Microbes from raw milk for fermented dairy products

International Dairy Journal 12 (2002) 91–109

Microbes from raw milk for fermented dairy products Jan T.M. Wouters*, Eman H.E. Ayad, Jeroen Hugenholtz, Gerrit Smit NIZO food research, P.O. Box 20, 6710 BA Ede, Netherlands Received 2 September 2001; accepted 11 October 2001

Abstract Milk has a high nutritive value, not only for the new-born mammal and for the human consumer, but also for microbes. Raw milk kept at room temperature will be liable to microbial spoilage. After some days, the milk will spontaneously become sour. This is generally due to the activity of lactic acid bacteria. A flora of these bacteria may develop, which can be transferred deliberately to fresh milk in order to maintain or even strengthen it. This principle is the basis for controlled acidification of milk towards products, sustainable and safe, with most often an attractive flavour. Various types of fermented milks and derived products have been developed in all parts of the world, each with its own characteristic history. Their nature depends very much on the type of milk used, on the pre-treatment of the milk, on the temperature (climate) and the conditions of fermentation and on the subsequent technological treatments. Most fermented dairy products contain lactic acid bacteria, but other bacteria, yeasts and moulds may be involved as well. In optimising the manufacturing processes, starter cultures for fermented dairy products have been developed. They are composed of selected microorganisms, propagated as multiple-strain starters consisting of a defined mixture of pure cultures or as mixed-strain starters consisting of an undefined mixture of different types of bacteria. The use of starters, on the one hand, has been tremendously positive with respect to the quality of the product, but, on the other hand, it has diminished the diversity of fermented dairy products. Since the dairy industry is keen to explore new possibilities for enhancing the diversity of its product range, there is a new interest nowadays in searching for potential starter organisms from the pool, which existed at the time of raw milk fermentation. This contribution reviews some potential opportunities and recent developments in this search. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Dairy starters; Lactococci; Mesophilic lactobacilli; Thermophilic lactic acid bacteria; Yeasts and moulds

1. Introduction The preparation of acid milk products has long been known in all countries and is probably as old as cattle breeding. The most natural way to obtain acid curdled milk is actually spontaneous acidification. This process relies on lactic acid bacteria, naturally present in milk as adventitious contaminants, which grow and produce the lactic acid required to coagulate the milk. Sometimes, the lactic acid bacteria are accompanied by yeasts or moulds, which give special features to the fermented product. These natural cultures are subsequently used to inoculate fresh milk the following day. In this way, several tribes around the world developed their own characteristic fermented milks. Kefir is an alcohol*Corresponding author. Tel.: +31-318-65-9511; fax: +31-318-659522. E-mail address: [email protected] (J.T.M. Wouters).

containing milk from Caucasian countries, fermented by a co-culture of yeasts and lactic acid bacteria. In south Russia and Siberia, the fermentation of horse milk results in koumiss, also alcohol-containing and with yeasts and lactic acid bacteria as biological agents. A third alcoholic acid milk is mazun from Armenia. Furthermore, there is leben from Egypt and yoghurt from Turkey and Bulgaria. The Italians have their gioddu and in Western Europe, buttermilk is well known. Scandinavian fermented milks are viili and langfil. The development of cheese fermentation also relied on the spontaneous development of lactic acid bacteria. Traditionally, farmers and shepherds made cheese from raw cows’, goats’ or ewes’ milk on a small scale, using naturally occurring lactic acid bacteria. Cultures of these bacteria were produced by incubating the milk or whey from the previous day under specific conditions. Today, this traditional way of producing cheese is still common

0958-6946/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 0 1 ) 0 0 1 5 1 - 0

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in many Southern European countries and these cheeses are generally designated as ‘artisanal’ (Cogan et al., 1997). The lactic acid bacteria used in the dairy fermentations can roughly be divided into two groups on the basis of their growth optimum. Mesophilic lactic acid bacteria have an optimum growth temperature between 201C and 301C and the thermophilic have their optimum between 301C and 451C. It is not surprising to discover that the traditional fermented products from sub-tropical countries harbour mainly thermophilic lactic acid bacteria, whereas the products with mesophilic bacteria originate from Western and Northern European countries. Most dairy industries use today starter cultures for rapid acidification, because the relatively small amounts of lactic acid bacteria in raw milk acidify the milk only slowly. These starter cultures are selected and maintained by subcultivation in milk. This method of working has reduced the number of strains present. The use of these starter cultures, which is also necessary when fermenting pasteurised milk, is responsible for a certain uniformity of the products, fermented milks as well as cheeses. The introduction of new fermentation techniques has the drawback that the raw milk floras are in danger of being lost. The nonstarter lactic acid bacteria in these floras, however, are believed to contain strains, which are essential for producing the characteristic flavours of traditional cheeses. Therefore, there is a strong need to study these raw milk floras more closely and to understand their importance in flavour formation. These studies may yield strains with promising and useful properties, which will make them applicable as starters for product innovation. In the following sections, several examples of these raw milk microorganisms will be presented. The first group to be discussed comprises the lactococci, the most prominent group of mesophilic lactic acid bacteria applied in dairy fermentations. Special emphasis will be put on the wild lactococci. Another group of mesophilic lactic acid bacteria that will be discussed is that of the lactobacilli, which appear as adventitious flora during the ripening of various cheese types and are designated as non-starter lactic acid bacteria. The thermophilic lactic acid bacteria are best known for their role in yoghurt-type products and as ripening agents in Swiss-type and Italian cheeses. There are now several studies, which show the broad spectrum of strains used for the manufacture of a great variety of products and which point to an interesting pool of potentially useful starters. Attention will also be paid to the secondary flora potentially emerging in milk fermentation after the primary acidification phase, particularly to the eucaryotic yeasts and moulds. Finally, a synopsis of possible future developments will be presented.

2. Lactococci 2.1. Starter cultures If raw milk is left at room temperature for some time, a microflora will develop in which mesophilic lactococci generally predominate. After maintaining this flora by subculturing it in milk, the number of different strains in the culture will decrease and a culture with only lactococci may eventually emerge. These bacteria acidify the milk and, as a consequence, the growth of the other indigenous bacteria is largely inhibited. This principle has already been practised in the manufacture of fermented dairy products for centuries, even before it was known that bacteria were actually involved at all. Inoculating the milk with some of the previous day’s product was the basis for a successful fermentation. The discovery of the role of lactococci in this success paved the way for their isolation, characterisation and exploitation. This started in 1878 (Lister, 1878) and has resulted in the development of starter cultures for the manufacture of fermented dairy products. Their use provides microbiologically safe products with reproducible organoleptic and structural properties. Both industrial and small-scale manufacture of fermented dairy products now almost always relies on industrially prepared starters. Starter cultures of mesophilic lactococci are used in the manufacture of a broad array of cheese types, of butter and of various fermented milks, irrespective of the type of milk. The selection of these cultures has been based on their performance in the fermentation and on the desired product properties. Also, the handling properties and the stability during the production have been a selection criterion for the starters (Marshall, 1991). The number of starter producers in the world is limited and, therefore, the possibilities of choice of starter cultures for the fermentation of milk are restricted. In the dairy industry, starters can be divided into two groups: undefined and defined starters. The undefined starters are a mixture of an unknown number of lactic acid bacteria types, which are derived from an artisanal production practice. For cheese manufacture, these mixtures of strains evolved from artisanal cultures that produced good-quality cheese. They were propagated in the laboratory under controlled conditions and commercialised as inocula for industrial cheese manufacture. Mesophilic starter cultures are commonly used for the manufacture of Cheddar, Gouda and other cheese types. Their use is based on their consistent performance, especially their well-recognised phage resistance (Stadhouders & Leenders, 1984; Limsowtin, Powell, & Parente, 1996; M.ayr.a-Makinen & Bigret, 1998). The mixed-strain starters for the manufacture for Gouda cheese are composed of acid-forming lactococci,

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Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris, together with citrate-utilizing strains, L. lactis subsp. lactis biovar diacetylactis and Leuconostoc spp. These starters are thus composed of complex mixtures of strains, forming a bacterial population, which is equipped with the properties suitable for the production of the desired cheese. Since their composition would change depending on the conditions of cultivation (Hugenholtz, 1986), their subculturing is minimised and the cultures are preserved by freezing or lyophilisation. They are propagated in the dairy only once before inoculation in the cheese milk. Nowadays, even starters for direct inoculation are available, which eliminates the need for subculturing in the dairy and reduces the risks of changes in composition (Sandine, 1996). Defined-strain starters are blends of two of more strains; in the case of Cheddar cheese, they are composed of a limited number of L. lactis strains. Since the risk of phage attacks is greater here than with the use of undefined mixed cultures, cultures with different phage sensitivity profiles are used in rotation (Heap & Lawrence, 1988; Limsowtin et al., 1996; Heap, 1998). In these systems with a limited number of strains, a phagesensitive strain may be replaced by a secondary resistant strain, derived from the original strain, as soon as a phage attack occurs (Daniell & Sandine, 1981; Hull, 1983; Timmons, Hurley, Drinan, Daly, & Cogan, 1988). 2.2. Starter functions It is interesting to consider that the industrial starters for the majority of cheeses are based on a single species, namely Lactococcus lactis. There are many strains of this species employed for the manufacture of various cheese types and, although they exhibit different characteristics, they have several biochemical attributes in common (Crow, Coolbear, Holland, Pritchard, & Martley, 1993; Desmazeaud & Cogan, 1996). The most important properties are their ability to produce acid in milk and to convert milk protein into flavour components. During ripening of cheese, proteolysis is the first biochemical step in the latter process toward the desired flavour and texture. Lactococci possess a proteolytic system that, together with other protein-hydrolysing enzymes like chymosin, is responsible for the conversion of casein into peptides and amino acids (Fox, O’Connor, McSweeney, Guinee, & O’Brien, 1996; McSweeney & Fox, 1997). Amino acids are the key precursors for the essential cheese flavour. They are metabolised by the action of amino acid-converting enzymes to aldehydes, alcohols, ketones, amines, acids, esters and sulphurcontaining compounds, which all contribute to the cheese flavour (Hemme, Bouillanne, Me! tro, & Desmazeaud, 1982; Urbach, 1993, 1995; Engels & Visser, 1996; Engels, 1997; Yvon, Berthelot, & Gripon, 1998). In all these conversions, the lactococci are involved.

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Various groups of volatile flavour components have been identified as being responsible for the final aroma and flavour of cheese. Based on sensory evaluation and chemical analysis of cheeses, the compounds mentioned above have been found in most cheeses, although a large diversity occurs in the relative concentrations among the cheese varieties (Badings, 1991; Bosset & Gauch, 1993; Urbach, 1995). We found that the differences in flavour between the types of cheese correspond largely with the distinct character of the starter used and are therefore highly strain-specific (Smit et al., 2000). The use of standardised starters (whether undefined or defined), of strictly hygienic processing methods and of well-controlled ripening conditions has had an enormously positive effect on cheese quality. However, as a consequence of the constant quality, the diversity of flavour varieties in any one type of cheese has diminished. The consumer market of today requires instead an increase in product diversification and this calls for innovations, for which novel starter strains are needed. These strains may be found in the original niches for lactococci, the raw milk environment (Steele . & Unlu, 1992). 2.3. Wild lactococci Mesophilic lactococci are generally considered to be associated with the milk environment (Sandine, Radich, & Elliker, 1972), but they can also be isolated from other sources (Klijn, Weerkamp, & de Vos, 1995). Lactococci isolated from artisanal manufacture of fermented dairy products without the application of industrially prepared starter cultures and from non-dairy environments are generally referred to as ‘wild’ lactococci. In an international project funded by the European Community, many wild strains of lactic acid bacteria were isolated and partially characterised (Cogan et al., 1997). Initial studies showed that this pool of lactic acid bacteria contained many L. lactis strains, which differ in a number of phenotypic properties from the strains commonly present in industrial starters. L. lactis subsp. lactis in the latter are characterised by their ability to hydrolyse arginine, to metabolise a number of sugars and to grow at 401C and/or in the presence of 4% NaCl. Members of the subspecies cremoris, however, are not able to grow under these conditions (Salama, Sandine, & Giovannoni, 1991; Cogan, 1996). Although most of the isolated wild lactococci matched this pattern, the phenotypic properties of some strains were not in line with this expected distinction (Ayad, Verheul, de Jong, Wouters, & Smit, 1999). Also, some strains that were phenotypically L. lactis subsp. lactis appeared genotypically L. lactis subsp. cremoris and vice versa (Salama, Sandine, & Giovannoni, 1993; Godon, Delorme, Ehrlich, & Renault, 1992; Salama et al., 1991; Klijn et al., 1995). The standard tests for phenotype characterisation

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can thus give ambiguous results with lactococcal strains, especially with those isolated from natural and hostile niches. This may lead to misclassification at the species level (Corroler, Mangin, Desmasures, & Gueguen, 1998). L. lactis subsp. lactis strains are commonly isolated from natural niches, whereas strains with a L. lactis subsp. cremoris phenotype are isolated only from a dairy environment. The habitat of the latter strains remains uncertain, because they may not survive in nature and may be exclusively confined to the milk environment (Klijn et al., 1995). Genetic studies using RAPD classification revealed that wild lactococci had profiles different from those of reference strains (Corroler et al., 1998). It is thus possible or quite likely that natural habitats, including raw milk, may harbour interesting novel strains of lactococci with potential application in producing new fermented dairy products. Lactococci require various amino acids for growth because of their limited biosynthetic capacity. The requirement of a certain amino acid for growth can result either from the absence of functional genes for specific biosynthetic reactions or from specific regulatory mechanisms (Chopin, 1993). The number of essential amino acids is strain-dependent and may vary from six for L. lactis subsp. lactis up to 14 for certain L. lactis subsp. cremoris strains (Reiter & Oram, 1962). At NIZO food research, Ayad et al. (1999) studied the amino acid requirements of wild lactococcal strains and compared them with those of industrial strains. Whereas the industrial cremoris subspecies required nine or ten amino acids, the wild L. lactis strains, lactis as well as cremoris subspecies, needed only one to three amino acids. The absence of some amino acid biosynthetic pathways in dairy lactococci might be a consequence of their adaptation to the milk environment (Deguchi & Morishita, 1992). In milk, amino acids are readily available by the proteolysis of caseins, and therefore, the need for such biosynthetic enzymes in starter culture strains is limited. Wild strains generally lack a rich medium like milk, which makes them more dependent on their own biosynthetic capacity than the industrial strains. Thus, wild strains are generally less demanding than industrial strains with respect to the amino acid supply. Wild strains probably harbour more and more active amino acid convertases and since these enzymes may play a key role in the formation of amino acidderived flavour components, this pool of strains is probably also interesting with respect to novel flavour formation in the manufacture of fermented dairy products. 2.4. Role in flavour formation Wild lactococci isolated from dairy and non-dairy environments were screened in our laboratory for their flavour-producing capacity in milk and in cheese models

(Weerkamp, Klijn, Neeter, & Smit, 1996; Ayad et al., 1999). Several strains exhibited more pronounced sensory characteristics than the reference industrial starter strains. The flavours were described as unusual by the sensory panel for 57 out of the 79 strains examined (Ayad et al., 1999). The unusual flavour profiles were described as cocoa, chocolate, malty, burnt, acid, yeasty, sweet, fruity, etc. The production of volatile compounds during growth in milk and cheese paste was examined for some characteristic strains using purge and trap thermal desorption cold-trap gas chromatography mass spectrometry (GC-MS) (Neeter & De Jong, 1992). An example of the GC-MS profile of a wild lactococcus strain as compared to that of an industrial strain is presented in Fig. 1. The relatively high levels of the methyl alcohols and methyl aldehydes containing four or five carbon atoms in the culture with the wild strain were striking. These compounds are most likely derived from the branched-chain amino acids, leucine, isoleucine and valine (Morgan, 1976; Molimard & Spinnler, 1996). The formation of methyl aldehydes in milk by the metabolic activity of L. lactis subsp. lactis biovar maltigenes has been recognised as the cause of off-flavours in Cheddar cheese (Morgan, 1976). On the other hand, 3-methylbutanal has been characterised as an important volatile compound formed during the ripening of Parmesan and Proosdij-type cheese, which is responsible for a spicy cocoa flavour (Barbieri et al., 1994; Neeter, de Jong, Teisman, & Ellen, 1996). Apparently, this aldehyde may have positive as well as negative effects on the sensory perception of a cheese. The contribution of 3-methylbutanal to the overall perception of the cheese probably depends on the other volatiles present and the composition of the matrix. In recent years, it has become clear that a number of enzymes are involved in the conversion of amino acids into flavour components. They have been identified in several strains of starter cultures (Lee, Lucas, & Desmazeaud, 1985; Alting, Engels, van Schalkwijk, & Exterkate, 1995; Yvon, Thirouin, Rijnen, Fromentier, & Gripon, 1997; Yvon & Rijnen, 2001). These enzymes may catalyse various reactions including deamination, transamination, decarboxylation and cleavage of the amino acid side chain. The first step in, for instance, the conversion of branched-chain amino acids by lactococci is their transamination into keto acids (Engels, 1997; Yvon et al., 1997; Gao & Steele, 1998; Roudot-Algaron & Yvon, 1998; Engels et al., 2000). These keto acids may undergo spontaneous degradation (Gao, Oh, & Steele, 1997) or may be enzymatically converted into the corresponding aldehydes or carboxylic acids (Smit et al., 2000). Most of these biochemical activities have been demonstrated in in vitro experiments, but how and whether they proceed in vivo is not easy to establish. The conditions in a ripening cheese are generally far from optimal for the enzyme reactions and their

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Fig. 1. GC-MS profiles of volatile flavour compounds from a cheese model (Ch-easy) inoculated with the Lactococcus lactis subsp. lactis strains, B1152 (wild strain) and SK110 (industrial strain), respectively. Relative peak areas are expressed in arbitrary units (from Ayad et al., 1999).

progress is therefore slow. Also, the enzymes for the formation of a specific flavour component are probably not all present in one strain. In that case, more than one strain is required to perform the complete conversion. This co-operation between strains in a milk environment has elegantly been shown in our laboratory by Ayad, Verheul, Engels, Wouters, and Smit (2001b). We studied the formation of the flavour component 3-methylbutanal in milk by a mixture of two L. lactis subsp. cremoris strains, one industrial and one wild. The proteolytic industrial strain was able to release leucine from casein and to transaminate this amino acid into a-ketoisocaproic acid. The non-proteolytic wild lactococcus performed the transamination reaction as well, but more importantly, could complete the subsequent decarboxylation step towards 3-methylbutanal. This phenomenon was confirmed by demonstrating the presence of the corresponding enzyme activities in the cell-free extracts of both strains. The strains complement each other with respect to their enzyme activities and this results in an enhanced production of the chocolate flavour component 3-methylbutanal (Fig. 2).

3-Methyl butanal production proteolysis

(A)

casein

transamination leucine

decarboxylation

α-ketoisocaproic acid

3-methyl butanal

(B) SK110

—

(C) B1157

—

(D)

SK110 + B1157

+

Fig. 2. Proposed pathway of flavour fromation from leucine by enzymes from individual and combined lactococcal starter cultures B1157 and SK110: (A) general pathway for the breakdown of caseins to 3-methyl butanal; (B) enzymatic steps in SK110; (C) enzymatic steps in B1157; and (D) enzymatic steps in the defined mix SK110+B1157. In the decarboxylation step, the dashed arrow represents very low activity while the thick arrow represents high activity (from Ayad et al., 2001b).

The production of 3-methylbutanal is characteristic in cheeses made with thermophilic lactic acid bacteria, like Parmesan and Proosdij-type cheese (Bosset & Gauch,

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1993; Barbieri et al., 1994; Neeter et al., 1996). The latter type is essentially produced like a Gouda cheese, but with the addition of an extra mixed-strain thermophilic culture. This culture contains mainly Streptococcus thermophilus and Lactobacillus helveticus (APS culture), which are precultivated in such a manner that they do not contribute to the acidification of the cheese milk. There are several variants of the APS culture available and one of them produces sufficient 3-methylbutanal, but tends to decarboxylate glutamic acid to g-aminobutyric acid and carbon dioxide (Zoon & Allersma, 1996). This decarboxylation occurs later on during the ripening of the cheese, and due to the visco-elasticity of the cheese matrix at that stage, this may cause undesired crack formation. An alternative APS culture, on the other hand, does not show this deficiency, but produces insufficient 3-methylbutanal. By supplying in the latter case, a wild lactococcus strain able to synthesise the typical flavour component 3-methylbutanal, we successfully manufactured a Proosdij cheese without cracks and with the right flavour (Ayad, Verheul, Bruinenberg, Wouters, & Smit, 2001a). Not all of the 3-methylbutanal-producing strains were suitable in this respect, because the production of excess 3-methylbutanal was experienced as an off-flavour. This indicates clearly that the right flavour of a cheese is the result of a subtle balance between various flavour attributes. 2.5. Bacteriocins Different lactic acid bacteria are able to produce bacteriocins, which are proteinaceous substances with bactericidal activity against microorganisms closely related to the producer strain (Klaenhammer, 1988; Jack, Tagg, & Ray, 1995); this property has been reviewed by several authors (Daeschel, 1989; Piard & Desmazeaud, 1992; Klaenhammer, 1993). Lactic acid bacteria are generally regarded as safe microorganisms and so are their bacteriocins. Thus, these bacteriocins can potentially be used to control the growth of spoilage and pathogenic organisms in food (Hoover & Steenson, 1993; De Vuyst & Vandamme, 1994). Bacteriocinproducing lactococcal strains have been used successfully in starter cultures for cheesemaking in order to improve the safety and quality of the cheese (Lipinska, 1973; Maisnier-Patin, Deschamps, Tatini, & Richard, 1992; Delves-Broughton, Blackburn, Evans, & Hugenholtz, 1996; Ryan, Rea, Hill, & Ross, 1996). We recently checked the production of bacteriocins and bacteriocin-like compounds by wild lactococci and found that 32 out of 79 strains were antimicrobially active (Ayad, Verheul, Wouters, & Smit, 2002). In 17 of these strains, the well-known antimicrobial peptide nisin was found, whereas the other produced diplococcin (2), lactococcin (3) or an unidentified bacteriocin-like compound (10). This percentage of bacteriocin-positive

strains is relatively high if compared with previously published data (Klijn et al., 1995; Cogan et al., 1997; Estepar, Sa! nchez, Alonso, & Mayo, 1999). It is furthermore in line with the general observation that lactococci isolated from non-dairy natural niches show a higher incidence of antimicrobial production than strains isolated from a dairy environment, including industrial starters. This phenomenon can possibly be explained by the fact that the ability to produce antimicrobial compounds offers these wild strains the power to withstand the competition of other microorganisms and thus to survive in their hostile natural environment. The bacteriocin-producing wild lactococcal strains may be useful as starters in cheesemaking, not only because of their antimicrobial activity but also because of their potential to synthesise interesting flavour compounds. These strains should then be combined with other strains, which are bacteriocin-resistant.

3. Mesophilic lactobacilli 3.1. Non-starter lactic acid bacteria Although mesophilic lactobacilli are undoubtedly inhabitants of raw milk and the dairy environment, upon acidification of raw milk, they are frequently overgrown by strong acidifiers of the genus Lactococcus. However, they do gain access to the cheesemaking process, because they are often found as secondary flora during the ripening of different cheese varieties. This is especially true for raw-milk cheese, but mesophilic lactobacilli are also common in cheese manufactured with modern technologies, using pasteurisation of the milk, defined-strain starters and hygienic processing. The starter is responsible for the acidification during the first stages of cheese manufacture and may reach up to 109 colony-forming units (cfu) per gram of cheese. During ripening, however, the number of starter cfu generally decreases rather quickly to lower than 107 g 1. Non-starter adventitious lactobacilli, which apparently originate from the milk or the environment, grow out subsequently and may reach numbers higher than those of the starter (Shakeel-Ur-Rehman, Fox, & McSweeney, 2000). The secondary flora in Cheddar cheese has been examined most extensively; it consists mostly of mesophilic lactobacilli and sometimes also pediococci (Peterson & Marshall, 1990). These bacteria are collectively referred to as non-starter lactic acid bacteria (NSLAB). Isolates from this group belong to the species Lactobacillus paracasei, Lb. plantarum, Lb. rhamnosus and Lb. curvatus. The composition of the NSLAB in the cheese varies with the day of manufacture and with the age of the cheese (Williams & Banks, 1997; Fitzsimons, Cogan, Condon, & Beresford, 2001). Adventitious

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NSLAB have also been reported for Emmental-type cheeses. They develop following the acidification by the thermophilic flora and together with the propionic acid bacteria. They may reach levels up to 5  107 per g of cheese (Gilles, Turner, & Martley, 1983; Turner, Morris, & Martley, 1983; Demarigny, Beuvier, Dasen, & Duboz, 1996). Mesophilic lactobacilli are also detected as a dominant non-starter microflora in Comte! cheese (Grappin, Beuvier, Bouton, & Pochet, 1999; Berthier, Beuvier, Dasen, & Grappin, 2001). Reports on the occurrence of mesophilic lactobacilli in Gouda cheese are scarce, but their presence has been established (Kleter, 1976). The number of homofermentative mesophilic lactobacilli in Spanish artisanal starter-free cheeses made from ewes’ and goats’ milk was 107 to even ! more than 109 cfu g 1 (Lopez & Mayo, 1997). The cheeses examined included hard, semi-hard, and blueveined varieties. The lactobacilli were classified as Lb. plantarum, Lb. casei subsp. pseudoplantarum, Lb. ! curvatus and Lb. casei subsp. casei (Lopez & Mayo, 1997). Cogan et al. (1997) characterised 4379 strains of lactic acid bacteria isolated from artisanal products and classified 12% of them as mesophilic lactobacilli. Bizzarro, Torri Tarelli, Giraffa, and Neviani (2000) reported recently on the phenotypic and genotypic characterisation of mesophilic lactobacilli occurring in Pecorino Toscana cheese, a semi-hard cheese made from pasteurised ewes’ milk with the Designation of Protected Orgin from Tuscany. The strains were identified as Lb. curvatus, Lb. paracasei subsp. paracasei, Lb. plantarum and Lb. brevis. The NSLAB have the unique ability to grow under the highly selective conditions prevailing in a ripening cheese. Lactose is largely depleted in the first hours of cheese manufacture by the fermentation of the starter bacteria. The pH is between 4.9 and 5.3, the temperature below 131C, the moisture content o50%, the salt concentration in moisture is 4–6% and oxygen is barely available. All in all, the ripening cheese seems a hostile environment for microorganisms. Yet, the adventitious lactobacilli manage to grow, obviously with a low rate, but the generally long ripening period allows them enough time to reach considerably high levels of cfu per gram of cheese. They apparently consume compounds other than lactose such as lactate, citrate, glycerol, amino sugars, amino acids and other metabolites (Peterson & Marshall, 1990). Even growth on material released by autolysing starter bacteria or from the membrane of fat globules has been proposed (Thomas, 1987). Although the NSLAB, like other lactobacilli, exhibit fastidious nutritional requirements, they clearly find ample opportunities for growth in ripening cheese. They possess a wide range of hydrolytic enzymes and are able to effect proteolysis and lipolysis (Khalid & Marth, 1990; Williams & Banks, 1997).

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3.2. Role in ripening of cheese Since NSLAB dominate the microflora of many longripened cheeses, they are believed to contribute to the maturation of cheese. The numbers of NSLAB are reported to be higher in Cheddar cheeses made from raw milk than in those from pasteurised milk (McSweeney, Fox, Lucey, Jordan, & Cogan, 1993). Differences in flavour between these cheeses, with a more intense flavour in raw milk cheeses, suggest that the indigenous NSLAB play an important role in flavour development. Indeed, they have been shown to contribute to the formation of small peptides and amino acids, which are the precursors for the flavour components (Fox, 1998). It thus seems likely that the indigenous NSLAB are at least partly responsible for this difference. Several studies have shown that the NSLAB consist of a wide variety of strains and that the composition of their population may vary between cheeses and with the age of the cheese. During ripening of Cheddar cheese, for most cheeses, no single NSLAB strain predominates, with up to 20 strains forming the NSLAB population (Crow, Curry, & Hayes, 2001). The population dynamics of NSLAB is clearly rather complex, and to understand the ripening process, the interactions that occur between the various bacteria in the flora should be elucidated. It is already known that one NSLAB strain can affect the development of another. The outgrowth of a Lb. rhamnosus strain, added on purpose as an adjunct in a Cheddar cheese trial, could be retarded by a simultaneously added Lb. casei strain (Martley & Crow, 1993). The development of a secondary flora depends also on the properties and composition of the starter used. The presence of a Leuconostoc species in the starter appears to affect the development of adventitious NSLAB (Martley & Crow, 1993). This may explain why the reports on the occurrence of NSLAB in Gouda cheese are rather limited. Most of these cheeses are manufactured with starters containing Leuconostoc species (Stadhouders & Leenders, 1984; Walstra, Noomen, & Geurts, 1999). There are further indications that the homofermentative NSLAB are most prevalent in Cheddar cheese, but heterofermentative strains may also develop, usually during the later stages of ripening. These strains appear to be associated with the generation of undesirable flavours (Peterson & Marshall, 1990). An example of such a strain is Lb. brevis. The presence of mesophilic lactobacilli may also have some other negative effects on the quality of the cheese. The mesophilic starters in Cheddar and Gouda cheese produce l(+)-lactate, whereas NSLAB produce d( )or dl-lactate, and racemise l(+)-lactate into the d( ) isomer (Thomas & Crow, 1983). The calcium salt of d( )-lactic acid tends to precipitate as white crystalline

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deposits on the surface of the cheese, which is detrimental for consumer acceptance (Johnson, Riesterer, & Olson, 1990). Heterofermentative representatives of NSLAB may further decarboxylate free amino acids, formed from degradation of protein and peptides during cheese ripening, to amines and carbon dioxide. Some of these amines are potentially toxic and outbreaks of food poisoning after consumption of cheese have been reported (Taylor, Keefe, Windham, & Howell, 1983). These outbreaks have been associated with Swiss-type cheese containing large amounts of histamine. Its production was believed to be caused by the heterofermentative Lb. buchneri. The formation of another amine, tyramine, in a Gouda cheese made from raw milk has been related to the presence of Lb. brevis in that cheese (Joosten & Northolt, 1989). Also, the formation of slits in Cheddar cheese has been correlated with heterofermentative NSLAB in the secondary flora (Martley & Crow, 1993).

3.3. Adjunct starters The observation that the presence of NSLAB in cheese on the one hand leads to a desirable flavour, and on the other hand may induce possible defects or spoilage, makes it a delicate choice for the cheesemaker to use a certain lactobacillus as adjunct starter. This strain should be selected with care, because only a limited number of the NSLAB present in cheese combine all the required properties with the concomitant lack of imperfections. Adjunct cultures may be defined as those added to cheese for purposes other than acid formation. Selected adjunct NSLAB cultures can be added to accelerate ripening and to produce desirable flavour. They may eliminate defects by adventitious NSLAB, since they inhibit their outgrowth. Several trials have been done with adjunct lactobacilli in the manufacture of Cheddar cheese. McSweeney et al. (1994) were successful in improving the cheese flavour by using strains isolated from raw milk cheese. This improvement was believed to be due to increased formation of amino acids. Cheese made from milk inoculated with strains of Lb. plantarum or Lb. casei subsp. pseudoplantarum received the best gradings (Lynch, McSweeney, Fox, Cogan, & Drinan, 1996). It is very likely that adjuncts with improved ripening properties will be developed, not only for Cheddar cheese but also for other cheeses with a long ripening period. New systematic approaches for selecting the right combination of starter and adjunct for improvement in the quality of the cheese should soon become available (Beresford, Fitzsimons, Brennan, & Cogan, 2001; Swearingen, O’Sullivan, & Warthesen, 2001).

4. Thermophilic lactic acid bacteria 4.1. Fermented milks The thermophilic lactic acid bacteria are best known as starters for fermented milks. Several varieties of fermented milks originate from countries in Asia Minor and the Balkans, like Armenia, Turkey and Bulgaria. These products have emerged from spontaneous acidification of raw milk by indigenous organisms. Although these organisms have by no means been exhaustively characterised, they consist largely of thermophilic lactic acid bacteria, probably due to the relatively high incubation temperature determined by the prevailing climate. The first description of milk fermentations by these bacteria can be found in the literature of some hundred years ago (Weigmann, 1905). Several attempts were made at that time to identify the bacteria dominating the flora in yoghurt-like products and they were given the names Bacillus bulgaricus and Diplostreptococcus. These spontaneous fermentations of milk into yoghurt have now been developed into microbiologically well-controlled industrial processes. The two most frequently used starter bacteria are now classified as Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus, generally shortened as Lb. bulgaricus and S. thermophilus, respectively. Yoghurt is usually made by inoculating a 1:1 mixture of S. thermophilus and Lb. bulgaricus in milk and incubating the milk at 37–451C. S. thermophilus grows optimally at these temperatures and hydrolyses lactose via a b-galactosidase. S. thermophilus is nutritionally fastidious and requires a complex mixture of amino acids for growth. Since it is weakly proteolytic, the coupling with the proteolytically more sophisticated Lb. bulgaricus stimulates its growth in milk. The production of formic acid and carbon dioxide from lactose by S. thermophilus stimulates the growth of Lb. bulgaricus (Driessen, Kingma, & Stadhouders, 1982). The thermophilic lactic acid bacteria offer yoghurt and other fermented milks a number of nutritive advantages as compared to the original milk. Most importantly, the product is better preserved by the lowering of the pH. Furthermore, the milk protein is partly degraded by the action of the bacterial proteolytic system and thus more easily digestible for the consumer. Also, lactose is partly hydrolysed, which is certainly beneficial to those consumers who are lactase-deficient and may suffer from some form of lactose intolerance. Some strains of lactic acid bacteria may synthesise vitamins and enrich the milk accordingly (Forsse! n, . 2000). Finally, yoghurt J.agerstad, Wigertz, & Witthoft, has good organoleptic properties with respect to flavour as well as texture. The most characteristic flavour component is acetaldehyde, produced by the metabolic

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activity of the starters. The texture of yoghurt is largely the result of choice of the technology of production process, but even here, the lactic starters may play a crucial role by producing exocellular polysaccharides, which positively affect the rheological properties of the yoghurt. 4.2. Proteolysis and flavour formation The attributes mentioned above will be elaborated in order to evaluate further the potential added value of the thermophilic lactic acid bacteria for yoghurt. The starters for yoghurt are known to be responsible for the hydrolysis of milk proteins. Especially, Lb. bulgaricus has a highly competent proteolytic system, consisting of an extracellular proteinase (Khalid & Marth, 1990) and several peptidases (Bockelmann, Fobker, & Teuber, 1991; Bockelmann, Schulz, & Teuber, 1992). The hydrolysis of milk protein by this proteolytic system not only stimulates the growth of the co-cultivated S. thermophilus, but may also liberate interesting peptides from the caseins and serum proteins. These proteins are known to include some amino acid sequences which upon liberation from the protein molecule exert a specific biological activity on the physiology of the consumer (Meisel & Schlimme, 1996). During the ripening of cheese, the hydrolysis of casein has been shown to liberate such a biologically active peptide, which has the potential to affect the blood pressure by regulating an angiotensin-converting enzyme (Meisel, Goepfert, & Gunther, . 1997). Whether similar or other functional peptides are produced during the fermentation of yoghurt is not known, but is certainly worth examining. There are reports of the presence of functional peptides in milk fermented with Lb. helveticus (Yamamoto, Akino, & Takano, 1994; Masuda, Naka-

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mura, & Takano, 1996), but a possible role of Lb. bulgaricus in this respect is not yet established. The right choice of the starter from the available pool of thermophilic lactic acid bacteria apparently offers enough possibilities for the production of yoghurt with an added health value. Another important feature of the yoghurt starter organisms is the synthesis of the characteristic flavour component, acetaldehyde, which is essentially a product of the protein degradation. It is produced from threonine, which is converted into glycine and acetaldehyde by threonine aldolase. Glycine is subsequently converted into serine by serine hydroxymethyltransferase, both compounds being major sources of one-carbon units necessary for the further biosynthesis. Interestingly, these reactions are also involved in the metabolism of folic acid (Ogawa, Gomi, & Fujioka, 2000). Until now, these reactions have not been studied extensively in the yoghurt organisms, but both bacteria appear to have an active threonine aldolase. Preliminary studies have shown that there are major differences among the various species of S. thermophilus and Lb. bulgaricus with respect to the activity and regulation of this enzyme (Mierau, Kleerebezem, Lerayer, Hugenholtz, & Chaves, 2001). Studies on the metabolic regulation in the strains studied clearly revealed that the right choice of strains and cultivation conditions offers ample opportunities to optimise the flavour development in yoghurt. 4.3. Folic acid production An interesting observation for the health of the consumer is the presence of a higher concentration of folic acid in yoghurt than in milk. S. thermophilus is known to produce folic acid during growth in milk (Rao, Reddy, Pulusani, & Cornwell, 1984; Rao &

160 140

Folic acid production by S. thermophilus

ng/g milk

120 100 80 60 40

0

B133 B119 B1128 B886 B126 B1122 B127 B883 B124 B130 B885 B132 B123 B128 B131 B125 B115 B121 B106 B109 B117 B116 B112 B118 B105 B114 B110 B108 B104 B113 B107 B111 B103 B129

20

Fig. 3. Folic acid production by different strains of Streptococcus thermophilus, grown in milk (A. K. C. Mertens & L. Tijsseling, unpublished results).

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Shahani, 1987), but the extent of production is straindependent (A. K. C. Mertens & L. Tijsseling, unpublished results, see Fig. 3). The amount of folic acid found in cows’ milk ranges from 20 to 60 mg/L, whereas its concentration in yoghurt may be increased depending on the strains used for the fermentation and on the storage conditions to values above 200 mg/L. This level also appears to depend on the strain of Lb. bulgaricus used, because the latter organism has been shown to use and to degrade folic acid during its growth (N. Peerlkamp & J. Hugenholtz, unpublished results, see Fig. 4). It is therefore of utmost importance to select the optimal combination of S. thermophilus and Lb. bulgaricus strains leading to an organoleptically acceptable yoghurt with a concomitantly increased folic acid concentration. The pools of available strains will undoubtedly harbour the desired strains. 4.4. Exopolysaccharides By producing exopolysaccharides, both S. thermophilus and Lb. bulgaricus contribute to the viscosity and the smooth texture of yoghurt. They stabilise the yoghurt gel and decrease its tendency to synerise (Schellhaas, 1983). The exopolysaccharides of the yoghurt bacteria exhibit a wide variety of chemical structures. The main monomers found are glucose, galactose and rhamnose, but the presence of fucose, Nacetylglucosamine and N-acetylgalactosamine has also been described (De Vuyst & Degeest, 1999; Looijesteijn, 2000; Laws & Marshall, 2001). The molecular mass of these polymers is up to around 1500 kg/mol and the amount formed in yoghurt is limited, up to a few hundred mg/L (Van Marle & Zoon, 1995). The amount

Folate breakdown by L. bulgaricus 120 100

ng/g milk

80 60 40

and sometimes also the structure of the exopolysaccharides are influenced by the growth conditions and the composition of the growth medium (Garcia-Garibay & Marshall, 1991; Gancel & Novel, 1994; Grobben, Sikkema, Smith, & de Bont, 1995). There are indications for some of the thermophilic strains that the biosynthesis of the polymers increases under suboptimal growth conditions (Gancel & Novel, 1994; Grobben et al., 1995), but for other strains the biosynthesis appears to be growth-related (Kimmel & Roberts, 1998; De Vuyst, Vanderveken, van de Ven, & Degeest, 1998). The chemical structure of the polymer apparently has an effect on the rheological properties of yoghurt, because the use of different starters results in differences in its microscopic structure and viscosity (Van Marle, 1998). The underlying mechanism of this effect, however, is not well understood. The emerging knowledge on the genetics of exopolysaccharide biosynthesis by S. thermophilus (Stingele, Neeser, & Mollet, 1996; Stingele et al., 1999) will offer opportunities to modify the structure of the polymer and to regulate the amount synthesised. Studying the effect of the use of a S. thermophilus strain, modified in this respect, in the fermentation of milk will reveal clues about the function of the exopolysaccharide structure and amount on the physical characteristics of the yoghurt obtained. Since the fat fraction of milk is always involved in the structural properties of yoghurt, the use of the same strategy may be useful in examining the effect of the polymer on the phenomenon of phase separation between the fat and water phase in yoghurt (Tuinier, 1999). All in all, yoghurt has several properties, which are a consequence of the presence of the specific organisms used as starter. Both S. thermophilus and Lb. bulgaricus occur in the dairy environment in multiple strain varieties, each with its particular effect on the sensory and health-promoting characteristics of the yoghurt produced. Basic knowledge about the background of the development of these characteristics will open avenues for innovations in the production of fermented milks. Although they undoubtedly opt for an important role as carrier to add nutritive value to fermented milk products, the probiotics are not included in this discussion, because their natural niche is generally not milk. 4.5. Cheese

20 0 milk

milk+S.t.

+L.b.B198

+L.b.B203

+L.b.B1124

Fig. 4. Folic acid breakdown by different strains of Lactobacillus bulgaricus, grown in milk enriched with folic acid by pre-growth with S. thermophilus and subsequent pasteurisation and pH adjustment. The bars represent from left to right: the folic acid concentration in milk, in milk after growth of S. thermophilus, followed by subsequent growth of L. bulgaricus strains B198, B203 and B1124, respectively (N. Peerlkamp & J. Hugenholtz, unpublished results).

The thermophilic lactic acid bacteria also play an essential role in the manufacture of some cheese types. The starters of Swiss-type and Italian cheeses consist mainly of S. thermophilus, Lb. helveticus and Lb. bulgaricus (Steffen, Eberhard, Bosset, & Ruegg, . 1999; Battistotti & Corradini, 1999). Also, in the ripening of Greek hard cheese types made from ewes’ and goats’ milk, the thermophilic lactic acid bacteria play a

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dominant role (Kalantzopoulos, 1999). Their niche in these cheese types is created by the specific high cooking temperature used in the manufacture. They convert lactose to lactic acid as in all dairy fermentations and this conversion is usually completed within about 24 h. Lactic acid plays its usual role as preservative and, for the Swiss-type cheese, is the appropriate substrate for the subsequent propionic acid fermentation, which is important for the characteristic eye formation. The lactobacilli in the thermophilic starters possess an elaborate proteolytic system capable of degrading milk proteins to amino acids, which are essential for their growth and that of the streptococci. In addition, these amino acids are the precursors for the cheese flavour as in other cheese types (Fox, Law, McSweeney, & Wallace, 1999). Italian cheeses are often produced from raw milk according to traditional technology in a restricted region, and very often they are legally designated by protected origin (DPO). It is not surprising that above all, the cheeses made from raw milk and without the use of starters harbour interesting pools of thermophilic lactic acid bacteria. Dairy microbiologists have realised this and have recently initiated several attempts to isolate and characterise these bacteria. The collection of Cogan et al. (1997), largely derived from artisanal fermented products, contains around 1000 strains of thermophilic lactic acid bacteria. Coppola, Parente, Dumontet, and La Peccerella (1988) studied the composition of 16 natural whey cultures for the manufacture of Mozzarella in the Province of Caserta and found several thermophilic streptococci and lactobacilli. These bacteria have also been found in Comte! cheeses, made from raw milk and designated of protected origin, and further characterisation revealed that, besides the starter streptococci, wild S. thermophilus were present (Bouton, Guyot, & Grappin, 1998). For Pecorino Toscano cheese, it was even possible to correlate the specificity of the flora including S. thermophilus, with the origin of the milk (Bizzarro et al., 2000). Randomly amplified polymorphic DNA (RAPD)-PCR offered the opportunity to type several S. thermophilus isolates from Italian DPO cheeses and this also allowed them to be discriminated according to the cheese origin. Furthermore, the presence of S. macedonicus in Italian cheeses could be shown (Andrighetto, Borney, Barmaz, Stefanon, & Lombardi, 2002). An elegant study by Randazzo, Torriani, Akkermans, de Vos, and Vaughan (2001) demonstrated the population dynamics in the artisanal Sicilian Ragusano cheese using the technique of denaturing gradient gel electrophoresis of PCR-amplified bacterial 16S ribosomal DNA fragments. In these cheeses, which are produced from raw milk without the addition of starter cultures, members of the genera Streptococcus and Lactobacillus clearly became the dominant microbiota during the ripening.

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5. Yeasts and moulds 5.1. Yeasts Although yeasts play a minor role in dairy fermentations, several fermented milk products with a natural yeast-containing microflora have been described. These products differ considerably in their physico-chemical and microbiological properties from those prepared with pure cultures of lactic acid bacteria. A distinctive feature of these products is that, in addition to the lactic acid fermentation induced by lactic acid bacteria, a slight alcoholic fermentation due to yeasts takes place (Koroleva, 1988b). The history of such products is centuries old and the first scientific descriptions of the occurrence of yeasts as cause of alcoholic fermentation in milk date from around the beginning of the 20th century (Weigmann, 1905). The best known examples of fermentation of milk by a combination of yeasts and lactic acid bacteria are kefir and koumiss, both originating from countries of Eastern Europe and Asia. The conventional process of kefir manufacture uses kefir grains as the basic inoculum. These grains are small, white bodies of some 2–15 mm diameter and are composed of a mixture of microorganisms, held together in an organised pattern. The peripheral layers of the granules are dominated by various rod-shaped bacteria, while towards the centre, yeasts become the major component of the microflora (Robinson & Tamime, 1990; Rea et al., 1996). Some of the microorganisms that have been reported as associated with kefir grains are mesophilic lactococci, both homofermentative and heterofermentative, thermophilic lactobacilli and yeasts. Also, the occurrence of Leuconostoc species and even of acetic acid bacteria has been described (Marshall, 1987; Koroleva, 1988b). The predominant species of yeast in kefir grains are Kluyveromyces marxianus, Candida kefir, Saccharomyces cerevisiae and Saccharomyces delbrueckii. The heterogeneity of the species found can be explained by the different techniques of kefir grain cultivation. Yeasts play an important role in promoting symbiosis among the microorganisms present, CO2 formation and development of the characteristic taste and aroma (Koroleva, 1988a). Koumiss is an ancient drink traditionally prepared from mares’ milk, although variants made from cows’ milk are also known (Koroleva, 1988b). The microflora of koumiss consists mainly of thermophilic lactic acid bacteria and species of Saccharomyces. The lactic acid bacteria acidify the milk and the yeasts perform the alcoholic fermentation. The final pH is around 4 and the alcohol percentage may reach 2%. The yeasts consist of lactose-fermenting and lactose-non-fermenting species. The latter ferment galactose, which is secreted by most of the lactobacilli (Hickey, Hillier, & Jago, 1986). These

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yeast species are Saccharomyces lactis, Kluyveromyces marxianus and Saccharomyces unisporus. The latter yeast ferments galactose, but not lactose. In traditional koumiss produced in Khazakstan, S. unisporus is the principal alcohol-fermenting yeast, especially at higher altitudes, where this yeast apparently meets less competition from other species (Montanari, Zambonelli, Grazia, Kamesheva & Shigaeva, 1996). Considering the complex microflora present in kefir and koumiss, it is obvious that a number of metabolic pathways are involved. Lactose can be utilised by the lactic acid bacteria through different pathways, yielding lactic acid and galactose. The yeasts metabolise lactose and galactose through alcoholic fermentation. The end products of lactic and alcoholic fermentation are very important for the formation of the typical flavour and refreshing taste of kefir and koumiss. The most important volatile components that contribute to the flavour of kefir are acetaldehyde, propionaldehyde, acetone, ethanol, 2-butanone, n-propanol, diacetyl and . amyl alcohols (Gorner, Palo, & Segin, 1972). Proteolysis by kefir and koumiss starters has received limited attention hitherto, although the accumulation of free amino acids has been observed (Meril.ainen, 1984). Both kefir and koumiss are believed to possess healthpromoting properties, which are mainly related to the ability of the starter to produce vitamins of the B-group (Glaeser, 1981) and antimicrobials. Especially, the antimicrobial activity of koumiss against Mycobacterium tuberculosis has been addressed in Russian research (Stojanowa, Ponomarjowa, & Spiridonow, 1982; Koroleva, 1988a). The presence of yeasts has also been reported in several traditional African fermented milks. The predominant yeasts encountered are Saccharomyces cerevisiae, Kluyveromyces marxianus and Candida kefir. Examples are the Nigerian fermented milk nono (Okagbue & Bankole, 1992), mbanik from Senegal (Gningue, Roblain, & Thonart, 1991), ergo and ititu from Ethiopia (Gonfa, Foster, & Holzapfel, 2001) and rob, mainly produced in rural areas of Sudan (Abdelgadir, Hamad, Mller, & Jakobsen, 2001). The possible role of yeasts in, for example, the aroma formation in this type of product has been reviewed by Jakobsen and Narvhus (1996). All these products harbour possibly interesting starter strains, not only several yeast varieties but also various mesophilic and thermophilic lactic acid bacteria. They should be considered as a useful pool of potential starters for industrially manufactured fermented dairy products. Relatively little is known about the role of yeasts in the process of cheese ripening. They have been found in the smear of surface-ripened cheese and previously, it was considered that they are involved in the lowering of the acidity of the surface, which allows the development of a secondary microbial flora (Reps, 1999). Positive

interactions between yeasts and starter organisms are well documented for semi-soft cheeses, like Limburger and Tilsit (Fleet, 1990). The yeasts metabolise lactate, which causes an increase of the pH, and may secrete factors that promote the growth of Brevibacterium linens, which is essential for the ripening of these cheeses. Yeasts are also believed to promote the development of Penicillium roqueforti in blue cheeses. They may contribute to the open structure of these cheeses by formation of gas (Coghill, 1979). On the surface of Camembert, the dominating yeast flora inhibits the growth of the spoilage moulds. The most prominent yeasts in cheese are Debaryomyces hansenii, Kluyveromyces marxianus, Kluyveromyces lactis, Yarrowia lipolytica, Saccharomyces cerevisiae and Zygosaccharomyces rouxii (Baroiller & Schmidt, 1990; Besanc-on et al., 1992). They are selected by the environmental conditions prevailing in the cheese and the brine, on the one hand, and the technological processes used during the cheese manufacture, on the other hand. It is important to appreciate the capabilities of these yeasts to become established in the cheese conditions and to function as a spontaneous starter. Their technological characteristics, such as proteolytic and lipolytic activity, formation of aroma components, fermentation of lactose and breakdown of lactate, positive interaction with mould starters (Penicillium) and with secondary flora (brevibacteria) and osmotolerance may make them very suitable as potential starters for these cheeses (Fox & Law, 1991; Bockelmann, 1999). Yeasts are worth testing for use as starter cultures for development of new productsFcheeses as well as fermented milks. Their technological properties, their positive microbial interactions and inhibitory effects against spoilage organisms should ensure successful application of appropriately selected strains as starters. 5.2. Moulds Moulds are mainly used in the manufacture of semisoft cheese varieties together with the lactic acidifiers. Their major role is to enhance the flavour and aroma and to modify the body and the structure of cheese. On the basis of their colour and growth characteristics, they can be divided into white and blue moulds. The former type grows on the outside of the cheese, e.g. Camembert and Brie, and is known as Penicillium camemberti. The blue mould is named Penicillium roqueforti and grows inside the cheese. Examples of ‘blue’ cheeses are Roquefort, Blue Stilton, Danish Blue and Gorgonzola (Tamime, 1990). Several varieties of these two Penicillium species have been described in the past, but they should all be considered as biotypes. The characteristic feature of mould-ripened cheese is the extensive proteolysis and lipolysis. These biochemical activities ultimately result in the formation of precursors for the

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typical volatile flavour components of the cheeses. Methyl ketones have a key role in the typical flavour. There is a positive correlation between the free fatty acid level and the amount of methyl ketones formed, and cheeses with limited lipolysis score lower in flavour (Gripon, 1999). Other typical compounds besides methyl ketones are secondary alcohols, esters, aldehydes and lactones. The aroma of these cheeses is of course completed by various compounds arising from the proteolysis and amino acid conversion. Sulphur components are clearly also present, and for ripe Camembert, they are usually thought to be generated by coryneform bacteria, although Geotrichum candidum may be active in this respect as well (Gripon, 1997). The floral note in traditional Camembert is caused by 2-phenylethanol, produced from phenylalanine by yeasts (Lee & Richard, 1984). Clearly, the flavour of mould-ripened cheese is a delicate balance of several compounds, produced by a succession of microorganisms, each of which perform their particular activity. Not only is the choice of the Penicillium strain important for the successful production of the soft surface mould cheeses; the selection of the concomitant starters is also crucial. Mucor species are known to be the ripening agent in the Norwegian cheese Gamalost made from skim milk. The species Mucor mucedo and M. racemosus grow on the surface and throughout the interior of the cheese and are responsible for a thorough decomposition of the protein fraction (Rage, 1993). 5.3. Geotrichum candidum Although the representatives of the genera Pencillium and Mucor described above are essential in the ripening of certain cheese types, they are not recognised as real milk moulds. This designation has been reserved for Geotrichum candidum. If raw milk is incubated at room temperature, it will acidify, and after some days, a mouldy layer will develop on the surface of the milk. This layer consists of branched hyphae, which divide at their tips into rectangular cells. This fungus has already been isolated from milk as early as 1850 by Fresenius, who classified it as Oidium lactis. It was reclassified as Oospora lactis by Saccardo in 1880 and, finally, it was placed in the genus Geotrichum and received its present name (Wouters, 1966). This small historical description clearly points to the close connection of G. candidum with milk and, therefore, it is not surprising that this fungus plays a role in the manufacture of some dairy products. There are two well-known examples of fermented products with G. candidum as one of the starters: the Finnish fermented milk viili and Camembert cheese. The starter of viili consists of mesophilic lactococci and Leuconostoc strains with G. candidum. Viili is made from non-homogenised milk and the fungus forms a mouldy layer on the cream. Its growth is generally limited due to

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the restricted amount of oxygen present in the container with the fermenting mixture. This limited growth keeps the pH well under 5, and the mild taste and the silky texture are well-appreciated attributes (Marshall, 1986). G. candidum is one of the contributors to the flora that plays a key role in the ripening of Camembert cheese. It starts to grow on the surface of the rind of Camembert, Pont l’Ev#eque and Livarot cheeses at the beginning of the ripening process and contributes to typical cheese flavours (Hemme et al., 1982). The value of this organism was recently emphasised by renewed studies (Gueguen & Schmidt, 1992). G. candidum consumes lactate and as a result of this activity, the pH of the cheese rises, allowing the development of other important organisms like P. camemberti and Brevibacterium linens (Molimard, Vassal, Bouvier, & Spinnler, 1995). It also causes a significant inhibition of fungal contaminants and undesirable microorganisms (Nielsen, Frisvad, & Nielsen, 1998). G. candidum excretes d-3phenyllactic acid, which inhibits the growth of Listeria monocytogenes (Dieuleveux, Lemarinier, & Gueguen, 1998). It produces several enzymes for the breakdown of protein and fat, resulting in important aroma compounds (Jollivet, Chataud, Vayssier, Bensoussan, & Belin, 1994). The lipolytic activity of G. candidum was first established by Nelson (1952) and studied in detail by Wouters (1966), Tsujisaka, Iwai, and Tominaga (1973) and Jacobsen, Olsen, Allermann, Poulsen, and Hau (1989). G. candidum produces two lipases and one of these is specific for esters of unsaturated fatty acids. The hydrolysis products of these lipases are the precursors of various volatile compounds like alcohols, fatty acids, methyl ketones and esters (Jollivet et al., 1994). Many strains of G. candidum have been described and they could be differentiated by their ability to produce these volatiles. G. candidum is not only able to produce amino acids from protein, but also able to convert sulphur-containing amino acids into various sulphides, which contribute to the typical flavour characteristics of certain dairy products. The main precursor of these compounds is methionine and this fungus is known to convert it into methanethiol and dimethyl sulphide by two distinct pathways (Demarigny, Berger, Desmasures, Gueguen, & Spinnler, 2000). Other sulphides formed are dimethyl disulphide and dimethyl trisulphide. Also, significant quantities of S-methyl thioesters are produced by this organism (Berger, Khan, Molimard, Martin, & Spinnler, 1999). Several G. candidum strains have been described and it is clear that they differ greatly in their biochemical capacity to contribute to flavour formation in dairy products (Spinnler, Berger, Lapadatescu, & Bonnarme, 2001). The potential application of these strains in starters for the production of cheese and other fermented dairy products with desirable organoleptic

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properties is certainly great, but should be elaborated further to facilitate product innovation. 6. Other bacteria Some other groups of bacteria, which have not been covered above, are nevertheless worth mentioning. The propionic acid bacteria constitute the essential secondary flora in Swiss-type cheese. After the homofermentative lactic acid fermentation, they convert lactate into propionate, acetate and carbon dioxide. The latter is responsible for the characteristic eye formation in these cheeses. Interest in the role of propionic acid bacteria in flavour formation has recently been renewed (Gagnaire, Molle! , Srhaug, & Le! onil, 1999). Also, for this group of bacteria, the raw milk environment appears to be a versatile and interesting source of strain variation (Fessler, Casey, & Puhan, 1999). A second group of bacteria, which play a major role in the maturation of surface-ripened cheese, comprises the brevibacteria. They are obviously present in the smear of these cheeses and are strongly proteolytic, which results in high levels of sulphur-containing volatiles. They also show some lipolytic and esterolytic activity, and produce distinct red-orange pigments (Rattray & Fox, 1999). Several strains of these alkalophilic and salt-tolerant bacteria have been isolated and characterised. Their variation in proteolytic activity, antimicrobial activity and pigment biosynthesis may offer opportunities for the selection of an appropriate variant for a specific application in the manufacture of a smear-ripened cheese or even another type of cheese (Bikash, Ghosh, Sienkiewicz, & Krenkel, 2000; Dufosse! , Mabon, & Binet, 2001). It is becoming apparent that the flora of smear-ripened cheese contains, besides brevibacteria, other coryneform bacteria, which contribute to its final organoleptic characteristics. These should of course not be excluded in defining starter cultures for this cheese type (Bockelmann & Hoppe-Seyler, 2001). The final group of bacteria to be mentioned here, the enterococci, occurs frequently in large numbers in dairy products. Their presence has generally been considered as a consequence of insufficient sanitary conditions during the processing. Several strains, however, show biochemical properties that make them possibly useful as starters for technological application in food fermentation. Since strains of enterococci have been recognised as potential pathogens, any selection should take virulence factors into account (Sarantinopoulos et al., 2001; Andrighetto et al., 2002). 7. Future perspectives Fermented products made from raw milk tend to have broader flavour profiles as compared to products made

from pasteurised milk. Perfectly aged raw-milk cheese, for instance, brings the consumer a sense of where the cheese comes from. Several of these cheeses have received the status of Designation of Protected Origin (Appellation d’Origine Prote! ge! eFDenominazione di Origine Protetta). They can be considered as cultural markers of society just as much as fine art products (Cimons, 2001). The consumption of raw-milk cheese, however, has lately been associated with health hazards and the associated exaggerated concern for food safety has driven agency officials to consider the possibility of requiring that the milk from which cheeses are made must be pasteurised. If this were to become the policy, the artisanal cheeses would no longer taste the same and their uniqueness would be in danger of being lost. Whatever the outcome of this discussion will be, the dairy microbiologist has understood his duty, as we have seen in the sections above, and has collected the microbes responsible for the unique flavour in the artisanal cheeses. Several pools of microbes are now available for the cheesemaker to choose from for product innovation. In the description of the collections of dairy-relevant microorganisms originating from the raw milk environment, the main emphasis has been on lactococci, mesophilic lactobacilli, thermophilic lactic acid bacteria and some yeasts and moulds. These collections were examined for the occurrence of interesting properties for application in dairy fermentations. Not surprisingly, they appeared to contain strains with promising features for novel dairy product development. Some of them would be very useful as adjunct starters in the manufacture of cheese and others in adding value to fermented milks like yoghurt. We do not claim to be complete and realise that there are other microorganisms, which may be of even more importance for specific cheese varieties. The important message is that the interest in the microflora of traditional fermented dairy products, often produced from raw milk, should be encouraged and that the pool of microbial strains from these floras should be saved for potential future applications. The product innovator is challenged to use strains from them in his trials to create the flavour or other attributes as found in raw-milk products, even after applying the safe pasteurisation process. These pools of microbes are sometimes huge in size and the selection of a desired strain in the traditional trial and error way is an elaborate and time-consuming task. Fortunately, microbial genomics has developed progressively in the last half decade and the genomes of several microbes have been sequenced. These developments have rejuvenated microbiology and new tools enable investigators to address basic questions (Nelson, Paulsen, & Fraser, 2001). Genome sequencing of several lactic acid bacteria is completed or under way. With the extensive knowledge on the network of pathways

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prevailing in these bacteria which are active in the flavour formation in cheese, bioinformatics tools are available to search in genomes for specific genes or gene clusters. This should lead to the design of probes for screening collections of microbes (pools of genes) very effectively for the presence of certain desired traits. The selection of strains for the innovation of fermented dairy products with an added value would thus be facilitated enormously (Van Kranenburg et al., 2002).

Acknowledgements The authors thank Andrea Mertens, Linda Tijsseling and Nienke Peerlkamp for communicating results prior to publication. This work was partly supported by the Foundation ‘Zuivel, Voeding en Gezondheid’, Utrecht, The Netherlands.

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