Screening Of Lipopeptide Producing Strains From Mangrove Forestsand And Optimization Of Fermentation Conditions

Antimicrobial lipopeptides have strong antibacterial activity against many pathogenic microorganisms,especially fungi,bacteria and mycoplasma.They have great potential in many fields,such as medicine and biological control.However,some strains from terrestrial sources have some problems,such as weak production capacity of lipopeptide and weak specificity of antimicrobial activity for specific pathogenic strains,which limit the development of lipopeptide production to a certain extent.It is necessary to screen lipopeptide-producing strains with strong lipopeptide-producing ability and significant antimicrobial targeting from new microbial resources.The microbial communities in mangroves live in salt-tolerant,oligotrophic,anaerobic,frequent tidal and other special living environment,which may be the resource pool of lipopeptide-producing strains.Because microorganisms in the marine environment often have special physiological and biochemical metabolic pathways,it is difficult to screen high-lipopeptide-producing target strains efficiently by routine isolation and screening methods;even if the target strains are screened,they may not be able to produce high-lipopeptide in the conventional lipopeptide fermentation medium.Therefore,in this study,the lipopeptide-producing strains were screened by antimicrobial activity,hemolytic activity and surface activity,and the lipopeptide synthase gene was re-screened by PCR amplification.The lipopeptide synthase gene-carrying strains were also screened by TOF-MS.The ability and homologous species were identified,and the fermentation medium and fermentation conditions were optimized by LC-MS and inhibition rate method.This will lay a foundation for further application of marine derived lipopeptide producing strains.1.In this study,mangrove leaves,bark,sea mud and sea water were used as samples to isolate bacteria with strong antibacterial activity and broad antibacterial spectrum.The isolates were studied by medium selection,antibacterial spectrum determination,16 S rDNA and phylogenetic tree establishment.The results showed that only 17 strains had stable antimicrobial activity when the antimicrobial spectrum was determined by dot-blot method,and only 6 strains could synthesize antimicrobial substances in a few medium.HY-5 showed wide antibacterial spectrum and strong activity against 26 strains,which could be used as a follow-up study strain.The 17 isolates were identified as Bacillus cereu?Pseudomonas?Bacillus amyloliquefacien?Vibrio fiuvialis ?Bacillus pumilus and Bacillus flexus by 16 S rDNA and Phylogenetic tree derived.2.In order to screen lipopeptide-producing bacteria quickly and accurately from the above-mentioned different strains,the blood plate method,oil drainage method,PCR technique and TOF-MS analysis were used to study the hemolytic activity of 6 strains.The results showed that 3 strains showed surface activity,and the diameters of transparent circles of 2 oil layers were 32 mm and 45 mm.On the basis of this screening,PCR amplification showed that strains HY-5,HY-4 and HN-9 detected lipopeptide synthase genes,in addition to both of them had iturin synthase regulatory genes,HY-5 and HY-4 also had surfactin synthase regulatory genes,HY-5 also contained Bacillomycin D synthase regulatory genes.HY-5 could produce surfactin,iturin and Bacillomycin D,HY-4 could produce surfactin and iturin,HN-9 could produce iturin.Three strains of bacteria carrying lipopeptide coding genes were cultured in Landy medium.Finally,crude lipopeptide extracts were analyzed by TOF-MS.HY-5 detected three kinds of mass spectrometry peaks,including three kinds of antimicrobial lipopeptide surfactants(1031/1047,1061,1059/1075),iturin(1043,1057,1071)and bacillomycin(1032/1054,1079,1093).HY-4 also detected three types of MS peaks,including three antimicrobial lipopeptides surfactin(1044,1058),iturin(1043,1057,1071)and bacillomycin(1065/1087/1103),while HN-9 detected molecular weight(1054.5917,1087.6058)was obviously on the corresponding peak of iturin.The results showed that PCR combined with TOF-MS could detect lipopeptide-producing bacteria quickly and accurately,and suggested that different parts of mangrove were a resource for lipopeptide-producing bacteria.3.Fermentation conditions such as inoculation amount of fermentation broth,dissolved oxygen,fermentation time,pH value of fermentation broth and rotational speed of shaking table have always been a significant factor affecting microbial lipopeptides production.It will result effect of lipopeptides production of the same strain.landy-based medium was used to fermentation of Bacillus amyloliquefaciens HY-5.the optimal fermentation conditions were 6% inoculum,150 r/min,36 h fermentation time and 40 mL/250 mL bottled.After optimization,After optimization,the content of HY-5 lipopeptides increased from 0.245g/L to 1.6g/L,and the yield of lipopeptide increased by 6.8 times.The optimization of fermentation conditions was a significant factor affecting the yield of HY-5 lipopeptides.