Screening Of Brevibacillus Laterosporus Producing Antimicrobial Peptides And Its Antimicrobial Mechanism Against Salmonella Typhimurium

Antimicrobial peptides,synthesized in organisms,are low molecular weight peptides with high antibacterial activity,low risk of drug resistance and green environmental protection.It is of great significance in the research and application of alternative antibiotics.Brevibacillus laterosporus,an important microorganism for the biosynthesis of natural antimicrobial peptides,generally exists in soil,insects,plants and other environments.In recent years,a variety of antimicrobial peptides and enzymes with different functions have been isolated and identified from the secondary metabolites of B.laterosporus.It is expected that B.laterosporus has a broad application in the fields of food processing and storage,agricultural biological control and drug development.Therefore,it is of great importance to develop new antimicrobial peptides in B.laterosporus.Salmonella is one of the most common foodborne pathogens in the world.In China,food poisoning caused by Salmonella accounts for 70%to 80%of bacterial food poisoning every year.The study of the mechanism of new antimicrobial peptides on Salmonella will provide theoretical basis for the application of antimicrobial peptides in food preservation.In this study,a strain of B.laterosporium with broad-spectrum antimicrobial activity was screened from soil.The chemical structure and composition of antimicrobial substances were isolated,purified and identified.Furthermore,the antimicrobial activity,stability and tolerance of the substances were tested.Finally,the antimicrobial mechanism of the substances against Salmonella typhimurium was explored.The main results are as follows:1?A strain of B.laterosporus was screened from the soil and named fmb70.The culture conditions and the preliminary screening of the culture medium were carried out,and the optimal fermentation medium was Luria-Bertani medium and the optimal fermentation conditions were obtained.The culture was performed at 30?,180 rpm,and 24 hours,and the yield of the antimicrobial substance was the highest at this time.After antimicrobial activity experiments,it was found that it has a good antimicrobial effect on gram-positive bacteria,gram-positive bacteria and fungi.2?Through n-butanol extraction,Sephadex LH-20 column chromatography,and semi-preparative liquid phase separation,two antimicrobial substances with purity higher than 90%were obtained.MS and MS/MS analysis were performed to obtain molecular weight and amino acid sequence,respectively.One of the antimicrobial substances was identified as the antimicrobial lipopeptide brevibacillin(MW1583.75 Da),and the other newly discovered lipopeptide was named brevibacillin V(MW1570.12 Da).They all consist of 13 amino acids and a C6 fatty acid chain.It is a cationic antibacterial lipopeptide because of one ornithine and two lysines.3?The pH and thermal stability of brevibacillin and brevibacillin V and their application potential in skim milk were explored.The results show that brevibacillin and brevibacillin V have excellent thermal stability and no loss of activity at 100? for one hour.There is a certain loss of activity at pH 3 and 9,and the activity is relatively stable between the value of pH 3-9.At the same time,the hemolytic activity of the two lipopeptides is low,and the hemolytic rate is less than 10%at a concentration of 512 ?g/ml.The inhibitory effects of Staphylococcus aureus and Listeria monocytogenes were simulated in skim milk.It is shown that 16 ?g/ml brevibacillin and brevibacillin V can effectively inhibit Staphylococcus aureus and Listeria monocytogenes in skim milk,and 64?g/ml brevibacillin and brevibacillin V can effectively kill Staphylococcus aureus and Listeria monocytogenes in skim milk samples.4?The antimicrobial mechanism of brevibacillin and brevibacillin V on Salmonella typhimurium CICC 21493 was studied.The addition of LPS,Mg2+and Ca2+partially eliminated the antimicrobial activity of brevibacillin and brevibacillin V against Salmonella typhimurium,suggesting that these two cationic lipopeptides can bind to LPS and replace divalent cations on the LPS network.The results of NPN uptake experiments and ET ELISA kit assay showed that as the concentrations of brevibacillin and brevibacillin V increase,part of the LPS will be gradually released into the environment,resulting in an increase in outer membrane permeability.Calcein exudates completely from the liposomes and releases DiSC3-5 from the cytoplasmic membrane,suggesting that brevibacillin and brevibacillin V may destroy cytoplasmic membrane through the carpet model.These results indicate that brevibacillin and brevibacillin V exert their antimicrobial activity through membrane damage.The membrane damage is a dose-dependent outer membrane permeability,with a concentration of 64-256 ?g/mL,and the cytoplasmic membrane carpet damage concentration is 4 ?g/mL.