Screening Of Bacteriocin-Producing Lactic Acid Bacteria And The Probiotic Properties Evaluation

Lactic acid bacteria(LAB)is an internationally recognized probiotic in the intestines,which has various physiological functions such as lowering cholesterol,promoting digestion and absorption,and maintaining the balance of gastrointestinal microbiota.The probiotic effect of lactic acid bacteria in the intestine is closely related to the various beneficial metabolites produced by their metabolism.Some lactic acid bacteria produce antimicrobial peptides during their growth process,which can exert efficient antibacterial or bactericidal effects by forming pores on the cell membrane or directly inhibiting certain cell functions,and have received widespread attention in intestinal health.Research has shown that bacteriocin-producing lactic acid bacteria can promote the colonization of themselves and other probiotics in the intestine by inhibiting the growth of pathogenic microorganisms,competitive rejection,or competitive adhesion,thereby improving the disordered gut microbiota environment and stabilizing the gut microbiota.Therefore,further exploration of bacteriocin-producing lactic acid bacteria with efficient antibacterial effects and analysis and evaluation of their intestinal probiotic activity play an important role in promoting the application of lactic acid bacteria in regulating intestinal microbiota and intervening or treating diseases.In this study,a bacteriocin-producing Lactiplantibacillus plantarum ZW1 with strong antibacterial activity was screened from 60lactic acid bacteria preserved in the laboratory.Its beneficial activity was evaluated in vitro,and its antibacterial peptide gene cluster was analyzed by whole genome sequencing and comparative genomics and initially expressed.The main research contents include:(1)By excluding organic acids,hydrogen peroxide interference,and other experiments,a bacteriocin-producing L.plantarum ZW1 with strong antibacterial activity was screened.The fermentation conditions of ZW1 were optimized using antibacterial activity as an indicator.When the carbon source was 100 g of sucrose and the nitrogen source was 25 g of yeast powder,the initial pH was 8.0,the temperature was 37℃,and the cultivation time was 24 hours,ZW1had the highest antibacterial activity,with a diameter of 30 mm.(2)The probiotic performance evaluation of ZW1 based on an in vitro model.ZW1 has good gastrointestinal digestive fluid tolerance,and the survival rate remains above 80%after 7hours in artificial gastrointestinal fluid;The 24-hour survival rate still reached 56.2%in 0.12%bile salt.ZW1 can significantly inhibit Gram-positive bacteria S.epidermidis ATCC 12228 and Gram-negative bacteria E.coli ATCC 25922.The growth and antibacterial activity gradually increase with the prolongation of culture time and the antibacterial activity against E.coli ATCC25922 is slightly stronger than that against S.epidermidis ATCC 12228,with a diameter of 25mm.Meanwhile,ZW1 has strong hydrophobicity,self aggregation,and antioxidant activity.In the anaerobic fermentation model of in vitro gut microbiota,the addition of high concentration ZW1 significantly enriched the mouse fecal microbiota,and the OTU number of the microbiota was significantly higher than the addition of probiotics such as L.rhamnosus LGG and non-bacteriocin-producing L.plantarum PL4.The Chao1,Shannon,and Simpson indices of the L.plantarum PL4 group were significantly improved compared to the control group;Principal component analysis showed that high concentrations of ZW1 and LGG could significantly regulate the structure of mouse fecal flora.ZW1 could significantly increase the relative abundance of Firmicutes and reduce the relative abundance of Proteobacteria,Proteus and Shigella.PL4 has no significant effect on the gut microbiota.(3)Complete genome sequencing of ZW1 and comparative genomic analysis with the genome data of 14 bacteriocin-producing L.plantarum strains in the NCBI database.ZW1 and L.plantarum 14917,20174,NC8,and STⅢhave ANI values of more than 98.85%,close genetic relationships,but low collinearity,and a large number of gene rearrangements such as insertion,deletion,inversion,and translocation.There is a significant difference in the number of unique genes among each strain,indicating that these 15 strains have high homology(>96%)and also have some evolutionary differences.Among them,ZW1 has the most unique genes,including transcriptional regulation,DNA templating,etc.The ZW1 genome has the highest number of genes related to energy metabolism and amino acid transport.Compared to the other14 strains of L.plantarum,which have a unique glycoside hydrolase family 8 gene,it can produce two Ⅱ_b class bacteriocins plnFE,plnJK,and one Ⅱ_c class bacteriocins plnA,and share a conserved Gly-Gly sequence with other bacteriocins genes.The bacteriocin structure gene in the ZW1 bacteriocin gene cluster was expressed,and six engineering strains were constructed.Among them,E.coli BL21-pET-32a(+)-plnIFEGH was partially expressed in cells.The activity verification experiment showed that the E.coli ATCC 25922 culture system was added with the final concentration of 2 mg/m L of cell broken supernatant crude protein,and the inhibition rate of E.coli ATCC 25922 was 50.8%after 1 h fermentation.