Analysis Of Potential Antimicrobial Peptide Genes In Traditional Vinegar Fermentation Process And Their Relationshi With Microbial Succession

Chinese traditional vinegar adopts open fermentation process,and the microbial community composition is complex,and the fermentation process continues to evolve.The interaction between microorganisms is one of the main factors of its community succession.The microbial composition of traditional vinegar fermentation process is complex,and the expression of antimicrobial peptides may exist and have an impact on the succession of microbial communities.In this study,bioinformatics methods were used to analyze the potential antimicrobial peptide genes in the metatranscriptome of Shanxi aged vinegar(SAV)fermentation process and their relationship with microbial community succession,and the heterologous expression analysis of possible novel antimicrobial peptide genes.And screened to obtain microorganisms with antibacterial activity against vinegar-contaminated microorganisms.The research results have important scientific value and potential application value for further understanding the succession mechanism of traditional vinegar microbial community and mining new antimicrobial peptides.Using the antimicrobial peptide database CAMP to analyze the metatranscriptomic data of vinegar and acetic acid fermentation,it was found that there were 52 potential antimicrobial peptides in the vinegar fermentation stage,including 368 potential antimicrobial peptide genes.Microbial traceability analysis of antimicrobial peptide genes showed that Lactobacillus and Bacillus were the main microbial sources of antimicrobial peptides.The correlations between 13 bacteriocins and 42 genera of microorganisms with high abundance and 8 genera of common polluting microorganisms in the process of vinegar fermentation were analyzed.The results showed that the correlation between bacteriocin and the main functional microorganisms Lactobacillus,Acetobacter,Bacillus was low(p<0.8),but it was negatively correlated with Staphylococcus,Clostridium,Escherichia,Serratia,the common contaminating microorganisms in vinegar fermentation process.Bioinformatics analysis of 368 potential antimicrobial peptide genes was carried out,and two potential novel antimicrobial peptide genes,gene-150024 and gene-325276,were mined.The amino acid sequence similarity with the antimicrobial peptide Mcd A1 was88.68% and 69.81%,respectively.The results of gene sequence alignment showed that gene-150024 and gene-325276 were derived from Streptococcus macedonicus.Heterologous expression of gene-150024 and gene-325276 was carried out in Escherichia coli BL21,but the expression products had no antibacterial activity.The recombinant plasmids p NZ8148-150024 and p NZ8148-325276 were constructed,and gene-150024 and gene-325276 were heterologously expressed by L.lactis NZ9000,the expression products have antibacterial activity against Bacillus subtilis,but against Escherichia coli,Staphylococcus aureus,Sphingobacterium,Bacillus badius,Clostridium,Serratia,Rummeliibacillus pycnus,Rummeliibacillus stabekisii,and Acetobacter pasteurianus have no antibacterial activity.Scanning electron microscope analysis and zeta potential analysis showed that antimicrobial peptides 150024 and 325276 may exert antibacterial activity by destroying the cell envelope of Bacillus subtilis.The antibacterial properties of 91 strains of Bacillus and 44 strains of Lactic acid bacteria isolated from the fermentation stage of vinegar were analyzed,and a strain of Bacillus amyloliquefaciens qu6 with broad-spectrum antibacterial activity was obtained.It has a good inhibitory effect on the contaminating microorganisms in the vinegar fermenting process and products,such as Staphylococcus aureus,Escherichia coli,Sphingobacterium,Bacillus badius,Serratia,Rummeliibacillus pycnus,Rummeliibacillus stabekisii,but had no inhibitory activity on the vinegar fermenting microorganism Acetobacter pasteurianus.The characteristics of antimicrobial peptides produced by Bacillus amyloliquefaciens qu6 were preliminarily analyzed,it had good antibacterial activity in the range of 80 ℃,8 g/100 m L ethanol and p H 5-8,indicating that it has the potential to prevent microbial contamination during vinegar fermentation.Preliminary analysis by PCR method showed that the antimicrobial peptides produced by Bacillus amyloliquefaciens qu6 may be Amylocyclicin and Bacilysin.