Chapter 2 Complex microbial communities as part of fermented food ecosystems and beneficial properties Imran M., Desmasures N., Vernoux* J-P *corresponding author:
[email protected] Unité des Micro-organismes d’Intérêt Laitier et Alimentaire, EA 3213, IFR146 ICORE Université de Caen Basse-Normandie, Esplanade de la paix, 14032 CAEN cedex, FRANCE Tel. : +33231565621 Fax: +33231566179
1. Introduction 1.1. Microbial classification Complex microbial ecosystems are composed of different microorganisms, which can be classified based on the inferred evolutionary relationships among microorganisms, i.e. upon similarities in their genetic characteristics. Using such an approach and by comparing rRNA sequences, most microorganisms appear in the universal phylogenetic tree of life in three domains: Bacteria, Archaea and Eucarya. Microbial classification can also be based on global similarity (morphological, physiological, ecological, molecular, metabolic and genetic characteristics) between microorganisms. Classification of prokaryotic organisms starts from two domains (e.g. “Bacteria” and “Archaea”), which are then divided into phyla (29 and 5 for “Bacteria” and “Archaea”, respectively) (Euzeby 2011). Further subdivision is organized 1
from the “Class” rank to the “species” rank, sometimes to the “subspecies rank” (Figure 1). The smallest level of differentiation is the “strain” level, which could be more or less compared to the “individuum” at Human scale. The main difference remains that, even at strain level, microorganisms, at the opposite of individuum, are always considered as populations of cells. Classification of Fungi (eukaryotic organisms) follows the same rules. Yeasts and moulds are members of fungi distinguished according to specific morphology, respectively a predominant mononocellular or pluricellular state; they are not true taxonomical groups. According to the rules of the International Committee on Taxonomy of Viruses, virus classification starts at the “order” level but they are excluded from the phylogenetic tree of life. Besides the scientific classification of microrganisms, a technological classification is often used, based on general microbial properties or activities of groups of microorganisms expressed along the food chain, from agricultural and fishery resources to the gastro-intestinal tract of the consumer. Classification can be based, sometimes disregarding taxonomical proximity, on properties positively or negatively linked with food processing, as fermentation activity (e.g. Lactic Acid Bacteria, Butyric Acid Bacteria, Acetic Acid Bacteria) or role during maturation of fermented food (e.g. ripening bacteria). It can be based on temperature or salt tolerance/requirement for growth, e.g. psychrotrophic, thermophilic/thermoduric, or halophilic microorganisms. Microorganisms can also be classified as risk indicators regarding hygiene of the process or health of the consumer (e.g. coliform bacteria, anaerobic sporeforming bacteria, “total” plate counts or aerobic counts, coagulase-negative and coagulasepositive staphylococci). None of these classifications is completely satisfying. Firstly, members of a given taxonomic rank are susceptible to fall within different technological categories and vice-versa. Secondly, the technological classification is often true in a given
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food but not transposable to another. Thirdly, such grouping is done at genus or species level while lot of food-related properties are strain specific. 1.2. Complex microbial ecosystems A complex microbial ecosystem is composed of a microbial community living in a matrix. It is defined as multi-species assemblage, in which microorganisms live together in a contiguous environment and interact with each other (Konopka 2009). A microbial community can be defined as a group of microorganisms that has different functions and activities distinguishing it from any other. From an ecological point of view, the study of a single isolated microorganism of one species is far from the reality of a microbial ecosystem. To approach the ecological reality, behaviour of strains should be analysed in the corresponding community. This point is very critical in order to be able to anticipate correctly the functionality of strains in a complex ecosystem. Microbial communities have impact from environment to human well being. Evolution, disease, corrosion, degradation, bioremediation, and global cycling are few of the many thousands of ways that microbial communities affect our lives. Microbial layers on the insides of household water pipes, the rolling tanks of bioreactor grains, the microorganisms that extract nutrients from streams, soil microbial communities, ocean plankton microbes, gastrointestinal tract biofilms, oral cavity microbial population and food related microbial communities are few examples. Complex microbial communities, such as biofilms in oral and gastrointestinal tract play an important role in health and diseases (Potera 1999). Microbial communities in human contain 100 times as many genes as the human genome (Versalovic and Relman 2006). Microbes in the form of biofilms are being linked to common human diseases ranging from tooth decay to prostatitis and kidney infections (Potera 1999). Microbial communities are major constituents of fermented foods and they contribute to the preservation of nutrients and to increasing the shelf life of food. Different fermented 3
foods and the associated microorganisms, bacteria and fungi, are known for a wide diversity of matrices, e.g. milk, yoghurt, cheese, beer, wine, sauerkraut, bread, cider etc. (Table 1). Fermented food related microbial communities have a direct effect on human life regarding safety and health effects. Not only food product but also processing surfaces are sight of multi species biofilm developments which could include pathogenic microbes (Kumar and Anand 1998). The role of microbial communities in human health, industrial processes, and ecological functions is under discussion and special attention is given to these microbial ecosystems, which are evolving ecosystems. In fact, they modify progressively with time and in space, due to changes in available metabolites and in physicochemical parameters of their own in situ environment. To illustrate this statement, cheeses especially smear and mould surface ripened cheeses were chosen as example of such evolutive complex microbial ecosystem and described in paragraph 3. 2. Microorganisms involved in manufacturing of fermented food Microorganisms involved in fermented food correspond mainly to bacteria, fungi (yeasts and moulds) but also to viruses including bacteriophages, which can have strong negative impact on fermentation process by destroying a specific strain. These bacteriophages could also contribute to microbial ecology and succession of LAB species in vegetable fermentations (Lu et al. 2003). Among bacteria and fungi encountered in fermented food, most are chemioorganotrophic organisms, which can be either thermophilic, mesophilic, psychrotrophic or psychrophilic. A non-exhaustive list of food-borne microorganisms described in fermented food is shown in Table 2.
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3. Composition of complex microbial communities: Examples of smear- and soft-cheeses microflora Cheese making began about 6000-9000 years ago originating from the middle East (Fox et al. 2000) and now there are about 1400 cheese varieties manufactured worldwide (Beresford et al. 2001). The primary objective of cheese manufacturing was to extend the shelf life and to conserve the nutritious components of milk. Manufacturing of most of varieties of cheese involves the combination of four components/ingredients: milk, rennet, microorganisms and salt, which are processed in the following steps during fermentation: acid production, gel formation, whey expulsion, and salt addition followed by a specific period of ripening (Figure 2). Fermented dairy food products like cheeses are examples of complex microbial communities, involving many strains of different species and genera grown together. Some of their roles are presented in Table 3. Cheese microflora can be divided into two main functional groups according to their functions: lactic acid bacteria, which contribute to acid production, bringing on the curd making, and ripening microbiota. Ripening of smear and mould-ripened cheeses starts with the growth of a large number of yeasts, which increase surface pH. As a result, a salt-tolerant, usually very complex and undefined bacterial consortium begins to develop and eventually covers the entire surface of the cheese, including coryneform bacteria, i.e. Arthrobacter, Brevibacterium (Cogan et al. in press) and some Gram negatives (Larpin et al. 2011), depending on cheese varieties and on dairies (Figure 2).
3.1. Lactic Acid Bacteria Lactic acid bacterias are present in variety of food and participate in the development of texture, flavour and safety quality of many fermented products, including cheeses. They have a common characteristic of lactic acid production from lactose. Starter bacteria encountered most often in cheese technology are members of the genera
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Lactococcus, Lactobacillus, Streptococcus, Leuconostoc, Pediococcus and Enterococcus (Beresford et al. 2001). These microorganisms are Gram positive, catalase-negative, nonspore forming, microaerophiles or facultative anaerobes bacteria. DNA of lactic acid bacteria has less than 55% G+C contents (Stiles and Holzapfel 1997). The use of starter cultures for cheese and sour milk production was introduced by Weigmann in 1890 (Stiles and Holzapfel 1997). The primary function of lactic acid bacteria is to produce acid during the fermentation process; however, they also contribute to cheese ripening in which their enzymes are involved in proteolysis and conversion of amino acids into flavour compounds (Fox and Wallace 1997). Lactic acid bacteria are either added deliberately at the beginning of cheese making or may be naturally present in raw milk and are called non-starter lactic acid bacteria (NSLAB). Generally, starter bacteria can be distinguished into two groups: mesophilic starters (i.e. Lactococcus, Leuconostoc) used for the cheese types in which temperature of the curd is not raised more than 40°C during acidification and thermophilic starters (Lactobacillus helveticus, Lactobacillus delbrueckii, Streptococcus salivarius ssp thermophilus) mostly used for cheese types where curd temperature may rise above 40°C (Fleet 1999). Both mesophilic and thermophilic cultures can be subdivided into mixed (undefined) cultures in which the number of strains is unknown and defined cultures (with known microbial composition). Starters are normally added to milk at initial population of between 10 5 and 107 cfu/ml, but these bacteria develop rapidly and are concentrated by whey expulsion to attain a concentration of 109-1010 cfu/g in the curd after one day of inoculation. Regarding diversity of lactic acid bacteria in soft cheeses, several studies have been done on the Camembert of Normandy, France. Lactococcus sp. is the dominant microflora with about 109 cfu/g during ripening with predominance of Lactococcus lactis (Desmasures et al. 1997; Richard 1984). Lactobacillus is the second dominant group of lactic acid bacteria found in camembert with
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up to 3x107 cfu/g during ripening. Lactobacillus paracasei and L. plantarum were the two species more frequently found in Camembert cheese (Henri-Dubernet 2004).
3.2. Ripening Microflora 3.2.1. Yeasts and moulds Fungi contribute to the organoleptic quality of cheese mainly by the phenomena of proteolysis, lipolysis and by consumption of lactic acid and production of alkaline metabolites such as ammonia and also for few yeasts species by fermentation of lactose (Addis et al. 2001). Their role in surface deacidification is an important phenomenon which allows further growth of acid sensitive bacteria (Guéguen and Schmidt 1992; Lenoir et al. 1985). Yeasts have ubiquitous characteristics and can be found in different ecological habitats. Their acid tolerant characteristics justify their presence in soft cheeses, where low pH, moisture content, temperature and high salinity favour their growth, numbers on the surface reaching rapidly 105-108 cfu/g (Fleet 1999; Larpin et al. 2006). Yeasts are found in wide variety of cheeses, but diversity is particularly high in those made from raw milk. They are capable of degradation of different organic substances and their role in curd deacidification and in formation of metabolites such as ethanol, acetaldehyde and CO 2 is beneficial. Near 1500 yeasts species from about 100 genera are documented (Barnett et al. 2000), among them about 50 species have been described in ripened cheeses. The yeasts genera frequently isolated from different cheese types includes: Candida, Debaryomyces, Geotrichum, Kluyveromyces, Pichia, Rhodotorula, Saccharomyces, Trichosporon, Torulaspora, Yarrowia and Zygosaccharomyces spp (Beresford and Williams 2004). Investigations on microbial diversity of the surface of Livarot, Limburger and Munster cheeses have shown that G. candidum, D. hansenii, Kluyveromyces lactis and K. marxianus are the yeasts species more frequently present and added as fungal starter. Geotrichum 7
candidum and D. hansenii develop at the start of ripening on the surface of a number of soft cheeses including Camembert, Pont l’Evêque, Tilsit, Limburger, Reblochon and Livarot (Eliskases-Lechner and Ginzinger 1995b; Goerges et al. 2008; Guéguen and Schmidt 1992; Larpin et al. 2006). Some species of Candida, like C. natalensis, C. catenulata, C. intermedia, C. anglica, C. deformans, C. parapsilosis are also found on the surface of either Livarot, Reblochon or Gubbeen cheeses (Cogan et al. in press; Larpin et al. 2011; Mounier et al. 2009). Some moulds are also found as technological agents in several cheese varieties, including Penicillium camemberti, Penicillium roqueforti, Trichothecium domesticum (Choisy et al. 1997; Lenoir et al. 1983) but generally, these are not used in smear-cheese production. In other cheese varieties, P. roqueforti or other moulds like P. expansum, P. janthinellum, P. viridicatum and Rhizomucor spp. can affect the cheese characteristics by acting as spoilage agents (Bockelmann et al. 1999). Fungi are transferred to cheese generally from fabrication premises (air, soil, walls, humans, and raw milk, water brine solution) (Baroiller and Schmidt 1990; Mounier et al. 2005; Mounier et al. 2006b; Viljoen et al. 2003).
3.2.2. Surface bacterial flora Technological non lactic acid bacteria have been found on the surface of different cheeses. This flora is usually aerobic, mesophilic and halotolerant but is acid sensitive. The main groups are coryneform bacteria and Staphylococcaceae (Goerges et al. 2008; Larpin et al. 2011; Maoz et al. 2003; Mounier et al. 2005) and these two groups represents 61 to 71 % of bacterial isolates (Mounier et al. 2008). Gram negative bacteria can also develop on the surface of smear ripened cheeses (Bockelmann et al. 1997; Bockelmann et al. 2005; Feurer et al. 2004b; Feurer et al. 2004a; Larpin et al. 2011; Rea et al. 2007). For example, it has been
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shown that Livarot surface flora consisted of about 34% Gram negative isolates (Larpin et al. 2011).
3.2.2A. Gram positive bacteria Coryneform bacteria The term coryneform is dedicated to bacteria whose characteristic feature is their tendency to arrange themselves in a V-like pattern or lined up; much like logs stacked one against the other. These are Gram positive, non-mobile, mostly aerobic. They are a branch of Actinobacteria (G+C contents > 50 %). In last fifteen years these have been deeply studied and are grouped into the sub orders Micrococcineae and Corynebacterineae composed of 9 and 6 families respectively (Stackebrandt et al. 1997). The coryneform genera found in cheese more frequently include Arthrobacter, Brevibacterium, Brachybacterium, Corynebacterium, Microbacterium and Micrococcus. Arthrobacter is a dominant genus on the surface of certain cheeses like Tilsit, Ardrahan, Durrus, and Milleen. Many Arthrobacter species including A. nicotianae, A. citreus, A. globiformis, A. variabilis and A. mysorens have been isolated from cheese (Eliskases-Lechner and Ginzinger 1995a; Feurer et al. 2004a; Mounier et al. 2005). Arthrobacter arilaitensis and A. bergerei have been described on the surface of French cheeses (Feurer et al. 2004a; Irlinger et al. 2005; Larpin et al. 2006; Rea et al. 2007). These bacteria, especially A. arilaitensis can come from commercial ripening culture and environment (Goerges et al. 2008). The source of these bacteria is not yet clearly understood, but A. arilaitensis has been isolated from raw milk (Mallet et al. 2010). Brevibacterium is the unique genus of the family Brevibacteriaceae. The most frequently described species in cheese was B. linens (Fergal and Patrick 1999). It has been isolated from the surface of various cheeses including Gruyère (Kollöffel et al. 1999) and
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Gubbeen (Brennan et al. 2002). In 2002, by using molecular techniques a large diversity was observed in the B. linens species (Alves et al. 2002). By using DNA/DNA hybridization, B. linens was divided into the three new species B. aurantiacum, B. antiquum, and B. permense (Gavrish et al. 2004). The reference strain B. linens ATCC9175, which was frequently found in cheese, was very similar to B. aurantiacum. So, large number of strains isolated from cheese identified as B. linens would be now reclassified in B. aurantiacum. The Brachybacterium genus contains 12 species, including Br. nesterenkovii, Br. faecium, Br. alimentarium and Br. tyrofermentans that have been isolated from many cheeses such as Salers (Duthoit et al. 2003), Livarot (Larpin et al. 2011), Gruyère (Schubert et al. 1996), Beaufort (Ogier et al. 2004), Saint Nectaire (Delbès et al. 2007). Corynebacterium is the dominant genus on the surface of many cheeses. Five species have been isolated from smear ripened cheeses. Corynebacterium variabile, C. casei, C. flavescens and C. ammoniagenes represented about 32 % of all coryneform isolated from Brick (Valdès-Stauber et al. 1997). In Gubbeen, C. casei represented about 50 % of isolates (Brennan et al. 2002) and C. mooreparkense - subsequently identified by Gelsomino et al. (2005) as a later synonym of C. variabile - was also described. Most Corynebacterium isolates were obtained after three weeks of ripening (Larpin et al. 2011; Rea et al. 2007). The genus Microbacterium is found in very low number on the surface of smear ripened cheeses (Eliskases-Lechner and Ginzinger 1995a; Valdès-Stauber et al. 1997). In Gubbeen, 12% coryneform bacteria were identified as new described species, one being Microbacterium gubbeenense (Brennan et al. 2001; Brennan et al. 2002). Later, this species was again found in Gubbeen (Mounier et al. 2005) and Domiati (El-Baradei et al. 2007). Micrococcus is a genus of the Micrococcaceae family, phylogenetically very close to Arthrobacter but with identical morphology to Staphylococcus. The only species of this genus detected in cheese was Micrococcus luteus (Bockelmann et al. 1997; Mounier et al. 2005).
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Various other coryneform bacteria have been described recently on the surface of smear cheeses, such as Leucobacter spp. (Larpin et al. 2011), Mycetocola reblochoni (Bora et al. 2008) and Agrococcus casei (Bora et al. 2007). Staphylococcaceae Among the five genera included in the Staphylococcaceae family, two have been associated with cheese: Macrococcus and mainly Staphylococcus, while Jeotgalicoccus and Salinicoccus were detected in raw milk (Callon et al. 2007; Mallet et al. 2010). For long time Staphylococcus has remained associated to Micrococcus from a taxonomical point of view. Since 1997, a new classification of Actinobacteria has been proposed in order to redefine the family Micrococcaceae, so Staphylococcus was classified separately (Stackebrandt et al. 1997). This genus is a sub branch of Clostridium-Bacillus, which consists of Gram-positive bacteria having G+C < 50 %. Some strains produce coagulase and/or enterotoxins and are potentially pathogenic or opportunistic pathogens (like S. aureus), contrary to the coagulase negative species found in cheese ecosystem: Staphylococcus equorum, S. vitulinus and S. xylosus which are the main species found in smear cheese (Hoppe-Seyler et al. 2004; Irlinger et al. 1997). In Gubbeen the Staphylococcus strains represented about 2.5% of total bacterial isolates (Brennan et al. 2002), while in Livarot about 9.5 % of bacterial isolates were Staphylococcus spp. (Larpin et al. 2011).
3.2.2B. Gram Negative Bacteria While literature data is abundant regarding the presence of yeasts/moulds or Grampositive bacteria in cheese, Gram-negative bacteria (GNB) have been studied rarely. However, a wide diversity of GNB can be found at relative high population level in raw milk (Desmasures et al. 1997; Lafarge et al. 2004) and in various cheeses including smear cheeses (Larpin et al. 2011; Maoz et al. 2003). GNB usually represent from 18 to 60 % of the bacteria 11
isolated from the surface of European smear cheeses (Larpin et al. 2011; Maoz et al. 2003; Mounier et al. 2005). GNB present on the surface of ripened soft cheese belong mainly to the Moraxellaceae, Pseudomonadaceae and Enterobacteriaceae families (Bockelmann et al. 2005; Maoz et al. 2003; Mounier et al. 2005; Tornadijo et al. 1993). Recently, a study of the GNB associated with French milk and cheeses indicated the existence of a large biodiversity of at least 26 different genera, represented by 68 species including potential new species, identified amongst the 173 studied isolates (Coton et al. submitted). Pseudomonas, Chryseobacterium, Enterobacter and Stenotrophomonas were the genera most frequently found in cheese core and milk samples; while Proteus, Psychrobacter, Halomonas and Serratia were the most frequent genera amongst surface samples. Alcaligenes sp., Hafnia alvei, Marinomonas sp., Raoultella planticola, Ewingella americana were also described on the surface of Livarot cheese (Larpin et al. 2011). Until now, the presence of Gram negative bacteria, and particularly coliform bacteria, in food was considered as an indicator of bad handling, which can spoil the product. There are now evidences of their positive contribution to cheese organoleptic qualities, as demonstrated for Proteus vulgaris (Deetae et al. 2007) or by the use of H. alvei as commercial ripening culture (Alonso-Calleja et al. 2002). Pseudomonas spp. is also able to produce a variety of volatile compounds which may contribute positively to cheeses sensory qualities (Morales et al. 2005a), including sulfur compounds as demonstrated for P. putida (Jay et al. 2005).
3.3. Pathogenic microflora in cheese Cheeses are currently considered to be safe foods for consumers, as they have been implicated in only 1.8% of verified foodborne outbreaks due to zoonotic agents in the EU in 2008 (EFSA 2010). Historically there have been outbreaks of diseases associated with the consumption of cheeses and the predominant organisms responsible have included 12
Staphylococcus aureus and zoonotic bacteria such as Salmonella, Listeria monocytogenes, and verocytotoxin producing Escherichia coli (VTEC) (De Buyser et al. 2001; EFSA 2010; Little et al. 2008; Zottola and Smith 1991). Nearly 2500 serovars have been identified inside the Salmonella genus. Among them, most (about 2000) belong to the subspecies Salmonella enterica ssp enterica. The two main ubiquitous serovars involved in foodborne outbreaks worldwilde are Salmonella Enteritidis and S. Typhimurium, which are responsible for gastroenteritis. Staphylococcus aureus is the bacteria most frequently associated to foodborne outbreaks due to dairy products (85.5% of these outbreaks between 1992-1997 were due to S. aureus according to De Buyser et al. (2001). Due to enterotoxin production by some strains, it can be responsible for diarrhea, and sometimes causes vomiting. About twenty different enterotoxins have been described (Hennekinne et al. 2003), which are thermostable. While raw milk was reported as an important food vehicle in foodborne Campylobacter outbreaks in 2007 (EFSA 2009), cheeses are rarely associated with campylobacteriosis. Listeria monocytogenes is the causative agent of listeriosis. It is a rare pathology in Europe; its incidence in 2008 was 0.3 per 100,000 compared to campylobacteriosis at 40.7 per 100,000 (EFSA 2010). However, because its pathogenicity with around 20-30% fatalities is an important public health concern, a focus is done below on L. monocytogenes. Food ecosystem like cheese is composed of biotic and abiotic components. These components are determining factors in the L. monocytogenes growth in cheese. The presence of L. monocytogenes in a food environment is favored by its ability to grow at refrigeration temperatures (2 to 4oC) with a survival range of 0-45oC, tolerance to pH values as low as 4.5 and to high sodium chloride concentration (up to 10%). Thus, it is very difficult to control L. monocytogenes in a cheese environment during ripening (Farber and Peterkin 1991).
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During ripening of bacterial surface-ripened cheeses (red smear cheeses), the increase in pH on the surface creates a favorable environment for the growth of microorganisms, including contaminants such as L. monocytogenes. Rind of these cheeses is usually considered edible; the accidental presence of L. monocytogenes on surface-ripened cheeses can pose a potential health risk for certain consumers. Because some outbreaks occurred, where cheese was found to be the source, several investigations have been conducted for testing the occurrence of L. monocytogenes in different smear ripened cheeses (Beckers et al. 1987; Breer and Schopfer 1988; Eppert et al. 1995; Loncarevic et al. 1998; Loncarevic et al. 1995; Pini and Gilbert 1988; Rudolf and Scherer 2001; Terplan et al. 1986). While Listeria was the emerging food-borne pathogen of the 1980s and has gained public attention, recent investigations on the hygienic status of red-smear cheese are rarely available. It is therefore unknown whether the lack of outbreaks in recent years is related to the improved hygienic status of these cheeses or due to other factors. The presence of L. monocytogenes in soft and semi-soft cheese made from raw or low heat-treated cow’s milk was detected in three out of seven qualitative investigations. For those investigations with positive findings, the proportions of positive samples ranged from 0.5% to 3.6%. Findings of levels above 100 cfu/g, which is the upper limit in cheese according the European regulation (EC 2073/2005), were not reported. For the batch-based sampling at retail about 2.8% non-compliance was reported for soft and semi-soft cheese (EFSA 2009). It is generally considered on the basis of investigations that L. monocytogenes was found more frequently in high moisture than in low moisture cheese (Ryser 1999). Incidence of Listeria monocytogenes in soft cheeses was found to be surprisingly higher in those made from pasteurized milk (8%) than from raw milk (4.8%) by Rudolf and Scherer (Rudolf and Scherer 2001). Comparable data have been recently obtained by the European Food Safety Agency for European cheeses (EFSA 2009) with respectively 4.2-5.2% for pasteurized and 0.3-0.4% raw milk cheeses and this tendency
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was confirmed recently (EFSA 2010). Low incidence of L. monocytogenes in raw milk cheeses may be due to various factors. One of them is the strong monitoring of raw milk quality encountered in raw milk processing dairies. Another one is the presence of some natural factors in raw milk and thereafter in raw milk cheeses, which might be destroyed during pasteurization (Gay and Amgar 2005), e.g. the lactoperoxidase system, and raw milk microbial communities.
4. Important microbial metabolic pathways in cheese ripening Many studies have been done on the physicochemical parameters of smear ripened cheeses (Choisy et al. 1997; Fox and Wallace 1997; Leclercq-Perlat et al. 2004a; Lenoir et al. 1985). In the cheese, lot of biochemical reactions coexist or occur successively, the major components of the curd being lactose, lactate, proteins, fat and their derivatives. The summary of all processes and their succession is explained in Figure 2, with main metabolites shown in Figure 3. 4.1. Degradation of lactose and lactic acid The lactose is transformed into D-, and principally, L-lactate by lactic acid bacteria during fabrication of curd, and part of lactose is eliminated with whey. Some yeasts also have the capacity of degrading lactose at start of the ripening process (Corsetti et al. 2001; Leclercq-Perlat et al. 1999; Leclercq-Perlat et al. 2000) by the action of a β–galactosidase which has been identified in K. lactis, K. marxianus and D. hansenii (Fleet 1999; Roostita and Fleet 1996). Lactic acid in cheese is usually found in form of lactate, which is the principal carbon source for yeasts and moulds. The degradation of lactate is achieved on surface of cheese during ripening mainly by G. candidum and/or D. hansenii up to negligible concentration (
