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2.9.2.1Beta Casein

Studies by Hébert et al. (2002) on four L. helveticus strains (ATCC 15009, CRL 1062, CRL 1178 and CRL 1177) revealed the hydrolysis patterns of purified β-CN. Each of these strains showed a different hydrolysis pattern, with the exception of CRL 1062 and ATCC 15009, which showed a similar pattern of protein hydrolysis. Tables 2.4, 2.5 and 2.6 provide further information on several CEP cleavage bonds common for three L. helveticus strains assessed on β-CN whereas other bonds are strain specific (Sadat- Mekmene et al., 2011a).

Figure 2.2 General schematic of the proteolytic systems identified in Lactobacillus

helveticus. M indicates the cell membrane and CW denotes the cell wall membrane

(Sadat-Mekmene et al., 2011a)

Further, other studies revealed various proteolytic activities and specificities among 15 strains of L. helveticus examined on pure caseins or on casein micelles directly in milk (Sadat-Mekmene et al., 2011a). The results showed more rapid hydrolysis of β-CN for all 15 strains compared to the kinetics involved in the hydrolysis of αs1-CN taking place in the presence of the two CEPs. The number of AA residues in the resulting peptides

varied from 6 to 33, showing vast specificity in cleavage bonds (Sadat-Mekmene et al., 2011a). In contrast, the growth of bacteria in milk provides varying results revealing massive reduction in the number of peptides released, indicating the accessibility of CEPs on casein micelle in milk compared with pure caseins.

Table 2.4 Amino acid cleavage sites for Lactobacillus helveticus CNR303 cell envelope proteinases in beta casein

1 RELEEL↓NVPG EIVESLSSSE ESITRINKKI EKFQSEEQQQ TEDELQDKIH PFAQTQSLVY 61 PFPGPIPNSL PQNIPPLTQT PVVVPPFLQP EVMGVSKVKE AMAPK↓HKEMP FPKYPVQPF↓T

121 ESQSLTLTDV ENLHLPPLLL QSWMHQPHQP LPPTVMFPPQ SVLSLSQSKV LPVPE↓KAVPY

181 PQ↓RDMPIQAF↓L↓L↓YQQPVLGP VRGPFPIIV

Table 2.5 Amino acid cleavage sites for Lactobacillus helveticus CP790 envelope proteinases in beta casein

1 RELEELNVPG EIVES↓LSSSE ESITRINKKI EKFQSEEQQQ TE↓DELQDKIH PFAQTQSLVY 61 PFPGPIPNS↓L PQNIPPLTQT PVVVPPFLQP EVMGVSK↓VKE AMAPK↓HKEMP FP↓KYPVQPF↓T

121 ES↓QSLTL↓TDV ENLHLPPLLL Q↓SWMHQPH↓QP LPPTVMF↓PPQ SVLSLSQ↓SKV LPVPE↓K↓AVPY

Table 2.6 Amino acid cleavage sites for Lactobacillus helveticus CP53 envelope proteins in beta casein

1 RELEELNVPG EIVESLSSSE ESITRINKKI EKFQSEEQQQ TEDELQDKIH PFAQTQSLVY 61 PFPGPIPNSL PQNIPPLTQT PVVVPPFLQP EVMGVSKVKE AMAPKHKEMP FPKYPVQPFT 121 ESQSLTLTDV ENLHLPPLLL Q↓SWM↓HQPH↓QP LPPTVMFPPQ SVLSLSQSKV LPVPEKAVPY

181 PQ RDMPIQAF↓LL YQQPVL↓GP VRGPFPIIV

2.9.2.2Alpha s1 Casein

Tables 2.7 and 2.8 show the locations of cleavage sites for some CEPs on pure αs1-CN in L. helveticus. The bonds indicate cleavage sites (Ile6–Lys7, Gln9–Gly10 and Leu142– Ala143) shared among some L. helveticus strains, while other bonds are strain specific. Further, Hébert et al. (2002) reported the ability of four L. helveticus strains (ATCC 15009, CRL 1177, CRL 1178 and CRL 1062) to release peptides from αs1-CN as a substrate after 3 h of incubation. In addition, Scolari et al. (2006) found that the proteinase of L. helveticus Zuc2 exhibits a low affinity for αs1-CN, cleaving the Arg22–Phe23 bond after incubation for 2 h. Martín-Hernández et al. (1994) showed that αs1-CN (1–9) was the main peptide released from this type of casein using L. helveticus L89 CEP, while αs1-CN (1–14) occurred to a lesser extent. Oberg et al. (2002) observed four patterns of specificity for eight L. helveticus strains on αs1-CN (1–23). Hydrolysis of the fragment αs1-CN (1–23) by the whole cell of L. helveticus CNRZ 32 and the PrtH mutant reveals that this strain has the ability to synthesise two active CEPs. Each has a particular specificity in terms of cleavage: for example, PrtH cleaves for αs1-CN (1–16, 18–23, 1– 17 and 17–23), whereas PrtH2 releases αs1-CN (1–9, 9–23, 1–16 and 10–23) (Pederson et al., 1999). Sadat-Mekmene et al. (2011b) reported that the cleavage sites on purified

αs1-casein are located not only between 1 and 40 residues of the N-terminus, but also in the middle of the sequence of residues ranging from 80 to 150. The hydrolysis of residues 170 and 199 at the C-terminus of αs1-CN was also observed by Jensen et al. (2009).

2.9.2.3 Alpha s2 Casein

Hydrolysis of purified αs2-CN has not been reported, which may be due to the level of difficulty involved in αs2-CN purification. The specificity of CEPs to this casein in assembled micelles in milk has been demonstrated (Sadat-Mekmene et al., 2011a). Cleavage sites are restricted to certain regions (positions 1–25 and 97–162), indicating that other cleavage sites are probably not accessible.

Table 2.7 Amino acid cleavage sites for Lactobacillus helveticus CNRZ303 cell envelope proteinases in alpha S1-casein

1 RPKHPI↓ KHQ↓G LPQEVLNENL LRFFVAPFPQ VFGKEKVNEL SKDIGSESTE DQAMEDIKQM

61 EAESISSSEE IVPNSVEQKH IQKEDVPSER YLGYLEQLLR LKKYKVPQLE IVPNSAEERL 121 HSMKEGIHAQ QKEPMIGVNQ EL↓AYFYPE↓LF RQFYQLDAYP SGAWYYVPLG TQYTDAPSFS

Table 2.8 Amino acid cleavage sites for Lactobacillus helveticus CP790 cell envelope proteinases in alpha S1-casein

1 RPKHPI↓KHQ↓G LPQEVLNENL LRF↓FVAPFPQ V↓FGKEKVNEL SKDIGSESTE DQAMEDIKQM

61 EAESISSSEE IVPNSVEQKH IQKEDVPSER YLGYLEQLLR LKKYKVPQLE IVPNSAEERL 121 HSM↓KEGIHAQ Q↓KEPMIGVNQ EL↓AYFYPE↓LF RQFY↓QLD↓AYP

S↓GAWYYV↓P↓L↓G TQYTDAPSFS 181 DIPNPI↓GSEN↓SEK↓TTMPLW

2.9.2.4Kappa Casein

The degree of hydrolysis of κ-casein by L. helveticus CEPs provides little or no activity compared to the other caseins as reported by Hébert et al. (2000) and Scolari et al. (2006). After 30 minutes of κ-casein incubation with L. helveticusZuc CEP, κ-casein was cleaved into glycomacropeptides, that is, κ-CN (106–169). The released peptides included κ-CN (147–169), κ-CN (106–111), κ-CN (112–116), κ-CN (112–149), κ-CN (106–113) and κ- CN (117–146) (Scolari et al., 2006). In contrast, this hydrolytic pattern was not observed by CEP of L. helveticus CP790, again indicating diversity in CEPs (Laloi et al., 1991; Yamamoto et al., 1993).