The Good, the Bad, and the Ugly: Tales of -Ripened SISTER NOËLLA MARCELLINO, O.S.B.,1 and DAVID R. BENSON2 1Abbey of Regina Laudis, Bethlehem, CT 06751; 2Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269-3125

ABSTRACT The history of cheese manufacture is a “natural cheese both scientifically and culturally stems from its history” in which , microorganisms, and the environment ability to assume amazingly diverse flavors as a result of interact to yield human food. Part of the fascination with cheese, seemingly small details in preparation. These details both scientifically and culturally, stems from its ability to assume have been discovered empirically and independently by a amazingly diverse flavors as a result of seemingly small details in preparation. In this review, we trace the roots of variety of human populations and, in many cases, have and its development by a variety of human cultures over been propagated over hundreds of years. centuries. Traditional cheesemakers observed empirically that have been made probably as long as mam- certain environments and processes produced the best cheeses, mals have stood still long enough to be milked. In unwittingly selecting for microorganisms with the best principle, cheese can be made from any type of mam- biochemical properties for developing desirable aromas and malian . In practice, of course, traditional herding textures. The focus of this review is on the role of fungi in cheese animals are far more effectively milked than, say, moose, ripening, with a particular emphasis on the yeast-like . Conditions that encourage the growth of although moose cheese can be found in Sweden today. problematic fungi such as Mucor and Scopulariopsis as well as Unfortunately, the price of moose cheese, not to mention Arachnida (cheese ), and how such contaminants might be the scarcity of cooperative moose, dooms such enter- avoided, are discussed. Bethlehem cheese, a pressed, uncooked, prises to a niche market. The more common mammals semihard, Saint-Nectaire-type cheese manufactured in the whose milk is used to produce cheese include cows, United Sates without commercial strains of bacteria or fungi, was sheep, goats, and, to a lesser extent, bison, camels, used as a model for the study of stable microbial succession horses, yaks, and llamas, depending on where one lives during ripening in a natural environment. The appearance of fungi during a 60-day ripening period was documented using in the world. light and scanning electron microscopy, and it was shown to be Words associated with cheesemaking have equally remarkably reproducible and parallel to the course of ripening of ancient roots. The ancient Greeks called the wicker authentic Saint-Nectaire cheese in the Auvergne region of cheese basket used for draining whey from the curds a . Geotrichum candidum, Mucor, and Trichothecium formos, which became forma to the Romans, from roseum predominate the microbiotas of both cheese types. which the modern Italian formaggio and French Geotrichum in particular was shown to have high diversity in fromage were derived (1). The English “cheese” is different traditional environments, suggesting that traditional manufacturing techniques selected for particular fungi. This and other studies suggest that strain diversity arises in Received: 13 April 2011, Accepted: 2 September 2011, relation to the lore and history of the regions from which these Published: 31 October 2013 types of cheeses arose. Editor: Catherine W. Donnelly, University of Vermont, Burlington, VT Citation: Marcellino N, and Benson DR. 2013. The good, the bad, and the ugly: tales of mold-ripened cheese. Microbiol Spectrum 1(1): CM-0005-2012. doi:10.1128/microbiolspec.CM-0005-2012. The history of cheese manufacture is a “natural history” Correspondence: Sister Noëlla Marcellino, O.S.B., mnoella@ in which animals, microorganisms, and the environment sbcglobal.net © 2013 American Society for . All rights reserved. interact to yield human food. Part of the fascination with

ASMscience.org/MicrobiolSpectrum 1 Downloaded from www.asmscience.org by IP: 190.151.168.196 On: Sun, 01 Mar 2015 12:31:56 Marcellino and Benson derived from the West Germanic reconstruct *kasjus via domesticated animals. Of the 237 pot shards analyzed, the Old English cyse and Latin caseus. Their roots are in 22% yielded dairy fat, indicating that dairying was in- the proto-Indo-European reconstruct *kwat-, to ferment deed an important aspect of farming in the British Iron or become sour. Age. In other work, Craig et al. (15, 16) characterized Human survival has a link to fermentation, the pro- lipids and proteins adsorbed to late Bronze Age and Iron cess by which yeasts and bacteria degrade organic Age ceramics from the island of South Uist in the Outer compounds to by-products such as lactic acid and al- Hebrides. The authors used gas chromatography-mass cohol and transform perishable foods into preserved spectrometry and gas chromatography combustion iso- products. In the case of cheese, the final fermented tope ratio mass spectrometry to identify the source of product is safe, durable, and, due to a reduced volume, lipids on the pottery. Twenty-five of 39 shards tested transportable, a feature upon which nomadic cultures showed adsorbed lipids with a high abundance of satu- depend (2). Historically, cheese was often reserved for rated fatty acids, especially 18:0, indicating that the use as a source of protein and fat during periods of low lipids were of origin. The ratio of ∂13C18:0 to milk production due to lactation cycles of animals (3)or ∂13C16:0 values further indicated that the animals were changes in climatic conditions (4). ruminants. They also developed an immunological de- Cheesemaking emerged as a by-product of dairying. tection method, the digestion-and-capture immunoas- Domesticated animals played a role in Neolithic (∼4000 say, to extract and characterize the 2,500-year-old BCE) farming communities worldwide, but there has proteins adsorbed to the ceramics. Their finding of bo- been much conjecture as to whether they were used only vine α-casein adsorbed to pottery shards was correlated as sources of meat or were also exploited for “secondary with the large number of neonatal cattle remains at the products” such as milk (5–7). Clear evidence of site, evidence that calves were culled to sustain a dairy cheesemaking is found in southern Mesopotamia, an economy. Maintaining lactating cattle with only a few area long recognized as the “cradle of civilization,” calves present in a herd implies that the cows were where great centers of religion and culture emerged in milked and a large milk supply was transformed into the late fourth millennium BCE (8). Mesopotamia is the dairy products that could be stored and preserved. The Greek word for “between the rivers,” and it was here authors note the example of bog butter that has been along the banks of the Tigris and the Euphrates in the preserved and found in other sites where there is also Fertile Crescent that many believe that dairying evidence of calf culling. Bog butter, a whitish, grey waxy progressed to actual cheesemaking (9), as evidenced by substance dating back to 400 BCE to 500 CE, has been depictions of milking and processing of milk products found in bogs in Ireland and Scotland. During the Iron (10). Eighteen to twenty varieties of cheese have been Age, butter was preserved in two-piece wooden kegs documented in the ancient Near East, including fla- buried in the anaerobic and cool conditions of the bogs vored, sweetened, sharp, crushed, and white (11). (17). Cronin et al. (18) analyzed the fatty acid content of Evidence for dairying in other ancient cultures has prehistoric bog butter in comparison to present-day Irish been found in temperate Europe and in Mediterranean fresh butter using gas-liquid chromatography and gas regions, in the form of pottery. Shards of ceramic sieves chromatography-mass spectrometry. They found that have been found at Linear Pottery sites distributed fatty acids are present in modern butter in the form of across temperate Europe from the Ukraine to France and triglycerides, while free fatty acids, in particular free from Hungary to the Baltic Sea (12, 13). The sieves were long-chain saturated fatty acids, predominated in the probably used to drain whey from curds and suggest bog butter due to hydrolysis. Proteolysis in the butters that dairying took place in temperate Europe as early was also studied using size exclusion high-performance as ca. 5400 BCE, the radiocarbon date for the Linear liquid chromatography and ion-exchange chromatog- Pottery culture in Hungary (14). More direct evidence raphy. Not surprisingly, the bog butter consisted pri- for prehistoric dairying has been obtained through the marily of small peptides (<0.5 kDa) and free amino acids field of biomolecular archaeology, which has allowed with little or no intact proteins, in comparison to mod- scientists to move beyond speculation based only on ern butter, in which caseins and whey proteins, ranging reconstructed pottery vessels. Copley et al. (5) analyzed in size from 14 to 30 kDa, remain. The authors con- fat residues from pottery from prehistoric sites using cluded that proteolysis in the bog butter was probably fatty acid composition, double-bond positions, tri- due to microbial activity in the bog environment. acylglycerol distribution, and ∂13 enrichment values In ancient Greece and Rome, cheese was an essential to evaluate the origins of the fats in relationship to and popular food. Fresh milk was never imbibed; in fact,

2 ASMscience.org/MicrobiolSpectrum Downloaded from www.asmscience.org by IP: 190.151.168.196 On: Sun, 01 Mar 2015 12:31:56 The Good, the Bad, and the Ugly: Tales of Mold-Ripened Cheese the barbarians were called, somewhat derisively, “milk- in Europe that reflect the cultural and environmental drinkers” (19). The Scythians, the horse-riding tribes of characteristics of particular locales (30). In the developed the Eurasian steppes, were called “mare-milkers” by world, there has been a trend towards decentralizing Homer (ca. 1184 BCE) and consumed fermented mare’s food production and a renewed interest in local foods milk and cheese called hippake (20). Because of the that are identified with particular rural areas and eth- abundance of olive oil in the Mediterranean, butter was nicity, to revitalize agriculture in economically depressed not used. The Greek geographer Strabo (66 BCE–25 CE) or depopulated regions (31). viewed the mountain people of the Iberian Peninsula Legislation to guarantee the territorial authenticity with distaste because they lived on goat meat and acorn and vintage of a product first arose in France, where, in bread, drank beer rather than wine, and used butter 1411, King Charles VI granted the people of - instead of olive oil (21). Varro (116–28 BCE), in his sur-Soulzon a monopoly on Roquefort-style cheese and treatise on Roman farm management, wrote, “Cheese in 1666 the court of Toulouse applied a judicial text to made of cow’s milk is the most agreeable to the taste, but Roquefort cheese, making it illegal for other villages to the most difficult to digest: next, that of ewe’s milk, assert that they produced the cheese. These declarations while the least agreeable in taste, but the most easily simply recognized a cheese type that had been produced digested, is that of goat’s milk” (22). Columella (4 BCE– since at least Roman times. Subsequent legislation ca. 70 CE) wrote of all aspects of cheesemaking in his created the appellation d’origine contrôlée (AOC) to work on agriculture, Rei Rusticae (23). The principles he protect traditional products that arose within the enunciated in the 1st century could be applied today. He boundaries of a rural community and its specific envi- described whey as the “acid liquid” which should be ronmental context (32). Many European artisanal pressed out of the cheese as quickly as possible (23). cheeses are now marked with the seal of the protected Today, we know that excess whey that contains lactose designation of origin (PDO), a denomination established in pressed cheeses can cause “postacidification" of the by the European Union to recognize and protect agri- curd, resulting in bitterness (24). Homer (ca. 1184 BCE) cultural products “whose quality or characteristics are was familiar with aspects of cheesemaking, as evidenced essentially or exclusively due to a particular geographi- by the detail with which he describes the scene in the cal environment with its inherent natural and human cave of the cyclops Polyphemos in his epic poem the factors and the production, processing and preparation Odyssey. Upon entering the cave, Odysseus notices of which take place in the defined geographical area” vessels “swimming with whey” and “willow baskets (33). As early as 500 CE, Cassiodorus (480–575 CE), that were heavy with cheese” (25) which he and his men Praetorian Praefect of the Ostrogothic king Theodoric, eat while awaiting their host. However, the cyclops, a evoked terroir when he described the banquets in Rome “giant dairy farmer” (26), is also “…a savage monster which included cheeses made from the milk of herds who despises the laws of hospitality to the point that he grazing in the lush pastures and forests on Mount Sila in devours guests in his own home, if indeed one can call southern Italy: his cave a home” (27). While his prisoners cower, the cyclops performs his dairy chores twice a day: he …and we praised the wines of Bruttii and the cheese of the his sheep, adds fig juice to form a curd (fig juice contains district around Mount Sila. The cheese, which retains in its the protease ficin, which acts to coagulate the milk), and pores the milk which has been collected there, recalls by its drains the curd in willow baskets. Odysseus and his taste the fragrant herbs upon which the cattle have fed; by its texture it reminds us of the softness of oil, from which it surviving men eventually escape by blinding the cyclops differs in colour by its snowy whiteness. Having been carefully and riding out of the cave underneath the bellies of pressed into a wide cask and hardened therein, it retains per- his rams. manently the beautiful round shape which has thus been given Given the coincidence between dairying, cheesemaking, to it. and civilization, it is not surprising that Kamber and Variae Epistolae, Book XII (34) Terzi regard cheese as “one of the primary symbols of mankind’s passage into civilization” (28), and tech- The concept of a geography of taste (31) has now niques for its production, unique to each culture, were taken hold on both sides of the Atlantic. Reacting to the handed down from generation to generation as a cultural globalized food market and suspicious of industrial food inheritance (29). In modern times, the establishment of processing techniques, consumers increasingly demand the European Economic Community has prompted a to know the source of their food and are seeking a more new self-awareness of each country’s regional products direct link to the producers (35). Hence, “sustainable

ASMscience.org/MicrobiolSpectrum 3 Downloaded from www.asmscience.org by IP: 190.151.168.196 On: Sun, 01 Mar 2015 12:31:56 Marcellino and Benson agriculture,” which includes the concept of reversing the of the bacteria and fungi growing in and on the trend of disappearing farmland, is on the rise. cheese that use various components of milk for their own nutritional requirements. The normal microorganisms involved in cheese rip- CHEESE RIPENING ening include lactic acid bacteria naturally present in the milk and/or added in commercial starter cultures. Lactic Cheese is milk that has grown up…it is preeminently the food of man—the older it grows the more manly it becomes, and in acid bacteria, the live cultures with which most people the last stages of senility it almost requires a room to itself. who eat yogurt are familiar, quickly catabolize lactose to The Epicure’s Companion, Edward Bunyan (1872–1939) (36) lactic acid and lesser amounts of acetic acid in the critical step of lowering the pH of the curd during cheese pro- Throughout the centuries, a blend of empirical obser- duction. Other microorganisms play a more or less im- vation and isolated cultural development has helped portant role in ripening depending on the type of cheese. sculpt the diversity of cheeses we know today. Flavor They include nonstarter indigenous lactic acid bacteria, diversity is created by the biochemical activities of other bacteria such as the aerobic surface-dwelling microorganisms degrading the curd and providing Brevibacterium linens, yeasts, and filamentous fungi flavors and aromas absent from the initially bland young (46). The microbial populations that develop on the cheese (37, 38, 39). Pasteurizing the milk used for surface of cheese can be quite complex depending on cheesemaking allows for more uniformity of the finished where they are ripened and how they were prepared. product and improves food safety but removes much of Some cheeses have traditionally been aged in jars and the indigenous microbiota of milk that imparts distinc- earthenware vessels buried in sand and soil (28), sealed tive flavors (40). The large-scale industrialization of pits called fosse dug in volcanic rock (47, 48), and/or in cheesemaking has caused a loss of traditional techniques caves—each environment bringing the cheese in prox- and ripening venues, including natural caves where di- imity to soil and diverse types of microorganisms (49, verse populations of fungi and bacteria have developed 50). Members of soil fungal genera such as Alternaria, sometimes over centuries. In response, some micro- Chrysosporium, Geotrichum, Mucor, and biologists have been isolating and characterizing the are commonly found on cheese ripened in cave en- members of microbial populations originating in tradi- vironments (51, 52). tional cheeses unique to specific geographical regions Scott observed that “Fermentation organisms are (39, 40, 41, 42). ambient in the environment, whether humans make use In one such project, 4,379 isolates of wild-type lactic of them or not” (53). And indeed humans have. If a acid bacteria were characterized from 35 products, in- cheesemaker observed empirically that certain con- cluding 24 artisanal cheeses made from cow, sheep, ditions produced the good cheeses, he or she tried to goat, and buffalo milk using traditional techniques in reproduce or maintain the environment that by default southern Europe. Not surprisingly, tremendous vari- selected for strains with the best biochemical properties. ability in acid and exopolysaccharide production, pro- Affineur Pierre Androuët says it this way: “Our pre- teolytic activity, and bacteriocins of the isolates was decessors thought with reason that the natural agents in discovered. A conclusion was that “each cheese was an the environment conditioned the personality of cheeses ecosystem” in itself (43). Cheesemakers for centuries and marked them with the indelible sign of vintage and have exploited such extraordinary diversity in the cheese territory” (54). cave or cellar where a cheese is aged. Even with this diversity, cheese microorganisms tend to follow a similar script during ripening. Three primary THE STRAIN YOU LOVE MAY BE YOUR OWN biochemical processes take place: fermentation of milk A case study for how novice cheesemakers can come to sugar (lactose) to lactic acid; hydrolysis of lipids to fatty terms with the cheesemaking process, indigenous acids; and breakdown of casein to peptides, amino acids, microorganisms, and the terroir of a region may be told and ammonia. Amino acids and fatty acid derivatives through the experiences of one of us (N.M.), who are precursors to flavor compounds. Their metabolism established a small artisanal cheesemaking facility at the plays a key role in determining the aroma of a cheese Benedictine in Litchfield (44). These processes lead to the desirable, and some- County, CT. times undesirable, sensory qualities of flavor, texture, When one thinks of dairying in New England, Con- and appearance (45), and they are mainly due to the necticut is not the first state to come to mind.

4 ASMscience.org/MicrobiolSpectrum Downloaded from www.asmscience.org by IP: 190.151.168.196 On: Sun, 01 Mar 2015 12:31:56 The Good, the Bad, and the Ugly: Tales of Mold-Ripened Cheese

Connecticut’s farmland is disappearing faster than that similar microbial successions during ripening. Bethlehem of any other state; approximately 9,000 acres of farm- cheese thus was used as a model for the study of stable land are lost to development every year. microbial succession during ripening in a natural envi- As an aside, we note that the loss of farmland is at odds ronment (58). with Connecticut history. Contributing to the westward Microbial succession on the ripening cheese over 60 expansion of agriculture in the United States, “The days was monitored by scanning electron microscopy Connecticut Yankee brought a cheese hoop with him and and light microscopy; the latter used stained paraffin wherever he went made cheese” (55). The Statistical sections to view cross-sections of the rind at various Account of the Town of Bethlehem for 1812 by the stages (58). When the cheese is removed from the cheese Reverend Azil Backus reports that “there are commonly press, it has a rubbery consistency, very little flavor, and sent to market 7,000 lb. of butter and 30,000 lb. of no discernible rind; indeed, no surface microorganisms cheese” (56). The first cheese factory in America was were visible by scanning electron microscopy. In the built by Lewis Mills Norton in 1844 in Goshen, another curd, however, pockets of gram-positive cocci and yeasts town in Litchfield County known for its lush farmland had developed, indicating that bacterial growth and and pastures. The “pineapple cheese” produced there fermentation begin within the curd soon after formation. earned its name from the pineapple-shaped wooden The morphology of all cells within an individual pocket mold into which Norton pressed a soft Cheddar-type was the same, suggesting derivation from single cells. cheese (57). By the middle of the 19th century, Litchfield At 48 h, the cheese surface is covered with a lawn of County was making nearly 3 million pounds of cheese a budding yeast (Fig. 1A). The number of yeast CFU on year. Pineapple cheese was first transported to the port the nascent rind increases at least 40-fold by day 4 and at New Haven for distribution, but by 1884 the demand stabilizes from day 10 to the end of ripening. The for the cheese was so great that the plant was eventually number of lactic acid bacteria on the rind and in the curd moved to Attica, NY, for better access to the railroads. remains fairly constant throughout ripening. The sharp From this period, cheesemaking in Connecticut began its increase of yeasts by day 4 is probably due to their decline. ability to use some of the lactic acid produced by the Cheesemaking returned in earnest at Regina Laudis in lactic acid bacteria, leading to an increase in the pH of 1977 when a third-generation cheesemaker from the rind. In many cheeses, this deacidification by yeast Cézallier in the Auvergne, France, came as a visitor. She prepares the surface for the growth of acid-sensitive brought with her the traditional methods of making a aerobic bacteria such as Brevibacterium linens (59). Saint-Nectaire-type cheese that she had learned from her Strains of Streptococcus cremoris are the most abundant grandmother. After 2 years and many disasters, a con- lactic acid bacteria within Bethlehem cheese, probably sistently good cheese was developed, which the abbey derived from the raw milk used in production. In con- named Bethlehem cheese in deference to the AOC des- trast to the case with the rind, yeast counts remain low ignation of Saint-Nectaire. Bethlehem cheese is a pressed, and fairly stable within the curd throughout the ripening uncooked, semihard, mold-ripened cheese made from the process. Strains of Debaryomyces and Torulopsis were fresh milk of the abbey’s Dutch Belted cows, a heritage identified, with the latter predominating in the interior of breed whose milk, high in protein, fat, and total solids, is the cheese. Torulopsis ferments lactose to the neutral excellent for cheesemaking. The milk is not pasteurized, ethanol that is important in low-acidity cheeses such as and commercial cultures of starter or fungi are not added Saint-Nectaire (60, 61). during production or ripening. By day 6 of ripening, the surface of Bethlehem cheese is Traditionally, the cheeses ripen on straw mats for 60 covered by the whitish-gray Mucor, identified by its days in the cellar of a house—the “cave.” During this typical sporangiospores in spherical, black sporangia. time, the appearance of fungi growing naturally on the Fig. 1B shows Mucor sporangia and hyphae on the sur- cheese rinds is remarkably reproducible and predictable face of the cheese. By day 9, the sporangiospores release and parallels the course of ripening of authentic Saint- from the sporangia and the cheese takes on a grayish- Nectaire cheese in the Auvergne (58). brown velvety coat composed of mycelia and spores from Fungal genera from Bethlehem cheese and raw milk Mucor entwined with Geotrichum candidum and drying Saint-Nectaire cheeses from the Auvergne region of of the cheese curd surface. G. candidum begins to dom- France were found to be similar and included Geotrichum inate around day 4 of ripening and covers the cheese with candidum, Mucor,andTrichothecium roseum.Thecon- a white powdery coat. G. candidum plays an important clusion was that similar means of preparation lead to role in the ripening of soft cheeses such as

ASMscience.org/MicrobiolSpectrum 5 Downloaded from www.asmscience.org by IP: 190.151.168.196 On: Sun, 01 Mar 2015 12:31:56 Marcellino and Benson

and semihard cheeses such as Saint-Nectaire and Re- blochon. Its lipases and proteases release fatty acids and amino acids and peptides involved in flavor develop- ment, and its aminopeptidases reduce bitterness im- parted by low-molecular-weight peptides in cheese (62). While the growth of Mucor is typical on traditional Saint-Nectaire and Tomme de cheeses (38, 63), it can be a disaster on pâte molle-type cheeses such as and Camembert. By day 60, patches of pink mold can be seen high- lighting the grayish coat left from the growth of Mucor. This pink fungus has been termed the “flower of the ” and is valued by makers of Saint-Nectaire cheese. Microscopically, the two-celled conidiospores that de- velop from the tip of the vertical conidiophore are di- agnostic of Trichothecium roseum (Pers.) Link 1809. Identification T. roseum is confirmed by isolation of the fungus and observation of conidiospore development as occurring in short chains in basipetal succession from the conidiophore using light microscopy (64). The contri- bution of T. roseum to the taste of cheeses like Bethlehem or Saint-Nectaire has not been studied, although the fungus is known for its lipolytic activity (65). By day 59 of ripening, a more complex microbial community has settled in and stabilized on the surface of the cheeses, including filamentous fungi and coryneform bacteria that are first evident by day 20 of ripening. of Brevibacterium and Arthrobacter spp. can be isolated from Bethlehem cheese based on cell morphol- ogy and colony color. Fig. 1C shows the distinctive V shape of coryneform bacteria that have developed be- neath the fungal debris on the cheese surface. Brevibacterium linens, the bacterium that predominates in the mixed culture on the surface of smear-ripened cheeses such as Époisses, Mont d’Or, Limburger, and Taleggio, gives cheeses a characteristic reddish-orange color (66). B. linens produces volatile sulfur compounds from the degradation of L-methionine, which contributes to the unique, sometimes pungent aroma of this class of cheeses and, by the way, to foot odor (67). The ripening of Bethlehem cheese is similar in pro- gression to that of other mold-ripened cheeses, with FIGURE 1 Scanning electron micrographs of Bethlehem some of the same organisms involved, including the cheese during ripening. (A) Micrograph of cheese at day 2 of lactic acid bacteria, Geotrichum candidum, and various ripening showing multilateral budding yeasts embedded yeasts. The difference in taste and consistency is due to within the cheese surface. Buds and bud scars are clearly the differences in microbial communities, which, in turn, μ visible. Bar, 1 m. (B) Micrograph of the cheese surface at day 6 are selected for by differences in handling of the curd as showing sporangia and hyphae of the zygomycete Mucor. Bar, it is being prepared. The selection often occurs at the 10 μm. (C) At day 59, the V shapes of coryneform bacteria can be distinguished at a high magnification beneath a mass of level of salting of the cheese. Inhibition of G. candidum fungal debris on the cheese rind. Bar, 1 μm. Panels A and B are can occur at 1.0 to 2.0% salt and of Mucor at 2.0 to reproduced with permission from reference 58. doi:10.1128/ 3.0%. Penicillium species are the most salt tolerant of microbiolspec.CM-0005-2012.f1.

6 ASMscience.org/MicrobiolSpectrum Downloaded from www.asmscience.org by IP: 190.151.168.196 On: Sun, 01 Mar 2015 12:31:56 The Good, the Bad, and the Ugly: Tales of Mold-Ripened Cheese the three genera, growing well at 5.0% salt. Saint- Nectaire, , and , and the class Nectaire-style cheeses are dry salted, and after 24 h in of smear cheeses to which Livarot, Limburger, and the cheese press, the residual salt is rinsed off, allowing Taleggio belong (69, 70). Pottier et al. (71) evaluated the for the growth of Geotrichum candidum, followed by importance of G. candidum to the French dairy industry, Mucor. In the production of the Spanish blue-veined calculating that G. candidum was present in about cheese Cabrales, during which no starter or fungal 600,000 tons of French cheese, one-third of total pro- cultures are added, the drained cheeses are covered with duction. They concluded that in 1 year, French consumers a layer of coarse salt which is not washed off and is kept ate close to 8 kg (18 lb) per person of cheese containing at room temperature for 10 to 15 days. The cheeses are G. candidum. Fifteen years ago, G. candidum may not subsequently placed in natural caves, where the salt have been commonly discussed in the United States, yet content and high humidity in the environment encour- now at the annual Society conference, it age the growth of Penicillium roqueforti (68). Hence, the is not unusual to hear artisanal cheesemakers comparing cheesemaker directs the microbial population to differ- their strains of G. candidum,or“geo.” Both wild-type ent outcomes. strains and commercial cultures affect the appearance and aroma of cheeses. In catalogues, starter strains are presented according to characteristics that might interest FRENCH LESSONS IN the cheesemaker: colony color, morphology (“yeasty” or “moldy”), biochemical properties (having known pro- The fact that the same types of fungi are found on tra- teolytic or lipolytic activity), and capacity for neutralizing ditional Saint-Nectaire from France and Bethlehem the curd, which affects the subsequent microbial ecology cheese from the United States indicates that the critical of the cheese. microorganisms are present naturally in the environ- Growth of G. candidum is generally observed on ment and that the traditional methods of preparation the cheese surface around the third day of ripening. In and ripening determine the finished product. At the some cheeses, such as Saint-Marcellin, the main role of same time, the genetic and biochemical diversity of G. candidum is in the presentation of the finished Geotrichum populations in the milk and curd and on product: the organism covers the cheese surface with a cheeses ripened in traditional caves of France in six uniform white velvety layer, sometimes called a jolie robe major cheesemaking regions was found to be immense (pretty dress) (72). On other cheeses, G. candidum is not (38). Saint-Nectaire, Reblochon, Mont d’Or, and Tamié obvious at the end of the ripening period. On ripened were looked at with regard to strain diversity in relation Camembert or Brie, G. candidum is covered by a white to the lore and history of the regions from which they layer of which first appears arose. The conclusion from that study indicated that a around day 6 of ripening (73, 74). G. candidum is the tremendous amount of biochemical and genetic diversity only filamentous fungus on Reblochon and, mixed with existed within the Geotrichum candidum clade. Further, Brevibacterium linens, creates an orange-white crust (67, such diversity can partly be explained by the empirical 75). In contrast, G. candidum is part of the more com- selection of cheesemaking techniques that favored the plex microbiotas on the surfaces of Saint-Nectaire and development of the “best” strains in local contexts. A Tomme de Savoie (62). Figure 2A shows a layer of corollary is that manufacturing practices aimed at pro- G. candidum on the rind of a 10-day-old Tomme de ducing a uniform cheese type would necessarily lead to Savoie that will be covered by a gray layer of Mucor later the loss of specific local strains. in ripening. Morphologically, G. candidum is described as a Geotrichum candidum: The Good “yeast-like fungus” because it appears microscopically The loss of diversity has implications for cheesemakers. with both single-cell stages and filaments (72). Figure 2B Among “the good, the bad, and the ugly” fungi, shows a light micrograph of a G. candidum strain iso- Geotrichum candidum stands as one of the best and thus lated from a 4-day-old Bethlehem cheese. Two cell types receives particular attention in this review, but it should can be seen: arthrospores and short septate hyphae. be noted that other microbial groups would paint a During the process of asexual reproduction, the hyphae similar picture. fragment and this process, called disarticulation, G. candidum contributes to the ripening of nearly all produces thallic-arthric conidia, called arthrospores (76) surface mold-ripened cheeses: pâte molle such as Brie —the cylindrical cells seen interdispersed among the and Camembert, semihard cheeses which include Saint- hyphae in Fig. 2B. The scanning electron micrograph in

ASMscience.org/MicrobiolSpectrum 7 Downloaded from www.asmscience.org by IP: 190.151.168.196 On: Sun, 01 Mar 2015 12:31:56 Marcellino and Benson

Fig. 2C(77) shows G. candidum arthrospores, roughly 10 μm in length, on the surface of a wooden Reblochon ripening shelf. Yeast-like strains are generally characterized by cream-colored colonies, very similar to yeast colonies on agar medium. On a microscopic level, these strains pro- duce many arthrospores and very few vegetative hyphae (62). They generally have an optimal growth tempera- ture of 22 to 25°C, show acidifying activity, and are weakly proteolytic (78, 79). More filamentous strains are characterized by white colonies, more or less “furry” or felt-like, which have vegetative hyphae predominating with very little sporulation (62). These fungus-like strains grow best at 25 to 30°C, have alkalizing activity, and are highly proteolytic. Classification of G. candidum may not necessarily interest cheesemakers, yet the commercial strain biotype may strongly influence the texture, cohesiveness, and thickness of the rind. The of Geotrichum has a complex history and is still evolving. About 100 synonyms have been used for the same taxon since 1809, when Link designated the genus as Geotrichum, with G. candidum as the only species (79). Previous names, such as Oidium lactis, assigned in 1850 when it was isolated from milk by Fresenius, reveal a historical con- nection to milk and dairy products (80). Traditional French cheesemakers to this day sometimes refer to G. candidum as “Oidium.” The microorganism was re- classified as Oospora lactis in 1880 and finally placed in the genus Geotrichum. G. candidum was included in Barnett’s first and second editions of Yeasts: Char- acteristics and Identification and classified as a “fungal- like yeast” (81, 82). Until the 1990s, fungal and yeast classification was based on observable (phenotypic) characteristics such as morphology, physiology, and biochemical properties (83). Together with its distinctive morphology, growth on D-xylose and lack of growth on cellobiose and maltose as sole sources of carbon are used in identifying Geotrichum to the species level (72). Despite its ability to live on cheese, G. candidum cannot use lactose as a FIGURE 2 The yeast-like fungus Geotrichum candidum. (A) carbon source, whereas assimilation of lactic acid is G. candidum on the rind of a 10-day-old Tomme de Savoie. variable (62, 84). Phenotypic variability presents a dif- (B) Light micrograph of arthrospores and septate hyphae of a ficult task for identifying each new strain (85). wild-type G. candidum strain isolated from a 4-day-old Over the past 10 years, advances in molecular Bethlehem cheese. Bar, 10 μm. (C) Scanning electron micro- methods for identifying and typing strains have led to graph of the surface of a Reblochon ripening shelf showing revisions in classification. G. candidum had been clas- G. candidum μ cylindrical arthrospores and bacteria. Bar, 5 m. sified as the imperfect form, or anamorph, of the yeast Reproduced with permission from reference 77 (micrograph Galactomyces geotrichum in 1986 by de Hoog et al. by Romain Briandet, UMR-UBHM, Institut National de la Re- fi cherche Agronomique et Ecole Nationale Supérieure des In- (86). However, taxonomic revisions of lamentous dustries Agricoles et Alimentaires, Massy, France). doi:10.1128/ fungi that reproduce by arthric conidiogenesis, based on microbiolspec.CM-0005-2012.f2.

8 ASMscience.org/MicrobiolSpectrum Downloaded from www.asmscience.org by IP: 190.151.168.196 On: Sun, 01 Mar 2015 12:31:56 The Good, the Bad, and the Ugly: Tales of Mold-Ripened Cheese internal transcribed spacer ribosomal DNA sequences pact discs and corrupted the information stored in them and nuclear DNA (nDNA)/DNA reassociation data, led (98). to renaming of the teleomorph (perfect form) Galac- G. candidum is not an agent of food poisoning and is tomyces candidus (87, 88). Isolates of the anamorph generally regarded as safe. No food-borne disease has genus Geotrichum were identified to the species level by been linked to the consumption of products containing using randomly amplified polymorphic DNA-PCR G. candidum, and food technologists handling the (RAPD-PCR) (89). In our investigation into the biodi- microorganism are not are risk (71). However, some versity of G. candidum in French cheese, we differenti- G. candidum strains can act as opportunistic pathogens, ated strains within species using RAPD-PCR (38). Other causing superficial or internal mycosis known as geo- approaches have used chromosomal pulsed-field gel trichosis (90, 99). electrophoresis to correlate genetic data with pheno- Lipolytic enzymes contributed by milk, starter bac- typic characteristics such as morphology (90). The latter teria, and fungi help develop texture and release free approach showed that the fungus-like strains and those fatty acids, precursors to flavor compounds in many with intermediate morphology had larger genomes than cheeses (100). From a biochemical standpoint, G. yeast-like strains. A standardized protocol has been candidum’s lipases are unique and contribute to flavor proposed for identifying G. candidum that allows and texture development in several classes of cheese identification and comparison between laboratories (101, 102). One isoform of G. candidum’s lipases (83). It uses PCR to distinguish between species of exhibits a preference for cis (Δ-9) unsaturated fatty acids Geotrichum and then differentiates strains within the in triglyceride —fatty acids such as oleic or linoleic species using RAPD-PCR in conjunction with random acid with a double bond at the ninth carbon position. amplified microsatellite PCR. The GATA4 primer used Fresno et al. (103) attribute the high concentration in random amplified microsatellite PCR allowed strains of oleic acid in Armada to the fact that to be grouped according to the ecological niche from G. candidum predominates on the cheese during the first which they were isolated, such as food, plants, the en- 2 months of ripening. In addition, the lipase has been vironment, and human sources. The protocol can be used for biotransformations in the oil industry, such as used to trace strains to their provenance and facilitates the synthesis of (S)-atenol and (R)-propanolol through tracking of strains throughout cheese production and lipase-catalyzed hydrolysis of the intermediate O-acetyl ripening. esters, and in pharmaceutical production for the G. candidum strains not only are part of the stereoinversions of arylethanols (104, 105). microbiota of raw milk but also are found in en- Of the three main biochemical activities involved in vironments as diverse as soil, barley, grass, and silage. cheese ripening—proteolysis, glycolysis, and lipolysis— The optimum pH for growth is around 5.5, but they can proteolysis is considered the most complex and impor- grow across pH values of 3.5 to 9.0 (91). G. candidum tant (106). While proteolysis is a key to flavor develop- strains are generally acid-tolerant microorganisms that ment, it is also responsible for bitterness, a common can create costly problems for citrus fruit and tomato defect in cheese (107). Mold-ripened cheeses are espe- industries (92, 93). The organism is part of the natural cially prone to bitterness (108), as are cheeses with low microbiota of barley and is used as an inoculum in malt salt concentrations. High salt concentrations in cheese production to improve quality as well as to inhibit the may promote the aggregation of large peptides, making growth of Fusarium species. The latter cause the defect them less accessible to degradation by proteinases into of “gushing” in beer and can produce mycotoxins (94). bitter peptides (109). G. candidum possesses an array of enzymes with great Aminopeptidases of G. candidum strains have been potential for biotechnological applications. Its lactate correlated with reduced bitterness in Camembert through oxidase has been used to determine lactate levels impli- the breakdown of low-molecular-weight peptides released cated in heart disease (95). In toxic waste management, by the proteinases of Penicillium camemberti (74). The G. candidum is used to decolorize azo dyes in industrial synergistic effects of the two fungi on protein catabolism waste water (96), and its peroxidases have shown high in Camembert are summarized in Fig. 3. Proteolysis in efficiency in decolorizing and biodegrading olive mill cheese begins when the proteinases (chymosin) in coagu- wastewater, a pollutant generated by olive oil extraction lant, milk (plasmin), and starter cultures hydrolyze casein (97). In 2001, G. candidum gained notoriety as the to high-molecular-weight peptides (Fig. 3A). The high- “fungus that eats CDs” when its enzymes bored holes molecular-weight peptides are then catabolized to low- in the aluminum and polycarbonate layers of com- molecular-weight peptides by proteinases of starter

ASMscience.org/MicrobiolSpectrum 9 Downloaded from www.asmscience.org by IP: 190.151.168.196 On: Sun, 01 Mar 2015 12:31:56 Marcellino and Benson

Casein The size of peptides (molecular mass < 6,000 Da), the nature of the N-terminal amino acids, and the higher Chymosin mean hydrophobicity of amino acids in a peptide chain PROTEINASES (coagulant) are factors implicated in bitter taste (109, 116). Peptides A (Lactic Acid Bacteria containing proline have been associated with bitterness in Plasmin starter cultures) cheese, as have peptides with basic amino acids such as (milk) lysine and arginine in the N-terminal position (117, 118, 119). In one study, a synthetic peptide with arginine in the N-terminal position was ∼250 times more bitter than High Molecular Weight Peptides caffeine (116). A high content of the hydrophobic leucine in a peptide also increases bitterness (120). PROTEINASES increasing Aminopeptidases, in cleaving the N-terminal amino acids B Penicillium camemberti ( ) bitterness of a hydrophobic peptide, can reduce bitterness (121, 122). To that end, aminopeptidases from Aeromonas caviae and the fungi Rhizopus oryzae and Aspergillus Low Molecular Weight Peptides sojae have been used to debitter protein hydrolysates (120, 123). The released free amino acids are precursors to fla- vor compounds, and their catabolism is significant in AMINOPEPTIDASES (Geotrichum candidum) mold-ripened cheeses (114). Through reactions such as reduction of deamination, transamination, and decarboxylation, the C bitterness CARBOXYPEPTIDASES amino acids are converted to alcohols, carbonyls, or short- (Penicillium camemberti, chain hydrocarbons (Fig. 3D) (106). Deamination of Geotrichum candidum ) glutamate, aspartate, leucine, phenylalanine, and methi- onine by G. candidum has been reported (124). The breakdown of sulfur-containing amino acids such as me- Amino Acids thionine by G. candidum produces desirable garlic or cabbage aroma notes characteristic of some pâte molle and smear cheeses (125, 126). Molimard (127) correlated Deamination D the cabbage aroma of traditional Camembert with the Decarboxylation Transamination concentration of isovaleric acid, a derivative of leucine via Strecker degradation, which has also been found in Swiss cheese (128). Jollivet et al. grouped eight G. candidum strains according to their production of methyl , secondary alcohols, phenol, phenylethanol, and dimethyl Volatile Aroma Compounds disulfide, when grown in milk and cheese media (129). All FIGURE 3 strains produced the compound 2-methylpropan-3-ol, an Schema for proteolysis in Camembert cheese. fi doi:10.1128/microbiolspec.CM-0005-2012.f3. alcohol previously not identi ed in cheese. We studied the extracellular aminopeptidases of 13 wild-type G. candidum strains isolated from bacteria and secondary microorganisms (110, 111). Camembert, Mont d’Or, Reblochon, Saint-Nectaire, G. candidum presumably does not have to hydrolyze Coulommiers, and Bethlehem cheeses chosen as rep- caseins into peptides, as these can be provided by the resentatives of major groups in our RAPD-PCR study. proteolytic activity of other organisms. Boutrou et al. We found that differences in the activity (the hy- (112) have pointed out that although G. candidum is ca- drolysis of p-nitroanilide derivatives of lysine, arginine, pable of using casein and peptone, in cheese its role is leucine, methionine, and alanine) correlated with the mainly peptidolytic rather than proteolytic. Bitterness complexity of microbial populations characteristic of in Camembert emerges at this step when the proteinases of these types of cheeses (unpublished data). Three strains P. camemberti degrade high-molecular-weight peptides to isolated from Saint-Nectaire and 1 from Bethlehem cheese low-molecular-weight peptides (Fig. 3B) (113). The αs1- clearly had the highest activity of the 13 isolates tested. and β-caseins have hydrophobic regions that, when re- The strain with the highest activity of the 13 tested in the leased by proteinases, increase bitterness (106, 114, 115). release of leucine, alanine, and methionine was the only

10 ASMscience.org/MicrobiolSpectrum Downloaded from www.asmscience.org by IP: 190.151.168.196 On: Sun, 01 Mar 2015 12:31:56 The Good, the Bad, and the Ugly: Tales of Mold-Ripened Cheese

American strain, isolated from a 45-day-old Bethlehem arthrospores per gram of cheese after 21 days of ripening cheese. The activity of strains isolated from cheese with at 10°C. The threshold for a yeasty taste is between 4 × complex fungal populations was in sharp contrast to that 103 and 104 G. candidum spores per gram of curd. of the 4 strains isolated from Reblochon from two dif- The neutralization of the cheese surface by G. ferent cheesemaking facilities. Reblochon has a less com- candidum and by other yeasts prepares the cheese sur- plex microbiota than Saint-Nectaire. The gray covering face for acid-sensitive bacteria, such as Brevibacterium seen on Saint-Nectaire and Bethlehem cheeses is due to the linens, which follow in the later stages of ripening (67). zygomycete Mucor, strains of which are known to be B. linens grows best at a neutral pH of 7.0 (135). In highly proteolytic. Indeed, the acid proteinases produced experimental Camembert-type cheeses inoculated with by Mucor pusillus and Mucor miehei which cleave the G. candidum, the pH of the rind reached 7.0 at the end Phe105–Met106 peptide bond of κ-casein have been used of ripening, whereas the pH of control cheeses remained commercially for microbial and genetically engineered at about 4.8 (112). rennet for years due to the shortage of calf rennet (130). The increase in pH is generally attributed to the Saint-Nectaire strains may thus be selected for by the ac- catabolism of lactic acid produced by starter bacteria cumulation of peptides released by Mucor proteinases to carbon dioxide (CO2) and H2O(72, 136). In our that are, in turn, metabolized by the aminopeptidase of diversity studies, the four wild-type G. candidum strains G. candidum. In contrast, a strain that was isolated from isolated from Reblochon were all positive for assimila- Saint-Nectaire curd before Mucor had developed showed tion of lactate as the sole source of carbon. The four little activity. producers used yogurt as the source of starter culture, Besides having a less complex fungal population, Re- perhaps selecting for strains capable of using lactate as a blochon is dominated by the bacterium Brevibacterium source of carbon. linens, which is highly proteolytic, possessing both Release of ammonia also plays a role in neutralizing proteinases and aminopeptidases, and has been used the cheese environment. Studies of the growth kinetics of as an adjunct in cheesemaking to accelerate ripening G. candidum on peptone-based medium with and (106, 131). The pathways of methionine catabolism without lactate showed that G. candidum preferred by B. linens have been investigated because the by- peptones as a carbon source, even when lactate was product, methanethiol, gives Reblochon and other present in the medium. Since lactate consumption was surface-ripened cheeses their characteristic flavor (132, significant only at the end of growth, it was concluded 133). Since B. linens has aminopeptidases, strains of that peptone was metabolized for cellular biosynthesis G. candidum with high aminopeptidase activity may not and lactate for cell maintenance (137, 138). An increase be selected for in Reblochon. in pH in the curd then results from both the assimilation Not all strains of G. candidum are equal. Some cause of lactate as a carbon and energy source and the release defects in cheese described as greasiness, bitterness, and of ammonia through the metabolism of amino acids. a strong yeasty flavor (134). On some cheese rinds, the Ammonia is a significant part of the aroma compounds proteolytic activity of G. candidum causes a wrinkly of Camembert (124). In the ripening of soft cheese, appearance that is referred to as peau de crapaud,or ammonia in the atmosphere can reduce Penicillium “toad skin,” by French cheesemakers. This texture is growth, thus lowering the level of proteolysis and bit- desirable on some cheeses, such as goat cheeses; but in terness (84). excess causes a slippery rind. When the cheese is turned The role played by G. candidum in controlling path- during ripening, the skin can separate from the cheese ogens and contaminants in cheese continues to be surface, thus ruining the integrity of the rind. To stop explored. The safety of raw milk mold-ripened soft excess growth, the cheesemaker may lower the temper- cheeses has been called into question due to the vulner- ature and/or humidity of the production area and cave. ability of these cheese types to contamination, particu- Due to G. candidum’s sensitivity to salt (inhibition can larly by Listeria monocytogenes, due to a high moisture occur at 1.0 to 2.0% salt), using a higher concentration content and high pH of the rind (139). It has long of salt or changing the salting method from brining to been assumed that pathogens will not survive in these dry salting may resolve the problem of excessive growth cheeses after a 60-day ripening period at a temperature (108). Attention must also be paid to the amount of of ≥1.67°C (>35°F). However, recent studies and epi- inoculum. Le Bars-Bailly et al. (108) note that one spore demiologic data have shown that neither a 60-day rip- of G. candidum per kilogram of curd introduced during ening period nor, surprisingly, pasteurization ensures cheese production can lead to a population of 105 the safety of this class of cheeses (140). Dieuleveux et al.

ASMscience.org/MicrobiolSpectrum 11 Downloaded from www.asmscience.org by IP: 190.151.168.196 On: Sun, 01 Mar 2015 12:31:56 Marcellino and Benson

(141) have shown that G. candidum can inhibit the growth of L. monocytogenes. The inhibitors were iden- tified as D-3-phenyllactic acid and D-3-indollactic acid; these compounds were stable over a wide pH range and could be heated at 120°C for 20 min. D-3-Phenyllactic acid altered the cell wall of an -resistant strain of L. monocytogenes, producing depressions and holes that were visualized with scanning electron microscopy (142, 241). G. candidum also inhibits the growth of the zygomycete Mucor, whose aerial hyphae (cat hair) are responsible for the cheese defect poil de chat (cat hair). The inhibition is strain and culture dependent (143). G. candidum may inhibit the sporulation of certain strains of the mycotoxin producer Aspergillus flavus (144) and undesirable species of Penicillium such as P. commune and P. caseifulvum, which can overtake fungal starters (145). More research is needed on the potential of G. candidum for microbial competition. The results of our own studies of G. candidum sug- gest that cheese technology and the microbial ecology of a cheese rind select for populations of G. candidum strains with different biochemical properties. The variables in cheese technology and ripening—salting, humidity, temperature, and materials in the cave such as wood or rye straw—selected for strains with unique characteristics. The fact that a strain from Bethlehem cheese, a Saint-Nectaire-type cheese made in the United States, would have biochemical properties similar to those of strains from Saint-Nectaire in Auvergne, France, tends to confirm the hypothesis that technology breeds the strains.

The Price of Innumerable Failures: The Ugly

One will never know the patience and groping it took to put into place the recipes for the fabrication of cheeses. It is the principal merit of the humble farmers and silent monks to have succeeded at the price of innumerable failures to refine and perfect the assortment of cheeses of which we are justifiably very proud. FIGURE 4 Challenges for the cheesemaker. (A) Scanning Les Fromages, Pierre Androuët (54) electron micrograph of a cheese . Left unchecked, cheese mites can lead to a 25% reduction in the weight of a ripened Ask anyone who has tried making cheese and he or she cheese. Photograph by William R. McManus, generously pro- will probably tell a tale of “innumerable failures” before vided and used with permission of William R. McManus and and sometimes after success is achieved. The natural Donald J. McMahon, Utah State University. (B) Spoilage of a succession of microorganisms (and sometimes fauna) on cheese by contamination and invasion of the rind by the fungal cheese during ripening has its benefits and risks. A genus Scopulariopsis. (C) Scanning electron micrograph of Mucor novice affineur may wonder if the pile of dust he saw on the zygomycete showing sporangia among collapsed hyphae. When the fragile outer membrane, the peridium a cheese or shelf an hour earlier really had moved to (arrowhead), of the sporangium breaks open, hundreds to fi another location. This observation would mark his rst thousands of spores can be released, contaminating a cheese encounter with cheese mites, the microscopic Arachnida cave overnight. Bar, 10 μm. doi:10.1128/microbiolspec.CM- Acarus farris and neiswanderi (146) 0005-2012.f4.

12 ASMscience.org/MicrobiolSpectrum Downloaded from www.asmscience.org by IP: 190.151.168.196 On: Sun, 01 Mar 2015 12:31:56 The Good, the Bad, and the Ugly: Tales of Mold-Ripened Cheese

(Fig. 4A). In the Auvergne, cheesemakers call mites Not only do cheese mites cause economic loss, but artisans, and in Haute-Savoie, they call them cirons. also their presence can cause enteritis, diarrhea, and Cheesemakers may notice that mites are abundant after a damage to the urinary tract (148). They are the source humid season, following heavy fungal growth on the of allergens implicated in asthma, rhinitis, and atopic rind. They have asked, “Are the mites eating the cheese or dermatitis. The storage mites Acarus siro, A. farris, the fungi?” Tyrophagus mites isolated from the Spanish and Tyrophagus putrescentiae can cause occupational mold-ripened cheese Cabrales are indeed fungivores allergies for farmers and cheesemakers (152), and the (147). Tyrophagus putrescentiae commonly infest affineur who spends long hours without a mask in a foods with high fat and protein contents (148), which mite-infested cave is vulnerable to a condition known as would certainly include cheese. The compounds cis- and “cheesemaker’s lung” caused by inhaling mites and their trans-octa-1,5-dien-3-ols produced by Trichothecium dust (153). Mites can be the vectors of microorganisms roseum have been found to be strong attractants for the (154), and some have even suggested that hay mites may mite (149). There is a relationship between mites and be a self-sustaining reservoir for the prion which causes fungi from which both may benefit. Fungal spores can be bovine spongiform encephalopathy, or mad-cow disease transported to new substrates, and mites can promote a (155). compensatory growth of fungal mycelium as new Control of mites continues to be a challenge for surfaces are created for the aerobic fungi (150). The cheesemakers. Generally, conditions in a cave which are mites’ choice of species of fungi upon which to feed best for cheese ripening, with temperatures ranging from depends upon their capacity to digest the fungus. Cell 10 to 15°C and relative humidity near 90%, are also walls of fungal mycelium are composed of chitin and favorable for mite populations (156). In the past, methyl chitosan, but in experiments on the digestibility of fungi bromide fumigation and organophosphate insecticides by various mite species, chitin passed through the di- were used to control mites, but these have been banned in gestive tracts of mites undigested. Trehalose in the my- consideration of food safety (156, 157). Many affineurs celium, however, was digested by the mites, indicating still find that mechanically removing mites from cheese that the mites possess the digestive enzyme trehalase by brushing or vacuuming is the best and safest method. (150). Of the mite species tested, trehalase activity was Other measures have been tried, such as washing cheeses highest in the Tyrophagus. with hydrogen peroxide after vacuuming and introduc- The presence of mites affects the appearance of the ing ozone into the cave for a few hours a day (158). The rind and certainly the taste of the cheese. According to Bleu Mont Dairy Company in Wisconsin has used dia- Sánchez-Ramos et al., “Mites feed and reproduce on tomaceous earth (DE) as a successful material for mite cheese surfaces producing an accumulation of dead control. DE does not leave harmful residues, nor is it mites, faeces, exuvia, eggs and bits of food, which ap- toxic to mammals (159). DE injures mites through a pear as a light brown dust that can reach a depth of 2 cm physical mode of action, rather than chemical. The mite’s or more in heavy infestations” (146). This may not protective cuticle is damaged through abrasion and ad- appeal to everyone’s tastes, yet Tyrophagus casei mites sorption of the cuticular waxes to the DE, which leads to are deliberately introduced into the German cheese dehydration of the insect’s body (160). Altenburger (151). The very hard cheese from Other approaches to control include the use of Lille, France, which can be aged for 2 years, develops its modified-atmosphere (MA) technology that alters the distinctive flavor with the help of mites. Yet on the whole natural ratio of the atmospheric gases, oxygen (O2), mites are a nuisance, especially for Cheddar makers, and nitrogen (N2), and CO2 (157). Mobile stages of larvae, their removal is time-consuming, expensive, and often nymphs, and adults are controlled quickly, whereas frustrating. preventing egg hatching is more difficult. One study At 25°C and a relative humidity of 80 to 90%, one reported 100% mortality in the egg stage using 99.5% generation of fungivore mites takes 10 days. Adult mites CO2 at 85% relative humidity after 6 days of exposure, can live for about 2 to 5 months, and Acarus farris, the while the mobile stages of the mites were killed in 1 day predominant species in Cabrales cheese, can reach a under the same conditions. Preventing egg hatching population density of 260 mobile mites/cm2. It should should therefore be the goal of any technology. Mono- come as no surprise, then, that these artisans carving and terpenes, including eucalyptol, showed acaricidal activ- eating their way through the cheese surface, if left un- ity against mobile stages of Tyrophagus putrescentiae checked in a cave, can be responsible for a loss of up to through vapor action but adversely affected the flavor 25% by weight of the final product (146). and odor of the treated cheese, so this approach was

ASMscience.org/MicrobiolSpectrum 13 Downloaded from www.asmscience.org by IP: 190.151.168.196 On: Sun, 01 Mar 2015 12:31:56 Marcellino and Benson abandoned (161). Other approaches include altered Scopulariopsis species are commonly found associat- ripening temperature and humidity levels; a lower rip- ed with soil, plants, and animal dwellings (166, 167). ening temperature used with Cabrales cheese reduced They are considered anamorphs of the fungal class mite density (156). Microascaceae (240) and microscopically can be con- Problems with fauna aside, some fungi are considered fused with Penicillium species. The conidia of contaminants that lead to defects in appearance and Scopulariopsis are oval and form in chains, with a flavor (162) and, on occasion, the loss of entire batches of truncated base attached by a ring to a vial-shaped co- cheese. The American mycologist Charles Thom de- nidiophore (165). The conidiospores often have rough scribed the filamentous fungus Scopulariopsis brevicaulis surfaces and are hyaline. Though not food-borne path- growing on Camembert in 1930 while working for the ogens, five species have been associated with human U.S. Department of Agriculture at the Agricultural Ex- diseases, and they can be serious emerging pathogens periment Station in Storrs, CT. He described how for immunocompromised individuals (167, 168, 169). S. brevicaulis “attacks Camembert cheese, overgrowing The fungus possesses an array of enzymes that increase the Camembert mold, producing an ammoniacal odor its capacity to grow and flourish within the environment and imparting an ammoniacal taste to the cheese” (163). of a ripened cheese rind. It is highly proteolytic: its In 1951 Keilling et al. described what they called a keratinases, which break down keratins, the principal “cheese canker” in Emmental, Gruyère, Edam, and proteins in hooves, claws, and feathers, are being studied Gouda, which “manifests itself during ripening and for use in the field of bioremediation (170). Its mode of storage through the appearance of little holes dug into invasion of a substrate is specialized boring hyphae that the rind of the cheese, containing a white, yellow, or penetrate fibers perpendicularly to the surface (171). brown floury substance.” They attributed the canker to Considering that the fungus can penetrate the spines of yeast and a fungus, Penicillium brevicaulis, a previous hedgehogs and cattle hair, it should not be surprising that name for Scopulariopsis brevicaulis (164). Unlike most Scopulariopsis species bore their way through cheese fungi, which prefer an acidic medium for growth, rinds. Since it prefers an alkaline environment, from an Scopulariopsis grows best in alkaline and high-nitrogen ecological standpoint it follows that Scopulariopsis would substrates, with an optimum growth pH of 9.0 (163). grow late in the ripening process. On a ripened cheese Scopulariopsis reportedly is more apt to grow on pas- surface, yeasts and fungi raise the pH through lactate teurized rather than raw milk Camembert and Carré de metabolism and ammonia production via proteolysis (44). l’Est, because the lactic acid bacterium population in the Bothast et al. noted that S. brevicaulis breaks down more pasteurized version is less complex, leading to a weaker protein than it uses for synthesis when insufficient car- fermentation and lower acidity (165). Scopulariopsis bohydrate is present in the substrate (163), which is spp. can contaminate many classes of cheese, with probably the case in ripened cheeses, which contain only Scopulariopsis fusca predominating, followed by S. trace amounts of residual carbohydrate (172). The fungus brevicaulis (108, 134). Samples of the aged pâte molle then liberates nitrogen as ammonia from the excess and cheese Pont-l’Évêque collected in four different de- utilizes carbon for energy production (163). Atmospheric partments of showed contamination by the conditions in a cheese cave, including concentrations of fungus, as did aged artisanal goat cheeses and pressed CO2, oxygen, and ammonia, determine microbial growth hard cheeses such as and Gruyère (108, 134). and enzyme activity (173). In their study of atmospheric S. fusca appears as powdery brown or mauve spots on a conditions during ripening of Camembert, Leclercq-Perlat cheese rind and is a common contaminant along with et al. found that at high levels of CO2 and low levels of Penicillium species (108, 134). Artisanal cheesemakers oxygen, the yeast-like fungus Geotrichum candidum grew recognize only too well the “cankers” in the rind and the well, whereas Penicillium roqueforti was negatively af- strong smell of ammonia produced by Scopulariopsis in fected (174). It would seem that an efficient ventilation their caves. The holes in the rind created by system in a cave which would allow for adjustments in Scopulariopsis allow it and other fungi to penetrate into atmospheric conditions, particularly removal of ammo- the curd. To add insult to injury, Keilling et al. warned nia, would play a role in controlling Scopulariopsis.A that “mites invade the cavities which they extend con- low-tech natural solution used by some cheesemakers to siderably deeper” (164). Many cheesemakers have ob- slow down Scopulariopsis growth is to wipe the cheese served that Scopulariopsis, besides destroying the surface and shelves with vinegar. integrity of the rind as shown in Fig. 4B, creates defects in Among its other enzymes, S. brevicaulis has an ex- flavor such as bitterness and a musty taste. tracellular chitin deacetylase, which enables it to use

14 ASMscience.org/MicrobiolSpectrum Downloaded from www.asmscience.org by IP: 190.151.168.196 On: Sun, 01 Mar 2015 12:31:56 The Good, the Bad, and the Ugly: Tales of Mold-Ripened Cheese chitin as a sole source of carbon (175). This enzyme may (loosely translated as cheese disasters) is the zygomycete enable S. brevicaulis to feed on fungi of preceding Mucor. As mentioned earlier, Mucor is part of the nor- populations on the cheese rind, using chitin contained in mal microbiota of Saint-Nectaire fermier (farmstead) their cell walls. Other enzymes of Scopulariopsis, such as cheese and Tomme de Savoie (58, 63). However, when xylanases, are of interest to the paper and pulp industry Mucor appears on a pâte molle cheese, it can instill fear for their bleaching capacity and the breakdown of lignin in the heart of a cheesemaker or affineur. A cheese such (176). Scopulariopsis is often carried into the ripening as Camembert, meant to be covered with a uniform environment on wrapping paper, so such paper should white coat of Penicillium camemberti, can become cov- be stored in a separate area (134, 177). ered with tufts of grayish-white hairs, a defect known as Scopulariopsis species are halotolerant, halophilic, poil de chat. Spore germination is the critical step in the and xerophilic; they grow well in salty foods such as growth of food spoilage fungi because “a product is cured fish and smoked bacon (178), in environments of spoiled as soon as visible hyphae can be observed” high salt concentration (179), and in salt-based media (183). At the tip of each sporangiophore in Mucor is a (180). During cheese ripening, water evaporates from the sporangium that appears as a black or gray ball to the surface, thus lowering the water activity of the rind. Fox unaided eye (143). The electron micrograph of the sur- et al. report that after 10 days of growth in nutrient broth face of a 9-day-old Bethlehem cheese in Fig. 4C shows of pH 6.6 at 25°C, Scopulariopsis fusca grew at 14% of two sporangia among collapsed Mucor hyphae. One can its maximum development at a salt concentration of see the fragile outer membrane, called a peridium, of the 20% (water activity, 0.880). At the same time, Penicil- larger sporangium breaking apart, revealing the spores. lium candidum grew at 1.1% and Mucor mucedo and One sporangium can liberate thousands of spores (108) Geotrichum candidum were completely inhibited (172). that will spread over the cheese surface and eventually Keilling et al. suggested that on Comté and Gruyère-type impart a grayish coat to the cheese. This may explain cheeses, meticulous wiping and care of the rind are crit- why Mucor has earned the name la bête noire (the black ical during the first 15 days of ripening, when the cheese beast) by some traditional cheesemakers. It grows 5 to rind is still acidic and the desired morge, or smear, is still 10 times faster than Penicillium species (108), giving it a developing on the surface. When the morge does not competitive advantage over Penicillium camemberti. develop quickly enough, patches of S. brevicaulis can When Mucor racemosus was grown in potato dex- appear (164). The members of the microbiota compris- trose agar medium at 25°C, its hyphal extension rate, ing the morge, including Brevibacterium linens, are salt based on the measurement of colony radial growth, was tolerant and from the 15th to 30th day of ripening reach 13.3 mm per day (183). a maximal growth of 109 to 1010 microorganisms per Mucor is ubiquitous in the environment and, because cm3 on the rind (181). Adjusting salt concentrations and/ of its proteolytic and saccharolytic activities, is respon- or care of the rind may inhibit undesirable fungi such as sible for decomposing fruit, meat, and bakery products Scopulariopsis and select for desirable species to compete (184, 185). Though not a food-borne pathogen, for nutrients on the cheese surface. Mucor can cause mucormycosis, an infection of the Considering what a widespread problem Scopulariopsis sinuses, brain, or lungs to which immunocompromised is for the cheese industry and the lack of dairy-related populations are most vulnerable (186). Zygomycetes research data currently available, cheesemakers would have rarely been reported to produce mycotoxins (183). benefit from investigations into modes of its inhibition. Mucor racemosus is the most widespread cheese con- In a recent investigation into antifungal activity by bacte- taminant, but other Mucor species associated with cheese ria, eight strains of lactobacilli isolated from vegetables and are M. plumbeus, M. hiemalis, M. globosus, M. fuscus, one strain of Propionibacterium jensenii of dairy origin and M. mucedo (108). A characteristic that distinguishes strongly inhibited the growth of Scopulariopsis brevicaulis Mucor from other genera of the zygomycetes is its ca- (182). It is of interest to note that while many classes of pacity for dimorphism (187), allowing it to adjust its fungi were inhibited, Geotrichum candidum was not, morphology to changing environmental conditions. In which is good news for cheesemakers. Testing of fermen- an anaerobic environment and in the presence of a fer- tative bacteria that exhibit antimicrobial activity while not mentable sugar, this filamentous fungus can assume a affecting sensorial quality within a cheese environment yeast-like morphology (185). In 1876, Louis Pasteur could be of interest. described the dimorphic capability of Mucor racemosus, The fungus that is probably the most common cause which was implicated in the fungal contamination of of what French cheesemakers call accidents de fromages beer (184). Le Bars-Bailly et al. (108) noted that in a

ASMscience.org/MicrobiolSpectrum 15 Downloaded from www.asmscience.org by IP: 190.151.168.196 On: Sun, 01 Mar 2015 12:31:56 Marcellino and Benson cheesemaking facility, the filamentous morphology of tion of Mucor can be partially inhibited by good growth Mucor racemosus predominates on the aerated surface of of G. candidum on the rind of a pâte molle cheese during a cheese, whereas under a cheese, where less oxygen is the early stages of ripening. This competitive inhibition is available, cells may assume a yeast-like morphology. strain dependent for G. candidum (144, 191). Le Bars- Zygomycetes grow poorly under conditions of low Bailly et al. observed that since Mucor needs access to water activity (183). With Mucor’s preference for high nutritive medium to grow, a previously implanted thick, humidity, the dairy environment presents many oppor- uniform layer of G. candidum creates a physical barrier tunities for contamination to which cheesemakers must between Mucor and the cheese substrate (108). As be alert. Its optimum growth conditions include a tem- mentioned earlier, tolerance to salt plays a large a role in perature of 20 to 25°C, a pH of 5.0 to 6.0, relative hu- growth and survival in or on cheese. Cheeses of the pâte midity of 90 to 95%, and water activity of >0.95 (188). molle type, such as Camembert and Brie, generally are The spores of Mucor are described as “myxospores” due salted within the range of 1.5 to 2.0% (wt/wt). The to the tendency of their cell walls to absorb moisture, cheesemaker may have to adjust the degree and method facilitating their adherence to various surfaces. The of salting if a good implantation of G. candidum does not spores stick to clothes, cheese molds, utensils, and the occur. Lactobacillus coryniformis subsp. coryniformis cheesemaker’s hands, thrive in liquids such as whey and Si3 has displayed a broad spectrum of antifungal activity stagnant water, and are dispersed in moist air (188, 189). against Mucor hiemalis as well as Aspergillus and Peni- Once contamination has occurred, it is difficult to get cillium species (192), and Brevibacterium linens has under control, so prevention is the first goal. shown antifungal activity via thiol production (143). During cheese production, newly formed cheeses are Using modified atmospheres for preventing fungal extremely vulnerable to contamination during the growth and mycotoxin production to extend the shelf life draining of whey. The high humidity in the draining of some kinds of food was evaluated (183). Taniwaki et room and the high moisture content of the undrained al. found that the growth of Mucor plumbeus and other curd favor Mucor development. The temperature of the fungi could be reduced in atmospheres containing 20 to room ideal for drainage (24 to 27°C) is also ideal for 40% CO2 with 1 to 5% O2 (193). Tormo and Barral germination of Mucor spores (189). Whey contains report that Mucor is sensitive to ammonium and UV light abundant lactose (about 70% of dry solids) (172), and (188). through its β-galactosidase activity, Mucor is able to Penicillium roqueforti and Penicillium camemberti, break down lactose into glucose and galactose (190). growing naturally or added as secondary starters, con- The presence of lactose in the whey allows for growth of tribute to the flavor and unique characteristics of blue- the yeast-like form of Mucor in the anaerobic environ- veined and pâte molle cheeses, respectively (52, 91). ment of the interior of the curd (189). Acidification of However, when they or other species of Penicillium grow the curd in production enhances the expulsion of whey on the wrong type of cheese, they are considered and reduces drainage time. If cheese is made with milk contaminants that can cause great economic loss (108). with a high fat content, good drainage may not occur P. roqueforti can contaminate Camembert and other (188, 189). The use of fresh milk for cheesemaking pâte molle cheeses (194) as well as hard cheeses such as minimizes the time that psychotropic bacteria can break Emmental and Parmesan (195), and P. caseifulvum can down the casein in milk, providing polypeptides as a cause yellow spots in blue-veined cheeses (196). Other substrate for Mucor (189). It is also recommended that Penicillium species—P. commune, P. solitum, P. palitans, draining cheeses be covered to avoid airborne spores. and P. crustosum—are especially problematic because Tormo and Barral suggest that if Mucor growth is visible they are closely related genetically to P. camemberti. on cheese 1 to 2 days after the curd is placed in the P. camemberti, the widely used starter in Camembert forms, one should suspect that the milk used in pro- and Brie, is considered a domesticated species derived duction was the original source of contamination. When from P. commune (195). The taxon P. commune was first Mucor appears 3 to 4 days after draining and the entire introduced by Thom in 1910 because it was the major cheese is covered with hyphae, then the curd, cheese source of cheese contamination in the United States (197). forms, and utensils should be suspected; if growth According to Lund et al. (198), it remains the most appears on only one side of the cheese, the atmosphere in widespread spoilage fungus, causing discoloration and the room is the source (188). flavor defects in cheese. The authors have observed that Mucor can be controlled by fungi and bacteria in- one conidium of P. commune on a cheese can develop into volved in cheese production and ripening. The sporula- a colony of 10,000 CFU in 4 days. Therefore, great care

16 ASMscience.org/MicrobiolSpectrum Downloaded from www.asmscience.org by IP: 190.151.168.196 On: Sun, 01 Mar 2015 12:31:56 The Good, the Bad, and the Ugly: Tales of Mold-Ripened Cheese must be taken to prevent the dispersal of contaminating is said to have introduced it to the court of King Louis conidia in the air and on hands, clothes, and packaging. XIV (203). The Bellonte family has made Saint-Nectaire Biocontrol of penicillia has also been noted. G. candidum for eight generations in the town of Farges, 3 km from can inhibit the growth of P. roqueforti in blue-cheese Saint-Nectaire, a village located on the top of Mont medium without NaCl, and some strains of P. camemberti Cornadore and renowned for its Romanesque church inhibit the growth of P. roqueforti, P. caseifulvum,and and ancient Roman thermal baths. Farges was built in P. commune (196). the volcanic crater of the Massif du Sancy, the oldest mountain in the Auvergne, and is part of the Regional Park of the Auvergne Volcanoes. The decline in population and number of farms in TALES OF MOLD-RIPENED CHEESE Farges since 1870 reflects Alphonse’s sentiment: “Mod- Difficulties arise when one attempts to describe linkages ern agriculture has made French countryside a desert.” In between natural and human history of cheesemaking in 1870, there were 90 inhabitants and 22 farms; in 1945, the context of its science. Perspectives range from the 46 inhabitants and 10 farms; in 1995, 23 inhabitants and small farmer interested in terroir—that elusive property one 474-ha farm, that of the Bellonte family. During the associated with the specificity of space, including cli- summer, cows graze in mountain pastures. The herd is mate, soil, and people—and the industrialist who wishes composed of Montbéliardes, a breed originating in the to control all aspects of a ripening process to control Jura region of France. In the past, milk of native breeds of product consistency. An example of these differing the Auvergne, such as Ferrandaise and Salers, was re- perspectives plays out in the Auvergne region of France. quired for making Saint-Nectaire with the AOC desig- The Auvergne is a volcanic region in the Massif nation (204). The Salers, a red cow whose ancestors were Central dominated by the Chaîne des Puys, a series of depicted in cave art near the town of Salers in Cantal in volcanic cones, domes, and craters west of Clermont- the Haute-Auvergne, is considered one of the oldest of all Ferrand (199). On the route from Montaigut-le-Blanc to European breeds (http://www.ansi.okstate.edu/breeds/ Champeix in Puy-de-Dôme, the arched openings to the cattle/salers/index.htm). This dual-purpose cow is well caves of Saint-Julien where wine was aged before the adapted to the rugged terrain and climate of the high powdery mildew (Oidium tuckeri) and phylloxera mountains. These cows are often pictured on labels for epidemics of grapevines, can be seen from a distance. Saint-Nectaire cheese (203), yet many farmers no longer Some of those caves belong to the Bellonte family. milk them because a calf must be tethered to the mother The elusive link of cheese to its environment is im- during milking (205). This requirement makes for a mediately apparent as one enters the Bellonte caves and fairly chaotic pastoral scene not necessarily practical for sees hundreds of Saint-Nectaire cheeses laid out on rye dairy operations today. straw on the floor. The owner, Alphonse Bellonte, speaks The Bellonte Montbéliarde cows are fed only hay passionately about the patrimony of the Auvergne. He is and grain, never silage. Silage is not allowed in the pro- fiercely attached to his land and cheese. The pride and duction of most AOC cheeses since it may contaminate spirit of resistance expressed in his words are common to milk with spores from Clostridium species, particularly the people of the Auvergne. The region is named for the C. tyrobutyricum and C. butyricum. These strains pro- Celtic tribe Arverni, best known for their chieftain duce carbon dioxide and hydrogen gases during the bu- Vercingétorix, who led his troops to victory over Julius tyric acid fermentation that cause openings, splitting, and Caesar’s forces at Gergovia in 52 BCE (200). Although “blowing” defects in cheese, particularly in large cheeses he was later defeated and taken in chains to Rome, he such as the Swiss types (206, 207). Silage can also be a remains a symbol of heroism to the region (201). source of mycotoxin-producing fungi and the food- Saint-Nectaire is an uncooked semihard surface borne pathogen Listeria monocytogenes when silage has mold-ripened cheese that has been made since at least undergone deterioration aerobically (208, 209, 210). the 17th century in two departments, southwestern Puy- The Bellonte farm makes Saint-Nectaire fermier cheese de-Dôme and northern Cantal. It is a PDO cheese, and using raw milk produced only on their farm. The produc- the 69 communes of origin are characterized by the tion of Saint-Nectaire laitier (industrial Saint-Nectaire) geographical and geological diversity found in the Sancy using pasteurized milk collected from many producers be- Mountains, the plateaus of Cézallier and Artense, and gan in the region in 1964, and at the time it was feared that the valleys known as Pays coupés (42, 202). The cheese farmsteadproductionwouldbethreatened(211). How- was named after Henri de Senecterre (1573–1662), who ever, Saint-Nectaire fermier is alive and well: of the 13,676

ASMscience.org/MicrobiolSpectrum 17 Downloaded from www.asmscience.org by IP: 190.151.168.196 On: Sun, 01 Mar 2015 12:31:56 Marcellino and Benson metric tons of Saint-Nectaire produced in 2005, 6,194 The cheesemakers of the Auvergne have observed for metric tons were fermier and 7,482 metric tons were laitier centuries that rye straw placed under ripening Saint- (http://www.fromages-de-terroirs.com/fromage-detail. Nectaire seems to produce the best cheeses. From a mi- php3?id_article=1634&lang=en). The Bellontes make 45 crobiological standpoint, the use of rye straw is a factor metric tons of Saint-Nectaire fermier annually. in fungal succession on the cheese rind: namely, it The bulk of Saint-Nectaire produced by the Bellontes encourages the growth of Trichothecium roseum, the is aged in the caves of Saint-Julien that were dug from “flower of the molds.” T. roseum is a saprophyte as well tuff (porous volcanic rock) in 1813; some also is aged in as a plant pathogen (215). The fungus is associated with caves in Farges dating to medieval times (742–814 CE). wheat, especially during seasons of high precipitation The medieval caves were dug from stone, called pierre de (216), and is among the fungi that can use wheat gluten Farges, that is very porous and retains moisture, thus as a substrate in the fermentation of minchin, a Chinese providing an excellent natural environment for the cereal-based food (2). T. roseum can overwinter in soil growth of fungi. Until the 1890s, the caves of Saint-Julien or crop debris, while under conditions of high humidity, were used to store wine, but by then the effects of phyl- such as one would find in a cheese cave, it grows well loxera (Daktulosphaira vitifoliae), the grapevine louse and sporulates (217). Proteases of T. roseum may war- that destroyed the vines of France, had reached the rant investigation to clarify the role of T. roseum in the Auvergne. As Gale describes it, “…the entire wine- microbial ecology of cheese ripening. growing world was not only affected but transformed— Some French cheeses have emerged bearing a mix of agriculturally, economically and socially—by the plague. natural and political history that includes universal Effects of the disaster rippled out from the vineyards into truths about the struggles and ingenuity of small farms their embedding cultures, invoking such large-scale and partly explains the passion with which traditions are consequences as rural depopulation and massive held. Reblochon, for example, is a cheese that originated emigration” (212). Until that time, the Auvergne had in the 14th century in the Haute-Savoie region in the been the third-largest winemaking region of France, but Chaîne des Aravis, a mountain range of the pre-Alps it would never again regain that stature. (218). It received its AOC appellation in 1958 and is Many of the caves in the countryside of Saint-Julien among the first French cheeses to do so. The story behind are abandoned, but others are used to store root vege- its inception is often recounted with pride by the French. tables or ripen cheese. In compliance with regulations of Reblochon is derived from blocher, which in Savoyard the European Union, the cheeses are now ripened on dialect literally means to “pinch a cow’s teat.” According plastic mats on wooden shelves and not directly on rye to the peasants hand-milking a cow, the sound of a straw on the cave floor (213). In many Saint-Nectaire stream of milk hitting a pail is similar to ploutch, which caves, the cheesemaker slips stalks of rye straw between developed into the verb blocher (http://projetbabel.org/ the mats and shelves or into the plastic crates of aging forum/viewtopic.php?t=9246). Re-blocher means to cheeses. This modification may be seen as resisting “milk again,” and a cow which holds back her milk and the regulations, or as Vollet et al. suggest, the cheese needs to be milked a second time is called une vache is presented on a bed of rye straw as a marketing tool reblochnieuse (219). In the 13th century, farmers were for tourists, to accent terroir (203). However, rye straw, taxed according to the quantity of milk produced by their or paille de seigle, cannot be viewed apart from the herds, so they began the practice of not milking the cows Celtic roots of the Auvergne and the history of the cheese. out completely on the day of the tax collector’s visit. The name Cézallier has a Celtic origin; the French After the tax collector departed, the cows were milked term for the region is pays du seigle,or“country of rye” again. To protect the ruse, the small quantity of rich milk (214). Rye grain was used to make bread in communal obtained from the second milking was quickly ovens in mountain hamlets and rye straw was placed transformed into cheese. There is a play on words in under the ripening cheeses. As Greliche has noted, “Rye Savoyard between reblocher (to milk again) and rablasse straw was so important that it gave its name to the or rablache (a fraudulent action), and thus Reblochon cheese that was produced locally called le gleo which is came to be known as the cheese of fraud or deception considered as the ancestor of St. Nectaire. The Celtic (220). Fresh raw whole milk is still required for pro- words gleo, glahou, and glui signify the rye straw on ducing AOC Reblochon today, and the cheese is made which the cheese was ripened” (213). During the Middle immediately after milking, twice a day. Reblochon Ages, peasants paid their lords “gléo cheese” as a form fermier (with a green identification label) is made on of currency. a farm with milk produced on that farm only,

18 ASMscience.org/MicrobiolSpectrum Downloaded from www.asmscience.org by IP: 190.151.168.196 On: Sun, 01 Mar 2015 12:31:56 The Good, the Bad, and the Ugly: Tales of Mold-Ripened Cheese whereas Reblochon laitier (with a red label) is made in a century BCE (222). The Celtic root jor, later Latinized to dairy from milk collected from many farms. In both juria, means “forest,” which aptly describes these “forest cases, the cheese is made from milk of three breeds: mountains” dominated by forests of Norwegian spruce Abondance and Tarentaise, both native to Haute-Savoie, (223). It was here near the source of the Doubs River that and Montbéliarde. Reblochon is considered a semihard the cheese which perhaps best expresses the landscape cheese, but Mariani (75) notes that it could be classi- from which it arose, Mont d’Or, was created. There are fied technologically as a transition between pâte molle two versions: the French-called Mont d’Or or (moist) cheese, to which it is similar at the beginning of du Haut-Doubs, which is made from raw milk, and the ripening, and a pressed cheese at the end of ripening. Swiss version, Vacherin Mont d’Or, made from It is washed twice during the 4- to 6-week ripening thermized milk. Mont d’Or is a seasonal cheese which period, encouraging the growth of the coryneform according to French law can be made only from 15 Au- Brevibacterium linens and Geotrichum candidum, which gust to 31 March (224). Cheesemonger and writer is the only filamentous fungus on Reblochon (221). Patricia Michelson describes well the seasonal quality of On Reblochon, Mucor is considered a contaminant. the cheese: “The first true taste of autumn for me comes Bärtschi et al. (221) suggested that contamination of when the cheese table in my shop displays Vacherin Reblochon with Mucor may be less common than in Mont d’Or. Not for me the bland taste of early Septem- pâte molle types because the curd is pressed, thus ber cheeses: I prefer to wait until the end of October—or shortening the draining period, when the cheeses are the weekend when we turn back the clocks in Britain— most vulnerable to contamination. Washing the rind until the most seductive and sensual of cheeses is avail- during ripening may also reduce the risk. However, able” (225). The timing has its roots in the ancient Reblochon is not immune to contamination, and some rhythms of mountainous regions. In late spring, the fête stories illustrate the vigor with which Mucor can foment de la transhumance or l’estive, the day the herds and disaster. One cheesemaker in Haute-Savoie recounted a flocks pass through the villages on their way to the higher time in the 1950s when on a Wednesday she returned elevations for the summer, is celebrated with music and from her cheese cellar and exclaimed to her husband, dance (226). Traditionally, the herds came down the “How beautiful the Reblochons are!” The ripened mountains just after 15 August. A cow decorated with a Reblochons were ready to go to market on Saturday. But wreath of flowers on her head leading a herd down the by Friday morning, the cheeses were covered with poil mountain may be the cow that has given the most milk de chat (Mucor), and in the evening, they were com- that summer. Once the cows returned to their winter pletely covered with black sporangia. The dairy con- quarters and were no longer grazing on lush pastures, sultant from La Roche-sur-Foron did not find Mucor in they gave less milk, and thus, Mont d’Or, a small cheese their milk and concluded that such a drastic contami- in comparison to other celebrated cheeses of Franche- nation could only be due to a mauvais sort—someone Comté such as Gruyère de Comté and Morbier, was had put a spell on their cheese. He suggested that they created. It is often called the Christmas cheese because it contact the priest to perform an exorcism. The outcome is available during the holiday season, at which time it is of the exorcism is unknown. The competition between in great demand throughout France. Mucor and Geotrichum can be unpredictable and may Mont d’Or is a creamy croûte fleurie (flowered be one-sided in new caves that have not had time to crust) cheese that must be made at an altitude of 700 to establish appropriate microbial populations. For that 1,400 m. At Métabief, the cheese is produced with the reason, newly established Reblochon caves are some- milk of local herds of Montbéliardes, the popular breed times watered down and seeded with Geotrichum that has been exported from her native Franche-Comté candidum (75). to many regions in France. The cheese is made in copper In the area of the Haut-Doubs in the region of vats, and Patrick Sancey-Richard explained that since Franche-Comté, the Sancey-Richard family has been milk contains very little copper, the vats encourage the making cheese since 1961. At 1,463 m of altitude, the growth of microorganisms that require copper for their Fromagèrie du Mont d’Or of Métabief is located 10 km growth. from the Swiss border at the base of Mont d’Or, a summit This master cheesemaker’s observation about the use in the rugged Jura Mountain range. The Jura Mountains of traditional materials provides an example of the em- traverse the Franco-Swiss border north of the Alps be- pirical knowledge of microbial ecology gleaned through tween the Rhône and Rhine rivers and were named by the centuries of hands-on experience, the complexity of Celtic tribe the Sequani, who inhabited them in the 4th which scientific research is only beginning to elucidate.

ASMscience.org/MicrobiolSpectrum 19 Downloaded from www.asmscience.org by IP: 190.151.168.196 On: Sun, 01 Mar 2015 12:31:56 Marcellino and Benson

European cheesemakers have long considered copper into strips, or sangles, by an artisan called a sanglier. The vats essential to Emmental production. Sieber et al. sangles are rolled into packets of 10 that are dried, speculate that in the evolution of humans’ use of metal, soaked in salt water for 24 to 48 h, boiled for 2 h, and the extraction of copper from ores brought an end to the stored in salt water until use. After the cheese is removed Stone Age 9,000 years ago. Copper is a prized metal from the mold, a sangle is placed around the fragile because it is malleable and, second to silver, the best newly formed cheese to hold it together and secured with conductor of electricity and heat (227). Copper is re- a pine toothpick. During ripening, Penicillium candidum quired as a micronutrient to serve as a cofactor for grows on the bark, presumably because Penicillium is metalloproteins and enzymes involved in microbial me- salt tolerant. The use of salt in preparing the bark may tabolism (228, 229). Cow milk contains very little copper have selected for Penicillium in years gone by, although (0.1 to 0.2 ppm) (228). However, the use of copper vats most producers now add a commercial strain. Early in for cheesemaking brings the copper content of the fin- ripening, G. candidum grows on the cheese surface. G. ished cheese to 7.6 and 16.5 ppm due to the leaching of candidum and the coryneform bacteria B. linens and copper ions by the casein in milk (230). As copper vats Arthrobacter create a whitish-orange crust characteristic have been replaced by stainless steel in the production of of Mont d’Or (66). At 21 days of ripening, the cheese, Emmental, the addition of CuSO4 salt to the cheese milk still encircled by the strip of bark, is placed in the spruce has become standard practice. However, in organic box. The cheese is served in the box, and its creamy pale cheesemaking operations, the supplementation with yellow interior is often eaten with a spoon. The aroma is copper sulfate is not allowed (231). Likewise, the con- dominated by the smoky flavor of terpenes, compounds centration of added copper must be finely tuned, as some contributed by the wood. Among the antimicrobial authors have noted that supplementary copper can in- terpenes isolated from Mont d’Or are p-cymene, limo- hibit the growth of starter and adjunct cultures such as nene, γ-terpinene, borneol, and terpineol (75). Streptococcus, Lactobacillus, and Propionibacterium Several types of cheeses owe their origins, continu- species critical to Emmental production (230). Inves- ance, or development to various abbeys in Europe. tigations into the inhibition of food-borne micro- These include common varieties such as Munster (from organisms by copper are promising. Mato Rodriguez the Latin Monasterium), Cheddar, Saint-Nectaire, and and Alatossava (231) report that the use of copper vats in Maroilles. St. Benedict, the founder of Western monas- cheese production inhibits the germination, vegetative ticism (ca. 500 CE) describes the enclosure of the mon- growth, and sporulation of strains of Clostridium astery as a “workshop” in his Rule for monks (233). His tyrobutyricum, the bacterium known to cause the late- emphasis on manual labor as well as the stability and blowing defect in Swiss-type cheeses. Noyce et al. (232) rhythm of monastic life created an environment for de- found that food surfaces composed of a copper alloy veloping knowledge based on experimentation. An ex- inhibited the growth of the food-borne pathogen ample is the creation of , which is Escherichia coli O157:H7, whereas stainless steel and attributed to the Benedictine Abbey of Maroilles in aluminum surfaces did not. When they inoculated seven northern France in 960. A monument in the town bears different types of cast copper alloys with 103 CFU of the inscription “Stile created to commemorate the crea- E. coli O157:H7, all of the cells were killed in 20 min or tion of Maroilles by the monks of the celebrated Abbey: less at 22°C. Likewise, the food-borne pathogens Sal- 960–1960.” The cheese was first called craquegnon but monella enterica and Campylobacter jejuni were in- was known in the Middle Ages as “the marvel of hibited by contact with copper surfaces, while surfaces Maroilles” (234). comprised of stainless steel and a synthetic polymer The Order of Cistercians of the Strict Observance was showed no antibacterial activity (229). This research founded in the 11th century and as the name implies was underscores the complexity of microbial inhibition and a reform movement within the Benedictine tradition. may cause us to reconsider “advances” in food safety A subsequent even stricter reform movement of the such as the use of stainless steel in certain food produc- Cistercian Order took place at the abbey La Grande tion environments. Trappe in Normandy in 1664, and thus the monks were The use of natural materials can be required for called Trappists. The Cistercians were known for their producing some PDO cheeses. The 2006 regulations for technological innovation and for transforming wilder- Mont d’Or production stipulate that the cheese must be ness and malaria-ridden marshy land into farmland. The surrounded by a strip of spruce bark and placed in a monks reintroduced water wheels to Europe, technology spruce box (75). The inner layer of spruce bark is cut which had been abandoned by the Romans (235). For

20 ASMscience.org/MicrobiolSpectrum Downloaded from www.asmscience.org by IP: 190.151.168.196 On: Sun, 01 Mar 2015 12:31:56 The Good, the Bad, and the Ugly: Tales of Mold-Ripened Cheese the sustenance of the monastic community, pilgrims, and professional and monastic training to cheesemaking. surrounding population, the monks developed food The monks keep their artisanal cheese “local” by using technologies, including beer brewing and cheesemaking milk from breeds native to the Alps, the Tarentaise and (236). Cheese was an important part of the monastic diet Abondance, which, when not grazing in alpine pastures because according to Benedict’s Rule, eating the flesh of in the summer, eat feed from the region. Frère Nathanaël “quadrupeds” was forbidden except by the infirm (237). extends safeguarding the patrimony of the region to the Since monastic meals were eaten in strict silence, the microbiota. Like other master cheesemakers, Frère monks used sign language to communicate serving Nathanaël wants to make a consistently uniform and needs. The Code of Signs found in records from the safe product yet does not want to lose the biodiversity of abbey of St. Thomas the Martyr in Dublin, founded in local microorganisms. Studies have shown that com- 1177, provides us with evidence that there were mercial cultures of starter bacteria such as Streptococcus cheesemakers at the monastic table: thermophilus and Lactococcus lactis quickly outnumber the wild-type species initially present in raw milk (42). Pro signo casei, utramque manum con junge per obliquum, Thus, in collaboration with a commercial culture com- quasi qui caseum premit. pany, he isolated bacteria from hay and milk of the re- [For the sign of cheese, join both hands obliquely, as one who gion and developed a collection of starter cultures that presses cheese.] (238) could be alternated in Tamié production in case the starters became infected by bacteriophage. Tamié thus is Tamié cheese is made at the Trappist Abbaye Notre- a cheese that stands at the crossroads of scientific and Dame de Tamié, founded in 1132 in Haute-Savoie in the traditional methods of cheese fabrication. Alps at 900 m. Since 1863, the monks have made the Frère Nathanaël’s latest innovation involves the cheese we now know as Tamié, but presumably other recycling of whey, a by-product of cheesemaking and a varieties were made far into the past before the aban- potential pollutant. Cheese production at the abbey donment of the abbey during the French Revolution in produces 1,000 m3 of whey annually, and Frère 1792 (239). From the archives of 1600 we read that Nathanaël developed and installed a methane-generat- “someone lost the key to the cheese cave.” ing system that transforms the whey into enough energy Tamié cheese was trademarked in 1946 and is made to heat water for the domestic needs of the abbey and its and sold as a means of sustainability for the monastic dairy for a year. The Abbey of Tamié was awarded the community, yet its production involves a social obliga- Rhône-Alpes 2005 Energies of Today prize. tion on the part of the monks. In 1132, the abbey was The cheese cave of the abbey, with its arched vaults founded to provide food for the peasants of the area as and limestone walls, lies beneath the Monastic Choir. well as travelers passing through the Alps from Geneva Standing in the cave, Frère Nathanaël shared the secret to the Little Saint Bernard Pass. During the 12th century, of knowing when a Tamié cheese is ready to be eaten. the monks helped the peasants clear land for pastures One gently taps the surface, and if there is no indentation, and roads and aided them in organizing agricultural it is ready. One then smells it underneath. If “ça sent la granges for large-scale cheese production (239). The vache” (it smells like the cow), it is good. In another obligation to local farmers is continued today; the context, he compared the smell to a less acceptable by- monks use milk from 11 local farms for the production product of the cow. Everyone may not be at ease with of Tamié. Clary (239) has noted that the supplier-pro- Frère Nathanaël’s analogy, yet it reminds us of the ducer relationship between the monks and farmers is earthiness associated with the origins of cheese. In com- maintained through professional contracts yet is built paring the language used to describe wine “consisting upon hundreds of years of establishing trust. Many of generally of associations with various fruit and flowers” these farmers are descendants of those who maintained to that of cheese, cheesemaker and philosopher Jim the abbey property during and after the French Revo- Stillwaggon mused, “Cheese on the other hand is less lution, and some are descendants of the original farmers discreet. If we address frankly what is evoked by cheese I whose land was surrendered for the building of the think it becomes clear why so little is said—usually little original abbey. more than sounds of approbation, and global adjectives: Frère Nathanaël, the monk in charge of cheese pro- ‘Mmmmmm, good! Interesting! Fantastic!’ So what does duction, attended dairy school and worked in the in- cheese evoke? Damp dark cellars, molds, mildews and dustry for 9 years before entering monastic life. He calls mushrooms galore, dirty laundry and high school locker cheesemaking a “complex alchemy” and brings his rooms, digestive processes and visceral fermentations,

ASMscience.org/MicrobiolSpectrum 21 Downloaded from www.asmscience.org by IP: 190.151.168.196 On: Sun, 01 Mar 2015 12:31:56 Marcellino and Benson

he-goats which do not remind of Chanel ….Insum,cheese 21. Garnsey P. 1986. Mountain economies in southern Europe. Itinera reminds of dubious, even unsavory places, both in nature 5-6:7–29. and within our own organisms. And yet we love it” 22. Harrison F. 1913. Roman Farm Management: The Treatises of Cato and Varro. Macmillan Company, New York, NY. (personal communication). 23. Forster ES, Heffner EH. 1956. Lucius Junius Moderatus Columella. Harvard University Press, Cambridge, MA. ACKNOWLEDGMENT fi 24. Choisy C, Desmazeaud M, Gripon JC, Lamberet G, Lenoir J, The authors declare that they have no commercial af liations, Tourneur C. 1987. Microbial and biochemical aspects of ripening, consultancies, stock or equity interests, or patent-licensing p62–99. In Eck A (ed), Cheesemaking, Science and Technology, 2nd ed. arrangements that could be considered to pose a conflict of in- Lavoisier, New York, NY. terest regarding the manuscript. 25. Oldfather WA. 1913. Homerica: I. akrhton gala, i 297. Classical Philol 8:195–212. REFERENCES 26. Bolling GM. 1958. Nuktos Amolgwi. Am J Philol 79:165–172. 1. Elkort M. 1991. The Secret Life of Food: A Feast of Food and Drink ’ 27. Hernández PN. 2000. Back in the cave of the Cyclops. Am J Philol History, Folklore, and Fact. St. Martin s Press, New York, NY. 121:345–366. 2. Blandino A, Al-Aseeri ME, Pandiella SS, Cantero D, Webb C. 2003. 28. Kamber U, Terzi G. 2008. The traditional cheeses of Turkey: Central Cereal-based fermented foods and beverages. Food Res Int 36:527–543. Anatolian Region. Food Rev Int 24:74–94. 3. McCormick F. 1991. Evidence of dairying at Dún Ailinne? Emania – 29. Alichanidis E, Polychroniadou A. 2008. Characteristics of major 8:57 59. traditional regional cheese varieties of East-Mediterranean countries: 4. Abdelgadir WS, Ahmed TK, Dirar HA. 1998. The traditional fermented a review. Dairy Sci Technol 88:495–510. milk products of the Sudan. Int J Food Microbiol 44:1–13. 30. Bérard L, Marchenay P. 1995. Lieux, temps et preuves: la construction 5. Copley MS, Berstan R, Dudd SN, Straker V, Payne S, Evershed RP. sociale des produits de terroir. Terrain 24:153–164. 2005. Dairying in antiquity. I. Evidence from absorbed lipid residues 31. Montanari A, Staniscia B. 2009. Culinary tourism as a tool for re- dating to the British Iron Age. J Archaeol Sci 32:485–503. gional re-equilibrium. Eur Plann Stud 17:1463–1483. 6. Copley MS, Berstan R, Mukherjee AJ, Dudd SN, Straker V, Payne V, 32. Bertozzi L, Panari G. 1993. Cheeses with Appellation d’Origine Evershed RP. 2005. Dairying in antiquity. III. Evidence from absorbed Contrôlée (AOC): factors that affect quality. Int Dairy J 3:297–312. lipid residues dating to the British Neolithic. J Archaeol Sci 32:523–546. 33. Bérard L, Marchenay P. 2008. From Localized Products to 7. Sherratt AG. 1981. Plough and pastoralism: aspects of the secondary – Geographical Indications. Awareness and Action. Centre National de la products revolution, p 261 305. In Hodder I, Isaac G, Hammond N (ed), Recherche Scientifique, Bourg-en-Bresse, France. Pattern of the Past: Studies in Honor of David Clarke. Cambridge Uni- fl versity Press, Cambridge, England. 34. Semple EC. 1922. The in uence of geographic conditions upon ancient Mediterranean stock-raising. Ann Am Assoc Geogr 12:3–38. 8. Zettler RL. 1998. Early dynastic Mesopotamia, p. 1–7. In Zettler RL, Horne L (ed), Treasures from the Royal Tombs of Ur. University of 35. Morris C, Kirwan J. 2010. Food commodities, geographical Pennsylvania Museum, Philadelphia, PA. knowledges and the reconnection of production and consumption: the case of naturally embedded food products. Geoforum 41:131–143. 9. Fox PF. 1993. Cheese: an overview, p 1–36. In Fox PF (ed), Cheese: Chemistry, Physics and Microbiology, 2nd ed. Chapman and Hall, 36. Shepard S. 2001. Pickled, Potted, and Canned: How the Art and Science London, England. of Food Preserving Changed the World. Simon & Schuster, New York, NY. 10. Edwards EE. 1949. Europe’s contribution to the American dairy 37. Beresford TP, Fitzsimons N, Brennan NM, Cogan TM. 2001. Recent – industry. J Econ Hist 9:72–84. advances in cheese microbiology. Int Dairy J 11:259 274. 11. Curtis RI. 2001. Ancient Food Technology. Brill Academic Publishers, 38. Marcellino N, Beuvier E, Grappin R, Guéguen M, Benson DR. 2001. Leiden, The Netherlands. Diversity of Geotrichum candidum strains isolated from traditional cheesemaking fabrications in France. Appl Environ Microbiol 67:4752– 12. Bogucki P. 1984. The antiquity of dairying in temperate Europe. 4759. Expedition 28:51–58. fi 39. Serhan M, Cailliez-Grimal C, Borges F, Revol-Junelles A-M, Hosri C, 13. Green eld HJ. 1988. The origins of milk and wool production in the Fanni J. 2009. Bacterial diversity of Darfiyeh, a Lebanese artisanal raw Old World: a zooarchaeological perspective from the Central Balkans. goat’s milk cheese. Food Microbiol 26:645–652. Curr Anthropol 29:573–593. fi 40. Nieto-Arribas P, Seseña S, Poveda JM, Palop L, Cabezas L. 2010. 14. Bogucki P, Grygiel R. 1993. The rst farmers of Central Europe: a Genotypic and technological characterization of Leuconostoc isolates to survey article. J Field Archaeol 20:399–426. be used as adjunct starters in Manchego cheese manufacture. Food 15. Craig O, Mulville J, Pearson MP, Sokol R, Gelsthorpe K, Stacey R, Microbiol 27:85–93. Collins M. 2000. Detecting milk proteins in ancient pots. Nature 408:312. 41. Montagna MT, Santacroce MP, Spilotros G, Napoli C, Minervini F, 16. Craig OE, Taylor G, Mulville J, Collins MJ, Parker Pearson M. 2005. Papa A, Dragoni I. 2004. Investigation of fungal contamination in sheep The identification of prehistoric dairying activities in the Western Isles of and goat cheeses in Southern Italy. Mycopathologia 158:245–249. – Scotland: an integrated biomolecular approach. J Archaeol Sci 32:91 103. 42. Delbès C, Ali-Mandjee L, Montel MC. 2007. Monitoring bacterial 17. Earwood C. 1997. Bog butter: a two thousand year history. J Irish communities in raw milk and cheese by culture-dependent and -indepen- Archaeol 8:25–42. dent 16S rRNA gene-based analyses. Appl Environ Microbiol 73:1882– 18. Cronin T, Downey L, Synnott C, McSweeney P, Kelly EP, Cahill M, 1891. Ross RP, Stanton C. 2007. Composition of ancient Irish bog butter. Int 43. Cogan TM, Barbosa M, Beuvier E, Bianchi-Salvadori B, Cocconcelli Dairy J 17:1011–1020. PS, Fernandes I, Gomez J, Gomez R, Kalantzopoulos G, Ledda A, Medina 19. McGee H. 1986. On Food and Cooking: The Science and Lore of the M, Rea MC, Rodriquez E. 1997. Characterization of the lactic acid Kitchen. Scribner’s, New York, NY. bacteria in artisanal dairy products. J Dairy Res 64:409–421. 20. Jones HL, Sterrett JRS. 1960. The Geography of Strabo. Harvard 44. Mounier J, Rea MC, O’Connor PM, Fitzgerald GF, Cogan TM. University Press, Cambridge, MA. 2007. Growth characteristics of Brevibacterium, Corynebacterium,

22 ASMscience.org/MicrobiolSpectrum Downloaded from www.asmscience.org by IP: 190.151.168.196 On: Sun, 01 Mar 2015 12:31:56 The Good, the Bad, and the Ugly: Tales of Mold-Ripened Cheese

Microbacterium, and Staphylococcus spp. isolated from surface-ripened 67. Arfi K, Landaud S, Bonnarme P. 2006. Evidence for distinct L-me- cheese. Appl Environ Microbiol 73:7732–7739. thionine catabolic pathways in the yeast Geotrichum candidum and the – 45. Picque D, Guillemin H, Mirade PS, Didienne R, Lavigne R, Perret B, bacterium Brevibacterium linens. Appl Environ Microbiol 72:2155 Montel MC, Corrieu G. 2009. Effect of sequential ventilation on cheese 2162. ripening and energy consumption in pilot ripening rooms. Int Dairy J 68. Florez AB, Mayo B. 2006. Microbial diversity and succession during 19:489–497. the manufacture and ripening of traditional, Spanish, blue-veined 46. Jany J-L, Barbier G. 2008. Culture-independent methods for identi- Cabrales cheese, as determined by PCR-DGGE. Int J Food Microbiol – fying microbial communities in cheese. Food Microbiol 25:839–848. 110:165 171. 47. Pascu G, Gus C, Beletti N, Rotar MA, Semeniuc C. 2004. Technology 69. Corsetti A, Rossi J, Gobbetti M. 2001. Interactions between yeasts and microbiological quality of Fossa cheese “Il Sogliano al Rubicone.” Bul and bacteria in the smear surface-ripened cheeses. Int J Food Microbiol – Univ Stiinte Agric Med Vet Cluj-Napoca Ser Agric 60:388–391. 69:1 10. 48. Fontana C, Cappa F, Rebecchi A, Cocconcelli PS. 2010. Surface 70. Oulahal N, Adt I, Mariani C, Carnet-Pantiez A, Notz E, Degraeve P. microbiota analysis of Taleggio, Gorgonzola, Casera, Scimudin and 2009. Examination of wooden shelves used in the ripening of a raw milk – Formaggio di Fossa Italian cheeses. Int J Food Microbiol 138:205–211. smear cheese by FTIR spectroscopy. Food Control 20:658 663. 49. Torsvik V, Ovreas L. 2002. Microbial diversity and function in soil: 71. Pottier I, Gente S, Vernoux JP, Guèguen M. 2008. Safety assessment of from genes to ecosystems. Curr Opin Microbiol 5:240–245. dairy microorganisms: Geotrichum candidum. Int J Food Microbiol 126:327–332. 50. Hansel CM, Fendorf S, Jardine PM, Francis CA. 2008. Changes in bacterial and archaeal community structure and functional diversity along 72. Guéguen M, Schmidt JL. 1992. Les levures et Geotrichum candidum, – a geochemically variable soil profile. Appl Environ Microbiol 74:1620– p 165 219. In Hermier J, Lenoir J, Weber F (ed), Les Groupes Microbiens ’ 1633. d Intérêt Laitier. CEPIL, Paris, France. 51. Demirel R, Ilhan S, Asan A, Kinaci E, Oner S. 2005. Microfungi in 73. Aziza M, Couriol C, Amrane A, Boutrou R. 2005. Evidences for cultivated fields in Eskişehir provience (Turkey). J Basic Microbiol synergistic effects of Geotrichum candidum on Penicillium camembertii – 45:279–293. growing on cheese juice. Enzyme Microb Technol 37:218 224. 52. Florez AB, Alvarez-Martin P, Lopez-Diaz TM, Mayo B. 2007. 74. Boutrou R, Aziza M, Amrane A. 2006. Enhanced proteolytic activities Morphotypic and molecular identification of filamentous fungi from of Geotrichum candidum and Penicillium camembertii in mixed culture. – Spanish blue-veined Cabrales cheese, and typing of Penicillium roqueforti Enzyme Microb Technol 39:325 331. and Geotrichum candidum isolates. Int Dairy J 17:350–357. 75. Mariani C. 2007. Ecologie microbienne des biofilms présents à la ’ fi ’ 53. Scott R. 2008. Ecology of fermented foods. Hum Ecol Rev 15:25–31. surface des planches d af nage en bois de l AOC Reblochon de Savoie et effet inhibiteur vis à vis de Listeria monocytogenes. Ph.D. thesis. Agri- 54. Androuët P. 1987. Préface, p 5–6. In Courtine RJ (ed), Les Fromages, culture, Alimentation, Biologie, Environnements et Santé, Ecole Nationale 2nd ed. Larousse, Paris, France. Supérieure des Industries Agricoles et Alimentaires, Paris, France. 55. Jenkins EH. 1925. Agriculture in the Nineteenth Century. The States 76. Kendrick B. 1992. The Fifth Kingdom, 2nd ed. Mycologue Pub- History Company, New York, NY. lications, Waterloo, Ontario, Canada. 56. Backus A. 1812. Statistical Account of the Town of Bethlehem. Connecticut Academy of Arts and Sciences, Bethlehem, CT. 77. Mariani C, Briandet R, Chamba J-F, Notz E, Carnet-Pantiez A, Eyoug RN, Oulahal N. 2007. Biofilm ecology of wooden shelves used in ripening 57. Cutter T. 2008. Cheese history in Western New York. Heritage – the french raw milk smear cheese Reblochon de Savoie. J Dairy Sci 10:20 21. 90:1653–1661. 58. Marcellino N, Benson DR. 1992. Scanning electron and light micro- 78. Guéguen M, Jacquet J. 1982. Etudes sur les caractères culturaux et la scopic study of microbial succession on Bethlehem St. Nectaire Cheese. morphologie de Geotricum candidum Link. Lait 62:625–644. Appl Environ Microbiol 58:3448–3454. 79. Boutrou R, Guéguen M. 2005. Interests in Geotrichum candidum for 59. Dolci P, Cocolin L, Rantsiou K, Ambrosoli R, Alessandria V, Barmaz cheese technology. Int J Food Microbiol 102:1–20. A, Zenato S, Pramotton R. 2009. Maturing dynamics of surface micro- flora in Fontina PDO cheese studied by culture-dependent and -indepen- 80. Wouters JTM, Ayad EHE, Hugenholtz J, Smit G. 2002. Microbes dent methods. J Appl Microbiol 106:278–287. from raw milk for fermented dairy products. Int Dairy J 12:91–109. 60. Vergeade J, Guiraud J, Larpent JP, Galzy P. 1976. Étude de la flore de 81. Barnett JA, Payne RW, Yarrow D. 1983. Yeasts: Characteristics and levure du Saint-Nectaire. Dairy Sci Technol 56:275–285. Identification. Cambridge University Press, Cambridge, England. 61. Devoyod J-J. 1990. Yeasts in cheese-making, p 228–237. In Spencer 82. Barnett JA, Payne RW, Yarrow D. 1990. Yeasts: Characteristics and JFT, Spencer DM (ed), Yeast Technology. Springer-Verlag, New York, Identification, 2nd ed. Cambridge University Press, Cambridge, England. NY. 83. Gente S, Sohier D, Coton E, Duhamel C, Guéguen M. 2006. Identi- 62. Marcellino, SN. 2003. Biodiversity of Geotrichum candidum Strains fication of Geotrichum candidum at the species and strain level: proposal Isolated from Traditional French Cheese. Ph.D. thesis. University of for a standardized protocol. J Ind Microbiol Biotechnol 33:1019–1031. Connecticut, Storrs, CT. 84. Picque D, Leclercq-Perlat M-N, Corrieu G. 2006. Effects of atmo- 63. Michel V, Hauwuy A, Chamba J-F. 2001. La flore microbienne de laits spheric composition on respiratory behavior, weight loss, and appearance crus de vache: diversité et influence des conditions de production. Dairy of camembert-type cheeses during chamber ripening. J Dairy Sci 89: Sci Technol 81:575–592. 3250–3259. 64. Cole GT. 1986. Models of cell differentiation in conidial fungi. 85. Gente S, Desmasures N, Panoff JM, Guéguen M. 2002. Genetic di- Microbiol Mol Biol Rev 50:95–132. versity among Geotrichum candidum strains from various substrates 65. Sipahioglu HN, Heperkan D. 2000. Lipolytic activity of studied using RAM and RAPD-PCR. J Appl Microbiol 92:491–501. Trichothecium roseum on hazelnut. Food Microbiol 17:401–405. 86. de Hoog GS, Smith MT, Guého E. 1986. A revision of the genus 66. Galaup P, Gautier A, Piriou Y, de Villeblanche A, Valla A, Dufossé L. Geotrichum and its teleomorphs. Stud Mycol 29:1–131. 2007. First pigment fingerprints from the rind of French PDO red-smear 87. de Hoog GS, Smith MT. 2004. Ribosomal gene phylogeny and spe- ripened soft cheeses Epoisses, Mont d’Or and Maroilles. Innov Food Sci cies delimitation in Geotrichum and its teleomorphs. Stud Mycol 50:489– Emerg Technol 8:373–378. 515.

ASMscience.org/MicrobiolSpectrum 23 Downloaded from www.asmscience.org by IP: 190.151.168.196 On: Sun, 01 Mar 2015 12:31:56 Marcellino and Benson

88. Bockelmann W, Heller M, Heller KJ. 2008. Identification of yeasts of 110. Steele JL. 1995. Contribution of lactic acid bacteria to cheese rip- dairy origin by amplified ribosomal DNA restriction analysis (ARDRA). ening, p 209–220. In Malin EL, Tunick MH (ed), Chemistry of Structure- Int Dairy J 18:1066–1071. Function Relationships in Cheese. Plenum Press, New York, NY. 89. Prillinger H, Molnar O, Eliskases-Lechner F, Lopandic K. 1999. 111. Fox PF, McSweeney PLH. 1996. Proteolysis in cheese during Phenotypic and genotypic identification of yeasts from cheeses. Antonie ripening. Food Rev Int 12:457–509. – van Leeuwenhoek 75:267 283. 112. Boutrou R, Kerriou L, Gassi J-Y. 2006. Contribution of Geotrichum 90. Gente S, Desmasures N, Jacopin C, Plessis G, Beliard M, Panoff J-M, candidum to the proteolysis of soft cheese. Int Dairy J 16:775–783. Guéguen M. 2002. Intra-species chromosome-length polymorphism in 113. Molimard P, Lesschaeve I, Bouvier I, Vassal L, Schlich P, Issanchou fi Geotrichum candidum revealed by pulsed eld gel electrophoresis. Int J S, Spinnler HE. 1994. Amertume et fractions azotées de fromages à pâte – Food Microbiol 76:127 134. molle de type camembert: rôle de l’association de Penicillium camemberti 91. Arteau M, Labrie S, Roy D. 2010. Terminal-restriction fragment length avec Geotrichum candidum. Lait 74:361–374. polymorphism and automated ribosomal intergenic spacer analysis pro- 114. Fox PF, Singh TK, McSweeney PLH. 1995. Biogenesis of flavour fi – ling of fungal communities in Camembert cheese. Int Dairy J 20:545 554. compounds in cheese, p 59–98. In Malin EL, Tunick MH (ed), Chemistry 92. Bartz JA, Eayre CG, Mahovic MJ, Concelmo DE, Brecht JK, Sargent of Structure-Function Relationships in Cheese. Plenum Press, New York, SA. 2001. Chlorine concentration and the inoculation of tomato fruit in NY. – packinghouse dump trucks. Plant Dis 85:885 889. 115. Morales P, Gaya P, Medina M, Nunez M. 2002. Hydrophilic and 93. Nakamura M, Suprapta, DN Iwai H. 2008. Differentiation of path- hydrophobic peptides produced in cheese by wild Lactococcus lactis ogenic and nonpathogenic isolates of Geotrichum candidum sensu strains. Lett Appl Microbiol 35:518–522. Suprapta et al. (1995) on citrus fruit based on PCR-RFLP analysis of 116. Habibi-Najafi MB, Lee BH. 1996. Bitterness in cheese: a review. Crit fi rDNA ITS and PCR using speci c primers designed in polygalacturonase Rev Food Sci Nutr 36:397–411. genes. Mycoscience 49:155–158. 117. Vafopoulou-Mastrojiannaki A, Litopoulou-Tzanetaki E, Tzanetakis 94. Linko M, Haikara A, Ritala A, Penttilä M. 1998. Recent advances in N. 1994. Proteinase, peptidase and esterase activity of crude cell-free the malting and brewing industry. J Biotechnol 65:85–98. extracts of Pediococcus pentosaceus isolated from cheese. Lebensm-Wiss 95. Sztajer H, Wang W, Lünsdorf H, Stocker A. 1996. Purification and Technol 27:342–346. some properties of a novel microbial lactate oxidase. Appl Microbiobiol 118. Izco JM, Irigoyen A, Torre P, Barcina Y. 2000. Effect of the activity Biotechnol 45:600–606. levels of the added proteolytic enzyme mixture on free amino acids in 96. Maximo C, Amorim MTP, Costa-Ferreira M. 2003. Biotransforma- ripening Ossau-Iraty cheese. J Chromatogr 881:69–79. tion of industrial reactive azo dyes by Geotrichum sp. CCMI 1019. 119. Macedo AC, Tavares TG, Malcata FX. 2003. Purification and Enzyme Microb Technol 32:145–151. characterization of an intracellular aminopeptidase from a wild strain of 97. Asses N, Ayed L, Bouallagui H, Ben Rejeb I, Gargouri M, Hamdi M. Lactobacillus plantarum isolated from traditional Serra da Estrela cheese. 2009. Use of Geotrichum candidum for olive mill wastewater treatment Enzyme Microb Technol 32:41–48. in submerged and static culture. Bioresour Technol 100:2182–2188. 120. Izawa N, Tokuyasu K, Hayashi K. 1997. Debittering of protein 98. Bosch X. 2001. Fungus eats CD. http://www.nature.com/news/2001/ hydrolysates using Aeromonas caviae aminopeptidase. J Agric Food Chem 010627/full/news010628-11.html. 45:543–544. 99. Guéguen M. 1988. Affections pathologiques dues à Geotrichum 121. Engel E, Nicklaus S, Septier C, Salles C, Le Quere JL. 2000. Taste candidum. Microbiol Aliment Nutr 6:119–124. active compounds in a goat cheese water-soluble extract. 2. Determination 100. Leclercq-Perlat MN, Corrieu G, Spinnler HE. 2007. Controlled of the relative impact of water-soluble extract components on its taste production of camembert-type cheeses. Part III. Role of the ripening mi- using omission tests. J Agric Food Chem 48:4260–4267. croflora on free fatty acid concentrations. J Dairy Res 74:218–225. 122. Sousa MJ, Ardo Y, McSweeney PLH. 2001. Advances in the study of 101. Holmquist M. 1998. Insights into the molecular basis for fatty acyl proteolysis during cheese ripening. Int Dairy J 11:327–345. fi speci cities of lipases from Geotrichum candidum and Candida rugosa. 123. Chien H-CR, Lin L-L, Chao S-H, Chen C-C, Wang W-C, Shaw C-Y, Chem Phys Lipids 93:57–65. Tsai Y-C, Hu H-Y, Hsu W-H. 2002. Purification, characterization, and 102. Das S, Holland R, Crow VL, Bennett RJ, Manderson GJ. 2005. genetic analysis of a leucine aminopeptidase from Aspergillus sojae. Effect of yeast and bacterial adjuncts on the CLA content and flavour of a Biochim Biophys Acta 1576:119–126. washed-curd, dry-salted cheese. Int Dairy J 15:807–815. 124. Molimard P, Spinnler HE. 1996. Review: compounds involved in the 103. Fresno JM, Tornadijo ME, Carballo J, Bernardo A, Gonzalez-Prieto flavor of surface mold-ripened cheeses: origins and properties. J Dairy Sci J. 1997. Proteolytic and lipolytic changes during the ripening of a Spanish 79:169–184. craft goat cheese (Armada variety). J Sci Food Agric 75:148–154. 125. Demarigny Y, Berger C, Desmasures N, Gueguen M, Spinnler HE. 104. Damle SV, Patil PN, Salunkhe MM. 2000. Biotransformations with 2000. Flavour sulphides are produced from methionine by two different Rhizopus arrhizus and Geotrichum candidum for the preparation of (S)- pathways by Geotrichum candidum. J Dairy Res 67:371–380. atenolol and (S)-propranolol. Bioorg Med Chem 8:2067–2070. 126. Gente S, La Carbona S, Guéguen M. 2007. Levels of cystathionine Y 105. Nakamura K, Fujii M, Ida Y. 2001. Stereoinversion of arylethanols lyase production by Geotrichum candidum in synthetic media and cor- by Geotrichum candidum. Tetrahedron Asymmetry 12:3147–3153. relation with the presence of sulphur flavours in cheese. Int J Food 106. McSweeney PLH, Sousa MJ. 2000. Biochemical pathways for the Microbiol 114:136–142. production of flavour compounds in cheeses during ripening: a review. 127. Molimard P, Lesschaeve I, Issanchou S, Brousse M, Spinnler HE. Dairy Sci Technol 80:293–324. 1997. Effect of the association of surface flora on the sensory properties of 107. Fallico V, McSweeney PLH, Horne J, Pediliggieri C, Hannon J, mould-ripened cheese. Lait 77:181–187. Carpino S, Licitra G. 2005. Evaluation of bitterness in Ragusano cheese. 128. Yvon M, Rijnen L. 2001. Cheese flavour formation by amino acid J Dairy Sci 88:1288–1300. catabolism. Int Dairy J 11:185–201. 108. Le Bars-Bailly S, Bailly JD, Brugere H. 1999. Mold-related failings in 129. Jollivet N, Chataud J, Vayssier Y, Bensoussan M, Belin J. 1994. cheesemaking. Rev Med Vet 150:413–430. Production of volatile compounds in model milk and cheese media 109. McSweeney PLH. 1997. The flavour of milk and dairy products. III. by eight strains of Geotrichum candidum Link. J Dairy Res 61:241– Cheese: taste. Int J Dairy Technol 50:123–128. 248.

24 ASMscience.org/MicrobiolSpectrum Downloaded from www.asmscience.org by IP: 190.151.168.196 On: Sun, 01 Mar 2015 12:31:56 The Good, the Bad, and the Ugly: Tales of Mold-Ripened Cheese

130. Beldarrin A, Acosta N, Montesinos R, Mata M. 2000. Character- 148. Aygun O, Yaman M, Durmaz H. 2007. A survey on occurrence of ization of Mucor pusillus rennin expressed in Pichia pastoris enzymatic, Tyrophagus putrescentiae (: ) in Surk, a traditional Turkish spectroscopic and calorimetric studies. Biotechnol Appl Biochem 31: dairy product. J Food Eng 78:878–881. – 77 84. 149. Van Asselt L. 1999. Interactions between domestic mites and fungi. 131. Rattray FP, Fox PF. 1998. Recently identified enzymes of Brevi- Indoor Built Environ 8:216–220. — – bacterium linens ATCC 9174 a review. J Food Biochem 22:353 373. 150. Hubert J, Jarosík V, Mourek J, Kubátová A, Zdárková E. 2004. 132. Dias B, Weimer B. 1998. Conversion of methionine to thiols by Astigmatid mite growth and fungi preference (Acari: Acaridida): com- lactococci, lactobacilli, and brevibacteria. Appl Environ Microbiol parisons in laboratory experiments. Pedobiologia 48:205–214. – 64:3320 3326. 151. Overstreet RM. 2009. Presidential address: flavor buds and other 133. Bonnarme P, Psoni L, Spinnler HE. 2000. Diversity of L-methionine delights. J Parasitol 89:1093–1107. catabolism pathways in cheese-ripening bacteria. Appl Environ Microbiol – 152. Solarz K, Senczuk L, Maniurka H, Cichecka E, Peszke M. 2007. 66:5514 5517. Comparisons of the allergenic mite prevalence in dwellings and certain 134. Guéguen M. 1988. Moisissures responsables de défauts d’affinage outdoor environments of the Upper Silesia (southwest Poland). Int J Hyg en fromagerie (à l’exclusion des Mucoraceae). Microbiol Aliment Nutr Environ Health 210:715–724. 6:31–35. 153. Renaud J, Gueugnot J, Petavy AF, Guillot J, Coulet M. 1977. Les 135. Lecocq J, Guéguen M. 1994. Effects of pH and sodium chloride on acariens du fromage: étude analytique—préparation d’extraits anti- the interactions between Geotrichum candidum and Brevibacterium géniques. Rev Fr Allerg Immunol Clin 17:247–250. linens. Dairy Sci 77:2890–2899. 154. Collins DA, Cook DA. 2006. Laboratory evaluation of diatomaceous fl 136. Fox PF, Wallace JM. 1997. Formation of avor compounds in earths, when applied as dry dust and slurries to wooden surfaces, against – cheese. Adv Appl Microbiol 45:17 85. stored-product insect and mite pests. J Stored Prod Res 42:197–206. 137. Adour L, Couriol C, Amrane A, Prigent Y. 2002. Growth of 155. Wisniewski HM, Sigurdarson S, Rubenstein R, Kascsak RJ, Carp R. Geotrichum candidum and Penicillium camemberti in liquid media in 1996. Mites as vectors for scrapie. Lancet 347:1114. relation with the consumption of carbon and nitrogen sources and the 156. Sánchez-Ramos I, Castañera P. 2009. Chemical and physical methods release of ammonia and carbon dioxide. Enzyme Microb Technol 31:533– 542. for the control of the mite Acarus farris on Cabrales cheese. J Stored Prod Res. 45:61–66. 138. Aldarf M, Amrane A, Prigent Y. 2002. Reconstruction of the 157. Conyers ST, Bell CH. 2003. The effect of modified atmospheres on biomass history from carbon and nitrogen substrate consumption, am- monia release and proton transfer during solid cultures of Geotrichum the survival of the eggs of four storage mite species. Exp Appl Acarol 31:115–130. candidum and Penicillium camembertii. Appl Microbiol Biotechnol 58:823–829. 158. McKnight Q. 2006. The Art of Farmstead Cheese Making in the 139. Linton M, Mackle AB, Upadhyay VK, Kelly AL, Patterson MF. British Isles. Dairy Artisan Series. The Babcock Institute for International Dairy Research and Development, University of Wisconsin—Madison, 2008. The fate of Listeria monocytogenes during the manufacture of Camembert-type cheese: a comparison between raw milk and milk treated Madison, WI. with high hydrostatic pressure. Innov Food Sci Emerg Technol 9:423– 159. Collins DA, Cook DA. 2006. Laboratory studies evaluating the ef- 428. ficacy of diatomaceous earths, on treated surfaces, against stored-product – 140. D’Amico DJ, Donnelly CW, Druart MJ. 2008. 60-day aging re- insect and mite pests. J Stored Prod Res 42:51 60. quirement does not ensure safety of surface-mold-ripened soft cheeses 160. Collins DA. 2006. A review of alternatives to organophosphorus manufactured from raw or pasteurized milk when Listeria monocytogenes compounds for the control of storage mites. J Stored Prod Res 42:395– is introduced as a postprocessing contaminant. J Food Prot 71:1563– 426. 1571. 161. Sánchez-Ramos I, Castañera P. 2000. Acaricidal activity of natural 141. Dieuleveux V, Van Der Pyl D, Chataud J, Guéguen M. 1998. monoterpenes on Tyrophagus putrescentiae (Schrank), a mite of stored Purification and characterization of anti-Listeria compounds produced food. J. Stored Prod. Res. 37:93–101. by Geotrichum candidum. Appl Environ Microbiol 64:800–803. 162. Bachmann HP, Bobst C, Bütikofer U, Casey MG, Dalla Torre M, fi 142. Dieuleveux V, Gueguen M. 1998. Antimicrobial effects of D-3- Fröhlich-Wyder MT, Fürst M. 2005. Occurrence and signi cance of phenyllactic acid on Listeria monocytogenes in TSB-YE medium, milk, Fusarium domesticum alias Anticollanti on smear-ripened cheeses. LWT and cheese. J Food Prot 61:1281–1285. Food Sci Technol 38:399–407. 143. Cholet O. 2006. Etude de l’écosystème fromager par une approche 163. Bothast RJ, Lancaster EB, Hesseltine CW. 1975. Scopulariopsis biochimique et moléculaire. Ph.D. thesis. UMR de Génie et Microbiologie brevicaulis: effect of pH and substrate on growth. Appl Microbiol des Procédés Alimentaires INRA/INA P-G. Institut National Biotechnol 1:55–66. Agronomique Paris-Grignon, Paris, France. 164. Keilling J, Casalis J, Mauro N. 1951. Sur le chancre superficiel des 144. Guéguen M, Jacquet J, Allain-Garnier M, Pineau A, Kogbo W. 1984. fromages à pâte ferme. Dairy Sci Technol 31:353–360. Sur les interactions microbiennes de Geotrichum candidum Link. 165. Jacquet J, Desfleurs M. 1966. Scopulariopsis brevicaulis Bainier ou Microbiol Aliment Nutr 2:139–152. mieux Scopulariopsis fusca Zack, agents des taches superficielles brun- 145. Nielsen MS, Frisvad JC, Nielsen PV. 1998. Protection by fungal violettes des fromages a pâte molle. Dairy Sci Technol 46:241–253. starters against growth and secondary metabolite production of fungal 166. Matkovic K, Vucemilo M, Vinkovic B. 2009. Airborne fungi in spoilers of cheese. Int J Food Microbiol 42:91–99. dwellings for dairy cows and laying hens. Arh Hig Rada Toksikol 60: 146. Sánchez-Ramos I, Alvarez-Alfageme F, Castanera P. 2007. Devel- 395–399. opment and survival of the cheese mites, Acarus farris and Tyrophagus 167. Salmon A, Debourgogne A, Vasbien M, Clément L, Collomb J, neiswanderi (Acari: Acaridae), at constant temperatures and 90% relative Plénat F, Bordigoni P, Machouart M. 2010. Disseminated Scopulariopsis humidity. J Stored Prod Res 43:64–72. brevicaulis infection in an allogeneic stem cell recipient: case report and 147. Sánchez-Ramos I, Alvarez-Alfageme F, Castanera P. 2007. review of the literature. Clin Microbiol Infect 16:508–512. Reproduction, longevity and life table parameters of Tyrophagus 168. Wagner D, Sander A, Bertz H, Finke J, Kern W. 2005. Breakthrough neiswanderi (Acari: Acaridae) at constant temperatures. Exp Appl Acarol invasive infection due to Debaryomyces hansenii (teleomorph Candida 43:213–226. famata) and Scopulariopsis brevicaulis in a stem cell transplant patient

ASMscience.org/MicrobiolSpectrum 25 Downloaded from www.asmscience.org by IP: 190.151.168.196 On: Sun, 01 Mar 2015 12:31:56 Marcellino and Benson

receiving liposomal amphotericin b and caspofungin for suspected as- the filamentous fungus Mucor miehei NRRL 3420. World Appl Sci J pergillosis. Infection 33:397–400. 4:578–585. 169. Cuenca-Estrella M, Gomez-Lopez A, Buitrago MJ, Mellado E, 191. Guéguen M. 1984. Contribution à la connaissance de Geotrichum Garcia-Effron G, Rodriguez-Tudela JL. 2006. In vitro activities of 10 candidum et notamment de sa variabilité, conséquences pour l’industrie combinations of antifungal agents against the multiresistant pathogen fromagère. Ph.D. thesis. UER des Sciences de l’Alimentation et de la Scopulariopsis brevicaulis. Antimicrob Agents Chemother 50:2248–2250. Nutrition. Université de Caen, Caen, France. 170. Brandelli A, Daroit D, Riffel A. 2010. Biochemical features of mi- 192. Kabak B, Dobson ADW, Var I. 2006. Strategies to prevent myco- crobial keratinases and their production and applications. Appl Microbiol toxin contamination of food and animal feed: a review. Crit Rev Food Sci Biotechnol 85:1735–1750. Nutr 46:593–619. 171. Blyskal B. 2009. Fungi utilizing keratinous substrates. Int Biodeter 193. Taniwaki MH, Hocking AD, Pitt JI, Fleet GH. 2001. Growth of Biodegr 63:631–653. fungi and mycotoxin production on cheese under modified atmospheres. 172. Fox PF, Guinee TP, Cogan TM, McSweeney PLH. 2000. Fun- Int J Food Microbiol 68:125–133. damentals of Cheese Science. Aspen Publishers, Inc, Gaithersburg, MD. 194. Guéguen M, Desfleurs M, Lemarinier S. 1978. Penicillium roqueforti 173. Mirade P-S, Daudin J-D. 2006. Computational fluid dynamics Thom responsable d’un nouvel accident en fromageries de pâtes molles. prediction and validation of gas circulation in a cheese-ripening room. Dairy Sci Technol 58:327–335. – Int Dairy J 16:920 930. 195. Giraud F, Giraud T, Aguileta G, Fournier E, Samson R, Cruaud 174. Leclercq-Perlat, MN, Picque D, Riahi H, Corrieu G. 2006. Micro- C, Lacoste S, Ropars J, Tellier A, Dupont J. 2010. Microsatellite loci to biological and biochemical aspects of camembert-type cheeses depend recognize species for the cheese starter and contaminating strains as- on atmospheric composition in the ripening chamber. J Dairy Sci 89: sociated with cheese manufacturing. Int J Food Microbiol 137:204– 3260–3273. 213. 175. Cai J, Yang J, Du Y, Fan L, Qiu Y, Li J, Kennedy JF. 2006. Purifi- 196. Decker M, Nielsen PV. 2005. The inhibitory effect of Penicillium cation and characterization of chitin deacetylase from Scopulariopsis camemberti and Geotruchum candidum on the associated funga of white brevicaulis. Carbohydr Polym 65:211–217. mould cheese. Int J Food Microbiol 104:51–60. 176. Aboellil AH, Geweely NS. 2005. An enrichment of xylanolytic 197. Polonelli L, Morace G, Rosa R, Castagnola, M, Frisvad JC. 1987. organism with high pH optima. Biotechnology 4:49–55. Antigenic characterization of Penicillium camemberti and related common 177. Choisy C, Guéguén M, Lenoir J, Schmidt JL, Tourneur C. 1987. cheese contaminants. Appl Environ Microbiol 53:872–878. Les phénomènes microbiens, p 259–290. In Eck A (ed), Le Fromage. 198. Lund F, Nielsen AB, Skouboe P. 2003. Distribution of Penicillium Lavoisier, Paris, France. commune isolates in cheese dairies mapped using secondary metabolite 178. Pitt J, Hocking A. 2009. Fungi and Food Spoilage, 3rd ed. Springer, profiles, morphotypes, RAPD and AFLP fingerprinting. Food Microbiol New York, NY. 20:725–734. 179. Frisvad JC. 2005. Halotolerant and halophilic fungi and their 199. Nowell D. 2008. The Chaîne des Puys volcanoes of the Auvergne, extrolite production, p 425–439. In Gunde-Cimerman N, Oren A, France. Geol. Today 24:231–238. Plemenitaš A (ed), Adaptation to Life at High Salt Concentrations in 200. Dietler M. 1998. A tale of three sites: the monumentalization of Archaea, Bacteria, and Eukarya. Springer, Dordrecht, The Netherlands. Celtic oppida and the politics of collective memory and identity. World 180. Yoder JA, Benoit JB, Zettler LW. 2003. Effects of salt and temper- Archaeol 30:72–89. ature on the growth rate of a -associated fungus, Scopulariopsis 201. Meltzer F. 2009. Reviving the fairy tree: tales of European sanctity. brevicaulis Bainier (Deuteromycota). Int J Acarol 29:265–269. Crit Inq 35:493–520. 181. Piton-Malleret C, Gorrieri M. 1992. Nature and variability of the 202. Cornu A, Kondjoyan N, Martin B, Verdier-Metz I, Pradel P, microbial flora in the Comté and smear. Dairy Sci Technol Berdague JL, Coulon JB. 2005. Terpene profiles in Cantal and Saint- 72:143–164. Nectaire-type cheese made from raw or pasteurised milk. J Sci Food Agric 182. Ho P, Luo J, Adams M. 2009. Lactobacilli and dairy propioni- 85:2040–2046. bacterium with potential as biopreservatives against food fungi and yeast 203. Vollet D, Candau J, Ginelli L, Michelin Y, Ménadier L, Rapey H, contamination. Appl Biochem Microbiol 45:414–418. Dobremez L. 2008. Landscape elements: can they help in selling ‘Protected 183. Dantigny P, Guilmart A, Bensoussan M. 2005. Basis of predictive Designation of Origin’ products? Landscape Res. 33:365–384. mycology. Int J Food Microbiol 100:187–196. 204. Verrier E, Bouffartigue B. 1993. Les AOC, element du maintient des 184. Orlowski M. 1991. Mucor dimorphism. Microbiol Mol Biol Rev races regionales? L’exemple des Alpes du Nord et reflexion sur d’autres 55:234–258. situations. Ethnozootechnie 52:1–20. 185. Lubbehusen TL, McIntyre M, Nielsen J. 2004. Aerobic and anaer- 205. Gostling FM. 1911. Auvergne and Its People. Macmillan Company, obic ethanol production by Mucor circinelloides during submerged New York, NY. growth. Appl Microbiol Biotech 63:543–548. 206. Martin B, Verdier-Metz I, Buchin S, Hurtaud C, Coulon J-B. 2005. 186. Schwarz P, Lortholary O, Dromer F, Dannaoui E. 2007. Carbon How do the nature of forages and pasture diversity influence the sensory assimilation profiles as a tool for identification of Zygomycetes. J Clin quality of dairy livestock products? Anim Sci 81:205–212. Microbiol 45:1433–1439. 207. Daly DFM, McSweeney PLH, Sheehan JJ. 2009. Split defect and 187. Serrano I, Lopes da Silva T, Roseiro JC. 2001. Ethanol-induced secondary fermentation in Swiss-type cheeses—a review. Dairy Sci dimorphism and lipid composition changes in Mucor fragilis CCMI 142. Technol 90:3–26. Lett Appl Microbiol 33:89–93. 208. Driehuis F, Oude Elferink SJ. 2000. The impact of the quality of 188. Tormo H, Barral J. 2004. Accidents de fromagerie. http://www. silage on animal health and food safety: a review. Vet Q 22:212–216. accident-fromagerie.fr/spip.php?rubrique2. Accessed 2 October 2013. 209. Coulon J-B, Delacroix-Buchet A, Martin B, Pirisi A. 2004. 189. Brenet M, Centeleghe JL, Milliere JB, Ramet JP, Weber F. 1972. Relationships between ruminant management and sensory characteristics Etude d’un accident en fromagerie de type Camembert causé par des of cheeses: a review. Dairy Sci Technol 84:221–241. mucorales. Dairy Sci Technol 52:141–148. 210. Garon D, Richard E, Sage L, Bouchart V, Pottier D, Lebailly P. 2006. 190. De Lima CJB, Cortezi M, Lovaglio RB, Ribeiro EJ, Contiero J, Mycoflora and multimycotoxin detection in corn silage: an experimental De Araújo H. 2008. Production of rennet in submerged fermentation with study. J Agric Food Chem 54:3479–3484.

26 ASMscience.org/MicrobiolSpectrum Downloaded from www.asmscience.org by IP: 190.151.168.196 On: Sun, 01 Mar 2015 12:31:56 The Good, the Bad, and the Ugly: Tales of Mold-Ripened Cheese

211. Ricard D. 1994. Les Montagnes Fromagères en France. CERAMAC, 228. Lonnerdal B, Bell J, Keen C. 1985. Copper absorption from human Université Blaise Pascal, Clermont-Ferrand, France. milk, cow’s milk, and infant formulas using a suckling rat model. Am J – 212. Gale G. 2003. Saving the vine from Phylloxera: a never-ending battle, Clin Nutr 42:836 844. p70–91. In Sandler M, Pinder R (ed), Wine: A Scientific Exploration. 229. Faúndez G, Troncoso M, Navarrete P, Figueroa G. 2004. Antimi- Taylor and Francis, Inc, London, England. crobial activity of copper surfaces against suspensions of Salmonella – 213. Greliche F. 1993. Science et Tradition du Saint-Nectaire. Morillat, enterica and Campylobacter jejuni. BMC Microbiol 4:1 7. Besse, France. 230. Mato Rodríguez L, Alatossava T. 2008. Effects of copper supplement 214. Bonnaud P. 2005. Survol géohistorique de la diversité de la Haute on growth and viability of strains used as starters and adjunct cultures for – Auvergne. http://geo.cybercantal.net/php/lire.php?id=39. Accessed 29 manufacture. J Appl Microbiol 105:1098 1106. April 2010. 231. Mato Rodriguez L, Alatossava T. 2010. Effects of copper on ger- mination, growth and sporulation of Clostridium tyrobutyricum. Food 215. Shamsi S, Ultana R. 2008. Trichothecium roseum Link: a new record – of hyphomycetous fungus for Bangladesh. Bangladesh J Plant Taxon Microbiol 27:434 437. 15:77–80. 232. Noyce JO, Michels H, Keevil CW. 2006. Use of copper cast alloys to control Escherichia coli O157 cross-contamination during food 216. Ishii K, Kobayashi J, Ueno Y, Ichinoe M. 1986. Occurrence of processing. Appl Environ Microbiol 72:4239–4244. trichothecin in wheat. Appl Environ Microbiol 52:331–333. 233. McCann J. 1952. The Rule of St. Benedict: in Latin and English. 217. McPartland JM, Hillig KW. 2008. Differentiating powdery mildew Burns & Oates, London, England. from false powdery mildew. J Ind Hemp 13:78–87. 234. Dion R, Verhaeghe R. 1987. Le Maroilles: le plus fin des fromages 218. Espinasse I. 2001. Les Fromages des Alpes. Libris, Seyssinet, France. forts. Histoire et Géographie des Fromages: Actes du Colloques de 219. Les Amis du Val de Thônes. 1987. Le Reblochon de la Vallée de Géographie Historique, Caen, 1985. Centre de Recherches sur l’Evolution Thônes. Amis du Val de Thônes, Thônes, France. de la Vie Rurale, Centre de Publications de l’Universite de Caen, Universite 220. Delforno G. 1977. Technology and chemical composition of Re- de Caen, Caen, France. blochon cheese. Mondo Latte 31:694–696. 235. Dubos R. 1972. A God Within. Scribner, New York, NY. 221. Bärtschi C, Berthier J, Valla G. 1994. Inventaire et évolution des 236. Fraser EDG. 2011. Can economic, land use and climatic stresses lead flores fongiques de surface du Reblochon de Savoie. Dairy Sci Technol to famine, disease, warfare and death? Using Europe’s calamitous 14th 74:105–114. century as a parable for the modern age. Ecol Econ 70:1269–1279. 222. Tierney JJ. 1959. The Celtic ethnography of Posidonius. Proc R Irish 237. Bazell DM. 1997. Strife among the table-fellows: conflicting attitudes Acad Sect C 60:189–275. of early and medieval Christians toward the eating of meat. J Am Acad 223. Mathez EA, Webster JD. 2004. The Earth Machine: The Science of Relig. 65:73–99. a Dynamic Planet. Columbia University Press, New York, NY. 238. Berry HF. 1892. On the use of signs in the ancient monasteries, with 224. Barjolle D, Chappuis J-M. 2000. Coordination des acteurs dans deux special reference to a code used by the Victorine Canons at St. Thomas’s filières AOC. Une approche par la théorie des coûts de transaction. Econ Abbey, Dublin. J R Soc Antiq Irel 2:107–125. Rurale 258:90–100. 239. Clary BJ. 2007. Broadening the concept of rational economic be- 225. Michelson P. 2002. Cheeses for all seasons (from The Cheese Room), havior: a case study of cheese making at the Abbey of Tamié. Rev Soc p13–19. In Hughes H (ed), Best Food Writing 2002. Marlowe & Com- Econ 65:165–186. pany, New York, NY. 240. Abbott SP, Sigler L, Currah RS. 1998. Microascus brevicaulis 226. Raveneau A. 1996. Le Livre de la Vache. Éditions Rustica, Paris, sp. nov., the teleomorph of Scopulariopsis brevicaulis, supports placement France. of Scopulariopsis with the Microascaceae. Mycologia 90:297–302. 227. Sieber R, Rehberger B, Schaller F, Gallmann P. 2006. Technological 241. Dieuleveux V, Lemarinier S, Guéguen M. 1998. Antimicrobial aspects of copper in milk products and health implications of copper. ALP spectrum and target site of D-3-phenyllactic acid. Int J Food Microbiol Sci 493:1–15. 40:177–183.

ASMscience.org/MicrobiolSpectrum 27 Downloaded from www.asmscience.org by IP: 190.151.168.196 On: Sun, 01 Mar 2015 12:31:56