Hydrolytic Properties Of Milk Proteins By Lactobacillus Helveticus And Its Cell Envelope Proteinases

Lactobacillus helveticus is widely used in fermented dairy products and has an efficient protein hydrolysis system to produce bioactive peptides during fermentation.It has been shown that the protein hydrolysis system of L.helveticus has a high intraspecific variability,but the effect of genetic heterogeneity of the protein hydrolysis system of L.helveticus on its milk protein utilization capacity,hydrolysis product diversity and angiotensin-converting enzyme-inhibiting peptide production properties still need to be further investigated.In this study,37 L.helveticus strains from different habitats were subjected to evolutionary analysis based on homologous genes and differential analysis of protein hydrolysis-related genes;representative L.helveticus strains were selected and their growth and acid production characteristics,casein utilization capacity and CEP activity were measured during fermentation.Subsequently,the differences in peptide composition in fermented products and hydrolysis patterns of representative L.helveticus strains were analyzed;the composition of ACE inhibitory peptides in the fermented products and ACE inhibitory activity of the fermented products were investigated.In addition,the crude proteinases were extracted and its hydrolysis products were used to investigate the effect of CEP on the peptide composition of fermentation products.Analysis of protein hydrolysis genes and their correlation with homologous evolution in37 L.helveticus strains showed that the genotypes of CEPs and the peptide transport system changed as the strains evolved,and the CEPs differed in structural domains and amino acid sequences.The strains with the closest root affinity to the homologous gene evolution tree commonly contained Prt H₄,followed by Prt H₁ variant,Prt H₂ as the main CEP genotypes in the remaining branches,and the strains with the most distant root affinity commonly contained the Prt H₃ gene,accounting for 56%of all strains.Lactobacillus helveticus strains showed differences in protein utilization and cell envelope proteinase activity in skim milk,with 4 L.helveticus strains containing Prt H₃ showing faster growth and acid production and higher enzymatic activity.The strains showed differences in hydrolysis preferences for?_s1-casein and?-casein,with most strains preferentially hydrolyzing?_s1-casein,but the preference was independent of the cell envelope proteinase genotypes.The composition of peptides with molecular weights below 3000 Da in the fermented milk of the L.helveticus strains differed,the differential peptides accounted for about half of the total peptides in all strains.The peptides in the fermentation products of strains containing Prt H₃were similar and clustered,and the peptides in the fermentation products of strains containing other CEPs were similar in composition.The hydrolysis of?_s1-casein by the five L.helveticus strains was not significantly distinct,all concentrated in the f1-40,f80-120 and f175-200regions,while there was strain variability in the preferred hydrolysis of the?-casein sequence,taking the region of?-casein f45-110 as an example,7M3 and M108 preferentially hydrolyzed f70-100,M98 preferentially cleaved f45-75,while R0052 had no significant preference,and32M42 had a similar cleavage pattern to M108 for this region.The ACE inhibitory peptide composition of the fermentation products of the 5 L.helveticus strains varied,and the strains showed a preference for specific sites.The ACE inhibitory activity of M108 and 32M42fermentation products was higher,but not proportional to the number of ACE inhibitory peptides.Correlation analysis of ACE inhibitory activity with peptide species showed that the tripeptide VPP correlated most significantly with the ACE inhibitory activity of the samples,and strains containing Prt H₃ were able to produce more VPP.32M42 and M108 digested samples still showed over 60%ACE inhibitory activity,and the amino acid composition and properties of the anti-digestion peptides followed a pattern.The crude extract of L.helveticus cell envelope proteinases showed a similar hydrolysis pattern to that of L.helveticus.The cell envelope proteinases also showed no cleavage preference for?_s1-casein,concentrating on cutting the N-terminal,C-terminal and f80-120regions;while the hydrolysis regions for?-casein were significantly different and roughly coincided with the preferred hydrolysis regions of the strains,exhibiting both M108(Prt H₃)and the corresponding CEP Prt H₃ preferred to hydrolyze f160-180,and strain R0052(Prt H₄)and the corresponding CEP Prt H₄ preferred to hydrolyze f1-70.In summary,the cell envelope proteinases of Lactobacillus helveticus strains exhibit a diversity with evolution,endowing the strains with different protein hydrolysis abilities and cleavage patterns,with Lactobacillus helveticus with Prt H₃ being able to hydrolyze the two major caseins in the milk system more rapidly and with higher enzymatic activity,and its cleavage pattern favoring the production of more peptides with ACE inhibition potential.The cell envelope proteinases and corresponding Lactobacillus helveticus strains showed similar hydrolysis regions for milk proteins,suggesting that the CEPs type has a major contribution to the peptide composition of the fermentation products of the strains.