Diversity Analysis Of Lactobacillus Helveticus Based On Genomics And Protease Characteristics

Lactobacillus helveticus,one of the lactic acid bacteria,is widely used in fermented dairy products,and has received the attention due to its proteolytic ability among lactic acid bacteria.Studies have shown that Lactobacillus helveticus exhibits diversity on the proteolytic system and ability,but the correlation between the genomics and phenotype needs to be further investigated.In this thesis,the traditional fermented foods and fecal samples from Sichuan,Yunnan,Qinghai and other regions were collected,isolation,purification,identification and preservation of Lactobacillus helveticus was conducted.The 38 isolated stranrs and another12 sequenced strans from NCBI were subjected to comparative genomics,including core-and pan-genome analysis,functional annotation,etc.The result showed that the genome of Lactobacillus helveticus varied among the strains.Lactobacillus helveticus had 794homologous genes,but 6-196 unique genes for different strains.Based on the phylogenetic tree of homologous genes,most of the strains clustered with those from the same sampling site.The strains from Qinghai Xining and Sichuan Aba are clustered in different evolutionary branches.The pan-genome analysis of the strains were“open”,and there was still a possibility of horizontal gene transfer.The result of Clusters of Orthologous Groups?COG?annotation indicated that amino acid transport and metabolism of Lactobacillus helveticus strains were different from each other,and strains exhibited significant difference in amount of cell wall protease encoding gene,but there was not much difference in amount of peptidases encoding gene.Then 30 strains of Lactobacillus helveticus were selected to determine cell envelope proteinase,amino acid peptidase?PepN?PepX?PepC?and aromatic aminotransferase activities,and the acidification ability were determined in MRS and skim milk medium.The result showed that proteolytic enzyme activities of strains were significantly different from each other,with the lowest CEP activity for V-GZTR-132-M1 from pickle.The intracellular amino acid peptidase and aromatic aminotransferase activities of the strain FSDJN10M6 from feces were significantly lower than those of other strains.The results indicated that CEP activity was related to PrtH₃ gene combined with genomics analysis.Correlation analysis and principal component analysis?PCA?showed that there was a significant positive correlation between three aminopeptidases activities of Lactobacillus helveticus,and the actitivity of CEP showed a significant positive correlation with PepN,PepC,aromatic aminotransferase activities and the acidification in skim milk.In addition,according to the result of CEP activity and PCA,it was found that Sichuan Aba and Qinghai Xining strains can be clustered by their geographical origin.Four isolated Lactobacillus helveticus strains with different proteolytic activities,including high,middle and low activities,DQHXN08M33,DQHXN-32M42,DSCAB11L3,V-GZTR-132-M1 and one commercial Lactobacillus helveticus ATCC15009 were fermented in skim milk.The results showed that CEP,three aminopeptidases,aromatic aminotransferase activities,protein and peptide molecular weight distribution,pH value,lactose,galactose,glucose,lactic acid and acetic acid content were different among Lactobacillus helveticus.Moreover,protein and peptide molecular weight distribution,pH value,loss of lactose,content of galactose and lactic acid were found to have a significant positive correlation with each other.In summary,Lactobacillus helveticus strains exhibited significant genotypic and phenotypic differences,and there was a certain relationship between both.The acitivity of cell envelope protease activities and acidification ability induced by Lactobacillus helveticus in skim milk were concerned with PrtH₃ gene.Thus,the acidification ability of Lactobacillus helveticus during fermentation in skim milk is not only affected by the utilization of carbohydrates,but also related to the proteolytic activities of strains.This study provides a theoretical support for the exploitation and application of Lactobacillus helveticus in dairy industry.