd El-Salam et al., 1976; Abou Donia, 1991).

acterized by a relatively high pH (6.0–6.5), and high levels of moisture (60–65%) and NaCl (5–8%). Changes in the gross composition of Domiati during storage are summarized in Table 1. The developed acidity strongly determines the changes in the gross composition of Domiati during storage. Acidification brings the pH of cheese close to the isoelectric point of casein and partially solubilizes colloidal calcium phosphate, which causes shrinkage of the cheese matrix and exudation of cheese serum into the brine (Hamed, 1955). The pH of ripened Domiati reaches as low as 3.3 as a result of two factors: the high lactose content of fresh cheese and the continuous supply of lactose from the salted whey used as brine for bacterial fermentation (Ahmed et al., 1972; Tawab et al., 1975). The use of salted whey diluted with aqueous NaCl solution as brine reduces acid development in Domiati (El-Abd et al., 1975). The available lactose (in cheese and brine) is more than that of which the cheese microflora can utilize, which explains the high level of residual lactose in ripened Domiati (Table 2). Lactose and galactose are found in Domiati even after 6 months of storage, but glucose is not detected (Abd El-Salam, unpublished). It seems that the pathway for lactose fermentation by starter organisms in Domiati is similar to that used by yoghurt starters (Dellaglio, 1988). Several interacting variables affect the changes in the general composition and acid development in Domiati during storage. These can be summarized as follows. Type of milk. Domiati is made mainly from buffalo’s milk or mixtures of buffalos’ and cows’ milks. However, the use of reconstituted or recombined milks, goat, sheep and even camel milk for Domiati cheese have been described (Abou Donia, 1991; Kandeel et al., 1991; Mehaia, 1993).

Table 1 Summary of changes that occur in Domiati cheese during ripening

Constituent

Changes during storage

Responsible factor(s)

Moisture

Decrease (about 2–3%) Increase (3–6%)

Changes during ripening

Fat-in-drymatter Acidity

Increase (1.0–1.5%)

General composition

pH

Decrease (2–2.5)

Lactose

Decrease (1.5–2.0%)

Exudation of cheese serum Decrease in solidsnot-fat Lactic acid fermentation Lactic acid fermentation Lactic acid fermentation

Extensive data are found in the literature concerning the moisture, fat, salt, pH and acidity of Domiati cheese during storage in brine. Fresh Domiati is char-

Cheese Varieties Ripened in Brine 229

Table 2 Changes in the carbohydrate content of Domiati cheese during ripening (%, as lactose) Storage period (days) Fresh

15

30

120

180

Reference

3.5 ND

3.40 2.09

2.85 1.84

1.65 ND

0.54

Ahmed et al. (1972) Tawab et al. (1975)

ND, not determined.

Domiati from buffalo milk (unstandardized) contains more fat in dry matter (FDM), less moisture and lower developed acidity than cheese made from cow or goat milk. The use of reconstituted or recombined milk reduces the moisture content of Domiati (El-Safty, 1969; Hagrass, 1971). However, raising the reconstitution ratio (i.e., higher total solids in cheese milk) increases the moisture content of the cheese (Moneib et al., 1981). Ripening at a low temperature reduces the rate of biochemical and microbiological changes in Domiati as apparent from the slow rate of acid development compared to cheese stored at room temperature (Abou Dawood, 1964; Teama, 1967). The moisture content of Domiati increases during early storage at a low temperature. This has been attributed to the relatively high pH of fresh cheese, which increases the hydration of the caseins at low temperature.

Ripening temperature.

There is no standard period for the ripening of Domiati cheese. However, 3–4 months storage in brine at room temperature gives a good quality product. The composition of Domiati changes continuously during storage with the highest rate during the first month, which coincides with the growth of the cheese microflora (Abou Dawood, 1964; El-Koussy, 1966; Ahmed et al., 1972; Naguib et al., 1974).

Ripening period.

Method of ripening. Storage in pouches or cans without

brine has been suggested for Domiati, during which the acidity develops faster than cheese stored in brine (Abd El-Salam et al., 1981; Al-Khamy, 1988; Gomaa, 1990).

Variable levels of NaCl are added to the milk for Domiati cheesemaking depending on season, quality of the milk and duration and temperature of ripening. The higher the percentage of salt added to the milk, the higher the moisture and the lower the extent of acid development in the cheese during ripening (Table 3).

Salt content.

Heat treatment of milk. Pasteurization of the cheese

milk has little effect on the gross composition of Domiati (Sharara, 1962; Teama, 1967; Naguib et al., 1974); a slight increase in moisture content and a slight decrease in acid development are apparent. The manufacture of UF-Domiati has been described (Abd El-Salam et al., 1981, 1982; Abd ElSalam, 1988; Gomaa, 1990; Hofi et al., 2001). The moisture content of UF-Domiati is usually higher and the fat content lower than those of traditional Domiati (Abd ElSalam et al., 1981; Al-Khamy, 1988; Gomaa, 1990) due to the poor syneresis and the high water-holding capacity of whey proteins retained in UF cheeses (Table 4).

Ultrafiltration.

Addition of lecithin to cheese milk increases the moisture content and acid development in Domiati (El-Abbassy et al., 1991). The partial replacement of NaCl by KCl has no significant effect on the composition or pH of Domiati cheese (Ramadan, 1995).

Additives.

Proteolysis

Domiati cheese undergoes continuous proteolysis during ripening in brine. Generally, the total nitrogen (TN) content of cheese decreases gradually due to the transfer of degradation products to the brine by diffusion, while the soluble nitrogen fractions of cheese increase continuously, indicating continuous proteolysis. Several factors affect proteolysis in Domiati cheese, as summarized in Table 5. The rennet contributes much to proteolysis in Domiati. This is due to the high concentration of rennet needed to coagulate salted milk (compared to most cheese varieties), the high level of rennet retained in cheese curd and to the storage in salted whey, which contains rennet. In Domiati, s1-casein is hydrolysed rapidly, while -casein is resistant to hydrolysis (Abd El-Salam and El-Shibiny, 1972; El-Shibiny and Abd

Table 3 Gross composition of Domiati cheese as affected by the level of NaCl in milk (Zaki et al., 1974) 8% NaCl

Moisture, % FDM, % Acidity, %

10% NaCl

Fresh

3 months

Fresh

58.6 34.6 0.27

51.4 49.7 2.24

59.2 35.0 0.24

FDM, fat in dry matter.

12% NaCl 3 months 52.2 48.2 2.02

15% NaCl

Fresh

3 months

Fresh

3 months

60.9 32.8 0.21

54.5 48.7 1.42

61.7 31.8 0.11

55.8 45.5 1.00

230 Cheese Varieties Ripened in Brine

Table 4 Gross composition of 30-day-old Domiati cheese made from cows’ or buffaloes’ milk by conventional or ultrafiltration techniques (Abd El-Salam et al., 1981) Conventional

Moisture, % FDM, % pH

Ultrafiltration

Cow

Buffalo

Cow

Buffalo

55.77 45.35 4.55

54.82 49.71 4.70

58.72 42.88 5.15

57.29 46.82 4.91

FDM, fat in dry matter.

El-Salam, 1974; Abd El-Salam et al., 1983; Mehanna et al., 1983). This pattern of changes arises from the action of rennet on cheese as affected by salt content (Fox and Walley, 1971). The high ionic strength and high ripening temperature seem to enhance the polymerization of -casein in Domiati via hydrophobic interactions and render it less susceptible to rennet action. The / s1-casein ratio in Domiati increases continuously during ripening (Abd El-Salam et al., 1983) and, after extended ripening, the water-insoluble proteins are mainly -caseins which explain partially the soft body and texture of ripened Domiati, as reported for Cheddar cheese by Creamer and Olson (1982). A number of degradation products with high or low electrophoretic mobility are apparent in the electrophoretic pattern of the proteins and polypeptides of Domiati, including s1-casein (f24–199) (Ramos et al., 1988) produced from s1-casein by the action of chymosin, and the -caseins produced from -casein by the action of plasmin (Eigel, 1977). The use of milk clotting enzymes other than calf rennet alters the pattern of proteolysis in Domiati (Abdou et al., 1976), but the use of different starters has only a slight effect (Abd El-Salam et al., 1983). Analysis of the soluble nitrogenous compounds by gel permeation chromatography (Abd El-Salam and El-Shibiny, 1972)

Table 5 Factors that affect proteolysis in Domiati cheese Increases proteolysis

Retards proteolysis

Cow milk  buffalo milk Homogenization Addition of denatured whey proteins, phosphate, citrate, capsicum tincture, cheese slurry Ultrafiltration

Low temperature storage Heat treatment of cheese milk H2O2-catalase treatment of milk

Direct acidification Salt-tolerant starters Storage in pouches

Use of reconstituted/recombined milk Increase in NaCl content

showed that this fraction consists mainly of low molecular weight components (amino acids and small peptides). Comparison of the free amino acid pattern in Domiati (El-Erian et al., 1974) with the amino acid profile of cow and buffalo caseins reveals a marked reduction in the concentration of glutamic acid and the formation of

-amino butyric acid through deamination reactions. Also, arginine is almost absent, having been converted to ornithine. The ripened cheese has a high concentration of ammonia, which indicates the significance of deamination reactions in Domiati and which contributes to flavour development in this type of cheese. The concentration of biogenic amines in Domiati is very low (Mehanna et al., 1989). Tyramine is the principal biogenic amine found in Domiati, together with low concentrations of histamine, tryptamine, phenylethylamine and putrescine. Proteins of Domiati cheese seem to undergo three levels of proteolysis, as illustrated in Fig 1. The key point is that the soluble products diffuse into the brine to attain equilibrium with their concentration in the cheese. Lipolysis

Data on the volatile acids in Domiati cheese have been recalculated as acetic acid (Table 6) which is the principal volatile acid in Domiati (El-Shibiny et al., 1974). Most of the changes in the volatile acids occur during the first month of ripening, which coincides with maximum bacterial growth (Naguib et al., 1974; Shehata et al., 1984). The pattern of free fatty acids in Domiati is comparable to the fatty acid profile of triglycerides in milk fat (Table 7), suggesting non-specific lipolysis. Analysis of glycerides from ripened Domiati cheese also indicates lipolysis (Precht and Abd El-Salam, 1985). However, the origin of lipases responsible for fat hydrolysis in Domiati is not clear. The contribution of free fatty acids to flavour development in Domiati has been confirmed from the analysis of cheese of different fat contents (El-Shibiny et al., 1974). Measurements of peroxide and thiobarbituric acid values indicate that fat oxidation occurs in Domiati cheese during storage (Hamed et al., 1987). Vitamin content

Almost all the vitamin A in milk is retained in Domiati and remains stable during ripening (Sabry and Guerrant, 1958). On the other hand, variable percentages of thiamine, riboflavin, niacin (Sabry and Guerrant, 1958), biotin, vitamin B12 and folic acid (Khattab and Zaki, 1986) are retained in fresh cheese. According to Sabry and Guerrant (1958), the level of biotin, vitamin B12 and folic acid remain unchanged

Cheese Varieties Ripened in Brine 231

First level

Fresh cheese proteins (αs1, αs2, β, para-κ-casein)

Salted whey (brine)

Proteinases –mainly residual rennet –milk proteinase (plasmin)

Diffusion Equilibrium

Water-soluble components (mainly peptides) Insoluble cheese proteins (αs1, αs2, β, para-κ-casein, large peptides)

Second level Bacterial enzymes –peptidases –carboxypeptidases –aminopeptidases

Diffusion Equilibrium

Small peptides

Diffusion

Amino acids

Equilibrium

Third level Bacterial enzymes –deaminases (mainly) –decarboxylases

Diffusion

Ammonia, carboxylic acids, amines, CO2

Equilibrium Figure 1 Schematic representation of proteolysis in Domiati cheese.

during storage while changes in riboflavin and niacin depend on storage conditions. Volatile flavour compounds

In addition to the volatile fatty acids, the concentrations of acidic and neutral carbonyls increase in Domiati dur-

ing storage (Magdoub et al., 1983). Several aldehydes, ketones, alcohols, esters, sulphur compounds and hydrocarbons are found in the volatiles of Domiati cheese. Forty-four of these compounds have been identified using a dynamic headspace GC–MS technique (Collin et al., 1993). Most of these volatiles are formed

232 Cheese Varieties Ripened in Brine

Table 6 Production of volatile fatty acids in Domiati cheese made from buffaloes’ milk during ripening (expressed as acetic acid, %) Ripening period (days) Coagulant

Fresh

15

30

60

90

Renneta R. pusillus proteasea Calf rennetb Bovine pepsinb R. meihei proteaseb C. parasitica proteaseb

0.073 0.073 0.034 0.044 0.052 0.029

0.150 0.121 0.089 0.092 0.084 0.078

0.158 0.142 0.107 0.097 0.105 0.107

0.173 0.184 0.109 0.107 0.108 0.118

0.179 0.219 0.111 0.109 0.111 0.120

a El-Safty and El-Shibiny, 1980. b Abdou et al., 1976.

after 2 months of maturation. Acrolein, butan-2-one, propan-1-ol, butan-2-ol, ethyl propionate, propyl acetate, ethyl butyrate, propyl propionate and propyl butyrate are found in good quality Domiati cheese. Various sulphur compounds are also found at low concentrations in good quality ripened Domiati cheese, but high concentrations of these compounds are associated with inferior quality cheese (Collin et al., 1993). Changes in the concentration of brine

The composition of salted whey used for the ripening of Domiati cheese changes continuously during maturation. This is attributed to the chemical and biochemical changes that occur in cheese and the diffusion of soluble constituents to attain equilibrium in their partition between cheese and brine. The following factors control the changes in the composition of brine: • Composition of the fresh cheese and brine. • Rate of the biochemical changes in Domiati, which are controlled by several factors. • Ratio of cheese to brine (usually 5–6:1). The volume of brine surrounding Domiati stored at room temperature increases (12.5%), and about 70% of the solids lost from the cheese during the first month of storage appear in the brine (Hamed, 1955). A further 5.9% of cheese solids are lost during the

Table 7 Average proportion* of free fatty acids in Domiati cheese (Ramos et al., 1988) C4:0

C6:0

C8:0

C10:0

3.80

8.72

5.83 3.32

Total free fatty acids (mg/kg) * % of total free fatty acids.

C12:0 C14:0 4.03

C16:0

C18:0

12.55 31.53 8.34 2308

C18:1 21.66

subsequent 2 months of ripening due to partial exudation of cheese serum into brine as a result of acid production and shrinkage of the cheese matrix. On the other hand, the volume of brine decreases during early ripening of Domiati cheese at a low temperature (Teama, 1967) through the hydration of the caseins. However, further ripening at low temperature is accompanied by an increase in the volume of brine and changes in its composition. The TN content of brine increases continuously during storage. However, the rate and extent of this increase is affected by the salt content and storage temperature (Teama, 1967) and homogenization (Ahmed et al., 1972). The concentrations of Ca and PO4 in the brine also increase initially but remain almost constant after 2 months of storage coinciding with changes in pH and acidity. About 25–30% of the Ca and PO4 in cheese are released into the brine (Ahmed et al., 1972). The salted whey used as a brine for Domiati cheese contains a small amount of fat, the level of which increases slightly with increasing salt content and heat treatment of the milk used for cheesemaking. Both factors weaken the cheese matrix and increase the loss of fat in the whey (Teama, 1967). However, the fat content of the brine changes very little during storage. The NaCl content of the brine changes during early storage to attain an equilibrium between brine and cheese. Equilibration also occurs in the distribution of lactose and lactate. The brine used for Domiati usually contains a significant amount of lactose even after 4 months of storage (Ahmed et al., 1972). Texture and microstructure

The combined effect of decreasing pH and adding NaCl to milk reduces significantly all textural parameters (elasticity, hardness, brittleness, adhesiveness, chewiness and gumminess) of Domiati cheese and increases its water retention (Elzeny, 1991). However, the textural parameters increase significantly with increasing rennet concentration, renneting temperature and addition of CaCl2 to the milk (Elzeny, 1991). Maximum textural parameters (elasticity modulus, 1.1  10 N/m2; hardness, 0.9 kg; brittleness, 0.6 kg; elasticity, 30.9%; cohesiveness, 59.5; chewiness, 0.4 and gumminess, 1.2) were obtained at a milk pH of 6.6, a clotting temperature of 39 °C, a rennet concentration of 0.09% (15 000 SU) and a CaCl2 level of 0.02% (Elzeny, 1991). The effect of composition and pH on textural parameters are in the following descending order: pH  NaCl  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 233

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 s1-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 fresh UF-Domiati can be controlled by changing the homogenization pressure, heat treatment and pH of the pre-cheese (Al-Khamy, 1988). Increasing the homogenization pressure and heat treatment, and reducing the pH of the pre-cheese increase the hardness of fresh UF-Domiati cheese (Al-Khamy, 1988). Electron microscopy of ultra-thin sections of Domiati (Abd El-Salam and El-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 Ca2 weakens the strong interactions in the casein aggregates.

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; ElZayat 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 et al., 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 et al., 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 (ElGendy et al., 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% NaCl (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% NaCl should be added to milk to suppress the growth of coliforms in Domiati made from raw milk (El-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 (El-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, NaCl content and storage temperature. Storage in brine for 60 days at 20–25 °C 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 Cl. tyrobutyricum and Cl. perfringens (Naguib and Shauman, 1973). Bacillus cereus has been isolated from Domiati (ElNawawy et al., 1981). Staphylococcus aureus can tolerate up to 15% NaCl 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% NaCl (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; El-Shibiny et al., 1988) or electrolytic corrosion of tins by NaCl and developed acidity (Abo Elnaga, 1971).

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 °C  15–20 s or 65 °C  30 min). 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 °C and, if pasteurized, a 40% solution of CaCl2 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

Feta Cheese 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 ultrafiltrated cows’ milk. Of course, the flavour and other sensory qualities of these cheeses does not equate to those of the original Feta cheese.

Coagulation is performed at 32–34 °C. 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 °C without pressing for 2–3 h

Cheese Varieties Ripened in Brine 235

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% NaCl 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 °C 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-

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 °C) 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 °C. 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 et al., 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 s1- 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

-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 °C, after which the cheese is transferred to a cold room (⬃4 °C) 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 -lactoglobulin and -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 °C). 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 (600 Da) and free amino acids soluble in 5% phosphotungstic acid (PTA-SN).

25

% of total nitrogen

20

15

10

5

0 0

20

40

60

80

100

120

Ripening time (days) Figure 2 Changes in the level of nitrogen soluble in water (䊊), 12% (w/v) trichloroacetic acid (䊉) or 5% (w/v) phosphotungstic acid (䉭) as percentages of the total nitrogen during ripening of Feta cheese.

Cheese Varieties Ripened in Brine 237

The amino acid (AA) content of mature Feta varies from 2 to 7 g/kg cheese, depending on the culture used and on the age of the cheese. Leucine, valine, lysine and phenylalanine are the major AAs. Feta also contains significant amounts of non-casein AAs, such as -aminobutyric acid and ornithine, while citrulline and -aminobutyric acid are present in very small amounts (Alichanidis et al., 1984; Valsamaki et al., 2000; Katsiari et al., 2000a). Biogenic amines are generated by the decarboxylation of amino acids, mainly by adventitious micro-organisms ( Joosten and Stadhouders, 1987). Investigations have shown that the average amine content of Feta is 400 mg/kg cheese (Valsamaki et al., 2000). Market samples (15) analysed for their amine content had somewhat higher values (⬃480 mg/kg) (Alichanidis, unpublished results). In both investigations, tyramine constituted about 40% of the total amines, followed by putrescine, histamine and cadaverine. Electrophoresis in polyacrylamide gels containing urea has shown that the rates and extents of degradation of s1- and -caseins in Feta cheese are different. During the first 20 days, about 50% of the s1-casein is

hydrolysed, while more than 80% of the -casein remains intact. In well-ripened cheeses (120–240-day-old), only ⬃20% of the initial content of s1-casein remains intact compared to ⬃65% of -casein (Katsiari et al., 2000a). The extensive hydrolysis of s1-casein early during ripening is probably due to the residual rennet, which is much higher in Feta than in other cheese varieties (Samal et al., 1993), and due to the low pH, which favours chymosin activity. -Casein is also a good substrate for chymosin, but its degradation is strongly retarded by salt (Fox and Walley, 1971), the concentration of which is about 6% in Feta. Later in ripening, the degradation of large peptides and their parent caseins could be attributed to the synergistic action of chymosin, the cell-bound proteinases of starter bacteria, and, possibly, the proteolytic enzymes of the non-starter lactic acid bacteria (NSLAB). The important role of residual rennet in proteolysis and its high activity on s1-casein are confirmed from the identification of the major peptides found in the water-soluble fraction of Feta (Michaelidou et al., 1998). The majority of the peptides identified (Fig. 3)

4

100

0.4

5

80

0.3

60 0.2

6 1

3

2

%B

A214 nm

7

10 40

5A 9

0.1 8

20

0 0

10

20

30

40

50

60

70

80

90

Elution time (min) Figure 3 Reversed-phase HPLC profile of the water-soluble fraction of 6-month-old Feta showing the peaks collected and identified. Eluent A was 1 ml of trifluoroacetic acid (TFA)/l of deionized water. Eluent B was 0.9 ml of TFA, 399.1 ml of deionized water and 600 ml of acetonitrile/l. Gradient: 0–10 min, eluent A; 10–90 min, 0–80% eluent B; 90–100 min, 100% eluent B. The flow rate was 0.8 ml/min. The absorbance of the eluate was monitored at 214 nm. Peak numbers correspond to the following compounds: 1, Tyr; 2, Phe; 3, s1CN (f4–14) and s1-CN (f40–49); 4, s1-CN (f1–14); 5, -CN (f164–180), s1-CN (f102–109) and s1-CN (f24–30); 5A, -CN (f96–105) and s1-CN (f91–98); 6, s1-CN (f24–32); 7, -CN (f191–205); 8, -LA; 9, -LG (f16–?); 10, -LG (from Michaelidou et al., 1998, courtesy of the Journal of Dairy Science).

238 Cheese Varieties Ripened in Brine

originated from the N-terminal half of s1-casein. Cleavage of Phe239Val24, Phe329Arg 33, Leu989Leu99, Leu1019Lys102 and Leu1099Glu110 can be attributed to chymosin action. The two peptides identified as originating from the C-terminal half of -casein resulted from the cleavage of Leu1909Tyr191 and Ile2059Leu206 by chymosin. One peptide, Ala969Phe105, originated from the C-terminal part of para--casein resulting from the cleavage of Met959Ala96 and Phe1059Met106. The second cleavage site is a bond well-known to be cleaved by chymosin during renneting. The bond Met959Ala96 could be a cleavage site of chymosin (Reid et al., 1997) or lactococcal proteinase (Reid et al., 1994). Lipolysis

The flavour of Feta cheese is critically affected by the high levels of short-chain carboxylic acids. All reports agree that acetic acid is the major free volatile acid (FVA) (Vafopoulou et al., 1989; Katsiari et al., 2000b; Kandarakis et al., 2001; Kondyli et al., 2002). It is worth noting that acetic acid is not produced from triglycerides by lipase activity. In Feta, acetic acid can be produced in high amounts during the early stage of ripening mainly from lactose and, perhaps, from the catabolism of citrate (Sarantinopoulos et al., 2002) and from amino acids (Kondyli et al., 2002). The concentration of free fatty acid (FFA) plus acetic acid in mature Feta usually ranges between 2 and 4 g/kg cheese. The accumulation of the FFAs varies with the heat treatment of the milk, the rennet used (home-made rennets from kid and lamb abomasa are rich in lipases), the starter culture, the kind of microbial contaminants and, also, the temperature at draining. Higher draining and warm room temperatures (21 °C versus 15 °C) favour the formation of all short-chain (C29C8) volatile acids. Most of the difference (90%) is due to the higher amount of acetic acid produced mainly during the early lactate fermentation (Kandarakis et al., 2001). Free volatile acid constitutes 30–50% of the total acids (except lactic) in mature cheese, acetic acid being the dominant (40–75%) FVA (Alichanidis et al., 1984; Georgala et al., 1999; Katsiari et al., 2000b; Kandarakis et al., 2001; Kondyli et al., 2002). Butyric and higher volatile fatty acids are vital for the development of the characteristic, slightly rancid, flavour of Feta cheese (Vafopoulou et al., 1989). Volatiles

While data on the concentration of acetic and other volatile acids have been provided by a number of studies, data on other volatile compounds are limited. From the data available (Horwood et al., 1981; Kondyli et al., 2002; Sarantinopoulos et al., 2002), it seems that ethanol, followed by butan-2-ol, are the

main volatiles in mature Feta cheese. Also, volatiles such as 3-hydroxy-butan-2-one, diethyl ether, 3methyl-butan-1-ol, acetone, pentan-2-one, propanal, ethyl acetate and acetaldehyde are present in appreciable amounts. Rheological and sensory properties

Data on the rheological properties of Feta cheese are limited, since this cheese easily breaks into pieces when compressed. From the data available, it seems that hardness (kg) varies from 2.7 to 7.0, force to fracture (kg) from 1.5 to 2.4 and compression to fracture (%) from 18.7 to 21.5 (Katsiari and Voutsinas, 1994; Katsiari et al., 1997; Kandarakis et al., 2001). Feta has a short, firm and smooth texture, snow-white colour, a moist surface without rind, and is sliceable. Irregular mechanical openings, distributed throughout the cheese mass, are desirable but the presence of small round holes is regarded as a defect. The flavour (taste and aroma) of Feta can be generally described as slightly acid, salty and mildly rancid. Although the flavour of Feta has not been studied extensively, from the data available it seems that the medium- and, especially, the short-chain volatile acids contribute to its flavour (Vafopoulou et al., 1989; Georgala et al., 1999). Besides these, several other volatile compounds are found in Feta cheese (Horwood et al., 1981; Kondyli et al., 2002; Sarantinopoulos et al., 2002), some at relatively high concentrations, which may contribute to the aroma of this cheese. Some of these compounds are produced solely from the catabolism of proteins or lipids or lactose and citrate, but several of them are common end products of metabolism of more than one of the above constituents. Experiments have shown that besides glycolysis and citrate catabolism, proteolysis should be accompanied by a certain amount of lipolysis and vice versa for a balanced flavour development (Vafopoulou et al., 1989; Katsiari et al., 2000b; Michaelidou et al., 2003). Microbiology

The microbiological quality of Feta cheese can be influenced by various factors, the most important of which are: the quality of the raw milk, the thermal treatment of the milk (no treatment, thermization, pasteurization) and the extent of microbial contamination during processing, especially during dry salting. As mentioned earlier (see ‘Manufacture’), the early acidification of the curd is a crucial step in Feta cheese manufacture. This, together with the rate of salt absorption and its final concentration in the cheese moisture, are the most important features of the manufacture of high quality and safe cheese.

Cheese Varieties Ripened in Brine 239

Several starters are used for Feta cheese, which are mostly combinations of mesophilic cocci, thermophilic cocci and thermophilic rods (Vafopoulou et al., 1989; Litopoulou-Tzanetaki et al., 1993; Katsiari et al., 1997; Pappa and Anifantakis, 2001). Their counts increase rapidly during the first days, remain relatively high during ripening in the warm room and, later on, decline significantly, especially those of the mesophilic cocci. This is probably due to the low pH and high salt-inmoisture content of the cheese (Litopoulou-Tzanetaki et al., 1993; Sarantinopoulos et al., 2002; Manolopoulou et al., 2003). The enzymes of the starter culture are probably responsible for the high rate of production of small peptides and amino acids during the early stage of ripening (Bütikofer and Fuchs, 1997). However, the environment of Feta seems to favour the growth of NSLAB, especially lactobacilli, which in a 30-day-old cheese represent about 90% of lactic acid bacterial isolates (Tzanetakis and Litopoulou-Tzanetaki, 1992). Among them, Lactobacillus plantarum is the predominant species, followed by Lb. paracasei subsp. paracasei and Lb. brevis. The dominant group of bacteria found in the brine throughout the maturation of Feta cheese are also NSLAB, and the principal species identified are Lb. paracasei subsp. paracasei and Lb. plantarum (Bintsis et al., 2000). Enterococci (Ec. faecalis and Ec. durans) and pediococci are also found in mature Feta cheese but at low numbers (Tzanetakis and Litopoulou-Tzanetaki, 1992). Investigations have shown that some strains of Ec. durans (LitopoulouTzanetaki et al., 1993) or Ec. faecium (Sarantinopoulos et al., 2002) improve the sensory properties of Feta when used as an adjunct culture. P. pentosaseus, used as adjunct in Feta cheese manufacture, also improves cheese quality and shortens the ripening time by 1 month (Vafopoulou-Mastrojiannaki et al., 1990). Salt-resistant yeasts grow to high numbers (6–8 log cfu/g) on the surface of Feta during dry salting but their number decreases significantly with ripening time (Tzanetakis et al., 1998). Among the species found, Saccharomyces cerevisiae is predominant, followed by Debaryomyces hansenii and Pichia farinosa. Defects Early blowing

This defect appears mainly during drainage but also during curd salting. It is characterized by the presence of small and/or large gas holes in the cheese mass, which gives it a spongy texture. This defect is due to the excessive growth of coliforms and/or yeasts. Coliforms can multiply very rapidly in the curd during the first few hours, when the pH and temperature are favourable. However, the problem is rare in modern dairies, provided that efficient pasteurization and good

manufacturing practices are followed. Furthermore, the activity of the starter culture is crucial for the control of coliforms by reducing the pH and the amount of lactose in the curd (Bintsis and Papademas, 2002). Late blowing

This defect, which is not very common, causes blowing of the containers, and is attributed mostly to heterofermentative lactic acid bacteria and very rarely to some species of clostridia. Usually, this defect occurs when the containers are sealed before the completion of the intense fermentation in the warm room and before the pH drops to the normal values (4.8). Mouldiness

When the cheeses are not completely immersed in the brine, various species of mould grow on the cheese surface, causing visible defects and reduced quality. Softening of the cheese body

In cheeses with normal pH and moisture, softening of the body is very rare; it occurs only when the concentration of salt in the brine in the final containers is lower than the salt-in-moisture content of the cheese. The problem is more frequent when cheeses with insufficient acidity are stored at a low temperature and when the salt concentration of the brine in the package is low. Ropiness of the brine

This is actually a defect of the brine, which makes the appearance of the cheese unsightly; the cheese itself is edible and, in most cases, its flavour is normal but the brine is ropy. This defect is caused mainly by some strains of Lb. plantarum, but also by Lb. casei subsp. casei and Lb. casei subsp. rhamnosus and is due to the production of exopolysaccharides (Samaras, 1994).

Gibna Bayda (Beida) Gibna bayda is the Arabic name for ‘white cheese’. It is produced in Sudan following a method similar to that for Domiati cheese (Tannous, 1991; Abdelgadir et al., 1998) by adding salt to milk before renneting and the cheese is brined in salted whey for at least a month. It is made from cows’ milk or variable mixtures of cows’ and sheep’s or goats’ milks (Ahmed and Abdel-Razig, 1998). Gibna bayda can also be made from recombined milk (Ahmed and Khalifa, 1989).

Mish Cheese Mish is one of the oldest known cheeses in Egypt (Abou Donia, 1991). It is made from naturally

240 Cheese Varieties Ripened in Brine

fermented, partially skimmed milk remaining after the separation of sour cream by gravity. Salt is sprinkled on the curd ladled onto cheese mats for whey drainage and then cut into suitable pieces. The fresh cheese (Kariesh) is either consumed directly or brined in concentrated salted buttermilk (laban zier) in earthenware containers for more than 1 year to obtain Mish (El-Gendy, 1983; Abou Donia, 1991). Usually, morta (the heat-coagulated protein-rich precipitate left after the manufacture of butter oil (ghee/samna) by the boiling-off method), red pepper and paprika are added to buttermilk used as brine for Mish cheese. The Mish cheese and brine are served together for consumption. Ripened Mish has a yellowish-brown colour with a distinctly salty, sharp and pungent flavour. Commercially, Mish-like products are made from pieces of different Egyptian cheese varieties (Domiati/Ras) of variable degree of maturity. They are mixed in a barrel with water, NaCl and emulsifying salts, spices and ripened Mish (about 2%) as a starter. The water and the different additives do not exceed 25% of the mixture. The container is kept sealed for 1 year and its contents are then heated to 70 °C with agitation to give a homogeneous, spreadable mass, which is then packaged into retail packages (0.5–1 kg). No specifications have been issued for Mish. Therefore, wide variations are found in the composition of market samples of this cheese (Nassib and El-Gendy, 1974; Abou Donia and ElSoda, 1986; Zaki and Shokry, 1988), as shown in Table 8. Marked proteolysis and lipolysis occur in Mish during ripening. Amino acid nitrogen represents a major part of the total N (El-Erian et al., 1975). Also, the levels of total volatile fatty acids and butyric acid increase as Mish cheese ripens (Taha and Abdel-Samie, 1961). Bacillus spp., Clostridium mishii, Cl. mishami, micrococci, arthrobacteria are found in Mish (Taha and Abdel-Samie, 1961; El-Erian and El-Gendy, 1975). Coliforms have not been detected in Mish.

Table 8 Chemical composition of Mish cheese (Abou Donia and El-Soda, 1986; Zaki and Shokry, 1988) Constituent

Minimum %

Maximum %

Moisture Fat Protein Ash Calcium Phosphorus NaCl

54.76 0.5 6.95 11.13 0.229 0.180 10.0

75.68 4.60 13.13 19.79 0.403 0.215 15.20

Mudaffara Cheese Mudaffara cheese is a braided, semi-hard cheese originating in Middle Eastern Countries and in Sudan. In Syria, it is named ‘Medafara’ or ‘Magdula’. Both Mudaffara and Magdula are Arabic names, which denote ‘braided’. The technique of making Mudaffara cheese in Sudan (Ahmed, 1987) differs slightly from that for Magdula cheese made in Syria (Abou Donia and Abdel Kader, 1979). In Sudan, Mudaffara is usually made from raw cows’ milk, but mixtures of cows’ and sheep’s or goats’ milk are also used. Mudaffara has been made from cows’, goats’ and buffaloes’ milk supplemented with skim milk powder or from reconstituted milk (Ahmed, 1987). Raw milk is renneted and left for 30–60 min after coagulation to ripen. After cutting, whey is partially removed and the curd is cut into small cubes. The curd cubes are left in whey for another 40–60 min to develop the proper acidity. The acidified curds are then cooked (5–10 min) in hot water (75 °C). Black cumin (Nigella sativa) is added to the hot paste, which is kneaded and pulled quickly while hot to form cords (2 m long). Three cords are braided to form a tress, cut into suitable pieces and immersed in brine or salted whey for 2 days. The cheese is packaged in brine or salted whey in sealed containers until consumed. Table 9 shows the composition of Mudaffara cheese made from different milks (Ahmed, 1987). Salt concentration and storage temperature affect the quality of Mudaffara cheese. The hardness of the cheese decreases significantly with continued storage and is more pronounced in cheese stored at 39  2 °C than in cheese stored at a lower temperature (19  2 °C). Storage of Mudaffara in 10% salted whey gives a product of superior quality than cheese stored in whey containing 15 or 20% NaCl (Abdel Razig et al., 2001). Syrian Magdula cheese differs from Mudaffara in that it is made from sheep’s milk and the cheese is brined for 1 week, sun-dried for 2–3 days and then kept in tight containers until consumed. It is eaten after soaking in water for 24 h (Abou Donia and Abdel Kader, 1979). Table 9 Composition of Mudaffara cheese from milk of different species (Ahmed, 1987)

Cows’ milk

Reconstituted Goats’ Buffaloes’ cows’ milk milk milk (20% TS)

Total solids, % 47.22 46.96 Fat, % 9.20 9.88 pH 5.14 5.47 Total N (TN), % 3.76 3.83 Soluble N (SN), % 0.448 0.27 SN/TN 11.9 7.05

49.0 10.6 5.1 4.1 0.48 11.8

48.0 5.8 5.4 4.2 0.3 7.2

Cheese Varieties Ripened in Brine 241

Nabulsi Cheese Nabulsi cheese is one of the most popular cheeses produced in Jordan. It is usually made in springtime when enough sheep’s and goats’ milk are available. However, the method of cheese preservation allows for its consumption throughout the year. Traditionally, Nabulsi is made without the intentional addition of a starter (Tannous, 1991). Fresh sheep’s milk or mixture of sheep’s and goats’ milks is warmed to about 35 °C and coagulated with rennet. The coagulum is pressed, cut into pieces (about 4  8 cm) and sprinkled with salt. The cheese pieces are then boiled in brine and flavoured with the spices mastic (Pistacia lentiscus) and mahlab (Prunus mahlab) during boiling. Boiling imparts the characteristic texture of the cheese and improves its shelf-life. Boiling usually continues for 5–15 min, after which the cheese pieces become soft and float to the surface of the brine. The cheese pieces are taken from the brine, re-shaped by slight pressing and packaged in tightly closed cans and covered with brine in which the cheese was boiled. Based on its moisture content, Nabulsi is considered a semi-hard cheese. Jordanian Standards (1991) stipulate a moisture content for Nabulsi of 50%. Typical market samples of Nabulsi have a moisture content in the range 36.1–51% (Humeid and Tukan, 1986, 1991). This variation has been attributed partly to the variable casein/fat (C/F) ratio of the cheese milk. The C/F ratio in the milk of Awassi sheep in Jordan varies from 0.7:1 to 1.1:1 with an average of 0.9:1 (Haddadin et al., 1995), which changes the chemical composition and sensory properties of the final cheese. Nabulsi cheese from milk with a C/F ratio of 0.7:1 is preferred, while cheese made from milk with a C/F of 0.5:1 or 1.0:1 is of poor quality. Attempts to manufacture Nabulsi cheese from pasteurized milk (Haddadin et al., 1995) with the use of commercial starter cultures yielded a cheese with strong, sharp flavour, which was not appreciated by the consumer. The use of salt-tolerant Lb. paracasei, Lb. rhamnosus, Lc. lactis subsp. lactis, Ec. faecalis, Ec. faecium and Ec. durans isolated from sheep’s and cows’ milks gives Nabulsi cheese made from pasteurized milk sensory properties as acceptable as the traditional product (Yamani et al., 1998). Staphylococcus aureus, E. coli, Bacillus cereus and Proteus vulgaris have been detected in retail Nabulsi cheese, but Listeria monocytogenes has not been detected (El-Sukhon, 1993).

Akawi Cheese Akawi is a popular cheese in several middle eastern countries, especially in Lebanon and Syria. It is made from cows’, sheep’s or goats’ milk. Traditionally, Akawi

is made from flash-heated (70–75 °C) milk, cooled to about 35 °C and renneted. After 1 h, the coagulum is cut, the whey is drained and the curd pieces are removed and quickly wrapped in cheese cloth in small portions (12  12  3 cm), layered on the top of each other and pressed. Care is taken with respect to the temperature of the curd before pressing (24–26 °C) to avoid excessive fat losses or poor cheese texture. The curd is then brined (10% NaCl). The cheese is sometime used as a filler in traditional sweets after desalting for several hours. Akawi cheese has a close texture with no gas holes and can be sliced. The typical composition of Akawi is 49.1% total solids, 22.5% protein, 21.6% fat and 5% ash (Tannous, 1991). The manufacture of an Akawi-type cheese in Denmark has been described (Kristensen, 1983). The cheese was made from pasteurized cows’ milk. A mixture of yoghurt starter and Cheddar cheese starter was used; 30–40 ml rennet, 10 g KNO3 and 10 g CaCl2 were used per 100 l milk. The cheese is sold in brine-filled casks or cans or, alternatively, wrapped in aluminium foil or plastic films. The manufacture of Akawi has also been mechanized (Olsansky et al., 1979), with no appreciable effects on the yield, composition or quality.

Telemea or Telemes Cheese This cheese originated in Romania from where its manufacture spread to other Balkan countries and Turkey. Traditionally, it was made from sheep’s milk, but for many years now it is made also from cows’, buffaloes’ or, even, goats’ milk or mixtures thereof. In this case, milk with a high fat or total solids content (sheep’s or buffaloes’) is usually blended with cows’ or goats’ milk. The colour of the cheese is very white when made from sheep’s, buffaloes’ or goats’ milk, and yellowish when cows’ milk is used. It has no rind and its texture is smooth when sheep’s milk is used but harder when goats’ milk is used alone. Cows’ milk sometimes gives a crumbly cheese. The flavour of Telemes can be described as slightly salty and acid; when this cheese is made from goats’ milk (alone or mixed with sheep’s milk) it has a piquant flavour, especially when it is very ripe. The manufacturing procedure (Table 10) varies from country to country to adapt to the local climate and to the type of milk used (Anifantakis, 1991; Mallatou et al., 2003). The technology has some similarities with that used for Feta, but differs considerably in the procedure of salting and of curd draining. In Feta, whey drains under gravity and by the action of coarse salt spread on the surface of the curd, while the moulded curd of Telemes is subjected to pressure to expel whey. Concerning salting,

242 Table 10 Technological features of some cheeses ripened in brine

1. Type of milk 2. Heat treatment 3. Starter

4. Ripening time of milk, min 5. Rennet 6. Renneting temperature, °C and time, min 7. Cutting 8. Ladling 9. Pressing 10. Salting brine concentration, % temperature, °C time, h 11. Ripening brine concentration, % temperature, °C time, days 12. Storage temperature, °C

Telemes

Beyaz Peynir

Bjalo Salamureno Sirene

Beli Sir U Kriskama

Ewes’, cows’, goats’, mixtures Optional or HTST or 63–65 °C for 30 min Yoghurt*, Lc. lactis subsp. lactis  Lb. casei, Lc. lactis subsp. lactis  Lb. debrueckii subsp. bulgaricus (0.1–1%) 15–30 Calf 30–32 60 Cubes, 2–3 cm Into hoops 1–1.5 kg/kg curd for 1–2 h

Ewes’, cows’, goats’, mixtures HTST or 65 °C for 30 min Yoghurt*, Lc. lactis subsp. lactis  Lc. lactis subsp. cremoris or Lb. casei (0.15–1%)

Ewes’, cows’, mixtures 68 °C for 10 min or HTST

Ewes’, cows’, mixtures HTST

Lc. lactis subsp. lactis  Lb. casei (0.1–0.4%)

Yoghurt*, Lc. lactis subsp. lactis  Lc. lactis subsp. cremoris or Lb. casei (0.1–0.3%)

– Calf 28–32 70–135 Cubes, 3  3 cm Into frames and cheese cloth 10 kg/kg curd for 30 min

30 Calf 28–32 60–90 Cubes, 2–3 cm Into frames and cheese cloth 1–1.5 kg/kg curd for 4–6 h

– Calf 28–32 60–90 Cubes, 1.5–2 cm Into frames 2 kg/kg curd for 1–2 h then 4 kg/kg curd for 1 h

14–18 14–18 12–16

14–16 15–16 6–12

22–24 14–16 12–24

20–24 12–15 8–20

8–12 14–18 15–20 4–8

14–16 12–15 30–60 5

10–12 14–15 12–15 3–4

10 14–16 15 10

* Yoghurt prepared the previous day or yoghurt-type starters (Str. thermophilus and Lb. delbrueckii subsp. bulgaricus, 1:1).

Cheese Varieties Ripened in Brine 243

Feta pieces are dry-salted for several days before being placed in the final container filled with brine containing 7–8% salt. Thus, the salt penetration into Feta blocks is slow. In contrast, after draining, the curd of Telemes is cut into pieces (usually 11  11  8–10 cm but sometimes smaller 7  7  7 cm), which are placed immediately in a brine containing ⬃18% salt for 20 h or even 22–24% salt for ⬃16 h. By this practice, salt quickly penetrates into the curd and, evidently, interrupts the biochemical activities in the ripening cheese and, primarily, the utilization of lactic acid, which accumulates in the curd following the initial fermentation of lactose by the starter culture. Consequently, the acidity of Telemes remains high for a long time, especially if it is refrigerated soon after manufacture (Efthymiou, 1967). The gross composition of Telemes cheese varies as, in different countries, milk of various species is used for cheesemaking. Additionally, in many cases, the C/F ratio is not standardized, a fact which, among others, affects the fat content of the final product. When Telemes is made from sheep’s milk, its gross composition is comparable to that of Feta cheese. Electrophoresis of samples of Telemes made from sheep’s milk (Alichanidis et al., 1981) or cows’ milk (Kalogridou-Vassiliadou and Alichanidis, 1984) shows that the hydrolysis of s1-casein is much more extensive than that of -casein. From the analysis of 56 Feta and 120 Telemes samples obtained from the Greek market (Alichanidis, unpublished results), it was concluded that proteoly-

sis, as indicated by %WSN/TN, is lower in Telemes than in Feta cheese (13.8 and 19.9%, respectively). Also, other indices of proteolysis, e.g., %TCA-SN/TN and %PTA-SN/TN, show similar trends. Results from several experiments (Polychroniadou and Vlachos, 1979; Alichanidis et al., 1981; Kalogridou-Vassiliadou and Alichanidis, 1984; Zerfiridis et al., 1989) showed that all the above-mentioned indices and the FAA content were lower in Telemes aged for less than 90 days than in Feta cheese, but in older Telemes (120 days), their levels became comparable to those of Feta cheese. However, the rate of formation of products of casein degradation differs between the two cheeses. In Feta, the rate is high during the early ripening period, slowing down later (Fig. 2). In contrast, in Telemes, the initial rate is much slower but it continues nearly unchanged throughout ageing (Fig. 4), probably because of the differences in the cheesemaking procedure described above. The free fatty acid (FFA) content of Telemes is lower than that of Feta, ranging from 1 to 2.5 g/kg cheese. Although acetic acid is not a product of lipolysis it is, by far, the principal volatile acid, ranging from 55 to 86% of the total (Efthymiou, 1967; Alichanidis et al., 1981; Buruiana and El-Senaity, 1986). In a recent study (Mallatou et al., 2003), batches of Telemes were produced using the same procedure but different kinds of milk, sheep’s, cows’, goats’ or a 1:1 mixture of sheep’s and goats’ milk. The outcome of this study was that, irrespective of the milk used, acetic acid accounted for

20

% of total nitrogen

16

12

8

4

0 0

20

40

60 80 Ripening time (days)

100

120

Figure 4 Changes in the level of nitrogen soluble in water (䊊), 12% (w/v) trichloroacetic acid (䊉) or 5% (w/v) phosphotungstic acid (䉭) as percentages of the total nitrogen during ripening of Telemes cheese.

244 Cheese Varieties Ripened in Brine

more than 70% of the total volatile acids. In ripe cheese (60- and 180-day-old), the acid degree value was significantly higher in cows’ milk cheese, as also was the sum of FFA produced by lipolysis.

Beyaz Peynir (Turkish White Cheese) Beyaz peynir is the most popular cheese made and consumed in Turkey. Originally, it was made from sheep’s and/or goats’ milk, but cows’ milk is now widely used for its production. It is a typical cheese ripened in brine with a salty and acid taste. Its colour is very white when sheep’s and/or goats’ milk is used. It has no rind and its texture varies from soft to semi-hard, depending on the milk used and on the stage of maturity. The shape of the cheese is cubic, 7  7  7 cm, or rectangular, 7  7  10 cm. It can be consumed while fresh, but mostly is consumed after ripening in brine. In modern factories, the cheesemilk is pasteurized and starters are used for curd acidification. However, artisanal production is still very large and significant amounts of cheese are made from raw milk without starter addition (Erkmen, 2000). The technology of Beyaz peynir is given briefly in Table 10. Details of the manufacture and the microbiological and biochemical properties of this cheese are given in the review by Hayaloglu et al. (2002). It is difficult to give a reliable average gross composition for this cheese, because the reported data vary widely (Tekinsen, 1983; Turantas et al., 1989; Yildiz et al., 1989; Hayaloglu et al., 2002). These differences can be attributed to the lack of standardization of the C/F ratio in the cheese milk and to the significant variations in the manufacturing procedure, concerning the heat treatment of the milk, the use of starters, the clotting time, the pressure applied to the curd during draining, the salting level, etc. (Tekinsen, 1983; Turantas et al., 1989; Yildiz et al., 1989). Besides composition, pH also varies substantially in mature cheese, ranging from 4.11 to 5.65 (Turantas et al., 1989) or even higher (Yildiz et al., 1989; Erkmen, 2000). For this type of cheese, a pH value higher than 5.0 could be detrimental for its keeping quality, when the salt-in-brine content is not very high. Proteolysis in Beyaz peynir has not been studied in detail. As with other cheeses ripened in brine, data from the electrophoresis of cheese samples show that s1casein is hydrolysed more extensively than -casein (Saldamli and Kaytanli, 1998). In mature cheese, the ripening index (WSN/TN) varies from 13 to 22.7%, with an average value of 19.5% (Hayaloglu et al., 2002). Free amino acid (FAA) development was studied by Ücüncü (1981), who found that mature cheese (120day-old) made from sheep’s milk contained 853 mg FAA/100 g cheese, while the same cheese made from cows’ milk contained 698 mg FAA/100 g cheese.

The total biogenic amine content of commercial Beyaz peynir (22 samples) was found to be 179 mg/kg in cheeses made with starters and 442 mg/kg in cheeses made without the deliberate use of starter. In both groups, tyramine, putrescine and cadaverine were the predominant amines (Durlu-Özkaya et al., 1999). The FFA content in cows’ milk Beyaz peynir has been studied by Akin et al. (2002). It was found that in 30-day-old cheeses, the total FFA content was 638 mg/kg cheese and, as in other brine cheeses, acetic acid was the dominant volatile acid, accounting for 58.6% of the total volatile acids (C2–C8).

Bjalo Salamureno Sirene (White Brined Cheese) Bjalo salamureno sirene (Belo salamureno sirene or Bjalo sirene or, simply, Sirene) is the traditional and the most popular cheese in Bulgaria. It is a white, semi-hard sheep’s milk cheese with a slightly salty and acid taste, and a smooth texture with no rind. Its shape is rectangular (12  12  10 cm) or cubic with a side of 10 cm. A variant of this cheese is made from pasteurized cows’ milk or a mixture of cows’ and sheep’s milks. The manufacturing technology, in brief, is given in Table 10 (Dimov et al., 1975). As a result of the high fat content (7.5–8.5%) of sheep’s milk, when cheese is made from unstandardized milk, it contains FDM ranging from 56 to 59% and protein from 14 to 16.2%. Usually, the C/F ratio in cheese milk is adjusted to 0.65–0.68:1 and the gross composition of the resulting mature cheese (60 days) is 49–51% moisture, 50–53% FDM and 17% total protein. The salt content varies between 3.5 and 4.5% and the pH between 4.35 and 4.7. Depending on the age of cheese and other factors, the ripening index (WSN/TN) ranges from 18 to 25% (Peichevski and Iliev, 1986; Mikov et al., 1996; Kafedjiev et al., 1998; Stankov et al., 1998).

Beli Sir U Kriskama Beli sir u kriskama means ‘white cheese in pieces’ in Serbo-Croatian. This type of cheese is produced in the countries of the former Yugoslavia under specific names, e.g., Bel Sprski, Travnincˇki, Sjenicˇki, Sarplaninski, ˇ Homoljski, etc. (Zivkovi´ c, 1963). Most of the cheeses were, traditionally, made from sheep’s milk but nowadays some of them are also made from cows’ milk or mixtures of milk. The manufacturing technology (Table 10) of all these cheeses is similar, with very few ˇ differences (Zivkovi´ c, 1971). All have the typical characteristics of white cheeses ripened in brine: sour-salty taste and tender but firm texture. Their surface is moist with no rind, although some of them have a very thin,

Cheese Varieties Ripened in Brine 245

greasy rind. Their shape is, usually, rectangular with typical dimensions of 10  10  10–12 cm or smaller.

Halloumi Halloumi is the traditional cheese of Cyprus. For hundreds of years, it has been produced from raw sheep’s milk or mixtures of raw sheep’s and goats’ milks; nowadays, large factories use also pasteurized cows’ milk for its manufacture. Normally, the colour of the cheese is white, but that made from cows’ milk is yellowish. It is a semi-hard cheese with no rind and no gas holes, and its texture is elastic and compact. The specific characteristic of its manufacturing procedure (Anifantakis and Kaminarides, 1983) is that the blocks of pressed curd (⬃10  10  3 cm) are heated at 90–95 °C in heat-deproteinated whey for at least 30 min. After cooling on a table, the curd blocks are salted with dry salt, often mixed with dry chopped leaves of mint (Mentha viridis). Halloumi is sold fresh (immediately after production) or is stored at 4 °C in the whey previously used to heat-treat the curd, containing ⬃120 g/l salt. The severe heat treatment, together with the use of salted whey, makes this cheese suitable for storage without refrigeration for a short period of time. The average gross composition of Halloumi from the Cyprus market is: 42–53% moisture, 44.52% FDM, 24.46% protein (TN  6.38), 3.54% NaCl (Anifantakis and Kaminarides, 1983). The severe heating of the curd reduces substantially the microbial population in the cheese (Papademas and Robinson, 2000). Also, the residual rennet is probably inactivated under these conditions. Consequently, proteolysis is limited. In fresh ovine and ovine/caprine market samples, the WSN/TN is about 4.0% (Anifantakis and Kaminarides, 1983; Kaminarides et al., 2000). According to Papademas and Robinson (2000), in both ovine and bovine Halloumi, acetic acid is the dominant volatile acid in fresh and mature cheese, accounting for about 40% and over 80% of the total, respectively.

Acknowledgements Prof. E. Alichanidis wishes to thank Prof. Ivan Stankov and Prof. Todor Dimitrov for providing literature on Bjalo Salamureno Sirene.

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Reid, J.R., Coolbear, T., Pillidge, C.J. and Pritchard, G.G. (1994). Specificity of hydrolysis of bovine -casein by cell envelope-associated proteinases from Lactococcus lactis strains. Appl. Environ. Microbiol. 60, 801–806. Reid, J.R., Coolbear, T., Ayers, J.S. and Coolbear, K.P. (1997). The action of chymosin on -casein and its macropeptide: effect of pH and analysis of products of secondary hydrolysis. Int. Dairy J. 7, 559–569. Sabry, Z.I. and Guerrant, N.B. (1958). Vitamin content of pickled cheeses as influenced by production and ripening. J. Dairy Sci. 41, 925–930. Saito, Z. and Hirose, M. (1972). Some properties of centrifugally fractionated casein micelles of cow’s milk. Bull. Fac. Agric. Hirosaki Univ. Japan 18, 35–45. Saldamli, I. and Kaytanli, M. (1998). Utilisation of Fromase, Maxiren and Rennilase as alternative coagulating enzymes to rennet in Turkish white cheese production. Milchwissenschaft 53, 22–25. Samal, P.K., Pearce, K.N., Bennett, R.J. and Dunlop, F.P. (1993). Influence of residual rennet and proteolysis on the exudation of whey from Feta cheese during ripening. Int. Dairy J. 3, 729–745. Samaras, P. (1994). The problem of ropiness of Feta cheese brine. Proc. Seminar on Dairy Technology, Ioannina. National Dairy Committee of Greece, Athens. pp. 145–155. Sarantinopoulos, P., Kalantzopoulos, 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. Scott, R. (1986). Cheese Making Practice, 2nd edn, Elsevier Applied Science, London. Seham, M., Sheleih, M.A. and Saudi, A.M. (1982). Occurrence of yeasts in some Egyptian dairy products. Egypt. J. Vet. Med. Assoc. 42, 5–11. Sharara, H.A. (1962). Effect of milk pasteurization and addition of starter on the yield and composition of Domiati cheese. Alex. J. Agric. Res. 10, 127–134. Shehata, A.E., Magdoub, M.N.I., Fayed, E.O. and Hofi, A.A. (1984). Effect of salt and capsicum tincture on the properties of pickled Domiati cheese. III. Bacteriological quality. Egypt. J. Dairy Sci. 12, 47–54. Sousa, M.J., Ardö, Y. and McSweeney, P.L.H. (2001). Advances in the study of proteolysis during cheese ripening. Int. Dairy J. 11, 327–345. Stankov, I., Dimitrov, T., Ilev, T. and Miteva, T. (1998). Milk yield, composition and cheesemaking properties of ewe’s milk produced from South-Bulgarian Corriedale breed. Bulgarian J. Agric. Sci. 4, 699–705. Taha, S.M. and Abdel-Samie, M. (1961). Mish spore-forming anaerobes. II. Volatile fatty acids production. Ann. Agric. Sci. 6, 65–74. Tannous, R. (1991). Miscellaneous white brined cheeses, in, Feta and Related Cheeses, Robinson, R.K. and Tamime, A.Y., eds, Ellis Horwood, London. pp. 209–227. Tawab, G.A., El-Koussy, L.A. and Hofi, A.A. (1975). Studies on Domiati cheese. II. Changes in lactose content during pickling. Egypt. J. Dairy Sci. 3, 84–88. Tawfik, N.F. (1993). Growth and inactivation of Listeria monocytogenes in Domiati cheese. Egypt. J. Dairy Sci. 21, 1–9.

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Pasta-Filata Cheeses P. Kindstedt, Department of Nutrition and Food Sciences, University of Vermont, Burlington, VT, USA M. Cari´c and S. Milanovi´c University of Novi Sad, Faculty of Technology, Bulevar Cara Lazara 1, Serbia and Montenegro

Introduction The pasta-filata cheeses are a diverse group that originated primarily in the greater northern Mediterranean region, encompassing Italy, Greece, the Balkans, Turkey and eastern Europe. Traditionally, pasta-filata cheeses have been produced from the milks of the cow, goat, sheep or water buffalo. Some are soft or semi-soft cheeses that are, typically, consumed fresh or after only a brief period of ageing (e.g., fresh Mozzarella, low-moisture Mozzarella, Scamorza). Others are hard or semi-hard ripened cheeses that may undergo considerable ageing before being consumed (e.g., Caciocavallo, Kashkaval, Provolone, Ragusano). The term pasta-filata, which is derived from an Italian phrase that literally means ‘spun paste’ or ‘stretched curd’, refers to a unique plasticization and stretching process that is shared by all pasta-filata cheeses and which gives this diverse group their common identity. This chapter is divided into two major sections. The first section is dedicated specifically to low-moisture Mozzarella cheese (LMMC), often referred to as Pizza cheese, which is consumed fresh or after only a brief period of ageing, and which is the most economically important of the pasta-filata cheeses. The second section focuses on Kashkaval, a pasta-filata cheese that is aged extensively.

Low-Moisture Mozzarella (Pizza) Cheese Global production of LMMC has experienced unprecedented growth during the last two decades and now exceeds that of all other pasta-filata cheeses because of its premier status as a pizza topping. Rapid market growth and keen competitive pressures have given rise to impressive increases in the production capacity of cheese plants that produce LMMC. Consequently, it is not unusual now to find cheese plants that routinely produce 100 000 kg or more of LMMC cheese per day. Cheesemaking on this scale requires precise control over all aspects of the manufacturing process, which has created a pressing need for a better scientific understanding of key manufacturing parameters and their influence on cheese composition, structure, function and yield. It is within this context that LMMC has

attracted considerable interest among cheese scientists. Indeed, the past decade has seen a remarkable increase in research aimed at understanding the effects of manufacturing parameters on LMMC and at elucidating the chemical, physico-chemical and microbiological factors that influence structure and functional properties. This section will focus primarily on the literature and scientific advances that have accrued during the past 10–15 years. Earlier research on LMMC was reviewed by Fox and Guinee (1987), Kindstedt (1991, 1993a,b) and McMahon et al. (1993). Overview of manufacturing technology

The basic manufacturing scheme and equipment lines that are commonly used in the industrial production of LMMC are very similar to those used for Cheddar cheese as far as the milling stage (Bylund, 1995; Kosikowski and Mistry, 1997a; Johnson and Law, 1999). The fat-in-dry matter (FDM) content of LMMC typically falls in the range of 30–45% (w/w). Therefore, the cheese milk is normally standardized to a higher casein to fat ratio by adding non-fat milk solids (e.g., non-fat dry milk, condensed skim milk or ultrafiltered milk protein concentrate) or, less often, by removal of cream (Barbano, 1996; Wendorff, 1996). The standardized milk is pasteurized and then inoculated with starter culture as the milk is pumped into horizontal or vertical enclosed vats. Low-moisture Mozzarella cheese can be manufactured using mesophilic (e.g., Lactococcus lactis subsp. lactis, cremoris) or thermophilic (e.g., Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lb. helveticus) lactic acid bacteria. In either case, the principal role of the starter culture is to produce lactic acid in sufficient quantity to transform the curd into one that will plasticize and stretch in hot water. This is accomplished by attaining a suitable combination of pH and calcium content in the curd at the time of stretching. Furthermore, in order to obtain the correct moisture content in the final cheese, generally between 45 and 52% (w/w), the starter must produce acid much more rapidly than in the making of Cheddar cheese. Rapid acidification allows the total manufacturing time to be shortened, which reduces the total

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252 Pasta-Filata Cheeses

amount of syneresis during cheesemaking and enables a higher moisture content to be achieved in the final cheese (Barbano et al., 1994). Thus, the typical manufacturing time for LMMC is much shorter than that for Cheddar, averaging about 2.5 h or less from coagulant addition to the start of stretching (McCoy, 1997). In general, thermophilic starters are used much more widely throughout the world than mesophilic starters for LMMC cheese, although there are some noteworthy exceptions. The inoculated cheese milk is coagulated with rennet, the coagulum is cut and then cooked to a temperature of about 41 °C if a thermophillic starter is used, after which part of the whey is drained off. The remaining whey and curds are then pumped to a large enclosed conveyor belt system where draining, matting and cheddaring of the curds proceed until the proper level of acidity is developed. By the time the cheddared curd reaches the end of conveyor belt, it should have attained an optimum pH value, usually between 5.3 and 5.1 if the curd is to be milled and then stretched immediately. Alternatively, the curd may be milled at a slightly higher pH and then dry-salted to incorporate a portion or all of the salt in the final cheese. The milled curds, salted or unsalted, are then plasticized and stretched mechanically in hot brine or hot water. The hot plastic curd is forced under pressure into a chilled mould which gives the cheese its shape and which precools the block sufficiently so that it will retain its shape when removed from the mould. The block may then undergo further cooling and salting by immersion in cold brine, although salting by brining is increasingly being replaced in part, and in some cases totally, by direct salting. Many variations of the basic process for making LMMC cheese are found in commercial practice but the underlying principles are common to all make procedures. A more detailed discussion of the manufacturing technology of LMMC can be found in a recent review by Kindstedt et al. (1999).

A

B

Plasticization and stretching

In the industrial manufacture of LMMC, plasticization and stretching are usually performed using continuous single or twin screw mechanical mixers that recirculate hot water at a temperature that is precisely controlled by steam injection. An example of a commercial mixer for LMMC is shown in Fig. 1. Stretching is a two-stage process. During the first stage, the milled curd enters a reservoir of hot water at the front of the mixer, where it is warmed as it settles to the bottom of the mixer (1A). When the curd temperature increases to approximately 50–55 °C, the curd is transformed into a plastic and workable consistency. In the second stage, the

C Figure 1 An example of a commercial mixer that is used to plasticize and stretch low-moisture Mozzarella cheese. (A) milled curd enters a reservoir of hot water at the front of the mixer; (B) plasticized curd is kneaded and stretched by counter-rotating augers; (C) hot (c. 60 °C) plastic curd exits the mixer under pressure and proceeds to the moulding machine.

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plasticized curd is kneaded and stretched by either a single pair of counter-rotating augers housed in an inclined barrel or a series of horizontal and inclined single or twin augers (1B). The action of the augers reorients the amorphous curd structure into a unidirectional fibrous ribbon of hot plastic curd that exits the mixer under pressure (1C). Physico-chemical characteristics of the curd

The ability of cheese curd to plasticize in hot water and reorganize into a unidirectional fibrous structure is presumed to be governed primarily by the amount of casein-associated calcium (more correctly calcium phosphate) that is available to crosslink the amorphous para-casein matrix at the time that heat is applied to the curd (Lawrence et al., 1987; Kimura et al., 1992; Lucey and Fox, 1993; Kosikowski and Mistry, 1997a). Furthermore, the hydration of paracasein increases as the level of casein-associated calcium decreases (Sood et al., 1979), which probably contributes strongly to the ability of the curd to plasticize. Curd that contains too much casein-associated calcium fails to attain a smooth, stretchable consistency upon heating and tears during stretching. Curd with too little casein-associated calcium becomes excessively soft and fluid-like during stretching. Two parameters determine the amount of casein-associated calcium in the curd at the time of stretching: (1) the total calcium content of the curd and (2) the distribution of total calcium between the soluble and the insoluble (i.e., casein-associated) states. The total calcium content of the curd is determined by the amount of calcium that is lost to the whey up to the time of stretching. It is well established that caseinbound calcium dissociates from the para-casein matrix to the water phase of cheese curd as the curd pH decreases, and is subsequently released from the curd along with the whey during syneresis (Lucey and Fox, 1993). Consequently, the ratio of total calcium to total protein in Mozzarella curd decreases progressively during cheesemaking as the pH decreases and syneresis progresses (Kindstedt, 1985; Kiely et al., 1992; Kimura et al., 1992). The amount of calcium lost depends on the timing of acidification relative to syneresis. When the pH decreases (and thus casein-associated calcium is solubilized) during the early stages of syneresis (i.e., up to draining), more calcium is lost than when the pH decreases during cheddaring, after most of the syneresis has already occurred (Kindstedt, 1985; Kiely et al., 1992; Kimura et al., 1992). Calcium losses are greatest when acidification occurs before the onset of syneresis (i.e., before coagulation and cutting), as in the making of preacidified and directly acidified Mozzarella. Therefore, directly acidified Mozzarella cheese characteristic-

ally contains a very low level of calcium relative to total protein (Kindstedt and Guo, 1997; Paulson et al., 1998; Metzger et al., 2000; Guinee et al., 2002). Thus, the total calcium content of Mozzarella curd may vary substantially at the time of stretching, depending on the conditions of acidification. Furthermore, the distribution of total calcium between the insoluble (i.e., casein-associated) and the soluble states may also vary substantially and, indeed, it is the combination of the total calcium content and its distribution that determine the level of casein-associated calcium and thus the capacity of the curd to plasticize and stretch. The distribution of calcium in Mozzarella curd at stretching is determined primarily by the pH of the curd, as evidenced by the strong pH-dependence of calcium distribution in the final cheese (Lucey and Fox, 1993; Guinee et al., 2000b; Kindstedt et al., 2001; Watkinson et al., 2001; Metzger et al., 2001b; Ge et al., 2002). Declining cheese pH favours a shift in calcium distribution from the casein-associated to the soluble state. Consequently, curd that contains a high total calcium content at stretching (e.g., c. 30 mg/g protein, as in the making of conventional cultured LMMC (Kiely et al., 1992)), must have a low pH in the range of c. 5.1–5.3, in order to attain a casein-associated calcium level that is low enough to allow the curd to plasticize and stretch. In contrast, curd with a low total calcium content (e.g., c. 22 mg/g protein, as in the making of directly acidified Mozzarella (Kindstedt and Guo, 1997)), is optimally stretched at a higher pH value (e.g., pH 5.6–5.7), the curd becoming excessively soft and fluid-like when stretched at a lower pH due to insufficient casein-associated calcium (Larson et al., 1967; Keller et al., 1974). Indeed, by demineralizing skim milk by electrodialysis before renneting, Kimura et al. (1992) demonstrated that curd with a very low calcium content (c. 19 mg/g protein) and very high pH (pH  6.15) can be plasticized and stretched to produce string cheese. Mozzarella cheese analogues made from rennet casein, which combine very low calcium content (c. 18.5 mg/g protein) with a very high pH (c. 6.3), plus the addition of calciumsequestering salts, presumably are based on this same principle (Guinee et al., 2000b; O’Sullivan and Mulvihill, 2001). Thus, plasticization and stretching can be achieved over an extraordinarily broad range of combinations of total curd calcium content and pH. Thermo-mechanical treatment of the curd

Stretching is a thermo-mechanical treatment that involves the application of mechanical energy in the form of shear stress as the plasticized curd moves down the barrel of the stretcher and its temperature increases. Mulvaney et al. (1997) studied the effects of two key operating parameters of a twin-screw stretcher

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(i.e., the temperature of the stretching water and the speed of the augers (screw speed)) on the thermomechanical treatment of the curd and the chemical and functional characteristics of the final cheese (Yun et al., 1994a; Renda et al., 1997). Increasing the screw speed from 5 to 19 rpm at a constant and relatively low stretching water temperature of 57 °C resulted in a shorter residence time in the stretcher and a lower curd temperature at the exit. Thus, the intensity of the heat treatment of the curd (in terms of both time and temperature) decreased with increasing screw speed. Furthermore, a higher screw speed resulted in a higher specific mechanical energy (a measure of the mechanical energy that was consumed during stretching), indicative of a more intense thermomechanical treatment. At the highest screw speed (i.e., 19 rpm), the curd temperature did not increase quickly enough in the 57 °C stretching water to completely plasticize the curd before it was subjected to the shearing forces of the screws. Consequently, the curd underwent extensive tearing which resulted in higher fat loss in the stretching water and substantially lower fat and moisture levels in the final cheese (Mulvaney et al., 1997; Renda et al., 1997). The cheeses stretched at 19 rpm also had higher initial shear modulus (Go) in stress relaxation, which indicated a more extensive elastic network structure that probably resulted from their lower levels of moisture and fat. Increasing the stretching water temperature from 57 to 74 °C at a constant mid-range screw speed of 12 rpm resulted in a shorter residence time in the stretcher, a higher curd temperature at the exit (ranging from 54.4 °C (57 °C water) to 66.5 °C (74 °C water)), a lower specific mechanical energy during stretching, a lower fat loss in the stretching water and cheese with higher FDM content and a higher Go immediately after manufacture. Also, during the 50 days of ageing at 4 °C, cheeses stretched at the highest temperature had much higher apparent viscosity and TPA-hardness and lower meltability values (Yun et al., 1994a). Thus, the temperature of the stretching water had a major impact on the time–temperature profile of the curd and the intensity of the mechanical treatment during stretching, and on the rheological and functional properties of the final cheese. The authors did not attempt to explain why a higher stretching water temperature resulted in the formation of a much more elastic network structure (in spite of a higher FDM in the cheese), as indicated by a substantially higher Go value in the final cheese immediately after manufacture. However, it seems reasonable to postulate that a higher stretching temperature may have favoured more extensive hydrophobic-mediated aggregation and contraction of para-casein into stronger, more elastic fibers. If this

were the case, then one might also expect to find greater phase separation of protein and water within the cheese structure (Kindstedt and Guo, 1998; Pastorino et al., 2002). The investigators did not specifically evaluate changes in the water phase of the cheese but they did observe that the highest stretching temperature produced cheese that released an unusually large amount of serum upon melting throughout the 51 days of ageing (Kindstedt et al., 1995b). In a different study, Kindstedt et al. (1995b) varied the screw speed from 5 to 19 rpm at a constant, relatively high, stretching water temperature of 74 °C. Stretching at a slow screw speed resulted in a longer residence time and a higher curd temperature at exit, ranging from 62 °C at 19 rpm to 66 °C at 5 rpm screw speed. Cheeses that were stretched at a slow screw speed and which, therefore, attained a higher temperature during stretching had a higher apparent viscosity and TPA hardness value throughout the 112 days of storage at 4 °C, indicative of a more elastic network structure. Furthermore, the amount of serum that was expressed from the cheeses upon centrifugation (12 500  g for 45 min at 25 °C) during the first 12 days after manufacture increased, and the concentrations of intact caseins and calcium in the expressible serum decreased with increasing curd temperature during stretching. These results suggested that a higher stretching temperature may have favoured increased hydrophobically mediated aggregation of para-casein and a shift in calcium distribution to the casein-associated state, resulting in a more highly calcium-crosslinked, elastic, fibrous structure and a greater phase separation of protein and water. However, in this and the other studies cited, it is impossible to separate the effect of stretching temperature on the initial network structure that was established during stretching from other effects that occurred concurrently, such as on the rate of proteolysis and microbiological activity, as discussed below. Thus, further study is needed to differentiate and elucidate the multiple effects of stretching temperature on the structure and functional properties of the final cheese. Thermal effects on starter bacteria and coagulant

The heat treatment of the curd during stretching presumably influences the viability and activity of the starter culture bacteria in the final cheese. Yun et al. (1995a) reported that both Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus survived and remained metabolically active when stretching was performed at the low end of the stretching temperature range (e.g., 55 °C curd temperature), as measured directly by plate count enumeration and indirectly by

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increases in titratable acidity and in the level of N soluble in 12% (w/v) trichloroacetic acid in the cheese during ageing. Petersen et al. (2000) also enumerated starter Sc. thermophilus and Lb. helveticus in LMMC before and after stretching (water temperature, 83 °C, curd temperature unknown) and reported little or no change in the number of viable starter bacteria. However, starter activity appeared to be reduced substantially at higher stretching temperatures (e.g., 62–66 °C exit temperature), as evidenced indirectly by a steep decline in titratable acidity and 12% TCA-soluble N in the final cheese (Yun et al., 1994a; Kindstedt et al., 1995b). Thus, the survival of metabolically active thermophilic starter bacteria appears to be highly temperature dependent within the normal range of stretching temperature employed in commercial manufacture. Residual coagulant activity in LMMC also may vary depending on the extent of heat inactivation during stretching (Gangopadhyay and Thakar, 1991). Indirect evidence of chymosin activity in LMMC during ageing, observed as either the breakdown of s1-casein by electrophoresis or the accumulation of N soluble in water at pH 4.6, has been reported in numerous pilot-scale cheesemaking experiments (Yun et al., 1993a,c,e, 1995a,b, 1998; Barbano et al., 1994; Kindstedt et al., 1995a,b; Renda et al., 1997; Guinee et al., 1998, 2000a, 2002; Hong et al., 1998; Walsh et al., 1998; Chaves et al., 1999; Feeney et al., 2002; Somers et al., 2002) In these studies, the curd temperature during stretching did not exceed c. 55–60 °C, i.e., the low end of the normal stretching temperature range. Microbial coagulants derived from Rhizomucor miehei (formerly Mucor miehei) and Cryophonectria parasitica (formerly Endothia parasitica) also remained active in LMMC stretched at 55 °C (Yun et al., 1993c). However, chymosin activity appeared to be reduced substantially at higher stretching temperatures (e.g., 62–66 °C exit temperature), as evidenced indirectly by a steep decline in the level of pH 4.6-soluble N in the final cheese (Yun et al., 1994a; Kindstedt et al., 1995b). More recently, Feeney et al. (2001) demonstrated conclusively that residual chymosin activity in LMMC decreased progressively when the curd temperature during stretching was increased from 55 to 66 °C, with the largest decrease occurring between 62 and 66 °C. From their results, one can conclude that stretching at a temperature higher than 66 °C would have little effect on residual chymosin activity since very little activity remained after stretching at 66 °C. This was confirmed by Mayes and Sutherland (2002), who reported that the rate of proteolysis (measured as increases in pH 4.6- and 12% TCA-soluble N during ageing) decreased in LMMC when the stretching temperature was increased from 60 to 67 °C. However, increases from 67 to 75 °C had little further effect

on proteolysis. The impact of curd pH at stretching has not been specifically investigated, but it is possible that a higher curd pH at a given stretching temperature may result in greater inactivation of chymosin and less residual chymosin activity in the final cheese (Guinee et al. 2002). Reorganization of curd structure

Several different microscopy techniques have been used to elucidate the changes in curd structure that occur during stretching. Using scanning electron microscopy (SEM), Oberg et al. (1993) and McMahon et al. (1999) observed that the amorphous curd structure before stretching was completely disrupted by the shearing forces of the screws, resulting in a reorganization of the aggregated para-casein matrix into roughly parallel aligned para-casein fibers. Furthermore, fat globules and starter bacteria became concentrated in longitudinal columns that separated the fibers. This striking reorganization is illustrated in Figs 2–4. Immediately after stretching and while the cheese was still hot, the para-casein had the appearance of smoothwalled fibers and there was little evidence of the fat, which had been removed during sample preparation for SEM (Fig. 3). However, after the cheese had cooled overnight, the walls of the fibers showed extensive spherical imprints left by the fat droplets as they solidified and acted as a template around which the pliable para-casein fibers moulded (Fig. 4). A similar transformation of curd structure during stretching was observed by Auty et al. (1998, 2001) using confocal scanning laser microscopy (CSLM). Furthermore, it was possible to directly observe both the protein and the fat phases of the curd, as well as non-fat, non-protein (presumably serum) regions. The CSLM images confirmed that fat globules became redistributed and concentrated in the channels that separated the para-casein fibers (Auty et al., 1998, 2001; Guinee et al., 1999). This striking reorganization of curd structure was also readily observed at much lower magnification by Taneya et al. (1992) using light microscopy and differential staining of the fat and protein phases of the cheese. The same researchers demonstrated, using cryo-SEM, that bulk phase water (serum) as well as fat occupied the columns that separated the protein fibers in newly stretched cheese. Functional properties

Cheese that is used as an ingredient in prepared foods must satisfy certain performance requirements that are determined by the function of the cheese in the particular food application in which it is used. As

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Figure 2 Scanning electron micrograph of low-moisture, part-skim Mozzarella curd taken prior to dry salting and stretching. Scale bar equals 10 m (reproduced with permission from Oberg et al., 1993).

noted earlier, LMMC is used mostly as an ingredient in pizza and other prepared foods that contain melted cheese. Therefore, functional properties are essential determinants of the quality and acceptability of LMMC, and considerable research has been directed towards developing and applying new methods to measure functional characteristics and fundamental rheological

properties that determine and affect functional behaviour. A summary of recent developments relating to analytical testing for functional and rheological properties is presented here. For a more complete overview of the topic of LMMC functionality, the reader is directed to the recent comprehensive reviews by Rowney et al. (1999), Fox et al. (2000) and Guinee (2002b).

Figure 3 Scanning electron micrograph of low-moisture, part-skim Mozzarella curd taken immediately after stretching, longitudinal view. Scale bar equals 10 m (reproduced with permission from Oberg et al., 1993).

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Figure 4 Scanning electron micrograph of low-moisture, part-skim Mozzarella curd taken after stretching and following 1 day of storage, longitudinal view. Scale bar equals 10 m (reproduced with permission from Oberg et al., 1993).

Functional properties before heating

LMMC is usually produced in block form, ranging in weight from c. 2.3 to 9.5 kg, and must therefore be comminuted (shredded or diced) before it can be used as an ingredient in prepared foods such as pizza. In the industry, the term ‘shreddability’ is used in reference to several important functional characteristics, including: the ease with which the cheese block is processed through a shredding machine (also referred to as ‘machinability’); the geometry and integrity of the shreds (i.e., the extent to which shreds of uniform dimensions with cleanly cut edges are obtained); the susceptibility of the cheese to shatter and form fines during shredding; and the ability of the shreds to resist matting and remain free-flowing. Problems related to shreddability may occur when the body of the cheese is soft and pasty or wet, causing the shredding machine to become clogged with cheese and resulting in shreds with ragged edges and deformed geometry, along with the formation of fines and gummy balls of cheese. Such cheese is also likely to undergo excessive matting after shredding, which makes it difficult to handle, store and apply uniformly on the product in which it is used with portioncontrolled precision. At the opposite extreme, cheese that is excessively firm and dry, as is often the case with low-fat Mozzarella, may take longer to process through the shredding machine and fracture excessively to produce shattered shreds and fines, which also make handling and portion control more difficult (Kindstedt, 1995).

Only a few attempts to quantify aspects of shreddability directly by empirical and imitative tests have been reported. Apostolopoulos and Marshall (1994) used quantitative image analysis data to calculate a shreddability index as a function of the size, shape and number of shredded fragments obtained upon shredding. The shreddability index values obtained by image analysis were highly correlated with sensory panel visual assessments made on the basis of the length of the shreds, amount of fragments present and degree of stickiness between the shreds. An empirical test to measure matting behaviour was proposed based on the ability of cheese particles to penetrate down though a stack of vibrating sieves of decreasing mesh size (Kindstedt, 1995). Sticky cheese that mats excessively is retained by the larger sieves, whereas cheese that remains free-flowing may penetrate to the bottom of the stack. The aggregation index value of Mozzarella cheese, calculated as a weighted average of sieve size  mass of cheese retained by each sieve, increased during storage and with higher fat content, indicating increased susceptibility to matting (Kindstedt, 1995). Many researchers have used texture profile analysis (TPA) and similar uniaxial compression tests at a temperature ranging from c. 10 to 25 °C to characterize the hardness or firmness of Mozzarella cheese. Generally, these studies showed that Mozzarella became significantly softer or showed a trend towards softening with increasing age and level of proteolysis (e.g., Tunick et al., 1993, 1995; Yun et al., 1993b,d,e, 1995a,b, 1998; Barbano et al., 1994; Kindstedt et al., 1995a,b; Renda

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et al., 1997; Guinee et al., 1998, 2000a, 2001, 2002; Hong et al., 1998; Walsh et al., 1998), with increasing fat and/or moisture content (e.g., Tunick et al., 1991, 1993, 1995; Yun et al., 1993e; Rudan et al., 1999) and with decreasing calcium content (Yun et al., 1995b; Guinee et al., 2002) and pH (Guinee et al., 2002). Fundamental rheological testing methods, such as stress relaxation (Diefes et al., 1993; Yun et al., 1994b) and small amplitude oscillatory shear (dynamic) tests (Diefes et al., 1993; Tunick et al., 1993, 1995; Ak and Gunasekaran, 1996), have also been used to characterize the softening of LMMC during refrigerated storage. Testing has been performed in the temperature range of c. 10–26 °C. Of particular interest are the results of Tunick et al. (1995) and Ak and Gunasekaran (1996), who used dynamic testing to measure changes in G (the elastic or storage modulus) and G (the viscous or loss modulus) of Mozzarella cheese during storage. The elastic modulus (G) decreased during storage, as expected, indicating a softening of the cheese caused by a weakening of the para-casein matrix by proteolysis. However, the viscous modulus (G) did not increase as expected but, instead, decreased, indicating a reduction in viscous dissipation as the cheese aged. Ak and Gunasekaran (1996) suggested that the decrease in the viscous modulus (G) may have been caused by increased binding of water resulting from proteolysis. Changes in the state of water in Mozzarella cheese during ageing have been the focus of considerable study, as will be discussed later. The impact of such changes on rheological and functional properties as differentiated from the direct effect of proteolysis on the network structure of the para-casein fibers is not completely understood and warrants further investigation. Heat-induced functional properties (melting)

Heat-induced functional properties are essential determinants of the quality and acceptability of LMMC that is used as an ingredient in cooking applications such as for pizza. Important heat-induced characteristics include: meltability (more correctly, flowability, i.e., the extent to which melted cheese flows and spreads upon heating); stretchability (the ability of the molten cheese to stretch and form strings when extended); elasticity (the ability of the cheese strings to resist deformation during extension, which is related to chewiness); oiling-off (the release of free oil); and blistering and browning (the formation of dark-coloured patches of varying size and colour intensity). In general, LMMC for ingredient use in pizza should, upon melting, flow readily to form a continuous melt with complete loss of shred identity, possess a stretchable and slightly to moderately elastic, chewy consist-

ency, and display limited blister formation, limited intensity of browning and a glistening, but not greasy, surface. Meltability. The meltability of LMMC has been evaluated extensively by empirical tests such as the Schreiber (Kosikowski and Mistry, 1997b) and Arnott (Arnott et al., 1957) tests, which measure the increase in diameter or decrease in height of a cylinder of cheese upon melting under standard conditions. Muthukumarappan et al. (1999a) proposed several modifications to improve the efficacy of the Schreiber test. More recently, Wang and Sun (2002a,b) applied computer vision image analysis to quantify the increase in the area of cheese samples upon melting as an index of meltability. Other widely used empirical approaches to measure flow properties or melted consistency include the method described by Olson and Price (1958), which measures the distance melted cheese flows in a horizontal glass tube, and helical viscometry (Kindstedt et al., 1989; Kindstedt and Kiely, 1992). The latter method measures the resistance on a rotating t-bar spindle as the spindle is drawn through a column of molten cheese, referred to as apparent viscosity. A high apparent viscosity generally corresponds to a fibrous, elastic, chewy melted consistency whereas a low apparent viscosity indicates a viscous fluid-like consistency. A more direct empirical method for measuring post-melt chewiness was described by Metzger and Barbano (1999). Post-melt chewiness was determined by blending melted cheese, that had partially cooled, with water in a stomacher, passing the contents through a series of sieves of decreasing mesh size, and determining the percentage of cheese solids retained in the largest mesh sieve (4.75 mm). Results of this empirical test were highly correlated with sensory ranking of chewiness. Guinee et al. (1998) described an empirical test for melt time, defined as the time required for a fixed weight of shredded cheese to melt and fuse into a molten mass, free of shred identity, on heating at 280 °C. Numerous researchers, who have used one or more of these empirical methods to study Mozzarella cheese, have generally reported that the melt time and apparent viscosity decreased and the meltability (flowability) increased with increasing age and extent of proteolysis (e.g., Oberg et al., 1991, 1992; Tunick et al., 1993, 1995; Yun et al., 1993b,d,e, 1995a,b, 1998; Barbano et al., 1994; Kindstedt et al., 1995a,b; Fife et al., 1996; Renda et al., 1997; Guinee et al., 1998, 2000a, 2001, 2002; Hong et al., 1998; Madsen and Qvist, 1998; Walsh et al., 1998), with higher fat and moisture contents (Tunick et al., 1991, 1993, 1995; Yun et al., 1993e; Perry et al., 1997; Rudan et al., 1999; Petersen et al., 2000), with

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lower calcium content (Yun et al., 1995b; Metzger et al., 2001a; Guinee et al., 2002) and with lower pH (Guinee et al., 2002). Reducing the calcium content of low-fat Mozzarella resulted in lower values for post-melt chewiness (Metzger et al., 2001b). Several different fundamental rheological tests have been used to study the melting process of LMMC. Guinee et al. (1999, 2002) used dynamic testing to characterize changes in the viscoelasticity of LMMC on heating from 20 to 80 °C. They reported that G (the elastic or storage modulus) remained relatively constant from c. 20 to 25 °C but then increased rapidly with increasing temperature to 45 °C, indicating a softening of the cheese which the authors have attributed, in part, to liquefaction of the fat phase. At temperatures above 45 °C, G decreased more slowly because the fat is fully liquid at c. 40 °C. Diefes et al. (1993) also reported substantially lower G values for LMMC at 60 °C than at 20 °C, indicative of thermal softening. Furthermore, Guinee et al. (1999, 2002) showed that the phase angle ( ) increased gradually when the temperature was raised from 20 °C to between 45 and 60 °C, and then increased steeply with increasing temperature to c. 80 °C. These results indicated that the cheese underwent a phase transition from largely elastic to largely viscous in nature when the temperature exceeded the range of c. 45–60 °C. Consistent with these results, Taneya et al. (1992) reported that thermal softening occurred in string cheese over the temperature range 5–45 °C, as measured by compression and stress relaxation tests. The same investigators reported a strong temperature dependency of flow properties over the range of 45–75 °C, as measured by capillary rheometry. In summary, melting of LMMC is characterized by an initial thermal softening, resulting from the liquefaction of fat, followed by a phase change from solid-like to liquid-like as the para-casein matrix collapses, liquid fat globules coalesce and flow, and adjacent planes of para-casein are displaced (Guinee, 2002b). Much interest has developed around lubricated squeezing flow as an approach to profiling melting behaviour. Ak and Gunasekaran (1995b) used a lubricated squeezing flow test to characterize the thermal softening of LMMC over the temperature range 30–60 °C, and softening during ageing. Later, this group (Wang et al., 1998) developed a test device based on lubricated squeezing flow, named the UW Meltmeter, to objectively measure the melt/flow behaviour of cheese at different temperatures. They reported that the meltability of Mozzarella cheese, as profiled by several fundamental rheological parameters measured by the Meltmeter, increased with higher fat content in the cheese and higher melting temperature. Kuo et al.

(2001a) used the UW Meltmeter to characterize the flow behaviour of LMMC as a function of cheese age and holding time at 60 °C for up to 20 min. They reported that the meltability of 1-week-old LMMC was not affected by holding at 60 °C for up to 20 min. However, the meltability of older cheeses (6 and 12 weeks) decreased sharply with longer holding time at 60 °C, which they attributed to an apparent redistribution of moisture in the cheese caused by increased hydrophobic interactions in the para-casein structure. A different test device, called the UW Melt Profiler, which is also based on the principles of squeeze flow rheometry, was developed by Muthukumarappan et al. (1999b) and later modified by Gunasekaran et al. (2002). This device is used to measure the softening point of cheese, which is defined as the temperature at which cheese begins to flow under constant force. Using the UW Melt Profiler, Muthukumarappan et al. (1999b) demonstrated that the softening point of Mozzarella cheese decreased with increasing age and fat content of the cheese. Both empirical and fundamental test methods have been proposed to evaluate the stretchability or elongational properties of Mozzarella cheese. Apostolopoulos (1994) and Guinee and O’Callaghan (1997) developed empirical tests to measure the distance to which the melted cheese could be stretched vertically or horizontally, respectively, before complete strand breakage. Authors who used the method of Guinee and O’Callaghan (1997) have generally found that the stretchability of molten LMMC increases with storage time at 4 °C up to c. 15–20 days and thereafter remains relatively constant up to 50–75 days (Guinee et al., 1998, 2000a, 2001, 2002; Walsh et al., 1998). However, the stretchability deterioriates fairly rapidly as the level of pH 4.6-soluble N (as % of total N) exceeds c. 14% (Feeney et al., 2001; Guinee et al., 2001), as, for example, occurs on prolonged holding of cheese at 4 °C (e.g., 130 days) or holding for a shorter time (e.g., 70 days) at higher storage temperature (e.g., 10–15 °C) (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 °C, 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 et al., 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

Stretchability.

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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).

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

Browning.

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 et al., 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

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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-melt 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,

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 °C 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 °C), 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 et al., 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

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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 et al., 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 -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 (NaCl), 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., 6%, w/w, NaCl) with a low calcium content (Geurts et al., 1972). Further evidence of microstructural swelling was obtained using several different microscopy techniques. McMahon (1995) and McMahon et al. (1999) confirmed using SEM that the microstructure of LMMC changes substantially during the first weeks after manufacture, as seen in Figs 5–7. Initially, the fatserum channels appear to be very open, as evidenced by shallow imprints of fat globules on the walls of the para-casein fibers (Fig. 5). However, by day 7 the fat globules appear to be partly engulfed by the para-casein matrix (Fig. 6), and by day 21 they appear to be completely encased by swollen protein (Fig. 7). A similar process of microstructural swelling was observed by Auty et al. (1998, 2001) using CSLM. Also, Cooke et al.

Figure 5 Scanning electron micrograph of low-moisture, part-skim Mozzarella cheese 1 day after cooling and brining. Fat-serum channel walls show indentations formed by solidified fat globules of varying size and starter bacteria. Bacterial cells and residual fat globule membrane material adhere to the fat-serum channel walls. Scale bar equals 10 m (reproduced with permission from McMahon et al., 1999).

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Figure 6 Scanning electron micrograph of low-moisture, part-skim Mozzarella cheese after 7 days of storage at 4 °C. Fat globule indentations are more pronounced than at day 1, indicating that the cheese protein matrix has expanded into the fat-serum channels. Scale bar equals 10 m (reproduced with permission from McMahon et al., 1999).

(1995) reported that the average size and space between electron-dense clusters shown by transmission electron microscopy increased in Mozzarella cheese during ageing. The change in spacing of the electron-dense clusters, which corresponds to the sub-aggregates of the

para-casein matrix, is consistent with the swelling phenomenon that has been observed by SEM and CSLM (McMahon et al., 1999). Furthermore, Paulson et al. (1998) demonstrated that NaCl has a striking effect on the microstructure of non-fat directly acidified

Figure 7 Scanning electron micrograph of low-moisture, part-skim Mozzarella cheese after 21 days of storage at 4 °C. The hydrated cheese protein matrix fills the spaces between the solidified fat globules. Impressions of discrete fat globules attest to the completeness of the cheese protein matrix hydration and subsequent expansion into the fat-serum channels. Starter bacterial cells embedded in the matrix are evident. Scale bar equals 10 m (reproduced with permission from McMahon et al., 1999).

264 Pasta-Filata Cheeses

Mozzarella cheese; salted cheese had a more homogeneous protein matrix that was free of serum pockets, whereas unsalted cheese contained pockets of free serum throughout the para-casein matrix and more widely spaced protein sub-aggregates. In summary, the above results strongly suggest that a NaCl-mediated redistribution of water, characterized at the microstructural level by swelling of the para-casein fibers, occurs in cultured Mozzarella cheese during the first few weeks after manufacture. The increase in solvation and decrease in calcium crosslinking of the swollen para-casein fibers during the first few weeks of ageing enable adjacent planes of para-casein to displace more readily and fat globules to coalesce and flow more freely upon melting. Consequently, meltability (flowability), stretchability and oiling-off characteristically increase, and the apparent viscosity, melt time and chewiness decrease as cultured LMMC ages. However, as noted earlier, it is possible to pre-empt many of the age-related changes in structure and functional properties by substantially reducing the initial level of casein-associated calcium in the cheese. This can be accomplished by directly acidifying the cheese milk, so as to attain a more highly solvated and less highly crosslinked para-casein matrix in the cheese immediately after manufacture (Kindstedt and Guo, 1997; Metzger et al., 2001a; Guinee et al., 2002). Such cheese may have functional properties immediately after manufacture that are largely similar to those attained by cultured LMMC after several weeks of ageing (Kindstedt and Guo, 1997; Guinee et al., 2002).

Kashkaval General characteristics

Kashkaval is one of the most popular hard cheeses in many Mediterranean countries, its production dating back to the eleventh and twelfth centuries. However, historical references suggest that Kashkaval has an even older tradition. According to the Roman writer Columella, a cheese named ‘manum pressum’ was produced in the Roman Empire by a method similar to that for Kashkaval. It has been assumed that Kashkaval was brought to the Mediterranean by nomadic tribes from the East during the second century BC through the seventh century. During Roman times, this technology was brought from Italy to Great Britain, adapted to the English conditions, modified, and resulted far later in a new type of cheese, named Cheddar, one of the most popular cheeses worldwide today. While in ancient times Kashkaval production was limited to the Greek and Roman empires, as well as their colonies, today it is produced in an area extending

from Crimea, South Ukraine, the Caucasus and Turkey, through Greece, Bulgaria, Romania, Yugoslavia, Albania and Hungary to Italy, Algeria, Tunisia, Egypt and Morocco. This entire area is characterized by a relatively hot and dry climate, a hilly terrain, with welldeveloped sheep breeding. International trade and globalization have led to the expansion of Kashkaval production to other parts of the world. The following Kashkaval-type cheese varieties, which differ in name and various aspects of manufacture, are produced in the Mediterranean region: Kashkaval Balkan, Kashkaval Preslav, Kashkaval Vitosha (Bulgaria), Kachkavalj (Yugoslavia), Kachkaval, Kachekavalo (USSR), Kasseri (Greece), Kasar (Turkey, Albania) and Cascaval Dobrogen (Romania). Kashkaval has also been given different commercial names according to the production district, e.g., Pirdop in Bulgaria, Epir in Greece, or Sarplaninski and Pirotski Kaskaval in Yugoslavia (Peji, 1956). The Italian version of Kashkaval is called Caciocavallo and in Egypt the name Romy is commonly used. Kashkaval is manufactured from cow, sheep, goat or mixed milk, which may be raw or pasteurized. For example, in Bulgaria, Kashkaval Preslav is produced from mixed milk, Kashkaval Balkan is produced from sheep’s milk, while Kashkaval Vitosha is produced from cows’ milk; in Romania, Cascaval Dobrogen is produced from sheep’s milk only. The typical form of Kashkaval is flat, cylindrical, with a smooth, amber-coloured rind, 30 cm in diameter, 10–13 cm in height and 7–8 kg in weight. However, sausage-like or pear-like shapes are also produced (Cari´c, 1993; Cari´c and Milanovi´c, 1994). The typical composition of Kashkaval produced in several countries is shown in Table 1. The European Economic Community (1990) specified the characteristics of Kashkaval as follows: ‘Kashkaval cheese of sheep’s milk, matured for at least 2 months, of a minimum fat content of 45% by weight in the dry matter, and a dry matter of at least 58%, in whole cheeses of a net maximum weight of 10 kg, whether or not wrapped in plastic’ (Cari´c, 1993). The National Committee for Standardization in Yugoslavia has defined the National Standard for Kashkaval cheese (Yugoslav Standard, 1997). The Standard takes into account traditional manufacturing practices, as well as modern trends in the industrial production of the cheese. According to the Standard, ‘Kashkaval is a semi-hard or hard, pastafilata cheese which is available in two types: Kashkaval (weigh 5–10 kg, minimum 56% dry matter, minimum 45% fat in dry matter and minimum 8 weeks ripening period) and Kashkaval Krstaˇs (weigh up to 3 kg, minimum 54% dry matter, minimum 45% fat in dry matter and minimum 4 weeks ripening period)’. Both cheese

Pasta-Filata Cheeses 265

Table 1 Composition of various types of Kashkaval cheese Type of Kashkaval

Fat (%)

Dry matter (%)

Total protein (%)

Salt (%)

Ash (%)

pH

Author

Bulgaria

30.0

60.14

19.60

4.0

5.69

5.0

Greece

33.88

66.36

25.14

2.2

4.38

5.1

25–29 25.5–31.4 27–32 20.5–21.2 14.5 21.1–26.3

58–65 48.8–60.1 60–65 47.9–49.4 49.5–51.7 49.9–61.2

26.0 19.0–22.2 – 22.0–23.1 30.3–33.5 21.4–25.2

2.6–3.2 1.6–2.3 2.0–3.5 1.2–1.5 2.5–2.7 –

– – – 2.8–3.4 5.0–5.7 –

– – 4.9–5.0 5.5–5.6 5.5–5.6 5.2–5.6

Kosikowski and Mistry (1997) Kosikowski and Mistry (1997) Robinson (1995) Kocak et al.(1996) Peji´c (1956) Alrubai (1979) Milanovi´c (1993) Omar and El-Zayat (1986)

Turkey Yugoslavia

Egypt

types can be produced from cows’, sheep’s, goats’ or mixed milk, which may be raw or pasteurized. Kashkaval, together with Mozzarella and Provolone, belongs to the pasta-filata group of cheeses. Kashkaval undergoes a texturization process that involves soaking the acidified curd in hot brine until a plastic consistency is achieved. The hot plastic curd is then kneaded and stretched to produce a homogeneous cheese with a fibre-like structure. The specific technology in the production of Kashkaval results in the characteristic structure of the final cheese: laminar, elastic, very close with visible layers and random slits, but no gas holes. Manufacturing technology

The manufacture of Kashkaval cheese consists of two independent stages: 1. production of the curd and its acidification (cheddaring); 2. texturizing of the acidified curd which involves heating and mechanical kneading and stretching by soaking in hot water or brine. The steps in the manufacturing process are shown in Fig. 8. Traditionally, Kashkaval cheese is produced from raw milk, which is generally of poor quality, without the addition of a starter culture. Heat treatment of the curd during texturizing has a preservative effect on the final cheese, enabling raw milk with a higher acidity, i.e., poor microbiological quality, to be processed. During the last two decades the use of pasteurized milk and starter cultures has been introduced gradually into commercial practice to standardize Kashkaval cheese quality (Cari´c, 1993; Cari´c and Milanovi´c, 1994; Milanovi´c and Cari´c, 1998; Pudja and Milanovi´c, 2000). Starter cultures for Kashkaval cheese usually consist Str. thermophilus, Lc. lactis subsp. diacetylactis (cit Lc. lactis subsp lactis), Leuc. mesenteroides subsp. dextranicus, Lb. delbrueckii

subsp. bulgaricus, Lb. helveticus, Lb. casei, used in various combinations at a level ranging from 0.1 to 0.5%, w/w (Cari´c, 1993; Cari´c and Milanovi´c, 1994). Rennet is added in sufficient quantity to coagulate milk within 30–40 min. In addition to calf rennet, coagulating enzymes of various origins are now used. Recent studies comparing the effects of calf rennet, recombinant chymosin and protease from Rhizomucor miehei on the microstructure of Kashkaval curd demonstrated that recombinant chymosin as well as microbial coagulant may be used successfully in the manufacture of Kashkaval cheese from milk or UF retentate (Milanovi´c, 1993, 1996; Milanovi´c and Cari´c, 1998; Milanovi´c et al., 1998). The protein matrias of curds obtained by coagulating milk using calf rennet, recombinant chymosin or microbial protease are shown in Figs 9 and 10. Milk coagulated with standard calf rennet contained smaller casein particles compared to those obtained using recombinant chymosin or microbial rennet, the latter showing more advanced fusion of casein micelles into chains and clusters (Fig. 9). The microstructure of the Kashkaval curds obtained by coagulating UF retentate differs from those made from milk (Fig. 10). Chains of casein particles predominated over clusters in all UF curds, while clusters predominated in gels from non-ultrafiltered milk. The curd matrix obtained using recombinant chymosin or microbial rennet was considerably finer than the matrix obtained using standard calf rennet. The microstructure of conventional and UF Kashkaval curds differed from each other to an extent depending on their water and fat contents and the origin of enzymes used. The coagulum is usually cut finely into particles, 6–8 mm, stirred at 32 °C for 5 min (Peji´c, 1956; Scott, 1981), and then scalded at 42 °C for 35 min in the making of Russian or Italian Kashkaval; in contrast, no scalding is used in the making of Balkan Kashkaval.

266 Pasta-Filata Cheeses

Milk Pasterurization 75 °C, 15–20 s Cooling 32 °C Starters Inoculation Rennet Coagulation 32 °C, 30–40 min Cutting 32 °C, 5 min Stirring 32 °C, 5 min Scalding 42 °C, 35 min Whey Pressing 30–40 min Whey Fresh curd Cutting in blocks Curd acidification 1–4 days, 20 °C Ripe curd Salt Texturing 72–75 °C, 1 min Salt Dry salting Moulding Cooling 20 °C, 24 h Pre-ripening 15–18 °C, RH 80–85%, 15 days Foils Packaging Ripening 10–12 °C, RH 80–85%, 2–3 months Storage 2–4 °C, 1 year Kashkaval

Figure 8 Manufacturing procedure for Kashkaval cheese (Cari´c and Milanovi´c, 1994).

The curds are then ladled into moulds and allowed to drain and self-press for 30 min. Alternatively, the whey may be drained from the vat and the curds allowed to fuse in the vat for 30–40 min. The bed of fused curds is then sliced into blocks and cheddared to allow the fermentation of lactose to continue. The length of the cheddaring process depends on the bacterial activity and curd acidity. During the warm season, cheddaring is usually completed in 1 day, while during winter, it may last 4 or 5 days, in both commercial and home-made procedures. The introduction of pasteurization and the use of starter cultures have improved the cheddaring process (Cari´c, 1993; Cari´c and Milanovi´c, 1994; Pudja and Milanovi´c, 2000). Proteolysis commences during cheddaring of the curd, which constitutes the first stage of Kashkaval ripening (Peji´c, 1956; Djordjevi´c, 1962; Cari´c, 1993). Proteolysis as measured by the Van Slyke method (Djordjevi´c, 1962), during cheddaring is 25% of the total proteolysis during ripening in the Balkan procedure, 33% in the Russian and even 46% in the Italian procedure. During cheddaring, lactic acid fermentation takes place, with a concomitant increase in acidity to pH 5.4–5.5 when cows’ milk is used, or pH 5.2–5.3 when sheep’s milk is used. The difference in acidity can be attributed to the relative differences in the proportion of individual caseins between sheep’s and cows’ milk. Acidification causes an increase in the concentration of soluble calcium (the curd after cheddaring contains 53% more soluble calcium than the unacidified fresh curd) and results in the formation of monocalcium para-caseinate (Cari´c, 1993). Acidification results in the characteristic fibre-like structure of the finished product. Additionally, the growth of lactic acid bacteria and the production of lactic acid inhibits the growth of many species of microorganisms (gas-forming, proteolytic, lipolytic) that can cause defects in the cheese. The ripened curd is then texturized, which is accomplished by soaking the blocks of curd (20–25 cm length; 5–10 cm width; 0.5–1 cm high) in a hot (72–75 °C) brine (5% NaCl) for 1 min (Peji´c, 1956; Cari´c and Milanovi´c, 1994) or 12–18% NaCl for 35–50 s (Cari´c, 1993), in either the traditional or mechanized production technique. The traditional method for Kashkaval production required substantial manual labour. After forming and cheddaring the blocks by hand, the ripened blocks were placed into metal or wooden perforated baskets which were immersed in 5% NaCl solution at 72–75 °C. The curd mass was then agitated with a strong, wooden stick in order to obtain a compact structure. The hot curd was then transferred to a table and hand-kneaded like a dough. At this stage, the curd has become plasticized and elastic.

Pasta-Filata Cheeses 267

CHR

CHR

GENC

GENC

REN

REN

0.5 μm

Figure 9 Protein matrices (dark areas) of Kashkaval curds obtained by coagulating milk using: (a) rennet (Ha-Bo, Chr.Hansen’s Lab. A/s, Denmark – CHR; (b) fermentation chymosin (Kluyveromyces lactis, Maxiren 15 l, Gist brocades, The Netherlands – GENC); (c) microbial protease (R. miehei, Rennilase 50l, Novo Industri A/S, Denmark – REN) (reprinted from Milanovi´c et al., 1998, with permission from Elsevier Science).

2.5 μm Figure 10 Protein matrices of Kashkaval curds obtained by coagulating UF milk retentate using: (a) rennet (Ha-Bo, Chr.Hansen’s Lab. A/s, Denmark – CHR; (b) recombinant chymosin (Kluyveromyces lactis, Maxiren 15 l, Gist brocades, The Netherlands – GENC); (c) microbial protease (R. miehei, Rennilase 50l, Novo Industri A/S, Denmark – REN). Small arrows point to casein particle chains, large arrows point to clusters (reprinted from Milanovi´c et al., 1998, with permission from Elsevier Science).

268 Pasta-Filata Cheeses

Texturizing has an additional advantage, i.e., a pasteurizing effect which suppresses undesirable microbial growth and encourages desirable fermentation and ripening, resulting in high-quality cheese. Microbiological evaluation of Kashkaval curd before and after texturizing showed that Escherichia coli and other coliform bacteria, which were present in the curd at the end of cheddaring, were absent from the final cheese due to heat inactivation during texturizing. Thus, texturizing enables the manufacture of Kashkaval in hot climates and the use of milk with high acidity in its production (Cari´c, 1993). In traditional manufacture, Kashkaval cheese is partially salted with dry salt during kneading of the texturized curd. After moulding (24 h in metal or wooden hoops), a small amount of salt is applied 4–5 times to the cheese during the first 2–3 weeks of ripening. However, salting of Kashkaval is also performed by brining (18–20% NaCl, 10–12 °C, 5–6 days) (Peji´c, 1956; Scott, 1981). In general, salt concentration depends on the specific variety of Kashkaval cheese (Table 1). Kashkaval is ripened at 15–18 °C and at a relative humidity (RH) of 80–85% for 15 days and then at 12–16 °C and 85% RH for 2–3 months. The shelf-life of Kashkaval is 10–18 months at 2–4 °C. During the past 20 years, the manufacturing process has been mechanized, replacing the manual labourintensive operations of slicing, heat treatment with agitation, salting and moulding (Cari´c and Milanovi´c, 1994; Tomatis, 1996). An example of processing equipment designed not only for Kashkaval but also for other pasta-

filata cheeses is shown in Fig. 11. The incorporation of mechanized texturing, milk pasteurization and starter cultures in the manufacture of Kashkaval has resulted in a more controlled process and better and more uniformquality cheeses. The development of Kashkaval structure at different stages of a mechanized process line is shown in Fig. 12 (Cari´c, 1993). It is evident that the para-casein curd is changed from an amorphous structure (Fig. 12a) to a fibrous material (Fig. 12b) after texturizing. At the end of the process, casein fibres form a compact, characteristic, laminar texture. Scanning electron microscopy micrographs of Kashkaval and other cheese varieties showed distinct differences in the microstructure of the cheeses, which helped to explain their textural properties (Hassan, 1988). Cheese hardness was highly correlated (R  0.764) with its chemical composition: increased protein and NaCl resulted in higher cheese hardness, while higher contents of water and fat and pH, as expected, reduced hardness. Kashkaval was ranked fourth according to its hardness, after Provolone, Ras and Gouda (Hassan, 1988). Although the manufacturing protocol for Kashkaval and Cheddar share many features, they differ in two fundamental aspects: 1 The main difference in the manufacture of these two hard cheeses is that Kashkaval has a specific operation: plasticization (heat treatment of cheddared curd with agitation in hot brine), resulting in a plasticized, homogeneous mass, which is afterwards formed into

ˇ Yugoslavia). Figure 11 Continuous texturing and moulding machine used in Kashkaval manufacture (Courtesy IMLEK Dairy, Sid,

Pasta-Filata Cheeses 269

a

c

2.5 μm

b

25 μm

d

25 μm

5 μm

Figure 12 Structure of Kashkaval cheese at different production steps: (a) acidified curd showing bacteria aggregated in nests; (b) textured curd with protein matrix having uniform orientation; (c) final product; (d) final product from another plant (Cari´c, 1993).

cheese, with no pressing. During the manufacture of Cheddar there is no plasticization, and moulding is followed by pressing (Milanovi´c and Tamime, 1990). 2 Traditionally, Kashkaval is produced from raw milk, without starter, while in Cheddar is usually produced from pasteurized milk, which is inoculated with a starter. The use of a starter increases the rate of acid production which affects the cheddaring time and, consequently, the time necessary for the complete manufacturing process. However, the use of pasteurized milk has been introduced recently for the manufacture of Kashkaval. Quality characteristics

The ripening process continues in the formed and salted cheese. During ripening, the distribution pattern of free amino acids (FAA) and free fatty acids (FFA) changes due to the complexity of the maturation process, resulting in the formation of the characteristic flavour of Kashkaval cheese (Omar and El-Zayat, 1986). The concentrations of glutamic acid, serine, aspartic

acid, threonine, proline, alanine and lysine were higher in 4-month-old Kashkaval than in young cheese but the concentrations of valine, methionine, isoleucine, leucine and tyrosine were lower (Table 2). The increasing amount of total FAA in mature cheese, as well as the presence of particular FAAs, especially glutamic acid, leucine, valine and tyrosine at high concentrations, may be correlated with the typical flavour of Kashkaval. The concentration of FFAs increases c. 3-fold after 2 months and 6.5 times after 4 months of ripening. The main FFA are in the following order: C16, C18:1, C14 and C16. The volatile fatty acids, C49C10, as well as C2 which contribute to cheese aroma, are present in low concentrations. Differences in the quality attributes of the numerous Kashkaval variants arise from the fact that its technology is subject to many variations with respect to curd composition, added cultures, degree of ripening and intensity of heat treatments. The chemical, rheological and sensory characteristics of Kashkaval cheese manufactured with different

270 Pasta-Filata Cheeses

Table 2 Concentrations of amino acids in Kashkaval cheese (Omar and El-Zayat, 1986) Ripening period Young

Amino acid Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Total

mg/100 g cheese 2.92 – – 3.25 1.76 1.01 8.94 3.48 1.39 3.53 6.48 3.27 3.76 8.19 4.21 7.17 59.40

2 months Per cent of total free amino acids 4.92 – – 5.48 2.96 1.70 15.10 5.86 2.34 5.94 10.92 5.51 6.33 13.80 7.11 12.10

mg/100 g cheese 5.91 1.79 2.12 8.68 4.93 2.73 26.4 9.16 3.07 6.53 9.92 4.53 6.36 13.20 8.15 16.20 130.00

coagulating enzymes and ripened for up to 1 year have been investigated in detail (Milanovi´c, 1993, 1995, 1996; Milanovi´c and Cari´c, 1993, 1994a, 1995a,b, 1998). A progressive increase in non-protein nitrogen (NPN) is evident in all samples of Kashkaval during ripening, but the rate of increase varies depending on the type of coagulant used. The level of NPN in Kashkaval produced with fermentation chymosin is similar to that in control cheese made with calf rennet up to 150 days, but the values differ by about 25% in advanced ripening. Lower values for NPN were observed in samples made with R. miehei protease compared to cheese with standard rennet (i.e., 20% lower at the start and 10% after 1 year). These results are possibly related to the lower retention of microbial rennet in the curd and consequently in the cheese. In the same study, PAGE was used to evaluate protein degradation throughout ripening. s1-Casein was degraded more rapidly than -casein, which agrees with numerous earlier published data that residual rennet causes more intense hydrolysis of s1- than -caseins, the latter being hydrolysed primarily by plasmin (Hassan and El Deeb, 1988; Cari´c and Milanovi´c, 1994). Calf chymosin and fermentation chymosin caused a very similar pattern proteolysis of casein, with more intense degradation of s1- than -casein, as has also been reported for Cheddar (Bines et al., 1989). Similar results were also obtained for Kashkaval made with Rennilase, which contrast with previous reports

4 months Per cent of total free amino acids 4.66 1.38 1.63 6.69 3.80 2.10 20.40 7.06 2.37 5.03 7.65 3.49 4.90 10.20 6.28 12.50

mg/100 g cheese 12.61 6.22 8.03 18.25 10.94 4.17 40.10 17.37 6.77 19.11 10.43 6.65 8.15 26.00 13.09 29.50

Per cent of total free amino acids 5.31 2.62 3.38 7.69 4.60 1.76 16.90 7.31 2.85 8.05 4.39 2.80 3.43 11.00 5.52 12.40

238.00

of higher breakdown of -casein by Rennilase than by chymosin in Cheddar cheese (Creamer et al., 1988; Cari´c and Milanovi´c, 1994). The production of volatile aroma components in Kashkaval during ripening was evaluated by Milanovi´c (1993) and Cari´c and Milanovi´c (1994) using capillary gas chromatography of samples by a simultaneous distillation/extraction (SDE) method (De Frutos et al., 1988) The volatile components identified in 360-day-old Kashkaval are shown in Fig. 13. Based on retention times and mass spectrum, seven aroma components were identified: caproic, caprylic, capric and lauric acids and the ethyl esters of caproic, caprylic and capric acids. The presence of volatile fatty acids of the homologous series, C89C12, and their role in Kashkaval aroma was reported by Hassan and El Deeb (1988) and is typical for semi-hard and hard cheese varieties (Scott, 1981). Caprylic and capric acids dominate the fatty acid profile in all Kashkaval samples throughout ripening (Milanovi´c, 1993; Cari´c and Milanovi´c, 1994), which agrees with the findings of Omar and El-Zayat (1986). The distribution of volatile aroma components is influenced by the type of coagulant used in cheesemaking. The content of volatile fatty acids varied throughout the investigation, emphasizing the complexity of Kashkaval ripening. Differences in the formation of fatty acid ethyl esters show that they are formed in various ways during the degradation of cheese proteins and lipids (Gonzàlez de Llano et al., 1990;

Pasta-Filata Cheeses 271

CHR IS

4

GENC 5

IS

7

6

5

6

4

3

7

1

2

3

1

Time (min)

Time (min)

REN IS

5

6

4

7

1

3

Time (min) Figure 13 Chromatogram of the volatile aroma components of 1-year-old Kashkaval cheeses: (a) CHR – cheese produced with standard rennet; (b) GENC – cheese produced with fermentation chymosin; (c) REN – cheese produced with R. miehei protease. Volatile components are: 1-ethyl caproate; 2-ethyl caproate; 3-ethyl caproate; 4-caproic acid; 5-caprilic acid; 6-capric acid; 7-lauric acid (Milanovi´c , 1993).

Martinez-Castro et al., 1991). The use of fermentation chymosin (Maxiren) or microbial protease (Rennilase) resulted in different aroma profiles from that of control cheese made with standard calf rennet. Kashkaval cheeses produced from 1.8-fold concentrated retentate differed markedly from the conventional cheese in chemical composition, profile of proteolytic

degradation products, pattern of volatile aroma components and sensory characteristics (Milanovi´c and Cari´c, 1994b,c). In general, it may be concluded that conventional or UF Kashkaval made using fermentation chymosin is not significantly different from that made using calf rennet, confirming numerous published data that

272 Pasta-Filata Cheeses

recombinant chymosin is a satisfactory alternative to calf rennet in cheese manufacture. However, significant differences were noted in the proteolytic pattern of Kashkaval made using R. miehei protease compared to that made with standard calf rennet. Addition of an enzyme ‘cocktail’, containing protease (Acelase AHC 100) and lipase (Palatase M 1000 l), to the curd after 75% of the whey had been drained off to accelerate the ripening of conventional and UF Kashkaval cheeses has been assessed. Accelerated ripening by the added proteolytic and lipolytic enzymes was most evident during the early stages of ripening of both UF and traditional Kashkaval cheeses (Milanovi´c, 1993; Cari´c, and Milanovi´c, 1994; Milanovi´c and Cari´c, 1995c). Fungal lipase (R. miehei-Palatase M 200 l) added at a concentration of 5 or 15 ml/100 l milk before renneting, accelerated the ripening of Kasar cheese (a Kashkavallike cheese manufactured in Turkey) (Kocak et al., 1996). Titratable acidity, water soluble N and total volatile fatty acids level were significantly higher in lipase-treated cheese than in control cheese during 90 days of ripening. Cheeses with added enzymes had a stronger flavour during the first month of ripening, but developed excessive rancidity at higher amounts of added enzyme. The flavour of low-fat Kashkaval cheese (23% fat) can be enhanced by adding heat- or freeze-shocked Lb delbruecki var. helveticus cultures at a level of 2% to cheese milk prior to renneting. Incorporation of heat- or freezeshocked culture greatly enhanced proteolysis and slightly increased the content of FFAs in low-fat Kashkaval cheese during 6 months of ripening. Low-fat cheese without the added cultures did not develop typical Kashkaval flavour and had poor body and texture (Aly, 1995). The relationship between the level of biogenic amines and the level of hygiene during the production of Caciocavallo and other typical Sicilian cheeses were studied by Lanza et al. (1994). The content of biogenic amines, determined using HPLC, in Caciocavallo cheese was generally high and for histamine, tyramine and putrescine amounted to 2.8–119.1, 3.8–110.6 and 0.6–37.4 mg/kg, respectively. Differences were attributed to variations in the hygienic quality of the original milk and in the manufacturing procedure, including storage. Other relevant changes during Kashkaval cheese ripening were described by Cari´c (1993) and will not be discussed here. Many of the quality defects encountered by Kashkaval cheese are of microbial origin (see Cari´c, 1993).

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Yun, J.J., Barbano, D.M., Larose, K.L. and Kindstedt, P.S. (1994a). Effect of mixer stretching temperature chemical composition, microstructure, proteolysis, and functional properties of Mozzarella cheese. J. Dairy Sci. 77(Suppl. 1), 34(Abstract). Yun, J.J., Hsieh, Y.L., Barbano, D.M. and Kindstedt, P.S. (1994b). Draw pH and storage affect rheological properties of cheese. J. Food Sci. 59, 1302–1304. Yun, J.J., Barbano, D.M., Kiely, L.J. and Kindstedt, P.S. (1995a). Mozzarella cheese: impact of rod to coccus ratio

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Cheeses Made from Ewes’ and Goats’ Milk M. Medina and M. Nuñez, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) Madrid, Spain

Southern European countries account for most of the production of ewes’ and goats’ milk cheeses. Traditional cheesemaking procedures are strictly followed in some cases and there are also examples of cheese varieties in which they co-exist with modern industrial technology. The manufacture of many ewes’ and goats’ milk cheeses is regulated by a Protected Designation of Origin (PDO) at the national level, established mainly in Mediterranean countries to define and protect highquality traditional products against imitations (Nuñez et al., 1989). The European Union adopted this system in 1992 to promote and protect food products. A PDO (Europa, 2002) covers the term used to describe foodstuffs which are produced, processed and prepared in a given geographical area using recognized know-how. Most ewes’ and goats’ milk cheeses with PDO are named after the region in which they are produced. Ewes’ and goats’ milk cheeses have special tastes and flavours, very distinct from those of cheeses made from cows’ milk. Compositional differences of ewes’ and goats’ milk with respect to cows’ milk, mainly in proteins and fat, account for differences in the sensory characteristics of the cheeses. Genetic, physiological and environmental factors are responsible for variations in milk composition within a single species. Thus, the influence of breed, lactation stage, feeding regime, breeding conditions and milking system on the composition of ewes’ and goats’ milk has been dealt with in numerous studies. For reviews on the chemical composition of ewes’ and goats’ milk see Anifantakis (1986) and Juárez and Ramos (1986), respectively.

Ewes’ Milk Cheeses General aspects of ewes’ milk cheeses

The production of ewes’ milk in the European Union was 2 181 382 tonnes in 2001. Italy, Greece, Spain and France account for 95% of that production (FAOSTAT, 2002). The technology, microbiology and chemistry of ewes’ milk cheeses were reviewed by Nuñez et al. (1989). Six main ewes’ milk cheese families were con-

sidered: white or fresh, brined or pickled, hard and semi-hard, blue-veined, stretched curd and whey cheeses. The seasonal nature of ewes’ milk production results in large variations in cheese production. Cheeses in European Mediterranean countries are produced mainly between December and June, from a milk production that increases sharply in the spring and decreases from July to November. Different procedures to regulate milk supply, including freezing of unconcentrated ewes’ milk (Anifantakis et al., 1980), concentration of milk by ultrafiltration (Voutsinas et al., 1995), freezing of pressed curds (Fontecha et al., 1994; Sendra et al., 1999) and freezing of fully ripened cheeses (Tejada et al., 2002) have been investigated. Genetic polymorphism of ovine milk proteins and their relationships with the technological properties of milk have been reviewed by Amigo et al. (2000). Although there is considerable information on the effects of the genetic polymorphism of caseins in cows’ or goats’ milk on their composition and cheesemaking potential, the results obtained in ewes’ milk are still preliminary. Technological consequences in the final product are not as important as for goats’ milk, due to the higher casein content of ewes’ milk. The effects of s1-casein CC, CD and DD genotypes on cheesemaking properties were investigated by Pirisi et al. (1999b). The CC milk had a higher casein content than CD or DD milk, a higher protein:fat ratio and a smaller casein micelle diameter. Cheesemaking trials with CC milk demonstrated its better renneting properties and cheesemaking characteristics than DD milk, while CD milk was intermediate. Three genetic variants of -lactoglobulin (-LG; A–C) have been described. The AA phenotype of -LG seems to be more efficient than other -LG phenotypes for cheese manufacture (Amigo et al., 2000). Some ewes’ and goats’ milk cheese varieties are manufactured with specific rennets. Pecorino Romano and Fiore Sardo are traditionally made with rennet paste prepared from macerated stomachs of lambs slaughtered immediately after suckling, which also contain pregastric esterases (PGEs). These cheese varieties

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280 Cheeses Made from Ewes’ and Goats’ Milk

have a sharp ‘picante’ flavour due to the high levels of short-chain fatty acids produced by PGE activity. Thus, these rennets play an important role in the development of cheese aroma and flavour. Vegetable rennet from Cynara cardunculus is used in the manufacture of many Portuguese and some Spanish cheeses made from ewes’ milk. C. cardunculus is a thistle that grows wild and abundantly in dry, stony and uncultivated areas in Mediterranean regions. Aqueous extracts are prepared from flowers that are picked and dried in the shade in the open air. Proteinases present in the flowers of C. cardunculus were initially termed cynarases or cyprosins, and currently cardosins. Three acid proteinases have been partly characterized (Campos et al., 1990; Heimgartner et al., 1990; Faro et al., 1992). An acid proteinase was first isolated and shown to induce milk clotting via cleavage of the Phe1059Met106 bond in bovine -casein (Faro et al., 1992). Two new aspartic proteinases, cardosins A and B, isolated from fresh stigmas of C. cardunculus have been reported to be similar to chymosin and pepsin, respectively (Veríssimo et al., 1995, 1996). Extracts from C. humilis are also used in the manufacture of ewes’ milk cheeses (Fernández-Salguero and Sanjuán, 1999; Vioque et al., 2000). A powdered coagulant from crude aqueous extracts of C. cardunculus has been patented (Fernández-Salguero et al., 2000) and used in cheese manufacture with similar results (Fernández-Salguero et al., 2002). Experiments on the use of C. cardunculus for cows’ milk coagulation showed that cheeses tended to be bitter and to have textural defects (Vieira de Sá and Barbosa, 1972). Cardosin exhibits a preference for bonds between hydrophobic amino acids of bovine s1-casein (Ala163!Val167) and -casein (Ala189!Tyr193), which are less susceptible to attack by chymosin. Several bitter peptides were identified in the digests (QueirozMacedo et al., 1996).

Roquefort, the most important ewes’ milk Blue cheese, is of very ancient origin and has been protected by a PDO since 1925. It is manufactured from raw ewes’ milk in southern France and Corsica. Milk at 28–32 °C is usually inoculated with a mesophilic lactic starter. Spores of Penicillium roqueforti are added to the milk or sprinkled, as a suspension, on to the curds when they are put into the moulds. After draining and salting, the cheeses are transported to the natural damp, aired caves of Roquefort-sur-Soulzon, where ripening must take place. Cheeses with a veined body are cylindrical in shape, around 10 cm high and weigh between 2.5 and 2.9 kg (see ‘Blue Cheese’, Volume 2). Ossau-Iraty is manufactured in south-western France from raw or pasteurized ewes’ milk. After renneting, curds are heated at 36–44 °C. Cheeses are ripened for at least 3 months. Ossau-Iraty is similar to Roncal cheese made south of the Pyrenees, although produced in two different forms, 2–3 and 4–7 kg. The effects of lipolytic (L1, Lipomod) and proteolytic (PP, Promod) enzyme preparations on proteolysis and the sensory characteristics of Ossau-Iraty cheese have been investigated (Izco et al., 2000). A bitter after-taste was detected in cheeses made with the PP preparation, with higher levels of free amino acids (FAAs) than in control cheese. Cheeses made with the L1 preparation had a more pungent flavour. Broccio is a whey cheese produced in Corsica and received a PDO status in 1988. Fresh whey is mixed with ewes’ milk and heated to 80–90 °C. The resulting mass is placed into moulds and allowed to drain. The cheese, in the form of a ball of curd that has been flattened and drained, is presented in returnable wicker baskets known as ‘canestres’. Broccio is usually eaten fresh, within 48 h of manufacture, and has a mild flavour. If drained and salted it can be ripened for at least 15 days. Greek ewe cheeses

French ewe cheeses

About 250 000 tonnes of ewes’ milk were produced in France in 2001. Out of a total of 37 PDO cheeses made in France, only three varieties, Roquefort, Ossau-Iraty and Broccio, are made from ewes’ milk. Their production in 2000 was 18 135, 2610 and 466 tonnes, respectively, representing approximately 40% of all cheeses produced in France from ewes’ milk. In the same year, PDO cheeses made from goats’ milk cheeses accounted for only 5%, and PDO cheeses made from cows’ milk for 9.8%, of total cheese produced from milk of the respective species (CNIEL, 2002).

Greece has a very long tradition in breeding small ruminants, and consequently in cheese production. Of the total milk produced in Greece, 39% is cows’ milk, 36% is ewes’ milk and 25% is goats’ milk (Zerfiridis, 1999). About 670 000 tonnes of ewes’ milk were produced in Greece in 2001, and approximately 88% of Greek cheese production is from ewes’ and goats’ milk. Twenty-three of the twenty-six traditional PDO cheeses are made from ewes’ milk mixed with goats’ milk (http://www.greece.org/hellas/cheese.html). Soft cheese production in 2000 was 84 240 tonnes, including 77 894 tonnes of Feta cheese. Semi-hard Kasseri cheese represented 4886 tonnes. Hard cheeses

Cheeses Made from Ewes’ and Goats’ Milk 281

accounted for 15 786 tonnes, including Graviera (8188 tonnes), Kefalograviera (3346 tonnes) and Kefalotyri (2753 tonnes). Whey cheese production was 13 245 tonnes (Greek National Dairy Committee, personal communication). Cheese consumption in Greece is 22 kg per caput, 8 kg of Feta and the remainder of Kasseri, Kefalograviera and different types of Graviera and Mizithra. Feta is the most famous traditional Greek cheese. It is a white soft cheese, ripened and kept in brine (10–12% NaCl) for at least 2 months. Feta is manufactured from pure ewes’ milk or a mixture with up to 30% of goats’ milk. Traditionally, Feta cheese was manufactured from raw milk in small family dairies. Nowadays, the greater part is produced from pasteurized milk with commercial mesophilic and thermophilic starter cultures. Feta has a salty, slightly acid taste, natural white colour, with a firm and smooth texture and pleasant organoleptic characteristics. It is marketed in barrels, in tinned boxes or as plastic-wrapped pieces. For further information on Feta and other cheese varieties ripened under brine see ‘Cheese Varieties Ripened in Brine’, Volume 2. Galotyri is a soft variety, with a creamy and spreadable texture, produced from ewes’ or goats’ milk or their mixture. The milk is heated to boiling point and left in containers at room temperature for 24 h. NaCl (3–4%) is added and the milk is left for another 2–3 days at room temperature with occasional stirring. Rennet may be added to favour coagulation. The curds are put into containers which are held at 8 °C for not less than 2 months when raw milk has been used. Kopanisti is a soft cheese produced from cows’, ewes’ or goats’ milk or their mixtures. The milk is coagulated at 28–30 °C in about 2 h. The coagulum is left to stand in the vat for 20–24 h, broken up and put into cloth sacks for draining. The drained curd is mixed with salt, put into containers and placed in a cool place with a high relative humidity to promote the growth of an abundant surface mould. Afterwards, it is mixed every 10 days for 30–40 days to facilitate even distribution of the mould. Kopanisti has a soft, spreadable texture and a salty and piquant flavour. High numbers of yeasts and moulds are found during ripening, with P. commune identified in 90% samples (Tzanetakis et al., 1987). When combinations of yeasts and Penicillium spp. were used to accelerate ripening, higher proteolysis was detected in cheeses containing Penicillium spp., (FFAs) and organoleptic characteristics were not affected by inoculation with selected cultures (Kaminarides et al., 1992). Pichtogalo Chanion is a soft cheese with a creamy texture, made from goats’ or ewes’ milk or their mixture. The milk is coagulated at 18–25 °C within 2 h.

The coagulum is left to acidify for 24 h and placed in cheese cloths to drain. Salt is added to 1%. Kasseri is a semi-hard cheese made from ewes’ milk, to which up to 20% goats’ milk may be added. Raw or pasteurized milk is used for manufacture (Moatsu et al., 2001). The coagulum is broken up and allowed to stand for 5–10 min. The curds are heated to 38–40 °C under constant stirring and left to drain until the pH falls to about 5.2. The block of curd is cut into thin slices, which are stretched in water at 70–80 °C for 15 min, and put into moulds for 2–3 days. The cheese is dry-salted 12–14 times during ripening at 18 °C for not less than 3 months. Formaella Arachovas Parnassou is a semi-hard variety made from ewes’ or goats’ milks or a mixture. The milk is coagulated at about 32 °C for about 2 h. The coagulum is broken up and heated to 40 °C for 10 min. The curds are left to settle and then divided into large pieces which fit in special moulds (hoops or wicker containers). The moulds are immersed in whey at 60 °C for 1 h. Afterwards, the cheeses are inverted and re-immersed in whey at 75–80 °C for 1 h. Cheeses are salted and left to dry for 24 h. Then, they are placed on shelves for 4 days to dry. Formaella cheese can be consumed fresh or ripened for not less than 3 months. Graviera Agrafon is a hard cheese produced traditionally from ewes’ milk or from a mixture of ewes’ and goats’ milk. The milk is coagulated at 34–36 °C with rennet. The coagulum is broken up after 25–35 min and the curds heated to 48–52 °C. Cheeses are pressed for several hours, left to dry on wooden shelves for up to 2 days and then placed in brine for 2–4 days. The cheeses are ripened initially at 12–15 °C, continued at 16–18 °C and completed at 12–15 °C. The minimum ripening time is 3 months. Graviera Kritis is a hard cheese made in Crete from ewes’ milk to which a low percentage of goats’ milk may be added. It is a Gruyère-type cheese which undergoes a limited propionic acid fermentation that gives the cheese a slightly sweet taste. Milk is heated up to 68–70 °C. The coagulum is cut and the curds are scalded at 50–52 °C. Cheese wheels, with a diameter of 40 cm and a weight of 14–16 kg, are salted in 16–20% brine for 4–5 days and ripened for 90 days at 15–16 °C. Its composition and microbiological characteristics were described by Kandarakis et al. (1998). Inoculation of milk with starter cultures resulted in a faster increase of medium and small molecular mass nitrogeneous fractions (Moatsou et al., 1999). Kefalograviera is a traditional hard variety manufactured from ewes’ milk or a mixture of ewes’ and goats’ milk, mainly in Western Macedonia. The coagulum is broken up, heated to about 48 °C, transferred to

282 Cheeses Made from Ewes’ and Goats’ Milk

moulds and pressed. The cheese is held at 14–16 °C for 24 h, and then brine-salted for 2 days. Ripening begins at 14–16 °C and during this period the surface of the cheese is dry-salted about 10 times. Afterwards, cheeses are held at less than 6 °C for at least 3 months. Kefalograviera is circular in shape, with numerous holes, a pleasant salty flavour and a rich aroma. Lowfat Kefalograviera cheese, with the same body, texture and flavour as control cheeses made from full-fat milk (6%), was produced from milk containing 3% fat (Katsiari and Voutsinas, 1994). Cold storage of milk accelerated flavour development of full-fat cheese and enhanced flavour of low-fat cheese (Lalos and Roussis, 2000). Partial replacement of NaCl by KCl did not affect proteolysis or lipolysis of cheeses (Katsiari et al., 2001). Kefalotyri is a hard, heavily salted cheese, with a strong flavour and small irregular eyes. It is manufactured from ewes’ milk, mixed ewes’ and goats’ milk or from cows’ milk, without a starter culture. Kefalotyri is considered to be the ancestor of many hard Greek cheeses. It has a salty and piquant taste and a rich aroma after ripening for at least 3 months. Lactobacilli and enterococci counts are high, with Enterococcus faecium, Lactobacillus plantarum and Lb. casei as the predominant species. Leuconostocs and Streptococcus thermophilus disappear early in ripening (LitopoulouTzanetaki, 1990). Ladotyri Mytilinis is a hard cheese manufactured from ewes’ milk or from a mixture of ewes’ and goats’ milk on the island of Lesvos. The coagulum is broken up and heated to 45 °C. The curds are pressed in the bottom of the vat, cut to their final cheese size and placed in special moulds. Cheeses are salted and ripened for not less than 3 months. After ripening, cheeses are immersed in olive oil or paraffined. Manouri is a soft whey cheese manufactured from whey obtained from ewes’ or goats’ milk or mixtures thereof. The mixture is heated to 88–90 °C over 40–45 min with constant stirring. At 70–75 °C, salt is added together with 25% ewes’ or goats’ milk or cream. When the temperature reaches 88–90 °C, the curds are left for 15–30 min and then transferred to cloth sacks for draining for 4–5 h. After this, the cheese is kept at 4–5 °C until consumed. Xynomyzithra Kritis is another soft whey cheese, with a sharp to sweetish taste and a granular to creamy texture, produced in Crete. The whey is filtered and heated to 90 °C over 30 min while stirring. At 68–70 °C, a small quantity of full cream is usually added. The curds are left to stand for 30 min and then transferred to moulds for draining for 3–5 h. Salt is added and the cheese is put into cloth sacks. The

cheeses are pressed for 1 week before being placed at 10 °C for at least 2 months. Italian ewe cheeses

Production of ewes’ milk in Italy was 850 000 tonnes in 2001 and ewes’ milk cheese varieties have great economic significance, particularly in central and southern Italy and in Sardinia. Varieties with PDO status are Pecorino Romano (33 650 tonnes in 2000), Pecorino Siciliano (735 tonnes), Pecorino Toscano (1328 tonnes), Pecorino Sardo (600 tonnes), Fiore Sardo (380 tonnes), Canestrato Pugliese (60 tonnes), Casciotta d’Urbino (230 tonnes) and Murazzano (62 tonnes). Cheese consumption per caput in Italy in 2001 was 19.8 kg. Pecorino Romano, the best-known Italian hard ewes’ milk cheese, is produced in regions around Rome and in Sardinia. Milk, raw or thermized, at less than 68 °C for not more than 15 s, is inoculated with a natural starter culture (scotta-fermento or scotta-innesto) obtained from the residual whey from Ricotta cheese manufacture. Starters consisting of Sc. thermophilus, Lactococcus lactis subsp. lactis and Lb. delbrueckii subsp. bulgaricus are also used for Pecorino Romano manufacture. Coagulation with lamb rennet paste takes place at 37–39 °C in 14–16 min. The coagulum is cut, left to settle for 2–3 min and cooked at 45–46 °C. After 30 min, the curds are moulded, pressed and trenched to facilitate whey drainage. Cheeses are dry-salted periodically for 30–60 days and ripened at 10–14 °C for 5–8 months (Battistotti and Corradini, 1993). Cheeses are cylindrical in shape, 25–32 cm high and 25–35 cm in diameter, and weigh 20–35 kg. Flavour is slightly piquant for cheeses ripened for 5 months, and piquant and very strong for older cheeses. The average composition is: dry matter (DM), 68–70%; fat, 28–30%; protein, 28.0–29.5%; NaCl, 3.2–4.5% (Battistotti and Corradini, 1993). Changes in the main microbial groups during ripening have been studied (Deiana et al., 1984). Staphylococcus, Micrococcus and yeasts are the main secondary microflora of traditional cheeses, with Debaryomyces hansenii and Kluyveromyces marxianus as the dominant yeasts (Deiana et al., 1997). Cheese pH 24 h after manufacture is 4.9–5.0 and DM, 58.6–59.6%; after 8 months, the pH is 5.6–5.9 and DM is 63.7–67.6%. Proteolysis proceeds slowly due to its high NaCl content and its low moisture, with 23% pH 4.6-soluble N and 18% TCA-soluble N as a percentage of total N in 8-month-old cheeses. Sensorial characteristics of Pecorino Romano depend mainly on the lipolysis caused by the PGE in the lamb rennet paste. Free fatty acids range from 467 mg/kg (C8) to 1181 mg/kg (C6), distributed in the order C6C4C10C8. Butanoic and hexanoic acids provide

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intensity to the fatty acid flavour, 2-ethyl butanoic provides the butyric acid flavour notes and 3-methyl butanoic acid the sweat-like and fatty-acid like flavour notes. 4-Methyl octanoic and 4-ethyl octanoic acids, together with p-cresol, m-cresol and 3,3-dimethyl phenol, appear to be responsible for ewe notes in Pecorino Romano cheese (Ha and Lindsay, 1991b). Pecorino Sardo is a semi-cooked cheese produced in Sardinia from ewes’ milk inoculated with a commercial or natural whey starter culture. Calf rennet is used to coagulate milk. The curds are heated to 41–42 °C and held at this temperature for approximately 10 min. Cheeses are brine-salted for 48 h. Mild Pecorino Sardo is ripened for 20–60 days, weighs from 1 to 2.3 kg and has an aromatic and slightly acid flavour. The mature type, which is ripened for at least 4 months, acquires a consistent structure and a strong and piquant flavour during ripening. Its weight varies from 1.7 to 4 kg. Thermophilic lactic acid bacteria (LAB) (Lb. delbrueckii subsp. bulgaricus, Lb. helveticus and Sc. thermophilus) predominate in industrial Pecorino Sardo manufactured from thermized milk with natural starter cultures (Mannu et al., 2002). Enterococci and lactococci, the main microbial groups in farm-made raw-milk Pecorino Sardo (Mannu et al., 1999), show a high genetic diversity (Mannu and Paba, 2002). The volatile components of Pecorino Sardo were described by Larráyoz et al. (2001). Pecorino Siciliano is a hard cheese, cylindrical in shape, with plane or slightly concave faces and a weight of 4–12 kg. It is manufactured from ewes’ milk to which lamb rennet paste is added. The cheese surface is dry-salted twice in 10 days. Pecorino Siciliano is consumed at different stages of ripening, either fresh, dry-salted for 1 week or ripened for up to 4 months. The mature cheese has a pungent aroma and a characteristic sharp taste. The composition and physico-chemical properties of Pecorino Siciliano cheese were reported by Gattuso et al. (1995), and its microbiological characteristics by Giudici et al. (1997) and Migliorisi et al. (1997). Fiore Sardo is a hard cheese manufactured from raw ewes’ milk in Sardinia. Coagulation takes place at 35–37 °C in 20–30 min with lamb rennet paste. After 30 min, the coagulum is cut into rice-grain sized particles and left to settle. Cheeses are brined or dry-salted. After brining, the cheeses may also be slightly smoked and ripened for 6–12 months. Cheeses weigh around 1.5–4 kg. The flavour is flowery and fragrant, and the mature type is more piquant. Lc. lactis subsp. lactis predominates during most of the ripening period, but E. faecium and occasionally E. faecalis are the dominant species in 4-month-old

cheeses (Ledda, 1996). Lb. plantarum, Lb. casei and Lb. paracasei are the main mesophilic lactobacilli (Mannu et al., 2000). Cheeses produced from thermized milk and inoculated with Lc. lactis subsp. lactis and E. faecium had sensory characteristics similar to cheeses made from raw milk without starters (Ledda et al., 1994). Milk thermization did not influence secondary proteolysis, but resulted in changes in rheological characteristics (Pirisi et al., 1999a). Short- and medium-chain FFAs were significantly higher in rawmilk cheese than in cheese made from thermized milk, whereas long-chain FFAs, C18:1 and C18:2, were not affected by milk thermization (Pinna et al., 1999). Volatile components of 8-month-old Fiore Sardo were described by Larráyoz et al. (2001). Canestrato Pugliese is a hard cheese produced in Foggia and Bari. Its name derives from the reed baskets (canestri) used as moulds. It is cylindrical in shape, 25–34 cm in diameter, 10–14 cm high and weighs 7–14 kg. Milk is coagulated at 38–45 °C with animal rennet for 15–25 min and placed in moulds. Cheeses are dry- or brine-salted and ripened for 2–10 months. Its flavour is strong, salty and piquant. Ripening has been characterized by Santoro and Faccia (1998). Raw-milk Canestrato Pugliese cheese has higher values of water-soluble and pH 4.6-soluble N, total FAAs (40 mg/g), FFAs (1673 mg/kg) and a greater diversity of NSLAB than pasteurized milk cheese (Albenzio et al., 2001). Pecorino Toscano is a soft or semi-hard cheese produced mainly in Toscany. Ewes’ milk is usually inoculated with starter cultures and sometimes lipolytic enzymes are added. Coagulation with calf rennet takes place at 35–38 °C in 20–25 min. The curds are heated to 40–42 °C for 10–15 min. The cheeses are brine-salted for 24 h. The cheeses are cylindrical and weigh 1–3 kg. The minimum ripening period is 20 days for soft Pecorino Toscano and at least 4 months for the semi-hard variety. The taste is fragrant and slightly spicy. The technological and microbiological characteristics of pasteurized milk Pecorino Toscano cheese are described by Neviani et al. (1998). A wide diversity of LAB was described by Bizzarro et al. (2000) in raw and pasteurized milk Pecorino Toscano cheeses. Caciotta D’Urbino is a soft cheese manufactured from a mixture of 70–80% ewes’ milk and 20–30% cows’ milk. It is cylindrical in shape and weighs 0.8–1.5 kg. Milk is coagulated at 35–38 °C with liquid or powdered lamb rennet. Cheeses are usually drysalted and ripened for 20–30 days at 10–14 °C and 80–90% RH. The taste is sweet. Murazzano is a soft cheese variety manufactured from ewes’ milk or mixtures of ewes’ (60% minimum) and

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cows’ milk. Milk is coagulated at 37 °C with animal rennet. The cheese, cylindrical in shape, is dry-salted and ripened for 4–10 days. Its weight varies from 0.3 to 0.4 kg. The body is soft and finely grained. Ricotta is a well-known whey cheese. The whey, usually from Pecorino cheese manufacture, is heated to 80 °C. The curd rises to the surface and is collected in shallow conical baskets where it drains for 12–14 h (Coni et al., 1999). Portuguese ewe cheeses

Most Portuguese cheeses are manufactured following traditional methods, the majority from raw milk without addition of a starter culture. The best-known Portuguese cheeses are Serra da Estrela, Serpa, Azeitao and Castelo Branco. These varieties, together with Evora, Nisa and Terrincho, have PDO status. The main characteristic that distinguishes them from other ewes’ milk cheeses is their soft or semi-soft texture, due not only to the milk or the technology used, but also the highly proteolytic vegetable rennet used as coagulant. In 2001, ewes’ milk production in Portugal was 98 000 tonnes, and 16 602 tonnes of ewes’ milk cheeses were produced. Azeitao is a buttery, semi-soft cheese, with few or no holes, made near Lisbon. Coagulation of salted milk by vegetable rennet takes place at 30–35 °C. The cheese weighs 0.1–0.25 kg and is ripened in two stages. Initially, the cheese is held at room temperature for 10 days at 90–95% RH. During the second stage, the cheese is kept at 10–15 °C and 85–90% RH for 2–3 weeks (Freitas et al., 2000). Castelo Branco is a semi-soft variety manufactured in central western Portugal (Freitas et al., 2000). It is related to Serra da Estrela cheese, with a stronger and slightly piquant flavour and a more compact rind. The cheese is obtained by slowly draining the curd after milk coagulation by vegetable rennet at 20–30 °C. Cheeses, 1 kg in weight, are ripened for 40–50 days at 8–14 °C and 80–90% RH. Evora, a semi-hard to hard variety with a strong acidic and slightly piquant flavour is produced in the Alentejo region of southern Portugal from raw ewes’ milk. Cheeses, 0.06–0.3 kg in weight, are ripened for 30 days in the case of the semi-hard type and a minimum of 90 days for the hard type (Freitas et al., 2000). Nisa is a ripened cheese produced in the middle region of Alentejo. It is manufactured in two sizes, 0.2 and 1.3 kg, and has a semi-hard consistency, with a white-yellow colour and small holes. It is ripened for at least 45 days at 8–14 °C and has an intense and slightly acidic flavour (Freitas et al., 2000).

Serpa is a buttery, semi-soft cheese with few or no holes, manufactured in the region of Serpa from Merino ewes’ milk (Freitas et al., 2000). Cheeses are produced in four sizes, 0.2–2.5 kg in weight and have a strong, slightly hot and spicy flavour. Ripening takes place in two stages, at 6–10 °C and 95–100% RH and at 7–11 °C and 75–90% RH for 30–40 days. Serra da Estrela is the most traditional Portuguese cheese, produced in the Serra da Estrela mountains. It has a strong aroma, a slightly acidic flavour and a soft buttery texture with few eyes. Cheese is made twice daily from raw ewes’ milk coagulated at 27–30 °C for 1–2 h with vegetable rennet. The coagulum is cut manually and usually stirred by hand. The curds are poured into moulds and are slightly pressed, either by hand or mechanically. Cheeses are dry-salted and ripened for 30–45 days, at 6–12 °C and 85–90% RH. They have a flat cylindrical shape, a diameter of 15–20 cm and weigh 1–1.7 kg. Changes in the microbiological and physico-chemical characteristics of the cheese have been reported by Macedo et al. (1993). A lower level of primary proteolysis occurs in cheeses made from milk coagulated with vegetable rather than animal rennet (Sousa and Malcata, 1997). -Casein is less susceptible to proteolysis than s-casein, and animal rennet is more proteolytic on both fractions than vegetable rennet. Higher levels of water-soluble N were observed in cheeses made with vegetable rennet, although levels of TCA- and PTAsoluble N were lower. Different patterns of peptides were detected at all stages of ripening (Sousa and Malcata, 1998). The main volatile compounds in Serra da Estrela cheese are derived from the degradation of sugars, FAAs and glycerides (Dahl et al., 2000). The short-chain carboxylic acids detected were acetic, propionic, iso-butyric and iso-valeric acids. High levels of 2,3-butanediol were attributed to the activity of spoilage bacteria, and high concentrations of ethyl octanoate and ethyl decanoate to yeast activity. Volatile short-chain fatty acids increased throughout ripening, with maximum levels of octanoic and decanoic acids detected at 150 days. The profile of volatiles in cheese was more complex if refrigerated milk was used instead of fresh milk, with higher concentrations of ethyl esters. Lactic acid bacteria and Enterobacteriaceae are the predominant microbial groups in Serra da Estrela cheese (Macedo et al., 1995; Tavaria and Malcata, 1998). Numbers of Enterobacteriaceae decrease whereas those of lactobacilli, lactococci and enteroccoci increase throughout ripening. Yeasts and staphylococci were 101–105 cfu/g and 104–107 cfu/g, respectively. Lc. lactis subsp. lactis, Lb. paracasei subsp.

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paracasei, Leuc. lactis, Leuc. mesenteroides and Lb. plantarum were the most frequently isolated LAB. A broad spectrum of yeasts are found in Serra da Estrela cheese. Terrincho is a semi-hard variety similar to other Portuguese ewes’ milk cheeses, except that milk is coagulated with animal rennet (Freitas et al., 2000). Milk is coagulated at 35 °C and the coagulum is cut to rice-grain size. Cheeses are pressed manually, drysalted and ripened at 5–12 °C and 80–85% RH for 30 days. Terrincho cheese weighs 0.8–1.2 kg and has a slightly oily texture with small eyes. Spanish ewe cheeses

Production of ewes’ milk in Spain was 306 000 tonnes in 2001. Six PDO ewes’ milk cheeses are made: Manchego (6122 tonnes in 2000), Roncal (380 tonnes), Idiazábal (1131 tonnes) and Zamorano (448 tonnes) are hard, uncooked cheeses; La Serena (186 tonnes) and Torta del Casar are semi-hard cheeses manufactured from milk coagulated with vegetable rennet. Manchego cheese is made in La Mancha (central Spain) from raw or pasteurized milk of Manchega ewes. Cheeses weigh 2.5–3.5 kg and are cylindrical in shape. The rind is imprinted with a design recalling the woven esparto grass bands used traditionally as moulds. Rawmilk cheese is manufactured with or without addition of starter cultures, whereas mesophilic lactic cultures are used in the production of pasteurized milk cheese. Coagulation by animal rennet takes place at 30–32 °C in 30–40 min. The coagulum is cut into 4–6 mm cubes, and scalded at 36–38 °C for 15 min while stirring. Cheeses, 20–22 cm in diameter and 8–10 cm high, are pressed for 6–18 h, brine-salted for 24–48 h and held at 10–15 °C for at least 2 months. Occasionally, Manchego cheese is immersed in olive oil during the later stages of ripening. Minimum pH (4.9–5.0) is generally reached in raw-milk Manchego cheese within 24 h of manufacture, after which the pH increases to 5.5–5.7 on day 90. In pasteurized milk Manchego cheese, the pH decreases to 5.1–5.3 during the first week and subsequently rises to 5.3–5.5 after 90 days. Dry matter is 40–50% in fresh curd and increases to 60–65% in 90day-old cheese. The salt-in-moisture is 5–7% on day 90. Greater degradation of s-casein than -casein occurs during ripening. Mean levels for pH 4.6-soluble N, TCA-soluble N and PTA-soluble N in 4-month-old raw-milk cheese were 23.6, 16.9 and 10.4% of total N, respectively, whereas levels of 21.1, 14.7 and 7.1% were found in pasteurized milk cheese (Gaya et al., 1990). Free amino acids accumulated during the 11-month ripening period (Ordóñez and Burgos, 1980).

However, other workers (Marcos and Mora, 1982) reported a maximum level of FAAs after 4 months followed by a subsequent decrease. Lipolysis occurs only to a limited degree in Manchego cheese (Ramos and Martínez-Castro, 1976). Lower levels of FFAs are found in pasteurized milk than in raw-milk cheese, due to the inactivation of indigenous milk lipase by pasteurization (Gaya et al., 1990). The majority of volatile compounds are more abundant in raw-milk Manchego cheese than in pasteurized milk cheese. Alcohols and esters predominate in raw-milk cheese, while methyl ketones and 2,3-butanedione (diacetyl) are quantitatively important in pasteurized milk cheese. Branched-chain alcohols were much more abundant in the raw-milk cheese. Aroma intensity is correlated with esters, branched-chain aldehydes and branched-chain alcohols in raw-milk cheese, and with esters, branched-chain aldehydes, 2-methyl ketones and 2-alkanols in pasteurized milk cheese. Diacetyl was positively correlated with the aroma attribute ‘toasted’ and negatively correlated with aroma quality in pasteurized milk cheeses (Fernández-García et al., 2002). Lower concentrations of residual caseins were observed in pasteurized milk cheese made, with a commercial mixed-strain culture, than in pasteurized milk cheese made with a defined-strain culture made up of Lactococcus isolates from raw-milk Manchego cheese, or in raw-milk cheese made with any of the two cultures. The use of a commercial mixed-strain culture or a defined-strain culture did not affect flavour quality and intensity of raw- or pasteurized-milk Manchego cheeses (Gómez et al., 1999). Acceleration of the ripening of Manchego cheese by ripening at an elevated temperature (Gaya et al., 1990), addition of different enzymes to milk in liquid or solid form (Nuñez et al., 1991b; Fernández-García et al., 1994) or encapsulation in liposomes (Picón et al., 1994, 1995, 1996) has been investigated, with increases in proteolysis and reduction of ripening period without impairment of cheese flavour. Flavour quality was improved and flavour intensity enhanced by the cysteine proteinase from Micrococcus INIA 528 (Mohedano et al., 1998). Increased proteolysis enhanced the formation of volatile compounds derived from amino acids, such as acetaldehyde, 2-methyl-2-butanal and 3-methyl-1-butanal (Mariaca et al., 2001). The microbial flora of Manchego cheese made from raw ewes’ milk is well known (Nuñez et al., 1989). Lactococci, mainly Lc. lactis subsp. lactis, predominate during the first month of ripening. Thereafter, they are outnumbered by lactobacilli, mainly Lb. plantarum and Lb. casei. Enterococci, mainly E. faecium, leuconostocs, mainly Leuc. mesenteroides subsp. dextranicum

286 Cheeses Made from Ewes’ and Goats’ Milk

and Leuc. paramesenteroides, and pediococci, mainly Pediococcus pentosaceus, are found at lower numbers during ripening. Micrococci are commonly found in curd and 1-day-old cheese, and then decrease gradually during ripening. In pasteurized-milk Manchego cheese, starter lactococci predominate during the first month, but lactobacilli may reach 108 cfu/g in 30-day-old cheese. Idiazábal is a semi-hard or hard variety produced in the Basque country and Navarra from raw milk of Latxa ewes. Industrial production based on traditional methods and traditional production by small artisanal producers co-exist. Homofermentative starter cultures may be added to the milk before coagulation. Raw milk is coagulated with commercial bovine rennet or rennet paste at 30 °C. The coagulum is cut into 5–10-mm grains, stirred and heated to 37 °C. The curds are pressed in the vat, placed into moulds, pressed for 6 h and dry-salted or brined for 24–48 h. Cheeses are ripened at 10 °C and 80% RH for 2–12 months. Smoking of cheeses during the third month of ripening by combustion of Alnus glutinosa wood for 24 h at 15 °C is optional. Mean values of pH 4.6- and TCA-soluble N in Idiazábal cheeses were 11.5 and 14.4% of total N, respectively, indicating low levels of proteolysis. Glu, Leu, Val, Lys and Phe were the major FAAs detected (Ordóñez et al., 1998). Butanoic acid was the main FFA, accounting for 33% of the total over the ripening period. The other common fatty acids (C6:0–C18:1) were also present (Nájera et al., 1994). Differences in the amounts of short-chain FFAs were detected between winter and summer cheeses (Chávarri et al., 1999). Effects of brining time and smoking on the microbiological (Pérez-Elortondo et al., 1993), physicochemical (Ibáñez et al., 1995), FFA profile (Nájera et al., 1994) and sensory characteristics (PérezElortondo et al., 2002) of Idiazábal cheese have been investigated. Smoking caused a decrease in the numbers of LAB and Micrococcaceae (Pérez-Elortondo et al., 1999a). Smoking lowered the aw and increased the pH, proteolysis and release of amino acids. Brining and smoking led to increases in FFAs (Nájera et al., 1994). However, butanoic acid levels were lower in the smoked than in unsmoked cheeses. Brining had little effect on the sensory properties of Idiazábal cheese and smoking had a positive influence on the acceptability of rind, colour of the interior, aroma and texture. Cheeses made with rennet paste had higher contents of total and short-chain FFAs (Bustamante et al., 2000). Changes in LAB, Micrococcaceae, Enterobacteriaceae and psychrotrophic bacteria during ripening were

studied by Pérez-Elortondo et al. (1998, 1999b), with citrate-utilizing (Cit) strains of Lc. lactis and Lb. casei subsp. casei as the dominant LAB throughout ripening. Idiazábal cheese made from raw ewes’ milk with the addition of a mesophilic starter culture had higher numbers of LAB during pressing, although no differences were observed during ripening. Flavour was typical of ripened ewes’ milk cheese. Starter cultures also increased the levels of FAAs (Vicente et al., 2001), which reached higher levels in raw-milk cheese made with or without starter culture than in pasteurized milk cheese (Mendia et al., 2000). Roncal is a hard cheese manufactured in Navarra from raw ewes’ milk. Its production is similar to that of Manchego, except for a higher renneting temperature (32–37 °C) and a smaller size (1.8–2.0 kg) and a ripening time of at least 4 months. The composition, biochemical and microbiological characteristics of Roncal cheese have been published (Ordóñez et al., 1980; Millán et al., 1992; Arizcun et al., 1997). The use of commercial calf rennet and aqueous extracts of dried, sliced and salted lambs’ stomachs in Roncal cheese manufacture has been compared (Irigoyen et al., 2002). Physico-chemical parameters were not affected by the type of rennet, whereas cheese manufactured with lamb rennet showed higher levels of pH 4.6- and TCA-soluble-N and greater proteolytic activity on - but mainly on s1-casein. These changes did not result in any sensory differences. Alcohols are the predominant volatile compounds in 4-month-old Roncal cheese, with ethanol, 2-butanol and 1-propanol at high concentrations. Ketones were the next most prominent group, most of them being methyl ketones. 3-Hydroxybutan-2-one (acetoin) was present at high levels and influenced Roncal cheese aroma. Acetic and butanoic acids were the most abundant acids and ethyl esters were the predominant esters (Izco and Torre, 2000; Larráyoz et al., 2001). Lc. lactis subsp. lactis, Lb. casei, Lb. plantarum, Leuc. mesenteroides subsp. mesenteroides and Leuc. mesenteroides subsp. dextranicum were the predominant LAB in Roncal cheese (Arizcun et al., 1997). Starter cultures added to milk affected cheese flavour, with greater intensity for refreshing, astringent and sweet attributes and lower scores on bitterness. More homogeneous texture and higher elasticity were also observed in cheeses made with starter cultures (Ortigosa et al., 1999). Zamorano is a hard cheese produced in Zamora from raw milk of Churra and Castellana ewes. Milk is coagulated with rennet extract at 28–32 °C for 30–45 min. The coagulum is cut by hand or mechanically into small pieces of 5–10 mm and the curds are heated to 38–40 °C before moulding and pressing. Cheeses are dry-salted or

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brined for 36 h and ripened for at least 100 days. Zamorano cheese has a firm texture and a dark grey rind, a cylindrical shape, 8–12 cm high and 18–22 cm in diameter, and weighs 3.5–4 kg. Castellano cheese, which is not protected by a PDO, is another ewes’ milk hard variety made with similar technology (Román-Blanco et al., 1999). La Serena is a semi-hard variety made from Merino ewes’ milk in La Serena (Southern Extremadura). Raw milk at 25–32 °C is coagulated in 50–75 min with vegetable rennet prepared by soaking dried flowers of C. cardunculus in tap water at room temperature for 24 h. The coagulum is cut into pieces of 10–20 mm in size and stirred for 2–3 min without scalding. After 10 min, the whey is removed. In traditional processing, the curds are transferred to woven esparto moulds where they are pressed by hand. Moulds are turned repeatedly for 1–2 h to assist whey drainage and the cheeses are dry-salted for 10–24 h or brined for 24 h. In modern dairies, plastic moulds, mechanical pressing and brine salting are used. Cheeses are ripened for at least 60 days at 8–12 °C and 85–95% RH. La Serena is a flat round cheese, 18–20 cm in diameter and 4–8 cm high, which weighs 0.8–1.5 kg. The rind is brownish in colour, with convex sides due to the softening of the cheese texture during ripening by the proteolytic action of the vegetable rennet. The cheese has a creamy to semi-hard texture with small eyes and a pronounced, sometimes slightly bitter, flavour. Chemical changes, pH and moisture content during the ripening of artisanal La Serena cheese were described by Fernández del Pozo et al. (1988b). In fully ripened cheese, pH 4.6-soluble N was 38.5% of total N, and TCA-soluble N 10.3–14.6% (Fernández-Salguero et al., 1978; Fernández del Pozo et al., 1988b). Degradation of s-casein was faster than that of -casein. During the first month of ripening the texture of the cheese softens, due to hydrolysis of the casein, developing a more consistent body gradually as ripening progressed (Fernández del Pozo et al., 1988b). Levels of FFAs increase gradually during ripening. Cheeses produced with animal rennet had a higher moisture content and a lower pH than cheeses made with vegetable rennet (Nuñez et al., 1991a). Proteolysis was more rapid in cheeses made with vegetable rennet, but there was less lipolysis. Softening of cheese texture was more pronounced in cheeses made with vegetable rennet, which received higher scores for flavour quality and intensity. Lc. lactis, atypical lactococci, Lb. casei, Lb. plantarum, Leuc. mesenteroides and E. faecium predominated in the interior of La Serena cheese (Fernández del Pozo et al., 1988a). High yeast and mould counts

were found on the cheese surface from 30 days onwards. These were mainly lactic acid-utilizing species. Cheese flavour quality and intensity were significantly impaired in cheeses made with a strain of Lc. lactis subsp. lactis as a starter culture; the cheese had a firmer texture, which was attributed to retarded proteolysis and a lower pH (Medina et al., 1991). Alcohols were the major volatile compounds, with ethanol, propanol, 2-propenol, 2-propanol, 2-butanol, 2-pentanol and branched-chain 2-methyl propanol and 3-methyl butanol being the most abundant (Carbonell et al., 2002). Ethyl esters of acetic, butanoic, hexanoic and octanoic acids and 3-methyl-1-butanol acetate were the main esters. The concentrations of most esters increased dramatically during ripening and, because of their low perception thresholds, may be considered as key constituents of the aroma of this cheese variety. Torta del Casar is a semi-hard cheese made in Cáceres (northern Extremadura) from raw milk of Merino ewes, which is coagulated with vegetable rennet at 25–30 °C. Manufacturing and ripening conditions are very similar to those for La Serena cheese. Torta del Casar has a PDO status since 2002. Chemical composition of cheese ripened for 60 days is 53.6% DM, 26.1% fat, 24.3% protein, 34.1% pH 4.6-soluble N and 13.0% TCA-soluble N as percentages of total N, and pH 5.21 (Mas Mayoral et al., 1991). The microbiology of Torta del Casar has been investigated (Poullet et al., 1991). Maximum counts of lactococci, lactobacilli, leuconostocs and enterococci are reached during the first 15 days of ripening. Coliform counts are 105–106 cfu/g during the first month and decrease to 102–104 cfu/g on day 60, whereas numbers of coagulasepositive staphylococci are 102–103 cfu/g during the first month and decrease to 10 cfu/g on day 60. The predominant LAB species during ripening were Lc. lactis subsp. lactis, Leuc. mesenteroides subsp. dextranicum, Leuc. mesenteroides subsp. mesenteroides, Lb. curvatus, Lb. plantarum and E. faecalis (Poullet et al., 1993). Another non-PDO Spanish ewes’ milk cheese also made with vegetable rennet is Los Pedroches (Fernández-Salguero and Sanjuán, 1999).

Goats’ Milk Cheeses Technology and flavour of goats’ milk cheeses

Goats’ milk production in the European Union was 1 443 782 tonnes in 2001. Except for minor local production of goats’ milk yoghurt or pasteurized goats’ milk, all the milk is transformed into cheese, alone or mixed with cows’ and/or ewes’ milks. The textural characteristics of goats’ milk curd are distinct from those of cows’ milk curd produced under

288 Cheeses Made from Ewes’ and Goats’ Milk

the same conditions. A coagulum strength of 15.0–18.5 g was reported for goats’ milk with a casein content of 29.5–30.8 g/kg versus a coagulum strength of 18.2–45.8 g for cows’ milk with a casein content of 23.8–24.7 g/kg (Storry et al., 1983). The weaker mechanical properties of goats’ milk curd constrain the manufacturing procedures used in goats’ milk cheesemaking and limit the diversity of cheese types. Although there is some local production of goats’ Blue cheeses, most cheeses made from goats’ milk fall within the following groups: • Fresh or white unripened cheeses, with a low DM content, usually less than 25%. • Soft cheeses, traditionally made from predominantly lactic curd, of small size, cylindrical or pyramidal in shape, and generally with mould growth or ash on the surface. • Semi-hard or hard cheeses, made from predominantly rennet-coagulated curd, of larger size than the soft cheeses, flat cylindrical shape (wheel) and dry rind. The composition of goats’ milk casein is the main factor responsible for its technological limitations. Caprine casein contains a lower proportion of s-casein, especially s1-casein, and a higher proportion of -casein than bovine casein. The polymorphism of caprine s1-casein plays a role in these compositional differences. The A, B and C alleles are associated with high levels of s1-casein synthesis, while the E allele is with a medium level, the D and F alleles with a low level and the O allele with a null level of s1-casein synthesis (Grosclaude et al., 1994). Cheesemaking from goats’ milk with a low s1-casein content results in a less firm curd (Ambrosoli et al., 1988) and lower protein retention and cheese yield (Pirisi et al., 1994; Vassal et al., 1994) than when milk of a high s1-casein content is used. On the other hand, cheese made from A allele milk with high s1-casein content has a weaker goaty flavour than those from E or F allele milks, due to lower production of aroma compounds or due to the firmer texture of the A allele curd, which impairs the release and perception of volatile aroma compounds (Vassal et al., 1994). Pierre et al. (1998) found a weaker flavour in cheese made from A allele milk than in cheese from O allele milk, which was attributed to 50% lower levels of hexanoic, octanoic, nonanoic, decanoic, 4-methyl octanoic and 4-ethyl octanoic acids in the former cheese. The characteristic flavour of goats’ milk cheeses is mostly due to volatile compounds. For a long time octanoic acid was considered to be the main ‘goaty’ compound in dairy products but, according to Ha and

Lindsay (1991a), its aroma and flavour lack the highly characteristic goatiness found in goats’ milk cheese. Instead, 4-ethyl-octanoic acid was found by systematic assessment of the aroma of individual FA to be principally responsible for the goaty-type aromatic notes in goat-milk cheese (Ha and Lindsay, 1991a). Among medium-size FAs, the lowest perception threshold in water and in oil is that of 4-ethyl-octanoic acid (Brennand et al., 1989). Seven volatile compounds present in goat cheeses (hexanoic, octanoic, nonanoic, decanoic, 4-methyl-octanoic, 4-ethyl-octanoic, plus an unidentified compound co-eluting with decanoic acid) were characterized by olfactometry as having a specific goat cheese aroma (Le Quéré et al., 1998). Salles et al. (2002) also concluded that 4-ethyl-octanoic acid is the most potent medium-chain FFA in typical goat cheese flavour. The concentration of 4-ethyl-octanoic acid in goat-milk cheese is 0.1 mg/kg versus 0.8 mg/kg for 4-methyl-octanoic acid and higher values for the other medium-chain FFAs. However, the threshold value for perception of 4-ethyl-octanoic acid in a cheese model was 0.0039 mg/kg versus 0.322 mg/kg for 4-methyloctanoic acid and considerably higher values for other medium-chain FFAs. Technological treatments influence the typical flavour of goats’ milk (Morgan and Gaborit, 2001). Thus, skimmed goats’ milk had a very mild flavour (0.78 points on a 10-point scale) compared with full-fat raw milk (4.14 points on the same scale). Heating milk at 65 °C for 1 min led to a slight reduction in goaty flavour (3.06 points versus 4.06 points for control raw milk), accompanied by a decrease in the level of lipolysis. On the other hand, cold storage (4.68 points for milk stored 3 days at 6 °C versus 2.96 points for control milk) and homogenization (4.90 points versus 4.06 points for control milk) of milk increased goat flavour. The total content of FFAs was 40 g/ml fat in control milk, and 100 or 120 g/ml fat after cold storage or homogenization, respectively. Fresh cheeses made from goats’ milk with high levels of lipolysis showed high levels of lipolysis and an increase in organoleptic defects (Morgan et al., 2001). However, no relationship was found between the level of lipolysis and the sensory characteristics of the ripened cheeses. The water-soluble extract of goats’ milk cheese had an umami taste, although it was also regarded as salty, astringent and bitter, whereas the water-soluble extract from cows’ milk cheese was described as showing mainly salty, umami and sour tastes, while that of ewes’ milk cheese exhibited a strong umami taste, followed by a salty taste (Molina et al., 1999). Small peptides and FAAs in fractions of water-soluble extracts of goat cheeses had no direct impact on their taste, which was

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influenced mainly by mineral salts and lactic acid (Salles et al., 2000). Also, when small peptides (MW 1000 Da) or FAAs from the water-soluble extract of goat cheese were evaluated sensorially by incorporation or omission in a cheese model, no effects of those compounds on the taste descriptors, salty, bitter, sour, umami or astringent, were found (Salles et al., 2002). Saltiness of the water-soluble extract of goat cheese was explained by an additive contribution of sodium, potassium, calcium and magnesium ions, whereas sourness was mainly due to the synergistic effect of sodium chloride, phosphates and lactic acid, and bitterness to calcium and magnesium chlorides, partially masked by sodium chloride (Engel et al., 2000). The hydrophobic peptides present in the water-soluble extract of goat-milk cheese were not perceived as being bitter by panellists, probably due to their low concentrations (Sommerer et al., 2001). French goat cheeses

France produces more than 50 goats’ cheese varieties. Goats’ milk production was 460 000 tonnes in 2001. Total annual production of goat cheeses is close to 75 000 tonnes, of which 23% are farm-made cheeses. The nine varieties produced under a PDO are Chabichou du Poitou, Crottin de Chavignol, Picodon de la Drôme or Picodon de l’Ardèche, Pélardon, Pouligny Saint-Pierre, Rocamadour, Sainte-Maure de Touraine, Selles-sur-Cher and Valençay, which account for a combined production of over 5000 tonnes per year. Detailed cheesemaking procedures for French goat cheeses have been reviewed by Le Jaouen and Mouillot (1990). Most French goat cheeses can be made from pasteurized or raw milks, but some PDO varieties, such as Pouligny Saint-Pierre, Rocamadour and Valençay must be made exclusively from raw milk. Many French goat-milk cheeses are soft varieties. In their manufacture, milk coagulation is due mostly to lactic acid production by the starter, with small doses of rennet, a coagulation temperature of 18–24 °C, and clotting times ranging from 16 to 48 h. One exception of considerable economic importance is Camemberttype goat cheese, made in the Poitou region. Pasteurized milk heated at 32–33 °C undergoes a lactic-rennet type coagulation, with a rennet dose 2–3 times higher than that for soft cheeses, and a coagulation time of 45–50 min. Other goat cheeses with a lactic-rennet type coagulation are Blue des Aravis and artisanal Chevrotin, both traditionally made in the Alps. Some general microbiological studies have been carried out on varieties such as Crottin de Chavignol (Hosono and Shirota, 1994), Valençay (Hosono and Sawada, 1995) and Sainte-Maure (Masuda et al., 2000).

Nahabieh and Schmidt (1990) found that D. hansenii was the dominant yeast species in the main goat cheeses. However, detailed scientific and technical information on the microbiota of most French goat-milk cheeses is relatively scarce. The microbiology of a Camembert-type goat cheese made from raw milk was investigated by Sablé et al. (1997b), who found similarities between its microbiota and the microbiota of Camembert cheese made from raw cows’ milk (Lenoir, 1963). Lc. lactis was the dominant Gram-positive species, Hafnia alvei the dominant Gram-negative species and Staphylococcus the dominant salt-tolerant micro-organism throughout ripening. Chemical changes during ripening (31 days at 13 °C and 90% RH) of raw-milk Sainte Maure-type goat cheese have been investigated by Le Quéré et al. (1998). Cheese DM increased from 32.7% on day 2 to 53.6% on day 31. Cheese pH rose from 4.4 on day 2 to 5.1 on day 31 at the surface, but only from 4.3 on day 2 to 4.4 on day 31 in the centre, giving rise to a pH gradient. Proteolysis was slight, with 92% of the casein intact on day 2 and 87% on day 31, with none of the individual caseins being preferentially hydrolysed. Small peptides increased from 5% of total N on day 2 to 8.5% on day 31. This pattern of casein breakdown corresponded to the proteolytic activity of Geotrichum candidum, the surface mould. FFAs accounted for 1% of total FFAs on day 2, rose to 2% on day 18, and afterwards increased more rapidly, up to 6% on day 31. Lipolysis apparently occurred in a two-step process. A specific rise of saturated C6-C10 FFAs was observed in 2-day-old cheeses, which was attributed to the lipoprotein lipase of goat milk or the lipolytic activity of somatic cells. Afterwards, an increase in C18:1, and to a lesser extent in C16:1 and C18:2, was observed. Unsaturated FFAs were 35.7% of FFAs, and this specific activity was attributed to the Geotrichum lipase. The typical goat cheese aroma was attributed to mediumchain linear and branched-chain FFAs. From day 2 to day 31, 4-ethyl-octanoic acid increased from 0.001 to 0.027 mg/kg, 4-methyl-octanoic acid from 0.012 to 0.139 mg/kg, hexanoic acid from 2.77 to 23.38 mg/kg, octanoic acid from 7.45 to 32.96 mg/kg and decanoic acid from 3.04 to 19.87 mg/kg. Those increases in FFAs might explain why fresh goat cheeses when submitted to sensory analysis are perceived as different from ripened goat cheeses. Fifty-one volatile compounds were identified and quantified during the ripening of Camembert-type goat cheese (Sablé et al., 1997a). Ethanol, 3-methyl butanol, 2-methyl butanol, ethyl acetate and 2-methyl propanol were the most abundant volatile compounds in 2-day-old cheese, whereas the major compounds in ripe cheese were ethanol, 2-heptanone, 2-nonanone,

290 Cheeses Made from Ewes’ and Goats’ Milk

2-heptanol, 3-methyl butanal, acetaldehyde, 2-propanol and acetone. The volatile profile of the cheese resembled that of Camembert cheese made from cows’ milk and other surface mould-ripened cheeses (Molimard and Spinnler, 1996), but compounds such as limonene, 3-hexanone, 4-heptanone, 3-methyl 2-heptanone, 4-nonanone, 2-methyl butanol and acetaldehyde were considered by the authors to be characteristic of goat cheese. Yeast and mould strains used for ripening of French goat-milk cheeses have a remarkable influence on the flavour of both soft cheeses and Camembert-type cheeses (Gaborit et al., 2001). In soft cheeses, the use of G. candidum GC1, alone or associated with D. hansenii DH1 or Rhodosporidium infirmominiatum RI, resulted in the lowest levels of lipolysis and the highest scores for goat odour and goat flavour intensity. Goat flavour intensity increased when GC1 was used in conjunction with DH1 or RI. The use of Penicillium candidum PC1, alone or associated with DH1 or RI, was detrimental to the sensorial quality of the cheeses. In Camembert-type cheeses, lipolysis and scores obtained for most descriptors were lower than those found for soft cheeses. Lipolysis levels were higher for cheeses made using STRAIN GC1 with STRAINS PC1, or PC1 and DH1. The best sensorial quality cheeses were made with STRAIN GC1. Greek goat cheeses

Goats’ milk production in Greece was 450 000 tonnes in 2001. There are no PDO cheeses in Greece made exclusively from goats’ milk, which is generally added to ewes’ milk for the production of ewes’ milk cheese varieties. Feta cheese made from 100% goats’ milk, mixtures of goats’ and ewes’ milks, or 100% ewes’ milk have been compared (Mallatou et al., 1994). No clear relationship between milk used for manufacture and proteolysis was found, but significantly higher levels of lipolysis were found in cheese made from 100 or 75% goats’ milk. The microbiology of white-brined cheese made from raw goats’ milk has been studied (Litopoulou-Tzanetaki and Tzanetakis, 1992). Most microbial groups were at their highest levels in 15-day-old cheese, when the pH was 5.45. Coliforms, staphylococci and LAB declined by 4.0, 3.0 and 1.5 log cycles, respectively, from day 15 to day 90, when the pH was 4.50; in contrast, yeasts increased by 1.8 log cycles in the meantime and enterococci did not vary. Specific starters have been designed for the manufacture of white-brined cheese from pasteurized goats’ milk (Tzanetakis et al., 1995). Anevato cheese is made from raw goats’ milk which undergoes a lactic-rennet coagulation at 18–20 °C for 12 h. The coagulum is cut, left to raise to the surface of the whey for 4–5 h and drained in cheese cloths for

24 h. Salt is added and, after thorough mixing, the curds are packed in plastic containers which are held at 4 °C. In Anevato cheese, the highest counts of most microbial groups were reached in curd after draining, when the pH was 4.28 (Hatzikamari et al., 1999). During cold storage for 60 days, the pH of the cheese did not change and counts of most microbial groups declined, but yeast counts increased by 1.2 log units. Anevato cheeses made from raw, pasteurized or thermized goats’ milk inoculated with 0.5% of a Lc. lactis subsp. lactis culture were compared (Xanthopoulos et al., 2000). All microbial groups, except yeasts, were present at higher levels in raw-milk curd and declined during cold storage. Different patterns of hydrolysis of s1- and -caseins during cheese ripening were recorded, with reduced proteolysis in cheeses made from pasteurized milk. Flavour scores were higher for raw-milk cheeses, but aroma and texture were not influenced by heat treatment of the milk. Italian goat cheeses

Production of goats’ milk in Italy was 140 000 tonnes in 2001. There are no PDO goat cheeses in Italy, although some PDO cheeses (Bitto, Bra, Castelmagno, Raschera, Valle d’Aosta Fromadzo) are made from cows’ milk to which small amounts of goats’ milk are added. In the manufacture of PDO Robiola di Roccaverano cheese, a minimum of 15% goats’ and/or ewes’ milk must be included together with cows’ milk, although Robiola ‘classica’ cheese is exclusively made from goats’ milk. Technological aspects of Italian goatmilk cheeses have been reviewed (Emaldi, 1987). Studies on the microbiology and the biochemistry of Italian goat cheeses are scarce. Caprino tradizionale is a soft cheese made from raw goats’ milk, to which a whey culture is added. Coagulation takes place after 18–24 h at 20–22 °C. The microbiology of this cheese variety has been studied, with maximum microbial counts in fresh curd, which had a mean pH of 4.35 (Foschino et al., 1999). After 10 days at 4 °C, the pH had declined to 4.32, and counts of lactococci, enterococci, S. aureus, coliforms and yeasts had decreased by 0.7, 3.7, 2.9, 5.3 and 1.5 log cycles, respectively. Bastelicaccia is a soft cheese made in Corsica by coagulating goats’ or ewes’ milks at 24–28 °C for 60–90 min, cutting the coagulum to rice-grain size, draining part of the whey, stirring the curds, draining the rest of the whey, dry-salting the curds and ripening for 30 days. Ripe goats’ milk cheese had a pH of 5.5–5.7, a high population of leuconostocs (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 (Casalta et al., 2001).

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Cacioricotta cheese is made traditionally by heating goats’ milk at 95 °C, cooling it to 40 °C 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 Rabaçal 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 °C with animal rennet. The coagulum is cut manually into irregular pieces and pressed by hand. Cheeses (flat cylinders) are dry-salted and ripened at 5–18 °C 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). Rabaçal 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 °C with animal rennet in

45–60 min and the coagulum is cut by hand to irregular grains. Cheeses (flat cylinders) are pressed manually, dry-salted and ripened at 10–15 °C and 70–85% 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 °C is coagulated with animal rennet in 40–50 min. The coagulum is cut into 1–1.5 cm cubes and pressed by hand. Cheeses (flat cylinders) are dry-salted, stacked in groups of two or three and turned frequently. Ripening takes place at 10–18 °C 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 s- 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 of ⬃20 non-PDO traditional cheese varieties, or new varieties such as Ibérico 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

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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 °C 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 °C and a low RH. The surface is rubbed with oil, paprika or both during ripening. The shape is flat 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 s- and -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 (Martín-Hernández et al., 1992). Residual s- and -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 °C in 30–60 min, of flat 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 (Zárate 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 °C in 60–90 min, generally with animal rennet. The coagulum is cut to medium-size (1–2 cm) grains. Cheeses of flat 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 González 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 Aragón, 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 °C in 30–60 min, the coagulum is cut and stirred, and cheeses are pressed for 2–4 h. After brinesalting, cheeses (flat cylinders weighing 0.3–1.5 kg) are held at 4 °C. For ripened cheese manufacture, milk is coagulated at 32–33 °C in 45–60 min with animal rennet. The coagulum is cut, stirred and heated to 35–37 °C. Cheeses (flat cylinders weighing 1–2 kg) are pressed, brine-salted for 12 h and ripened at 12–14 °C 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 Tiétar or La Vera, is farm-made from raw milk, coagulated with animal rennet at 25–30 °C 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 °C 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 °C. 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 s- and -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 °C 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.

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Cheeses are dry-salted, and after 5 days are covered with oak ash. Ripening takes place at 10–15 °C 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, s-casein is almost completely degraded, but 50% -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 °C with animal rennet in 105–120 min. The coagulum is cut to 1-cm-size grains, moulded and pressed for 12 h. Cheeses, flat cylinders weighing 0.8–1.2 kg are dry-salted and ripened at 10–15 °C 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 °C 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 °C 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 s- and -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 °C 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 °C 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

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 et al., 2000). Recently, extensive studies on the effects of highpressure treatment on the microbiological (Capellas et al., 1996; Buffa et al., 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.

Conclusions 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.

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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 Universität Berlin, Faculty of Process Sciences, Department of Food Rheology, Berlin, Germany

Introduction 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 fresh 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 °C. 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 °C (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 material – 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 Käseverordnung (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 Frischkäse (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 à la crème 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

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302 Quark, Cream Cheese and Related Varieties

Acid/rennet- and acid-curd cheese varieties

Fresh cheeses

Standard varieties Paste-like consistency

Ripened acid-curd cheeses

Other varieties Mainly acid coagulated

Quark and quark-related varieties

Cottage cheese

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

Acid-heat coagulated

Harzer Mainzer Olmuetzer Quargel Topfkaese

Ricotta, Ricottone Mascarpone Queso Blanco, Queso Fresco

Gel-like consistency Layered white cheese (Schichtkaese) Figure 1 Fresh cheese varieties.

1990 to 3 662 000 tonnes in 1999). In 1999, 32% of the total cheese production was fresh cheese, compared to 30% 10 years ago (Sørensen, 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 EU – 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; Sørensen, 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 Cheese and Related Varieties 303

Table 1 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

1.8

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 11.3–18

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; Fernández-Albalat et al., 2001.

Table 2 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

18.0b 12.0c 10 1.8

19.0 11.3 10.0 1.9

20.0 10.5 20.0 4.0

22.0 9.7 30.0 6.6

24.0 8.7 40.0 9.6

25.0 8.2 45.0 11.3

27.0 8.0 50.0 13.5

30.0 6.8 60–maximum 87 18.0–26.1

German Fresh Cream cheesed (Rahmfrischkäse) Dry matter (%) Fat in dry matter (%) Fat, absolute (%) German Fresh Double Cream cheesed (Doppelrahmfrischkäse) Dry matter (%) Fat in dry matter (%) Fat, absolute (%) a Layered cheese (Schichtkäse) is defined as quarter-fat cheese. b Products with DM 18% are to be labelled as Fresh cheese (Frischkäse). c German Quark: whey protein must not exceed 18.5% of total protein. d Compositional specification varies greatly with country. Data from Käseverordnung (German Cheese regulations, Anon, 1986).

39.0 50.0 19.5 44.0 60–maximum 87 26.4–38.3

304 Quark, Cream Cheese and Related Varieties

Table 3 Annual Fresh Cheese Production and Consumption (Approximate values based on data available for period 1990–1999)

Region/Country Europe Germany France Poland Italy Czech and Slovak Republics Russia Spain UK Denmark Austria Hungary Belgium Greece Finland The Netherlands Switzerland Ireland Iceland Norway Sweden Estonia Asia Israel

Major types a

Mainly Quark

Production 1999 (’000 tonnes)

Per capita consumption 1999 (kg/head)

637in 1991 465 n.a. 307in 1992 56in 1992

748 558 264 380 44.8b

8.7 7.7 6.7 5.7in 1992 3.6in 1992

424 48 31 37 32 24 12 n.a. 13 4.0 n.a. 1.6 1 n.a. n.a.

n.a. 85 36 52 24 42 14–20d 18in 1998 4.8 11 13.1 5.3 1.8 1 12.1 7.0

4.5in ⬃1990 2.1 0.6c 1.0in 1996 n.a. 4.3 4.1 n.a. 2.5in 1996 2.2 0.2in 1998 0.5 6.4 0.2 n.a. 4.7

60.2

75.3in 1998

12.3in 1998

17 n.a.

48 n.a.

1.2 n.a.

51 1069

54 1585

0.7 5.4in 1998

1.7

3.4

0.08

Topfen (Quark) only

Quark only

Excluding whey cheese Fresh and soft cheese Quark only Including Quark, Cottage cheese and salted cheeses

Oceania Australia New Zealand America Canada USA

Production 1990 (’000 tonnes)

Includes Mozzarella, Ricotta, Cream and Cottage cheese

South Africa

a If not otherwise stated, mainly Quark/Tvorog, Cream cheese, Cottage cheese and Mozzarella. b Czech Republic only. c Based on estimated production and population figures. d In 1999, output has been affected downwards due to contamination with dioxin. n.a., Data not available. Compiled from Guinee et al., 1993; Anon, 1994a; Sørensen, 1995, 2001; Richarts, 2001.

Principles of Gel Formation during Combined Acidification and Renneting Many manufacturers produce Cream cheese and similar products without rennet. In some acid-curd cheeses rennet may be added, but in much smaller quantities (2–20 ml of standard strength rennet/1000 l of milk) than for rennet-curd cheeses (⬃200 ml of standard strength rennet /1000 l of milk). The weaker acid gel in

comparison to an acid-rennet gel requires modifications of, for example, agitators and pumps. The process of acid gelation will be covered in ‘Formation, Structural Properties and Rheology of Acid-coagulated Milk Gels’, Volume 1. The present chapter will focus on the combined acid-rennet gelation. Despite its importance in the technology of fresh cheeses, gel formation upon combined continuous

Quark, Cream Cheese and Related Varieties 305

Standardised milk Pasteurisation and homogenisation Culture

Acidification and pre-ripening

Rennet

Renneting Acidification and coagulation Stirring Thermisation

Separation/filtration

Whey/permeate

Curd

Cooling

Cooling

Mixing with cream, fruit, vegetables

Mixing with cream, fruit, vegetables, stabilisers

Mixing with salt, stabilisers

Pasteurisation

Heat treatment

Cooling

Homogenisation

Homogenisation

Whipping

Mixing with fruit, vegetables

Mixing with fruit, vegetables

Cooling

Packing

Quark, Topfen, Baker′s cheese, Tvorog, Skyr

Packing

Quark dessert

Mixing with salt

Cooling Packing

Packing

Hot-pack Cream cheese

Cold-pack Cream cheese

Figure 2 Generalised flow-chart for the manufacture of fresh cheese. Optional processing steps (---) (from Guinee et al., 1993; Ottosen, 1996).

acidification and renneting has been relatively underresearched until recently. A few studies were dedicated to the subject around 10 years ago (Lehembre, 1986; Noël, 1989; Dalgleish and Horne, 1991; Noël et al., 1991). Observations remotely related to acid-rennet coagulation or acid-rennet gels have been reported (Roefs, 1986; van Hooydonk et al., 1986a; Zoon et al., 1989; Roefs et al., 1990; Attia et al., 1993; Walstra, 1993; Gastaldi et al., 1996; Herbert et al., 1999; Kelly and O’Kennedy, 2001). Recently, there has been a renewed interest in the study of milk coagulation after

combined acidification and renneting using more sensitive rheological instruments (Schulz et al., 1999; Lucey et al., 2000; Schulz, 2000; Tranchant, 2000; Tranchant et al., 2001). Physico-chemical changes

Acidification promotes two major physico-chemical changes: a reduction of the negative surface charge on the casein micelles and solubilisation of micellar calcium phosphate. These changes (which influence other related

306 Quark, Cream Cheese and Related Varieties

physico-chemical properties) confer metastability on the casein system, which, through structural rearrangements, reaches a new stable state in the form of a gel network. Physico-chemical changes induced by acidification (and limited renneting) have been discussed comprehensively by Guinee et al. (1993). Physico-chemical changes in combined acid-rennet gelation are to a large extent similar to acid gelation. However, there are differences that are discussed briefly below. The dissociation of calcium phosphate upon acidification is not altered by the addition of a small amount of rennet (van Hooydonk et al., 1986b). The dissociation of casein from the micelles (mainly -casein) is at a maximum at pH 5.6 and 30 °C. This maximum is less pronounced during the acidification of renneted milk and is caused more by non-specific rennet-induced proteolysis of -casein (van Hooydonk et al., 1986b). Both voluminosity and solvation are reduced slightly between pH 6.7 and 4.6 during combined acidification and renneting (Fig. 3). Maximum voluminosity and solvation are at pH 5.3 during acidification. This maximum is less pronounced for renneted milk and is shifted to pH 5.6. In contrast to pure acidification, renneting reduces the solvation and voluminosity by 27% and 37% at pH 6.7 and 5.3, respectively (van Hooydonk et al., 1986b). Creamer (1985) measured a weak maximum at pH 5.1 using different renneting conditions (e.g., renneting at 6 °C).

Slow acidification leads to two ‘adverse’ reactions. On one hand, the casein micelles tend to aggregate due to the reduced negative surface charge and therefore reduced hydration and, hence, increased hydrophobic interactions; on the other hand, casein micelles disintegrate as a result of the solubilisation of colloidal calcium phosphate, which is completely in solution at pH 5.2 at 20 °C (Walstra and Jennes, 1984; Heertje et al., 1985; Roefs, 1986; van Hooydonk et al., 1986b; Gastaldi et al., 1996). As long as the pH is above the clotting pH (e.g., 5.3 at 30 °C), the disintegration processes dominate, i.e., no gel is formed. At a pH below 5.3, the aggregation forces are greater than the disaggregation forces (solubilisation of colloidal calcium phosphate), i.e., a gel is formed (Guinee et al., 1993). The mechanism during a combined acid-rennet gelation is even more complex. The effects of combined acidification and renneting are synergistic, with acidification potentiating the aggregating tendency of the renneted casein particles. The presence of renneted sites, i.e., supplementary reactive sites on the acid-modified casein particles, may mitigate the adverse effects of on-going casein demineralisation on gel cohesiveness and, ultimately, contribute to the structure of what may be regarded as a rennet-reinforced acid milk gel (Tranchant et al., 2001). A local maximum in complex viscosity (*) or storage modulus (G) around pH 5.6 has been reported by

4.0

A

B

3.0

3.0 ml/g

g H2O/g casein

4.0

Mechanism

2.0

2.0

1.0 4.6

5.0

5.4

5.8 pH

6.2

6.6

1.0

4.6

5.0

5.4

5.8

6.2

6.6

pH

Figure 3 Solvation (A) and voluminosity (B) of casein in three milk samples at 30 °C during acidification (—) or combined renneting and acidification (---) (reproduced from van Hooydonk et al., 1986a).

Quark, Cream Cheese and Related Varieties 307

several authors during combined acidification and renneting of milk, indicating a local maximum in gel strength or firmness (van Hooydonk et al., 1986a; Noël, 1989; Noël et al., 1991; Walstra, 1993; Schulz et al., 1999; Lucey et al., 2000; Schulz, 2000; Tranchant et al., 2001). On lowering the pH below 5.6, the firmness diminishes until pH 5.3–5.0 and, thereafter, increases again on further acidification towards the isoelectric pH (Fig. 4). Depending on experimental conditions, the local maximum and minimum have been found at different pH values; pH 6.0–5.5 and 5.5–5.0 at 40 °C (Tranchant et al., 2001); pH 5.60–5.00 and 5.00–4.95 at 30 °C (Schulz, 2000); pH 5.60 and 5.00 at 30 °C (Noël, 1989); pH 5.6 and 5.3 at 25 °C (van Hooydonk et al., 1986a), respectively. A local maximum has been reported at pH 5.3 at 30 °C (Dalgleish and Horne, 1991) and a local minimum at pH 5.10–5.20 at 30 °C (Lucey et al., 2000). The local maximum is most pronounced if the rennet concentration is high, the heat treatment is low and the pH at renneting is high. Addition of CaCl2 does not markedly influence the presence of the local maximum (Lucey et al., 2000; Schulz, 2000; Tranchant et al., 2001). The local maximum occurs only if the acid coagulation sets in at an advanced stage of rennet-induced coagulation (Schulz, 2000). Similar to Noël (1989) and Tranchant et al. (2001), Schulz (2000) divided the coagulation process into several distinct stages, to which the following fermentation processes can be related: (i) adaptation of the starter and acidification until the desired pH of renneting;

(ii) primary phase of rennet coagulation (enzymatic hydrolysis of -casein); (iii) onset of the secondary phase of rennet coagulation (aggregation and gel formation), i.e., complex viscosity increases; (iv) transition from rennet-type to acid-type gel and concominant microsyneresis (occurs if the gel is constrained and cannot shrink); segregation into dense and less dense gel network on a local scale, leading to wider pores on average (Walstra, 1993), i.e., complex viscosity decreases; (v) predomination of acid coagulation, formation of the final acid-rennet gel, i.e., a second increase in complex viscosity; (vi) syneresis; shrinking of casein strands, causes microsyneresis (no visible whey expression), followed by macrosyneresis (visible whey expression), i.e., complex viscosity decreases again. It is assumed that the decrease in firmness after the local maximum in the viscosity/time curve is due to demineralisation of the forming rennet gel, which is almost completed at pH 5.3–5.1 (Heertje et al., 1985; van Hooydonk et al., 1986b; Noël, 1989; Zoon et al., 1989; Gastaldi et al., 1996). Rheological properties of acid-rennet gels formed at pH values ⬃5.2 are essentially similar to those of rennet-induced gels, while at pH values ⬃5.2, the properties are similar to those of acid casein gels. At pH ⬃5.2, rennet-induced gels have a very high permeability coefficient and tan (loss tangent) which indicates increased relaxation behaviour (shorter lifetime)

pH

7.0 6.0 5.0

Complex viscosity (mPa s)

180 160 140 120 100

Max1

IP1

IP3

Max2

80 60 40 20 0 100

RA A 300

IP2 500

Min 700

900

1100

4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 –0.5 –1.0 –1.5 1300

Rate of complex viscosity increase (mPa s/min)

4.0

Time (min) Figure 4 Typical curves for complex viscosity (—䉱-) and rate of viscosity increase (—) during combined acid-rennet coagulation of skim milk. RA, rennet addition; A, aggregation; IP1, IP2 and IP3, inflection points 1, 2 and 3 of the complex viscosity/time curve (i.e., rate of complex viscosity increase is at a maximum or minimum); Max1 and Max2 donate the local (primary) and final maximum of viscosity/time curve (i.e., rate of complex viscosity increase is 0); Min donates a local minimum in the complex viscosity/time curve corresponding to a temporary reduction between the Max1 and Max2 (redrawn from Schulz et al., 1999).

308 Quark, Cream Cheese and Related Varieties

40 Temperature (°C)

of protein–protein bonds (Roefs et al., 1990; Lucey et al., 2000). In acid-induced gels, most bonds presumably are protein–protein salt bridges, and these are most abundant at pH 4.6, implying that the number of bonds keeping the gel together decreases on increasing the pH. At pH 6.7, the bonds may be mostly of the colloidal phosphate type, and lowering the pH leads to dissolution of colloidal calcium phosphate and, hence, to a reduced number (or strength) of bonds. At pH ⬃5.2, the resulting number of bonds presumably is at a minimum (Roefs, 1986; Roefs et al., 1990; Walstra, 1993; Lucey et al., 2000). The process of combined acidification and renneting marks a gradual transition from a rennet-type casein gel to an acid-rennet-type gel with a local minimum in complex viscosity or gel modulus due to microsyneresis which leads to a coarsening of the gel network (Lucey et al., 2000; Schulz, 2000). During microsyneresis, the gel network becomes more dense at some sites on a local scale and less dense at others. The surface-weighed average pore size and permeability increase (Walstra, 1993). Figure 5 shows that the permeability increases strongly with decreasing pH; the rate of change in the permeability over time is a measure of the tendency of a gel to exhibit syneresis (Walstra, 1993). The maximum in tan at the local minimum during acid-rennet gelation (e.g., Noël, 1989; Noël et al., 1991; Lucey et al., 2000; Tranchant et al., 2001) is an indicator of the increased susceptibility of the gel to rearrangements and (micro-) syneresis (van Vliet et al., 1991; Lucey et al., 2000). Figure 6 shows the tendency of rennet gels to synerese as a function of pH and temperature (Roefs et al., 1990). There is a very sharp transition from the region of rapid syneresis to that of no syneresis around

Rapid syneresis 30

No syneresis

sis

re

ow

Sl

20 4.5

5.5 pH

s

e yn

No syneresis 6.5

Figure 6 Tendency of rennet-induced gels to synerese as a function of pH and temperature (redrawn from Roefs et al., 1990).

pH 5.15, a transition that is hardly temperature dependent. Factors that influence the combined acidification and renneting process

The most important characteristics of acid gels (e.g., yoghurt) and acid-rennet gels (e.g., curd for Quark) are their gel strength and tendency to expel whey. Syneresis is desirable to a degree during Quark production at the separation step; however, spontaneous syneresis is undesirable in the finished product. Spontaneous syneresis is understood as the contraction of a gel without the application of any external force (e.g., separator or UF) and is related to instability of the gel network, i.e., large-scale rearrangements resulting in the loss of the ability to entrap all the serum phase (Walstra, 1993; Lucey et al., 2001). The acid (-rennet) gel structure determines quality aspects such as mouthfeel (i.e., smoothness and creaminess), appearance (coarseness) and physico-chemical stability (wheying-off) during processing and storage. Factors that influence the acidrennet gelation will be discussed below. Rennet concentration

Permeability (mm2)

pH 5.35 1.5

pH 5.75

1.0 pH 5.97 pH 6.33

0.5

pH 6.68

0 0

1

2 3 Time (h)

Figure 5 Permeability of rennet-induced skim milk gels as a function of time after renneting at 30 °C 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 (Bäurle 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 l 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

Table 4 Rennet concentration and renneting pH for Quark manufacture reported in the literature Rennet concentration (ml/1000 l) 0–20 (9–12 for Separator- and Thermoquark) 1.66–6.66 10 10

10–150

10–30 15.4 2.2–35.2 2.5 4.4–217

4.4–44

5–10 8 l concentrate 88.8

n.s., not specified.

Activity details (arbitary units)

Renneting pH

Fromase 220TL (DSM Food Specialties B.V., Dortmund) Standard rennet (Chr. Hansen) Standard rennet (Chr. Hansen) Commercial liquid rennet (CSK, Leeuwarden)

220 IMCU/ml

Double strength calf rennet (New Zealand Dairy Institute) Commercial rennet, Sochal (St.-Etienne de Chaumeil) Calf rennet (SKW, Baupte) Single strength calf rennet (Chr. Hansen) Chymosin (Boll, Arpajon) Powdered calf rennet (Chr. Hansen)

280 IMCU/ml

Rennet type

Single strength calf rennet (Horan-Lally Co. Ltd., Mississanga) Standard rennet High purity rennet P99 (Chr.Hansen) Powdered calf rennet (Chr. Hansen)

Temperature (°C)

Method of acidification

Reference

6.6–5.8 (control 6.45)

30

Starter culture

Schulz (2000)

n.s.

6.60

30

Starter culture

n.s.

⬃6.30

23

10800 Soxhlet units

Simultaneously with starter culture Simultaneously with GDL

25

Starter culture or GDL Starter culture

Dalgleish and Horne (1991) Mara and Kelly (1998)

30

GDL

Lucey et al. (2000, 2001)

van Hooydonk et al. (1986a)

520 mg chymosin/l

6.10

20

Starter culture

Gastaldi et al. (1994)

n.s. 610 IMCU/ml

⬃6.10 6.4

30 40

GDL Starter culture

Herbert et al. (1999) Tranchant et al. (2001)

520 mg chymosin/l 99.3% active chymosin (method FIL 110A, 1987) 88 rennin units/ml

6.30 5.98–6.62

25 32–34

Starter culture Starter culture

Attia et al. (1993) Noël et al. (1991)

1 h after culture addition

30

Starter culture

Sohal et al. (1988)

1:10 000 179 IMCU/ml

6.3 6.4

25–28 30

Starter culture Starter culture

Ramet (1990) Schkoda et al. (2001a,b)

99.3% active chymosin (method FIL 110A, 1987)

6.00

30

Starter culture

Noël (1989)

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--casein in comparison to the -casein (Roefs et al., 1990). Therefore, over-acidification can be prevented by rennet addition. If the heat treatment is severe (i.e., 95 °C 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 °C 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 °C 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  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  0.26). As tan 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 l of milk) than in high-rennet-GDL gels (150 ml/1000 l) 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 Horne, 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 l of milk (see Table 4 for rennet strength) at 30–40 °C 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 et al.,

c

200

6.5

160

d 6.0

140 120 e

100

5.5

pH

Complex viscosity (m Pa s)

180

80 60

b

40 20 0 200

a 400

600

800

1000

5.0 4.5 1200

Time (min)

Figure 7 Time-related change in complex viscosity (—) 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 l milk: a, 0; b, 0.5; c, 2; d, 9; e, 20) during slow and quiescent acidification at 31 °C (redrawn from Schulz, 2000).

Quark, Cream Cheese and Related Varieties 311

Table 5 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 °C

pH Elastic modulus, G at   0.01 rad s1 (Pa) Loss tangent, tan at   0.01 rad s1 Fracture stress (Pa) Fracture strain () Permeability B ( 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 (Noël, 1989; Noël 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 l, the pH at aggregation and the local maximum of the complex viscosity increased

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 °C. 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). Noël 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 °C causes denaturation of the whey proteins, -lactalbumin and -lactoglobulin, some of which may complex with micellar -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 Flüeler, 1974; RA

6.5 6.3

Enzymatic hydrolysis

A

6.1 5.9 pH

5.7

Initial aggregation

IP1

5.5 Rennet gel formation

5.3 5.1

Max1

Microsyneresis

4.9

IP3

4.7

Acid gel formation

4.5 0

2

4 6 8 Rennet concentration (ml/1000 l)

10

Figure 8 Phases during combined acid-rennet coagulation of skim milk as a function of rennet concentration. The skim milk was heat-treated at 72 °C for 30 s; gelation at 31 °C 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 °C, 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 °C, leads to protein aggregation which causes sandiness in the fresh cheese (Bäurle et al., 1984; Schkoda and Kessler, 1997a,b). Depending on the manufacturing method used, the heat treatment is usually in the range 88–95 °C for 3–6 min for Thermo- or UF Quarks (e.g., Bäurle et al., 1984; Röckseisen, 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 °C for 30 s) or high-heated milk (86 °C for 6 min), the pH at which aggregation begins remained constant at 6.3 but the pH for the local maximum (Max1 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

pH (–)

5.9 5.7 5.5

IP1

Max1

5.3 5.1

Rennet gel formation Microsyneresis IP3

4.9 4.7 4.5 1:0

Acid gel formation 1:1 Ratio Low-heated : high-heated skim milk

0:1

Figure 9 Phases during combined acid-rennet coagulation of skim milk as a function of the ratio of low-heat-treated skim milk (74 °C for 30 s) and high-heat-treated skim milk (86 °C for 6 min) in the milk blend used for gelation; gelation was initiated at 31 °C by a mesophilic starter culture and 9 ml/1000 l 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 313

The clotting time is also reduced at lower pH values during combined acidification and renneting (van Hooydonk et al., 1986a; Noël et al., 1991; Schulz et al., 1999; Schulz, 2000). There are discrepancies over the following stages of the coagulation process. Noël 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 (Max1 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

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). 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 -casein to para--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

pH (–)

6.0

sis

droly

tic hy

ma Enzy

Initial aggregation

IP1

5.5 Max1

Rennet gel formation Microsyneresis

5.0

IP3

Acid gel formation 4.5 5.8

6.0

6.2

6.4

6.6

pH at rennet addition Figure 10 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 °C for 30 s; gelation was initiated by a mesophilic starter culture and 9 ml/1000 l rennet (Fromase 220TL, DSM Food Specialties B.V., Dortmund) at 31 °C. 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 °C) or warm method (28–31 °C) 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; Roefs, 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; Horne, 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 et al., 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 °C 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 Kaláb, 1980; Modler and Kaláb, 1983; Modler et al., 1983; Ozer et al., 1999). Increasing the protein concentration in skim milk by nanofiltration 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 °C 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 °C for 15 s or 92 °C 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 315

(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 et al., 1985; van Hooydonk et al., 1986b; Dalgleish and Law, 1989). This suggests that the addition of CaCl2 to milk during fresh cheese production is not justified. If milk has been subjected to a high heat treatment, 500–800 ml of a liquid CaCl2 solution (33%, ww) per 1000 l milk can be added to improve its rennet coagulation properties (Spreer, 1998). The effect of CaCl2 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 CaCl2 is added (Schkoda, 1998). Schulz (2000) did not observe an effect of CaCl2 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 l (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 l. At a rennet concentration 20 ml rennet/1000 l, the clotting time and pH were reduced by calcium, i.e., calcium affects the acid-rennet gelation only when 10 ml rennet/1000 l are added and the gelation becomes more like rennet coagulation. Noël (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/hg) and then decreased (Noël, 1989).

Quark and Related Varieties – Manufacture The majority of acid- and acid/rennet-curd fresh 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).

Quark – 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). Quark – original (standard) separator process

Skim milk is pasteurised (72 °C for 40 s), cooled to 28–30 °C and coagulated with a mesophilic culture and a small amount of rennet within ⬃16 h. Rennet (⬃2–20 ml standard strength rennet/1000 l 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 °C), 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, ultrafiltration of whey). In the Centriwhey Process, the Quark whey is heated to 95 °C 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). Ultrafiltration can also be used to concentrate whey instead of separators (Herbertz, 1982; Knüpfer, 1982; Kreuder and Liebermann, 1983).

316 Quark, Cream Cheese and Related Varieties

Quark – Thermo process (Westfalia)

The milk is pasteurised at 95–96 °C 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 °C 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. Quark – 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; Bäurle 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 °C and ultra- or micro-filtered to the desired DM content, cooled, optionally homogenised and packed (e.g., Bäurle et al., 1984; Siggelkow, 1984; Dieu et al., 1990; Korolczuk and Mahaut, 1991a,b; Rogenhofer et al., 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 °C 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 °C 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 (Bäurle 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 et al., 1992a,b; Sharma et al., 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 für 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 (Hülsen, 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 317

Table 6 summarises the yield and whey protein recovery for the various methods. Quark – 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 Fromage 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 °C) 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

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 20 °C, to retard further acidification, may result in more destruction of the gel for a given degree of agitation (Guinee et al., 1993). Separation is generally carried out at a temperature between 34 and 40 °C (Senge, 2002a). Whey separation causes concentration and aggregation of the broken gel pieces. Collision during concentration may be expected to result in the formation of large irregularly shaped conglomerates of varying thickness and length, which are forced into close proximity. The moisture content of the curd is closely related to the degree of aggregation. All factors which enhance aggregation (coarser gel structure, higher separation temperature) reduce the water content and increase the coarseness and firmness of the resulting curd (Guinee et al., 1993; Senge, 2002a). Increasing coarseness of the gel structure before separation results in a product with a coarser/rougher appearance and grainier mouthfeel (separator-produced Quark versus Thermoquark). This is particularly important for products that are packed at this stage (cold-pack Cream cheese, Fromage frais, Quark). After leaving the separator, the Quark is subjected to further shearing (pumping, cooling, storing, optionally mixing with cream and condiments and packing). Such treatments will influence the structural, rheological

Table 6 Yield of Quark and whey protein recovery using various production methods for Quark

Method

Principle

Westfalia Standard separator process Westfalia Thermoprocess Centriwhey/ Westfalia-Lactal/ Meggle-Alcor Ultrafiltration Ultrafiltration Weihenstephan (FMLa) process

Separation of acidified milk Separation of acidified milk Separation/Decanting/ Ultrafiltration (UF) of whey Full UF of milk Full UF of acidified milk Nanofiltration of milk to a volume concentration factor (VCR) of 2. UF or separation of acidified retentate Microfiltration of milk (VCR  1.7–2.2). Separation of acidified retentate.

Hannover (Aubios) process

Typical yield (kg skim milk/kg Quark)

% Whey protein recovery in Quark

Flavour and texture

4.50–4.70

⬃15

Firm and sour

4.08–4.30

50–70

Firm, smooth and mild

3.98

50–100

Whey taste possible

⬃3.8 3.60–3.98 ⬃3.4

⬃100 ⬃100 100

Bitter taste Smooth Smooth, sweet mild flavour

4.00

60

As Thermoquark

a Forschungsinstitut für Milch und Lebensmittel (Research institute for dairy and food), Weihenstephan, Germany. Data from Dolle, 1981; Anon, 1984; Bäurle et al., 1984; Lehmann et al., 1991; Schkoda and Kessler, 1997a; Hinrichs, 2001; Hülsen, 2002.

318 Quark, Cream Cheese and Related Varieties

and syneretic properties of the final product. The texture of Quark as measured by the yield value (0, using the Bingham model) correlates with the extent of syneresis of the final product. The yield value (0) of Quark leaving the separator decreases gradually during further processing (Senge, 2002a). Syneresis of the final product increases as the temperature at which the Quark is pumped increases. Below 15 °C, the Brownian motion of the serum is restricted; the high viscosity of the product also inhibits reincorporation of the serum (Senge, 2002a). After packing, the yield value increases during storage for 30–40 days (Hawel and Heikal, 1994; Senge et al., 1998; Senge, 2002a). In fresh cheese products where the stirred gel is concentrated to obtain the correct level of DM, the application of a relatively high pressure has already led to large-scale syneresis, which is necessary for whey separation. However, once the stirred gel has been concentrated to the correct DM, syneresis may continue due to delayed network arrangements, which in this situation is undesirable. The level of syneresis will depend on several conditions (i.e., composition and further treatments of the concentrated gel), which affect structure and porosity, and on the absence/presence of hydrocolloids that bind the moisture phase (Guinee et al., 1993).

(0) and viscosity of the Quark decrease during this phase. During the second phase, the cream becomes gradually immobilised within the protein matrix and the yield value increases (Senge, 2002a). The addition of cream to Quark reduces the yield value and viscosity, especially in Quark with a high FDM (40%, w/w). This is due to a number of reasons: (i) the fat causes lubrication; (ii) adding cream up to 40%, w/w, FDM leads to an overall reduction in protein; (iii) the fat globules (0.5–10 m) further interrupt gel particle interactions (Senge, 2002a). In Quark concentrated by filtration, the fat can be added either before or after fermentation and whey separation. When full-fat milk is homogenised at 15 MPa prior to fermentation, fat globules react with the protein matrix and the serum-holding capacity and apparent viscosity of stirred fermented milks (7%, w/w, protein) increase with increasing fat content. The addition of cream to the fermented milk, however, does not lead to an increase in serum-holding capacity and even results in a slight decrease in viscosity as the fat globules do not serve a structure-building function but are present in the fermented milk structure only as a filling substance (Schkoda, 1998; Schkoda et al., 2001b). Thermisation of curd or fresh cheese

Addition of cream

In the manufacture of Quark using a separator, cream is added to the concentrated curd in order to reduce fat losses in the whey during separation. In some separator types, cream dosing takes place in the separator immediately after the Quark has left the separator bowl. Fat standardisation in Thermoquark, which is less sensitive to shearing, is usually done by continuous online mixing of the separated curd and cream using a dosing pump. Other additives, such as fruit, herbs, etc., can be incorporated simultaneously (Spreer, 1998; Senge, 2002a). Homogeneous mixing of Quark and cream is difficult due to a density difference of ⬃40 kg/m3. Sufficient mixing is necessary to fully incorporate the cream; however, if mixing takes too long, the fragile Quark structure can be damaged. Batch mixing of separator-produced Quark is obtained by a combination of a vertical screw pump and a scraped surface agitator. The latter ensures that all products are transported to the screw pump and cavities are created into which the cream can flow to achieve localised mixing. Two phases are described during batch mixing, which takes approximately 15 min: a dilution phase and a structuring phase. During the first phase, all ingredients are mixed homogeneously, so that the chemical composition is the same throughout the tank. The yield value

A second heat treatment of either the curd before whey separation (60 °C for 3 min) or the Quark-type product after separation (55–75 °C for 30–60 s) is called thermisation. In the case of Thermoquark, thermisation of the curd is necessary to ensure sufficient whey separation from the finer curd structure due to the co-precipitated whey proteins. The thermisation of Quark after separation greatly increases shelf-life stability, particularly by preventing off-flavour developments such as bitterness, stale or acidic. The production of lactic acid is stopped and the proteolytic activity contributed by rennet and starter bacterial enzymes is reduced (Bäurle et al., 1984; Zakrzewski et al., 1991; Sachdeva et al., 1993; Rogenhofer et al., 1994; Mara and Kelly, 1998). The firmness of the rather soft UF fresh cheeses can be increased by a heat treatment (75 °C for 2–3 min) after acid coagulation and whey removal if the heat treatment is carried out in the presence of undenatured whey protein. Native whey proteins are retained in the UF retentate, but not in microfiltration or separatorproduced Quark (Bodor et al., 1996). The firmness of Labneh is higher when UF is carried out at 50–55 °C instead of 35 °C (Tamime et al., 1991a,b). For Quark containing no stabilisers with up to 40%, w/w, FDM, the limits for thermisation are pH 4.2 and a heating temperature 60 °C, otherwise the protein coagulum

Quark, Cream Cheese and Related Varieties 319

would become too firm and sandiness/grittiness would develop (Spreer, 1998). This problem occurs particularly in low-fat cheeses, i.e., less than 10%, w/w, FDM (Bodor et al., 1996). Stabilisers can be added during thermisation. Thermisation can be followed by hot filling or aseptic cold filling. Starch is sometimes used in low-fat systems to build body and replace fat in Quark-based desserts. Instead of adding cream to skim milk Quark, microparticulated whey proteins (5–7.5%, w/w) can be used to obtain a creamier texture in flavoured Quark preparations (Hoffmann, 1994; Hoffmann and Buchheim, 1994). The milk can also be fermented with ropy culture strains, which produce exopolysaccharides (e.g., Desachy and Parmantier, 1998; Sebastiani et al., 1998). The combined use of acidifying strains and texturising strains is necessary to prevent the density of the acid gel being too close to that of the whey which would impair the separation process. The gel strength decreases significantly with increasing amount of texturising cultures (Sebastiani et al., 1998). Varieties directly related to Quark Tvorog and Tvarog (Eastern Europe), Topfen (Austria) and Baker’s cheese (Germany, US)

Tvorog/Tvarog/Twarog are Slavic translations for Quark. Topfen and Baker’s cheese with 22–24%, w/w, DM also belong to this group (18%, w/w, DM). For these high DM cheeses small amounts of rennet are necessary for good whey separation. Fromage Frais (France and UK)

Fromage frais is very similar to Quark, but has a lower DM content (⬃14%, w/w). Because of the lower DM, Fromage frais is produced increasingly by a recombination method similar to yoghurt fortification. Rennet is used only in a few cases. Buttermilkquark

Because of its high lecithin content, Buttermilkquark is used as a baking emulsifier and for nutritional reasons. It can be produced from unacidified (sweet cream) buttermilk in the same way as normal Quark (Spreer, 1998; Boone, 2001b). If produced from acidified buttermilk, the buttermilk is heat treated (65–70 °C for 40 s), held at 45–50 °C for 1–3 h under agitation, to facilitate deaeration and protein clotting, and then separated (Spreer, 1998; Boone, 2001a). Approximately 20%, w/w, of the fat is phospholipids of which ⬃30%, w/w, is lecithin (Boone, 2001a). Labneh (or Labaneh, Leben), tan (or than) and tulum (Middle East and Balkan regions)

Labneh, a concentrated yoghurt (22%, w/w, DM), is produced from full-fat milk acidified with a yoghurt

culture (optionally coagulated with rennet) at 42–45 °C for 3.5–4.0 h. It is then concentrated. Similar to Quark, the traditional method (cheesecloth bags for straining the cold yoghurt) has been replaced by mechanised processes (mechanical separation or ultrafiltration of the warm yoghurt immediately after fermentation) (Tamime et al., 1989a,b, 1991a,b; Lehmann et al., 1991; Ozer et al., 1998, 1999). If a Quark separator is used, cream is added to the concentrate after separation. Rennet addition increases the throughput of a separator by ⬃30% (Lehmann et al., 1991). Salt and other additives (e.g., dried herbs) are blended in after separation. Labneh can be stored in olive oil for several months. Traditionally, cows’, sheep’s and goats’ milks have been used for the manufacture of Labneh. Labneh made from cows’ milk has a more uniform structure and greater firmness than that made from goats’ or sheep’s milk. Homogenisation of Labneh markedly reduces the firmness of products from goats’ or sheep’s milk. The firmness of cows’ milk Labneh is less affected by homogenisation (Tamime et al., 1991b,c). The milk for goats’ milk Labneh can also be fermented with a mesophilic starter culture at 22 °C for 16–18 h (Mehaia and El-Khadragy, 1999). With increasing rennet level, the yield of Labneh and its DM content increase gradually. However, above a certain rennet concentration (depending on the milk type), an undesirable cheesy flavour was detected (El-Tahra et al., 1999). The effect of different salt levels on Labneh has been investigated by Ammar et al. (1999) and Mehaia and El-Khadragy (1999). Increasing the UF temperature from 35 to 55 °C doubles the firmness of Labneh and halves the processing time (Tamime et al., 1991a,b). Labneh produced by UF at 50–55 °C is very firm, similar to the traditional product. The increase in firmness has been attributed to enhanced formation of casein chains, as observed by transmission electron microscopy, at a temperature above 45 °C, similar to what happens to yoghurt when heated after fermentation (Tamime et al., 1991a,b). Ultrafiltration Labneh (50–55 °C) which is not homogenised is very granular and has a rough texture. Chakka and Shirkhand (India)

Chakka is produced from buffaloes’ milk and is very similar to Labneh. Mixed cultures of mesophilic and thermophilic starter bacteria are used. Shirkhand is produced by blending Chakka with cream, sugar and cardamom (Tamime and Robinson, 1988). Skyr (Iceland)

Skyr is a concentrated fermented milk product (thermophilic bacteria in combination with lactosefermenting yeasts, optionally rennet) having a composition very similar to that of skim milk Quark. The

320 Quark, Cream Cheese and Related Varieties

concentrated Skyr normally undergoes a further heat treatment, i.e., thermisation (Tamime and Robinson, 1988). Ymer (Denmark), Lactofil (Sweden)

The most common fresh cheese in Denmark, Ymer (14.5%, w/w, DM, 5–6%, w/w, protein, 3.5%, w/w, fat) is made from skim milk fermented with a mesophilic starter culture. As with Quark, cream is added after separation (Tamime and Robinson, 1988; Kosikowski and Mistry, 1997). As the protein is concentrated only to 5–6%, w/w, Ymer and Lactofil can easily be produced by fermenting a sweet UF retentate. Swedish Lactofil is similar to Ymer, the only evident difference being a higher fat content. Rheological and syneretic aspects of Quark-type cheeses

Rheological and microscopical investigations have shown that fresh cheeses can be described as dispersions or pastes of hydrated acid casein gel particles in whey (Korolczuk, 1993; Tscheuschner and Nimbs, 1993; Ozer et al., 1998; Senge et al., 1998; Senge, 2002a). Scanning electron microscopy (SEM) shows that acid-type cheeses and stirred yoghurts have similar structures. They are composed of irregular protein particles of varying dimensions, which consist of open, loose networks of casein micelles aggregated in branched chains and coarse clusters (Allan-Woitas and Kaláb, 1984; Ozer et al., 1999). Traditional set-type Labnehs show a continuous structure with the voids and protein matrix evenly distributed, whereas stirred-type Labnehs have a disrupted discontinuous structure with thicker casein clusters. Small thread-like structures are visible between strands in UF Labneh (Ozer et al., 1999). Ozer et al. (1998) described Labneh (DM 22.5%, w/w, 6.4–9.2%, w/w, protein, 6.1–9.2%, w/w, fat) as a weak visco-elastic gel with the storage modulus higher than the loss modulus over the amplitude range measured (0.015–0.150 mNm at 25 Hz). Fromage frais, as produced in France (DM 15–20%, w/w, protein 7–12%, w/w, FDM 0–58%, w/w), has a very soft consistency and is regarded as a visco-elastic liquid rather than a solid (Korolczuk and Mahaut, 1989; Korolczuk, 1996). Applying a frequency sweep, Senge et al. (1998) described Quark (DM 18%, w/w, protein 12%, w/w, FDM 0–40%, w/w) as a material with distinct solid-like properties. Fresh acid-curd cheeses of the Fromage frais and Quark type show shear thinning behaviour. The effect of shearing time shows that the material is thixotropic. The cheeses exhibit plastic flow which can be described very well by the Bingham model (Korolczuk and Mahaut, 1989;

Mahaut and Korolczuk, 1992a; Korolczuk, 1993; Hawel and Heikal, 1994; Senge et al., 1998; Senge, 2002a). With increasing shear rate, the structure progressively breaks down and at a sufficiently high shear, viscous flow can be observed. The stress decrease during shearing can be explained by a decrease in the extent of aggregation of the protein particles. The aggregates of casein gels are also capable of assuming some structural reformation by flocculation. The thixotropic behaviour suggests that under shear there is a continuous process of destruction and restoration, which is a function of the shearing time and shear rate (Korolczuk and Mahaut, 1989; Korolczuk, 1993; Senge, 2002a). Senge (2002a) described the rheological behaviour of Separator and Thermoquark during various stages of manufacturing. Of the various standard rheological models, the Bingham (linear, 2-dimensional) and Herschel-Bulkley (non-linear, 3-dimensional) are the most suitable models for separator-produced Quark and Thermoquark, respectively (Table 7). The rheological parameters, yield value and effective viscosity, are more temperature dependent for Thermoquark than separatorproduced Quark in the temperature range studied (5–30 °C). These differences are explained by the different microstructures and are reflected in different sensory properties of these two Quark types (Senge, 2002a). Proteolysis and bitterness in Quark

Bitterness may develop in Quark for a number of reasons and also depends on the production method. Schkoda and Kessler (1997a) attribute bitterness in UF Quark primarily to the composition of the retentate (mainly calcium) and the enzymes of the starter culture and rennet. The pH-sensitive solubility of calcium is the main cause of bitterness in Quark made from ultrafiltered sweet milk ( Jelen and Renz-Schauen, 1989). In both UF and non-UF Quarks, enzymes responsible for proteolysis are mainly from rennet (Sohal et al., 1988; Mara and Kelly, 1998), but also enzymes from lactic acid starter bacteria, and milk enzymes like plasmin and the acid proteinase, cathepsin D (Mara and Kelly, 1998; Hurley et al., 2000). Significantly higher proteolysis during storage was observed in Quark produced with rennet (Sohal et al., 1988; Zakrzewski et al., 1991; Mara and Kelly, 1998). Starter proteinases contribute little to primary proteolysis and bitterness (Sohal et al., 1988; Mara and Kelly, 1998), probably due to the fact that lactic acid bacteria are generally weakly proteolytic (Fox et al., 1996). In Thermoquark, starter numbers are also considerably reduced by the thermisation step (Mara and Kelly, 1998).

Quark, Cream Cheese and Related Varieties 321

Table 7 Rheological parameters of Thermo- and Separatorquark (Rheometer MC1; temperature 10 °C; Profile: (i) pre-shear 100 s1, 60 s; (ii) rest 0 s1, 60 s; (ii) shear rate sweep 0.1–100 s1, 60 s; (iv) holding shear rate 100 s1, 60 s; (v) shear rate sweep 100–0.1 s1, 60 s (used for regression) Model

Equation

Thermoquark

0 (Pa)

/K (Pa s/Pa sn)

n(–)

r (–)

s (Pa)

Herschel-Bulkley (HB)

.

s (Pa)

  √0  √CA  0 non-linear plastic, 2-dimensional   0  K  n non-linear plastic, 3-dimensional

r (–)

Casson (CA)

n(–)

  0  BH  linear plastic, 2-dimensional

/K (Pa s/Pa sn)

Bingham (BH)

0 (Pa)

  f(  ) in Pa

Separatorquark

115

3.39

1.0

0.974

21.06

135

2.37

1.0

0.998

4.59

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

12.7

5.11

, shear stress (Pa); 0, yield value (Pa);  , shear rate s1; , viscosity (Pa s); K, consistency factor (Pa sn); n, flow index; r, regression coefficient; s, standard deviation of shear stress (Pa) (Data from Senge, 2002a).

Cream Cheese 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 °C for 15–90 s) 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 °C), 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 °C in order to prevent over-acidification, heated (80 °C 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 °C or UF at 50–55 °C (Sanchez et al., 1996b). 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 et al., 1993). For fresh cheese, UF is normally carried out around 40–45 °C; for Cream cheese, 50–55 °C 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, 1981; 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; Kaláb and Modler, 1985a,b). Further treatments of curd after separation

In most cases, the curds are heat-treated (70–95 °C), mixed with salt (0.5–2.0%, w/w) and stabilisers (mainly hydrocolloids), homogenised (two-stage at 15–25 MPa at 65–85 °C) and either hot-packed or cold-packed after cooling to 10–20 °C in a scraped-surface heat exchanger (Walenta et al., 1988; Sanchez et al., 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 -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 °C (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): • Starting curd after centrifugal separation: Casein–fat globule aggregates are first produced during curd formation. • 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. • 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. • 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). • Cooling: Homogenisation clusters are destroyed by high shear stresses developed in the heat exchanger during cooling to 20 °C. This effect is even more pronounced when the final curd temperature is

Quark, Cream Cheese and Related Varieties 323

below 20 °C (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 (Kaláb et al., 1981; Kaláb 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 (Kaláb 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 Fresh cheese preparations (Frischkäsezubereitungen) 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 (Käseverordnung, 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 which are Ripened Acid-curd cheeses (Sauermilchkäse), typical examples of which are Harzer, Mainzer or Olmützer Quargel cheese, have a very strong flavour and odour, a white

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 °C in 15–20 h or warm: 2–5%, thermophilic culture at 40–45 °C in 1.5–3.0 h). The coagulum is cut and cooked at 35–45 °C 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 °C (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 °C for 15 h; sweating at 20–25 °C and 90% relative humidity for 2–3 days, ripening at 12–16 °C and 90% relative humidity for 3 days and further ripening at 10–15 °C. 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 (Kochkäse, Topfkäse), also manufactured from Quark, is a different type of cheese, usually with 20%, w/w, FDM. During ripening at 18–22 °C 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 °C for 10–20 min. The hot cheese is filled into cups or pots (Kessler, 1996; Spreer, 1998).

Other Varieties Layered cheese (Schichtkäse)

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 °C, 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 °C, 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).

Acknowledgement 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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van Hooydonk, A.C.M., Boerrigter, I.J. and Hagedoorn, H.G. (1986a). pH-induced physico-chemical 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 Hooydonk, A.C.M., Hagedoorn, H.G. and Boerrigter, I.J. (1986b). pH-induced physico-chemical changes of casein micelles in milk and their effect on renneting. 1. Effect of acidification on physicochemical properties. Neth. Milk Dairy J. 40, 281–296. 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. Walenta, W., Bäurle, H.W. and Kessler, H.G. (1988). Einfluß von Molkenproteinzusätzen auf die Strukturausbildung von Frischkäsezubereitungen. DMZ Deutsche Molkereizeitung 18, 538–543. Walstra, P. (1993). The syneresis of curd, in, Cheese: Chemistry, Physics and Microbiology. Vol. 1. General Aspects, Fox, P.F., ed., Chapman & Hall, London. pp. 141–191. Walstra, P. and Jennes, R. (1984). Dairy Chemistry and Physics. John Wiley & Sons, New York. Zakrzewski, E., Stepaniak, L., Abrahamsen, R.K. and Sørhaug, T. (1991). Effect of thermization on the quality of Quarg. Int. Dairy J. 1, 199–208. Zoon, P., van Vliet, T. and Walstra, P. (1989). Rheological properties of rennet-induced skim milk gels. 4. Effect of pH and NaCl. Neth. Milk Dairy J. 43, 17–34.

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

Introduction 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).

Specifications for Cottage Cheese 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, CaCl2 (maximum of 200 mg/kg milk), NaCl 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, NaCl, lactic acid, citric acid, phosphoric acid, hydrochloric acid, glucono- -lactone (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.

Principles of Cottage Cheese Manufacture 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 °C  30 min or 71.7 °C  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 °C. 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 et al. (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 Cheese

Table 1 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 135 min 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.

Incubation of Milk 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

0.5 4.0

80 80

5.2 5.2

less mesophilic lactic acid bacteria while for direct-set (or direct acid) Cottage cheese, acidification is done using a combination of organic acid, mineral acid or acidogen. Cultured Cottage cheese may be manufactured by three methods that are based on the length of time it takes from the addition of starter to cutting the coagulum. The three methods are designated short-, long- or intermediate-set methods (Emmons, 1963a,b; Emmons and Tuckey, 1967; Kosikowski, 1982; Scott, 1986). The temperature  time combinations for the method are given in Table 3. Regardless of the method used, the main principle behind Cottage cheesemaking is the coagulation of caseins at, or near, their isoelectric pH (4.6) and cooking of the curds obtained at a similar pH value. Also, the choice of method depends on the schedule of plant personnel. It is important to ensure that the incubation temperature remains constant and uniform throughout the entire mass for proper and uniform acid development. Sandine (1975) suggests that in the case of the long-set method, if the room temperature is above 22 °C, the incubation temperature should be 21 °C, and if room temperature is less than 21 °C, then the incubation temperature should be 22 °C. As a precautionary measure, and to ensure proper acid development, Reidy and Hedrick (1971) suggested that the titratable acidity of the skimmilk after incubation for 1.5 h should increase by 0.05–0.06% for the short-set method, and by 0.04–0.05% after 3.5 h for the long-set method. Another starter may be added if less acid has developed at that time.

Starters Strains of Lactococcus lactis subsp. lactis or subsp. cremoris, which are least susceptible to agglutination, are used as cultures for acid production during Cottage cheese manufacture. The culture may be an active bulk culture or a frozen concentrated culture. The level of bulk culture used is usually in the range of 1–8%, depending on the method used for manufacture (Table 3), although 0.25–1.0% for the long-set method has also been recommended (Watrous, 1997) to prevent over acidification. Agglutination of starter bacteria in Cottage cheese vats has been reviewed (Salih and Sandine, 1980). When starter bacteria agglutinate, they form clumps and settle to the bottom of the vat during manufacture (Emmons et al., 1966). This results in localized production of lactic acid to give a pH difference of about 0.5 between skimmilk at the bottom and the top of the vat after approximately 4 h of incubation (Salih and Sandine, 1984; Milton et al., 1990). Consequently, precipitation of caseins occurs and sludge is formed at the bottom of the vat.

Cottage Cheese 331

Table 3 Typical parameters used for short-, intermediate- and long-set methods for Cottage cheese

Method

Incubation temperature (°C)

Setting time (h)

Starter level (%)

Single strength calf rennet level (ml/454 kg skimmilk)

Short Intermediate Long

30–32.2 28.3 21.1–22.2

4–6 8–9 12–16

5–8 2–2.5 1–2

1.0–1.5 1.0–1.5 0.5–1.0

Brooker (1986) showed that starter agglutination causes minor sludge formation while major sludge, which results in total loss of the vat contents, is caused by slow acid production due to the destruction of starter bacteria by bacteriophage. This view is supported by electron micrographs of samples taken from the bottom of failed vats that show compacted casein micelles, starter bacteria at various stages of lysis and bacteriophage (Brooker, 1986). Minor sludge formation results in yield losses of 4–8% (Grandison et al., 1986). Agglutination can be prevented by homogenizing the skimmilk at 15.2 MPa (Emmons and Elliot, 1967) or bulk culture at 17.2 MPa (Milton et al., 1990) or the addition of de-oiled lecithin (0.5%) to the bulk culture (Milton et al., 1990). Homogenization of skimmilk destroys milk agglutinins while homogenization or addition of lecithin to the starter culture causes fragmentation of starter chains without affecting starter cell density or acid production. Low CO2-producing strains of citrate-positive Lc. lactis subsp. lactis (formerly Lactococcus lactis subsp. lactis biovar. diacetylactis) or Leuconostoc spp. are added for the production of diacetyl, which is important for flavour. However, most of the diacetyl produced (3.2 mg/kg) is lost in the whey at cutting (Mather and Babel, 1959). Therefore, some processors prefer to add diacetyl to the creaming mixture or culture the creaming mixture with diacetyl-producing organisms (Gilliland, 1972). The production of diacetyl and other flavour compounds in Cottage cheese is discussed later in this review. The use of strains that produce excessive amounts of CO2 (through citrate metabolism) causes the curds to float during cooking. Also, the production of CO2 leads to erroneous determination of titratable acidity of the whey due to the formation of H2CO3 but this can be corrected by boiling whey before measuring its acidity (Emmons and Beckett, 1984b).

Direct Acidification The use of food-grade acids or acidogen instead of starter bacteria is an alternate method for manufacturing Cottage cheese. The method of acidification is

based on patents issued to Hammond and Deane (1961), Corbin (1971) and Loter et al. (1975). Essentially, direct acidification is a two-step process in which a less-expensive acid (e.g., lactic or phosphoric acid) is added to cold (2–12 °C) skimmilk to give a pH of approximately 5.2. The cold skimmilk is warmed slowly to 32 °C. Then, glucono- -lactone (GDL) is added to the warm skimmilk to slowly reduce the pH to about 4.6 in an hour. In solution, GDL hydrolyses slowly to gluconic acid, thereby reducing the pH of the skimmilk (Deane and Hammond, 1960). Skimmilk at pH 5.2 and 26.7 °C requires between 4.8 and 5.4 g GDL per liter to reduce its pH to 4.7 in an hour (Satterness et al., 1978). The slow acidification of milk by GDL causes casein micelles to aggregate into a network with fewer links compared to clustered casein micelles observed (by electron microscopy) during rapid acidification with HCl or lactic acid (Harwalkar and Kalab, 1980, 1981). During acidification of milk, casein micelle coagulation does not start until pH 5.1 (Bringe and Kinsella, 1990) when virtually all the colloidal calcium phosphate is solubilized (Gastaldi et al., 1996). The obvious advantage of direct acidification is efficiency of cheesemaking and the elimination of possible problems associated with starter performance in cheese vats (i.e., agglutination, bacteriophage and antibiotics (Sharma et al., 1980)). The total processing time from raw skimmilk to the end of washing during continuous Cottage cheese manufacture by direct acidification with HCl is about 35 min (Ernstrom and Kale, 1975). However, Cottage cheese made by direct acidification using a continuous process has poor texture (Ernstrom and Kale, 1975), while direct-set Cottage cheese made by conventional methods usually has good texture (Geilman, 1981). Pre-culturing milk to pH 5.5 prior to direct acidification significantly improves the texture and body of the finished cheese (Ernstrom and Kale, 1975). A method for making Cottage cheese using a combination of starter bacteria and direct acidification has been patented (Reddy et al., 1990). Other patented methods for Cottage cheesemaking by direct acidification use HCl (Ernstrom, 1963), an aliphatic dione (C29C8 glyoxal) or H2O2 (Metz, 1980) as acidifying agents.

332 Cottage Cheese

Addition of CaCl2 and Rennet A very low level of milk-clotting enzyme (e.g., chymosin) is required for Cottage cheese manufacture. Suggested levels of single-strength rennet (chymosin) used for short-, intermediate- or long-set cultured Cottage cheesemaking are given in Table 3. The milk-clotting enzyme is added to skimmilk within 1 h of starter addition for cultured Cottage cheese. Some manufacturers prefer to add the milk-clotting enzyme along with the starter. Because the level of chymosin added is small, adequate dilution and stirring is necessary to ensure proper distribution in the skimmilk. After the milk-clotting enzyme is stirred in, the skimmilk is left quiescent for gel formation. The pH of coagulation increases with the amount of rennet used. For directset Cottage cheese, rennet is added simultaneously with GDL. Emmons et al. (1959) recommended using 0.2 ml single strength rennet/454 kg skimmilk for cultured Cottage cheese manufacture in order to achieve a satisfactory coagulum ready to cut at pH 4.8. For direct-acid Cottage cheese, more rennet is necessary – about 2.9 ml single strength/454 kg skimmilk, and attempts to manufacture direct-acid Cottage cheese without rennet have been unsuccessful because the coagulum is very weak and the curds shatter extensively on agitation. This is consistent with findings by Kim and Kinsella (1989) who reported that acid-induced gelation with GDL did not occur for 3 h at 35 °C. Bishop et al. (1983) observed that the average size of casein micelles in cultured Cottage cheese curd made with microbial rennet is twice as large as that made without rennet, indicating that rennet promotes aggregation of casein micelles and shortens coagulation time. The United States standards permit the addition of 0.02% CaCl2 to skimmilk to improve curd firmness at cutting during Cottage cheesemaking. However, Emmons et al. (1959) reported that the addition of CaCl2 has no effect on curd strength or the quality of Cottage cheese, indicating that CaCl2 does not play a significant role in the coagulation of casein micelles during Cottage cheesemaking because at pH values 5, most if not all, of the colloidal calcium phosphate in milk is dissolved from the casein micelles (Pyne and McGann, 1960) and the caseins are liberated into the serum phase (Roefs et al., 1985).

Cutting and Cooking Curd The pH at which the coagulum is cut is perhaps the most critical step in Cottage cheese manufacture. The desired cutting and cooking pH during Cottage cheese manufacture are 4.75–4.8 and 4.55–4.6, respectively

(Tuckey, 1964; Emmons and Beckett, 1984a). The size of wire knives used to cut the coagulum determines the size of Cottage cheese curd. Coagulum cut with 0.95 cm or larger wire knives produces large-curd Cottage cheese, while cutting with 0.64 cm knives gives small-curd Cottage cheese. At a cooking temperature in the range 46–60 °C, the firmness and cohesiveness of dry-curd Cottage cheese and its DM content increase with the pH at cutting in the range 4.6–4.9 (Perry and Carroad, 1980; Emmons and Beckett, 1984a). However, when the coagulum is cut at a high pH (e.g., 4.9), the curds have a tendency to matt during cooking (Emmons and Beckett, 1984a). Cutting at a pH lower than 4.6 results in curds that are soft and mushy, and that shatter during cooking. A soft coagulum (Emmons et al., 1959) that holds a high level of moisture after cutting and cooking (White and Ray, 1977) is obtained when heat treatment of milk prior to cheesemaking is more severe than normal pasteurization (72 °C  15 s). To obtain a firm coagulum that is easy to cut from milk that has been severely heated (e.g., 80 °C  30 min), more single strength rennet (15–20 ml/454 kg) is used (Emmons et al., 1959). Consequently, the pH at cutting is high (5.1–5.2) and the quality of the cheese is reduced (Emmons et al., 1959; Durrant et al., 1961). Some cheesemakers use time to indicate when the coagulum is ready to cut. However, this is not reliable. Other than pH measurement, titratable acidity measurement on the whey may be used. This is done by straining or centrifuging curd to obtain clear whey and titrating it with 0.1 N NaOH. The optimum titratable acidity at cutting ranges from 0.42 to 0.60% lactic acid or higher, depending on the DM content of the skimmilk (Table 4). Alternately, the A-C (acid coagulation) test (Emmons and Tuckey, 1967) is used to determine when the coagulum is ready for cutting. In the A-C test, a well-mixed sample of milk is taken from the vat after starter addition but before rennet or coagulant is added. The sample, contained in a stainless steel cup, is suspended in the vat so that it maintains the same Table 4 Effect of dry matter in skimmilk on titratable acidity at cutting during Cottage cheese manufacture Dry matter content of skimmilk (%)

Titratable acidity at cutting (%)

7.8 8.7 9.6 10.5 12.4 14.3

0.44 0.50 0.55 0.62 0.74 0.86

Cottage Cheese 333

temperature as the cultured and rennet-treated skimmilk in the vat. When whey appears in the sample as a knife is drawn through the coagulum in the A-C cup, then the coagulum in the vat is ready to cut. After cutting, the curds are left undisturbed in the whey for about 15 min to ‘heal’ (i.e., contract and expel some whey) before commencing agitation and heating (cooking) to 52–60 °C, over 1.5–2 h. Collins (1961) recommended cooking to a minimum temperature of 55 °C and holding at this temperature for at least 18 min to kill coliforms and psychrotrophic bacteria since D-values for E. coli, Pseudomonas fragi, Lc. lactis subsp. cremoris and Lc. lactis subsp. lactis in whey (pH 4.6) at 55 °C are 4.3, 1.88, 0.64 and 4.57 min, respectively. A high cooking temperature also increases the firmness and DM content of the curds (Chua and Dunkley, 1979; Emmons and Beckett, 1984a). However, at a constant cooking temperature in the range 46.1–60 °C, the DM in curd increases with cooking pH (4.5–4.8) (Emmons and Beckett, 1984a). When rennet is used to aid coagulation and the pH at cooking is 4.5, partial proteolysis of the caseins results in a soft curd even when cooked to a high temperature, e.g., 60 °C (Tuckey, 1964; Emmons and Beckett, 1984a). Provided that agitation is not too rapid to shatter the curd, the optimum heating rate is the fastest rate at which no matting occurs (Emmons and Tuckey, 1967). An increase in heating rate from 0.11 °C/min at the start to 0.3 °C/min at the end of cooking, so that a temperature of 51.6–54.4 °C is reached within 2 h, was suggested by Tuckey (1964) and Emmons and Tuckey (1967) for the production of high-quality Cottage cheese. The firmness and DM content of Cottage cheese curd increase with heating rate in the range 0.18–0.50 °C/min (Chua and Dunkley, 1979). Tuckey (1964) proposed that when the heating rate is too rapid, a surface protein film, which acts as a semipermeable membrane surrounding each cube of Cottage cheese curd becomes denatured, causing whey to be trapped in the curd. However, scanning electron microscopy (SEM) of Cottage cheese (Glaser et al., 1979; Kalab, 1979) failed to demonstrate such a protein film but showed that casein particles on the surface of curds are densely packed and unevenly distributed with pores between them to allow for free outward movement of whey. Similarly, SEM of the interior of a dehydrated curd granule shows clustered and unevenly distributed casein particles. By transmission electron microscopy (TEM), Glaser et al. (1979) showed a distinct difference between the surface and interior of Cottage cheese curd and observed that a surface skin appeared only after severe heat treatment (70 °C  1 h), which is unusual for Cottage cheese manufacture.

Drainage, Washing and Cooling of the Curd After cooking, the whey is drained and the curds are washed 2–3 times with water to remove excess lactose and lactic acid, thereby stabilizing curd pH, and to cool the curd. It is important to drain hot water from the vat jacket during whey drainage so that curd temperature does not rise during washing. Washing also cools the curds and retards bacterial growth. To achieve a final curd temperature 4 °C, wash water at 30, 16 and 7–4 °C or 10 and 4 °C for 3 or 2 successive washings, respectively, is used. The volume of wash water varies (4.7–15.2 l/kg curd, i.e., 30–100% of the skimmilk volume, depending on the equipment used) among manufacturers (Dunkley and Patterson, 1977). A typical value is 80% of the original volume of skimmilk. The water is added slowly down the sides of the vat or by sprinkling, and the curds are stirred gently during washing. After adding the required volume of water, stirring is continued for 10 min before draining. In an industry survey, Rosenberg et al. (1994) reported that washing time in the vat varied from 20 to 45 min. Cottage cheese whey and waste wash water are acidic (pH, 4.5–4.6) and contain proteins, lactose and salts at levels that create disposal problems. Nilson and LaClair (1975) reported BOD levels ranging from 34 500 to 42 167 mg/l and COD levels of 64 188– 67 213 mg/l during Cottage cheese manufacture from 550 to 600 kg skimmilk. In addition to protein and lactose, most of the minerals in milk, both colloidal and soluble, are lost in the whey and wash water; 87, 72 and 87% of Ca, P and Mg, respectively, are lost (Wong et al., 1976, 1977). To reduce the pollution load of effluent from Cottage cheese plants, a reduction in the volume and number of washings has been suggested (Nilson and LaClair, 1975; Emmons et al., 1978). Cross et al. (1977) reported an average loss of 4% of the curd as fines in whey and wash water. Curd solids are lost into wash water by diffusion, the rate of which increases with temperature (Emmons et al., 1978; Bressan et al., 1981) according to the equation: Deff  (0.0658T  1.72)  106 where, Deff is the effective diffusion coefficient in cm2/s and T is in °C (Bressan et al., 1981). Knowledge of Deff is useful for engineering calculations in designing equipment to optimize curd washing during Cottage cheese manufacture. The isothermal diffusion of whey components from dry small-curd Cottage cheese granules during washing is similar to that of a spherical geometric model (Bressan et al., 1981, 1982), suggesting that small-curd

334 Cottage Cheese

Cottage cheese can be modelled as spheres for mass transfer studies. The wash water obtained after the first 20 min of washing contains about 87 and 93%, respectively, of the DM and lactose present after 60 min of washing (Bressan et al., 1982). This suggests that diffusion of lactose from curds to wash water is more rapid than the diffusion of other soluble constituents like whey proteins, salts and lactic acid. Therefore, washing for 20 min may be economically and nutritionally advantageous. The quality of water used to wash curds affects the keeping quality of Cottage cheese. Washing curds with high pH water dissolves the casein on the surface of the curd particles, making them slick and slimy in appearance. Therefore, wash water is chlorinated (5–8 mg/kg), and then acidified (pH 5.5–6.0) before use (Angevine, 1959). Acid wash water would probably also cause less loss of protein due to solubilization. Wash water may also be pasteurized.

Adding Cream Dressing Cream dressing is added and mixed with dry curds after washing. A typical formula for cream dressing is 10.4% fat, 7.8% milk solids non-fat, 2.0% whey, 2.5% salt and 0.35% stabilizer (Morley, 1981); a typical curd to dressing ratio is 60/40 or 56/44 when some of the non-fat milk solids is replaced with whey solids (Morley, 1981). Methods for preparing cream dressing (Manus, 1957) and the ratio of dressing to curds needed to control the fat content of Cottage cheese have been described (Kemp and Schultz, 1979; Lundstedt, 1980). Essentially, the dressing is prepared by blending desired quantities of the ingredients, pasteurization at 74 °C  30 min or at least 82.2 °C  30 s, homogenization (double stage – 13.8 MPa first stage plus 3.4 MPa second stage) and then cooling to 3.3–4.4 °C. Single-stage homogenization of the dressing at 17.2 MPa results in increased viscosity due to fat clumping but whether this treatment leads to improved absorption of the dressing is questionable (Morley, 1981). Alternately, after pasteurization and homogenization, the cream dressing is cooled to 22 °C, inoculated with 2% of a culture containing citrate-positive (cit) Lc. lactis subsp. lactis, incubated for 6 h, and then rapidly cooled to 5 °C before use (Sandine, 1975). Patented procedures have been developed (Sing, 1976) for adding concentrated cells of cit Lc. lactis subsp. lactis to non-cultured chilled cream dressing just before mixing with dry-curd Cottage cheese for the purpose of flavour enhancement and to control the growth of psychrotrophic bacteria during storage. At the time of adding the cream dressing to Cottage cheese curds, flavour materials may be added. The desired final pH

of creamed Cottage cheese is ⬃5.1. This results from blending the dressing, which has pH of 6.4–6.6, with dry-curd Cottage cheese with a pH of 4.6. Whey separation and the appearance of whey in the package occur when the pH of creamed Cottage cheese is 5.0. On the other hand, creamed Cottage cheese with a pH 5.2 has a reduced shelf-life. Instead of adding cold cream dressing, Overcast and Mackens (1973) suggested the use of hot (⬃88 °C) cream dressing to improve shelf-life. Addition of ascorbic acid to a cream dressing as a preservative has been suggested (Custer, 1977). The cream dressing has several functions in finished Cottage cheese. Morley (1981) lists the functions of cream dressing as: • source of flavour in creamed Cottage cheese; • lubrication of curd to make the product easier to handle during pumping and packaging; • modifies perceived texture and improves palatability; • adds nutritive value to Cottage cheese; • helps to control the fat and moisture levels in creamed Cottage cheese to meet regulatory standards.

Physical Structure of Cottage Cheese The microstructure of direct set and cultured Cottage cheese are similar except for slight differences observed during the early stages of coagulation (Glaser et al., 1980). During Cottage cheesemaking, the numberaverage diameter of the casein particles increases from ⬃88 nm in milk to ⬃182 nm during early gelation, ⬃185 nm at the end of healing, ⬃206 nm during cooking and finally to 207 nm at the end of cooking (Glaser et al., 1980). However, the average diameter of the micelles is larger in cheese made with rennet than in that made without rennet (Bishop et al., 1983), suggesting that rennet aids in the fusion of the micelles. Typical electron micrographs of Cottage cheese curds show a porous mass of casein particles in the form of clusters and short chains.

Yield and Quality of Cottage Cheese The typical yields (kg cheese at 80% moisture per 100 kg skim milk) of dry-curd (uncreamed) Cottage cheese made from HTST-pasteurized skimmilk is 15–17%, depending on the protein (casein) content of the skimmilk. Except for the results of White and Ray (1977), yields reported for direct-set Cottage cheese are generally slightly higher (Satterness et al., 1978;

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Sharma et al., 1980; Geilman, 1981) than for cultured Cottage cheese. This is attributable, in part, to loss of soluble peptides produced from casein on proteolysis by starter bacteria. The use of proteinase-negative (prt) starters minimizes yield losses resulting from the proteolytic activity of starter bacteria (Stoddard and Richardson, 1986). Other methods reported to increase the yield of Cottage cheese include: 1. heating of skimmilk to a temperature higher than minimum HTST (Emmons et al., 1959; Durrant et al., 1961; White and Ray, 1977); 2. thermization (74 °C  10 s), followed by storage at 3 °C for 7 days, then HTST treatment (Dzurec and Zall, 1982); 3. addition of sodium hexametaphosphate (SHMP) (Dybing et al., 1982); 4. addition of iota-carrageenan  SHMP (Manning, 1985; Manning et al., 1985); 5. ultrafiltration (UF) of skimmilk to 6.4% (Mathews et al., 1976), 9% (Ocampo and Ernstrom, 1987) or 15% (Covacevich and Kosikowski, 1978) protein. Also, Kosikowski et al. (1985) described the use of retentate-supplemented skimmilk for Cottage cheese manufacture. It is not known whether the methods listed above have been adapted for the commercial production of Cottage cheese. The use of highly heated skimmilk, gums, polysaccharides and UF techniques is aimed at trapping more whey proteins in Cottage cheese.

Use of UF Skimmilk While the commercial production of some high-moisture, acid-coagulated cheeses (e.g., Quarg) from UF milk is promising, commercial production of Cottage cheese from UF skimmilk (UF Cottage cheese) is currently unattractive partially because of the difficulty in making good UF Cottage cheese with 20% DM retentate (Ocampo-Garcia, 1987). Ultrafiltration Cottage

cheese contains higher DM (24%) than conventional cheese (20% DM), which results in a higher (2%) moisture-adjusted yield in the former. However, the high DM in the UF cheese is not accompanied by increased firmness, suggesting the need for research to make a better quality UF Cottage cheese containing 20% DM. The most acceptable firmness of conventional Cottage cheese by a consumer panel is 25–35 g/cm (Mackie et al., 1989), as measured by the resistance to the movement of a wire through the curds (Voisey and Emmons, 1966). Cottage cheese made from UF skimmilk (pH 6.6) absorbs cream dressing poorly, giving it a gelatinous appearance (Covacevich and Kosikowski, 1978). However, this problem can be corrected by acidifying the skimmilk to pH 5.8 prior to UF (Ocampo and Ernstrom, 1987). Another problem encountered during the manufacture of Cottage cheese from UF skimmilk is that the coagulum is tough to cut with conventional cheese knives. Heat treatment (71.7– 82.2 °C for 7 s) of the UF skimmilk (Raynes et al., 1988) or the addition of 0.3% sodium citrate (Geilman, 1988) to it prior to cheesemaking decreases coagulum firmness at cutting.

Nutritional Quality of Cottage Cheese Nutritionally, Cottage cheese is a wholesome low-calorie food that supplies less than 110 kcal/100 g (Table 5). Cottage cheese contains less Ca (about 30 mg/100 g for dry-curd, 60–100 mg/100 g for creamed curd and 68 mg/100 g for low-fat creamed curd) than rennetcoagulated cheeses (700–900 mg/100 g). These values suggest that approximately 50% of the Ca in Cottage cheese comes from the cream dressing. The concentrations of Ca, Mg, K and Na in Cottage cheese vary seasonally (Bruhn and Franke, 1988), probably as a result of seasonal variations in their concentrations in milk. Increasing Ca and reducing Na content of Cottage cheese are major nutritional concerns for the dairy industry. Wong et al. (1976) reported a 5-fold increase in Ca and a nutritionally favourable P:Ca ratio of 1.5

Table 5 Proximate composition of Cottage cheese varieties Cottage cheese variety

Component

Creamed (large- or small-curd)

Low-fat (2% milk fat)

Low-fat (1% milk fat)

Dry-curd (large or small curd)

Moisture (g/100 g) Protein (g/100 g) Fat (g/100 g) Ash (g/100 g) Carbohydrate (g/100 g) Energy (kcal/100 g)

79.0 12.5 4.5 1.4 2.6 103.0

79.0 14.0 1.9 1.4 3.6 90.0

82.5 12.4 1.0 1.4 2.7 72.0

79.5 17.3 0.4 0.7 1.85 85.0

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in Cottage cheese when 0.2% of either sodium pyrophosphate, sodium tripolyphosphate or SHMP was added to skimmilk prior to cheesemaking. Addition of CaCl2 to milk does not affect the Ca content of Cottage cheese but the Ca content of large-curd Cottage cheese is about 60% higher than that of smallcurd Cottage cheese. The exposed surface area to water during washing for large-curd Cottage cheese is smaller than that of small-curd Cottage cheese. Consequently, more minerals leach out of the latter into the wash water during manufacture (Wong et al., 1976). Cottage cheese can be fortified with Ca (to 2  normal levels) through the addition of calcium salts (chloride, lactate or phosphate) to the cream dressing without adverse effects on sensory and microbiological qualities (Shelef and Ryan, 1988). The use of UF skimmilk retentate or addition of carrageenan to skimmilk did not affect Ca content of Cottage cheese (Craddock and Morr, 1988). The concentration of Na in Cottage cheese is about 4 mg/g (Demott et al., 1984; Bruhn and Franke, 1988), most of which is added with the cream dressing because the recovery of milk Na (approximately 0.5 mg/g) in curd is only about 3% (approximately 0.11 mg/g) after three washings during Cottage cheese manufacture (Wong et al., 1976). A 25% reduction of the sodium content of Cottage cheese dressing (Wyatt, 1983) or replacement of up to 50% of the NaCl by KCl (Demott et al., 1984) results in low-sodium Cottage cheese with sensory qualities similar to regular Cottage cheese. Generally, dairy products are poor sources of dietary iron. The concentration of Fe in Cottage cheese is ⬃174.1 g/100 g (Wong et al., 1977). Addition of Fe in the form of ferric ammonium citrate to skimmilk (to give 20 g Fe/ml milk) resulted in 58% Fe recovery in washed curds without adverse effects on cheese quality (Sadler et al., 1973). The concentrations of Zn, Cu and Mn in Cottage cheese are 482.6, 2.6 and 3.7 g/100 g, respectively (Wong et al., 1977). Fortification of Cottage cheese with vitamins A and C has been reported to produce satisfactory results (Sweeney and Ashoor, 1989).

Microbiological Quality An earlier review on the microbial quality of Cottage cheese was published by Emmons (1963). Cottage cheese has limited shelf-life (time from manufacture to unacceptability). Surveys of the shelf-life of Cottage cheese (Table 6) in three countries show that it starts to deteriorate within 2 weeks of storage at 5–7 °C, and is dependent on temperature (Schmidt and Bouma, 1992).

Table 6 Reported shelf-life of Cottage cheese stored at refrigerated temperatures in various countries Country

Shelf-life (days)

Reference

Canada USA UK

16 17.8 9–15

Roth et al. (1971) Hankin et al. (1975) Brocklehurst and Lund (1988)

The most frequently found spoilage organisms in Cottage cheese are psychrotrophic bacteria (Pseudomonas, Achromobacter, Flavobacterium, Alcaligenes, Escherichia, Enterobacter), yeasts and moulds (Witter, 1961; Cousin, 1982). However, Pseudomonas fluorescens, Ps. putida and Enterobacter agglomerans are the principal cause of spoilage (Brocklehurst and Lund, 1985). Bishop and White (1985) found no correlation (r  0.61) between bacterial numbers and Cottage cheese shelf-life but reported that proteolysis was inversely related to potential shelf-life. Psychrotrophic bacteria (Marth, 1970) and their proteases (White and Marshall, 1973) cause defects like surface discolouration, off-odours and off-flavours in Cottage cheese. Bitterness, resulting from increased proteolysis by psychrotrophic bacteria (Stone and Naff, 1967), occurs in Cottage cheese. The shelf-life of Cottage cheese can be extended by 75% by adding sorbic acid or potassium sorbate (0.075%, w/w) to the cream dressing to inhibit psychrotrophic bacteria (Bradley et al., 1962; Collins and Moustafa, 1969; Bodyfelt, 1979; Brocklehurst and Lund, 1985) and moulds without producing objectionable flavours. Sorbic acid is most effective as an anti-microbial agent in its undissociated form, which represents about 59–31% of the concentration used in Cottage cheese at pH 4.6–5.1 (Brocklehurst and Lund, 1985). Chen and Hotchkins (1991) found that dissolving CO2 in Cottage cheese prior to packaging in airtight containers inhibits the growth of gram-negative bacteria and extends shelf-life up to 60 days at 4 °C. Mannheim and Soffer (1996) also reported that modified atmospheric packaging by headspace flushing with CO2 extends the shelf-life of Cottage cheese stored at 8 °C by 150%. Other ‘natural’ ways of extending shelf-life are by the addition of bifidobacteria to inhibit Staphylococci (Brisolva, 1987) or a pre-cultured skim milk product, MicroGARDTM (Salih et al., 1990). The inhibitory effect of MicroGARDTM is due to a heat-stable, low-molecular weight (⬃700 daltons) peptide. It is claimed (Boudreaux et al., 1988) that the addition of 106–107 cfu/g Propionibacterium shermanii NRRL-B-18074 plus 106–107 cfu/g of either cit Lc. lactis subsp. lactis (formerly, S. lactis

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subsp. diacetylactis) NRRL-B-15005, NRRL-B-15006, NRRL-B-15018 or ATCC 15346 to Cottage cheese dressing inhibits psychrotrophic bacteria and moulds by producing propionic and acetic acids which act as antimicrobial agents. Mather and Babel (1959) showed that the addition of acetic or propionic acid to Cottage cheese dressing retards slime formation. Some lactic acid bacteria produce anti-microbial substances that inhibit bacteria of other genera (Branen et al., 1975). The patented (Gonzalez, 1986) strains of citrate-positive (cit) Lc. lactis subsp. lactis exert their preservative effect without metabolizing lactose or citrate because they are lac, cit; they lack a 41 MDa plasmid and a 5.5 MDa plasmid which control lactose and citrate utilization, respectively, in those organisms. The survival of Listeria monocytogenes (strains Scott A or V7) during the manufacture and storage of cultured Cottage cheese was studied by Ryser et al. (1985) who found that small numbers (100 cfu/g) of the organism survived the cheesemaking process. However, Listeria-free direct-acid Cottage cheese was made using GDL but not HCl because undissociated organic acids are more soluble in the bacterial cell membrane and are more bacteriostatic than dissociated acids (El-Shenawy and Marth, 1990).

Cottage Cheese Flavour The acidic taste of Cottage cheese is due largely to lactic acid, which is present at a concentration in the range 124–452 mg/kg; other acids present include formic (23–306 mg/kg) and acetic (11–292 mg/kg), while the concentrations of propionic and butyric acids are 1 mg/kg (Brocklehurst and Lund, 1985). Formic, acetic, propionic and butyric acids are volatile, thus contributing to the aroma of Cottage cheese. The most distinct flavour compound in Cottage cheese is diacetyl which is produced by oxidative decarboxylation of -acetolactic acid, an intermediate compound formed during citrate metabolism by microorganisms that contain citrate permease and citritase (Seitz et al., 1963a), or by condensation of acetyl CoA (from acetic acid) and acetaldehyde, as a C29thiamine pyrophosphate (TPP) complex (Collins, 1972). Diacetyl production is pH depedent, occurring at pH values 5.5 (Collins, 1972). Acceptable levels of diacetyl in Cottage cheese are estimated to be about 2 mg/kg (Hempenius et al., 1965) but a diacetyl:acetaldehyde ratio of 3–5 is desirable for good flavour (Lindsay et al., 1965). A ratio of diacetyl:acetaldehyde 5 or 3 results in harsh or green flavour defects, respectively. The lack of diacetyl flavour can be attributed partially to the oxidation

of diacetyl to acetoin by some starter or contaminating bacteria (e.g., coliforms, Pseudomanas and Alcaligenes) that contain diacetyl reductase (Seitz et al., 1963b). Since oxidation increases with temperature (Pack et al., 1968), storage of Cottage cheese at refrigeration temperatures is important for the retention of diacetyl flavour. The pathway for the conversion of citrate to diacetyl is very well understood. Further, the accumulating evidence on the central role of pyruvate in diacetyl synthesis, the diacetyl destructive enzymes, and procedures developed for selective isolation of mutants that favour the synthesis of the key intermediate, -acetolactate, have offered immense possibilities of ‘engineering’ high flavour-producing lactococci (Hugenholtz, 1993; Hugenholtz et al., 1994). Metabolism of citrate by citrate-fermenting lactic acid bacteria is dependent on the transport of citrate into the cell via a permease system, which functions optimally below pH 6.0. Genetic coding for the permease enzyme is found on a plasmid. Fermentation of citrate by citrate-fermenting lactococci starters also results in the generation of CO2. However, excessive gas production within Cottage cheese curds leads to floating curd cubes during cooking and causes improper, inadequate and non-uniform syneresis, resulting in defective texture and at times matting of individual curd particles, which is undesirable. The citrate-fermenting lactococci produce significant amounts of CO2 (some strains produce excessive amounts) and relatively high concentrations of acetaldehyde, which results in a ‘yogurt-like green apple flavour’.

Common Defects in Cottage Cheese and Possible Causes The following are common defects in Cottage cheese and their possible causes (Emmons and Tuckey, 1967; Sandine, 1975). 1. Mealy curd a. Cooling too fast b. Curd particles contacting hot surface during cooking (possibly due to inadequate stirring) 2. Matted curd a. pH too high, i.e., not enough acid at cutting 3. Shattered curd a. Excessive heat treatment of skimmilk b. Too much acid at cutting c. Rough handling of the curd, especially at cutting d. Too low solids e. Too much rennet 4. Rubbery curd a. Cooking temperature too high

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5. Weak pasty curd a. Excessive heat treatment of skimmilk b. Cooking temperature too low c. Low pH at cutting d. Too much acidity before and during cooking 6. Acid and unclean flavour a. Excess acid at cutting b. Cooking too rapid, especially during early stages c. Contamination during and after manufacture d. Poor quality skimmilk and/or starter 7. Gelatinous curd a. Spoilage bacteria b. Alkaline wash water 8. Poor shelf-life a. Unclean equipment b. Failure to keep cream, dry curd and creamed Cottage cheese cold (5 °C) 9. Bitterness a. Contamination by psychrotrophic bacteria such as Ps. putrefaciens b. Cooking too fast 10. Gassiness a. Contaminated milk b. Use of starters containing high gas-producing cit Lc. lactis subsp. lactis 11. Sediment in cheese vat at cutting a. Clumping of starter bacteria by agglutinins in milk; most common with mastitic and early lactation milk 12. Medicinal flavour a. Use of chlorinated wash water high in organic matter.

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Deane, D.D. and Hammond, E.G. (1960). Coagulation of milk for cheesemaking by ester hydrolysis. J. Dairy Sci. 43, 1421–1429. Demott, B.J., Hitchcock, J.P. and Sanders, O.G. (1984). Sodium concentration of selected dairy products and acceptability of sodium substitute in Cottage cheese. J. Dairy Sci. 67, 1539–1543. Dunkley, W.L. and Patterson, D.R. (1977). Relations among manufacturing procedures and properties of Cottage cheese. J. Dairy Sci. 60, 1824–1840. Durrant, N.W., Stone, W.K. and Large, P.M. (1961). The effect of increasing serum protein content of Cottage cheese curd on yield and quality. J. Dairy Sci. 44, 1171 (abstr.). Dybing, S.T., Parson, J.G., Martin, J.H. and Spurgeon, K.R. (1982). Effect of sodium hexametaphosphate on Cottage cheese yield. J. Dairy Sci. 65, 544–551. Dzurec, D.J. and Zall, R.R. (1982). Effect of on-farm heating and storage of milk on Cottage cheese yield. J. Dairy Sci. 65, 2296–2300. El-Shenawy, M.A. and Marth, E.H. (1990). Behavior of Listeria monocytogenes in the presence of gluconic acid and during preparation of Cottage cheese curd using gluconic acid. J. Dairy Sci. 73, 1429–1438. Emmons, D.B. (1963a). Recent research in the manufacture of Cottage cheese – Part I. Dairy Sci. Abstr. 25, 129–137. Emmons, D.B. (1963b). Recent research in the manufacture of Cottage cheese – Part II. Dairy Sci. Abstr. 25, 175–182. Emmons, D.B. and Beckett, D.C. (1984a). Effect of gas-producing cultures on titratable acidity and pH in making Cottage cheese. J. Dairy Sci. 67, 2192–2199. Emmons, D.B. and Beckett, D.C. (1984b). Effect of pH at cutting and during cooking on Cottage cheese. J. Dairy Sci. 67, 2200–2209. Emmons, D.B. and Elliot, J.A. (1967). Effect of homogenization of skimmilk on rate of acid development, sedimentation and quality of Cottage cheese made with agglutinating cultures. J. Dairy Sci. 50, 957 (abstr.). Emmons, D.B. and Tuckey, S.L. (1967). Cottage Cheese and Other Cultured Milk Products. Pfizer Cheese Monographs. Vol. 3. Chas. Pfizer and Co., Inc., New York. Emmons, D.B., Swanson, A.M. and Price, W.V. (1959). Effect of skimmilk heat treatment and methods of acidification on manufacture and properties of Cottage cheese. J. Dairy Sci. 42, 1020–1031. Emmons, D.B., Elliot, J.A. and Beckett, D.C. (1966). Effect of lactic streptococci agglutinins on curd formation and manufacture of Cottage cheese. J. Dairy Sci. 49, 1357. Emmons, D.B., Beckett, D.C., Campbell, N.J. and Humbert, E.S. (1978). Reducing washing of Cottage cheese and increased recovery of whey solids. Cult. Dairy Prod. J. 13 (2), 13–17, 22–24, 26–29. Ernstrom, C.A. (1963). Process for preparing cheese curd. US Patent 3089776. Ernstrom, C.A. and Kale, C.G. (1975). Continuous manufacture of Cottage cheese and other uncured cheese varieties. J. Dairy Sci. 58, 1008. Fedrick, I.A. and Houliman, D.B. (1981). The effect of lactose hydrolysis on the yield of Cottage cheese curd. Aust. J. Dairy Technol. 36 (3), 104–106.

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Ocampo-Garcia, J.R. (1987). Cottage Cheese Made from Ultrafiltered Skimmilk by Direct Acidification. MS Thesis, Utah State University, Logan, UT. Overcast, W.W. and Mackens, D. (1973). The use of hot cream as dressing for Cottage cheese. Cult. Milk Prod. J. 8 (1), 6–7, 31. Pack, M.Y., Vedamuthu, E.R., Sandine, W.E., Elliker, P.R. and Lessment, H. (1968). Effect of temperature on growth and diacetyl production by aroma bacteria in single and mixed strain lactic cultures. J. Dairy Sci. 51, 339–344. Perry, C.A. and Carroad, P.A. (1980). Influence of acid related manufacturing practices on the properties of Cottage cheese curd. J. Food Sci. 45, 794–797. Pyne, G.T. and McGann, T.C.A. (1960). The colloidal phosphate of milk. II. Influence of citrate. J. Dairy Res. 27, 9–17. Raynes, R., Ernstrom, C.A. and Hicks, C.L. (1988). Sensory and curd characteristics of Cottage cheese manufactured from 16% dry matter retentate. J. Dairy Sci. 71 (Suppl. 1), 81 (abstr). Reddy, M.S., Mullen, J., Washman, C.J., Brown, C.G. and Hunt, C.C. (1990). Cheese manufacture. US Patent 4,959,229. Roefs, S.P.F.M., Walstra, P., Dalgleish, D.G., and Horne, D.S. (1985). Preliminary note on the change in casein micelles caused by acidification. Neth. Milk Dairy J. 39, 119–122. Rosenberg, M., Tong, P.S., Sulzer, G., Gendre, S. and Ferris, D. (1994). California Cottage cheese technology and product quality: an in-plant survey. 1. Manufacturing process. Cult. Dairy Prod. J. 29 (1), 4–11. Roth, L.A., Clegg, L.F.L. and Stiles, M.E. (1971). Coliforms and shelf life of commercially produced Cottage cheese. Can. Inst. Food Sci. Technol. J. 4, 107–111. Reidy, G. and Hedrick, T.I. (1970). Highlights of Cottage cheese industry in the USA. Cult. Dairy Prod. J. 5 (3), 18–20. Reidy, G. and Hedrick, T.I. (1971). Highlights of Cottage cheese industry in the USA. Cult. Dairy Prod. J. 6 (1), 21–24. Ryser, E.T., Marth, E.H. and Doyle, M.P. (1985). Survival of Listeria monocytogenes during manufacture and storage of Cottage cheese. J. Food Prot. 48, 746–750. Sadler, A.M., Lacroix, D.E. and Alford, J.A. (1973). Iron content of Bakers and Cottage cheese made from fortified skimmilk. J. Dairy Sci. 56, 1267–1270. Salih, M.A. and Sandine, W.E. (1980). Lactic streptococci agglutinins: a review. J. Food Prot. 43, 856–858. Salih, M.A. and Sandine, W.E. (1984). Rapid test for detecting lactic streptococci agglutinins in cheese milk. J. Dairy Sci. 67, 7–23. Salih, M.A., Sandine, W.E. and Ayres, J.W. (1990). Inhibitory effects of MicrogardTM on yogurt and Cottage cheese spoilage organisms. J. Dairy Sci. 73, 887–893. Sandine, W.E. (1975). Quality Cottage cheese. Cult. Dairy Prod. J. 10 (2), 12–16. Satterness, D.E., Parson, J.G., Martin, J.H. and Spurgeon, K.R. (1978). Yields of Cottage cheese made with cultures and direct acidification. Cult. Dairy Prod. J. 13 (1), 8–13.

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Acid- and Acid/Rennet-curd Cheeses Part C: Acid-heat Coagulated Cheeses N.Y. Farkye, Dairy Products Technology Center, California Polytechnic State University, San Luis Obispo, CA 93407

Introduction

Manufacturing Methods

Several cheeses are manufactured throughout the world by a combination of acid and heat coagulation of milk. Notable are Queso Blanco (Central and South America), Paneer (India) and Ricotta (Italy), which are reviewed herein. Unlike Cottage cheese in which coagulation is induced by acidification, coagulation and curd formation in this group of cheeses is by a combination of acid and heat (Kosikowski, 1982). The high-heat treatment (70 °C  20 min) given to the milk causes denaturation of whey proteins which form a complex with -casein (Sawyer, 1969) via disulfide linkages and subsequently co-precipitate on acidification of the heated milk to pH 5.5. During acidification of the heated milk, there is a progressive removal of calcium phosphate from the casein micelles to form a soluble calcium salt (Rao et al., 1992) and at pH 5.2, most, if not all, of the calcium is solubilized (Pyne and McGann, 1960; Bringe and Kinsella, 1990; Gastaldi et al., 1996). Acid-heat coagulated cheeses are generally fresh, soft, unripened varieties manufactured from whole milk (e.g., Queso Blanco, Paneer), cream (e.g., Marscapone) or whey or whey blends (e.g., Ricotta, Ricottone).

Queso Blanco can be manufactured from whole, low-fat, skim or recombined milk by methods described by Chandan et al. (1979), Hill et al. (1982) and Kosikowski (1982). A continuous method for making Queso Blanco is described by Modler (1984, 1988) and Modler and Emmons (1989a,b). The main principle of Queso Blanco manufacture is heat-acid co-precipitation of milk proteins. When whole milk is used, there is a large variation (60–85%) in fat recovery (Hill et al., 1982; ParnellClunies et al., 1985a), resulting in a yield of 11.5–22% for milk containing 3–6% fat (Siapantas and Kosikowski, 1965, 1967; Hill et al., 1982). This suggests mechanical occlusion of fat in heat-acid coagulated milk protein and indicates an upper limit of 4.5% fat or a protein:fat ratio of 1:1.2 for the production of acceptable Queso Blanco with high yields (Hill et al., 1982). However, in India, Paneer is manufactured traditionally from buffalo milk standardized to 6% fat (see Torres and Chandan, 1981a,b; Kalab et al., 1988). Fat recovery increases to 93% when the cheese milk is homogenized (13.7 MPa) but a high-moisture (Parnell-Clunies et al., 1985a) soft cheese, lacking desirable slicing properties (Siapantas and Kosikowski, 1967), results.

Queso Blanco and Paneer Queso Blanco (White Cheese) is a soft mildly acid variety that is popular in Latin America and in Caribbean countries, where it is known by different names depending on the country of origin (USDA, 1978). Cheeses similar to Queso Blanco are also manufactured in other parts of the world, e.g., Paneer, a popular cheese in India and Pakistan. Queso Blanco (Queso del Pias or cheese of Puerto Rico) was first introduced in the USA by Weigold (1958). Since then, researchers in the USA and Canada have worked to standardize the manufacturing methods and elucidate the properties of Queso Blanco as made in North America.

Heat Treatment of Milk In the traditional methods for making Queso Blanco or Paneer, milk is heated to boiling and acid (sour) whey is added, with continuous stirring, until coagulation is completed. In industrial processes, several heat treatments have been reported – mostly 82–90 °C for 0–30 min (Weigold, 1958; Siapantas and Kosikowski, 1973; Chandan et al., 1979; Hill et al., 1982; Rao et al., 1992; Farkye et al., 1995). However, it appears that 85 °C for 5 min is the best heat treatment for the production of Queso Blanco with the most desirable qualities (Parnell-Clunies et al., 1985a).

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344 Acid-heat Coagulated Cheeses

For the manufacture of Paneer, buffalo milk is heated to 90 °C without holding (Kalab et al., 1988) or to 82 °C for 5 min (Rao et al., 1992), then cooled to 70 °C before acidification, to reduce curd firmness (see Kalab et al., 1988).

Acidification The hot milk is acidified using food-grade acids, e.g., HCl (Chandan et al., 1979), H3PO4 (Siapantas and Kosikowski, 1973), lactic, tartaric, citric or glacial acetic acid (Siapantas and Kosikowski, 1973; Chandan et al., 1979; Farkye et al., 1995), fruit juices (Kosikowski, 1982) or acid whey concentrate (Hirschl and Kosikowski, 1975). However, citric or glacial acetic acid is used most frequently. The amount of acid required for coagulation depends on the buffering capacity of the milk (Siapantas and Kosikowski, 1973; Hill et al., 1982). To achieve a final pH of 5.2–5.3 in Queso Blanco, 120 ml glacial acetic acid/45.5 kg milk (Siapantas and Kosikowski, 1967) or 0.34% (w/w) citric acid monohydrate (Hill et al., 1982) is added to the milk. Prior to addition to the milk, the acid is diluted with nine parts of water (Siapantas and Kosikowski, 1967; Chandan et al., 1979) but Parnell-Clunies et al. (1985b) indicate that dilution of the acid to 1–2% before adding to milk produces more cohesive curd and cheese with improved body and texture. Rao et al. (1992) suggested that 1.5–3 g citric or lactic acid per kg milk is needed for the manufacture of Paneer.

Drainage and Curd Handling After acidification, the curds are allowed to settle, then trenched in the vat to facilitate whey drainage. For Paneer, whey is drained off above 63 °C (Mistry et al., 1992). Following whey removal, salt (2–2.5%, w/w) is added to Queso Blanco curd, which is then hooped and pressed for a few hours. After removal from the press, Queso Blanco is stored at 8 °C. On the other hand, Paneer curd is not salted but hooped and pressed for 15–20 min. After removal from the press, Paneer is cut into small cubes that are immersed in chilled water (4–6 °C) for 2–3 h. Then, the cubes are removed from the water and allowed to drain or wiped with a dry cloth before cold storage and marketing (Vishweshwaraiah and Anantakrishnan, 1985; Rao et al., 1992). Fresh Queso Blanco has an average composition of 15–20% fat, 21–25% protein, 50–56% moisture, 2–2.5% NaCl, 2.5–2.7% lactose and a pH in the range 5.2–5.5. It contains approximately 341, 357 and 665 mg Ca, P and Na, respectively, per 100 g (Torres and Chandan,

1981b). The chemical composition of Paneer varies depending on whether it is made from bovine, buffalo or mixed milk. The typical composition of Paneer from milk containing 3.5% fat is ⬃55% moisture, 19% fat, 21% protein, 2% lactose, 1.6% ash and pH 6.0 (Mistry et al., 1992). The typical composition of Paneer made from buffalo milk is ⬃51% moisture, 18% protein, 27% fat, 2% lactose and 1.8% ash (Chandan, 1991; Rao et al., 1992).

Microstructure of Queso Blanco The microstructure of Queso Blanco is affected by the severity of heat treatment of the milk. Increasing heating/coagulation temperature in the range 62.8–98 °C increases compactness of the curd and results in cheese with a smoother mouth-feel (Kalab and Modler, 1985). Unlike curd from rennet-treated milk, which consists of distinguishable casein particles fused together in chains and clusters, the microstructure of fresh Queso Blanco shows relatively large protein particles composed of transformed and indistinguishable casein particles (Kalab and Modler, 1985). According to Harwalkar and Kalab (1980, 1981), curds obtained by acidification of hot milk to pH ⬃5.5 possess a so-called ‘core-and-lining’ structure in which a solid casein micelle core (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 -lactoglobulin and -casein, enhanced by the presence of calcium ions, results in the development of filamentous appendages. Caseins, particularly -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 °C is 0.1 m compared to 0.5–5 m when coagulation is at 96–98 °C (Kalab and Modler, 1985). Kalab et al. (1988) also observed coreand-lining structures in Paneer made from cow and buffalo milks.

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Physico-Chemical Properties

Microbial Quality

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 et al., 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 °C) 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 °C. 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 °C). 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.

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 °C) results in butyric acid fermentation due to the growth of spore-forming bacteria in the milk used for manufacture.

Ricotta 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 Ricotta – 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 skimmilk – 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 0.16% lactic acid and its pH 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 °C and NaCl is added. The mixture is heated continuously in large open kettles to 80–85 °C. 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 °C; 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 °C. Cream may be added at this stage. Next, NaCl (0.5%, w/v) is added and heating continued to 85–95 °C. Alternately, CaCl2 may be added. NaCl 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 ⬃88 °C.

Table 1 Proximate composition of different types of Ricotta cheese 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, NaCl (1.86 g/kg milk) is added. Also, stabilizer (0.23 g/kg milk) is added to prevent foaming of the milk during heating. When the temperature of the milk reaches ⬃76 °C, a wideblade spatula is passed through the milk to observe the initiation of curd formation. Heating is continued to 80 °C. 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 °C and adding granular citric acid (0.12 g/kg 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 14.45–15.11 kg/100 kg 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 °C 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 °C.

Acid-heat Coagulated Cheeses 347

References 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 AltaTM 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- -lactone-induced skim milk gels. J. Texture Stud. 11, 35–49. Harwalkar, V.R. 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 -lactoglobulin 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, F.V. (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, F.V. (1967). The making of Ricotta cheese. Proc. 4th Annual Marschall Invitational Italian Cheese Seminar, Madison, WI. pp. 1–7. Kosikowski, F.V. (1982). Cheese and Fermented Milk Foods, 2nd edn, Edward Bros, Inc., Ann Arbor, MI.

Maubois, J.L. and Kosikowski, F.V. (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 -lactoglobulin and -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, F.V. (1965). Acetic acid preparation phenomenon of whole milk for Queso Blanco cheese. J. Dairy Sci. 48, 764 (abstr.). Siapantas, L.G. and Kosikowski, F.V. (1967). Properties of Latin American white cheese influenced by glacial acetic acid. J. Dairy Sci. 50, 1589.

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Siapantas, L.G. and Kosikowski, F.V. (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. 10th 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. Asian J. 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´c, University of Novi Sad, Faculty of Technology, Bulevar Cara Lazara 1, Serbia and Montenegro M. Kaláb, 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.

Pasteurized PCPs 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 ⬃95% 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 Kochkäse. 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 Schachtelkäse, 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

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350 Pasteurized Processed Cheese and Substitute/Imitation Cheese Products

Pasteurized processed and analogue cheese products

Pasteurized process cheese products • manufactured by blending, heating and shearing mixtures of ingredients, mainly of dairy origin • 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

Analogue cheese products

• manufactured by blending, heating and shearing mixtures of ingredients of dairy and/or vegetable origin • not necessary to include natural cheese • 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

Figure 1 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: • 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, 1974a,b; Abou-El-Nour, 2001; Subak and Petranin, 2001).

• 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. • Their nutritive value (e.g., especially as a source of calcium and protein) as a food for children. • 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. • 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). • 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

Table 1 Optional ingredients permitted in pasteurized processed cheese products a,b Ingredient type

Main function/effect

Examples

• Standardization of composition • Contributes to flavour, texture and cooking characteristics • Standardization of composition • Assist in ‘creaming’ (thickening of blend during manufacture) and formation of product • Contribute to texture and rheological (e.g., fracturability, hardness) and cooking properties • 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

• Substitute for young cheese • Similar in behaviour to milk proteins, it contributes to thickening during manufacture, texture and cooking properties • Assist the formation of a physico-chemically stable product • Impart desired texture and cooking characteristics • Assist control of the pH of final product • Impart flavour to processed cheese foods and spreads, especially where much young cheese, cheese base, or milk proteins are used • Accentuate flavour • Affect appearance, flavour and texture, and product differentiation • Increase sweetness, especially in products targeted to young children • Impart desired colour • 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 NaCl, 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’.

• 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. • 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. • 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´áse (processed cheese), Schmelzkäsezubereitung (processed cheese preparation), Kasezubereitung (cheese preparation) and K´ásekomposition (cheese composition), as detailed in the Deutsche K´áseverordnung 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

Table 2 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 0.2%, w/w, of, and /or sodium propionates at levels 0.3%, w/w, of, the finished product) when product is in the form of slices or cuts in consumer packs As for pasteurized blended cheese, but with the following extra optional ingredients: emulsifying salts (such as sodium phosphates and/or sodium citrates at a level of 3%, w/w, of finished product), food-grade organic acids (e.g., lactic, acetic or citric) at levels such that the pH of the finished product is not less than 5.3 As for pasteurized processed cheese but with the following extra optional ingredients: dairy ingredients (milk, cream, skim milk, buttermilk, cheese whey, whey proteins – in wet or dehydrated forms) As for pasteurized processed cheese but with the following extra optional food-grade hydrocolloids (e.g., carob bean gum, guar gum, xanthan gum, gelatin, carboxymethylcellulose and/or carageenan) at levels 0.8% (w/w) of finished products, and food-grade sweetening agents (e.g., sugar, dextrose, corn syrup, glucose syrup, hydrolysed lactose) As for pasteurized process cheese spread, except that emulsifying salts are not permitted

Pasteurized processed cheese

Pasteurized processed cheese foods Pasteurized processed cheese spread

Pasteurized cheese spread

Moisture (%, w/w)

Fat (%, w/w)

FDM c (%, w/w)

43

–

47

43

–

47

Not less than 5.3

44

23

–

Not less than 5.0

40–60

20

–

Not less than 4.0

40–60

20

–

Not less than 4.0

pH

a Data presented are summarized from the Code of Federal Regulations (CFR, 1986). b For each category, there may be product variations for which compositional specifications differ from those presented. c FDM, fat-in-dry matter.

subsidiary body of the FAO/WHO, is endeavouring to draft a single standard for pasteurized PCPs which will be accepted globally. It is expected that a Codex standard will assume increased importance because such a standard will be used by the World Trade Organization in the resolution of trade disputes. In the United States, the code of Federal Regulations defines four types of PCPs based on permitted ingredients and composition (Table 2). Under this system, which is detailed in the Code of Federal Regulations, Food and Drugs, Part 133 (Edition 4-1-93), four main categories of PCPs are identified, namely pasteurized processed cheese, pasteurized PCF, pasteurized PCS and pasteurized blended cheese. The criteria for classification include permitted ingredients and compositional parameters; the main aspects of the different categories are summarized in Table 2. Pasteurized processed cheese is usually sold in the form of sliceable blocks (e.g., processed Cheddar) or slices; spreads and foods may be in the form

of blocks, slices, spreads or pastes (e.g., in tubes). Pasteurized blended cheese, which is the least common category, is usually sold in forms giving a natural cheese image. An arbitrary fifth category of ‘non-standarized processed cheese-like products’ may be considered as consisting of processed cheese-like, processed cheesebased products such as dips and sauces. Manufacturing protocol for PCPs

The manufacture of PCPs involves the following major steps (Fig. 2): • formulation and selection of the different types and levels of ingredients to be included; • cleaning and comminution of the cheese; • blending with water and other permitted ingredients; • processing (heating and shearing) of the blend; • homogenization of the molten blend (optional); • hot packing and cooling.

Pasteurized Processed Cheese and Substitute/Imitation Cheese Products 353

a Compiled from data by Hetzner and Richarts (1996), Sørensen (1997), ZMP (Zentral Markt- und Preisberichtstelle GmbH, 2001). b For countries for which data are available. c Data for Russian Federation, Canada and New Zealand are for 1994.

PCP. However, age (and hence level of proteolysis) appears to have major effects, as reflected by the use of cheese age as a major selection criterion for blend formulation at production level. Block processed cheeses with good sliceability and elasticity require predominantly young cheese (70–90% intact casein) whereas predominantly medium ripe cheese (60–75% intact casein) is used for cheese spreads (Meyer, 1973). Hence, the meltability of processed Cheddar, in which 4–6-month old Cheddar was substituted by ultrafiltered milk retentate (UMR) at a level of 50%, w/w, on cooking increased significantly as the degree of proteolysis in the UMR was increased by the addition of a proteinase from Aspergillus oryzae (Sood and Kosikowski, 1979). Owing to inter-cheese variation in microstructure, composition and level of proteolysis (Fox et al., 2000), different types of cheese give processed products with different consistency characteristics. Hence, it is generally recognized that hard and semi-hard cheese varieties, such as Cheddar, Gouda and Emmental, give firmer, longer-bodied processed products than mouldripened varieties such as Camembert and Blue cheeses (Meyer, 1973). The latter cheeses undergo more extensive proteolysis during ripening and therefore, have a lower degree of intact casein than the former cheeses (Gripon, 1993; Lawrence et al., 1993; Walstra et al., 1993; Steffen et al., 1993). The effects of cheese characteristics (type, age) and the other ingredients used in the manufacture of PCPs on its textural and cooking properties are discussed in ‘Influence of various parameters on the consistency and cooking characteristics of PCPs and ACPs’.

Formulation of blend

Cleaning and size reduction of cheese

Formulation involves selection of the correct type and quantity of natural cheeses, ES, water and optional ingredients to give a PCP with the desired composition, textural and functional properties. Cheese is the major constituent of PCPs, ranging from a minimum level of ⬃51%, w/w, in spreads and foods to ⬃98%, w/w, in processed cheeses. Consequently, the type, the blend and the degree of maturity of the cheeses selected for processing have a major influence on the consistency of the product. In some countries, pasteurized processed cheese made from only one type of cheese is very popular, e.g., Cheddar in the UK and Australia, Cheddar, Gruyère and Mozzarella in the USA and Canada and Emmental in France and Germany. However, PCPs, especially cheese foods and cheese spreads, are produced from a blend of various types of natural cheese. The mixed selection facilitates the procurement of the desired flavour and texture in the finished PCP. Little published information is available on how the attributes of the raw cheese impact on those of the

Cleaning generally involves the removal of surface contamination (e.g., adventitious mould growth) or rind using rapid motor-driven scrapers. The cheese is cut, using hydraulically operated blades, into segments, which are finely minced by passing through high-speed shredders or large mincing machines. The rind of the cheese may also be size-reduced, using counter-rotating stainless steel rollers, to particles sufficiently small (1 mm) to enable the adequate uptake of moisture during subsequent processing. Increasing the surface area of the cheese, by size reduction, increases the homogeneity of the formulated blend, maximizes the surface area of the cheese which facilitates heat transfer to the blend during subsequent processing and the interaction between the cheese and other ingredients (e.g., between ES).

Table 3 Production data for pasteurized processed cheese products in the period 1995–2000a,b

Region/Country EU 15 Belgium Denmark Germany Spain France Ireland Italy The Netherlands Austria Portugal Finland Sweden United Kingdom Norway Czech Republic Hungary Russian Federation USA Canada Australia New Zealand Japan Approximate Global

1996 (’000 tonnes)

1995 (’000 tonnes)

517 54 15 157 39 126 12 20 30 14 4 15 5 27 3 18 11 80c 1081 75c 50 12c 97 1,944

538 55 20 171 37 134 11 20 17 18 1 16 8 32

Mean annual change in period 1996–2000 (difference as % 1996 quantity) 1.0 0.8 8.1 2.2 1.3 1.5 2.1 0.1 10.9 8.3 18.4 1.4 13.0 4.2

Pre-mixing of formulation materials

In batch cooking, the finely ground cheese is conveyed directly to the cooker where it is blended with ES, water and optional ingredients. Alternatively, the cheese may

354 Pasteurized Processed Cheese and Substitute/Imitation Cheese Products

Hard cheese

Semi-hard cheese

Rindless cheese

Soft cheese

Derindering, cleaning

Derinding, cleaning

Removing, wrapping, cleaning

Washing in water, brushing, cleaning

Cutting machinery Additives, other ingredients, water

Rollers

Cutter

Mixer

Processing cooker

Homogenizer

Filling machines

Blocks

Portions

Ventilator

Trolleys

Conveyor belt

Pallets

Cool chamber

Cooling tunnel

Cooling tunnel

Cans

Warm room

Sterilizer Slowly cooling

Sorting, labeling machines

Cooling

Processed cheese

Figure 2 Schematic overview of the manufacturing process for pasteurized processed cheese products.

Pasteurized Processed Cheese and Substitute/Imitation Cheese Products 355

be pre-mixed at room temperature with ES (and some or all of the water and optional ingredients) using various types of pre-blenders (e.g., Drais, Blentech). Some cheese cookers, with interchangeable bowls, allow one batch to be filled and pre-mixed while another is simultaneously being cooked (processed). Pre-mixing has two main effects: • it allows the physico-chemical changes, which occur during processing (e.g., water uptake by the protein), to take place at a lower temperature prior to cooking; • it is conductive to a more uniform quality in the end product. For a given cheese variety, variations in cheese composition (e.g., pH, calcium-to-casein ratio and the level of intact casein) can occur owing to variations in milk composition, cheesemaking conditions and degree of maturation. In turn, such variations can affect its processability, e.g., how readily the para-casein in the cheese becomes hydrated and emulsifies free fat during subsequent processing. Pre-mixing evens out the effects of differences in composition and proteolysis, and thus processability, of the raw materials (e.g., cheese) on the consistency of the final product. The efficacy of pre-mixing depends on the type and the capacity of the pre-blender and the capacity of the cooker. However, the capacity of the pre-blender should not be so great as to cause a considerable difference in the premixing time between the first and the last sub-batches withdrawn for processing. Otherwise, time-related differences in the degree of physico-chemical changes (as discussed below) between the first and the last lots of a given pre-mix at processing could lead to differences in processability and ultimately in the consistency of the end product. The degree of physico-chemical change in a blend after a given time depends on the type of pre-blender which influences the level of shear applied, the shear rate and the degree of mixing and interaction between the different ingredients. Processing of the blend

Following pre-mixing, the blend is tipped into bins and carried on wheels or rails to a hoist, and discharged into the cooker, where it is processed. When pre-mixing is not practised, the ingredients are added directly to the cooker. The order of addition of ingredients varies with plant practices, cooker type, overall plant design and duration of cooking. A typical order of addition is: ground cheese, a dry blend of ES and optional dairy ingredients (e.g., skim milk powder), water and flavours. When the cooking time is relatively short, the ES may be dispersed in a portion of

the water prior to addition and only a portion of the water added at the beginning of processing. This approach minimizes the time required for the ES to dissolve during cooking, increases the concentration of ES during early cooking and thereby enhances the effectiveness of the ES in promoting the desired physico-chemical changes in the blend. After a pre-set time, the remaining water may be added manually, delivered by metering pump, or drawn in by vacuum inside the vessel. Flavours may be added later in the process to minimize the loss of volatile flavour compounds. Processing refers to the heat treatment of the blend, by direct or indirect steam, with constant agitation. Application of a partial vacuum during cooking is optional, but may be used to regulate moisture when using direct steam injection, and is also beneficial in removing air and thus preventing the presence of air openings in the finished, set product. Processing has two main functions: • to kill any potential pathogenic and spoilage microorganisms, and thereby extend the shelf-life of the product; • to facilitate the physico-chemical and microstructural changes which transform the blend to an end product with the desired characteristics and physico-chemical stability. Processing may be performed in batch (e.g., Stephan, Damrow, Blentech, Scanima) or continuous cookers (e.g., Kombinator, Votator, Choc-Steriliser) connected to supplies of water, steam and vacuum. The temperature–time treatment in batch processing varies (e.g., 70–95 °C for 4–15 min) depending on the formulation, the extent of agitation, the desired product texture, the body and the shelf-life characteristics. The heat treatments are generally sufficient to kill vegetative cells (Warburton et al., 1986); they are not adequate to eliminate microbial spores (see ‘Characteristics of different ES in the manufacture of PCPs and ACPs’). However, a temperature 130 °C may be required to kill some spores. A temperature of 140 °C can be achieved in continuous cookers by virtue of their design (Zehren and Nusbaum, 1992). For example, scraped surface heat exchangers which maximize the surface area of contact between the heating medium (e.g., stainless steel heated by steam, oil or hot water) and the blend, ensure sufficient agitation to prevent burn-on of the blend on the heat transfer surface. In continuous cookers, the blend is, typically, heated to and held at 140 °C for 5–20 s and then cooled to 70–95 °C by flash evaporation of moisture due to a pressure drop, or by passing through scrape surface tubular coolers. The product is then held at this temperature for 4–15 min to allow adequate time for interaction of

356 Pasteurized Processed Cheese and Substitute/Imitation Cheese Products

the different blend ingredients, the desired physicochemical changes to occur and the development of the desired textural characteristics. The blend thickens progressively with holding time at 70–95 °C. Processing may also be performed continuously using extrusion whereby the blend of all ingredients is pumped directly to a twin-screw extruder and worked at a temperature of 70–90 °C (Zuber et al., 1987; Blond et al., 1988; Tatsumi et al., 1989; Begueria, 1999). This form of cooking gives a high degree of protein hydration and emulsification very rapidly and may be used to produce ES-free PCPs. Extrusion cooking results in unidirectional alignment of the layers of the molten mass through die plates and is conducive to the formation of a fibrous texture. Hence, microscopic studies have shown that extrusion-cooked pasteurized processed Gouda and Cheddar (containing added whey protein concentrate (WPC)) have superior fibrousness compared to the corresponding kettle-cooked (control) PCPs (Ido et al., 1993).

tic film, e.g., saran-coated polyester, which is automatically flattened and crimped into a chain of individual wrapped slices using crimping conveyors and rotating crimping heads (Zehren and Nusbaum, 1992). The chain of slices is then passed through a water-cooling tank and cooled to 10 °C, dried by removing water using fans and/or scrapers, and finally cut into individual slices which are stacked and packed. Alternatively, the hot molten cheese may be pumped through a manifold with 8–12 nozzles which extrude it in the form of a continuous sheet onto the first of two or three counter-rotating refrigerated chill rolls which cool it rapidly from 80–70 °C to 30 °C. The cooled sheet is then automatically cut into parallel ribbons, each of which is cut into slices which are individually wrapped in plastic films, stacked and packed. Principles of manufacture of PCPs

However, since homogenization requires capital investment, increased operating and maintenance costs, it is practised mainly for high-fat PCPs only (Cari´c and Kaláb, 1993).

The principles of processed cheese manufacture have been reviewed extensively (e.g., Guinee, 1987; Zehren and Nusbaum, 1992; Cari´c and Kaláb, 1993; Fox et al., 1996, 2000; Cari´c, 2000). Processing in the presence of ES, such as sodium citrates and sodium phosphates, results in a number of physico-chemical changes, which bring about a structural transformation from a ‘coarse’ oil-in-water (o/w) emulsion physically encased within a particulate cheese para-casein matrix, as in natural cheese, to a ‘finer’ o/w emulsion in a concentrated para-casein(ate) dispersion, in PCPs. Moreover, the composition of the emulsifier differs; in natural cheese, it is the native fat-globule membrane which consists mainly of protein and phospholipids (cf., Fox and McSweeney, 1998) and in PCPs it is a reformed membrane of re-hydrated para-casein(ate) which interacts, to a greater or lesser degree, with the casein in the bulk phase. Since the structural change is central to the formation of a physico-chemically stable PCP and its functionality (e.g., rheological and cooking properties), the microstructures of both the natural and the processed cheeses are discussed below.

Hot packing and cooling

Microstructure of rennet-curd cheese

For most packaging formats, typically, the processed blend is conveyed (e.g., pumped directly or by gravity flow) from the cooker (e.g., by way of hopper) or homogenizer to the filling machine (sometimes via an intermediate buffer tank with gentle agitation). Numerous packaging formats are possible through the use of specialized filling machines (Meyer, 1973; Zehren and Nusbaum, 1992): individually wrapped portions (e.g., foil-wrapped triangles), blocks, sausageshapes, cans, tubes or slices. In the manufacture of slices, the hot molten cheese is pumped continuously into an endless ‘tube’ of plas-

The rennet-induced coagulation of milk is characterized by casein micelles aggregating into interconnected clusters and forming a network in which fat globules are interspersed as loose inclusions (Gavari´c et al., 1989). Electron microscopy (EM) is the method of choice to study this structure (Brooker, 1979; Kaláb, 1983, 1995), particularly during the early stages of its development before the minute para-casein particles fuse into larger clusters during cheesemaking. Optical or light microscopy (LM) using specific staining to distinguish different components (Flint, 1994) is used to study the general structure of various cheeses

Homogenization

The hot molten mass may be homogenized, with typical first and second stage pressures of 15 and 5 MPa, respectively. Homogenization has a number of effects (Meyer, 1973): • it assists in further mixing and size reduction of any coarse (e.g., rind) or undissolved particles (e.g., ES, dry ingredients), and thereby contributes to a more homogeneous and smooth end product; • it results in further shearing of the blend and interaction of blend ingredients; • promotes a finer dispersion of fat droplets (Walstra and Jenness, 1984); • generally promotes thickening.

Pasteurized Processed Cheese and Substitute/Imitation Cheese Products 357

(Awad et al., 2002). Confocal laser scanning microscopy (CLSM), which uses monochromatic laser light, is gaining popularity (Auty et al., 2001) for its ability to section the sample optically by focusing at predetermined levels below the surface of the sample. The resulting stack of images is then processed by a computer to produce a view of the three-dimensional structure. Both optical and electron microscopy have been used extensively to study the microstructures of milk gels and cheeses (Hall and Creamer, 1972; Knoop, 1972; Rüegg and Blanc, 1972; Kimber et al., 1974; Kaláb, 1977, 1979; de Jong, 1978; Green et al., 1981, 1983; Kiely et al., 1992, 1993; Mistry and Anderson, 1993; Bryant et al., 1995; Desai and Nolting, 1995; Guinee et al., 1998, 1999, 2000a; Auty et al., 2001). The protein matrices of both acid- and rennetinduced milk gels are particulate (Fig. 3; Gavari´c et al., 1989), being composed of entangled clusters of partially fused casein or para-casein aggregates. Ongoing aggregation of the casein, or para-casein, and whey expulsion lead to a gradual fusion of the para-casein network. Consequently, the matrix changes from being particulate to a highly fused aggregated structure (Fig. 4). The integrity of the matrix is maintained by various intra- and inter-aggregate electrostatic and

hydrophobic attractions between amino acid side groups on the para-casein molecules, and between calcium ions and organic serine phosphate groups or ionized carboxyl residues (calcium bridges; Walstra and van Vliet, 1986). The protein network is essentially continuous, extending in all directions, although some discontinuities exist at the macro- and micro-structural levels. Discontinuities at the macro-structural level exist in the form of curd granule junctions and, in Cheddar and related dry-salted cheese varieties, as curd chip junctions. Both kinds of junction are discernible by the naked eye in appropriately prepared sections (Kaláb and Emmons, 1978; Brooker, 1979; Kaláb, 1979; Lowrie et al., 1982; Rüegg et al., 1985; Rüegg and Blanc, 1987; Kaláb et al., 1988). Unlike the interior of the curd particles, which consists of protein and fat at a ratio corresponding closely to that of the overall cheese, the junctions are comprised mainly of casein, being almost devoid of fat. The difference in cheese composition between the interior and the surface of curd particles arises as a result of the cutting or breaking of the coagulated milk (gel) into curd particles, which leads to the loss of fat globules from the freshly cut surfaces into the surrounding whey. As the protein matrix contracts and adjoining curd particles

p

F

p

F

Figure 3 Rennet gel from non-homogenized milk, showing fat globules (F; retained by post-fixing the gels with osmium tetraoxide) encased within a particulate para-casein matrix (P). Bar  5 m (courtesy of M. Kalab).

358 Pasteurized Processed Cheese and Substitute/Imitation Cheese Products

a

5 μm 1800X

b

2 μm

in the interior and the exterior of curd particles and thus to differences in structure–function relationships. The protein matrix occludes fat globules (clumped or coalesced to varying degrees), moisture and its dissolved solutes (minerals, lactic acid, peptides and amino acids) and enzymes (e.g., residual rennet, proteinases and peptidases from starter and non-starter micro-organisms; Kimber et al., 1974; Laloy et al., 1996; Guinee et al., 2000a). The fat phase may be described as a concentrated o/w emulsion within a para-casein network. Evidence that clumping and coalescence of fat globules occur in stirred-curd cheeses is provided by both transmission (TEM) and scanning electron microscopy (SEM); in TEM micrographs (Fig. 5), the clumps retain their fat globule membranes. Scanning electron microscopy micrographs, obtained by examining cheese samples (e.g., Cheddar) from which the fat globules had been extracted during sample preparation, reveal irregularly shaped voids in the para-casein matrix (Mistry and Anderson, 1993; Bryant et al., 1995; Fig. 4). The occurrence of coalesced fat in the form of elongated pools between the para-casein fibres has been similarly demonstrated in Mozzarella and String cheeses by SEM (Taneya et al., 1992; Kaláb, 1993, 1995; McMahon et al., 1993, 1999; Tunick et al., 1993) and CLSM (Fig. 6; Auty et al., 2001; Guinee et al., 2002). The presence of fat clumps and pools suggests partial coalescence of denuded liquid fat droplets, probably formed as a consequence of damage to the native fat globule membrane during curd manufacture, handling and plasticization, while the curd is still warm. Transmission electron microscopy micrographs

7000X Figure 4 Scanning electron micrographs of full-fat (33.0%, w/w) Cheddar cheese at low (1800, a) or high (7000, b) magnification. The arrows correspond to the para-casein matrix and the arrowheads to the areas occupied by fat and free serum prior to their removal during sample preparation; bacteria (most likely starter lactococci) are visible in b, being concentrated mainly at the fat–para-casein interface ((a) reproduced with permission from the Society of Dairy Technology and adapted from Guinee et al., 1998 and (b) adapted from Fenelon et al., 1999).

mat through their fat-depleted surface layers, these fatdepleted areas become part of the internal cheese structure. Curd chips form another type of junction in Cheddar cheese and related dry-salted varieties (Lowrie et al., 1982). The difference in cheese composition between the interior and the surface of curd particles (or chips) probably leads to differences in the molecular attractions between contiguous para-casein layers

Figure 5 Transmission electron micrograph showing the protein matrix (M) of 1-day-old Cheddar cheese interspersed with fat globules (F) encased in fat globule membranes (arrows); B  bacterium (from Cari´c and Kaláb, 1993).

Pasteurized Processed Cheese and Substitute/Imitation Cheese Products 359

a

b

Bar = 25 μm Figure 6 Confocal scanning laser micrographs showing protein (a) and fat (b) as light areas against a dark background in 1-dayold unheated Mozzarella cheeses. Bar corresponds to 25 m (from M.A.E. Auty and T.P. Guinee, unpublished results).

taken during the course of Cheddar manufacture clearly show the aggregation of fat globules, which is first notable at maximum scald, increases with the progression of cheesemaking as the protein network shrinks and forces the fat globules into closer proximity (Kimber et al., 1974; Kaláb, 1995; Laloy et al., 1996). In the temperature range used for cheesemaking (⬃30–55 °C), most or all of the milk fat is liquid (Norris et al., 1973) and therefore flows on the application of stress. Some coalescence of the fat globules probably also occurs during the ripening of Cheddar, Mozzarella and other varieties, as reflected by the increases in level of fat that can be expressed from the cheese at room temperature when subjected to hydraulic pressure or centrifugation (Guinee et al., 1997, 2000a; Thierry et al., 1998) or by extraction of the melted cheese (at ⬃74 °C) with a water/methanol mixture, followed by centrifugation (Yun et al., 1993). The increase in the degree of aggregation of fat during ageing, as revealed by SEM (Tunick and Shieh, 1995), also supports this view. Coalescence is possible, as a large quantity of fat (⬃20–30% of total) is still expected to be liquid at the ripening temperature (⬃4–7 °C) used for Cheddar or Mozzarella (Norris et al., 1973). The agerelated increase in free (expressible) fat may be accentuated by a possible increase in the permeability of the fat globule membrane during maturation due to storagerelated hydrolysis of membrane components by lipoprotein lipase activity (Sugimoto et al., 1983; Deeth, 1997) and by proteolysis of the casein matrix, which holds the fat globules in place (Tunick and Shieh, 1995).

In contrast to Cheddar, Mozzarella and String cheeses, relatively little clumping and coalescence of fat globules is evident in other cheese types such as Cheshire, Gouda (Hall and Creamer, 1972) and Meshanger cheese (de Jong, 1978). Heating natural cheeses in the absence of ES

At the microstructural level, CLSM indicates that heating of Cheddar to 95 °C results in extensive clumping and coalescence of fat globules, and a less homogeneous distribution of the fat and the para-casein phase (Fig. 7; Guinee and Law, 2002). The ensuing partial phase separation increases with the fat content of the cheese and decreases with homogenization of the cheesemilk (Guinee et al., 2000b). Similarly, Paquet and Kaláb (1988) observed, using SEM, that heating Mozzarella cheese in a conventional or microwave oven resulted in extensive coalescence of fat globules and shrinkage of the protein matrix. The heat-induced effects were the most severe in high-fat cheeses, less in low-fat cheeses and the least in processed cheese (Paquet and Kaláb, 1988). Hence, application of heat (70–90 °C) and mechanical shear to natural cheese, as in processing, without the presence of stabilizers usually results in the formation of a heterogeneous, gummy, pudding-like mass which undergoes extensive oiling-off and moisture exudation during manufacture and, especially, on cooling. These defects arise from: (i) coalescence of the liquefied fat due to the shearing of the fat globule membrane and (ii) partial dehydration/aggregation and shrinkage of the

360 Pasteurized Processed Cheese and Substitute/Imitation Cheese Products

a

b

c

d

Bar = 25 μm Figure 7 Confocal scanning laser micrographs of 5-day-old unheated Cheddar cheese (a, b), and the same cheese after heating to 95 °C and then allowed to cool to room temperature (c, d). The micrographs show protein (a, c – long arrows) and fat (b, d – short arrows) as light areas against a dark background. Bar corresponds to 25 m (modified from Guinee et al., 2000b).

para-casein matrix induced by the relatively low pH of cheese (i.e., for most cheeses, 5.7) and high temperatures applied during processing. The modified structure, consisting of a shrunken para-casein matrix with large pools of free oil and free moisture, has impaired ability to occlude fat and free moisture. Consequently, free moisture and de-emulsified liquefied fat seep through the more porous, modified structure. The role of ES in the formation of a physicochemically stable product

In the presence of ES, high heat and shear result in the formation of a smooth, homogeneous, stable product. This transition is facilitated by the ES-induced partial hydration and solubilization of para-casein which emulsifies the dispersed droplets of free fat (Templeton and Sommer, 1936; Meyer, 1973; Rayan et al., 1980; Lee et al., 1986; Cavalier-Salou and Cheftel, 1991; Marchesseau et al., 1997; Bowland and Foegeding, 2001). The fat, released by heating, is emulsified into

small globules (Fig. 8; Rayan et al., 1980) and new membranes develop on fat particle surfaces (Cari´c and Kaláb, 1993). Extensive emulsification produces fat globules smaller than 1 m in diameter (Heertje et al., 1981; Tamime et al., 1990), particularly if the processed cheese is subsequently homogenized. The most commonly used ES include sodium citrates, sodium orthophosphates, sodium pyrophosphates, sodium tripolyphosphates, sodium polyphosphates (e.g., Calgon), basic sodium aluminium phosphates (e.g., Kasal) and phosphate blends (e.g., JOHA and SOLVA blends from BK Giulini Chemie & Co., OHG, Ladenburg, Germany; Kasomel blends from Prayon S.A., Siege Social, B-4480 Engis, Belgium). These salts generally have a monovalent cation (i.e., sodium) and a polyvalent anion (e.g., phosphate). While the salts are not emulsifiers per se, they promote, with the aid of heat and shear, a series of physico-chemical changes within the cheese blend which result in rehydration of the insoluble aggregated para-casein (matrix) and its conversion to an

Pasteurized Processed Cheese and Substitute/Imitation Cheese Products 361

active emulsifying agent. These changes include calcium sequestration, para-casein hydration and dispersal, upward pH adjustment and stabilization (buffering), emulsification and structure formation (Templeton and Sommer, 1936; Becker and Ney, 1965; Morr, 1967; Lee et al., 1986; Cavalier-Salou and Cheftel, 1991; Marchesseau et al., 1997), and are discussed briefly below. While ES are the main agents used to promote para-casein hydration and fat emulsification during the formation of PCPs and ACPs, other emulsifying agents, such as Tween 80, lecithin and protein hydrolysates, have been used experimentally (Marshall, 1990; Drake et al., 1999; Kwak et al., 2002). The ability to sequester calcium is one of the more important functions of the ES. The principal caseins in cheese ( s1-, s2-, -) are amphilic in nature, having both apolar lipophilic segments and polar hydrophilic segments which contain most of the sepine phosphate (Fox and McSweeney, 1998). This structure allows the caseins to function as emulsifiers (Mulvihill, 1992). In cheese, a large proportion (i.e., 65% of total in Cheddar at pH ⬃5.3) of the calcium is insoluble (Guinee et al., 2000c) and probably exists in the form of calcium–phosphate complexes precipitated onto the casein matrix, or in the form of calcium bridges which interlock and aggregate the para-casein molecules (Walstra and van Vliet, 1986). Precipitated calcium phosphate probably reduces the intermolecular repulsive effect of the negative charge on the casein molecules and, thereby, enhances the degree of casein aggregation (Horne, 1998). Consequently, the paracasein in cheese is essentially insoluble; it is estimated that only ⬃15%, w/w, of the total moisture in cheese is

100

Soluble para-caseinate, % total casein

Figure 8 Scanning electron micrograph showing the protein matrix (M) of processed cheese interspersed with fat globules (asterisks). Insoluble calcium phosphate crystals (white arrow), in the lower left, differ in appearance from imprints of soluble emulsifying salts (arrow heads). Bar corresponds to 25 m (adapted from Kaláb et al., 1987).

bound to the protein (Geurts et al., 1974). Calcium sequestration involves the exchange of the Ca2 (attached to casein via the carboxyl groups of acidic amino acids and/or by phosphoseryl residues, or precipitated on the matrix) of the para-casein for the Na of the ES. The sequestration of the calcium results in partial hydration of the insoluble para-casein and conversion to a sodium phosphate para-caseinate dispersion (sol; Morr, 1967; Nakajima et al., 1975; Sood et al., 1979; Wagner and Wagner-Hering, 1981; Lee et al., 1986; Marchesseau et al., 1997). The increase in the hydration of para-casein is paralleled by large increases in the levels of water-soluble N, and N that is non-sedimentable on ultracentrifugation. The degree of calcium sequestration and casein hydration (as determined by the proportion of total N that is nonsedimentable on ultracentrifugation of the processed cheese) is dependent on the processing conditions and the type and level of ES (calcium chelating strength, pH and buffering capacity; Irani and Callis, 1962; van Wazer, 1971; Thomas et al., 1980; Lee et al., 1986; Cavalier-Salou and Cheftel, 1991; Fig. 9). However, the mechanism by which added ES chelates calcium and increases para-casein hydration during manufacture is not entirely clear; this may differ with type and concentration of ES (cf. Nakajima et al., 1975). In the commercial manufacture of ACPs and PCPs, trisodium citrate, as the sole ES, is generally added at a much higher level (e.g., 3%, w/w) than orthophosphates (e.g., 1%, w/w). This suggests that added orthophosphates may largely sequester calcium bound

90 80 70 60 50 40 0

0.5

1 1.5 2 Emulsifying salt, % w/w

2.5

3

Figure 9 Influence of concentration of emulsifying salt on the percentage of total N, which was non-sedimentable on ultracentrifugation (300 000 g  1 h at 20 °C) of a dispersion prepared by blending a 5 g sample of analogue cheese in distilled water and different levels of trisodium citrate (䊉) or sodium tripolyphosphate (䉱; redrawn from Cavalier-Salou and Cheftel, 1991).

362 Pasteurized Processed Cheese and Substitute/Imitation Cheese Products

to phosphoserine residues (i.e., in the form of calcium bridges) whereas added citrate may additionally sequester calcium from precipitated calcium phosphate. Thus, it is noteworthy that the content of non-sedimetable soluble calcium in ACPs decreases with increasing level (0–3%, w/w) of added sodium phosphate ES (disodium monohydrogen phosphate, tetrasodium pyrophosphate and sodium tripolyphosphate) and increases marginally with trisodium citrate (CavalierSalou and Cheftel, 1991). The increase in para-casein hydration during processing is paralleled by a physical swelling of the caseinate dispersion (Nakajima et al., 1975) and an increase in the viscosity of the melting processed cheese mass (Wagner and Wagner-Hering, 1981). The increase in viscosity is frequently referred to as creaming in the processed cheese industry (see ‘Influence of various parameters on the consistency and cooking characteristics of PCPs and ACPs’). The use of the correct blend of ES usually shifts the pH of cheese upwards (typically from ⬃5.0–5.5 in the natural cheese to 5.6–5.9 in the PCP) and stabilizes it by virtue of their high buffering capacity (Gupta et al., 1984; Cavalier-Salou and Cheftel, 1991; Marchesseau et al., 1997). This change contributes to an enhanced calcium-sequestering ability of the sodium phosphate ES per se (Irani and Callis, 1962) and an increased negative charge on the para-caseinate. The latter changes lead to an increased casein hydration and a more open reactive sodium (phosphate) para-caseinate conformation with superior water-binding and emulsifying properties (Nakajima et al., 1975; Lamure et al., 1988; Marchesseau et al., 1997). Hence, the buffering capacity of the ES is a critical factor controlling the rheological, textural and melting attributes of PCPs and ACPs (Rayan et al., 1980; Thomas et al., 1980; Gupta et al., 1984; Cavalier-Salou and Cheftel, 1991; Savello et al., 1989; Guinee and Corcoran, 1994). The increase in hydration and dispersion of the paracasein, sometimes referred to as peptization, is enhanced by increasing the temperature and the shear within the normal limits applied during processing (see ‘Processing of the blend’). Under the conditions of cheese processing, the dispersed hydrated para-caseinate contributes to emulsification by coating the surfaces of dispersed free fat droplets, and to emulsion stability by immobilization of a large amount of free water (Phillips, 1981; Mulvihill, 1992). During the cooling of PCPs, the homogeneous molten viscous mass sets to form its characteristic body, which, depending on blend formulation, processing conditions and cooling rate, may vary from a firm sliceable product to a semi-soft spreadable consistency. Factors contributing to the setting probably

include fat crystallization, protein–protein interactions, and interactions between the dispersed emulsified fat globules and the bulk phase para-casein. The occurrence of rheological changes in PCPs during storage, to a degree dependent on storage time and temperature in the range 10–30 °C (Tamime et al., 1990), lends support to the contribution of the latter interactions. Comparison of the microstructure of natural and processed cheeses indicates that the latter differs markedly from the former by the absence of curd granule and/or curd chip junctions and the more homogeneous distributions of the fat and protein phases. Micro-structure of PCPs and ACPs

Micro-structural studies on PCPs or ACPs indicate that the structure typically consists of a concentrated emulsion of discrete, rounded fat droplets of varying size (typically ⬃1–10 m) in a hydrated protein matrix (Fig. 8; Kimura et al., 1978; Rayan et al., 1980; Taneya et al., 1980; Heertje et al., 1981; Lee et al., 1981; Kaláb et al., 1987; Savello et al., 1989; Tamime et al., 1990; Kaláb, 1995; Guinee et al., 1999; Auty et al., 2001). There is less clumping or coalescence of fat globules than in natural cheese. Consequently, the mean fat globule size tends to be generally smaller (Sutheerawattananonda and Bastian, 1995), although it varies depending on the type and level of ES, milk protein additions, processing time and extent of shear (Rayan et al., 1980; Kaláb et al., 1987, 1991; Savello et al., 1989; Tamime et al., 1990). Generally, for most ES, the fat globule size decreases as the processing time at a high temperature increases, e.g., up to 40 min (Rayan et al., 1980; Kaláb et al., 1987). The para-caseinate membranes of the emulsified fat globules appear to attach to the matrix strands, thereby contributing to the continuity of the matrix. The positive correlations between the degree of emulsification (DE) and firmness or elasticity, and the inverse relationship between DE and flowability of PCPs support this suggestion (Rayan et al., 1980; Cari´c et al., 1985; Savello et al., 1989). The incorporation of emulsified para-caseinate-coated fat globules, which can be considered as pseudo-protein particles, into the new structural matrix may be considered as increasing the effective protein concentration (van Vliet and Dentener-Kikkert, 1982; Marchesseau et al., 1997; Michalski et al., 2002). The fat globule size is important as it influences the firmness of the final PCP and the ability of the fat to become free and contribute to oiling-off when the PCP is subsequently cooked for consumption. When cheese is baked or grilled, some oiling-off is desirable, as it limits drying-out of the cheese and thus contributes to

Pasteurized Processed Cheese and Substitute/Imitation Cheese Products 363

the desired flowability, succulence and surface sheen of the melted product (see ‘Cheese as an Ingredient’, Volume 2). Generally, for a given formulation, a reduction in the mean diameter of the emulsified fat globules gives PCPs which are firmer and, which on cooking, exhibit a low tendency to oil-off and have poor flowability (Rayan et al., 1980; Savello et al., 1989). Comparative studies on the effect of different ES indicate that, for a given processing time, the mean fat globule diameter is generally smallest with tetrasodium pyrophosphate (TSPP) or sodium tripolyphosphates (STPP), largest with basic sodium aluminium phosphate (SALP) and intermediate with trisodium citrate (TSC) or disodium phosphate (DSP; Rayan et al., 1980; CavalierSalou and Cheftel, 1991). Hence, in practice, SALP is generally claimed to give PCPs and ACPs with good melting properties. Increasing the concentration of ES (1–4%, w/w) and processing temperature (80–140 °C) results in a progressive decrease in mean fat globule diameter and a concomitant increase in firmness. Generally, for most ES, the fat globule size decreases as the holding time at a high temperature increases, e.g., up to 40 min (Rayan et al., 1980; Kaláb et al., 1987); the resultant products become firmer, more elastic and less flowable. High-resolution TEM (e.g., 60 000 x) has been used to study the structure of the protein matrix in PCPs (Kimura et al., 1978; Taneya et al., 1980; Heertje et al., 1981; Klostermeyer et al., 1984; Lee et al., 1996). The protein phase consists of varying proportions of individual para-caseinate particles and strands, which are probably formed through end-to-end association of para-caseinate particles. The individual particles (20–30 nm diameter) may correspond to casein submicelles released from the para-casein micelles in the matrix of the natural cheese as a result of calcium sequestration by the ES (Kimura et al., 1978; Taneya et al., 1980; Heertje et al., 1981). The proportions of strands to individual particles vary with the rheolgy and the texture characteristics of the processed cheese. Hard PCPs contain a high level of long protein strands (e.g., ⬃100 versus 25 m) which form a matrix (Fig. 10) that is finer than the protein matrix of natural cheese. In contrast, the protein matrix of soft PCPs usually consists predominantly of individual particles. However, abundant protein strands were also found in a soft processed cheese made with a mix of orthophosphate and polyphosphate when direct steam heating was used (Cari´c and Kaláb, 1987). The presence of protein strands was apparently associated with the use of polyphosphates but no conclusive study has been reported on this subject. Apart from a few exceptions, including acid-heated coagulated Queso blanco-type cheese (Kaláb and Modler,

Figure 10 The protein matrices of soft (A) and hard (B) processed cheeses. The black string-like protein strands (highlighted by the arrows) are longer and thicker in the hard product than in the soft product. F  fat globule. Bar corresponds to 0.2 m (reproduced with permission from Kimura et al., 1978).

1985) and Paneer (Kaláb et al., 1988), it is not possible to identify the type of cheese used to make PCPs. The casein particles in the acid-heat type cheeses, above, have a characteristic core-and-shell ultrastructure (Harwalkar and Kaláb, 1981; Kaláb and Modler, 1985; Kaláb et al., 1988), which is very stable and withstands the conditions of cheese processing. Microstructural analyses of processed cheeses frequently reveal the presence of various crystalline species such as calcium phosphate. Crystallization, if excessive, may be give rise to visual defects, and is discussed in ‘Characteristics of different ES in the manufacture of PCPs and ACPs’. Properties of ES important in cheese processing

Differences in the functionality of ES offer the manufacturer of PCPs and ACPs a major lever with which to impart customized characteristics to the finished product, e.g., sliceability, spreadability and meltability (Thomas et al., 1980; Gupta et al., 1984; Abdel-Hamid et al., 2000a,b; Awad et al., 2002). The ES most commonly

364 Pasteurized Processed Cheese and Substitute/Imitation Cheese Products

used are sodium citrates, sodium hydrogen orthophosphates, sodium polyphosphates and sodium aluminium phosphates. Other potential emulsifying agents include gluconates, lactates, malates, ammonium salts, gluconic lactones and tartarates (Meyer, 1973; Price and Bush, 1974 a,b; Gupta et al., 1984). Tartaric acid from wine, added as an ingredient, acts as a calcium-sequestering agent in the manufacture of Swiss Cheese Fondue (Schär and Bosset, 2002). At the commercial level, ES are supplied increasingly as blends of phosphates (e.g., Joha C special, Solva 35S) or of phosphates and citrates (e.g., Solva NZ 10), tailormade to impart certain functionalities (e.g., different degrees of meltability, sliceability, spreadability) to different pasteurized products (e.g., blocks, slices, spreadable) manufactured under different conditions (e.g., from cheeses of varying degrees of maturity or using cookers with varying degrees of shear input).

Group Monomers, orthophosphates

Polymers, linearly condensed polyphosphates

Phosphoric acid Potassium dihydrogen orthophosphate Dipotassium hydrogen orthophosphate Tripotassium orthophosphate Sodium dihydrogen orthophosphate Disodium hydrogen orthophosphate Trisodium orthophosphate Tetrapotassium diphosphate Disodium diphosphate Triosodium diphosphate Tertrasodium diphosphate

Structure O MO

O MO

O

P

O

OM Pentapotassium triphosphate pentasodium triphosphate

P

OM

OM

O MO

O

P

O

OM

O

P

O

P

OM

OM

OM

Sodium tetrapolyphosphate

O MO

Major types of ES

Salts consisting of a monovalent cation and a polyvalent anion have the best emulsifying characteristics. Of the many citrates available, trisodium citrate (Na3C6H5O7) is used most commonly. Monosodium citrate, when used alone, gives over-acid PCPs which are mealy, acid and crumbly and show a tendency to oiling-off due to poor emulsification (Gupta et al., 1984). The use of disodium citrate as the sole ES also leads to high acidity and to water separation during solidification of the molten PCP (Cari´c and Kaláb, 1993). The dissociation constants (pKa) of citric acid at the ionic strength of milk are 3.0, 4.5 and 4.9 (Walstra and Jenness, 1984). Owing to their acidic properties, mono- and di-sodium citrates may be used to correct the pH of a processed cheese blend, for example, when using a high proportion of very mature, high-pH cheese or skim milk solids. The terminology and manufacture of phosphates has been reviewed by Bell (1971) and van Wazer (1971). The phosphates have a structure in which each phosphorus atom is surrounded tetrahedrally by four oxygen atoms. Neighbouring PO4 groups may react and share one or more oxygen atoms to form 9P9O9P9 bonds and form condensed phosphates with a phosphorus content, expressed as percentage P2O5, 72.5 (Zehren and Nusbaum, 1992). The condensed phosphates are called linear condensed phosphates when one oxygen atom is shared by neighbouring PO4 groups, and metaphosphates (or cyclic phosphates) when three oxygens are shared. The generic structures of some of the sodium phosphates are given in Fig. 11. The phosphate-based ES used in cheese processing are mainly the sodium salts of orthophosphates (e.g., disodium monohydrogen phosphate (Na2HPO4), trisodium

OM

P OM

P

O

P OM

O

OM

O O

P

OM

OM

O

O

P

MO

O

OM

OM Sodium hexametaphosphate (Graham's salt) Soluble sodium polyphosphate

O

O O

P

O

P

P

OM

OM

OM m

Insoluble sodium polyphosphate (Madrell's salt) M

(n+2)PnO(3n+1)

Cyclical polyphosphates

O

Sodium trimetaphosphate

OM P

O

O

P

P

O

MO

O

OM

O O

Sodium tetrametaphosphate

OM P O

O

O

MO P

P O

O

O

OM

P O

OM

M: metal ion (Ca, K)

Figure 11 Structure of different sodium phosphates used in the manufacture of pasteurized processed and analogue cheese products (courtesy of M. Cari´c).

monophosphate (Na3PO4)) which contain one PO4 group, and linear condensed phosphates such as pyrophosphates (two PO4 groups) and polyphosphates (3–25 PO4 groups, e.g., tripolyphosphate with three PO4 groups). Potassium and sodium aluminium phosphates may also be used in the manufacture of PCPs and ACPs (Karahadian and Lindsay, 1984), e.g., the latter ES is used widely in the manufacture of analogue pizza cheese

Pasteurized Processed Cheese and Substitute/Imitation Cheese Products 365

(APC). Of the orthophosphates, disodium hydrogen orthophosphate (Na2HPO4) is the most commonly used; when used alone, the mono- and trisodium salts tend to give over- or under-acid products, respectively (Templeton and Sommer, 1936; Scharf, 1971; Gupta et al., 1984). Comparative studies have shown that the potassium salts of orthophosphates, pyrophosphates and citrates give PCPs with textural properties similar to those made with the equivalent sodium salts at similar concentrations (Gupta et al., 1984; Karahadian and Lindsay, 1984); however, the use of the potassium salts reduced slightly the flowability of the melted PCs. Hence, while potassium ES may have potential in the preparation of reduced-sodium PCs, they are rarely used in practice as they impart a bitter taste to the finished product, which becomes more pronounced with storage (Templeton and Sommer, 1936), and are more expensive. Some of the characteristics of the commonly used ES in aqueous solution are presented in Table 4. Characteristics of different ES in the manufacture of PCPs and ACPs

The properties of different ES in both PCPs and ACPs have been studied and reviewed extensively (Swiatek, 1964; Scharf, 1971; van Wazer, 1971; Meyer, 1973; Tanaka et al., 1979; Rayan et al., 1980; Lee et al., 1986;

Cari´c and Kaláb, 1987; Savello et al., 1989; CavalierSalou and Cheftel, 1991; Fox et al., 1996, 2000; AbdelHamid et al., 2000a,b). Discrepancies between these studies vis-à-vis the influence of ES on different physico-chemical changes exist, probably because of inter-study differences in product formulation (e.g., levels of total protein and intact protein, pH), levels and combinations of ES and processing conditions (e.g., cooker type, degree of shear, time–temperature treatment). However, these studies indicate definite trends that are summarized in Table 5 and discussed below. In practice, individual salts are rarely used, with blends of two or more ES being used widely to combine the best effects of the individual salts (Cari´c and Kaláb, 1993; Abdel-Hamid et al., 2000a,b). Customized blends with specific functionality are prepared by suppliers of ES or developed in-house by manufacturers of PCPs and ACPs. Calcium sequestration. The ability to sequester cal-

cium is closely related to the ability to hydrate and solubilize protein. Calcium sequestration involves the exchange of the divalent calcium cations, which aggregate and interlock the casein molecules in the paracasein network of the cheese or rennet casein, for the monovalent cations of the ES. It is best accomplished

Table 4 Properties of emulsifying salts used in cheese processinga

Group

Emulsifying salt

Formula

Mol. mass (Da)

Citrates

Monosodium citrate monohydrate Trisodium citrate dihydrate Trisodium citrate undecahydrate Sodium dihydrogen phosphate (SDP) SDP monohydrate SDP dihydrate Disodium hydrogen phosphate (DSP) DSP dihydrate DSP heptahydrate DSP dodecahydrate Trisodium phosphate (TSP) TSP hemihydrate TSP-dodeca-hydrate Disodium pyrophosphate Trisodium pyrophosphate Tetrasodium pyrophosphate Pentasodium tripolyphosphate (PSTPP) PSTPP hexahydrate Sodium tetrapolyphosphate Sodium hexametaphosphate (Graham’s salt) Sodium aluminium phosphate

NaH2C6H5O7.H2O NaH2C6H5O7.2H2O 2NaH2C6H5O7.11H2O NaH2PO4 NaH2PO4. H2O NaH2PO4.2H2O Na2HPO4 Na2HPO4.2H2O Na2HPO4.7H2O Na2HPO4. 12H2O Na3PO4 Na3PO4.0.5H2O Na3PO4.12H2O Na2H2P2O7 Na3HP2O7.9H2O Na4P2O7.10H2O Na5P3O10

232 294 714 120 138 156 142 178 268 358 164 173 380 222 406 446 368

– – – 59 51 45 50 40 26 20 44 41 19 64 35 32 58

16.8 75 79.4 85.2 – 39.9 9.3 80 – 2.0 11 – – 13 32 10 14.6

3.75 8.55 7.95 4.5 4.5 4.5 9.1 9.1 9.1 9.1 11.9 11.9 11.9 4.1 6.7–7.5 10.2 9.7

Na5P3O10.6H2O Na6P4O13 (NaPO3)n

476 470 (102)n

45 60 70

– 170.0 157.0

9.7 8.5 6.6

Orthophosphates

Pyrophosphates

Polyphosphates

Aluminium phosphates

NaH14Al3(PO4)8.4H2O

a Compiled from Scharf and Kichline (1968), van Wazer (1971), Cari´c and Kaláb (1993).

P2O5 content (%)

Solubility at 20 °C (%)

pH (1% solution)

8.0

366 Pasteurized Processed Cheese and Substitute/Imitation Cheese Products

Table 5 Emulsifying salts commonly used in pasteurized processed cheese products and their properties during cheese processing Physico-chemical changes during processing

Group Citrates Orthophosphates Condensed phosphates Pyrophosphates

Polyphosphates

Commonly used forms

Calcium sequestration

Buffering action

Para-casein hydration

Fat emulsification

Trisodium citrate Disodium phosphate Trisodium phosphate

Low

High

Low

Low

Low

High

Low

Low

Medium

Medium

Very high

Very high

High to very high

Low to very low

High to low

Very high to low

Disodium pyrophosphates Trisodium pyrophosphates Tetrasodium pyrophosphates Pentasodium tripolyphosphate Sodium tetrapolyphosphate Long-chain polyphosphates

Compiled from various sources: Cari´c and Kaláb (1993), Fox et al. (1996, 2000), Guinee (2002a).

by ES with a monovalent cation and a polyvalent anion, and effectiveness generally increases with valency of the anion. The general ranking of the calcium sequestration ability of the ES used in PCPs is in the following order: polyphosphates  pyrophosphates  orthophosphates  sodium aluminium phosphate  citrates (Nakajima et al., 1975; Wagner and Wagner-Hering, 1981; Lee et al., 1986; Cavalier-Salou and Cheftel, 1991; Cari´c and Kaláb, 1993; Chambre and Daurelles, 2000). However, the sequestering ability, especially of the shorter chain phosphates, is strongly influenced by pH. The increased ion-exchange function at higher pH values is attributed to more complete dissociation of the sodium phosphate molecules resulting in the formation of a higher valency anion (van Wazer, 1971). Thus, for the short-chain phosphates, calcium binding generally increases in the following order: NaH2PO4, Na2HPO4, Na2H2P2O7, Na3HP2O7, Na4P2O7, Na4P2O7 (Cari´c and Kaláb, 1987). An appropriate pH value (e.g., 5.6–6.1) during processing is important for several reasons: it affects protein conformation and hydration, solubility of the ES, calcium sequestration by the ES and ultimately the DEE. It also affects the textural and melting characteristics of the final PCP or ACP. The effect of pH on the texture of processed cheese was clearly demonstrated by Karahadian and Lindsay (1984), using mono-, di- or trisodium phosphates for which the respective pH of a 1%, w/v, solution was 4.2, 9.5 or 13.0. Cheese made with NaH2PO4 (low pH) was dry and crumbly, whereas cheese made with Na3PO4 (high pH) was moist and plastic; the texture of cheese made with Na2HPO4 was intermediate. The buffering capacity of sodium phosphates, in the pH range normally encountered in PCPs (i.e., 5.5–6.0), decreases with increasing chain length and is effectively pH adjustment and buffering.

zero for the long-chain phosphates (n  10). This decrease in buffering capacity with chain length is due to the corresponding decrease in the number of acid groups per molecule; these occur singly at each end of the polyphosphate chain (van Wazer, 1971). The orthoand pyrophosphates have a high buffering capacity in the pH ranges 2–3, 5.5–7.5 and 10–12; thus, in cheese processing, they are not only very suitable as buffering agents but also as pH-correction agents. Within the citrate group, only the trisodium salt has buffering capacity in the pH range 5.3–6.0; the more acidic mono- and di-sodium citrates give over-acid, crumbly cheese with a propensity to oiling-off (Gupta et al., 1984). The pH of PCPs and ACPs is related to the pH of the ES (blend) used and to its buffering capacity. The pH of ACPs made with trisodium citrate or different sodium phosphate ES, at equal concentrations (3%, w/w), decreases in the following order: tetrasodium pyrophosphate ⬇ trisodium citrate ⬇ pentasodium tripolyphosphate  disodium hydrogen phosphate  sodium polyphosphate (Cavalier-Salou and Cheftel, 1991). The pH of cheese increases linearly with the concentration of ES in the range 0–3%, w/w, for trisodium citrate, tetrasodium pyrophosphate, sodium tripolyphosphate and disodium hydrogen phosphate (Cavalier-Salou and Cheftel, 1991). Similar observations have been made by others (Templeton and Sommer, 1936; Swiatek, 1964; Gupta et al., 1984) for PCPs. However, Swiatek (1964) reported that increasing the concentration of polyphosphate had little effect on the pH of PCPs. The increase in para-casein hydration during the manufacture of PCPs and ACPs is supported by the large increases in the levels of water-soluble N and non-sedimentable N (when a dilute mixture of cheese and water is centrifuged; Templeton and Sommer, 1936; Ito et al., 1976; Casein hydration and dispersion.

Pasteurized Processed Cheese and Substitute/Imitation Cheese Products 367

Thomas et al., 1980; Lee et al., 1986; Cavalier-Salou, 1991). While all the commonly used ES increase casein hydration, there are large differences between the effects of the different salts at the level (3%, w/w) typically used in cheese processing. The results of some studies (Templeton and Sommer, 1936; Lee et al., 1986) indicate that para-casein hydration and dispersion decrease with the chain length of sodium phosphates while the results of Cavalier-Salou and Cheftel (1991) suggest the opposite trend. Similarly, Thomas et al. (1980) reported that trisodium citrate gives lower casein hydration than ortho- or pyrophosphates while others (Templeton and Sommer, 1936; Cavalier-Salou and Cheftel, 1991) found it gave levels that were comparable or higher. These discrepancies may be related to differences in the blend formulation and composition, concentration of ES, processing time (Csók, 1982) and pH. Casein hydration for sodium phosphates and trisodium citrate decreases as the pH falls in the range 7.5–5.5, with the decrease at pH 6.5 being particularly large for orthophosphates and trisodium citrate (Lee et al., 1986); the more highly condensed phosphates are the least susceptible to a decrease in pH. The effectiveness of different ES in promoting emulsification in PCPs, as determined from electron microscopy and oiling-off studies, is in the following general order: sodium tripolyphosphates  pyrophosphates  polyphosphates (P 10)  orthophosphates ⬇ citrates (slightly)  basic sodium aluminium phosphates (Templeton and Sommer, 1936; Roesler, 1966; Rayan et al., 1980; Thomas et al., 1980; Cavalier-Salou and Cheftel, 1991). This trend generally coincides with that of calcium sequestration.

Ability to promote emulsification.

Linear condensed phosphates undergo varying degrees of hydrolysis and conversion to orthophosphates during processing and storage of PCPs (Glandorf, 1964; Roesler, 1966; Scharf, 1971; van Wazer, 1971; Meyer, 1973). Hydrolysis proceeds rapidly to tripolyphosphates and pyrophosphates and then more slowly to orthophosphates. The extent of degradation increases with processing time and temperature, product storage time and temperature, moisture level in the final product and phosphate chain length (Glandorf, 1964). Hydrolysis decreases as the concentration of added ES increases (Glandorf, 1964). In experiments with pasteurized processed Emmental, the level of polyphosphate breakdown (n  4) during melting at 85 °C varied from 7% of total for block product (processed for 4 min) to 45% for spreadable product (processed for 10 min; Roesler, 1966). While the breakdown of condensed phosphates to monophosphates was complete in the spreadable cheese after Hydrolysis (stability).

7 weeks, low levels were detectable in block processed cheese even after 12 weeks. The greater extent of polyphosphate degradation in the spreadable processed cheeses is also expected due to their higher pH and moisture values (Scharf, 1971; van Wazer, 1971). The effect of temperature on the hydrolysis of phosphates and polyphosphates in dilute solution (1%, w/v) has been demonstrated clearly by Berger et al. (1989). The consequences of hydrolysis probably include variations in the functionality (e.g., buffering capacity, calcium sequestration) of the ES blend with processing conditions as the ratio of short-chain to long-chain phosphates increases; ultimately, this may affect product pH and degree of casein hydration/emulsification. Continued hydrolysis may lead to several problems during storage. The change in hardness that is frequently observed in PCPs during storage, and which is more pronounced as the storage temperature is raised from 10 to 30 °C, has been attributed to the hydrolysis of polyphosphates to orthophosphates (Ney, 1988; Tamime et al., 1990; Chambre and Daurelles, 2000). This is because the continued effects (calcium sequestration, protein hydration and emulsification) of ES differ as the proportions of phosphates of different chain length change. Other problems associated with the hydrolysis of polyphosphates is an increased propensity to crystallization of ES in the PCP, due to precipitation of dodecahydrate disodium orthophosphate (Na2HPO412H2O) on product storage (Scharf and Kichline, 1969), and labelling difficulties in relation to declaration of the ES used. Tendency to crystallize during storage Occurrence and types of crystals. A common defect in

processed cheese, known at a commercial level as crystallization, is the formation of crystalline deposits on the surface and in the interior of processed cheese. The deposits are visible to the naked eye as a delicate white powdery covering on the surface of the cheese, and sometimes are referred to as a haze or a bloom. A coating of crystals imparts a dull grainy, uneven appearance to the surface, especially in slices, which is otherwise normally smooth and shiny; hence, products containing crystal deposits are not aesthetically pleasing and can sometimes be rejected by the retailer/consumer who may confuse the white deposits with mould contamination. The crystalline deposits are frequently observed by SEM or TEM in PCPs (Fig. 12; Pommert et al., 1988; Cari´c and Kaláb, 1993; Kaláb, 1995). Using various techniques such as electron microscopy, X-ray diffraction analysis, infrared spectroscopy and energy dispersive X-ray spectrometry analysis (EDSA), several crystal types have been identified, including monoclinic calcium

368 Pasteurized Processed Cheese and Substitute/Imitation Cheese Products

• The incomplete dissolution of the ES (especially when excess ES is added), the carryover of crystal inclusions from the natural cheese (e.g., insoluble tyrosine in Swiss cheese (Flückiger and Schilt, 1963), or calcium lactate from Cheddar cheese (Brooker et al., 1975; Brooker, 1979)). • The use of excess lactose (e.g., in added skim milk powder or whey) which leads to the supersaturation and formation of lactose crystals which may then act as nuclei for the crystallization of mineral species (e.g., Ca) which are present at supersaturated levels. The tendency to lactose crystallization may, however, be reduced if the lactose is first hydrolysed enzymatically (Patocka and Jelen, 1988).

Figure 12 Light micrograph of processed cheese crystals of insoluble calcium phosphate (P) and sodium citrate (C), emulsified fat in globules (F) and bacteria (b) dispersed in the protein matrix (M). Bar corresponds to 5 m (modified from Cari´c and Kaláb, 1993).

pyrophosphate dihydrate, disodium phosphate dodecahydrate, unreacted melting salts, tyrosine, calcium citrate, lactose and complexes of various materials such as calcium, fatty acids, protein and lactose (Scharf and Michnick, 1967; Scharf and Kichline, 1968, 1969; Morris et al., 1969; Scharf, 1971; Uhlmann et al., 1983; Klostermeyer et al., 1984; Yiu, 1985; Bester and Venter, 1986; Pommert et al., 1988; Kondo et al., 1990). The composition of crystalline structures in natural cheeses and PCPs was studied by Washam et al. (1985) using SEM, EDSA and X-ray diffraction. Yiu (1985) identified calcium phosphate crystals by optical microscopy using Alizarin Red as a stain specific for calcium. Calcium phosphate aggregates were observed to grow beyond the size of 30 m in diameter in PCP made with sodium diphosphate (Rayan et al., 1980). Other crystalline inclusions may be calcium lactate in the form of randomly arranged aggregates up to 80 m in diameter (Brooker et al., 1975; Brooker, 1979) or crystalline amino acids such as tyrosine, e.g., in Swiss cheese (Flückiger and Schilt, 1963). Careful standardization of the moisture content and the use of phosphates as ES were credited with the lower incidence of calcium lactate crystals in processed cheese than in natural cheese. Crystals of a tertiary sodium calcium citrate, NaCaC6H5O7, were identified in PCPs by Klostermeyer et al. (1984). Eliminating citrate from the ES blend may prevent their development. Major causes of crystallization. The main causes of

crystallization in PCPs and ACPs probably include: • The formation of insoluble calcium phosphate crystals (as a result of the interaction between the anion of the ES and the Ca of para-casein).

Treatments which lead to dehydration of the cheese, such as smoking, are conducive to crystal growth on the processed cheese surface. Other factors (e.g., pH) may contribute to the formation of crystal deposits involving ES in PCPs and ACPs, as discussed below. Effects of calcium phosphate level and pH. Hard rennet-curd cheeses tend to be supersaturated with calcium phosphate (cf., Morris et al., 1988). In Cheddar cheese, only ⬃38% of total calcium (i.e., ⬃750 mg/100 g) is soluble at pH 5.0. This is equivalent to a calcium concentration of ⬃720 mg/100 ml cheese serum (Guinee et al., 2000c), which is ⬃6-fold the total soluble Ca concentration in milk at pH 5.0 (van Hooydonk et al., 1986). Hence, it is not surprising that crystalline inclusions already noticeable in natural cheese (Brooker et al., 1975; Blanc et al., 1979; Bottazzi et al., 1982; Paquet and Kaláb, 1988), particularly those composed of insoluble calcium phosphate, which are not affected by processing, are subsequently also found in processed cheese. For a given formulation, the risk of crystal formation in the resultant PCP probably decreases as the levels of calcium and phosphate in the natural cheese used decrease. Hence, it would be expected that the risk would be in the following order: Camembert-type cheese  Blue cheese  Cheshire  Cheddar ⬇ Mozzarella  Emmental. The addition of relatively large quantities of sodium phosphate ES (e.g., up to 3%, w/w) accentuates the risk of precipitating salts containing phosphate. Moreover, much of the water in PCPs is bound and, presumably, not available for solution of salts or other solutes such as lactose and amino acids. The levels of bound and free water in PCP made by heating at 95 °C for 1–30 min varied from 1.4 to 1.6 g/g solidsnon-fat (SNF) and from 0.5 to 0.7 g/g SNF, respectively (Csók, 1982). For a given formulation and processing conditions, the pH and the concentration of ES have major effects on the susceptibility to crystal formation, especially where sodium orthophosphates are used as ES. This is

Pasteurized Processed Cheese and Substitute/Imitation Cheese Products 369

pH  pKa  log

salt(e.g. Na2HPO4) acid(e.g. NaH2PO4)

The salt and the acid forms differ markedly in their solubility in aqueous solution (Table 4) and hence their ratio determines the likelihood of crystallization at a given concentration of ES. At the pH of PCPs (pH 5.5–6.0), NaH2PO4 and Na2HPO4 are the major forms present, irrespective of the type of orthophosphate added, since the pKa values for H3PO4, are 2.14, 6.86 and 12.4 at 25 °C. The ratio of Na2HPO4 to NaH2PO4 varies from 0.1 to 0.15 in PCPs, depending on pH. At the temperatures used during processing (e.g., 75 °C), the disodium salt occurs mainly in the form of NaH2PO47H2O and is very soluble (80%, w/v; Scharf and Kichline, 1968). At temperatures 35 °C, the dodecahydrate disodium salt (Na2HPO412H2O) is the main form of the disodium salt in PCPs (Scharf and Kichline, 1968), and its solubility (⬇1.5–2.5%, w/v, Na2HPO4) is much lower than that of the monosodium salts (Table 4). Hence, in experimental PC slices made using sodium phosphates, Na2HPO412H2O is the predominant crystalline species (Scharf and Kichline, 1968), and its tendency to crystallize increases markedly with small increases in the pH of the PCP in the pH range 5.5–6.5. However, there is an inverse relationship between the pH required in the PCP to prevent the formation of crystals and the phosphate content of the PCP (Fig. 13). Trisodium citrate is normally used with phosphate in PCPs. However, at the pH of PCPs (pH 5.5–6.0), the forms of citrate present are Na2HC6H5O7 and NaH2C6H5O7 (e.g., at a molar ratio of ⬃8:1 at pH 6.0). In contrast to Na2HPO412H2O, the sodium citrates are highly soluble (Table 4). However, the resultant calcium citrates are relatively insoluble, e.g., the solubility of calcium citrate tetrahydrate, Ca(C6H5O7)2, at 18 °C is 0.08%, w/v, and decreases on reducing the temperature (cf., Scharf and Kichline, 1969). Hence, refrigerated storage of PC increases the susceptibility to the formation of calcium citrate deposits. Bacteriocidal effects. Cheese is the main ingredient used in PCPs. The occurrence of pathogens in cheese has been reviewed recently (Fox et al., 2000). While natural cheese is a relatively safe food, pathogenic bacteria may occur in cheese, especially in those made from raw milk (Fox et al., 2000). However, very few food-poisoning outbreaks have been attributed to cheese, e.g., a total of 32 outbreaks in western Europe, USA and Canada in the period 1970–1997 from an

7.5 pH of processed cheese

because the pH determines the level of dissociation and hence the ratio of salt-to-acid forms of the salt, according to the Henderson-Hasselbalch Equation:

7 6.5 6 5.5 5 1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

P2O5 content, %, w/w

Figure 13 Relationship between pH and phosphorus content in the development of crystals on processed cheese slices exposed to air. The cheese slices beneath the regression line (䊉) did not develop crystals whereas samples above (䊊) developed crystals (NaH2PO412H2O), as detected using X-ray diffraction analysis; the regression line (–) was fitted to the experimental data points (䊊, 䊉). (redrawn from Scharf and Kichline, 1968).

estimated cheese production of 235 million tonnes. The micro-organisms involved in these outbreaks of food poisoning included Listeria monocytogenes, Clostridium botulinum, Salmonella spp., Staphylococcus aureus and Escherichia coli O157. The main reasons for the occurrence of these bacteria, which apart from the spores of Cl. botulinum, are killed by pasteurization (72 °C  15 s), in cheese are poor starter activity, poor plant hygiene, gross environmental contamination and faulty pasteurization (Fox et al., 2000). Cheese processing normally involves the use of a temperature (70–95 °C for 4–15 min) that kills the vegetative cells of most bacteria, yeasts and moulds, but not spores. Hence, PCPs may contain viable spores and their vegetative cells, especially of the genus Clostridium, which may originate in the cheese (especially if cows are fed poor-quality silage) or the formulation ingredients and condiments (Meyer, 1973; Thomas, 1977; Wagner and Wagner-Hering, 1981; Sinha and Sinha, 1988; Warburton et al., 1986; Cari´c and Kaláb, 1987). Conditions favourable for the germination of spores (e.g., Cl. tyrobutyricum, Cl. sporogenes) in PCPs during storage include the heat activation of spores at the high processing temperatures, the anaerobic environment and the relatively high pH, water activity (Rüegg and Blanc, 1981; Csók, 1982) and moisture content of PCs, compared to most natural cheeses (Russell and Gould, 1991). This may lead to problems such as blowing of cans, protein putrefaction and off-flavours. Perhaps, more importantly, contamination with Cl. botulinum spores could lead to the growth and formation of toxin on storage at a high temperature (e.g., 30–35 °C) to an extent dependent on ES type, moisture level and pH (Kautter et al.,

370 Pasteurized Processed Cheese and Substitute/Imitation Cheese Products

1979; Tanaka et al., 1979; Eckner et al., 1994). Bacteria may also gain access to PCPs following manufacture. Various studies have been undertaken to determine the ability of PCPs to host pathogens and thereby simulate the effects of post-processing contamination. These studies have monitored the changes in populations of various pathogenic bacteria inoculated into the cooled PCP, or the development of bacterial toxin during storage (Kautter et al., 1979; Jaskulka et al., 1995). Glass et al. (1998) inoculated PCP slices (water activity, 0.926–0.918; pH, 5.61–5.78; NaCl, 2.5%, w/w) with various strains of pathogenic bacteria and followed their growth over 96 h incubation at 30 °C. The populations of the Listeria monocytogenes, Escherchia coli 0157:H7 and Salmonella serotypes which were inoculated at levels of 1.3  103 to 4  103 cfu/g slice decreased by 0.5–2.0 log counts. In contrast, the population of inoculated Staphylococcus aureus in PCP remained constant over the 96-h period. In a large predictive modelling study involving the inoculation of PCPs with Cl. botulinum, Jaskulka et al. (1995) developed a prediction equation to relate the composition of the spreads to clostridial toxigenosis after 6 months’ storage at 30 °C. Bacterial spoilage in PCPs is minimized by a number of factors:

Some ES also possess bactericidal properties. Polyphosphates and orthophosphates inhibit the growth of various Salmonella species and many Gram-positive bacteria, including Staphylococcus aureus, Bacillus subtilis, Clostridium sporogenes and Cl. botulinum, with the effect increasing with the level added (van Wazer, 1971; Tanaka et al., 1979, 1986; Wagner, 1986; Ter Steeg et al., 1995; Loessner et al., 1997). The inhibitory effect of sodium orthophosphates depends on the moisture and the sodium salt levels and pH of the PCP (Tanaka et al., 1986). However, polyphosphates were found to be much more bacteriostatic than orthophosphates in laboratory media (Wagner, 1986) and than both orthophosphates and pyrophosphates in PCPs (Eckner et al., 1994). The general bacteriostatic effect of phosphates may reflect their interactions with bacterial proteins and sequestration of calcium, which generally serves as an important cellular cation and cofactor for some microbial enzymes (Stanier et al., 1981). Citrates appear to have no bacteriostatic effect (Chambre and Daurelles, 2000) and may even be subject to microbial degradation, thus reducing product keeping quality (Cari´c and Kaláb, 1987). Tanaka et al. (1979) reported that the inhibitory effect of sodium orthophosphates on the growth of Cl. botulinum in pasteurized PCSs with moisture levels in the range 52–58%, w/w, was superior to that of sodium citrates.

• The addition of preservatives, such as nisin, ascorbic acid, sodium bisulphite, sodium sorbate, sodium propionate or sodium nitrite (Somers and Taylor, 1987; Gouda and El-Zayat, 1988; Ryser and Marth, 1988; Delves-Broughton, 1990; Russell and Gould, 1991; Roberts and Zottola, 1993; Plockova et al., 1996) or the use of natural cheese made using nisinproducing lactococci (Zottola et al., 1994). • Formulating to keep the pH, water activity and moisture level as low as possible and to keep the NaCl level moderately high (e.g., 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. • Using ES with bacteriostatic properties. • 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.

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 °C, to eliminate post-pasteurization contamination (Schär and Bosset, 2002).

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).

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 371

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: • the mixing and the uniform distribution of all ingredients throughout the blend; • 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); • 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); • dispersion of free (non-globular) fat/oil and moisture; • 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

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 °C (cf. Swiatek, 1964; Rayan et al., 1980; Kaláb 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 °C 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 °C 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 °C 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

Table 6 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 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

Elasticity

Spreadability

Heat-induced flowability

c b

c b

NA NA

c b

b c

b c

b c

b c

c c c

c c NA

b b NA

b b b

NA NA NA NA NA NA

NA NA NA NA NA c

b b b b c NA

NA NA NA c NA NA b b

b b

c c

NA c

c c

c c

b b

b b

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 qor decreases p. 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 ⬃60%, 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, Csøk (1982) reported that on holding a cooked processed cheese at 95 °C, the bound water increased to a maximum (e.g., at ⬃15 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 °C 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 (Rayan et 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 °C and held for 5 h, was markedly higher than in the control, which was cooled after 3 min at 85 °C (Kaláb et al., 1987). The electron-dense zones may correspond to regions of

Pasteurized Processed Cheese and Substitute/Imitation Cheese Products 373

Processing temperature and shear

Water in processed cheese, g/g solids-non-fat

1.65

1.35

1.05

0.75

0.45 0

10 20 Holding time, min

30

Figure 14 Changes in the level free water (䉭) and total bound water (䉱) in pasteurized processed cheese as a function of processing time at 95 °C (redrawn from Csøk, 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 °C but was absent, or markedly less, in fresh PCF held for 3 min at 82 °C (Kaláb 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; Kaláb 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´c 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.

According to Meyer (1973), processing at a temperature 95 °C 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 °C 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, Lee et al. (1981) observed a positive relationship between the firmness of processed Emmental and the processing temperature in the range 80–l40 °C. 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--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 °C gave a significant increase in flowability and decrease in the spreadability of fatfree PCP; the firmness was highest at 70 °C and lowest at 80 °C. 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 et al., 1980; Harvey et al., 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 al.,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 et al. (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 17.8 °C) 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 °C, 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 (10 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 Processed Cheese and Substitute/Imitation Cheese Products 375

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 (Kaláb 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 (Kaláb 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 °C 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 Kaláb 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 m in diameter with no creaming (melting time, 4 min), ⬃0.5 m with mild creaming (melting time,

Figure 15 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 °C. Bar corresponds to 0.2 m (adapted from Kaláb et al., 1987).

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, 65% total) cheese. Type B rework imparts firmness and elasticity to block processed cheese blends (Meyer, 1973). Unlike regular rework (type B), type C rework has a very strong creaming effect and can lead very quickly to over-creaming. Hence, the inclusion of type C rework should be avoided, except at very low levels (1%, w/w). Kaláb et al. (1987) evaluated the effect of the following types of commercially produced rework in an experimental PCF (45%, w/w, moisture) made with trisodium citrate or trisodium orthophosphate: regular cheese food slice (RPCF), quickly frozen cheese food slice – prepared by cooling from 82 to 4 °C in 10 min and then freezing (QFPCF), and hot melt cheese food slice (HMPCF), – prepared by slow cooling from 82 to 4 °C over 5 h and then freezing. Added at a level of 20%, w/w, all types of rework increased the apparent viscosity of the hot PCF (immediately after processing) and the firmness of the stored PCF, and reduced the flowability of the melted PCF (Fig. 16), with the apparent viscosity and flowability being the most affected. The effects were highest for the HMPCF and lowest for the QFPCF and increased with the level of HMPCF. The mechanism by which rework exerts its effects on the physical properties of PC is not clear. However, tentative explanations include: • Further heating of precooked cheese may cause a higher degree of thermally-induced dehydration and aggregation of the para-casein (especially if hot melt is used), thereby increasing the degree of product elasticity. • A more effective dissolution of ES in the rework (due to the longer contact time) which leads to a more rapid hydration of the fresh para-casein introduced to the new blend. • A higher effective concentration of protein in the processed blend due to the high DE and, hence, high level of pseudo-protein particles in the rework (especially in hot melt). A high protein concentration would give a high viscosity, which in turn would give a more efficient fat dispersion and emulsification in the fresh blend.

Apparent viscosity (Pas)

376 Pasteurized Processed Cheese and Substitute/Imitation Cheese Products

1000 800 600 400 200 0

Flowability of the melted cheese food (%)

Control

QFPCF

RPCF

HMPCF

QFPCF

RPCF

HMPCF

70 60 50 40 30 20 10 0 Control

Type of processed cheese foods

Figure 16 Apparent viscosity and flowability, on heating at 140 °C for 6 min, of different types of processed cheese food: control, made with a regular formulation with no added rework, stored at 4 °C after manufacture and tested at 24 h; QFPCF, as for the control except that it contained 20%, w/w, added rework which was a regular processed cheese food which was frozen/held at 10 °C immediately after manufacture; RPCF, as for the control except that it contained 20%, w/w, added rework which was a regular processed cheese food which was held at 4 °C immediately after manufacture; HMPCF, as for the control except that it contained 20%, w/w, added rework which was a processed cheese food which was held for an extended time (5 h) at 82 °C (drawn from data of Kaláb et al., 1987).

Blend ingredients: cheese base (CB), ultrafiltered milk retentate (UFMR), cheeses from high heattreated milks and whey proteins

Attempts to reduce formulation costs of PCPs and improve end-product consistency have led to extensive investigation on the development of, and study of the effects of, ingredients which are more cost-effective than cheese (Mann, 1970, 1981, 1984, 1990, 1997). In this regard it has been attempted to replace blend cheese by milk ultrafiltrate (Sood and Kosikowski, 1979; Anis and Ernstrom, 1984) or CB (Ernstrom et al., 1980; Park et al., 1992; Simbuerger et al., 1997). A major difference between these materials and rennet curd cheeses is that they contain whey proteins in addition to casein or para-casein (as in natural cheeses). Whey proteins may be also added to PCPs and ACPs in the form of WPCs (Schulz, 1976; Savello et al., 1989; Nishiya et al., 1990; Hill and Smith, 1992; Kaminarides and Stachtiaris, 2000), total milk proteinates (Abou El-Nour et al., 1996), co-precipitates (Thomas, 1970) and cheese with a high level of whey

protein, e.g., UF cheese or acid-heat coagulated curd (Kaláb and Modler, 1985; Collinge and Ernstrom, 1988; Collinge et al., 1988; Kaláb et al., 1991). Production of CB generally involves ultrafiltration and diafiltration of skim milk, inoculation of the retentate (typically 20–25%, w/w, dry matter) with a lactic culture, incubation to a set pH (5.2—5.8), pasteurization and scraped-surface evaporation to, typically, 60%, w/w, dry matter (Ernstrom et al., l985; Ganguli, 1991; Sutherland, 1991). However, rennet may be added to the retentate to form a curd from which a small quantity of whey is removed (compared to that in natural cheese manufacture) and which is dry-salted and pressed, and stored as natural cheese. The retentate may also be treated with lipase to enhance the flavour of the final PCP; it was claimed (Aly et al., 1995) that up to 80%, w/w, of Ras cheese solids could be replaced by the lipase-treated retentate, with the resultant PCPs having flavour and consistency considered to be superior to those of the control. A recent patent submission (Hyde et al., 2002) describes the preparation of CB by the acidification and cooling of a blend comprising of one or more powdered milk protein ingredients, milk fat, NaCl, edible acid and/or preservative. Increasing the level of substitution of natural cheese by CB, made in the conventional manner, or UFMR, normally results in a ‘longer-bodied’, firmer PCP which is less flowable on heating (Collinge and Ernstrom, 1988; Tamime et al., 1990; Younis et al., 1991). The lower flowability may be attributed to a number of factors, including: • a higher degree of intact casein in the CB; • the presence of whey proteins in the CB (⬃8.7%, w/w) which are denatured and complex with para--casein to form a pseudo-gel at the high processing temperature (85–90 °C for 3 min; cf., Doi et al., 1983a,b, 1985). The adverse effect of whey proteins on the functionality of PCPs is probably due to their ability to form thermally induced para--casein/-lactoglobulin aggregates or gels at the high temperature (typically ⬃98 °C) reached during baking/grilling, when present in significant quantities (e.g., 3–7%, w/w) in the cheese. The tendency to aggregate and gel is probably accentuated by the high levels of protein and soluble calcium in the cheese (Doi et al., 1983a,b; Jelen and Rattray, 1995). On setting, the gels would impede the flow of the cheese as the fat phase melts and coalesces (Sood and Kosikowski, 1979; Savello et al., 1989). However, the effects on flowability vary depending on the method of preparation of the CB and UFMR and the subsequent heat treatment during processing: (i) Decreasing the pH of milk, from 6.6 to 5.2, prior to UF resulted in CBs with lower calcium levels

Pasteurized Processed Cheese and Substitute/Imitation Cheese Products 377

and processed products with improved meltability (Anis and Ernstrom, 1984). (ii) Rennet treatment of the UF retentate results in poorer meltability (Anis and Ernstrom, 1984), an effect which may be attributed to the higher degree of interaction between -lactoglobulin and para--casein (than with native casein) during subsequent processing (Doi et al., 1983 a,b). (iii) Treatment of retentate with exogeneous proteinases (i.e., Savorase-A, and enzymes from Aspergillus oryzae and Candida cylindracea), which increase the level of proteolysis in the CB, yields PCPs which are softer and more meltable than those made with untreated CB (Sood and Kosikowski, 1979; Tamime et al., 1990, 1991). (iv) Increasing the processing temperature in the range 66–82 °C results in processed products with reduced meltability, an effect attributed to the gelation of whey proteins at the higher temperatures, especially when rennet-treated CB is used (Collinge and Ernstrom, 1988). Similarly, the addition of calcium co-precipitate (to a level of 5%, w/w) to processed Cheddar reduces flowability, with the effect decreasing as the level of proteolysis in the Cheddar cheese increases (Thomas, 1970). The direct addition of whey proteins to PCPs and ACPs, as a substitute for cheese or casein, generally has been found to increase the fracture stress and firmness, and reduce the flowability of the heated cheese (Savello et al., 1989; Gupta and Reuter, 1993; Abou-El-Nour et al., 1996; Gigante et al., 2001; Mleko and Foegeding, 2001). In this context, it is noteworthy that a PCP which is resistant to flow on cooking, can be prepared by adding a heat-coaguable protein (3–7%, w/w, lactalbumin or egg albumen), at a temperature 70 °C, to the PCP blend on completion of processing (Schulz, 1976). In contrast, Kaminarides and Stachtiaris (2000) reported that the hardness of PCPs with similar final composition decreased from 3- to 5-fold with the replacement of Kasseri cheese by added WPC (24%, w/w, protein, added at a level of 9–39%, w/w) and soybean oil. However, the quantity of ES used was added according to the quantity of added cheese in the blend and was, therefore, greatly reduced from 2.6%, w/w (control), to 1.5%, w/w, at the highest WPC level. French et al. (2002) investigated the effects of replacing sodium caseinate by a range of milk protein concentrates (MPCs, 72–82.5%, w/w, protein), whey protein concentrates (WPCs, 80 or 34%, w/w) or lactalbumin (80%, w/w, protein) on the hardness, cohesiveness and springiness of PCP. For each ingredient, the effects depended on the ratio of the different ES used, i.e., trisodium citrate and disodium orthophosphate. At both ES ratios, the

MPCs gave higher hardness than the control, while the lactalbumin and the WPCs gave lower hardness; however, experimental details are scarce. The use of WPCs as a replacement for cheese solids has also been found to accelerate storage-related flavour deterioration, which increased with the level of WPC added in the range 0–20%, w/w (Thapa and Gupta, 1992). In contrast to the above studies, the flowability of pasteurized processed Ras-Quark cheese was enhanced by the substitution of WPC for Quark, with the effect becoming more pronounced as the level of added whey protein was increased from ⬃3 to 6%, w/w (Abd El-Salam et al., 1996). A similar trend was reported by Al-Khamy et al. (1997) who found that the magnitude of the effect varied with the type of ES and storage time of the PCP at room temperature. Similar observations were made by Kaláb et al. (1991) who substituted a non-flowable acid/heat-coagulated white cheese (AHC) for Cheddar cheese in a PCP. The flowability of the PCP increased by ⬃40% as the level of AHC was increased from 0 to 16%, w/w, and then decreased to a value slightly higher than the control as the level of AHC was further increased to 33%, w/w; the addition of 16 or 33%, w/w, AHC was equivalent to adding 0.64 or 1.28%, w/w, whey protein. Discrepancies between the foregoing studies vis-à-vis the effect of added whey protein on the functional characteristics of PCPs and ACPs may be due to differences in the characteristics (e.g., level of denaturation, pH, levels of Ca and protein, particle size) and format (e.g., as AHC, WPC, WPI) of the whey protein added and in PCP formulations (i.e., type of casein, type of ES, cheese age) and processing conditions. These factors may determine the degree of aggregation of the whey proteins, aggregate size and interaction with the casein/para-casein; they may also influence the pH of the blend during processing which, as discussed below, has a major influence on casein hydration and the characteristics of the end-product. Blend ingredients: caseins

Caseinates and caseins (acid and rennet) are used widely in PCPs and ACPs, the main attractions being lower cost (relative to cheese protein), a consistent level of intact casein, good emulsifying capacity of caseinates and good stretching properties of rennet casein which makes it ideal for APC. Caseinates (especially sodium) find most applications in processed cheese spreads (PCSs) where their high water-binding capacity and good emulsifying properties promote a desired creaming effect. Gouda et al. (1985) reported that full replacement of cheese solidsnon-fat by calcium caseinate caused deterioration in spreadability of Cheddar PCS, probably due to an excessive creaming effect. However, partial replacement in a

378 Pasteurized Processed Cheese and Substitute/Imitation Cheese Products

formulation (with skim milk powder, calcium caseinate, ripe Cheddar, butter oil and ES at respective levels of 6–8, 5–7, 15, 14 and 3%, w/w) improved the meltability of the PCS, suggesting a desirable level of creaming. Caseinates may be used in spreadable ACPs (Hokes et al., 1989; Marshall, 1990). Rennet casein, despite its insolubility, is generally preferred in the manufacture of APC, which is the major imitation cheese product (McCarthy, 1990; Fox et al., 2000; Guinee, 2002b; ‘Analogue Cheese products (ACPs)’). Recently, the use of casein hydrolysates has been found to reduce the quantity of ES required for the emulsification and formation of a stable product (Kwak et al., 2002). When added at a level of 3%, w/w, to replace the ES completely, the hydrolysate gave a PCP which on cooking had a high flowability but excessive oiling-off.

(Piergiovanni et al., 1989; Kombila-Moundounga and Lacroix, 1991). However, Hong (1990) found that replacement of experimental cheeses by lactose at levels of 5–20%, w/w, reduced the firmness of PCP. Excess lactose may also increase the propensity to crystallization in PCPs during storage, with the formation of mixed crystals containing various species, e.g., Ca, P, Mg, Na, tyrosine and/or citrate. Owing to the relatively high level of bound water in PCPs (a maximum of 1.6 g/g solidsnon-fat; Csøk, 1982), the effective lactose concentration in the free moisture phase may easily exceed its solubility limit (⬃15 g/100 g H2O at 21 °C). This may result in the formation of lactose crystals which could serve as nuclei for the crystallization of mineral species which are supersaturated (Uhlmann et al., l983; ‘Characteristics of different ES in the manufacture of PCPs and ACPs’).

Blend ingredients: co-precipitates

Compositional parameters

Co-participates are protein products containing casein and whey proteins and are formed by heat treatment of the milk and subsequent precipitation of the protein complex by acidification and calcium addition (Mulvihill, 1992). Depending on the level of CaCl2 added, three types may be obtained, namely, high-, medium- and low-calcium co-precipitates containing 2.5–3.0, 1.0–2.0 and 0.5–0.8%, w/w, calcium, respectively. The use of various co-precipitates at a level up to 5%, w/w, of the blend material resulted in PCPs with increased firmness and sliceability, lower meltability/flowability and higher emulsion stability (Thomas, 1970). Because of their excellent emulsifying capacity, Thomas and Hyde (1972) reported that the level of ES can be reduced from 3.0 to 2.0–2.5%, w/w, if calcium co-precipitate is used in the blend at a level of 2–3%, w/w. However, a high level (3%, w/w) significantly reduced the flowability of the PC, especially when a high proportion of young cheese was used.

Although the rheological attributes of PCPs with the same moisture content can differ significantly due to variations in blend composition and processing conditions, increasing moisture content yields products which are softer, less elastic and viscous, sticky and spreadable (Kairyukshtene and Zakharova, 1982; Salam, 1988; Gupta and Reuter, 1993). Marshall (1990) studied the effect of varying moisture-in-non-fat substances (MNFS; 50, 55 and 60%, w/w) and fat level, which was varied from 5.0 to 20%, w/w, at each MNFS level, on the rheological properties of model ACPs. Rheological measurements by uniaxial compression at large deformation included maximum stress, max, deformation at max, DMS; work to max, WMS; other analyses included stiffness, measured by low deformation compression, and work to fracture (WF), analysed by measuring cutting force. There was an inverse relationship between the levels of MNFS and protein; however, details on the actual levels of dry matter and protein were not presented. Linear regression analysis indicated that DMS was negatively related to the MNFS content and positively to the protein content. Multiple regression analysis showed that an increase in the levels of both fat and MNFS resulted in marked decreases in DMS, WMS, stiffness and WF. However, as discussed earlier, the DE for a given fat content and protein-to-fat ratio has a major effect on the rheological and cooking properties of PCPs. Hence, as postulated by Shimp (1985), the protein-to-fat ratio is a major determinant controlling the rheological and cooking properties of PCPs, but only at levels of emulsification below the maximum, or the critical, DE. pH has a major effect on the texture of commercial and experimental PCPs (Scharf, 1971; Gupta et al., 1984; Shimp, 1985), in which the pH is varied by changing, among other factors, the type and level of ES.

Blend ingredients: skim milk powder

Addition of skim milk powder to PCP blends at a level of 3–5%, w/w, results in softer, more spreadable products (Kairyukshtene and Zakhrova, 1982). However, higher levels (7–10%, w/w) lead to textural defects such as crumbliness and lack of cohesiveness (Thomas and Hyde, 1972; Kairyukshtene and Zakhrova, 1982) and may remain undissolved. However, a high level may be added if the skim-milk powder is first reconstituted and then precipitated by proteolytic enzymes or citric acid, and the curd added to the blend (Thomas, 1970). Blend ingredients: lactose

Added lactose, in the range of 0–5%, w/w, results in lower spreadability, lower water activity and increased propensity to non-enzymatic browning in PCPs during processing (especially at a high temperature) and storage

Pasteurized Processed Cheese and Substitute/Imitation Cheese Products 379

Low pH (4.8–5.2), e.g., due to the use of monosodium citrate, monosodium phosphate or sodium hexametaphosphate alone, gives short, dry, crumbly cheese which shows a high propensity to oiling-off (Gupta et al., 1984). High pH values (6.0) give PCPs that tend to be very soft and flow excessively on heating (Gupta et al., 1984). Similar trends were noted by Lee et al. (1981), who noted that increasing the pH of PCP from 5.75 to 6.05, by increasing the level of added sodium polyphosphate, was accompanied by a 2-fold decrease in hardness (as measured by penetrometry). Marchesseau et al. (1997) studied the effect of pH (5.7, 6.1, 6.7) in experimental PCPs made using a standard formulation with the same type (commercial polyphosphate blend) and level of ES, by adding NaOH or HCl to the blend before cooking. Increasing the pH resulted in marked decreases in the elastic shear modulus (G, index of elasticity and firmness) and loss modulus (G, index of viscous component of stress; 12-fold) and an increase in the loss tangent (tan , from 0.25 to 1.39). Scanning electron microscopy analysis of the PCPs showed that increasing the pH from 5.7 to 6.1 led to a decrease in the level of the para-casein aggregation and a finer para-casein matrix, and a further increase to pH 6.7 led to a decrease in the continuity of the matrix (Marchesseau et al., 1997). These structural changes coincided with increases in the hydration (moisture of pellet obtained on ultracentrifugation of the cheese at 86 000 g  25 min) and solubilization (the ratio of supernatant N to total N on centrifugation of the cheese at 300 000 g for 45 min) of the para-casein. Since pH reduction in the region 6.1–5.7 (typical of commercial processed cheeses) markedly reduces the calciumcheating effects of ES (‘Characteristics of different ES in the manufacture of PCPs and ACPs’), the study probably does not reflect the direct effect of pH, but rather the combined effects of pH and degree of calcium sequestration. Similar to the results of Marchesseau et al. (1997), Lee and Klostermeyer (2001) reported that increasing pH caused reductions in hardness and viscosity and an increase in tan of ACPs prepared from sunflower oil and sodium caseinate. Cavalier-Salou and Cheftel (1991) reported that increases in the pH (⬃6.1–6.7) of ACPs, as affected by increases in the level of ES, caused a 1.5- to 2-fold increase in the flowability of the melted product when using NaH2PO4 and trisodium citrate as ES. pH had little, or no, effect when sodium phosphates with 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 et al., 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.

Analogue 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

Table 7 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

Milk proteins

Vegetable proteins

Starches Stabilizers Emulsifying salts

Hydrocolloids Acidifying agents Flavours and flavour enhancers Sweetening agents Colours Preservatives Minerals and vitamin preparations

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

Soya bean protein Peanut protein, wheat gluten

Native and modified forms of maize, rice, potato starches Sodium phosphates and sodium citrates 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 381

(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 El-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: • the use of many ingredients in common, including ES, stabilizers, non-cheese dairy ingredients, colours, flavours and flavour enhancers; • similar manufacturing technology, involving the application of heat and shear to the formulated blend, followed by hot filling, packing and cooling; • 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’); • 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) • the diverse range of textures, flavours, cooking properties and packaging formats; • 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.

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 ⬃300 000 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: • the sequestration of Ca from the rennet casein by added ES at the high temperatures (typically ⬃80–84 °C); • upward pH adjustment of the blend by the added ES; • concomitant hydration of the casein by the ES, shear and heat; • dispersion of added fat by the shear and its emulsification by the hydrated para-caseinate; • 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

Formulation of blend

A

B

C

2

1

4+5

2. Pre-blend of casein and oil

3

4

3

3. Dry ingredients, except casein

4

5

5

1. All dry ingredients

4. Oil

Cheese cooker

5. Water

Mix for ~1–2 min Process: heat to ~85 °C, shear continuously

Addition of part of oil (e.g. 5% total) optional

Homogeneous molten mass pH ~8.5 Acid regulator

Flavours

Homogeneous molten mass pH ~6.0–6.4 Mould and hot pack

and cooking to ⬃85 °C (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 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.

Store at 4 to–4 °C

Formulation Figure 17 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 383

Table 8 Typical formulation of low-moisture analogue Mozzarella cheesea 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

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-El-Nour et al., 1996). In a subsequent study, Abou-El-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 UF, indicated that the latter gave notably higher firmness in the unheated APC and lower flowability of the heated APC (Abou-El-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, 1981; Yang and Taranto, 1982; Yang et al.,

384 Pasteurized Processed Cheese and Substitute/Imitation Cheese Products

1983; Kim et al., 1992; Ortega-Fleitas et al., 2001), peanut (Chen et al., 1979), pea protein (El-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 balling

60

Flowability, %

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

50

Phase angle, δ, °

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, wheat) 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.

50 40

40

30

20

30 20

10

10 0 0

30

60

90

120

150

180

Storage time at 4 °C, days

Figure 18 Changes in the flowability, after heating at 280 °C for 4 min, of low-moisture Mozzarella (䉱) and low-moisture Mozzarella cheese analogues without (䊊) or with added native maize (䉭) or potato (ⵧ) starch during storage at 4 °C (modified from Guinee, 2002b).

0 0 2 4 6 8 10 Level of added pre-gelatinized maize starch, %, w/w

Figure 19 Effect of level of added pre-gelatinized maize starch on the fluidity of experimental analogue pizza cheese, after heating to 20 °C or 95 °C (drawn from data of Mounsey and O’Riordan, 1999).

Pasteurized Processed Cheese and Substitute/Imitation Cheese Products 385

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 4 °C, 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 °C for 7 days) is markedly influenced by formulation, processing conditions and product composition (cf., see ‘Properties of ES important in cheese processing’; Savello et al., 1989; Cavalier-Salou and Cheftel, 1991; Abou El-Nour et al., 1996, 2001; Ennis and Mulvihill, 1997; Bowland and Foegeding, 1999; Mleko and Foegeding, 2001). Few studies have considered the changes in casein-based APCs during ripening. Mulvihill and McCarthy (1994) reported a progressive increase in proteolysis (e.g., N soluble at pH 4.6 increased from ⬃3.5 at 0 days to 19.5% total N at 51 weeks) and reductions in elasticity and chewiness on storing at 4 °C for 51 weeks. However, the changes during the first 6 weeks were relatively small; normally, analogues are used within 1 month after manufacture. In general, the functionality of APCs is much more stable than that of LMMC and other natural rennet-curd cheeses, as reflected by the relatively small changes in flowability, stretchability and apparent viscosity on storage (4 °C) for 150 days (Kiely et al., 1991; Guinee, 2002b; Fig. 18).

Conclusions Pasteurized processed cheese and analogue cheeses are a diverse groups of products manufactured by comminuting, heating and shearing into a homogeneous product, a blend of various ingredients which, depending on the product category, may include cheese, dairy ingredients, vegetable oils, flavours, colours and ES. Heating of cheese and other ingredients to a high temperature (e.g., 90 °C), while shearing, usually results in protein dehydration and formation of free fat which result in the formation of an unhomogeneous mass which exudes moisture and fat. The addition of ES to the blend prior to heating and shearing orchestrates a number of physico-chemical changes such as calcium sequestration, pH adjustment, protein hydration, emulsification of free fat and structural re-organization. The re-hydrated protein behaves as a water-binding and emulsifying agent and transforms the structure, e.g., from a gel in cheese or an aggregate/precipitate in ingredients such as rennet casein, to a concentrated para-caseinate-stabilized o/w emulsion. The degree of para-casein hydration, or aggregation, and the size distribution of the emulsified fat droplets have a major influence on the rheology and cooking properties of the resultant products. Many factors influence the degree of para-casein hydration and degree of fat emulsification, e.g., emulsifying salt type and level, degree of hydration of protein in the blend which in turn is affected by the level of proteolysis and bound calcium, pH of the blend and extent of heat treatment (i.e., temperature and holding time), and level of shear which determines the surface area of the fat phase. Exploitation of the factors affecting para-casein hydration and degree of fat emulsification facilitate the creation of an extensive array of pasteurized PCPs and ACPs which offer diversity in appearance, texture, flavour and cooking properties. Such diversity has contributed greatly to the increased consumption of these products.

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Cheese as an Ingredient T.P. Guinee and K.N. Kilcawley, Dairy Products Research Centre, Teagasc Moorepark, Fermoy, Co. Cork, Ireland

Introduction There are a total of 500 (IDF, 1981) to 800 (Hermann, 1993) varieties of cheese. These differ to varying degrees in nutritive value, appearance, flavour, texture and cooking properties. Consequently, cheese is capable of satisfying a diverse range of sensory and nutritional demands and, therefore, has very wide appeal. The diversity of cheese is extended when subjected to secondary processing to create an array of cheese-based products, as shown in Fig. 1. While it is generally assumed that cheese was originally eaten on its own or with bread, its use as an ingredient has been recorded since Roman times (Ridgway, 1986). Typical uses included the blending of hard cheese with oil, flour and eggs in the preparation of cakes and the mixing of soft cheeses with meat or fish, boiled eggs and herbs in the making of pies. Cheese has long been used in the home and in hostelries as an ingredient, along with other foods and condiments, to create an extensive array of dishes; typical applications include toasted sandwiches, omelettes, sauces and lasagna. Today, much of the cheese purchased through the food service/catering sector is to a large extent used in the preparation of fast foods (e.g., pizza pie, burgers) and culinary dishes (e.g., quiche, lasagna, cheesecake, toasted sandwiches, Mexican dishes; Anonymous, 1993; Fig. 2). Natural cheese is also used extensively by the industrial sector for the commercial manufacture of a vast array of assembled foods (e.g., pizza pie, sandwiches) or formulated foods (e.g., gratins, prepared meals, processed cheese products (PCPs), co-extruded products, cheese cake, dairy desserts). Moreover, cheese is also used by the industrial sector for the production cheese ingredients, including ready-to-use grated cheeses, shredded cheeses, cheese blends, dried grated cheeses, freeze-dried cheese pieces, cheese powders (CPs) and enzyme-modified cheeses (EMCs). Rapid cure cheeses (Fig. 1) are semi-hard cheeses (e.g., Cheddar type) used to impart intense cheese flavour to formulated foods. They are typically produced by the addition of exogeneous enzymes (proteinases, peptidases, lipases) and/or starter culture adjuncts to the cheesemilk and develop a high flavour intensity after a short ripening time (Fig. 2). These ingredients are, in turn, used by the manufacturers of formulated foods

(e.g., soups, dried cheese sauces, dehydrated potato mixes, infant meals, pasta dishes, snack coatings, bakery products; Duxbury, 1991; Lewin, 1996; Missel, 1996; King, 1999), and to a lesser extent by the catering/food service sector in the preparation of culinary dishes (Fig. 2). Additionally, non-standard cheese curd-like products, such as substitute cheeses and cheese bases, are also used as substitutes for natural or processed cheeses in a number of applications, e.g., imitation cheese products for pizza cheese, or cheese base for PCPs or EMCs (‘see Pasteurized Processed Cheese and Substitute/Imitation Cheese Products’, Volume 2). The percentages of total cheese consumed via the retail, food service and industrial sectors are 70, 20 and 10, respectively, in the EU and ⬃33, 33 and 33, respectively, in the USA (Sørensen, 2001). This consumption pattern suggests that an estimated 35–45% of total cheese is consumed as an ingredient in other foods. Moreover, recent market analyses indicate that the consumption of cheese as an ingredient is growing rapidly, with a shift in the direction of the USA consumption pattern being evident, especially in Europe and Australia (Market Tracking International Ltd, 1998; Sutherland, 1998; Sørensen, 2001). Owing to the economic importance of cheese as a product per se and as an ingredient, research on different aspects of cheese, such as biochemistry and flavour, texture/rheology and cooking properties of individual varieties such as Mozzarella (‘Pasta-Filata Cheeses’, Volume 2), has been extensive. However, there have been relatively few reviews on cheese as a food ingredient (Fox et al., 2000; Guinee, 2002). In this chapter, we will review the functional requirements of cheese as an ingredient in assembled/formulated food, from the perspective of the rheological characteristics of the unheated cheese and the cooking performance of the heated cheese, and the properties of cheese ingredients, including EMCs and CPs.

Functional Requirements of Cheese as an Ingredient When used as an ingredient in other foods or in the preparation of cheese ingredients, cheese is subjected to an array of treatments such as comminution (e.g.,

Cheese: Chemistry, Physics and Microbiology, Third edition – Volume 2: Major Cheese Groups ISBN: 0-1226-3653-8 Set ISBN: 0-1226-3651-1

Copyright © 2004 Elsevier Ltd All rights reserved

396 Cheese as an Ingredient

MILK

Non-standard products

Natural cheese

Retail cheese, catering cheese, industrial cheese Substitute/imitation cheeses

Cheese bases • UF milk retentate-based products • Formulated products based on milk proteins

• Dairy analogues • Partial dairy analogues • Vegetable analogues

Cheese used to prepare cheese ingredients

Cheese used as an ingredient

Home/food service

Industry PCPs • PCs cheese • PCSs

Concentrated cheese flavours

Cheese powder

Comminuted cheese

• PCFs

• Natural • Extended • Grated cheese Rapid-cure cheeses

Enzyme-modified dairy Ingredients (EMDI)

• Semi-hard varieties

• Diced cheese

Club cheese

Cheese blends

Dried cheese

• Crumbed cheese • Shredded cheese

EMF

EMC

• Lipolysed fats

• Hydrolysed cheese

Figure 1 Cheese, cheese ingredients and cheese-like products. Cheese may be used: (a) directly, as an ingredient in the home, food service and industrial sectors for the preparation of a variety of culinary dishes, formulated foods or assembled foods or (b) indirectly, in the preparation of various cheese ingredients, such as cheese powders and enzyme-modified cheeses, which are then used in an array of food applications. Various types of processed cheese products (PCPs), including processed cheese spreads (PCSs) and processed cheese foods (PCFs), may be formulated from natural cheese.

portioning, shredding, grating, grinding), cooling, freezing, thawing, heating and/or re-heating. In the home and the food service sector, the cheese-containing dish, once prepared, is generally cooked and consumed immediately. In contrast, following assembly or formulation in the industrial sector, cheese-containing foods are frequently heated or pre-heated (pre-cooked) and frozen, and are then re-heated prior to consumption. The treatments of cheese during the manufacture of cheese-filled co-extruded products (e.g., cheese-filled croquette or meat-balls) are typical of those applied during the manufacture of many formulated foods; they include preparation of a cheese filling by dicing, grating, blending and/or processing (as in PCPs), co-extrusion of the cheese with the encasing material, deep-fat frying and freezing (prior to distribution and during retailing). Consideration of the various processes indicates that size reduction (comminution) and heating are the most common processes to which cheese is exposed when used as an ingredient. Hence, the behaviour of

the cheese during these processes is a major determinant of its functionality and its suitability as an ingredient. In the raw state, the cheese may be required to exhibit a number of certain rheological properties so as to facilitate its usage in the primary stages of preparation of various dishes, e.g., the ability to crumble easily, to slice or to shred cleanly, to bend when in sliced form. The rheological properties also determine the textural properties of cheese during mastication (cf. ‘Rheology and Texture of Cheese’, Volume 1). On grilling and baking, the cheese may be required to melt, flow, stretch, brown, blister, oil-off and/or stretch to varying degrees. The baked cheese may also be expected to be chewy (as in pizza pie) and contribute to certain mouth-coating characteristics (as in sauces and soups). In many dishes, e.g., sauces, the cheese is required to have the ability to interact with other food components such as water, carbohydrates, proteins and fats, during food preparation. The flavour of the unheated and the heated cheeses is important in

Cheese as an Ingredient 397

CHEESE

Processed cheese products

Secondary Processing

Cheese ingredients – Comminuted cheeses – Dried cheeses – Cheese powders – Enzyme-modified cheeses

Home and food service uses

Assembled and formulated foods

Uncooked Sandwiches Cheesecake Desserts Salads Sprinkling Cooked – Baked Camembert, – Cheese-filled meat, fish and vegetable products – Fried paneer – Fondues – Omelette – Pasta dishes – Pizza – Quiche – Rarebit – Raclette

Prepared or semi-prepared (consumer) foods – Chilled or frozen pizza – Burger insets – Cheesecake – Cheese-filled meat, fish and vegetable products – Cordon-bleu entrees – Baked Camembert in batter – Deep-fried cheese sticks in batter – Desserts – Gratins – Prepared meals

Dry foods – Biscuits – Convalescent meals – Infant meals – Pasta dishes – Cheese-coated snacks (potato crisps, nachos) – Sauce – Soups – Sprinklings

Figure 2 Uses of cheese and cheese ingredients.

almost all applications, and it should be noted that flavour can be affected by various unit operations. Heating may adversely affect flavour due to the loss of key volatile compounds, or thermal processing may induce the Maillard reaction, resulting in the production of different flavouring substances. The development of rancid off-flavours can occur where interaction of fat and oxygen is promoted by any physical increase in the surface area of the fat during processing. The flavour of other ingredients (such as sauces, condiments, added flavours and flavour enhancers) used along with cheese in some formulated (e.g., soups) and assembled (e.g., pizza, sandwiches) foods and culinary dishes may somewhat mask or alter the flavour of the cheese. From a generic perspective, the functionality of cheese as an ingredient may be defined as its behaviour during all the stages in the preparation and consumption of the food in which it is incorporated. A functional cheese may be described as one which exhibits the required properties when raw or heated, and thereby contributes to the formation of, and/or

enhances the quality of, the food product in which it is used. Conversely, the absence of the correct functional attributes, or the occurrence of undesirable functional attributes at any stage of preparation, may impede the formation of a physico-chemically stable product and impair the usage performance and appeal. More specifically, the functionality of cheese may be defined as a composite property comprising different functional attributes, which may be classified into four main types: • rheology-related properties of the raw cheese, i.e., those properties exhibited when the cheese is subjected to a stress (such as cutting, shearing, mastication) or strain (compression, extension). The rheological properties affect the textural attributes during consumption (see ‘Sensory Character of Cheese and its Evaluation’, Volume 1) and the deformation and fracture behaviour during formulation and processing, which in turn determines how the cheese grates, shreds, crumbles or slices (Table 1). • rheology-related properties of the heated cheese, i.e., those properties exhibited when the cheese is subjected to

398 Table 1 Functional requirements of unheated cheeses

Functional requirements Shreddability

Sliceability

Gratability

Spreadability

Crumbliness

Description Ability: –to cut cleanly into long thin strips of uniform dimensions (typically cylindrically shaped; 2.5 cm long, 0.6 cm in diameter) –low susceptibility to fracture or to form curd dust during shredding –to resist sticking, matting or clumping during shredding or when loosely packed Ability: –to cut cleanly into thin slices –to resist breakage or fracture at slice edges (on contacting packing equipment) –to undergo a high level of bending before breaking Ability: –to fracture easily into small hard particles –of particles to resist matting during shearing, crushing, fluidization or piling –of particles to exhibit free flow Ability: –to spread easily when subjected to a shear stress Ability: –to fracture easily into small irregularly shaped pieces when rubbed

Cheese type displaying property1

Related rheological properties

Large strain deformation characteristics2

Low-moisture Mozzarella, Swisstype, Gouda, Cheddar (y oung– medium aged), Provolone, some PCPs, ACPs

High elasticity, long, firm

 f –high max –medium–high

Shredded cheese for retail or catering, pizza

Swiss-type, Gouda, Cheddar, Provolone, some Quesco Blanco, some PCPs and ACPs

High elasticity, long, high fracture resistance, firm

 f –high max –medium–high

Slices for retail and food service

Hard brittle cheeses, e.g., Parmesan, Romano-type

Brittle, elastic fracturability, firm, low tendency to stick

f –high  f –low max –high

Dried cheese for sprinkling

Mature Camembert and Brie, some cream cheeses , processed cheese spreads, some ACPs Feta, Blue, Stilton, Cheshire

Long, plastic fracturability, soft, adhesive Medium-soft, brittle cheese which breaks into irregularly shaped pieces

 f –high f –low max –low  f –low f –medium–low max –medium–low

Cheese for spreading, e.g., on crackers and bread Tossed salads, crêpes au fromage, soup garnishes

Application

1 Abbreviations: PCPs, processed cheese products; ACPs, analogue cheese products. 2 Rheological terms relating to large strain deformation using uniaxial compression tests:  f, fracture strain; f, fracture stress; max, firmness. See Table 2 for explanations of rheology-related properties and large strain deformation characteristics; more detailed explanations given in ‘Rheology and Texture of Cheese’, Volume 1.

Cheese as an Ingredient 399

stresses throughout its mass as a result of the heatinduced physico-chemical and microstructural changes such as liquefaction of the fat, fat coalescence and changes in protein hydration and structural rearrangement of the matrix. These properties include the ability of the cheese to melt, flow and stretch. • physico-chemical- and microstructural-related properties induced by heating, including oiling-off, browning, blistering, fat coalescence and exudation/ separation, interaction of free amino groups with reducing sugars, moisture evaporation, para-casein aggregation and precipitation. • Flavour/aroma-related properties, which are characteristic of a given variety and may be positively or negatively altered during processing. Depending on the application, the combination of, and intensity of, individual attributes vary. Hence, a cheese which shreds well and melts and flows on heating, is ideal for the preparation of lasagna. However, a cheese which exhibits stringiness on heating is very unsuitable for the preparation of sauces, dips, gratins or cheese powders; a more suitable cheese would comminute to a sticky mass, blend easily with other ingredients, and, on heating, form a non-stringy, fluid, homogeneous mass.

Rheology-Based Functional Properties of Unheated Cheese In all applications, whether as a consumer product or as an ingredient, cheese is exposed to size-reduction operations involving a combination of shear and compressive stresses (e.g., 600 kPa) and strains (e.g., 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

Table 2 Rheological properties of unheated cheese which affect its functionality as an ingredient Types of properties

Description

Measurements1

Elasticity and related properties (springiness, toughness)

Tendency of cheese to recover to original dimensions following removal of the applied stress (, force per unit surface area)

Fracturability (and related terms)

Tendency of cheese to fracture into pieces when a stress () is applied, e.g., during compression or extension

Recovery of sample after compression, obtained using Texture Profile Analysis Fracture stress (f) – force to fracture Fracture strain ( f) – displacement at fracture

– brittleness

– longness – crumbliness Firmness (and related terms) – Firm – Soft Adhesiveness

Tendency to fracture into pieces at a low deformation or displacement (strain; , i.e., after a low-percentage compression). Low deformation at fracture, i.e., low  f Tendency to fracture at a large deformation, i.e., high  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 () High resistance to deformation, i.e., high max Low resistance to deformation, i.e., low max Tendency to be sticky and resist separation from a material it contacts

Firmness (max) – stress () required to achieve a given compression/extension

Texture Profile Analysis

References used in compilation: van Vliet (1991), Visser (1991), Fox et al. (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: • the magnitude of the stress required to fracture (fracture stress, f); • the degree of strain (e.g., change in dimensions) required to fracture (fracture strain, f); • the level of force or stress required to achieve a given deformation (max); • the type of fracture (i.e., clean or jagged); • the degree to which a piece of cheese recovers (in size dimensions) after being strained (e.g., compressed or sheared). The various rheological terms (f, f, max) described above are easily measured from the force (or stress, )/displacement (or strain, ) curve obtained during compression of a cheese sample, as described in ‘Rheology and Texture of Cheese’, Volume 1. On consideration of the forces operative during deformation, and the structure and the biochemistry of cheese, it can be inferred that relationships do exist between the functional and the rheological characteristics of unheated cheese. Similarly, cheese texture, which is a composite sensory attribute resulting from a combination of physical properties that are perceived by the senses of touch (including kinaesthesis and mouthfeel), sight and hearing, has been found to be related to rheological (stress–strain) characteristics of cheese (Szczesniak, 1963; Sherman, 1969; Brennan, 1988). The relationships between some common functional properties and the rheological parameters of the raw cheese, as described below, are given in Table 1. The rheological characteristics of the raw cheese have a major impact on how it behaves during comminution and its usability as an ingredient (Table 2). Thus, it is difficult to cleanly portion hard cheeses which have a relatively a low fracture strain (Parmesan) or which fracture in a jagged fashion (e.g., an overacid Cheddar or Cheshire) owing to their tendency to break at the edges. Similarly, these cheeses are unsuitable for applications where shredded cheese is required (e.g., pizza) because of their susceptibility to fracture/shattering and the resultant formation of a high level of curd fines/dust on the surface of the uncooked pizza, which is aesthetically unappealing. Conversely, other hard cheeses, such as Cheddar, lowmoisture Mozzarella (LMMC) and Gouda-type, are unsuitable for grating owing to their lack of brittleness and to their elasticity and relatively high f and f, which enables a relatively high degree of recovery to their original shape and dimensions following crushing. However, the latter cheeses generally shred very well to give pieces of uniform size which are relatively non-adhesive, which makes them ideal for distribution

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 shredded/diced cheese applications because of their tendency to stick to the shredding equipment and of the shredded cheese to balling and clumping. However, the ability of these cheeses to undergo plastic fracture and flow under shear (i.e., spread) makes them ideal for spreading on crackers and for blending with other materials such as butter, milk or flour in the preparation of fondues and sauces. The brittleness and tendency of hard cheeses, such as Parmesan and Romano, with low levels of moisture and fat-in-dry matter, to undergo elastic fracture (clean fracture without flow) endows them with excellent gratability (when crushed between rollers) and suitability as a free-flow condiment for sprinkling, e.g., onto pasta dishes. However, these properties render the latter cheeses unsuitable for food applications that require slices (e.g., filled sandwiches, cheeseburgers) or shredded cheese. The crumbliness of Feta and Stilton makes them very desirable for use in tossed salads and Greek salads as the irregularly shaped, curd-like particles create an image of ‘real’ cheese and are more visually appealing to the consumer than cheese shreds. Factors influencing the rheological (functional) properties of unheated cheese

Cheese rheology and the factors that affect it have been studied (Culioli and Sherman, 1976; Vernon Carter and Sherman, 1978; Chen et al., 1979; Creamer and Olson, 1982; Green et al., 1985; Luyten, 1988; Visser, 1991; Fenelon and Guinee, 2000) and reviewed extensively (van Vliet, 1991; Visser, 1991; Rao, 1992; Prentice et al., 1993; Fox et al., 2000; ‘Rheology and Texture of Cheese’, Volume 1). The rheology of cheese is a function of the combined effects of various factors, including its composition, micro-structure (i.e., the spatial arrangement of its components and the strength of attractions between the structural elements) and the physico-chemical state of its components (e.g., degree of casein hydrolysis). Moreover, it is difficult to quantify the direct effects of any of the gross compositional components (fat, protein or moisture) separately, owing to the fact that these tend to vary simultaneously, especially where large changes in the concentration of a particular component (e.g., fat) occur and in the absence of process interventions. However, for convenience, the effects of individual factors are discussed separately below. Protein level

The concentration and the type of protein have a major influence on the rheological properties, as

Cheese as an Ingredient 401

confirmed by the positive correlation between the volume fraction of the casein matrix and cheese firmness (max) and the 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 et al., 1983; Green, 1990b; Guinee et al., 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 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, / ), 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): G  CN

d2A dx2

1200 900

800

700

600 500 400

Firmness, σmax, N

1000 Fracture stress, σf, kPa

• the concentration of gel-forming protein; • 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 °C), 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 °C, where all the milk fat is liquid (Guinee and Law, 2002). High heat treatment (HHT) of milk and denatured whey proteins

where: N  number of strands per unit area of the gel in a cross section perpendicular to x, bearing the stress; C  coefficient related to the characteristic length

300 200 100

0 20

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:

25

30

35

40

Intact casein, %, w/w

Figure 3 Relationship between the content of intact casein and firmness (ⵧ) and the fracture stress (■) in Cheddar cheeses of varying fat content in the range 6–31%, w/w (reprinted from Guinee et al., 2000a with permission from Elsevier).

High heat treatment of milk increases the level of in-situ denaturation of whey proteins and their complexation with -CN at the micelle surfaces. The denatured whey proteins form appendages which protrude from the micelle surfaces and render the Phe1059Met106 bond of -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 °C for 15 s) has lower f and max than cheese made from milk pasteurized at a normal temperature (e.g., 72 °C for 15 s; El-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 LoTM) in reduced-fat Cheddar reduces f, f and max (Lucey and Gorry, 1994; Fenelon and Guinee, 1997). The whey proteins in these preparations, at least in the case of Dairy LoTM, 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 content

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 °C, increasing the level of fat in Cheddar cheese results in decreases in elasticity (E), f, f, max, cohesiveness, springiness, chewiness and gumminess and an increase in adhesiveness. The latter trends are expected because of the concomitant

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 °C. 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, f and 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 °C) 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. 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.,

Cheese as an Ingredient 403

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 f and max of 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 et al. (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).

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 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, f and max (Luyten, 1988; Visser, 1991); the f 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 f as the moisture content of Cheddar was increased from 34.0 to 39.7%, w/w, with f at the lower moisture level being almost twice that at the higher moisture level. Salt (NaCl) 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, f (from ⬃28 kPa at 0.4%, w/w, S/M to ⬃83 kPa at 11.3%, w/w, S/M) and max (Visser, 1991). The fracture strain,  f, 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., 6.5%, w/w, NaCl), para-casein in cheese absorbs water (Geurts et al., 1972; Guinee and Fox, 1986), an occurrence which is indicative of a salting-in effect on the protein matrix and a concomitant increase in casein hydration. Hence, the presence of NaCl, at a level of 5%, w/w, increased the degree of casein hydration in dilute suspensions of casein micelles, over the pH range 6.7–4.6, and especially at the pH of maximum hydration, i.e., ⬃5.2–5.3 (Creamer, 1985). Conversely, when cheese (para-casein) is placed in a brine of higher concentration (e.g., 6.5%, w/w, NaCl), the loss of moisture, especially in the rind region, suggests a salting-out of the protein matrix and concomitant

404 Cheese as an Ingredient

casein dehydration/aggregation. An increase in dehydration would be expected to cause an increase in max, e.g., as seen by comparing the hardness of cheese rind to that of the cheese interior. The effect of salt, inter alia other factors, on the f is apparent on comparison of the ‘long’ smooth body of low-to-medium salt cheeses (e.g., LMMC, Gouda or Swiss; 2–5%, w/w, S/M), with the ‘short’ crumbly body of high-salt varieties (e.g., Feta, Stilton, Blue; 8–12%, w/w, S/M; Table 1). pH

Small differences in cheese pH can have relatively large effects on its rheological properties which in turn may affect parameters (e.g., crumbliness, longness, shortness, softness, adhesiveness; see ‘Rheology and Texture of Cheese’, Volume 1) that are important in determining its suitability (e.g., portionability, sliceability, shreddability, gratability) for a particular ingredient application. The effects of pH on the rheological properties of cheese and its use as an ingredient are clearly manifest on comparing different varieties. Low-pH cheeses (e.g., Cheshire, Feta) generally tend to have low values of f and f and to crumble into many pieces on fracturing, whereas relatively high-pH cheeses (e.g., pH 5.35–5.50; Emmental and Gouda) exhibit higher values of f and f and tend to fracture into larger pieces. Inter-varietal differences in pH occur mainly as a result of differences in make-procedure, composition and the type and level of biochemical changes during ripening. However, intra-varietal differences in cheese pH occur also due to a variety of other reasons (see Fox et al., 1996) including differences in: • lactate level resulting from differences in lactose level in the cheesemilk (Huffman and Kristoffersen, 1984; Fox and Wallace, 1997) for non-washed curd varieties and moisture level; • pH at whey drainage due to variations in the starter activity, pH at rennet addition, and time between starter addition and pitching; • buffering capacity, as a result of variations in phosphate content (Lucey and Fox, 1993) due to differences in pH at whey drainage (Lawrence et al., 1984); • extent of biochemical changes (e.g., as affected by type and level proteolysis, glycolysis, lipolysis deamination) as influenced by, among other factors, residual rennet activity, salt level, starter type and cell density, and ripening period. Creamer and Olson (1982) studied the effect of cheese pH (4.9, 5.15, 5.4) on age-related changes in f and f in Cheddar cheeses in which the pH was varied by altering the pH at whey drainage and in which the levels of fat, moisture-in-non-fat substances (MNFS) and salt were relatively similar. Increasing the pH from 4.9 to

5.4 resulted in a linear increase in f, an effect which became more pronounced with time over the 50-day investigation period (Creamer and Olson, 1982). The f changed little as the pH was raised from 4.9 to 5.15 but increased markedly on further increase of pH to 5.4. Similar trends were noted for model Gouda cheeses in which an increase in pH from 5.0 to 5.2 led to a reduction in E and a marked increase in f but had little effect on f (Luyten, 1988; Visser, 1991). Moreover, increasing the pH from 5.2 to 5.6 led to a marked increase in f (to values much higher than those at pH 5.2), and an increase in E. The pH at which f was maximal increased with ripening time, e.g., from ⬃5.2 in a 1-week-old Gouda to ⬃5.4 in a 3-month-old Gouda cheese. The effect of pH probably ensues from its influences on: (i) the ratio of soluble-to-colloidal Ca (Guinee et al., 2000c), (ii) total calcium where pH is varied by reducing the pH at whey drainage (Lawrence et al., 1984), and, consequently, (iii) the degree of para-casein hydration or aggregation (Creamer, 1985). Model systems of rennettreated skim milk or casein suspensions have shown that the hydration of para-casein is maximal at pH ⬃5.2–5.3 (Creamer, 1985); the pH of maximum casein hydration may vary somewhat in different varieties due to differences in the degree of proteolysis and levels of calcium and NaCl. As aggregation of para-casein shows the opposite trend to para-casein hydration, it is expected that the degree of para-casein aggregation/fusion and the elasticity of the para-casein matrix would be minimal at pH 5.2–5.3. The effect of raising the pH above 5.2–5.3 is greater than that of reducing the pH below 5.2–5.3, an effect that may be attributed to the large increase in the calcium binding by the casein at the higher pH (van Hooydonk et al., 1986). The uptake of calcium reduces casein hydration (Sood et al., 1979) and greatly enhances cheese elasticity (Lawrence et al., 1987). Ripening and para-casein hydrolysis

For most varieties, the hydrolysis of s1-CN at the Phe239Phe24 peptide bond, by residual chymosin early during ripening, results in a marked weakening of the para-casein matrix and decreases in f and max (de Jong, 1976; Creamer and Olson, 1982; Fenelon and Guinee, 2000). The sequence of residues 14–24 of s1-CN is strongly hydrophobic and confers intact s1-CN with strong self-association and aggregation tendencies in the cheese environment (Creamer et al., 1982). It has been suggested that self-association of s1-CN in cheese, via these hydrophobic patches, leads to extensive cross-linking of para-casein molecules and thereby contributes to the overall continuity and integrity of the matrix (Creamer et al., 1982). Indeed, de Jong (1978b) reported a linear relationship between the content of intact s1-CN and the softness in Meshanger

Cheese as an Ingredient 405

cheese (a soft, internal bacterial-ripened Dutch variety) in which proteolysis was varied by altering the quantity of added coagulant. Moreover, at a microstructural level, proteolysis may result in discontinuities or ‘breaks’ in the para-casein matrix (de Jong, 1978a), a factor expected to reduce its stress-bearing capacity. Another factor contributing to the age-related weakening of the matrix structure is the increased hydration of the matrix, as reflected by the decrease in the level of expressible serum (on centrifugation or hydraulic pressing of the cheese) or, more appropriately, the increase in the level of non-expressible serum per gram of protein during maturation (Guinee et al., 2002). In contrast to the above, the firmness of some cheeses (e.g., brine-salted and/or surface dry-salted varieties that are not packaged for part of their ripening period) may increase initially even though proteolysis occurs during this period. The increase in firmness is a consequence of the loss of moisture and the concomitant increase in protein level. Other factors such as changes in pH and the increase in the salt content in the inner regions, as a result of inward diffusion from the surface rind zone, may also contribute to the initial increase in firmness. However, the softening associated with proteolysis becomes dominant when the composition has stabilized, and f and max decrease (de Jong, 1976, 1978b; Visser, 1991).

Functional Properties of Heated Cheese The heat-induced functional properties have been discussed in detail for Mozzarella cheese, and for pasteurized PCP and analogue cheese product (ACP) in ‘Pasta-Filata Cheeses’ and ‘Pasteurized Processed Cheese and Substitute/Imitation Cheese Products’ (Volume 2), respectively. Hence, this section will concentrate mainly on those of natural cheeses, other than Mozzarella. Types and definitions and principles of functional properties

Cheese is used extensively as an ingredient in cooking applications, e.g., grilled cheese sandwiches, pizza pie, cheeseburgers, pasta dishes and sauces; in these applications the cheese attains a temperature of ⬃80–100 °C. 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. These attributes, which have been defined previously (Kindstedt, 1995; Fox et al., 2000; Guinee, 2002; ‘Pasta-Filata Cheeses’, Volume 2), are summarized in Table 3; the number and the intensity of the attributes required are determined by the application.

Heat-induced softening or melting involves liquefaction of the fat phase. Heat-induced flow or spread and stretchability may be defined as heat-induced rheological changes, involving strain displacement as a result of stresses on the para-casein matrix. These stresses may be of two types: • those which occur spontaneously during heating of the cheese under quiescent conditions (e.g., baking); • those applied externally to the hot molten cheese mass after cooking, e.g., manually during consumption or instrumentally during testing (e.g., shear during viscometric testing, compression during squeeze flow evaluation or extension during stretchability testing). The changes in viscoelasticity on heating cheese help to explain the mechanism of the melting process (Fig. 5). Spontaneous heat-induced stresses arise when the fat globules, which at low temperatures are solid and reinforce the para-casein matrix that surrounds them, melt and flow on heating. Consequently, the surrounding viscoelastic matrix deforms to a degree dependent on the ratio of the elastic-to-viscous character, which is in turn a function of the degree of casein aggregation or hydration. Additionally, the free oil (FO), arising from the coalescence of liquefied, nonglobular fat droplets, lubricates the displacement of adjoining layers of the deforming matrix and thereby contributes to flow. Clear evidence for heat-induced coalescence of fat globules/droplets in natural cheese is provided by dynamic microscopy of cheese during heating (Paquet and Kaláb, 1988; Auty et al., 1999; Guinee et al., 1999; Fig. 4) and by the release of oil on baking (Rudan and Barbano, 1998; ‘Pasta-Filata Cheeses’, Volume 2). At the microstructural level, heating results in extensive clumping and coalescence of fat globules and a less homogeneous distribution of the fat and para-casein phase, at least in the case of Cheddar (Fig. 4) and Mozzarella (Paquet and Kaláb, 1988). From the foregoing, it is clear that the measures (e.g., levels of flow, stretchability, apparent viscosity, elastic shear modulus, phase angle) of the functionality of heated cheese are to a large extent controlled mainly by the concentrations of fat and protein and their microstructural distributions, and the level of casein hydration. Factors that affect the functionality of heated cheese

The functionality of heated cheese is influenced by many factors (Kindstedt, 1993, 1995; Rowney et al., 1999; Guinee, 2002; ‘Pasta-Filata Cheeses’, Volume 2), including variations in: • milk pre-treatments, e.g., pasteurization conditions, homogenization;

406 Table 3 Functional requirements of heated cheese which affect its functionality as an ingredient Types of properties Meltability

Flowability

Flow resistance

Stretchability

Description

Examples of measurements

Level required

Ability to soften on heating Empirical –dropping point is the High temperature at which first drop of melted sample falls from orifice of a sample holder under defined conditions Objective –change in magnitude G, G , on heating, as measured using lowamplitude strain oscillation rheometry Ability of heated cheese to Empirical – percentage increase in a dimen- High spread on heating sion (e.g., diameter of a disc, length of a (runny, low tube) of sample on heating; examples: viscosity) Schreiber test, Price-Olson test Moderate Objective –level of increase in or decrease in G on heating, as (moderately measured using low amplitude plastic strain oscillation rheometry consistency) Ability of heated cheese As for flowability Medium–high to resist flow and retain original dimensions on heating

Ability of heated cheese to form strings and/or sheets when extended uniaxially

Empirical – length of strings/sheets of heated cheese at failure, or force at string failure, when heated cheese mass subjected to a uniaxial extension

Medium–high

Cheese type and application • Most cheeses, apart from low-fat and skim milk cheeses • Most, if not all, applications

• Many mature full-fat cheeses such as Cheddar, Gouda, Raclette, Cheshire, Blue • Gratins, Cordon-blue products • Many young-medium mature full-fat hard/semi-hard cheeses, LMMC, half-fat Cheddar • Many culinary dishes, such as toasted sandwiches and pizza • Acid-heat coagulated cheese such as Paneer; rennet-curd and some acid-curd cheeses prepared from high-heat treated milk; low-fat hard/semi-hard types, especially if prepared from homogenized milk, some ACPs and PCPs • Fried cheese, deep fried cheese sticks, cheese for kebabs, cheese insets in burgers • Pasta-filata cheeses such as LMMC, Halloumi, Provolone, Kashkaval • Pizza

Low

Oiling-off (surface sheen)

Tendency of heated cheese to exude oil

Level of oil extracted from heated cheese under defined conditions; Diameter of oil ring that forms on melting a cheese disc on a filter paper

• Most cheeses, apart from pasta-filata types • Most applications, especially gratins, cordon bleu applications Low–moderate • Most rennet curd varieties, apart from those form milks (not forming homogenized at high pressures pools, but giv- • Most applications, ranging from moderate gratins to low for ing a surface omelettes and pizza sheen) Low–very low • Some ACPS and PCPs, low-fat hard and semi-hard rennet curd varieties • Flow resistant applications such as fried cheese

References used in compilation: Kindstedt and Rippe (1990), Rü egg et al. (1991), Guinee and O’Callaghan (1997), Fo x et al. (2000), Guinee et al. (2000b). ACPs, analogue cheese products; LMMC, low-moisture part-skim Mozzarella; PCPs, processed cheese products. See ‘Pasta-Filata Cheeses’, Volume 2 for further details of tests used in evaluating the functionality of heated cheese.

Cheese as an Ingredient 407

a

b

c

d

Bar = 25 μm Figure 4 Confocal scanning laser micrographs of 5-day-old unheated Cheddar cheese (a, b) and the same cheese after heating to 95 °C and then allowed to cool to room temperature (c, d). The micrographs show protein (a, c – long arrows) and fat (b, d – short arrows) as light areas against a dark background. Bar corresponds to 25 m (Modified from Guinee et al., 2000b).

• make procedure, e.g., set pH, cooking temperature, pH at whey drainage, plasticization conditions; • compositional parameters, e.g., concentrations of fat, protein and moisture, calcium-to-casein ratio, pH; • degree of proteolysis; • other factors (e.g., absence or inclusion of fat replacers or whey proteins). Most of these factors exert their effects indirectly by influencing the microstructural distributions, and physico-chemical properties, of the protein (e.g., level of calcium binding, degree of aggregation or hydration, degree of casein hydrolysis) and the fat (e.g., degree of fat emulsification, level of fat coalescence) phases. Some of the major factors affecting functionality are discussed separately below. Comparison of different varieties

Studies on the functional properties of retail samples of different varieties of natural cheese indicated that there

are considerable intra- and inter-varietal differences in melt time, flowability, stretchability and apparent viscosity (Park et al., 1984; Guinee et al., 2000a). These differences undoubtedly reflect inter- and intravarietal differences in the conditions of manufacture, composition, degree of maturity and/or formulation (e.g., levels and types of added ingredients and processing conditions) in the case of the PCPs and analogue pizza cheese (see ‘Pasteurized Processed Cheese and Substitute/Imitation Cheese Products’, Volume 2). Studies (Guinee, 2002) on the storage-related changes in different functional parameters in different cheese varieties confirm the inter-varietal variation and show that functionality is dynamic, changing with storage time and proteolysis (Fig. 6). Compared to other varieties, pasta-filata cheeses (e.g., Mozzarella, Provolone and Kashkaval) are differentiated by their superior stretchability, relatively high apparent viscosity and moderate flowability. These functional attributes endow the pasta-filata cheeses with the characteristics that are

408 Cheese as an Ingredient

(c) 70

(b) 60

(a) Phase angle, δ, °

50 40 30 20 10 0 20

30

40 50 60 70 Temperature, °C

80

90

Figure 5 Phase angle, δ, as a function of temperature for 5-day-old full-fat Cheddar cheese on heating from 20 to 82 °C. The temperature/δ curve may be divided into three regions: a) region of fat liquefaction, which is essentially complete at 40 °C; b) region of coalescence of liquefied non-globular fat, which overlaps region (a) and continues on further heating; c) region where the protein matrix deforms to a degree dependent on the level of para-casein hydration and temperature, as the interior elastic support of the encased fat globules diminishes with liquefaction of fat and coalescence of non-globular liquid fat. Heat-induced fat coalescence is shown in Fig. 4 and its gradual occurrence with rise in temperature is verified by the observations of Auty et al. (1999).

typically associated with the melted cheese on pizza, i.e., sufficiently rapid melt and desirable levels of stringiness, chewiness and flow. In contrast to the pasta-filata cheeses, other types of cheese, including analogue pizza cheese and Cheddar and Emmental, have relatively low stretchability, low apparent viscosity and high (e.g., Cheddar) or low (some analogue cheese) flowability characteristics. If such cheeses were used exclusively, or as a substantial part (e.g., 30%, w/w) of a pizza topping, the melted cheese topping would lack the desired stringiness, would flow excessively and lack the desired chewiness. Conversely, stringiness, which is typical for LMMC and other pasta-filata cheeses, such as Kashkaval and Provolone, is an undesirable attribute for applications such as sauces, gratins, cordon bleu applications or fondues. Cheeses with a high flowability, such as mature Cheddar, Emmental, Raclette and Gouda (see Fox et al., 2000), are more satisfactory for the latter applications because of their relatively high flavour intensity and their lack of stringiness when heated. The flowability of retail Cheddar and other natural cheeses is generally

superior to that of cheese analogues, probably as a consequence, inter alia, of the higher degree of fat emulsification in the latter (see ‘Pasteurized Processed Cheese and Substitute/Imitation Cheese Products’, Volume 2). The superior stretchability of pasta-filata varieties is largely attributable to plasticization (heating to ⬃58 °C and kneading) of the fermented curd (pH typically ⬃5.2) in hot water or dilute brine at ⬃80 °C. The relatively low curd pH and the high temperature are conducive to limited aggregation of the casein and the formation of para-casein fibres of high tensile strength (Taneya et al., 1992; Pagliarini and Beatrice, 1994; Guinee and O’Callaghan, 1997). Other factors that probably contribute to the high stretchability in these varieties are the low level of proteolysis, because of extensive inactivation of coagulant in the curd during plasticization (Feeney et al., 2001) and the relatively short storage time (at least for LMMC). A survey (Guinee et al., 2000b) of commercial cheeses indicated that the mean concentrations of pH 4.6 SN%TN in Cheddar and LMMC were 20.3 and 4.7, respectively). All other factors being equal, a low level of proteolysis in LMMC, compared to Cheddar (because LMMC is ripened for a short period), would be conducive to a more aggregated and intact casein matrix in the heated cheese, which when subjected to extension or shear stress would be less likely to fracture. The corollary to this is the general ability of unheated LMMC to withstand fracture when subjected to a high level (e.g., 75%) of compression. The rate of primary proteolysis in LMMC, as measured by urea-PAGE and the formation of pH 4.6-soluble N (pH4.6SN), is comparable to that in full-fat Cheddar stored at a similar temperature (7–10 °C) over 70 days (Fenelon and Guinee, 2000; Feeney et al., 2001; Fig. 7). Undoubtedly, the higher moisture and the lower salt content in LMMC, compared to Cheddar, are more favourable for proteolysis by residual rennet than in Cheddar. Moreover, plasmin activity appears to make a greater contribution to proteolysis in LMMC than in Cheddar (Creamer, 1976; Yun et al., 1993b; Fenelon and Guinee, 2000; Feeney et al., 2001). Despite the increase in proteolysis and the concomitant decrease in the level of intact casein, the stretchability of LMMC is not significantly impaired until the concentration of pH4.6SN exceeds ⬃16% of total N, e.g., after storing at 4 °C for 140 days or at 15 °C for 17 days (Feeney et al., 2001; Guinee et al., 2001). Similarly, the stretchability of experimental Kashkaval does not decrease with increasing level of pH4.6SN in the range 2–16% of total N. In contrast, the stretchability of full-fat Cheddar cheese, which is only slightly inferior to that of LMMC when young (e.g., stored for 30 days at 4–7 °C), deteriorates rapidly on ageing as the level of pH4.6SN increases to a value 6% of total N (Fig. 7). The different stretchtime/pH4.6SN profiles of Cheddar and LMMC probably

Cheese as an Ingredient 409

80

(A)

(B)

16 14

60 Flowability, %

pH 4.6-soluble N, % total N

18

12 10 8

40

6 20

4 2

0

0 0

30

60 90 Ripening time, d

140

120

150

0

30

60 90 120 Ripening time, d

150

(C)

Stretchability, cm

120 100 80 60 40 20 0 0

30

60 90 120 Ripening time, d

150

Figure 6 Changes in levels of primary proteolysis (a) and heat-induced functional characteristics (b, c) of different cheese varieties during storage. Cheeses were: Kashakaval (䉭), low-moisture part-skim Mozzarella (■), half-fat low-moisture Mozzarella (10%, w/w, fat; ⵧ), full-fat Cheddar (30.0%, w/w, fat; ●), half-fat Cheddar (17.2%, w/w, fat; 䊊) and Emmental-type cheese (▲). All cheeses were experimental (produced on pilot-scale) apart from the Emmental-type cheese which was factory-produced. The data presented are the means of replicate treatments: 5, Kashkaval; 5, low-moisture part-skim Mozzarella; 3, full-fat Cheddar; 3, half-fat Cheddar; 2, Emmental; 3, half-fat low-moisture Mozzarella. Flowability was defined as the percentage increase in the diameter of a cheese disc on heating in a convection oven at 280 °C for 4 min, and stretchability as the length of cheese strings at failure on uniaxial extension of molten cheese following heating in a convection oven at 280 °C for 4 min, as described by Guinee et al. (2000a,c).

reflect differences in the state of aggregation of paracasein (as affected by the inclusion/absence of a plasticization stage), the ratio of soluble-to-colloidal Ca, the pH and the type of proteolysis (i.e., hydrolysis of s1versus -CN; Guinee, 2003). Cheese varieties also differ markedly with respect to the change in flowability with ripening time and, consequently, the level of flow after any given storage time. Differences in flowability between different cheeses can result from differences in milk pre-treatment, make procedure, composition, proteolysis and ripening conditions (Kindstedt, 1993, 1995; McMahon et al., 1993a; Rowney et al., 1999; Guinee, 2002). The interactive

effects of these factors influence the degree of protein aggregation, or hydration, and the level of fat coalescence on heating, which in turn determine the level of heat-induced displacement. Varieties with low levels of fat and primary proteolysis, e.g., half-fat Cheddar, tend to have poor flowability. Differences in protein and fat contents

Increasing the protein content of Cheddar, by reducing the level of fat, impairs its functionality, as reflected by decreases in flowability, stretchability and an increase in the apparent viscosity of the melted cheese (Olson and Bogenrief, 1995; Guinee et al., 2000a,b). The extent of

410 Cheese as an Ingredient

25

160

120 100

15

80 10

60

Stretchability, cm

pH 4.6-soluble N, % total N

140 20

40 5 20 0 0

50

100

150 200 Storage time, d

250

0 300

Figure 7 Changes in the levels of pH 4.6-soluble N (closed symbols) and stretchability (open symbols) in experimental low-moisture Mozzarella cheese samples ripened at 4 (▲, 䉭) or 10 (■, ⵧ) °C, and full-fat Cheddar (●, 䊊) ripened at 4 °C for 30 days and at 8 °C thereafter. Data presented are the means of triplicate trials for the Cheddar, and of duplicate trials for the Mozzarella cheeses.

the effect increases with the degree of fat reduction and protein increase. Hence, the level of intact casein in Cheddar and LMMC is correlated positively with the apparent viscosity of the molten cheese and negatively with its flowability (Guinee et al., 2001). Similarly, increasing the protein content of LMMC, by lowering the fat content, reduces the flowability and increases the apparent viscosity (see ‘Pasta-Filata Cheeses’, Volume 2). The adverse effects of increasing protein content is due to a number of concomitant changes which impede displacement of adjoining layers of the matrix. The changes include an increase in the volume fraction of the casein matrix, decreases in the levels of MNFS and proteolysis and the lower degree of heat-induced fat coalescence and FO (Rudan and Barbano, 1998; Rüegg et al., 1991; McMahon et al., 1993a; Guinee and Law, 2002; ‘Pasta-Filata Cheeses’, Volume 2). Moreover, a reduction in the number of fat globules embedded in the casein matrix probably enhances the degree of fusion and aggregation of the rennet-altered micelles within the matrix during gel formation and post-cutting stages of cheese manufacture. Occluded fat globules limit the extent of contraction of the surrounding matrix and thereby physically impede casein aggregation. Storage time and proteolysis

Numerous studies have shown that the various functional attributes of cheeses such as Mozzarella and Cheddar change during storage to a degree depending on the composition and functional attributes of the

cheese, e.g., whether stretch or flow (Fig. 6). Changes in proteolysis (e.g., level of pH4.6SN) and protein hydration are major factors contributing to age-related changes in functionality. For a given level of protein, the functionality of cheeses such as Cheddar and LMMC is markedly influenced by the extent of proteolysis (Arnott et al., 1957; Bogenrief and Olson, 1995; Guinee et al., 2001). This effect is reflected by the positive curvilinear relationship between the magnitude of primary proteolysis, as measured by the level of total N soluble at pH 4.6, and the flowability for different varieties (Fig. 8). Hence, elevation of primary proteolysis using different means, e.g., the addition of exogeneous proteinases, the use of coagulants more proteolytic than chymosin (Cryphonectria parasitica proteinase) or elevation of storage temperature, enhances the flowability of different cheese types (Lazaridis et al., 1981; Yun et al., 1993a,b; Madsen and Qvist, 1998; Feeney et al., 2001; Guinee et al., 2001). The positive effect of proteolysis may be associated with a number of concomitant changes, including the increased water-binding capacity (Kindstedt, 1995) and an increase in the number of discontinuities or ‘breaks’ in the casein matrix at the micro-structural level (de Jong, 1978a). The latter factors are expected to reduce the degree of casein aggregation, which should enhance heat-induced displacement of adjoining layers of the casein matrix. The different relationships between flowability and primary proteolysis among varieties, as measured by

Cheese as an Ingredient 411

100 90 80

Flowability, %

70 60 50 40 30 20 10 0 0

3

6

9

12

15

18

pH 4.6-soluble N, % total N Figure 8 Relationship between flowability and primary proteolysis, as measured by pH 4.6-soluble N, for different varieties of cheese: Kashakaval (䉭), low-moisture part-skim Mozzarella (■), half-fat Mozzarella (ⵧ), full-fat Cheddar (30.0%, w/w, fat; ●), Emmental-type cheese (▲) and low-fat Cheddar (7.1%, w/w, fat; 䊊). Flowability was measured as described in Fig. 6.

pH4.6SN (Fig. 8), clearly highlights inter-varietal differences in the contribution of other factors (e.g., curd treatment, pH, levels of fat and casein, calcium-to-casein ratio) to flowability. Hence, for a given level of proteolysis, the flowability of reduced-fat cheeses is much lower than that in their full-fat counterparts (Fig. 7). Degree of fat emulsification (DE), fat coalescence and milk homogenization

Increasing the degree of fat emulsification (DE) in cheese by high-pressure homogenization of the cheesemilk (e.g., at first- and second-stage pressures of 25 and 5 MPa) impairs the flowability and stretchability of heated full-fat Cheddar (Guinee et al., 2000b), Halloumi (Lelievre et al., 1990) and full-fat Mozzarella (Jana and Upadhyay, 1991; Tunick et al., 1993). The effect of homogenization, which for Cheddar is similar to reducing its fat content from 30 to 1.3%, w/w (Guinee et al., 2000b), would be highly undesirable in applications such as pizza but highly desirable where a high degree of flow resistance is required, e.g., in frying. The adverse effects of milk homogenization coincide with marked reductions in the degree of fat coalescence in the unheated and the heated cheeses and in the release of FO on baking. Increasing the degree of emulsification in PCPs by the selective use of emulsifying salts and other blend ingredients and processing conditions has similar effects (see ‘Pasteurized Processed Cheese and Substitute/Imitation Cheese Products’, Volume 2).

In contrast to the above studies, Rudan et al. (1998) found that homogenization of milk or cream, at first- and second-stage pressures of 13.8 and 3.45 MPa, did not significantly affect the mean flowability of low-fat (⬃8.5%, w/w) LMMC over a 45-day ripening period. However, similar to the results of Jana and Upadhyay (1991), there was a significant reduction in the level of FO released on baking. The similar flowability of the control and the homogenized low-fat cheeses in the study of Rudan and Barbano (1998), despite the differences in FO, was probably due to the very low level of FO in all low-fat cheeses. The FO was ⬃0.25 or 0.6 and 3.9% of total fat for the homogenized-milk cheese, homogenized-cream cheese and the control low-fat cheeses at 40 days; the corresponding values for commercial LMMC ranged from ⬃10 to 40% of total fat, depending on FDM and age (Kindstedt and Rippe, 1990; Kindstedt, 1993). Thus, it is noteworthy that Tunick et al. (1993) reported that the interaction between fat content and homogenization pressure had a significant effect on the flowability of LMMC, ripened for 1 or 6 weeks. Homogenization of milk had little effect on the flowability of low-fat LMMC (⬃10%, w/w, fat) but impaired markedly that of regular LMMC with a higher fat level (⬃25%, w/w). The adverse effects of increasing the DE on the functional properties of heated cheese are due to the interaction of newly formed fat globules with the paracasein matrix. The effective protein concentration of, and the overall level of protein–protein interactions in,

412 Cheese as an Ingredient

the casein matrix are thereby increased (Guinee and Law, 2002). Consequently, it is expected that functional properties relying on displacement of contiguous layers of the casein matrix (e.g., flow and stretch) would be impaired by homogenization of the cheesemilk. Moreover, the recombined fat globule membrane stabilizes the newly formed fat globules to heat-induced coalescence (Guinee et al., 2000b). The consequent reduction in FO predisposes the cheese to dehydration during heating (Rudan and Barbano, 1998) and reduces the lubricating effect of oil on the surfaces of adjoining layers of the para-casein matrix during displacement. Thus, the adverse effects of homogenization on flowability and stretchability may be reduced (Lelievre et al., 1990) by: • lowering the homogenization pressure which has the effect of reducing the surface area of the fat phase and the number of newly formed fat globules; • preventing the casein micelles adsorbing at the fat–water interface by using a surface film of lecithin, which has the effect of making the newly formed globules more susceptible to heat-induced coalescence. Effect of whey proteins and casein–whey protein interaction on flowability

In some cheese applications, softening or melt is essential but very limited flow, or a high degree of flow resistance, is required so as to preserve the shape and identity of the cheese. Examples of the latter include fried Paneer or Quesco Blanco, grilled or fried burgers containing cheese insets, deep-fried breaded cheese sticks, kebabs and casseroles in which the identity of cheese pieces following cooking is desirable (Chandan et al., 1979; Anonymous, 1999). Most other natural cheeses, especially when mature, are unsuitable for these applications owing to their excessive flow and oiling-off on cooking. In the case of cheese insets in deep-fried burgers, the latter attributes result in the melted cheese piece permeating the interstices of the coarse meat emulsion, and hence the inset looses its shape and visual effect in the cooked product (Guinee and Corcoran, 1994). Flow resistance in natural cheese is generally conferred by the presence of whey proteins, which may be included by several means: • in-situ denaturation of the whey proteins by HHT of the cheese milk, e.g., ⬃65% of total whey proteins are denatured at 100 °C  120 s; • high concentration ultrafiltration (HCFUF) of milk, with or without HHT of the milk before UF or the retentate after UF, for cheese manufacture involving little or no whey expulsion (e.g., using the AL-curd coagulator);

• addition of PDWPCs, prepared by HHT and acidification of whey, to the cheese curd (see ‘High heat treatment of milk and denatured whey proteins’). High heat treatment of the milk (e.g., 95 °C  5 min) and the resultant incorporation of a high level of denatured whey proteins is a feature of the manufacturing process of acid-heat coagulated cheese types, e.g., Queso Blanco types and Paneer and some fresh cheeses such as Cream cheese (Guinee et al., 1993b; ‘Acid- and Acid/Rennet-Curd Cheeses: Part A Quark, Cream Cheese and Related Varieties, Part B Cottage Cheese, Part C Acid-heat Coagulated Cheeses’, Volume 2). In contrast, HHT of milk is not usually practised in the manufacture of rennet-curd cheese varieties, because of the impaired curdforming properties of the heated milk, higher moisture content of the cheese and, generally, higher fat losses (see Fox et al., 2000). Guinee (2002) showed that increasing the level of whey protein denaturation (WPD) from 3 to 13% total in the milk (⬃0.3–1.0%, w/w, denatured whey protein in the cheese) had little, or no, effect on the flowability of reduced-fat Cheddar. Even though the moisture content of the cheese increased with increasing level of WPD, there was a marked decrease in the flowability on further increasing the WPD to 30% total (⬇2.4%, w/w, denatured whey protein in the cheese). The stretchability of the molten reduced-fat Cheddar was reduced at all levels of WPD, with the effect increasing with the degree of WPD. Similarly, the flowability of Mozzarella cheeses prepared from HHT milk (e.g., ⬃5–35% WPD; Schafer and Olson, 1975) or from HCFUF milk retentates (e.g., 42–48%, w/w, dry matter; Covacevich, 1981; Madsen and Qvist, 1998), was markedly lower than the corresponding cheeses from control milk. Some cheeses may be produced from HCFUF retentates (often referred to as liquid pre-cheeses) which are treated with starter culture and/or rennet, and then placed in containers, or moulded either before (as in cast Feta) or after curd coagulation and further curd treatments (e.g., as in Blue- and Brie types, Cream cheese, Cheddar, Mozzarella). This method of production involves little or no loss of whey following treatment of the retentate and retention of most of the whey proteins (see ‘Application of Membrane Separation Technology to Cheese Production’, Volume 1). Maubois and Kosikowski (1978) described a method whereby Mozzarella cheese with stretch properties similar to those of a control cheese could be manufactured by HCFUF (to ⬃43% dry matter) and diafiltration at pH 5.8 (to reduce the calcium content); however, little information was given on experimental details or flowability. Covacevich (1981) described the manufacture of Mozzarella curd, which plasticized

Cheese as an Ingredient 413

satisfactorily, from HCFUF milk retentate (⬃42%, w/w, dry matter) which had been pre-acidified and salted prior to diafiltration to reduce its Ca content. However, the flowability of the UF Mozzarella at 1 week was less than half of that of commercial Mozzarella (Covacevich, 1981). The UF cheese had a markedly lower pH (⬃5.15 versus 5.9) and lower levels of moisture (⬃460 versus 510 g per kg) and MNFS than commercial Mozzarella, changes which could affect flowability adversely (Metzger et al., 2000a,b; Guinee et al., 2002). Similarly, Madsen and Qvist (1998) reported that the flowability of Mozzarella produced from a pre-cheese (48% dry matter) prepared by HCFUF of milk and containing 7%, w/w, whey protein was significantly lower than that of the control Mozzarella throughout a 5-week ripening period. The adverse effect was partly counteracted by increasing the level of casein breakdown in the cheese by treating the curd with a proteinase from Bacillus licheniformis or Bacillus subtilis (Neutrase®). The addition of denatured whey proteins (prepared by the Centriwhey process or by HHT of reconstituted WPC) at a level of 0.3–0.4%, w/w, to the cheese milk for LMMC, markedly reduced the flowability and/or the stretchability, and increased the apparent viscosity, of the melted cheese (Punidadas et al., 1999; Meade and Roupas, 2001). McMahon et al. (1996) reported different effects for added whey protein preparations, Simplesse® D100 and Dairy-Lo®, on the flowability of low-fat (4–5%, w/w) Mozzarella cheese. At most times during a 28-day storage period, the flowability of the cheese containing Simplesse® was numerically, but not significantly, higher than that of the control while that of the cheese with added Dairy-Lo® was lower than that of the control. The latter trend is probably a consequence of: • a higher level of whey protein in the Dairy-Lo®containing cheese (estimated at 0.6%, w/w, versus 0.23%, w/w); • the HHT of Dairy-Lo®-containing milk relative to the control and Simplesse®-containing milks; • differences in the size of whey protein particles and their spatial distribution in the cheese matrix (McMahon et al., 1996); • the degree of interaction of the whey protein particles in the different preparations with the para-casein matrix, as affected by factors such as pH and calcium level (Jelen and Rattray, 1995). The adverse effects of whey proteins on the functionality of heated cheese are probably due to their ability to undergo self-aggregation or aggregation with the para-CN in the concentrated acid cheese environment to form aggregated protein structures (pseudo-gels) at the

high temperature (typically ⬃98 °C) reached during baking/grilling. The tendency to aggregate and gel is probably accentuated by the high content of soluble calcium in the cheese (Doi et al., 1983; Jelen and Rattray, 1995). On setting, these structures would impede displacement of adjacent layers of the para-casein matrix and thereby flow of the molten cheese mass. The formation of some type of aggregate or pseudo-gel is supported by the results of dynamic viscoelastic analysis on heating reduced-fat Cheddar cheese from 20 to 90 °C. At temperatures 61 °C, the phase angle, , decreased and G increased uncharacteristically at a DWP level 1%, w/w, in the cheese (Guinee, 2002). A similar trend for was observed by Horne et al. (1994) for Cheddar cheese prepared from HHT milk (110 °C  60 s). These trends suggest an abrupt increase in elasticity, and a decrease in fluidity, as a result of some aggregation/gelation at a temperature 61 °C. Browning of cheese as a consequence of the Maillard reaction

Maillard browning essentially involves reactions between an aldehyde group (e.g., of reducing sugars such as lactose and galactose) and a free amino group (e.g., - or -amino groups of amino acids, peptides and protein) and other reactive N-groups (O’Brien, 1995). Browning may occur sometimes during storage of the unheated cheese (e.g., Parmesan, Romano) or processed cheese (Piergiovanni et al., 1989; Younis et al., 1991; Abd El-Salam et al., 1996; Gopal and Richardson, 1996) but more frequently on heating, e.g., Mozzarella and other cheeses made with a thermophilic culture and PCPs or ACPs. While slight browning of cheese may be desirable in some cooked applications (e.g., lasagna, pizza, crustinis), intense (dark) browning is unacceptable from aesthetic and nutritional viewpoints. Browning rarely occurs on cooking rennet-curd cheeses made with a mesophilic culture (e.g., Cheddar) since these have little or no residual sugars, even after a very short ripening time, e.g., 14 days (Torres et al., 1995). However, these cheeses may be susceptible to browning on heating if residual lactose persists in the cheese as a result of excessive salt, which inhibits starter metabolism (Thomas and Pearce, 1981; Jordan and Cogan, 1993). Moreover, in the absence of reducing sugars, aldehyde groups, resulting from degradation of amino acids or FFAs (Fox et al., 1996; Fox and Wallace, 1997), may pre-dispose the cheese to heatinduced browning, especially if the cheese is mature and has a high concentration of amino acids. Cheeses made using galactose-negative thermophilic starters (e.g., most strains of Streptococcus thermophilus and Lacobacillus delbrueckii subsp. bulgaricus) are very susceptible to Maillard browning, especially during

414 Cheese as an Ingredient

heating. Hence, Parmesan or Romano cheeses, which may contain residual galactose and free amino acids, are usually dried at a low temperature (e.g., 40 °C) to minimize the risk of brownish discolouration. Browning of Mozzarella cheese during cooking and its causes are discussed in ‘Pasta-Filata Cheeses’, Volume 2. Browning frequently occurs on cooking PCPs and ACPs, especially those with a high concentration of lactose or other reducing sugars added via ingredients such as maltodextrins, skim milk or whey powder, WPCs, milk protein or unfermented milk ultrafiltrates (Thomas, 1969; ‘Pasteurized Processed Cheese and Substitute/Imitation Cheese Products’, Volume 2). Owing to their generally higher pH, high concentration of lactose and buffering capacity (associated with added sodium phosphates), PCPs are probably more prone to Maillard browning during storage and cooking than natural cheeses.

Cheese Ingredients Cheese ingredients are prepared by subjecting cheese to either: • minimal primary processing involving a macrostructural change by the application of some physical form of comminution (e.g., as for diced, grated or shredded cheeses), or • more elaborate secondary processing involving processes (e.g., heating, shearing) and/or agents (e.g., enzymes, emulsifying salts) which lead to marked changes in microstructure, composition, levels of proteolysis and lipolysis, texture, flavour and/or physical form. The various types of cheese ingredients listed in Fig. 1 can be arbitrarily categorized as comminuted cheeses, dehydrated cheese ingredients (DCI) and concentrated cheese flavours (CCF) which comprise different types of enzyme-modified dairy ingredients (EMDI). Sometimes, EMDIs, such as EMC, may be dried for convenience of use (e.g., dry-blending with powdered formulations such as soups and cake mixes) and consequently fall into the category of DCI. The following sections focus mainly on DCIs and EMDIs. Little published information is available on the properties (e.g., free-flow, fines levels, shelf-stability) of comminuted cheese forms and how these are affected by the characteristics of the cheese. However, factors that affect the shreddability of Mozzarella cheese are discussed in ‘Pasta-Filata Cheeses’, Volume 2; moreover, much information on the physical properties of comminuted cheeses can be gleaned from an understanding of the fracture properties of cheese, which are discussed in ‘Rheology and Texture of Cheese’, Volume 1 and

‘Rheology-Based Functional Properties of Unheated Cheese’. Dehydrated cheese ingredients (DCIs): dried cheeses and cheese powders

The DCIs are industrially produced cheese-based ingredients which were developed during the Second World War as a means of preserving cheese solids under conditions to which natural cheese would not normally be subjected, e.g., temperature 21 °C for a long time period. There are currently three main types of DCIs, namely, dried grated cheeses, cheese powders and dried EMCs. More recently (Anonymous, 1999; King, 1999), freeze-dried cheese pieces (e.g., cubes) have been developed as commercial products. Today, DCIs are of major economic importance owing to their ubiquitous use as flavouring agents and/or nutritional supplements in a wide range of foods (Duxbury, 1991; Lewin, 1996; Missel, 1996; King, 1999). These include bakery products, biscuits, dehydrated salad dressings, sauces, snack coatings, soups, pasta dishes, savoury baby meals, cheese dips, au gratin potatoes and readyprepared meals. Other uses are their inclusion in processed and analogue products as flavouring agents or as a functional ingredient in powdered instant cheese preparations, which can be reconstituted by the consumer for the preparation of instant functional cheeses (e.g., pizza type) for domestic use. Advantages over natural cheeses as an ingredient in the above applications areas include: (i) convenience of use by fabricated food manufacturers. Dehydrated cheese ingredients can be applied easily to the surface of snack foods (e.g., popcorn, potato crisps, nachos) or incorporated into fabricated food formulations, e.g., by dry-mixing with other dry ingredients such as skim milk powder (e.g., as in dried soup, sauce or cake mixes) or blending into wet formulations. In contrast, natural cheeses require size-reduction prior to their use in these applications. (ii) their longer shelf-life, owing to their lower water activity (aw), than natural cheese. The aw for natural cheeses ranges from ⬃0.99 for Quarg to 0.917 for Parmesan (Rüegg and Blanc, 1981); from ⬃0.93 to 0.97 for PCPs (Kautter et al., 1979; Tanaka et al., 1979; Eckner et al., 1994), and from ⬃0.2 to 0.3 for various dairy powders (Spiess and Wolf, 1983). The relatively high stability of cheese powders allows them to be stored for a long period without alteration or deterioration in quality. In contrast, the changes which occur in natural cheese during storage may influence its processability (e.g., ability to be size-reduced, its inter-

Cheese as an Ingredient 415

action with other ingredients) and flavour profile and intensity. Hence, compared to natural cheese, cheese powders lend themselves to easier inventory management, set-manufacturing methods and end-products with consistent quality in large-scale manufacturing operations. (iii) the greater diversity of flavour and functional (e.g., mouth-feel) characteristics that can be obtained from a cheese powder, made possible by the use of different cheese types, EMCs and other ingredients in its preparation. Dehydrated cheese ingredients may be classified into three types, depending on the ingredients used: (i) Dried grated cheeses (e.g., Parmesan, Romano); (ii) Cheese powders, which may be natural (made using natural cheeses, emulsifying salts and, optionally, natural cheese flavours) or extended, which incorporate other ingredients, such as dairy ingredients (e.g., skim milk solids, whey, lactose), starches, maltodextrins, flavours, flavour enhancers and/or colours. Alternatively, cheese powders can be classified according to the proportion of cheese solids, as a % of total dry matter: high cheese solids (i.e., ⬃95%, w/w), medium cheese solids (50%, w/w) or low cheese solids (50%, w/w; Missel, 1996). (iii) dried EMCs. Dried cheeses

Dried grated cheeses are normally used as highly flavoured sprinklings (e.g., for pasta dishes) and in bakery products, e.g., biscuits. Essentially, the production of these products involves fine grinding of hard cheeses and conveyance of the ground cheese to a dryer (usually fluidized bed-type) where it is exposed to low humidity air (e.g., 15–20% relative humidity) at an air inlet temperature 30 °C. Under these conditions, the vibrated cheese is dehydrated rapidly and evaporatively cooled, thereby reducing the risk of fat exudation, the tendency to balling/clumping and browning. The dried grated cheese (typically 17%, w/w, moisture) is generally pulverized and packaged under nitrogen to reduce the risk of oxidative rancidity during distribution and retailing. High moisture levels (e.g., 21%, w/w) are conducive to clumping and offflavour development, especially if the powder is exposed to air (Hermann, 1993). Certain properties are required for the production of dried grated cheeses, i.e., relatively low levels of moisture (e.g., 30–34%, w/w) and fat-in-dry matter (e.g., 39%, w/w), brittleness and elastic fracture characteristics. These properties lend themselves to efficient size reduction on grinding, minimize the susceptibility

to fat exudation and sticking of the cheese particles and contribute to efficient drying and a homogeneous product free of clumps. An intense cheese flavour is also a desirable characteristic. Generally, dried grated cheeses are used in small quantities, as sprinklings, with the objective of imparting a strong cheese note to pasta dishes, soups and casseroles. The cheeses which meet these criteria best are Parmesan and Pecorino cheeses, because of their composition, fracture properties and their strong, piquant, lipolysed flavour. In the Pecorino cheeses the latter characteristic ensues mainly from the addition of pre-gastric esterases (PGEs; from kid, goat or lamb) to the cheese milk, which preferentially hydrolyse the short chain fatty acids (C4–C8, especially butyric) from milk fat triglycerides during cheese maturation. High levels of butyric acid (i.e., 1500–2000 mg per kg cheese) endow Romano cheese with its peppery piquant flavour (Fox and Guinee, 1987). Parmesan is produced from raw milk and ripened for a long time (⬃2 years) and lipolysis is due mainly to the indigenous lipase. Owing to their generally lower firmness and high levels of moisture and FDM, cheeses such as mature Cheddar (moisture and FDM – ⬃37 and 52%, w/w, respectively) or Gouda (moisture and FDM – ⬃41 and 48%, w/w, respectively) are unsuitable for drying. These characteristics render the cheese susceptible to fat exudation and clumping during grinding and drying. However, these cheeses also may be grated and dried provided that they are first shredded and blended with Parmesan or Roman-type cheeses before grinding. The moisture content of dehydrated grated cheese may be reduced further from 17%, w/w, by using the Sanders drying process (Kosikowski and Mistry, 1997). The grated cheese powder, placed on trays which are conveyed through a drying tunnel, is exposed to hot air which heats the cheese particles to 63 °C and reduces the moisture content to ⬃3–4%, w/w. High-moisture (82%, w/w) cheeses such as Cottage cheese may also be dried directly to 3–4%, w/w, moisture, by first pulverizing and then subjecting them to specialized spray-drying operations (e.g., Silo spray drying using the Birs Dehydration Process; Kosikowski and Mistry, 1997). These low-moisture, dried natural cheeses are generally used for nutritional supplementation of foods, e.g., dried baby meals. Freeze-dried formats of a number of different cheeses such as mature Cheddar, Gloucester, Stilton and oaksmoked Cheddar are now produced commercially (Anonymous, 1999; King, 1999). The benefits of freezedrying, compared to air-drying or spray-drying, include: • the retention of volatile flavour compounds (e.g., degradation products of FFAs and amino acids),

416 Cheese as an Ingredient

• the ability to dry cheeses in the form of croutons, cubes or pellets, which convey an image of cheese pieces and greater naturalness compared to powder and are convenient to use (such pieces can be individually wrapped and added to soups and other products by the consumer), • the crunchy light texture of the dried cheese pieces which makes them easy to disperse (by rubbing between the fingers) as a topping and to re-hydrate allowing fast flavour release in the mouth. The production of freeze-dried cheese involves size reduction to the required format, layering onto trays, freeze-drying to ⬃3%, w/w, moisture and packaging.

Cheese powders Manufacture. The manufacture of cheese powders

essentially involves the production of a pasteurized processed cheese slurry (40–45%, w/w) which is then spray-dried (Fig. 9). The individual processing steps have been described (Hedrick, 1981; Guinee et al., 1993a, 1994; Missel, 1996; Darrington, 1999) and are discussed briefly below. (a) Formulation of the slurry blend. The blend usually consists of comminuted natural cheese(s), water, emulsifying salts, flavouring agents, flavour enhancers, colours, anti-oxidants such as propyl gallate, butylated hydrozyanisole (BHA) and/or filling materials such as

Cheese Milk solids whey buttermilk skim milk caseinate

Maltodextrin starches

Comminution Water

Formulation

Flavours & flavour enhancers EMDIs NaCl MSG Yeast extract

Emulsifying salts

Colours antioxidants free-flow agents Dissolving tank (cooker) Heat and shear

Steam Hot molten slurry (35 – 45%, w/w, dry matter; 75 – 85 °C)

Homogenization Atomization Spray-dry

Cheese powder (>96%, w/w, dry matter) Figure 9 Outline of production process for cheese powder. Abbreviations: EMDIs, enzyme-modified dairy ingredients; MSG, monosodium glutamate.

Cheese as an Ingredient 417

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-

tion) to a temperature of ⬃75–85 °C 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 85 °C minimizes the loss of flavour volatiles and the risk of browning, especially in formulations containing high levels of lactose or high dextrose equivalent (DE) maltodextrins. The blend is worked until the hot fluid slurry is homogeneous in colour and consistency. The levels of fat and protein, pH and degree of hydration of the constituent ingredients are the main factors that control the viscosity of the cheese slurry. The viscosity in turn has a major influence on its tendency to foam and the levels of occluded and interstitial air in the resultant powder. High-viscosity slurries (e.g., 3.0 Pas) have less propensity to foam and thus give lower levels of air in the powder compared to low-viscosity (e.g., 0.3 Pas) slurries (Masters, 1976; Tetra Pak Processing Systems, 1995). Incorporation of air should be avoided as it affects the physical (bulk density) and instant (wettability, dispersability) characteristics of the powder and its susceptibility to oxidation on storage (Masters, 1976; Tetra Pak Processing Systems, 1995). The air content of the cheese powder is also influenced by the presence of ingredients which tend to promote (e.g., undenatured whey proteins) or depress (e.g., fat, food-grade antifoaming agents) foaming. However, for a given formulation, air incorporation may be minimized by de-aerating the slurry prior to spray-drying and by using the appropriate type of atomizer, e.g., a pressure nozzle rather than a rotary disc atomizer (J. Kelly, personal communication).

Table 4 Typical formulations for cheese powders with low (25.5%, w/w, of total), medium (54.3%, w/w, of total) and high (95.4%, w/w, % of total) levels of cheese solids Level of ingredients added during formulation (%, w/w, of total blend), prior to processing and drying

Ingredient

Low cheese solids powder

Medium cheese solids powder

High cheese solids powder

Mature Cheddar Extra-mature Cheddar Enzyme-modified cheese paste Enzyme-modified cheese powder Whey powder Skim milk powder Maltodextrin Emulsifying salt Sodium chloride Butylated hydroxyanisole Water and condensate

– 20.0 1.0 – 12.0 8.0 16.5 1.5 1.0 0.2 39.6

17.0 19.0 0.2 2.0 5.0 3.8 11.0 1.5 1.0 0.4 39.1

32.0 33.0 – – – – – 2.0 0.5 0.5 32.0

Based on data from Guinee et al. (1993a).

418 Cheese as an Ingredient

(c) Homogenization of the slurry. This step is optional but is commonly practised to ensure homogeneity of the slurry. The pressures applied (typically 15 and 5 MPa, first and second stages, respectively) have a major influence on the viscosity of the slurry, with higher pressures generally imparting higher viscosity for a given level of dry matter. (d) Spray-drying of the slurry. Several spray-drying processes (e.g., single stage or two stage) and dryer configurations (e.g., tall-form, filter mat, silo-form) may be used. The different processes and their operations (e.g., atomizer type and pressure, air-flow direction, air inlet and outlet temperatures, air humidity) influence the physical (e.g., bulk density, wettability and solubility) and flavour characteristics of the cheese powder. The wettability and solubility characteristics are important in applications requiring reconstitution of the cheese powder, e.g., ready-prepared soups, sauces and baby foods. Typical air inlet temperature ranges from 180 to 200 °C and air outlet temperature from 85 to 90 °C, depending on the dryer type. The powder is cooled to ⬃20 °C in an external fluidized bed, using dehumidified air before packaging. The moisture content of the dried powder is typically 3.0–4.0%, w/w, and generally decreases with increasing outlet air temperature. A high outlet temperature (95 °C for a tall form dryer) should be avoided to minimize Maillard browning, loss of flavour volatiles, loss of wettability characteristics, excessive oiling-off and free fat formation. Free fat in the cheese powder leads to lumpiness, flow problems and flavour deterioration. Commercially, cheese powders are normally manufactured using two-stage drying systems, e.g., filtermat (box) dryers are used frequently in the USA while tallform dryers with one or more integrated fluidized beds are commonly used in Europe. While the operating conditions of these dryers influence the quality of the final cheese powder, the tall-form dryer is generally considered to give better flavour retention, larger powder particles and better powder flowability. Conventional single-stage dryers are rarely used, except for experimental purposes, because of the high outlet air temperature (e.g., 95 °C) necessary to achieve the low moisture content (⬃4%, w/w) required. However, single-stage silo-dryers (with the drying tower ⬃70-m high compared to ⬃10 m for the tall-form dryer) may be used, as in the Birs Dehydration Process (Kosikowski and Mistry, 1997), where the drying air at 0–30 °C, is de-humidified. The moisture content of the powder emerging from the tower is ⬃10%, w/w, and is reduced to ⬃4%, w/w, in smaller drying chambers. The main advantages of this process over conventional

two-stage drying is that it results in improved colour stability and enhances flavour retention, especially in products with a high level of volatile flavour compounds (e.g., Cottage cheese, yoghurt). Cheese powders are generally used as flavouring ingredients in a wide variety of foods, especially snack coatings (e.g., pop corn, nachos, tortilla shells), extruded snacks, cheese sauces, soups, savoury dressings and savoury biscuits (Missel, 1996). The development of ‘bake-stable’ cheese powders is claimed to overcome problems (e.g., unpredictable rise during the baking of crackers, off-flavour development during the production of extruded pelleted snacks) encountered during high-temperature processing (Lewin, 1996). In snack foods, application involves dusting on the powder after the snack has been lightly sprayed with vegetable oil. In cheese sauces, the level of cheese powder is typically 5–10%, w/w, depending on the flavour intensity of the cheese powder and the types and levels of other flavouring ingredients in the formulation. Generally, the cheese powder, at these levels, has little influence on the rheological properties of the resultant sauces; the latter properties are controlled mainly by the types and the levels of starchy materials used (Guinee et al., 1994). Cheese powders may be also combined with various spices, such as cumin, garlic, chilli or onion, for the production of seasonings or savoury additives (Anonymous, 1993; Lewin, 1996; Mortensen, 1999). Applications of cheese powders.

Enzyme-modified dairy ingredients: enzyme-modified cheese

Natural cheeses have certain limitations as a food ingredient: (i) low flavour stability due to ongoing biochemical/microbiological changes during storage, (ii) flavour inconsistency (e.g., due to changes in cheese composition), (iii) insufficient flavour strength to enable small quantities to impart a strong cheese flavour, (iv) the high cost due to the relatively long ripening time for most cheese varieties and the high usage level required to impart a given cheese flavour intensity to foods and (v) the necessity to comminute cheese prior to its application and the fact that comminuted cheese is not suited to the bakery and the snack food industries which are large users of cheese. These deficiencies led to the development of EMC, by exploiting the natural biochemical cheese flavour development pathways through enzyme technology, which resulted in cheese flavour intensities of up to 30-fold that of the corresponding natural cheese (Kilcawley et al., 1998; Kilcawley, 2002). Enzyme-modified cheeses can be defined as concentrated cheese flavour ingred-

Cheese as an Ingredient

ients, which offer a cost-effective alternative to natural cheese as a source of cheese flavour. Enzyme-modified cheese variants of many natural cheeses, such as Cheddar, Blue, Romano, Parmesan, Colby, Gouda, Camembert, Mozzeralla, Gruyere, Brick and Emmental are available commercially (Freund, 1993; Holdt, 1996; Kilcawley et al., 1998). However, little scientific information is available on the types of enzymes and their specificities, enzyme dosage levels, substrate characteristics and process conditions. Most of the information relating to these parameters remain proprietary to individual manufacturers. Applications

Enzyme-modified cheeses are used principally as flavouring agents in industrial-based cheese products/ingredients such as PCPs, cheese substitutes/imitations, cheese powders, soups, sauces, dips, salad dressings, snack coatings, crackers and in prepared and semi-prepared foods. There has been a marked increase in the production of EMCs in recent years. This market continues to expand due to consumer demand, as modern life-styles have reduced the time available to prepare food in the home and increased the amount of food eaten outside the home (Cowan and Cronin, 1999). Legislation regarding the labelling of products containing EMCs varies between the USA and Europe. Enzyme-modified cheeses can be classified into two groups: EMCs per se which are produced by natural processes (e.g., enzymatic hydrolysis and fermentation) and EMC-WONF (with other natural flavours) which are similar to EMC but contain chemically synthesized nature-identical flavours, frequently referred to as ‘top notes’ in the industry. It is generally assumed that these substances are produced or isolated by chemical processes to mimic natural flavour compounds (Freund, 1995). In the USA, EMCs received GRAS (generally regarded as safe) status in 1969, and since 1970 have been added to specific categories of pasteurized process cheese, non-standard cheese products such a nontraditional reduced-fat or fat-free products and to various prepared foods. Non-standard cheeses include cheese products for which standards of identity do not exist (Code of Federal Regulations, 1986). There are no EU standards relating to the classification of EMDIs, including EMCs. However, a European Union Directive (European Union, 1998) classifies EMC as a ‘flavouring preparation’ which is defined as a substance obtained by physical, enzymatic or microbiological processes from material of vegetable or animal origin, either in the raw state or after processing for human consumption, by traditional food-preparation processes. Enzyme-modified cheeses with other natural flavours are classified as ‘flavouring substances’ which are defined

419

as: (i) chemical substances with flavouring properties obtained by either chemical synthesis or isolated by chemical processes and which are chemically identical to a substance naturally present in material of vegetable or animal origin and/or (ii) by chemical synthesis, but which are not chemically identical to a substance naturally present in material of vegetable or animal origin (European Union, 1998). Technology and manufacture

The general manufacture of EMC typically involves the following steps (Fig. 10): (i) Production of a cheese curd, as in conventional cheese manufacture; (ii) Formation of a paste substrate (typically, 400–600 g per kg dry matter) by blending the curd with water and emulsifying salts. Additional sources of fat and protein are often added as substrates for flavour generation or to enhance other characteristics, such as consistency and texture. Manipulation of compositional parameters is an integral feature of EMC production; (iii) The cheese paste substrate is usually pasteurized (e.g., 72–80 °C for 10–20 min) to inactivate the existing cheese microflora and enzymes. Pasteurization reduces the risk of flavour inconsistencies due to variations in strain composition and populations of starter culture, NSLAB and residual enzyme activities (e.g., chymosin) in cheese curds; (iv) Often, the pasteurized cheese paste is homogenized to increase the surface area of fat available for optimal lipolysis; first and second stage pressures vary, e.g., 15–25 MPa and 5 MPa, respectively; (v) Treatment of the pasteurized cheese paste with the desired enzymes (blend of proteinases, peptidases, lipases) to give the required flavour profile and intensity in the final EMC. Starter cultures may be used to give more authentic natural cheese flavours but they are usually added in combinations with enzymes; (vi) Incubation of the pasteurized cheese paste. Process parameters are typically 25–45 °C with constant slow agitation for ⬃1–4 days at pH 5–7; (vii) Pasteurization of the enzyme-treated paste to inactivate added enzymes and thereby to preserve the generated flavour characteristics with minimal change on storage; (viii) Homgenization of the hot pasteurized paste to reduce the tendency of phase separation on subsequent storage and thus to ensure product homogeneity. The homogenized cheese paste, known as EMC paste, may be then packaged, usually in opaque materials to minimize the risk of oxidative

420

Methods of Enzyme-Modified Dairy Ingredient EMDI Production

EMF

EMC

Integrated approach

Two-step approach

Component approach

Cheese, water, emulsifying salts

Cheese, water, emulsifying salts

Fat

Protein

Cheese slurry

Cheese slurry

Homogenize

Homogenize

Pasteurize Homogenize

Pasteurize Homogenize

Lipase/esterase

Proteinases/peptidases and/or starter cultures

Enzymes Starter cultures

Proteinases/peptidases and/or starter cultures

Incubate –agitate 25–45 °C for 1–4 days

Pasteurize

Homogenize Cool

Incubate –agitate 25– 45 °C for 1– 4 days

Blue cheese concentrate submerged fermentation Cream Milkfat UHT milk Butteroil Other fats/oils Homogenize

Lipase/esterase Incubate –agitate 25– 45 °C 1–2 days

Incubate –agitate 25– 45 °C 1–2 days

Pasteurize

Pasteurize

Incubate –agitate 25– 45 °C for <1–2 days

Pasteurize

Pasteurize Homogenize Cool

Lipases/esterases

Homogenize Cool

Homogenize Cool

Penicillium roqueforti inoculum

Blend Incubate –agitate Low aeration 25–30 °C for 2–6 days

Incubate –agitate 25– 45 °C for 1– 4 days Pasteurize Pasteurize

Homogenize Cool

Homogenize Cool

Figure 10 Methods of producing enzyme-modified dairy ingredients (EMDI), including enzyme-modified cheese (EMC) and enzyme-modified fat (EMF).

Cheese as an Ingredient 421

rancidity and off-flavour development and stored at refrigeration temperatures; (ix) The EMC paste may be dried to give an EMC powder, which has a longer shelf-life than the paste and can be stored at room temperature. Powdered products are better suited for applications involving dry blending with other ingredients. There are several different industrial approaches to EMC manufacture, which are outlined in Fig. 10. The one-step process involves a single substrate which is hydrolysed simultaneously by proteolytic and lipolytic enzymes to the desired final flavour (Fig. 10a). Alternatively, a two-step process may be used in which a single substrate is hydrolysed initially by proteolytic enzymes and subsequently by lipolytic enzymes to create the final product (Fig. 10b). The two-step process offers more controlled flavour production by reducing the possibility of lipase degradation (by proteinases) and preventing the pH from falling below the optimum for proteolytic activity by neutral proteinases. A high level of lipolysis is an integral feature of EMC production, which increases the concentration of FFA, leading to a marked reduction in pH (Kilcawley, 2002). The component process involves the independent hydrolysis of individual substrates by either proteinases or lipases and their subsequent combination (Fig. 10c). This process enables the creation of a range of different product types from two substrates. A further alternative approach is often used for Blue mould-type EMC flavours only. This method involves the submerged aerobic fermentation of various milkfat substrates, such as high fat cream, with Penicillium roqueforti (Nelson, 1970; Dwivedi and Kinsella, 1974). This technology is based on the development of a highly lipolysed product, which directly provides flavour and aroma components associated with Blue cheese flavours, such as butyric acid and precursors of key methyl ketones known to be critically important in Blue cheese flavour. Commercially, blue mould cheese flavour is produced primarily for use in Blue cheese-flavoured dips and salad dressings. Flavour generation in EMC production

It is recommended that natural cheese of the same variety as the type of flavour desired in the EMC should be used as the substrate so as to ensure product consistency and quality (West, 1996). The age of the cheese used as the substrate is also important as young cheese is cheaper but lacks flavour compounds associated with the development of a more authentic natural cheese flavour. Emulsification of the substrate optimizes enzyme activity, in particular lipolysis, which is significantly more important in EMC production than in most natural

cheese varieties (Kilcawley et al., 2001; Kilcawley, 2002). Lipolysis generates key volatile flavour compounds, i.e., compounds with a low flavour threshold such as fatty acids, esters, alcohols, lactones and ketones (Fox et al., 1996; Wallace and Fox, 1998; Curioni and Bosset, 2002) which contribute significantly to both the aroma and the flavour of cheese and are therefore directly related to its perceived ‘intensity’ of flavour. A highly emulsified substrate is generally achieved by homogenization and increases the surface area available for lipolysis. The use of emulsifying salts is necessary to provide a stable emulsion and the salts typically used are those that bind calcium weakly, such as disodium phosphate, trisodium phosphate and trisodium citrate. These salts are not actually emulsifiers per se, but with the aid of heat and shear convert insoluble calcium para-casein to an active hydrated form capable of binding water. Emulsifying salts can also be used to adjust the pH of the substrate to a level optimal for the enzymes used in the process, thereby further accelerating flavour development. In EMC production, an excessive level of hydrolysis may adversely affect emulsion stability and thus influence rate of flavour development. To alleviate this problem, preliminary work is often necessary to optimize the level of emulsifying salts, enzymes and substrate composition. Many EMC products are produced with the inclusion of flavour additives, often called ‘top notes’ in the industry, to create a diverse product portfolio or to match a particular requirement. These EMCs are classified as EMC-WONF. The most commonly used ‘top notes’ are monosodium glutamate and yeast extracts which are a source of free glutamic acid, which is known to mask bitterness and enhance the perception of flavour (Kilcawley, 2002). Organic acids such as acetic, butyric, propionic, valeric, lactic acids and diacetyl are also widely used to impart specific flavour notes to EMC products. These ‘top notes’ are used not only to impart specific flavours or enhance flavour, but also to harmonize the flavour of other ingredients, thus creating a more balanced overall flavour profile (Buhler, 1996). The widespread use of these compounds in EMC-WONFs, as evident from the work by Kilcawley et al. (2000, 2001), indicates that it may be difficult or uneconomical to match specific flavour requirements by enzyme technology alone. The relationship between the various commercially available EMC flavours and their corresponding natural cheese varieties is somewhat dubious. Hulin-Bertaud et al. (2000) carried out a detailed descriptive sensory analysis on 15 commercial Cheddar-type EMCs and were able to separate them into five separate clusters based on their sensory characteristics. This work highlighted major sensory differ-

422 Cheese as an Ingredient

ences between commercial EMC products marketed as having Cheddar flavour and also showed that none had a flavour identical to that of natural Cheddar cheese. The composition, flavour-forming reactions and flavour components of EMCs have been reviewed extensively (Talbott and McCord, 1981; Kilara, 1985; Moskowitz and Noelck, 1987; Grueb and Gatfield, 1989; Takafuji, 1993; Kilcawley et al., 1998). Enzymes used in EMC manufacture. The enzymes used in the EMC production process have major implications for the intensity and the type of flavour developed. These enzyme preparations contain proteinases, peptidases and lipases and act on casein and fat to produce flavour components and precursors of flavour components (peptides, free amino acids, amines, aldehydes, alcohols, ammonia, FFAs, ketones, lactones, esters and alcohols). A wide range of commercial enzymes are available, derived from plant, animal or microbial sources. Most of the enzymes used in EMC production are derived from microbial and/or animal sources. Plant enzymes are not generally used as they are comparatively impure and expensive, due to large volumes of plants required to produce sufficient amounts of enzyme. The choice of enzyme depends upon a number of factors, such as desired flavour, type and composition of substrate, cost and processing equipment available.

Quite a number of commercial microbial proteinases are available for EMC production, most of which are derived from either Bacillus or Aspergillus species and are therefore neutral or acidic in nature. Bacillus proteinases have been implicated in the development of bitterness in cheese and cheese products, and this has precluded their use to some extent in EMC manufacture (West, 1996). Studies by Kilcawley et al. (2002a) have shown that some commercial proteinase preparations from Bacillus spp. lack general aminopeptidase and post-proline dipeptidyl aminopeptidase activities, which are critical in the degradation of bitter peptides. However, the use of proteinases derived from Bacillus spp. is not a problem if used in conjunction with commercial peptidase preparations that contain general aminopeptidase and proline-specific peptidases. Peptidase preparations from Lactococcus lactis have been shown to contain high levels of post-proline dipeptidyl aminopeptidase activity, and peptidases from Aspergillus spp. contain high levels of general aminopeptidase activity (Kilcawley et al., 2002b). Therefore, a cocktail of peptidases derived from both sources provides suitable peptidase activities to prevent the accumulation of bitter peptides. Proteinases/peptidases.

Lipases. Lipolysis is very important in EMC production due to the production of volatile compounds, which are necessary in providing the perception of cheese flavour to a product to which EMC is added. Lipases are available from two main sources, animal and microbial. The most significant animal lipases are those isolated from bovine and porcine pancreatic tissues or the pre-gastric tissues of kid goat, lamb and calf (Nelson et al., 1977). Different-PGEs produce characteristic flavour profiles: calf PGE generates a ‘buttery’, slightly ‘peppery’ flavour, kid PGE generates a sharp ‘peppery’ flavour, often called ‘piccante’, whilst lamb PGE generates a ‘dirty sock’ flavour, often called ‘pecorino’ flavour (Birschbach, 1992). The use of animal lipases can often limit the application of products as they do not have vegetarian or Kosher status. Kosher status can be important, particularly in the USA, as these products are often perceived by consumers as being of higher quality than non-Kosher products. Microbial lipases are enzyme preparations derived from yeasts, moulds or bacteria, e.g., Rhizomucor miehei, Rhizopus arrhizus, Aspergillus niger, Aspergillus oryzae, Geotrichum candidum, Penicillium roqueforti, Achromobacter lipolyticum, Pseudomonas spp., Staphylococcus spp. and Candida cylindracea (Birschbach, 1994). Microbial lipases are generally cheaper than animal lipases due to lower production costs and have the added advantage of being suitable for use in vegetarian and Kosher foods and also do not contain amylases which may cause problems in foods to which EMCs are added (West, 1996). Commercial lipase preparations for potential use in EMC manufacture have been discussed by Kilcawley et al. (2002b). The selectivity of microbial lipases tends to vary with species and is an important facet in their choice in EMC flavour development, as differing ratios of FFAs impart characteristic flavours. However, it should be noted that extensive levels of lipolysis can lead to non-selective release of FFAs resulting in the development of similar lipolytic flavour profiles despite the use of lipases with different acyl selectivities. A major factor responsible for this is the migration of individual fatty acids on a glyceride, resulting in non-specific release of fatty acids. The effect of acyl migration can be markedly reduced by limiting the level of lipolysis or by using fat substrates of varying FA composition, e.g., the inclusion of vegetable fats with milk fat. It is now also possible to create structured lipids, i.e., lipids that contain specific fatty acids at precise positions on triglycerides. Interesterification catalysed by enzymes appears to offer the greatest potential and involves the exchange and redistribution of acyl groups among the lipid triacylglycerols. Using this approach, it is possible to enrich a fat substrate with specific fatty acids, known to impart flavour, nutritional

Cheese as an Ingredient 423

or functional characteristics (Balcao and Malcata, 1998; Bosley, 1999) and therefore possibly expand the use of EMC as an ingredient. Another problem associated with high levels of lipolysis is the production of surface active agents, e.g., mono- and diglycerides and soaps of FFAs, which inhibit lipolysis by blocking the interface between the lipase and the fat. This can reduce the rate of lipolysis, but can be alleviated by the addition of calcium salts which promote the formation of insoluble calcium soaps (Kilara, 1985). Starter cultures used in EMC production. Starter cultures are used in the commercial production of EMCs but details on the type, concentration and pretreatments of cultures used are not available publicly. It can be assumed that the use of starter cultures in EMC production is analogous to the role of attenuated starter cultures to accelerate the ripening of natural cheese, i.e., they are added as an additional source of flavour-generating enzymes (Kilcawley et al., 1998). Lee et al. (2001) demonstrated the potential of producing strongly flavoured Cheddar-type EMC using exogeneous enzymes in combination with Lactobacillus helveticus DPC 4571 using a two-step process. Dried EMC

Enzyme-modified cheeses may be dried to extend shelf-life and/or for ease of use in specific ingredient formulations. However, the preparation of dried EMCs requires the addition of other ingredients to aid the drying process, which in turn has a diluting effect on the flavour intensity of the final product. This problem is exacerbated by the heat used in the drying process, which results in losses in key volatile flavour compounds. While it is difficult to quantify the negative effect of drying on the flavour of EMCs, as drying procedures vary depending upon the ingredients and process parameters used, it is probably significant. Recent advances in spray-drying technology offer the potential to alleviate flavour loss associated with traditional spray-drying (Fischer, 2000). The possibility of adding functional coating layers through fluidized bed technology has the potential to add further functional characteristics, thereby creating more applications. Enzyme-modified dairy ingredients: enzyme-modified fat

These ingredients, although not cheese per se, are discussed briefly as they are used widely to impart cheese-like flavours and are produced by technology similar to that used in EMC production. Their impor-

tance as ingredients is due to their very high flavour intensity, which enables small amounts to deliver sufficient flavour to a product. High levels of flavour intensity are achieved via lipolysis of fat, resulting in the production of volatile compounds, e.g., short chain fatty acids. These can be degraded and contribute to the formation of other volatile flavour/aroma compounds such as methyl ketones, lactones, esters, alcohols and aldehydes, which have low flavour thresholds and can be perceived at minute concentrations. The importance of FFAs and their catabolic products are known to be critical in many dairy flavours, e.g., cheese-, butter-, cream- and yoghurt-type products (Urbach, 1993; Fox and Wallace, 1997). Hence, enzyme-modified fats (EMFs) find application in the baking, dairy, confectionery and savoury food sectors. The manufacture of EMFs generally involves producing an emulsion containing predominately fat or an oil-based substrate. Typically, butter-fat or cream is used as the oil substrate since they contain high levels of lower-chain volatile fatty acids, which are the most important flavour-producing fatty acids. However, depending on the application, certain vegetable oils may be used for cost-effectiveness functionality, flavour or nutritional purposes. The substrate is hydrolysed by starter cultures and/or lipases chosen on the basis of their acyl specificity/selectivity and incubated under defined conditions until the desired flavour is attained. The reaction is usually terminated by heat to inactivate the cultures and/or enzymes used. Reviews of EMFs include Arnold et al. (1975), Kilara (1985) and De Greyt and Huyghebaert (1995).

Conclusion Cheese is a highly versatile dairy ingredient, which is used directly or indirectly, in the form of cheese ingredients, in a vast array of culinary, formulated and prepared food products. The rheological, flavour and cooking properties are functional attributes, which have a major impact on the preparation and quality of these products. The interactive effects of various factors, including make procedure, cheese composition, degree of fat emulsification, proteolysis and lipolysis, affect cheese functionality. Many of the rheological and cooking functions may be viewed as displacement of adjoining layers of the casein matrix. Hence, these factors exert their effect mainly by affecting the microstructural distributions of fat and protein, and the degree of hydration (or aggregation) of the protein matrix in the raw and heated cheeses.

424 Cheese as an Ingredient

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Index

Acid-and Acid/Rennet-curd cheese, 17–18, 301–304 acid-heat coagulated, 343 Queso Blanco and Paneer, 343–5 Ricotta, 345–6 combined acidification/renneting: calcium chloride, 315 concentration, 308–11 heat treatment, 311–13 incubation temperature, 314 level/type of gel-forming protein, 314–15 pH at renneting, 313–14 rate of gelation, 314 Cottage cheese, 329–38 Cream cheese, 321–3 fresh cheese preparations, 323 gel formation during combined acidification/renneting: factors influencing, 308–15 mechanism, 306–308 physico-chemical changes, 305–306 other varieties, 323–4 Quark, 315–21 rheology: Cream cheese, 323 Quark, 320 unheated cheese, 399–405 ripened varieties, 323 see also Quark; Ricotta; Cottage cheese; Cream cheese Akawi, 241 Amarelo da Beira Baixa, 291 Analogue cheese products (ACPs), 21, 349, 379–85 composition/functionality, 384–5 formulation, 382–4 principles/manufacturing protocol, 382 see also Processed cheese products (PCPs) Anevato, 290 Appellation d’Origine Contrôlée (AOC), 1 Armada, 293 Arthrobacteriaceae, 206 Asiago, 55 Austrian cheese, 319 Azeitao, 284 Bacteriocins: smear-ripened cheese: anti-Listeria compounds of Geotrichum and Penicillium, 218 coryneform bacteria, 215–17 enterococci, 217–18 lactic acid bacteria (LAB), 214–15 Baker’s cheese, 319 Balkan, cheeses, 58–9, 319 Bastelicaccia, 290 Beli Sir U Kriskama, 244–5 Beyaz Peynir (Turkish White), 244

Bjalo Salamureno Sirene (White Brined), 244 Blue cheese, 6, 16–17, 175 micro-organisms contributing to ripening: contaminants, 183 lactic acid bacteria, 179 non-starter lactic acid bacteria, 182–3 Penicillium roqueforti, 179–80, 185 yeast, 180–2 microbial interactions, 183 microenvironment, 175–7 ripening: formation of aroma compounds, 189–90 lipolysis, 185–6 production/occurrence of mycotoxins, 190–1 proteolysis/amino acid catabolism, 186–9 selection of cultures, 191–2 Blue des Aravis, 289 Brevibacterium linens, 204 Brie, 199 Broccio, 280 Buttermilk Quark, 319 Cabra Transmontano, 291 Cacioricotta, 291 Caciotta D’Urbino, 283 Camembert, 157, 199 control of ripening, 169–71 flavours, 162–9 glycolysis, 160–1 goat-type, 289 lipolysis, 162 microbial flora, 158–60 proteolysis, 161–2 technology, 157–8 texture, 169 Cameros, 293 Canestrato Pugliese, 56–7, 283 Caprino tradizionale, 290 Castellano, 287 Castelmagno, 57 Castelo Branco, 284 Catabolism of amino acids: Blue cheese, 186–9 surface mould-ripened cheese, 165–7 Cendrat del Montsec, 292–3 Chakka/Shirkhand (India), 319 Cheddar, 14–15, 71 casein, 75 cheddaring, 71 chemical composition/quality: effect of FDM, 85 effect of MNFS, 83–4 effect of pH, 84–5 effect of S/M, 85

430 Index

Cheddar – contd. dry-salting, 71–2 flavour: milkfat, 89 proteolysis, 89–90 role of adjuncts, 92–3 role of non-starter lactic acid bacteria, 92 role of starter, 90–2 grading, 93–4 manufacture: acid production at vat stage, 76–7 cheddaring, 77–8 coagulant, 74–5 cutting, 75 heating/cooking curd, 75–6 milk composition/starter culture, 73–4 milling, 78–9 pressing, 81–3 salting, 79–81 texture: effect of pH, calcium, salt, 87 effect of protein, fat, moisture, 87 effect of ripening, 88 varieties: low-fat, 94–5 stirred-curd/granular cheese, 95 washed-curd, 95–6 Cheese analogues, 8, 379–85 Cheese as an ingredient, 18, 395–423 dehydrated cheese ingredients, 414–18 cheese powder application, 418 cheese powder manufacture, 415–18 dried cheeses, 414–15 enzyme-modified cheese (EMC): applications, 418–19 dried, 422 enzymes used in manufacture, 421–2 flavour generation in EMC production, 419–21 starter cultures, 422 technology/manufacture, 419 enzyme-modified milk fat, 422–3 functional properties of heated cheese: browning; Maillard reaction, 413 comparison of different varieties, 407–409 degree of fat emulsification, fat coalescence, milk homogenization, 411 differences in protein/fat contents, 409–10 storage time/proteolysis, 410 types, definitions, principles, 405 whey proteins/casein-whey protein interaction on flowability, 411–13 functional requirements, 396–9 rheology-based functional properties of unheated cheese: fat content, 402 high heat treatment of milk/denatured whey proteins, 401–402 homogenization of cheesemilk/fat emulsification, 402–403 moisture content, 403 pH, 404 protein level, 400–401 ripening/para-casein hydrolysis, 404–405 salt (NaCl) content, 403–404 Cheese categories, 1–22 Cheese technology: goats’ milk cheese, 287–9 overview, 24

post-vat stages: brine-salted varieties, 38–46 dry-salted varieties, 31–8 fresh cheese, 46–9, 315–19, 321–3, 343 Pasta filata, 47–9, 251–3, 265–6 processed cheese products, 353–6 Swiss cheese, 148–9 Vats, 25–31 Chevrotin, 289 Codex Alimentarius, 1 Colby, 95–6 Conjero, 292 Cooking: Cottage cheese, 332–3 curds, 75–6 Coryneforms, 204 Arthrobacteriaceae, 206–207 Brevibacterium linens, 204–206 Corynebacterium, 206 Cottage cheese: addition of CaCl2/rennet, 332 common defects/possible causes, 337–8 cream dressing, 334 cutting/cooking curd, 332–3 direct acidification, 331 drainage, washing, cooling of curd, 333–4 flavour, 337 incubation of milk, 330 manufacture, 329–30 microbiological quality, 336–7 nutritional quality, 335–6 physical structure, 334 specifications, 329 starters, 330–1 use of UF skimmilk, 335 yield/quality, 334–5 Cream cheese, 321 further treatments of curd after separation, 322–3 recombination technology, 322 rheological/syneretic aspects, 323 whey separation: using separators, 321 using UF, 321 Crottin de Chavignol, 289 Cultures see Starter cultures Cutting of milk gel: Cheddar, 75 Cottage cheese, 332–3 Feta, 234–5 Vats, 25–31 Danish cheese, 320 Dehydrated cheese ingredients (DCIs), 414–18 see also Cheese as an ingredient Domiati, 16, 227–8 microbiology, 233–4 ripening, 228–33 see also Ripened-in-brine cheese Dried cheeses, 414–15 Dry-salting, 71–2 Eastern European cheeses, 319 Emmental, 141 eye formation, 152–4 fermentations, 141–8 hygienic safety, 154 ripening, 149–52 technology, 148–9

Index 431

Emulsifying salts characteristics of: bactericidal effects, 369–7 calcium sequestration, 365–6 casein hydration, 367–8 crystallization, 367–9 emulsification, 367 flavour, 370 hydrolysis, 367 pH adjustment and buffering, 366–7 major types, 364 Enterococcus, 207 Enzyme-modified cheese (EMC), 418–22 applications, 418–19 dried, 422 enzymes used: lipases, 421–2 proteinases/peptidases, 421 flavour generation, 419–21 starter cultures, 422 technology/manufacture, 419 see also Cheese as an ingredient Equipment see Cheese technology Escherichia coli 0157:H7, 213–14 Evora, 284 Ewes’ milk cheeses: French, 280 general aspects, 279–80 Greek, 280–2 Italian, 282–4 Portuguese, 284–5 Spanish, 285–6 Extra-hard varieties, 14, 51 Balkan, 58–9 Italian, 52–7 main chemical/technological features, 52–9 nutrition, 67 ripening: changes in microflora, 59–60 lactose metabolism, 60 lipolysis, 60–2 proteolysis, 62–4 volatile compounds, 64–7 Russian, 58 Spanish, 58 Swiss, 57–8 Eye formation, 132–3, 152–4 Feta, 16, 234, 290 biochemistry of ripening: lipolysis, 238 proteolysis, 235–8 volatiles, 238 defects, 133, 239 manufacture, 234–5 microbiology, 238–9 rheological/sensory properties, 238 yield/gross composition, 235 see also Ripened-in-brine cheese Fiore Sardo, 56, 283 Flavour, 149–51 amino acid catabolism: aldehydes, 165–6 amine biosynthesis, 166 amines, 166 catabolism of amino acid side chains: indole ring, 166 sulfur compounds, 166–7

Cheddar, 88–92, 88–93 Cottage cheese, 337 development, 128–9 Domiati, volatile compounds, 231–2 Enzyme-modified cheese (EMC), 419, 421 Fatty acid catabolism, 162, 164, 165 Gouda, 128–9 miscellaneous compounds: esters, 167–8 pyrazines, 168–9 styrene, 167 terpenes, 168 volatile contaminants, 169 surface-ripened cheese: proteinases/lipases, 209–10 sulphur amino acids, 208 Formaella Arachovas Parnassou, 281 Fossa, 57 Fresh cheese, 323 Fromage frais, 319 Galotyri, 281 Gibna Bayda (Beida), 239 Glycolysis, surface mould-ripened cheese, 160–1 Goat’s milk cheese: French, 289–90 Greek, 290 Italian, 290–1 Portuguese, 291 Spanish, 291–3 technology/flavour: Blue cheese, 288 composition, 288 Gouda, 15 manufacture: brining, 113–14 cheesemaking, 108–13 control of pH/water content, 119–22 curdmaking, 108–109 draining/moulding, 109–10 milk standardisation, 116–17 milk treatment, 105–108 pressing, 110, 112–13 process principles, 104–105 rind treatment/curing, 114–16 starters, 122–4 maturation: cheese composition during ripening, 124–6 fermentation of lactose/citric acid, 126–7 flavour, 128–9 lipolysis, 128 possible microbial defects, 133–5 proteolysis, 127–8 texture, 129–33 origin/characteristics, 103–104 yield: bactofugation, 118–19 CaCl2, 119 cold storage of milk, 118 curd washing, 119 genetic variants of milk proteins, 118 inclusion of native whey proteins, 119 mastitis, 118 mechanical losses, 119 pasteurisation of milk, 118 rennet type, 118 salting, 119

432 Index

Gouda – contd. seasonal effects, 118 starter, 118 Grading, 93–4 Grana Padano, 55 Graviera Agrafon, 281 Graviera Kritis, 281 Gredos (Tiér, La Vera), 292 Gruyère, 7, 199 Halloumi, 245 Havarti, 199 Hygiene, 154 Ibérico, 291 Ibores, 291–2 Icelandic cheese, 319–20 Idiazabal, 58, 286 Imitation cheese see Analogue cheese products (ACPs) Indian cheese, 319 Ingredient, cheese as see Cheese as ingredient Italian-type cheese, 52–7, 282, 290 Jarlsberg, 141 Kashkaval: general characteristics, 264–5 manufacturing technology, 265–9 quality characteristics, 269–72 Kasseri, 281 Kefalograviera, 281–2 Kefalotyri, 282 Kopanisti, 281 La Serena, 287 La Vera, 292 Labneh (Labaneh, leben), 319 Lactic acid bacteria (LAB), 92, 179, 183–4 Lactofil (Sweden), 320 Ladotyri Mytilinis, 282 Layered cheese (Schichtkäse), 323–4 Leerdamer, 141 Limburger, 199 Lipolysis, 61 Blue cheese, 185–6 Camembert, 162 Domiati, 230 extra-hard cheeses, 60–2 Feta, 238 Gouda, 128 surface mould-ripened cheese, 162 Listeria monocytogenes, 212–13 Low-moisture Mozzarella cheese (Pizza cheese), 251 age-related changes in structure/function, 260–4 functional properties: before heating, 257–8 browning, 260 heat-induced (melting), 258–60 meltability, 258–9 oiling-off, 260 stretchability, 259–60 manufacturing technology, 251–55 plasticization/stretching, 252–4 physico-chemical characteristics of curd, 253 reorganization of curd structure, 255 thermal effects on starter bacteria/coagulant, 254–5 thermo-mechanical treatment of curd, 253–4

Maasdamer, 141 Mahón, 58 Maillard reaction, 413 Majorero, 291, 292 Manchego, 58, 285–6 Manouri, 282 Mascarpone, 18, 324 Microbial flora, 59, 158 bacteria, 160 interactions between micro-organisms, 160 Micrococcus spp., 203–204 Middle Eastern cheeses, 319 Milk: cold storage, 118 composition, 73–4 Domiati, 228–9 Feta, 234 genetic variants of proteins, 118 incubation for Cottage cheese, 330 preparation for cheese manufacture, 23–5 treatment: bactofugation, 107–108 pasteurisation, 105, 107, 118 quality, 105 standardisation, 108, 116–17 Mish, 239–40 Montasio, 55–6 Monterey, 95–6 Mould-ripened cheese see Surface mould-ripened cheese Mozzarella di Bufala, 15–16 Mozzarella see Low-moisture Mozzarella cheese Mudaffara, 240 Münster, 199 Murazzano, 283–4 Murcia, 292 Nabulsi, 241 Nisa, 284 Non-European cheeses, 19–21 Norwegian whey cheese, 18–19 Ossau-Iraty, 280 Palmero, 292 Paneer, 343 acidification, 344 drainage/curd handling, 344 heat treatment of milk, 343–4 manufacturing methods, 343 Parmigiano Reggiano, 52, 55 Pasta-filata varieties, 7, 15–16, 251 Kashkaval, 264–72 low-moisture Mozzarella (Pizza) cheese, 251–64 Pecorino Romano, 56, 282–3 Pecorino Sardo, 56, 283 Pecorino Siciliano, 56, 283 Pecorino Toscano, 283 Penicillium roqueforti, 179–80 interactions with: contaminants, 185 lactic acid bacteria, 183–4 yeasts, 184–5 Picante da Beira Baixa, 291 Pichtogalo Chanion, 281 Pizza cheese see Low-moisture Mozzarella cheese Pont l’Evêque, 199 Port du Salut, 199

Index 433

Portuguese cheese: ewe, 284–5 goat, 291 Post-vat stages of cheese manufacture, 31–48 see also Vats Pressing of curd, 81–2 rapid cooling, 82–3 vacuum, 82 Processed cheese products (PCPs), 7, 18, 21, 349–51 blend ingredients: caseins, 377 cheese, 374 cheese base/UF milk retentate, high heat-treated milk/whey proteins, 376–7 co-precipitates, 378 emulsifying salts, 373–4 lactose, 378 rework, 374–6 skim milk powder, 378 classification, 351–2 consistency/cooking characteristics: blend ingredients, 373–8 compositional parameters, 378–9 processing temperature/shear, 373 processing time, 371–3 stabilizers (binding agents)/hydrocolloids, 379 ‘Creaming’ in processed cheese products: creaming effect, 371 over creaming, 372–3 emulsifying salts, 360–71 effect on consistency, 373–4 effect on cooking properties, 373–4 major types, 364 properties, 365–70 role in formation of physico-chemically stable product, 360–3 manufacturing principles: heating natural cheese in absence of emulsifying salts, 359–60 microstructure of rennet-curd, 356–9 manufacturing protocol: cleaning/size reduction, 353 formulation of blend, 353 homogenization, 356 hot packing/cooling, 356 pre-mixing of formulation materials, 353, 355 processing of blend, 355–6 micro-structure, 362–3 see also Analogue cheese products Propionic acid fermentation, 135, 141, 142–4 effect of: facultatively heterofermentive lactic acid bacteria, 145, 147 feeding season, 144–5 Lactobacillus helveticus, 147–8 eye formation, 152–4 hygienic safety, 154 lactic acid, 141–2 ripening: flavour formation, 149–51 general aspects, 149 texture formation, 151–2 technology, 148–9 Protected Designations of Origin (PDO), 1 Proteolysis: Blue cheese, 186–9 Camembert, 161–2 Cheddar, 89–90

Domiati, 229–30 extra-hard cheese, 62–4 Feta, 235–8 Gouda, 127–8 Quark, 320 surface mould-ripened cheese, 161 Quark, 315 manufacture: acid or acid/rennet gel treatment, 317–18 addition of cream, 318 filtration methods, 316–17 original (standard) separator process, 315 recombination technology, 317 thermisation of curd/fresh cheese, 318–19 thermo process (Westfalia), 316 tradition batch methods, 315 proteolysis/bitterness, 320 rheological/syneretic aspects, 320 varieties directly related, 319–20 see also Acid- and Acid/Rennet-curd cheese Queso Blanco, 343 manufacture: heat treatment of milk, 343–4 methods, 343 milk acidification, 344 whey drainage/curd handling, 344 microbial quality, 346 microstructure, 344 physico-chemical properties, 346 see also Acid- and Acid/Rennet-curd cheese Rabaçal, 291 Ricotta, 18, 284, 345–6 part-skim, 345 shelf-life, 346 whey/skimmilk, 345 whole milk, 345 see also Acid- and Acid/Rennet-curd cheese Ripened-in-brine cheese, 16, 227 Akawi, 241 Beli Sir U Kriskama, 244–5 Beyaz Peynir (Turkish White), 244 Bjalo Salamureno Sirene (White brined), 244 classification/nomenclature, 227 Domiati, 227–34 Feta, 234–9 Gibna Bayda (Beida), 239 Halloumi, 245 Mish, 239–40 Mudaffara, 240 Nabulsi, 241 technological features, 242 Telemea/Telemes, 241, 243–4 see also Feta; Domiati Roncal, 58, 286 Roquefort, 280 Russian cheese, 58 Saanenkäse, 57 Sainte-Maure, 289 Salt, 119 Blue cheese, 176 Cheddar: equilibration, 80 mellowing, 79–80 milled curd, 79 seaminess/fusion, 81

434 Index

Salt – contd. Domiati, 229 effect on functional properties, 403–4 Feta, 235 surface-ripened cheese, 169–70, 201 Sbrinz, 57 Secondary/adjunct cultures, 92–3 Serpa, 284 Serra da Estrela, 284–5 Skyr (Iceland), 319–20 Smear cheeses: bacteriocins: anti-listerial compounds of Geotrichum and Penicillium, 218 Coryneform bacteria, 215–17 enterococci, 217–18 lactic acid bacteria, 214–15 defined cultures, 211–12 environmental factors, 200 manufacture, 199–200 micro-organisms: coryneforms, 204–207 flavour development, 208–10 pigmentation/colour development, 210 secondary flora, 207–208 staphylococci/micrococci, 203–204 yeast, 201–203 origin of surface microflora, 210–11 pathogens, 212 physical/chemical characteristics, 200–201 ripening, 199 ripening consortia, 218–19 Soft cheese, 6 Spanish cheese, 58 Staphylococcus, 203–204, 213 Starter cultures, 118 Blue cheese, 179, 191–2 Camembert, 157 Cheddar, 73–4 composition/handling, 122–4 Cottage cheese, 330–1 Domiati, 233 Emmental, 141 enzyme-modified cheese (EMC), 422 low-moisture Mozzarella (Pizza) cheese, 254–5 Mozzarella, 251 non-starter lactic acid bacteria (NSLAB), 92 Parmigiano Reggiano, 55 Pecorino Romano, 56 preparation, 25 role: enzymes, 91 other activities, 91–2 Sbrinz, 57 Smear, 199 Swiss, 141 Substitute cheese see Analogue cheese products; Processed cheese products (PCPs) Surface mould-ripened cheese, 6, 16, 157 control of ripening, 169–71 diversity, 157

flavour: catabolism of amino acid side chains, 166–7 fatty acid catabolism, 162–6 miscellaneous compounds, 167–9 glycolysis, 160–1 interactions between micro-organisms, 158–60 lipolysis, 162 proteolysis, 161–2 technology, 157–8 texture, 169 see also Camembert Swedish cheese, 320 Swiss cheese, 57–8 see also Emmental Tan (Than) cheese, 319 Technology see Cheese technology Telemea/Telemes, 241, 243–4 Tenerife, 292 Terrincho, 285 Texture, 3–4, 151–2 Cheddar, 85–8 Domiati, 232–3 eye formation in Swiss cheese, 132–3 Gouda, 129–33 structure, 129, 131–2 surface mould-ripened cheese, 169 Tiér, 292 Tilsit, 199 Tofu, 21 Topfen, 319 Torta del Casar, 287 Trappist, 199 Tulum, 319 Tvorog/Tvarog, 319 US cheese, 319 Valdetja, 293 Valençay, 289 Vats, 25–31 APV CurdMaster, 29–30 continuous processes, 30–1 Damrow Double-O, 27–8 Damrow horizontal, 28 OST vat, 26–7 Scherping horizontal cheese vat (HCV), 28 see also Post-vat stages of cheese manufacture Yeast: Blue cheese, 180–1, 184–5 brine/dairy environment, 181 occurrence/growth, 181–2 raw milk, 181 in smear cheese: de-acidification, 201–202 stimulatory compounds, 202–203 Ymer (Denmark), 320 Zamorano, 286–7

FRESH CHEESES (soft)

C

SHORT RIPENED CHEESES (soft)

E

QUARK

A3

RICOTTA (goat or sheep)

Whey derived A4

RICOTTA (cow)

A5

Cream derived MASCARPONE

A6

Buttermilk derived SKYR

A7

(Not available)

"LACTICINIA" fresh or ripened (soft or hard)

B

D

Milk derived A2

B1

PETITE SUISSE (acid curd)

B2

C1

CRESCENZA

C2

D1

CAMEMBERT (white mould rind)

E1

STILTON (cow)

PRIMO SALE

ASIAGO PRESSATO

B3

CAPRINO (goat or sheep)

B4 MOZZARELLA (plastic or kneaded; cow or buffalo)

B5

COTTAGE (cubes or flakes)

C3

CACIOTTA (goat or sheep)

C4 SCAMORZA (plastic or kneaded curd)

C5

FETA (Ripened under brine)

D2 LIVAROT (smeared surface)

TALEGGIO D3 TRONCHETTO DI CAPRA D4 (goat or sheep; smeared or (smeared or mould rind) mould rind)

E2

E3

Colostrum derived KOLOSTRUM KAESE (Not available)

SURFACE RIPENED CHEESES (soft)

CAMBOZOLA (white rind)

FORMATICA

A

LEBNEH

LACTICINIA

A1

ROQUEFORT (goat or sheep)

BLUE VEINED CHEESES (SOFT)

F1

MONTASIO (semicooked)

F2

FONTAL (washed curd)

F3 CANESTRATO (goat or sheep; ripened)

F4 CACIOCAVALLO (kneaded curd; ripened)

F5 MAASDAMER (cheese with eyes; ripened)

F6 CANTAL (structured; ripened)

F7

FONTINA (smeared rind; ripened)

G1

GRANA (cooked; ripened)

G2

EDAM (washed; ripened)

G3 PECORINO (goat or sheep; ripened)

G4 PROVOLONE (kneaded curd; ripened)

G5 EMMENTAL (cheese with eyes; ripened)

G6 CHEDDAR (structured; ripened)

G7

APPENZELLER (smeared rind; ripened)

H3

H4

H5

H6 SPECIAL TECHNOLOGY H7 IMITATION CHEESES CHEESES (vegetable Substitutes)

F SEMIHARD CHEESES

HARD CHEESES

H1 PROCESSED CHEESES H2 (melted cheeses) H

SMOKED

GRATED

MIXED (different ingredients)

FROMAGES FORT

(ripened under special conditions)

"MISCELLANEA"

MISCELLANEA

G

Plate 1 Examples of cheese from the principal groups of Ottogalli (1998, 2000a,b, 2001); see Table 7 for further details. (See page 13.)

Plate 2 APV Cheddarmaster belt system. Courtesy of NZMP Whareroa, New Zealand. (See page 33.)

Plate 3 Trommel salting system. Courtesy of NZMP Edendale, New Zealand. (See page 35.)

Plate 4 Laude block mould. Courtesy of Laude bv, The Netherlands. (See page 37.)

Plate 5 Curd distribution tank. Courtesy of NZMP Stirling, New Zealand. (See page 38.)

Plate 6 Blockformers with bag presenters. Courtesy of NZMP Edendale, New Zealand. (See page 39.)

Plate 7 Rapid cooling tunnel. Courtesy of NZMP Hautapu, New Zealand. (See page 40.)

Plate 8 Robot stacking of cheese blocks. Courtesy of NZMP Hautapu, New Zealand. (See page 40.)

Plate 9 APV SaniPress tunnel pressing system. Courtesy of Invensys APV, UK. (See page 43.)

Plate 10 Conveyor pressing system, with Casomatics in foreground. Courtesy of NZMP Lichfield, New Zealand. (See page 43.)

Plate 11 APV tray brining system. Courtesy of Invensys APV, UK. (See page 44.)

Plate 12 Deep brining system. Courtesy of NZMP Lichfield, New Zealand. (See page 45.)

Plate 13 Almac cooker/stretcher. Courtesy of Almac, Italy. (See page 48.)

Plate 14 SSF cooker/stretcher. Courtesy of Stainless Steel Fabricating, Inc., USA. (See page 49.)

Plate 15 CMT String cheese moulder. Courtesy of Construzioni Meccaniche E Technologia, Italy. (See page 49.)

Plate 16 Traditional Swiss Emmental cheese. (See page 142.)

Plate 17 Cutting of the curd. (See page 149.)

Plate 18 Filling of the curd into the cheese moulds. (See page 149.)