Proteolysis

The levels of free amino acids in milk are insufficient to support the growth of auxotrophic starter LAB, although there are pronounced inter- and intraspecies differences in amino acid requirements. The LAB possess a complex, albeit well-characterized, proteolytic system, the products of which enable the bacteria to meet their amino acid requirements from the hydrolysis of milk proteins. The peptides and amino acids released also contribute to, and act as precursors for, flavor development in cheese. In Cheddar cheese, primary proteolysis is affected by the added chymosin and endogenous milk enzymes; small peptides and free amino acids are released from the primary products by the action of the LAB proteolytic enzymes (102).

The proteolytic system of the lactococci has been examined extensively, many of the component enzymes have been purified and characterized, and the encoding genes se-quenced (103-105). The proteolytic systems of other LAB are less well characterized, but several enzymes in many strains have been studied. In general these enzymes closely resemble and complement their lactococcal counterparts. The proteolytic complex of Lc. lactis comprises a cell-envelope-associated 180-190 kDa molecular mass extracellular serine proteinase, peptide and amino acid transport systems, and a diverse range of intracellular peptidolytic enzymes. The membrane-anchored proteinase (CEP, lactocepin, Prt) is the only extracellular lactococcal proteolytic enzyme, although the strain specificity toward caseins resulted in the initial classification of different proteinase groups (e.g., Prt I, Prt III). The specificity of the CEP from different Lactococcus strains on as1-, as2-, h-and n-caseins has been established (102,103). The intracellular lactococcal proteinases are unlikely to be involved in peptide generation for growth in milk but may contribute to proteolysis in cheese as a consequence of starter cell lysis. Although less well studied, the principal cell wallassociated proteinase of lactobacilli resembles the lactococcal enzyme (102,103).

Casein degradation products are transported into the cell by specific amino acid transport systems, two distinct dipeptide/tripeptide systems (DtpT, DtpP), and an oligopeptide transport system (Opp) (103). The Opp transport system comprises two ATP-binding proteins, two membrane proteins, and a substrate-binding protein; it transports peptides containing four to eight amino acids. The Opp transport system, unlike the DtpT system, is essential for growth on casein: casein-derived oligopeptides, small enough to be transported by the Opp system, represent some 98% of the available nitrogen source for lactococci in milk.

The transported peptides are hydrolyzed intracellularly by a complex of peptidolytic enzymes with overlapping specificities. The types of peptide hydrolases present in LAB include both endopeptidases and N-terminal aminopeptidases, dipeptidases, and tripepti-dases expressing exopeptidase activity. Although lactococci would not appear to possess a carboxypeptidase, this activity has been reported once in some lactobacilli (106). An extensive array of peptide hydrolases is utilized to release the amino acids essential for LAB growth. The general peptidases characterized to date in lactococci include two endopeptidases (PepF, PepO), a tripeptidase (PepT), a dipeptidase (PepV), and the metallo-and thiol-type aminopeptidases (PepN and PepC, respectively). In addition, lactococci possess peptidases with more specific functions, and these include glutamyl aminopeptidase (PepA), pyrrolidene carboxylpeptidase (PCP), and the four proline-specific enzymes— aminopeptidase P (PepP), proline iminopeptidase (prolylaminopeptidase, PepI), prolidase (PepQ), and a post-proline dipeptidyl peptidase (PepX). The peptidase profile of the lactobacilli studied is broadly similar, although PepA, PepP, and PepT have been reported only in Lactococcus spp. However, leucylaminopeptidase (PepL), a dipeptidase (PepD), prolinase (PepR), and the endopeptidases (PepE and PepG) conversely have been characterized only in some species of mesophilic and thermophilic lactobacilli. The properties of these and other unassigned LAB peptidases have been extensively reviewed (e.g., 102-105). Although approximately 20 different peptidase hydrolases have been characterized in lactobacilli, the full complexity of the peptidolytic systems of most species of nonlactococcal LAB is not known. However, studies using diagnostic substrates indicate that most Lacto-bacillus spp., in common with Lc. lactis, possess a wide range of peptide hydrolase enzymes

(67.107). The similarity of the peptidolytic enzyme systems in the two LAB genera is not unexpected as both groups of bacteria have evolved to survive and proliferate in an ecosystem in which the level of free amino acids is insufficient to sustain abundant growth.

The contribution of the lactococcal intracellular proteolytic enzymes in the ripening of Cheddar cheese may be greater than that of the enzymes of the nonstarter lactobacilli, in view of the greater tendency of starter cells to autolyse in the cheese during ripening. Neither bacterial group contributes significantly to primary proteolysis in Cheddar cheese, and although the contribution of the starter lactococci to secondary proteolysis is greater

(43.108), the presence of both types of LAB is associated with increased proteolysis and higher levels of free amino acids in the cheese.

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