Analysis Of Antibacterial Properties And Preliminary Exploration Of Antibacterial Mechanism Of L₃ Of Lactobacillus Plantarum

Lactic acid bacteria are important microbial flora in fermented food.These microorganisms endorse fermented food with special color,aroma and taste through fermentation and protein decomposition processes.In addition,some lactic acid bacteria also inhibit the growth of other microorganisms,especially some spoilage microorganisms,by producing acid and secreting bacteriocin.Therefore,the screening of lactic acid bacteria with GRAS(generally recognized as safe)status and the in-depth excavation of their functions have important theoretical significance and application prospect.In this experiment,the strain L₃ screened in our group through the bacteriostatic experiment previously was used as the starting strain.Firstly,the strain was identified through physiological,biochemical characterization and 16S r DNA sequencing analysis.Then the bacteriocin secretion curve was plotted by measuring the bacteriostatic activity of the strain at different fermentation times.The antibacterial substances in the supernatant were verified by full wavelength scanning over the ultraviolet region and protease sensitive experiments.After determining its proteinous properties,the cell-free supernatant was extracted by ethyl acetate,followed by filtration and purification using Sephadex G-50 gel column to obtain the crude bacteriocin.Then the molecular weight was determined by Trinice-SDS-PAGE electrophoresis.The bacteriostatic effect of the bacteriocin on most Gram-positive bacteria,Gram-negative bacteria and fungi was determined by agar diffusion and agar cake method.The temperature and p H stability of the bacteriocin were determined;The antibacterial mechanism was preliminarily explored by measuring conductivity and PI staining technique;Finally,the whole genome was sequenced,and the bacteriocin related genes were identified and predicted.The experimental results showed that the strain L₃ was Lactobacillus plantarum,named Lactobacillus plantarum L₃,and the NCBI serial number was MT78360.Lactobacillus plantarum L₃ had the strongest antibacterial activity when cultured in MRS medium at 37?for 18?24 h.The antibacterial substance had the maximum absorption peak at 280 nm,and the protease sensitivity test showed that it was a kind of protein.The crude bacteriocin L3 showed molecular weight about 4?5 k Da through Trinice-SDS-PAGE electrophoresis analysis.The stability experiment showed that bacteriocin L3 still had good stability under high temperature and p H 2?10;It had antibacterial activities against Gram-positive bacteria,Gram-negative bacteria and some fungi.The preliminary exploration of the bacteriostatic mechanism showed that bacteriocin L3 destroyed the cell membrane integrity and led to the extravasation of the contents,resulting in the cell death.Lactobacillus plantarum L₃ contains more than four bacteriocin synthesis gene clusters.Although there is no leading peptide,it has transporters and immune proteins.