Cheese and MicrobesC he
es e
Microbes and
Washington, DC
C he
es e
Microbes and
Edited by Catherine W. Donnelly Department of Nutrition and Food Sciences and Vermont Institute for Artisan Cheese The University of Vermont Burlington, Vermont
Copyright © 2014 american Society for Microbiology. all rights reserved. No part of this publication may be reproduced or transmitted in whole or in part or reused in any form or by any means, electronic or mechanical, including photocopying and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
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library of Congress Cataloging-in-publication Data
Cheese and microbes / edited by Catherine W. Donnelly, Vermont institute for artisan Cheese, the University of Vermont, Burlington, Vermont. volumes cm includes bibliographical references and index. iSBN 978-1-55581-586-8 (hardcover) — iSBN 978-1-55581-859-3 (e-book) 1. Cheese—Microbiology. 2. Cheesemaking. i. Donnelly, Catherine W., editor of compilation. QR121.C47 2014 637’.3—dc23
2014000520
10 9 8 7 6 5 4 3 2 1 printed in the United States of america
address editorial correspondence to aSM press, 1752 N St. NW, Washington, DC 20036-2904, USa e-mail: books@asmusa.org Send orders to aSM press, p.O. Box 605, Herndon, Va 20172, USa phone: (800) 546-2416 or (703) 661-1593; fax: (703) 661-1501 Online: http://www.asmscience.org
doi:10.1128/9781555818593
v
CONTeNTS
Contributors vii Preface xi
1. From Pasteur to Probiotics: A Historical Overview of Cheese and Microbes / 1
Catherine W. Donnelly
3. Cheese Classification, Characterization, and Categorization: A Global Perspective / 39
Montserrat Almena-Aliste and Bernard Mietton
4. Mesophilic and Thermophilic Cultures Used in Traditional Cheesemaking / 73
Mark E. Johnson
5. The Good, the Bad, and the Ugly: Tales of Mold-Ripened Cheese / 95
Sister Noëlla Marcellino, O.S.B., and David R. Benson
6. The Microbiology of Traditional Hard and Semihard Cooked Mountain Cheeses / 133
Eric Beuvier and Gabriel Duboz
7. The Microfloras and Sensory Profiles of Selected Protected Designation of Origin Italian Cheeses / 151
Giuseppe Licitra and Stefania Carpino
8. Wooden Tools: Reservoirs of Microbial Biodiversity in Traditional Cheesemaking / 167
Sylvie Lortal, Giuseppe Licitra, and Florence Valence
vi n CONteNtS
10. Biodiversity of the Surface Microbial Consortia from Limburger, Reblochon, Livarot, Tilsit, and Gubbeen Cheeses / 219
Timothy M. Cogan, Stefanie Goerges, Roberto Gelsomino, Sandra Larpin, Markus Hohenegger, Nagamani Bora, Emmanuel Jamet, Mary C. Rea, Jérôme Mounier, Marc Vancanneyt, Micheline Guéguen, Nathalie Desmasures, Jean Swings, Mike Goodfellow, Alan C. Ward, Hans Sebastiani, Françoise Irlinger, Jean-François Chamba, Ruediger Beduhn, and Siegfried Scherer
11. Microbiological Quality and Safety Issues in Cheesemaking / 251
Dennis J. D’Amico
Benjamin E. Wolfe and Rachel J. Dutton
Index / 323
Montserrat Almena-Aliste Department of Nutrition and food Sciences, University of Vermont, Burlington, Vt 05405-0086, and green Mountain Coffee Roasters, Waterbury, Vt 05676
Ruediger Beduhn J. Bauer Kg, 83512 Wasserburg/inn, germany
David R. Benson Department of Molecular and Cell Biology, University of Connecticut, Storrs, Ct 06269-3125
eric Beuvier UR342 technologie et analyses laitières, institut National de la Recherche agronomique, 39801 poligny Cedex 1, france
Nagamani Bora School of life and Health Sciences, aston University, Birmingham B4 7et, United Kingdom
Stefania Carpino CoRfilaC, 97100 Ragusa, italy
Jean-François Chamba (deceased) institut technique français des fromages, 74801 la Roche-sur-foron Cedex, france
Timothy M. Cogan Moorepark food Research Center, teagasc, fermoy, ireland
Dennis J. D’Amico Department of animal Science, University of Connecticut, Storrs, Ct 06268
Nathalie Desmasures Unité des Micro-organismes d’intérêt laitier et alimentaire, ifR146 iCORe, Université de Caen Basse-Normandie, 14032 Caen, france
Catherine W. Donnelly Department of Nutrition and food Sciences and Vermont institute for artisan Cheese, the University of Vermont, Burlington, Vt 05405
viii n CONtRiBUtORS
Gabriel Duboz UR342 technologie et analyses laitières, institut National de la Recherche agronomique, 39801 poligny Cedex 1, france
Rachel J. Dutton faS Center for Systems Biology, Harvard University, Cambridge, Ma 02138
Roberto Gelsomino Sa Coca-Cola Services N.V., 1070 Brussels, Belgium
Stefanie Goerges Naturkost ernst Weber gmbH, 81371 Munich, germany
Mike Goodfellow Microbial Resources Centre, University of Newcastle, Newcastle upon tyne Ne1 7RU, United Kingdom
Micheline Guéguen Unité des Micro-organismes d’intérêt laitier et alimentaire, ifR146 iCORe, Université de Caen Basse-Normandie, 14032 Caen, france
Markus Hohenegger Bundesanstalt für alpenländische Milchwirtschaft, 6200 Rotholz, austria
Françoise Irlinger laboratoire de génie et de Microbiologie des procédés alimentaires, iNRa, agroparis- tech, 78850 thiverval-grignon, france
emmanuel Jamet actilait, 75314 paris Cedex 09, france
Mark e. Johnson Wisconsin Center for Dairy Research, Madison, Wi 53706-1565
Paul S. Kindstedt Department of Nutrition and food Sciences, University of Vermont, Burlington, Vt 05405-0086
Sandra Larpin Bioprocess Division, Millipore Corporation, 67120 Molsheim, france
Giuseppe Licitra Department of agriculture and food production, Catania University, 95100 Catania, italy
evanthia Litopoulou-Tzanetaki laboratory of food Microbiology and Hygiene, Department of food Science and technology, faculty of agriculture, aristotle University of thessaloniki, thessaloniki 57001, greece
Sylvie Lortal iNRa, agrocampus Ouest, UMR1253 Science et technologie du lait et de l’oeuf, 35042 Rennes, france
Sister Noëlla Marcellino, O.S.B. abbey of Regina laudis, Bethlehem, Ct 06751
Bernard Mietton expertise agroalimentaire, 39800 poligny, france
Jérôme Mounier laboratoire Universitaire de Biodiversité et Écologie Microbienne (ea3882), ifR148 ScinBioS, Université européenne de Bretagne, Université de Brest, eSMiSaB, technopôle de Brest iroise, 29280 plouzané, france
Mary C. Rea Moorepark food Research Center, teagasc, fermoy, ireland
CONtRiBUtORS n ix
Hans Sebastiani Bundesanstalt für alpenländische Milchwirtschaft, 6200 Rotholz, austria
Jean Swings BCCM/lMg Bacteria Collection, laboratorium voor Microbiologie, Universiteit gent, 9000 gent, Belgium
Nikolaos Tzanetakis laboratory of food Microbiology and Hygiene, Department of food Science and technology, faculty of agriculture, aristotle University of thessaloniki, thessaloniki 57001, greece
Florence Valence iNRa, agrocampus Ouest, UMR1253 Science et technologie du lait et de l’oeuf, 35042 Rennes, france
Marc Vancanneyt BCCM/lMg Bacteria Collection, laboratorium voor Microbiologie, Universiteit gent, 9000 gent, Belgium
Alan C. Ward Microbial Resources Centre, University of Newcastle, Newcastle upon tyne Ne1 7RU, United Kingdom
Benjamin e. Wolfe faS Center for Systems Biology, Harvard University, Cambridge, Ma 02138
xi
PReFACe
Cheese is a topic which makes science and microbiology highly tangible. in 2009, i had the pleasure of delivering a lecture on the topic “Say Cheese:
Understanding the living foods We eat” for the public program series “the Dish” at the Marian Koshland Science Museum of the National academy of Sciences. Before my lecture, Chris Condayan, the outreach manager in the Communications Department at the american Society for Microbiology, con- ducted an interview as well as a taping during my presentation. Chris is a superb interviewer, and we had no problem enthusiastically discussing the topic of cheese and microbes for an hour. the interview became the basis of the MicrobeWorld video entitled “Cheese and Microbes.” later that year, i received a call from eleanor Riemer of aSM press asking if i had interest in compiling an edited book, which became this work.
there has never been a better time to explore the relationship between cheese and microbes in fundamental new ways. i have been fortunate to serve as the codirector of the Vermont institute for artisan Cheese (ViaC) at the University of Vermont over the last 7 years along with paul Kindstedt. Over the past decade, there has been explosive growth in the artisan cheese industry in the United States. One of the first and most important activities we undertook at ViaC was to develop a comprehensive education curriculum for those new to cheesemaking; this curriculum focused on the science of cheese, principally to promote cheese quality and safety. Many of the chapters in this book focus on aspects of that requisite knowledge.
throughout our work at ViaC, we have been assisted by eminent scholars from europe who are extremely heartened (if not downright excited) by the development of a true culture of cheese appreciation in the United States. in this book they, along with our U.S. collaborators, graciously share their knowl- edge and insight as we scrape the surface of our collective knowledge of the role of microbes in cheesemaking. in order to fully understand this role, this
xii n pRefaCe
book was organized into the chapters which follow. the basic steps of chee- semaking are necessary to understand, as these steps select for the microbial communities that characterize the diverse cheese varieties which are made worldwide. the chapter on cheese classification shows how the many diverse cheese types can be logically ordered into families based upon the type of pro- cedures used for coagulation, cutting, cooking, and ripening of cheese. the role of starter cultures in cheesemaking is explored, followed by chapters on a vari- ety of cheeses which have been made in europe for centuries in countries such as italy, france, and greece and chapters on the roles which bacteria and fungi have in expressing the character and sensory properties of these fine cheeses. We explore the important role which wooden equipment plays in cheese pro- duction and consider whether its use enhances or harms microbiological safety. We explore the ever-changing landscape of cheese regulations and pathogens of concern to cheesemakers. in the last chapter, we explore where molecular biology will take our inquiries on cheese and microbes in the future. New tools of molecular biology are affording the opportunity to investigate the relation- ship between cheese and microbes in ways not previously possible, allowing the identification of a complexity of organisms whose role in cheesemaking was previously unrecognized.
i hope this book inspires those who love to eat cheese as much as those who enjoy learning about cheese. Microbiology is a fascinating topic and never more interesting than when explored through the delicious foods we consume. the emergence of a culture of cheese appreciation also offers the opportunity to expose a new generation of students to the exciting science that undergirds cheesemaking.
i am indebted to all of my colleagues who have contributed the chapters which made this book possible. i am also deeply grateful to Kenneth april, gregory payne, and eleanor Riemer of aSM press for their immense assistance, wisdom, and encouragement throughout this project. Without them, this book would not have been possible.
CatHeRiNe W. DONNellY
AND MICROBES
Catherine W. Donnelly1
1 INTRODUCTION Nowhere in the microbial world are microor- ganisms on more magnificent display than on the surfaces or in the interiors of the great cheeses of the world. Cheesemaking is inextricably linked to microbiology, which makes the study of cheeses, their history, and the vast science of cheese and micro- bes particularly fascinating. Over the past two decades, there has been explosive growth in the U.S. artisan cheese industry. The availabil- ity of artisan cheeses, made using traditional practices, has ignited renewed consumer in- terest in cheesemaking and cheese consump- tion. This affords a tremendous opportunity to educate a new population of students, scientists, cheesemakers, technologists, and cheese connoisseurs about the essential role which microorganisms play in the process of cheesemaking.
Many of the chapters in the book Cheese and Microbes (48) provide a scientific overview of the beneficial associations of microbes with cheese, through the lens of the numerous unique cheeses which result due to growth of bacteria, yeasts, and molds which play a
crucial role in cheesemaking. Whether due to surface or internal mold, yeast, or bacterial ripening, growth, or metabolism, a vast array of products are able to be produced through transformation of a single starting material: milk. Cheeses in general are microbiologically safe foods, but there are occasional outbreaks of illness linked to cheese consumption. The chapters in Cheese and Microbes have been authored by scientists who are the leading researchers and experts on the various aspects of the association of microbes with traditional cheeses. Many of the authors reside in Europe, where the traditional cheeses which they study have been continuously produced for centuries. In addition to the informative overview of the science of cheesemaking and the microorganisms involved, selected photographs capture the culture, tradition, and vast array of unique cheese varieties, all of which are dependent on the action of a di- verse population of bacteria, yeasts, and molds. New tools of molecular biology are informing the study of cheese microbiology in ways not previously possible, and this emerg- ing science is providing new insights into the complexity of the microbial biodiversity of traditional cheeses. This inquiry will further advance our knowledge of some of the oldest traditional foods known to humankind.
1Department of Nutrition and Food Sciences and Vermont Institute for Artisan Cheese, The University of Vermont, Burlington, VT 05405.
Cheese and Microbes, Edited by Catherine W. Donnelly, © 2014 American Society for Microbiology, Washington, DC, doi:10.1128/microbiolspec.CM-0001-12
1
A HISTORY OF CHEESE AND MICROBES The development of the microscope by two pioneering scientists, Robert Hooke and Antonie van Leeuwenhoek, was an advance- ment which greatly informed our understand- ing of microbiology in general and of cheese in particular. Of important note was the very first recorded observation of microbes associ- ated with cheese, described in 1665 by Robert Hooke in his book Micrographia (1). Hooke writes of “[t]he Blue and White and several kinds of hairy mouldy spots, which are observable upon divers kinds of putrify’d bod- ies, whether Animal substances, or Vegetable, such as the skin, raw or dress’d flesh, blood, humours, milk, green Cheese, etc….” Hooke provided the first published
depiction of a microorganism, a “hairy mold” colony which microbiologists have subse- quently identified as Mucor (Fig. 1). Shortly following Hooke’s description, in 1674 Antonie van Leeuwenhoek, in a letter to the Royal Society, affirmed Hooke’s findings, writing, “Examining this water…I found floating there- in divers earthy particles, and some green streaks, spirally wound serpent-wise…and I judge that some of these little creatures were above a thousand times smaller than the smallest ones I have ever yet seen, upon the rind of cheese, in wheaten flour, mould, and the like” (44).
From the earliest recognition of the role of microorganisms in cheesemaking, scientific inquiry has informed our understanding of the identities and roles of microorganisms so that
FIGURE 1 Robert Hooke’s 1665 depiction of a “hairy mold” colony which was subsequently identified as Mucor. doi:10.1128/microbiolspec.CM-0001-2012.f1
2 DONNELLY
cheesemaking has become a controlled, pre- dictable activity. The majority of cheese pro- duced around the globe today is made on an industrial scale. Industrial cheesemaking has been perfected over time to yield cheeses with consistently controlled functionality and char- acter, largely a result of use of highly specific and defined microorganisms as starter cultures, along with controlled production and aging. Such cheeses have strayed far from their origins. In contrast, and likely in response, consumer interest in artisan cheese is experi- encing a renaissance worldwide. The world of artisan cheese is truly an exciting one, particu- larly at this time, when global demands for cheeses and cheese products are creating new opportunities for artisan producers. Artisan cheeses are defined as cheeses made by hand on a small scale, normally using milk from heritage breed animals in a closed herd and utilizing traditional, time-honored practices such as bandage wrapping or traditional utensils (2). Artisan cheesemaking is typically characterized by small-scale production in limited volume by individual producers. There are over 1,400 named cheese varieties in the world today, yet most of these cheeses belong to one of 20 distinct cheese types which share key manufacturing conditions and compositional characteristics (35, 36). These cheese types comprise the cheese families we know today as fresh, bloomy rind, smear ripened, hard uncooked, hard cooked, and blue. The historic evolution of these cheeses was impacted by geography, climate, and cultural and economic condi- tions. Selection of indigenous microorganisms existing in raw milk or in the cheesemaking or aging environment became a function of the manipulation of milk by the cheesemaker. The local cheesemaking technology and envi- ronment shaped the chemistry and microbiol- ogy of local cheese, which, in turn, shaped the characteristics and identity of cheese (3). Some of the great cheeses of the world which we enjoy today, such as Parmigiano Reggiano, have been continuously produced for 700
years or longer using essentially the same pro- duction practices (Table 1) (4).
Alpine cheeses are examples of a unique family of cheeses which share similar manufac- turing technologies and chemical compositions. Alpine cheeses are large cheeses with hard rinds and are commonly referred to as Swiss cheeses. Appenzeller, Comté, Emmental, and Gruyère are among the best-known Alpine cheese vari- eties. Alpine cheeses are characterized by a smooth, tight knit and elastic texture with the presence of holes or eyes. Although Alpine cheeses are now produced worldwide, their production originated in the Alpine regions of Switzerland and eastern France (3). The condi- tions under which Alpine cheesemaking was perfected were most certainly shaped by the regional geography, which consisted of the re- mote mountainous regions of the Alps. It is thought that cheesemaking began in this region as early as the first century BCE. Till- able land was very limited, and thus, it was farmed intensively. The harsh Alpine winters created the need for nonperishable food. Fortunately, Alpine meadows provided suitable places to graze cows for milk produc- tion, and communal farming and cheese- making became a necessity for farmers in these remote, isolated locales. Small-scale cheesemakers worked collaboratively to make cheese from an entire herd of cows. Copper
TABLE 1 Years of origin of noted cheese varietiesa
Cheese variety Yr of first documentation
Gorgonzola. . . . . . . . . . . . . . . . . . . 879 Roquefort . . . . . . . . . . . . . . . . . . 1070 Grana . . . . . . . . . . . . . . . . . . . . . . 1200b
Cheddar . . . . . . . . . . . . . . . . . . . . 1500b
Parmesan . . . . . . . . . . . . . . . . . . . 1579 Gouda . . . . . . . . . . . . . . . . . . . . . 1697 Gloucester . . . . . . . . . . . . . . . . . . 1697 Stilton . . . . . . . . . . . . . . . . . . . . . 1785 Camembert . . . . . . . . . . . . . . . . . 1791
aAdapted from reference 4. bDate is approximate.
1. HISTORICAL OVERVIEW OF CHEESE AND MICROBES 3
cauldrons were used by cheesemakers as vats, and cheesemaking huts, or chalets, were built at different altitudes as cows moved up and down the mountains during the grazing season.
Wooden tools and utensils were also utilized (Fig. 2) (37). This remote production dictated the cheesemaking characteristics, which re- quired a hard, elastic durable cheese which was
FIGURE 2 Antique engraving by Charles-Joseph Panckoucke, 1784, depicting wooden cheesemaking tools for Gruyère production. From Recueil de Planches de l’Encyclopédie, par ordre de matières, Tome troisième (43) (author’s collection). doi:10.1128/microbiolspec.CM-0001-2012.f2
4 DONNELLY
low in moisture and had a long shelf life and was suitable for transport down the mountains. In order to achieve these characteristics, cheese- makers developed three key innovations: the curd was cut into small particles to facilitate whey expulsion; curds were cooked at high temperatures, which further drove out mois- ture; and curds were pressed, which facilitated additional whey expulsion (3).The impact of these technical innovations to produce a durable cheese further shaped the distinct Alpine cheese characteristics. The slow, delayed acid produc- tion facilitated a high mineral content and high pH, which created a sweet cheese. Because salt was scarce, it was used sparingly in production. The low-salt, high-pH environment, in turn, selected for the bacterium Propionibacterium freudenreichii subsp. shermanii, which ferments lactate to produce CO2, leading to the devel- opment of characteristic eyes in the cheese curd, in addition to acetate and propionate, which impart the sweet and nutty flavor char- acteristic of Alpine cheeses (3, 5, 6). The clas- sical dairy propionic acid bacteria (PAB) are important to the microbiology of Alpine cheeses. These organisms may have origins in unfermented feed, but they are rarely detected in samples other than milk and dairy products (7). Most strains isolated from cheese belong to P. freudenreichii subsp. shermanii, suggesting a high degree of heat resistance possessed by this species in comparison to other dairy PAB, which include Propionibacterium jensenii, Propionibacterium thoenii, Propionibacterium acidi- propionici, and Propionibacterium cyclohexanicum (6, 7). There is growing evidence that PAB such as P. freudenreichii subsp. shermanii may have important roles as probiotic cultures, serving as important modulators of the colon flora, and they may have a role in prevention of colon cancer (8, 9, 10).
Similar cultural and geographic forces shaped development of other cheese varieties, such as soft ripened cheeses, which include the bloomy-rind and smear-ripened/washed- rind cheeses. Bloomy-rind cheeses develop a white surface mold due to a complex ecosys- tem formed by the growth of Penicillium
camemberti, Geotrichum candidum, Kluyveromyces lactis, and Debaryomyces hansenii, which, as rip- ening fungi, become major contributors to the sensory properties of cheeses such as Brie and Camembert (11). Bloomy-rind cheeses had their origins in France. Because these cheeses were produced for home consump- tion or sale in local villages, there was no need to withstand transportation over long distances, nor the need for these cheeses to withstand extended storage. These cheeses were small and easy to make. Following coag- ulation of milk with rennet, the curd was drained in small molds. Acid develops quickly and pH declines rapidly to pH 4.6, thus favor- ing the selection of fungi such as P. camemberti, which forms a white surface mold upon stor- age in a damp cellar (3).
Smear-ripened/washed-rind cheeses are ripened by aerobic bacteria and include nota- ble varieties such as Muenster, Limburger, Taleggio, Beaufort, and Langres. Washed- rind/smear-ripened cheeses evolved far differ- ently than the bloomy-rind cheeses, having origins within the monasteries of Europe. In the Benedictine order, a ban on meat con- sumption existed, which favored consumption of cheese, and cheese thus became an integral part of monastic life. Because cheese was pro- duced for consumption within monasteries and thus did not require transportation, it did not need to be durable, nor did it require a long life. As cheesemaking became a source of income for the monasteries, low-tempera- ture cooking/pressing was incorporated into cheesemaking, resulting in lower-moisture, washed-rind cheeses. In these cheeses, acid de- velopment is slow and favors surface growth of yeasts which elevate the cheese pH, which, in turn, favors growth of Brevibacterium linens, Geotrichium candidum, or Debaryomyces hansenii following salting (3, 6). Rea and colleagues, in studies of a washed-rind cheese (12), noted that despite inoculation of cheese milk with starter cultures, only two of five commercial cultures were subsequently found in cheese, suggesting the presence of bacteria from sources other than inoculated cultures which
1. HISTORICAL OVERVIEW OF CHEESE AND MICROBES 5
become part of the dominant cheese flora during ripening. Importantly, results suggested the presence of a house microflora as evidenced by similar pulsed-field gel electro- phoresis patterns isolated from cheeses pro- duced at different times of the year (38).
Artisan cheesemaking traditions which evolved in England were much different from those in France or in the Alpine regions. English cheesemaking traditions had a tremen- dous impact on the establishment of farmstead cheesemaking in the United States. In England during the Middle Ages, agriculture was domi- nated by feudalism, with land owned by no- bility or by monasteries. Small farmers secured smallholdings from landowners but also had access to common land for grazing of live- stock. Farmers paid rent to landowners from proceeds of the sale of agricultural goods. Soft farmstead cheese made at home was common in this period. The bubonic plague significant- ly reduced the population in the 15th century, creating acute labor shortages. Tenant rights could now purchased by peasants. Henry VIII dissolved monasteries, making land for farm- ing available. Feudalism collapsed in the 16th and 17th centuries and was replaced by agri- cultural capitalism. In the 17th and 18th cen- turies, a few rich peasants became yeomen, chief tenants to landlords, who, in turn, raised rents and forced yeomen to become entrepre- neurial (3).
Yeoman dairy farmers moved away from home or local cheese production to cheese production for mass markets in London. The cheeses, made in East Anglia (Suffolk or Essex), had to be large and durable and have extended shelf life for transport to London. The manufacture of these cheeses was charac- terized by moderate to rapid acid production during manufacture. Curds were scalded at high temperatures and pressed for whey ex- pulsion. Salt, which was readily available, was added to further assist whey expulsion. Such manufacturing parameters yielded a low- moisture cheese which was durable during transport. These cheeses became characterized by fundamentally different chemistry (acidity,
mineral content, and salt) and microbiology than those of Alpine cheeses (3).
The microflora of Cheddar cheese is com- posed of both starter lactic acid bacteria (LAB) and nonstarter LAB (NSLAB). Bacterial starter cultures are well-defined and characterized strains which are utilized during cheesemaking to control the fermentation and ensure the consistency of cheese production. Starter LAB used to facilitate acid production during Cheddar making typically consist of defined mesophilic cultures such as Lactococcus lactis subsp. cremoris and Lactococcus lactis subsp. lactis. Despite the use of defined cultures, the popu- lation of microflora emerging during Cheddar cheese aging is different in composition than the defined strains added to milk. The main microflora consists of mesophilic lactobacilli and pediococci, commonly re- ferred to as NSLAB. The species most com- monly isolated include Lactobacillus paracasei, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus plantarum, and Lactobacillus curvatus (13). As Cheddar cheese ripens, NSLAB from milk or the cheesemaking environment devel- op and become important to the character of Cheddar cheese. Occasionally, strains of heterofermentative lactobacilli are identified. Isolated species vary between plants, between countries, and within cheese during ripening. NSLAB likely contribute to Cheddar cheese flavor, yet the specific role is as yet undefined. Previous authors (14, 15) have found NSLAB in association with good-quality Cheddar cheese. Somers et al. (16) determined that res- ident NSLAB biofilms contaminate the dairy environment, and resident niches, including floors, drains, the cheese vat, hoops, and pack- aging machines, serve as sources of these orga- nisms during cheesemaking.
English cheesemaking techniques were brought to America by Puritan reformers and would define American cheesemaking for three centuries. By 1849, cheesemaking was well established in New England and the mid-Atlantic region of the United States (Fig. 3) (17). However, with the develop- ment of railroads, artisan cheesemaking was
6 DONNELLY
abandoned in the 1900s in favor of produc- tion of fluid milk for pasteurization which could be shipped great distances. By the 1940s, large-scale industrialized cheesemaking replaced artisan cheesemaking, and Cheddar cheese was almost exclusively produced in the United States. Between 1940 and 2006, the number of dairy farms in the United States declined precipitously, but the volume of milk produced dramatically increased. The 3,000 cheese plants in 1940 declined in num- ber to approximately 400 in 2006; these plants produced 9.5 billion pounds of cheese on an industrial scale to feed an ever-expanding fast food market (http://www.nass.usda.gov/ Quick_Stats/Lite/result.php?FB4AA96A- 11A3-3D4D-80A1-D3F6DE2BE2EB).
MICROBIOLOGICAL SAFETY Today we see a tremendous revival of artisan cheesemaking in the United States and else- where around the globe. Farmers are opting for artisan cheese production as a means of di- versifying farm income. The book The Atlas of American Artisan Cheese profiles 345 of the 400 artisan cheese producers who were actively
working in the United States in 2007 (2). As of 2012, it was estimated that there were over 800 artisan cheese producers in the Unit- ed States. There is active adaptation of tradi- tional recipes to meet present-day regulatory standards. The microbiological safety of cheese is a topic of renewed interest as global demand for cheese and cheese products continues to grow. Current regulations which govern the use of raw, heat-treated, and pasteurized milk for cheesemaking in the United States were promulgated in 1949 (18). One of two options could be selected by cheesemakers to ensure the safety of cheese: pasteurize milk destined for cheesemaking or hold cheese at a temperature of not less than 2°C (35°F) for a minimum of 60 days (45). Research has shown that Salmonella enterica serovar Typhimurium, Escherichia coli O157: H7, and Listeria monocytogenes can survive well beyond the mandatory 60-day holding period in Cheddar cheese prepared from pas- teurized milk (19, 20, 21). Efforts have been under way in North America to exam- ine a regulatory change requiring mandatory pasteurization of all milk intended for
FIGURE 3 Map depicting U.S. locations of cheese made on farms in 1849. From reference 17; downloaded from Maps ETC (http://etc.usf.edu/maps). doi:10.1128/microbiolspec.CM-0001-2012.f3
1. HISTORICAL OVERVIEW OF CHEESE AND MICROBES 7
cheesemaking. The pathogens of concern to cheesemakers today, including E. coli O157: H7, Listeria monocytogenes, Salmonella enterica serovar Typhimurium DT104, and Staphylo- coccus aureus, were not the same pathogens of concern as in 1949. In 1997, the U.S. Food and Drug Administration (FDA) requested that the National Advisory Committee for the Microbiological Criteria for Foods review the 60-day aging rule for cheese production. Concern was expressed that a policy revision may be necessary as 60 days of aging may be insufficient to provide an adequate level of public health protection. At the same time, in 1996 in Canada, a proposed amendment would have required all cheeses to be made from pasteurized milk or the equivalent. Health Canada ultimately withdrew this amendment because a scientific expert com- mittee stated that the technical requirements could not be met in the manufacturing pro- cess by small-scale cheesemakers (22).
While many countries around the world view traditional cheeses made from raw milk as microbiologically safe products, other governments are demanding interventions such as pasteurization to ensure cheese safety. It is ironic that France, which has created most of the world’s great raw milk cheese, was also the country where the eminent sci- entist Louis Pasteur developed the concept known today as pasteurization. From his home laboratory in Arbois, Pasteur conducted experiments which have revolutionized our understanding of the role of microorganisms in food fermentation. Today, in Poligny, just a few miles from Pasteur’s home, contempo- rary scientists such as Eric Beuvier (39) are employing cutting-edge technologies to char- acterize a complex array of bacteria, yeasts, and both surface-growing and internal molds which impact cheese flavors and textures as well as contribute to the microbiological safe- ty of cheese. Originally developed as a mild heat process applied to prevent spoilage of wine, pasteurization has been applied to fluid milk to eliminate bacterial pathogens. In the early 1900s, raw milk was a major source of
human disease, including tuberculosis and scarlet fever. Numerous deaths were linked to raw milk consumption. Pasteurization has done more than any single intervention to protect public health from dangerous milk consumption. While pasteurization of milk intended for cheesemaking has also been ap- plied to protect public health, pasteurization of cheese milk has been done largely for reasons other than safety, mainly to ensure consistency and quality of produced products. Industrial equipment for pasteurization was available as early as 1895. A large number of dairies in Denmark were using milk pasteuri- zation in cheesemaking as early as 1908–1909, and milk pasteurization was promoted for hard cheeses produced in Denmark in order to eliminate pathogenic bacteria from milk (23). In France, Fromagerie Renard- Gillard was the first company to employ milk pasteurization for cheesemaking, using recommendations developed by Pierre Mazé of the Pasteur Institute (Fig. 4). Research in the United States on using pasteurization in the cheesemaking process began in 1907 in Wisconsin, with the primary goal of improv- ing cheese quality, although product safety was also a concern. Stevenson (24), working in New Zealand, reported on the advan- tages of pasteurized milk for cheesemaking, which included improved cheese flavor, supe- rior yield, more uniformity, extended shelf life, and simplification of the cheese manufac- turing process. If raw milk was of inferior quality, cheese made from pasteurized milk received preference scores during evaluation. Similar results were obtained by Hochstrasser and Price (25) when evaluating Camembert cheese manufactured from pasteurized milk. Brie cheese was first imported into the United States in 1936. Pasteurization was used to fa- cilitate export of Brie to the United States be- cause of the need to find a stable and safe way to distribute cheese. Pasteurized milk made it easy to produce a cheese that had a long enough shelf life for transport by ship and by rail. All of the aforementioned studies utilized holding pasteurization (145°F, 30 min). It
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should be noted that the pathogens of con- cern during these times were not the patho- gens about which we have concerns today.
Many artisan cheesemakers utilize raw milk in cheesemaking and, from this raw material, manipulate the cheesemaking process to select for desirable organisms. Cheesemakers argue that raw milk is a reservoir of a diverse micro- flora which imparts diverse organoleptic and sensory characteristics to cheese. In many tra- ditional cheesemaking procedures, milk is preripened (held overnight at room tempera- ture) to select for mesophilic bacteria, which are beneficial to the cheesemaking process, fa- cilitating the development of acidity as a result of lactic acid production during metabolism. This practice is either discouraged or not per- mitted in many countries, with regulations re- quiring instead the use of refrigeration of milk prior to cheesemaking. U.S. regulations (46) state that “if milk is held for more than 2 hours between time of receipt or heat treat- ment and setting, it shall be cooled to 45°F. or lower until time of setting.” Lafarge et al. (26) examined the impact of refrigerated stor- age of milk prior to cheesemaking on the
shifts in the composition of bacterial popula- tions in raw milk. These investigators conducted DNA analysis of bacterial popula- tions in refrigerated versus nonrefrigerated raw milk samples using temporal temperature gel electrophoresis (TTGE) and denaturing gradi- ent gel electrophoresis (DGGE). Lactococcus lactis was the major raw milk species identified via TTGE in unrefrigerated milk samples, along with Staphylococcus species, Streptococcus uberis, Listeria innocua, Listeria monocytogenes, Lactobacillus fermentum, and Enterococcus faecium. DGGE analysis revealed Klebsiella pneumoniae, Arthrobacter species, and Brevibacterium linens. Following incubation of raw milk samples for 24 h at 4°C, increases in psychrotrophic species, including Listeria (L. innocua and L. monocytogenes) and Aermonas hydrophila, along with Lactobacillus fermentum, Staphylococcus epidermidis, Pseudomonas fluorescens, Enterococcus faecium, and Serratia marcescens, were observed. Decreases in Lactococcus lactis, Brevibacterium linens, Lactobacillus plantarum, and Lactobacillus pentosus were observed, among others. The results illustrate that employment of refrigera- tion to enhance milk quality and safety prior
FIGURE 4 Antique Brie and Coulommiers label from Fromagerie Lorraine Renard-Gillard, located in Biencourt, near Montiers-sur-Saulx, France. Alfred Renard-Gillard worked from 1906 to 1922 with P. Mazé of the Pasteur Institute on improved tech- niques of cheese production, including the use of pasteurized milk for cheese manufacturing. (Author’s collection.) doi:10.1128/microbiolspec.CM-0001- 2012.f4
1. HISTORICAL OVERVIEW OF CHEESE AND MICROBES 9
to cheesemaking may actually select for bacte- rial populations which pose safety and quality issues. Quigley et al. (27) recently identified the presence of several microbial genera not previously associated with cheese, includ- ing members of the genera Faecalibacterium, Prevotella, and Helcococcus. The authors report detection of Arthrobacter and Brachybacterium from goat cheese. Through use of pyro- sequencing of bacterial populations associated with artisanal cheeses, the authors identified 21 different genera (Fig. 5). Marcellino et al., in their groundbreaking studies on biodiversi- ty of G. candidum strains, suggested that cheesemaking technologies play a role in strain selection, and the diverse strains con- tribute to the diversity of flavor found in arti- san cheeses (40). They state that “as traditional techniques for cheesemaking are threatened or abandoned, the collection, characterization and preservation of native strains of cheese ripening microorganisms is critical.”
Bachman and Spahr (28) assessed the safety of Swiss hard and semihard cheeses made from raw milk. Approximately 80% of the cheeses made in Switzerland are manufactured from raw milk without prior heat treatment. These
authors inoculated the pathogens Aeromonas hydrophila, Campylobacter jejuni, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella, Staphylococcus aureus, and Yersinia enterocolitica to raw milk at levels ranging from 104 to 106 CFU/ml prior to the manufacture of hard and semihard cheeses. In Swiss hard cheeses, no detection of pathogens beyond 1 day was recorded. This was attributed to the cooking temperature of 53°F to which patho- gens are exposed during cheesemaking. Fur- ther, the rapid decrease of the redox potential of Swiss cheese likely imparts additional in- hibitory effects. Similar results have been shown for Italian Grana cheeses (29). Thus, for certain cheese varieties, the term “raw milk” cheese is a misnomer, as this term does not reflect the high curd cooking temperatures used in the manufacture of aged Swiss and Italian cheeses. The Australian Food Safety Authority concluded, in recently completed comprehensive risk assessments, that raw milk hard Swiss cheese varieties, in- cluding Emmental, Gruyère, and Sbrinz, and extra hard grating cheeses, including Parmi- giano Reggiano, Grana Padano, Romano, Asiago, and Montasio, had microbiological
FIGURE 5 Microbial biodiversity of soft cheese (a), semihard cheese (b), hard cheese (c), and cheese rinds (d). Reproduced with permission from reference 27. doi:10.1128/microbiolspec.CM-0001-2012.f5
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safety equivalent to that of cheeses made from pasteurized milk due to manufacturing and aging parameters (30).
In tracing the origins of pasteurization in the United States, in 1924, the Public Health Service implemented the Standard Milk Ordinance to assist states in the volun- tary adoption of programs to control milk- borne disease. In 1939, milk pasteurization was adopted in the United States for the first time and was defined in a milk ordinance and code (31). In 1950, the U.S. Surgeon General invited regulatory agencies to estab- lish procedures for a voluntary Interstate Milk Shipper Certification Program. The Grade A Pasteurized Milk Ordinance estab- lished national uniform standards. Products covered under the Grade A Pasteurized Milk Ordinance included creams, concentrated milks, yogurts, and low-fat and skim milk. The FDA is also responsible for additional regulations to protect the safety of cheese, but these regulations are not part of the grade A program. Milk can be grade A or grade B, with grade A milk meeting the sanitary standards for fluid milk products (and usable for any dairy product). Grade B is considered a manufacturing grade. Milk in the United States is also classified, with classification used for pricing systems. Producers may participate in the Market Order Program, which estab- lishes prices according to milk uses. Class I is of the highest price and is used for fluid milk products. Class II is used for soft milk prod- ucts, like yogurt, cottage cheese, and ice cream. Class III is used for hard cheeses, and class IV is used for butter and for milk prod- ucts in dried form. The U.S. Code of Federal Regulations (47) states, “No person shall cause to be delivered into interstate commerce or shall sell, otherwise distribute, or hold for sale or other distribution after shipment in in- terstate commerce any milk or milk product in final package form for direct human con- sumption unless the product has been pasteur- ized or is made from dairy ingredients (milk or milk products) that have all been pasteur- ized, except where alternative procedures to
pasteurization are provided for by regulation, such as in part 133 of this chapter for curing of certain cheese varieties.” In the U.S. Code of Federal Regulations, cheese has been de- fined as belonging to one of four groups: very hard, hard, semisoft, or soft. The type of cheese depends on the type of milk used, the methods used for coagulation of the curd, the cooking and forming of the curd, the type of culture used, the salting method, and the rip- ening conditions. For instance, a soft cheese, like cottage cheese, is an unripened cheese with 80% moisture. Parmesan and Romano are very hard cheeses, referred to as grated or shaker cheeses.
In the 21st century, the global demand for artisan cheeses is creating new economic op- portunities. Consumers seeking distinctive products with regional flavor, or terroir, are becoming connoisseurs of hand-crafted prod- ucts with distinctive tastes and character. Such demands have created new concerns for food safety and international trade. In response, new technologies, such as microfiltration, are being proposed to increase cheese safety, but these technologies fundamentally alter the traditional artisan practices and may not enhance microbiological safety. European cheesemakers have protected their artisan cheese practices through programs such as protected designation of origin and appellation d’origine contrôlée (AOC). AOC establishes the authentication of content, method, and origin of production of a French agricultural item. In 1935, the Institut National des Appellations d’Origine (INAO) was created as a govern- ment branch developed to administer and manage the AOC process for wines. The INAO’s responsibilities were later broadened to protect other artisanal and traditional prod- ucts, such as cheese. Every AOC product has its own set of regulations based on the prod- uct’s unique history, area of production, and locally recognized practices. There are cur- rently 44 AOC cheeses representing approxi- mately 15% of the more than 600 cheeses produced in France. Since 1996, the European Union Protected Designation of
1. HISTORICAL OVERVIEW OF CHEESE AND MICROBES 11
Origin system has also protected regional foods, wines, and spirits on a European level.
Camembert de Normandie is an AOC product that must be made with unfiltered raw milk produced in Normandy from cows fed under strict conditions and have a fat con- tent of 38%. Corroler et al. (32) conducted an ecological study to determine the effect of geographic origin of specific strains on the manufacture and ripening of a traditional Camembert de Normandie cheese. The con- sistent and specific presence of wild-type strains of Lactococcus lactis subsp. lactis strains isolated from raw milk produced within the AOC Camembert region confirmed the dairy significance of the Camembert registered des- ignation of origin region. As stated by the authors, “It is well known that traditional cheeses made with raw milk ripen faster and develop a more intense flavor than cheeses made with pasteurized or microfiltered milk.” Understanding the biodiversity of the micro- bial population associated with artisan cheese affords a look into the uniqueness which arti- san production contributes to a biodiverse mi- croflora which, in turn, imparts unique sensory attributes. A variety of culture-depen- dent and culture-independent and molecular methods have been utilized for microbial characterization, but many of the traditional approaches are cumbersome and may miss unique strains which are difficult to culture and characterize. New advances in molecular biology offer some innovative approaches for rapid and comprehensive characterization of microbial communities (41).
As of 2013, it appears that efforts to require mandatory pasteurization of milk for cheese- making are being abandoned in favor of a risk-based approach to ensure cheese safety. This is due, in part, to a redefinition of pas- teurization which occurred as part of the 2002 Farm Security and Rural Investment Act (33). The U.S. legal definition of pasteurization is “[a]ny process, treatment, or combination thereof, that is applied to food to reduce the most resistant microorganism(s) of public health significance to a level that is not likely
to present a public health risk under normal conditions of distribution and storage” (33). At the University of Vermont, research has been conducted to examine the fate of patho- gens in cheeses legally manufactured under the 60-day aging rule (42). Microbiological risk varies depending on the specific characteristics of the cheese being manufac- tured. Of highest risk are the bloomy-rind soft cheeses, for which high-pH and high-mois- ture conditions facilitate growth of pathogens. The U.S. Code of Federal Regulations (21 CFR 133.182) permits manufacture of soft ripened cheeses from raw milk provided that these cheeses are aged for 60 days or longer at a temperature of not less than 35°F (34). Due to renewed interest in specialty cheeses, arti- san and farmstead producers are manufactur- ing soft mold-ripened cheeses from raw milk, using the 60-day holding standard to achieve safety. Lower-moisture soft ripened cheeses to be held for 60 days supported the growth of very low levels of L. monocytogenes as a postprocess contaminant independent of the milk type used for manufacture. The safety of cheeses within this category must be achieved through control strategies other than a 60-day holding period, and revision of current federal regulations is warranted (34).
The study by D’Amico et al. (34) is of par- ticular interest to the FDA and Health Canada as they embark upon a joint soft cheese risk assessment. The FDA and Health Canada have documented associations between con- sumption of certain soft cheeses and onset of listeriosis. They are therefore continuing to evaluate the safety of soft ripened cheeses, particularly those made from raw milk, and will do so through a joint FDA/Health Canada risk assessment. This risk assessment will assess the public health impact of L. monocytogenes in soft ripened cheese through focusing on sources of contamination, the im- pact of various manufacturing and processing steps, and the effectiveness of intervention strategies, including new technologies. The impact of consumer handling practices will also be evaluated and a model developed to
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assess predicted risk associated with manufac- turing processes, interventions, and handling practices. It is important to note that the ma- jority of cheese-related outbreaks are caused by postprocess recontamination of cheese; thus, employment of pasteurization of milk does not address this problem. A reevaluation of the safety of traditional artisan practices, validation thereof, and communication of the scientific principles which promote safety will therefore be necessary to enable the continued production of traditional artisan cheeses in global commerce.
ACKNOWLEDGMENT The author has no conflict of interest to declare.
CITATION Donnelly CW. 2013. From Pasteur to probiotics: a historical overview of cheese and microbes. Microbiol Spectrum 1(1):CM-0001-12. doi:10.1128/ microbiolspec.CM-0001-12.
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1. HISTORICAL OVERVIEW OF CHEESE AND MICROBES 15
THE BASICS OF CHEESEMAKING
Paul S. Kindstedt1
2 INTRODUCTION All cheeses share a set of principles that involve a complex matrix of interdependent chemi- cal, biochemical, and microbiological changes. Collectively, these changes first transform milk into fresh or unaged cheese. Although some varieties are consumed immediately after man- ufacture as fresh cheese, most undergo a subse- quent period of aging or ripening, ranging from weeks to years depending on the variety, during which the sensory characteristics un- dergo multifaceted and often quite dramatic changes.
The various steps performed during the first day of cheesemaking, or first few days for cheeses that require extended salting regimens, are especially critical because they establish the chemical characteristics of the cheese at the start of ripening, which, in turn, influence the ripening process. For most cheeses, the first day of cheesemaking is centered on the bacterial fermentation of lactose to lactic acid. The rate at which lactic acid is produced profoundly shapes the initial chemical characteristics of the cheese, which, in turn, exert powerful selective
influences on the complex microbial popula- tions that invariably find their way from the milk and surrounding environment into the cheese. Of the plethora of organisms that are present in newly made cheese, some will re- main viable and may even proliferate during aging, others will be suppressed completely, and others may be initially suppressed and then favored or vice versa, depending on the chem- ical environment to which they are subjected. To add to the complexity, the chemical envi- ronment of the cheese often changes dra- matically as ripening progresses. The mix of organisms that remain viable and their popula- tion densities, as well as the timing of cell death and lysis, directly and indirectly shape the chemical and biochemical reactions that drive flavor and texture development during ripening.
All of this sounds very complicated, and in- deed it is, but much of this complexity can be reduced to a handful of scientific principles that in practice can be controlled and system- atically varied (even if the science is not un- derstood) to achieve an almost limitless range of cheesemaking outcomes, for better or for worse. The historical development of distinctly different cheese varieties can be thought of as
1Department of Nutrition and Food Sciences, University of Vermont, Burlington, VT 05405-0086.
Cheese and Microbes, Edited by Catherine W. Donnelly, © 2014 American Society for Microbiology, Washington, DC, doi:10.1128/microbiolspec.CM-0002-12
17
modulations of these basic scientific princi- ples, or variations on a theme. Over the course of 9,000 years or so, cheesemakers in various places discovered these modulations through careful observation and trial-and- error experience, and they modified their craft and equipment as necessary to produce outcomes (i.e., wonderful cheeses) that met their needs in the time and place in which they lived. Although the science of cheese- making is very complex and incompletely un- derstood (despite having been the subject of systematic study for more than a century), much can be distilled down to a handful of principles. Therefore, this article presents the basics of cheesemaking by integrating the practical steps that are used by all cheese- makers with the scientific principles, in highly distilled form, upon which those practices are based. The specific goal is to paint a concep- tual picture in which the microbiology of cheese “fits together” with the basic cheese- making practices and the scientific principles that underpin those practices. It is hoped that this article will foster a better appreciation for how the bland raw material known as milk came to be transformed into the stunning ar- ray of cheese varieties that we have inherited.
THE BASICS OF MILK CHEMISTRY Milk is the raw material from which all cheeses are produced; therefore, the basics of cheesemaking begin with the basics of milk chemistry. The following brief review of the five fundamental components of milk (i.e., water, lactose, fat, protein, and salts) lays the foundation for understanding how each com- ponent contributes to the chemistry and struc- ture of cheese and their integration with one another.
Water Milk is approximately 85% water; therefore, water is milk’s most abundant component and serves as the continuous phase throughout which the solid components (lactose, fat, pro- tein, and salts) are dispersed (1). Because of their strong dipolar nature, water molecules
are attracted to one another and other polar molecules and ions; therefore, they tend to cluster together tightly though transient hy- drogen bonding. In contrast, water molecules shun nonpolar molecules and minimize their area of interface. The solid constituents of milk remain dispersed throughout the water phase because they either are polar in nature or, in the case of milk fat and casein, are packaged within macromolecular structures that contain a polar surface layer that enables the structure to interact with water molecules. Coagulation, the pivotal first step in cheese- making upon which all else depends, is ac- complished by converting proteins in milk (or cream, or whey or buttermilk depending on the cheese variety) from their native polar form to a nonpolar form. When this occurs, the protein is forced to separate from the wa- ter phase through a process that entraps fat and minerals and, initially, all of the water and dissolved substances. This phenomenon, re- ferred to as coagulation, and the process of syneresis (i.e., curd contraction and water [whey] expulsion) that follows coagulation give rise to discrete curd particles from which cheese is fashioned. Thus, the pivotal first step in cheesemaking centers on transforming milk proteins from a polar to a nonpolar state, thereby initiating coagulation. There are three different mechanisms by which this may oc- cur, which give rise to three fundamentally different cheese families: rennet-coagulated, acid-coagulated, and acid/heat-coagulated cheeses (2).
Lactose Milk contains about 5% lactose, which is a highly polar disaccharide that exists in true solution. Therefore, when the water in milk separates as whey from the curd during cheesemaking, it carries lactose with it in equal proportion. Only a small fraction (gener- ally around 5%) of the water and lactose in milk is ultimately retained in cheese. Lactose is vital to cheesemaking, nevertheless, because it is the substrate that lactic acid bacteria (LAB) ferment to lactic acid during the manufacturing
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process. The small amount of residual lactose that is retained in newly made cheese also impacts ripening in a range of ways depending on the microbial players that ultimately fer- ment the residual lactose and the fermentation pathways that they employ.
Milk Fat About 98% of the fat in milk consists of triglycerides (3). Triglycerides are very nonpo- lar and thus cannot remain dispersed in water unless they are packaged as an emulsion in the form of droplets that are coated with a polar surface layer. Milk fat exists as large triglycer- ide droplets or globules that are packaged in a polar phospholipid membrane that enables the globules to remain dispersed in milk (4). During cheesemaking, the milk proteins phys- ically entrap the fat globules when the pro- teins separate from the water phase during coagulation and syneresis. Therefore, almost all of the fat in milk (generally 90% or more) becomes concentrated in the cheese.
Milk fat strongly influences both the flavor and texture of cheese. Texture is influenced in a highly temperature-dependent manner because the triglycerides of milk fat possess a gradual melting range; that is, the proportion of noncrystallized (liquid) to crystallized (solid) triglycerides increases gradually with increas- ing temperature. At <5°C, the majority of triglycerides in milk fat globules are crystal- lized, forming hard solid spheres. The globules become less crystallized and progressively softer and more fluid-like with increasing temperature, attaining full liquidity around 38°C (5). In cheese, this transition translates into a softer and stickier texture. Milk fat con- tributes to cheese flavor by virtue of its char- acteristically high distribution of short-chain fatty acids within the triglycerides. When freed from the triglyceride structure through enzymatic hydrolysis (i.e., lipolysis), short- chain fatty acids (4 to 12 carbons in length) are highly volatile and possess strong piquant and pungent aroma and flavor notes that con- tribute to the sensory characteristics of many cheese varieties (6). The resulting flavors and
aromas may range from exquisite to obnox- ious depending on the concentrations and relative proportions of free fatty acids released. Bacteria, yeasts, and molds all serve as sources of esterases (i.e., lipases) capable of producing free fatty acids from milk fat; thus, the micro- bial ecology of cheese has profound implica- tions for free fatty acid flavor production, for better or for worse. Free fatty acids, in turn, may serve as the substrate for the production of highly volatile and flavorful methyl ketones by mold species during aging, which may produce sensory notes ranging from fruity and floral to mushroom and musty depending on concentrations and relative proportions.
Milk Protein The protein in milk is made up of two distinct families, known as the caseins, which account for about 80% of the total protein, and whey proteins, which account for the re- maining 20%. Two of the three major families of cheese (the acid-coagulated and rennet- coagulated families) arise through the coagula- tion of casein alone. For these cheeses, the whey proteins do not participate in coagula- tion and, as their name implies, are removed along with the whey during cheesemaking. In contrast, both whey proteins and caseins participate in coagulation and are incorporat- ed into the third major cheese family, those for which a combination of acid and heat is used to initiate coagulation at the start of cheesemaking.
CASEINS The caseins consist of four major components that are designated αs1-, αs2-, β-, and κ-casein. They are classified as phosphoproteins, mean- ing that they possess up to 13 negatively charged phosphate groups that are bonded to serine residues along the amino acid back- bones of the casein molecules, which range in length from 169 to 209 amino acids (7). Phosphoserine residues have the capacity to form ionic bonds with calcium ions, which are abundantly present in milk; ionically bound calcium, in turn, forms ionic complexes with
2. THE BASICS OF CHEESEMAKING 19
inorganic phosphate ions that also are abun- dant in milk, thereby creating nanocrystals of colloidal calcium phosphate. Because of this feature, the vast majority of caseins in milk are packaged as large spherical macromolecular structures referred to as casein micelles. The casein micelle can be thought of as a tangled spherical mass of thousands of individual casein molecules that are bonded together in part by calcium phosphate nanocrystals through ionic linkages with phosphoserine residues on adja- cent casein molecules (8). About two-thirds of the total calcium and one-half of the total phosphorus in milk are occluded within casein micelles in the form of colloidal or micellar calcium phosphate (1).
There are three characteristics of casein micelles that are essential to the making, rip- ening, and ultimate diversity of cheeses. First, the surface of the casein micelle is very polar due to its high concentration of κ-casein, which is unique among the caseins in that it contains highly polar carbohydrate side chains with charged acidic groups that protrude from the micelle surface and essentially form a hairy polar surface layer (1). In contrast, the interior of the micelle is comparatively nonpolar. The polar surface enables the micelle to interact with water molecules and remain dispersed in the water phase of milk. Under certain circumstances, however, the polar micelle sur- face can be neutralized or shaved off, thereby rendering the micelles less able to interact with water and causing them to separate in the form of a coagulum. This is the basis of coagulation, and cheese would not exist if this were not so.
Second, casein micelles have a prodigious buffering capacity (i.e., capacity to absorb hy- drogen ions) because of their high calcium phosphate content. One can think of casein micelles as spherical sponges that continually absorb and neutralize a fraction of the hydro- gen ions that accumulate in the water phase of the milk or cheese curd as LAB produce lactic acid during cheesemaking. As casein micelles absorb hydrogen ions, micellar calci- um phosphate is converted to a soluble form
and released from the micelles into the water phase (1). For some cheese varieties, the production of lactic acid during the first day of cheesemaking is characteristically rapid and extensive, which results in rapid and extensive demineralization of the curd and a final cheese that is depleted of calcium phosphate. For other cheeses, lactic acid production is slow and limited, resulting in limited demineralization and a final cheese that is rich in calcium phos- phate. The great diversity that exists among cheese varieties is partly related to differences in the rates and extents of acid production during the first day of cheesemaking and con- comitant demineralization of the curd. For this reason, the first day of cheesemaking is centered on the bacterial fermentation of lac- tose to lactic acid. Thus, it is not surprising that dairy microbiologists have directed much research towards gaining control over the pro- duction of lactic acid by LAB through ad- vanced starter culture technologies (9).
Finally, it is important to recognize that casein micelles possess strong water-binding and water-holding capacities. Water in cheese exists in several different physical and che- mical states that fall into two broad catego- ries: chemically bound or nonsolvent water, which is tied up and not available to support microbial growth and enzymatic processes, and bulk phase water, which is weakly im- mobilized or loosely held within the cheese matrix and therefore biologically available (10). The amount of bound water in cheese is directly influenced by the casein content; therefore, two cheeses with the same amount of water but different casein contents will pos- sess different levels of casein-bound water and, therefore, biologically available water, which has implications for microbiological and enzy- matic activities during ripening. The water- holding capacity of cheese, i.e., the ability to immobilize bulk phase water and prevent separation of the cheese serum (called “weep- ing”), is also directly influenced by casein con- tent. Thus, for every cheese variety there is a balance between water and casein contents that must be maintained within certain ranges;
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some cheeses are highly sensitive to shifts in this balance and others more forgiving, but large shifts invariably affect ripening and quality.
WHEY PROTEINS The whey proteins mostly exist as monomers or dimers that are folded into compact globu- lar three-dimensional arrangements (11). Their native folded states enable the whey proteins to remain dispersed in the water phase of milk because the polar regions of their amino acid backbones are oriented outwards towards the water phase and shield the nonpolar regions buried beneath. Therefore, like lactose, whey proteins are removed with the whey in equal proportion to water during the making of acid- and rennet-coagulated cheeses. However, whey proteins are heat sensitive and begin to denature and unfold at temperatures above about 79°C, thus exposing their nonpolar hy- drophobic regions. Whey proteins become even more heat sensitive when the pH of milk is decreased from its physiological value (around 6.7) to 6.0 or lower (e.g., due to lactic acid production by LAB or to the external ad- dition of an acidulant). Heat-denatured whey proteins lose their capacity to interact with water molecules due to their exposed hydro- phobic regions, and this causes them to aggre- gate with each other and with casein micelles. Casein micelles also become heat sensitive when the pH of milk is lowered, and this in- stability combined with whey protein dena- turation and casein-whey protein interactions is the basis for acid/heat coagulation, as dis- cussed below.
Salts Milk contains both inorganic and organic salts, but calcium and phosphate ions in the form of calcium phosphate are by far the most important from a cheesemaking standpoint. Calcium and phosphorus combined account for about one-half of the total mineral content in milk. The chief importance of calcium phosphate lies in its association with casein micelles, as already discussed.
THE BASICS OF COAGULATION Milk coagulation is the quintessential first step in cheesemaking because it initiates a process of selective concentration that results in the separation of most of the casein and fat in milk (along with lesser amounts of salts) as curd from most of the water and its dissolved solids as whey. Cheesemakers of old discovered three different ways to coagulate milk, which gave rise to three distinctly different cheese families: acid-coagulated, rennet-coagulated, and acid/ heat-coagulated cheeses.
Acid Coagulation Acid coagulation occurs when harmless LAB ferment lactose to lactic acid while growing and reproducing to high populations in warm (e.g., 20 to 32°C) milk. In traditional practice, LAB were naturally present in raw milk as ad- ventitious contaminants from environmental sources such as teat surfaces and surfaces of pails, vats, utensils, etc. In modern practice, the species and strains of LAB and their popula- tion densities are determined by the starter culture that is added to the milk at the start of cheesemaking. As lactic acid is produced and the milk pH declines towards the isoelectric point of casein at pH 4.6, the accumulation of hydrogen ions essentially neutralizes the polar surfaces of casein micelles, thereby rendering them incapable of interacting with water mol- ecules (12). The micelles are thus forced to in- teract with one another in a peculiar manner that results in the formation of aggregates and chains of micelles. As coagulation progresses, the micellar chains increase in length and inter- lock with one another to form a three-dimen- sional net-like matrix that initially entraps all of the water and solid components (mainly lac- tose, fat, whey proteins, and salts) of the milk, as illustrated in Fig. 1. Over the course of sev- eral hours, the milk is thus transformed from a liquid state to a soft fragile gel, or coagulum. The casein matrix that gives the coagulum its structure is highly demineralized because most of the micellar calcium phosphate is dissolved by the high concentration of lactic acid and low pH needed to initiate coagulation.
2. THE BASICS OF CHEESEMAKING 21
The demineralized casein matrix is very fragile and has limited ability to contract and expel whey, which impedes whey drainage dur- ing manufacture (12). Therefore, acid-coagulat- ed cheeses are generally quite high in water content (usually around 70 to 80% moisture) and very vulnerable to microbiological spoilage, especially by yeasts and molds because of their low pH values (around pH 4.6). Therefore, most acid-coagulated cheeses are consumed fresh (unaged) as soft, high-moisture varieties. Cottage cheese, quark, fromage frais, and cream cheese are well-known modern examples of fresh acid- coagulated cheese. A small amount of rennet, for example, 1 to 10% of the dosage used for rennet coagulation, may be added to the milk shortly after the start of fermentation to increase the firmness and syneresis capacity of the curd, pro- ducing a lower-moisture product with improved texture and stability (13). There are a few rip- ened versions of acid-coagulated cheese, such as traditional varieties produced in the Near East that are aged in sealed clay vessels or animal skins (14). Apart from these few exceptions, however, most acid-coagulated cheeses are consumed fresh (unaged) as soft, high-moisture varieties.
It should also be mentioned that a subcate- gory of acid-coagulated cheese is produced by adding moderate amounts of rennet (e.g., 30 to 50% of the level used for rennet coagula- tion) to the fermenting milk, resulting in a hybrid acid/rennet-coagulated cheese. Hybrid cheeses are lower in moisture than acid-coag- ulated types, and several cheese varieties made by this technology are surface ripened with the white mold Penicillium camemberti. Hybrid cheeses made from goat milk are discussed in detail by Le Jaouen (15) and are not re- viewed here.
Acid/Heat Coagulation Although whey proteins are readily denatured on heating, casein micelles in fresh milk are highly heat stable and remain in colloidal dis- persion at temperatures up to 140°C (11). However, milk that is moderately acidified (e.g., to pH 6.2 to 5.4), either through the production of lactic acid by LAB or through the addition of an external acidulant such as vinegar, becomes susceptible to heat-induced coagulation at relatively low temperatures (e.g., 85°C), and this gives rise to a second, rather
Lactic acid bacterium (LAB)
Extensive acidification by LAB to pH 4.6
Casein matrix, depleted of MCP
Extensive conversion of MCP
Soluble calcium phosphate
FIGURE 1 Diagrammatic representation of the process of acid coagulation. LAB ferment lactose to lactic acid and acidify the milk to around pH 4.6. Coagulation occurs when casein micelles aggregate to form a net-like matrix. During acidification, micellar calcium phosphate (MCP) is extensively converted to soluble form, resulting in a casein matrix that is highly depleted of MCP. doi:10.1128/microbiolspec.CM-0002-12.f1
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small cheese family known as the acid/heat- coagulated cheeses. Coagulation occurs be- cause the whey proteins unfold and lose their ability to interact with water under the com- bined influence of acid and heat. Concomi- tantly, the polar surfaces of the casein micelles become neutralized by an incompletely un- derstood mechanism that may involve calci- um-induced destabilization (16). This causes the denatured whey proteins to attach onto the micellar surfaces and the micelles to aggre- gate into clusters that entrap fat globules. The resulting flocs or curd particles then float to the surface and are separated from the whey with a sieve or perforated ladle. The curds are then allowed to drain and in some cases are pressed. Ricotta is a well-known example of a drained acid/heat-coagulated cheese; queso blanco is a pressed version.
The acid/heat-coagulated cheese family is characteristically high in moisture content (around 50 to 80%), and this, in combina- tion with their generally high pH values, renders most of these cheeses very suscepti- ble to microbial spoilage. There are a few aged versions of acid/heat-coagulated cheeses, produced traditionally in the Near East, which are ripened in sealed clay or skin ves- sels (14). However, like the acid-coagulated cheeses, acid/heat-coagulated types are mostly consumed fresh.
Rennet Coagulation “Rennet” refers to a group of aspartic pro- teinases that preferentially cleave κ-casein at the surface of casein micelles when added to milk, thereby initiating coagulation (17). In traditional and modern practices, rennet en- zymes are derived from a variety of animal, plant, and microbial sources. Technically, the term rennet is restricted to enzymes derived from the abomasa of ruminants; coagulating enzymes from other sources are referred to simply as coagulants or rennet substitutes (18). However, in common usage “rennet” refers to any milk-coagulating enzyme used in cheesemaking, and this broader meaning is used in this article.
Calf rennet is the most widely used animal rennet, but kid and lamb rennets are also used frequently in the making of traditional cheeses from goat and sheep milk. All three animal rennets consist of a blend of the enzymes chymosin and pepsin, with chymosin domi- nating (18). Plant-derived rennets have also been used in traditional cheesemaking for thousands of years. Enzymes extracted from the flowers of two Cynara species (Cynara cardunculus and Cynara humilus), commonly known as the cardoon and the globe arti- choke, are still used in the Mediterranean re- gion for cheesemaking, especially in Spain and Portugal. In West Africa, Calotropis procera is used as a rennet source, and numerous other plants have contributed rennet sources in tra- ditional practice, such as the sap of the fig tree (Ficus carica).
The microbial rennets, which are derived from fungal sources, are relative newcomers, developed in the second half of the 20th cen- tury. There are three major microbial rennets, derived from Rhizomucor miehei, Rhizomucor pusillus, and Cryphonectria parasitica, that from R. miehei being the most widely used (19). These fungi, which are propagated in large fermentation tanks, secrete aspartate protein- ase enzymes that are harvested, purified, and standardized for milk clotting activity and then marketed as microbial or so-called “veg- etable” rennets. The most recent variation on this theme, made commercially available in the early 1990s, is fermentation-produced chymosin. In the case of this product, the gene that codes for bovine chymosin is spliced into the DNA of a host microorganism, usually a yeast or a fungus, which then is propagated in large fermentation vessels to produce pure chymosin that is harvested, purified, and stan- dardized for milk clotting activity.
Rennet coagulation occurs according to a two-step process that is characterized by an enzymatic phase and a nonenzymatic phase (19). Regardless of source, all rennet enzymes initiate the enzymatic phase by hydrolyzing κ-casein, resulting in the release of the charged, carbohydrate-rich C-terminal region
2. THE BASICS OF CHEESEMAKING 23
of the molecule, referred to as the casein- omacropeptide. Rennet enzymes effectively clip off the carbohydrate-rich polar layer at the micellar surface. This exposes the micellar interior, which becomes nonpolar in the cal- cium-rich environment of milk, and this triggers the nonenzymatic phase. The spheri- cal micelles lose their ability to interact with water molecules, forcing them to interact with one another to form micellar aggregates and chains, similar to the process that occurs during acid coagulation. As coagulation prog- resses, the micellar chains increase in length and interlock with one another to form a three- dimensional net-like matrix that initially entraps all of the water and solid components (mainly lactose, fat, whey proteins, and minerals) of the milk, as illustrated in Fig. 2.
Although the illustrations for acid and ren- net coagulation in Fig. 1 and 2 look quite similar, there are two very import