prefers a higher pH to grow and is the only species able to transform the urea in milk into NH3 and CO2.
The position of S. thermophilus among other streptococci (in particular S. salivarius) has now been clarified. It is recognized as a separate species and specific probes have been developed (5). The taxonomic studies on the group L. delbrueckii led to the hypothesis that L. delbrueckii subsp. lactis is the common ancestral genotype from which two variants adapted to specialized ecological niches evolved: plants for L. delbrueckii subsp. delbrueckii and fermented milks for L. delbrueckii subsp. bulgaricus, respectively. Because of their differences in the sugar fermentation profiles, the differentiation between the three subspecies can be easily performed. On the other hand, L. helveticus is not phylogenetically related to the L. delbrueckii group (less than 40% of DNA-DNA hybridization), and several easy means of differentiation exist. For thermophilic lactobacilli, also, species-specific probes or other molecular methods were developed (7). Recently, convenient phenotypic methods based on zymogram profiles of peptidoglycan hydrolases (8) or cell wall protein profiles (by SDS PAGE) have been proposed to distinguish L. helveticus from other lactobacilli (9).
Various tools for strain typing have been developed for thermophilic LAB, including genomic macrorestriction profiles and random amplified polymorphic DNA (10-12) which allow collection typing and assessment of genotypic diversity.
The estimation of genome sizes (mainly by pulsed field gel electrophoresis) has produced similar values for the four thermophilic LAB discussed (i.e., 1.85-2 Mb). However the GC% of the L. delbrueckii group was markedly higher (49-51%) than that of S. thermophilus and L. helveticus (38-40%). Plasmids are described in the four species but their presence depends up species and strains (apparently less frequent in S. thermophilus and L. delbrueckii subsp. bulgaricus compared to the two other species of thermophilic LAB). In only a few cases, they have been associated with specific phenotypic traits (i.e., cell wall proteolytic activity in L. helveticus strains, stress protein in S. thermophilus, antibiotic resistance). Several plasmids have been completely sequenced and characterized (13,14). The advances in elucidating the genetics of thermophilic LAB has recently been reviewed (15). Briefly, genome exchange between S. thermophilus and the mesophilic LAB Lacto-coccus lactis was demonstrated to occur. Indeed, some of the insertion sequences found in S thermophilus strains are clearly of recent lactococcal origin. Evidence for similar transfer from L. helveticus to L. delbrueckii subsp. lactis has also been reported. Several genes related to sugar transport, glycolysis, proteolysis, stress response, or ability to produce exopolysaccharides were cloned and sequenced (15). Most important, complete genome sequencing projects are already on going for S. thermophilus (J. Delcour, Belgium; A. Bolotin, France and USA) and for L. delbrueckii subp. bulgaricus (E. Maguin, France; and by Danone). The sequence for L. helveticus has recently been completed (J. Steele, USA) but the data are not yet available. This complete sequencing will obviously allow significant advances in our knowledge of thermophilic LAB genetics and metabolism. However, few gene transfer methods are yet available, hampering the obtainment of mutants needed to assess the respective role of enzymes, in cheese in particular, and the potential future development of modified strains (GMO). The problem is particularly urgent for L. del-brueckii subsp. bulgaricus, for which no transformation protocol of any kind exists. In contrast, several strains of S. thermophilus, including industrial strains, can be electro-transformed, and a promising method of conjugal transfer of foreign DNA was recently described for L. helveticus (16).
B. Technological Properties of Thermophilic Starters Contributing to Cheesemaking
Cheese-making and ripening is a complex and time-consuming process involving the gradual breakdown of milk components (carbohydrates, proteins, and fat). The major biochemical processes are acidification, proteolysis, and subsequent transformation of the released amino acids into flavor compounds, in parallel with the hydrolysis of milk fat (i.e., lipolysis). The first criteria for selection of thermophilic LAB is their ability to produce lactic acid from lactose with a defined kinetic. Indeed the rate and extent of acid production and resulting demineralization of the curd are critical determinants of cheese structure and texture.
In thermophilic LAB, lactose is transported into the cell by permease systems and converted into glucose and galactose by a h-galactosidase. Glucose is metabolized through the glycolytic pathway; and galactose, when metabolized, through the Leloir pathway. Indeed, S. thermophilus, L. delbrueckii subsp. bulgaricus, and, depending on the strain, L. delbrueckii subsp. lactis are not able to metabolize galactose (Table 2). In this case, 1 mol of galactose is excreted in the medium per mol of lactose consumed. Although some strains of S. thermophilus can be galactose-positive, this property is highly unstable (the problem of residual galactose in cheese is discussed below). Depending on the species, the quantity and isomers of lactate produced may differ (Table 2). The kinetics of lactic acid production is highly strain dependent and must be tested under technological conditions, taking into account the temperature gradient for each technology (17-19). There is also considerable diversity between strains regarding temperatures at which maximum rates of acid production occured. When combined, initial acidification is due to S. thermophilus, which stops growth at about pH 5.2; the final acidification is essentially due to lactobacilli, because of their acid tolerance. In general, the resistance to heating of L. delbrueckii subsp. lactis strains is higher than the one of L. helveticus strains, explaining a later acidification with this last species (20,21). Recently, Cachon et al. (22) proposed screening LAB not only for acidification but also for reduction capacity, which can influence the oxydo reduction potential of the medium. Interestingly, acidifications with S. thermophilus and thermophilic lacto-bacilli finished before the end of the reduction phase. Oxido reduction potential creates conditions for a balanced flavor development in cheese, so this approach seems promising.
In mozzarella cheese, the impact of the rod/coccus ratio on acidification and subsequent chemical composition, proteolysis, and functional properties was determined by Yun et al. (23). Regardless of the initial ratio, cocci were dominant in the curd and resisted stretching at 57 °C. The main impact on the acidification schedule is the amount of inoculation. In Swiss cheese, streptococci also grow first and reached a maximum level after 4 to 6 hr of pressing (Fig. 2). It must be underlined that acidifying properties can be unstable in some strains of thermophilic lactobacilli after successive generations, in particular in L. helveticus (24).
In Swiss cheeses, lactose is normally completely removed at day one at the end of pressing (pH between 5.2 and 5.5). During the ripening, pH can increase (0.2 to 0.4 units)
due to proteolysis and consumption of lactate by other flora. The absence of residual sugars is regarded as a guaranty of cheese stability during ripening. In pasta filata cheeses, variation exists. In general, the residual starter (surviving to stretching) continues to ferment lactose during the first days of ripening, resulting in negligible residual levels. By contrast galactose is fermented very slowly and can persist in pizza cheese. The level of galactose declines, of course, more rapidly when the starter includes L. helveticus. Thus, the starter culture plays an important role in pizza cheese browning by affecting the lactose/galactose content. In general, high level of residual galactose is regarded as a risk as it can support the growth of undesirable flora or generate post-acidification.
Phage attack can compromise dramatically the acidification step, in particular phages attacking S. thermophilus. In the past few years, molecular tools have allowed a better understanding of the interaction between the thermophilic LAB starter and phage populations in a cheese factory (25). However, despite the increase of knowledge about phages of thermophilic LAB (several were completely sequenced)(26,27), good hygienic practices during the preparation of the starter, rotation of strains, and use of mixed strains (in particular of S. thermophilus) remain the current defenses against phage attack of ther-mophilic LAB.
Milk contains a large amount of proteins, of which 26 g/kg are caseins, but is relatively poor in free amino acids and small peptides. The presence of cell wall-associated proteases is thus essential for auxotrophic bacteria to cleave caseins into short peptides, which can then be imported and degraded intracellularly into free amino acids. Thermophilic lacto-bacilli have generally a strong cell wall proteolytic activity (PrtP), which is not the case with S. thermophilus, except in a few strains [about 3%; (28)] (Table 3). Thus, in a mixed
Table 3 Short Synthesis About Proteolytic and Peptidasic Activity of Thermophilic Starters
S. thermophilus subsp. lactis subsp. bulgaricus L. helveticus
Cell wall proteinases
Binding to cell wall Hydrolyze
Covalent aS1 and ß casein
PrtB Strong * Noncovalent ß casein
PrtB Strong Noncovalent aS1 and ß casein
PrtH, PrtY Strong Noncovalent aS1 and ß casein preferentially Other Observations
* inducible in milk, strongly repressed by peptides from casein
Peptidases® cloned PepC, pepN, and sequenced pepS, pepO
PepN, pepNi, pepC, pepT, pepV, pepD, pepO, pepQ*, pepX*, pepl*, pepR, pepT
peptidases a pepN, general aminopeptidase; pepC, thiol amino peptidase, pepT, tripeptidase; pepV, pepD, dipeptidases; pepL, leucyl aminopeptidase; pepO, endopeptidase; pepP, aminopeptidaseP; pepQ, prolidase; pepX, X-prolyldipeptidy-laminopeptidase; pepl, proline iminopeptidase; pepR, prolinase.
starter, the more proteolytic lactobacilli stimulate S. thermophilus growth by increasing free amino acids and small peptides, released by the PrtP activity, and that is one aspect of their symbiosis. In turn, S. thermophilus produces formate and CO2, which stimulate lacto-bacilli growth. Several proteases of thermophilic LAB have been cloned and sequenced and all belong to the serine proteases family, with an optimum activity pH and temperature of, respectively, 6.0-6.5 and 40-45°C. However, they differed in some characteristics such as specificity, regulation, and mechanism of cell wall binding (Table 3) [for review, see (15,29-32)]. The peptides are then hydrolyzed into shorter peptides and free amino acids by numerous peptidases. Various aminopeptidases, dipeptidases, and peptidases specific to proline-containing peptides have been isolated, cloned, and sequenced (Table 3). The pep-tidase activity of the four thermophilic LAB species was compared using the same 34 sequenced peptides issued from h-casein by Deutsch et al. (33). Regardless of strain, L. helveticus was the most efficient, whereas S. thermophilus was not able to release free proline. This is consistent with the absence of pepIP (removing proline in a N- terminal position) and a low activity of pepQ (prolidase hydrolyzing X-pro peptides) in that species (30). Peptidases of all four species were not able to hydrolyze significantly the three phosphopeptides of the h-casein hydrolysate, providing the first experimental evidence of the intrinsic resistance of those peptides. Last, the presence of at least one carboxypepti-dase was again suggested (33). As no carboxypeptidase was successfully isolated and purified, this controversial point should be clarified.
In Swiss cheese, the choice of lactobacilli has a major impact on the peptidic profiles and the final extent of proteolysis. Their cell wall proteases contribute to casein hydrolysis, in particular casein as1. However, they are especially active in the degradation of peptides resulting from rennet and plasmin activities, and in this way, they contribute to the final flavor by removing bitter peptides that are issued from the N-terminal of the as1 casein or the C-terminal part of the h-casein. This ability to remove bitterness is one of the most important traits in terms of cheese final flavor (34,35). The protease activity toward whole caseins or large peptides in cheese is largely strain and species dependent (36) and an in vitro assay was recently proposed for improving thermophilic lactobacilli selection (37,38).
In pasta filata cheeses, it was shown that the primary proteolysis was mainly due to the coagulant and that the contribution of thermophilic starters occurs in the subsequent hydrolysis of large peptides into small peptides and free amino acids. The extent of proteolysis is directly related to the ratio of rods to cocci in the starter and depends on the chosen strain of lactobacilli. The functional properties are also influenced by the starter proteolytic activity: Mozzarella cheese made with single strains of proteinase-deficient L. delbrueckii subsp. bulgaricus exhibits less browning, greater melting and less stretch than control (39).
As mentioned in the Introduction, thermophilic LAB, because of their high peptidasic activity, are also used as adjuncts in cheese to improve the flavor and or to reduce ripening time, as for example in Cheddar with a low inoculum of 102 or 103 cfu/mL (40). When added at high inoculum, the acidifying activity of thermophilic LAB cultures can be first reduced or suppressed by an ''attenuation'' treatment in order to avoid any effect of the adjunct on the acidification kinetics of the cheese concerned [for review, see (41)]. Of course, the attenuation treatment should not provoke any significant inactivation of the proteolytic/peptidasic activities of the strain. Lactobacillus helveticus is particularly valued as attenuated starter for many cheeses varieties because of its high ability to increase release of free amino acids, to reduce bitterness, and to improve flavor.
In Swiss cheese, as soon as the acidification step ends, the numbers of thermophilic lactic acid bacteria decreased continuously (Fig. 2) (42). It was recently demonstrated that ther-mophilic starters lyse early in the curd, at least in Swiss- and Grana-type cheeses, releasing intracellular enzymes, especially peptidases, which then contribute efficiently to cheese secondary proteolysis (35,43-45). The ability to lyse was shown to be strain dependent and the mechanisms involved and the impact on cheese proteolysis have been reviewed (46). Briefly, carbon starvation (lactose or galactose) induces lysis of thermophilic starters in cheese, and the variation in extent of lysis is related to a larger free amino acid content: this emphasizes the importance of lysis ability of thermophilic LAB as a new selection criteria, as it was evidenced for lactococci.
The catabolism of amino acids, particularly sulfur, branched chain, and aromatic ones, can lead to important cheese flavor compounds as demonstrated for the mesophilic LAB, Lactococcus lactis (47,48). Few similar data are available for thermophilic LAB. Aminotransferase activity was clearly detected in cell-free extracts of L. helveticus by Klein and Lortal (49), and in resting cells of the three thermophilic LAB species by Helinck et al. (50). From leucine, the main aroma compounds produced by intact cells were a-ketoisocaproic acid, isovaleric acid, and 3 methylbutanol. Methane thiol was produced from methionine, but the quantities produced can vary 10-fold depending on the strain. From trytophan, the risk of producing off-flavor compounds has been emphasized (51). Thermophilic lactobacilli are also known to produce acetaldehyde in milk (by cleaving threonine by a threonine aldolase) as well as various ketones, aldehydes, and sulfur compounds. The ability to decar-boxylate glutamic acid, producing CO2, gamma-amino-butyric acid (GABA) and alpha-amino-butyric acid exists in S. thermophilus and may have an impact on the opening and the taste of some cheeses (52). However, this property is highly strain dependent. The determination of the real impact of thermophilic starter on final flavor through amino acid catabolism requires further investigations, as no assay in cheeses have been published yet.
Thermophilic LAB exhibit rather low esterolytic and lipolytic activities, and very few studies have been devoted to these enzymes. Those of thermophilic lactobacilli were reviewed by Gobbetti et al. (53). The activity of S. thermophilus was shown to be at least twice that of mesophilic lactococci. A lipase from L. delbrueckii subsp. bulgaricus was characterized by El-Sawah et al. (54) and an aryl esterase of L. helveticus was recently cloned that is active in cheese conditions (55). Unfortunately, very few data are available about the contribution of thermophilic LAB in cheese lipolysis: in Swiss cheese, the role of thermophilic LAB seems minor compared to the impact of dairy propionibacteria on lipolysis (56).
Thermophilic LAB produce exopolysaccharides composed mainly of glucose, galactose, rhamnose, and sometimes N-acetylglucosamine residues (57,58). This property is chro-mosomally encoded but is relatively unstable. Variable amounts are produced depending on the species: values from 50 to 350 mg/L and from 60 to 425 mg/L were cited for S. thermophilus and L. delbrueckii subsp. bulgaricus, respectively. The EPS gene cluster was recently sequenced (15). In cheese, this production can have a major impact on texture and water content (59-61) and this has been successfully exploited to improve, for example, the texture of low-fat mozzarella. However, strains producing exopolysaccharides can also lead to draining defect and should be used carefully.
Among the technological properties of the thermophilic LAB, the most well characterized are acidification and proteolysis. Even so, however, these two essential properties cannot be fully controlled because interactions between strains and species can greatly modulate their expression in situ. The impact on ripening of their ability to produce exopolysaccharides and of their amino acid catabolism are promising fields of research and development.
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