Identification And Safety Of Active Components Of Bacillus Amyloliquefaciens

The application of probiotics in aquaculture has attracted much attention.When people study the beneficial effects of probiotics,they gradually realize that there may be safety risks in the application process.Because probiotics in the process of growth and reproduction will produce a series of metabolic product and part of the metabolites have antibacterial effect,this part of the metabolites of antibacterial composition,features and functions are not clear,this kind of probiotics in aquaculture production extensive use of whether adverse impact on aquaculture water flora,whether for the introduction of area caused by biological invasion,are poorly understood.Bacillus is one of the most widely used probiotics in aquaculture.A strain of B.amyloliquefaciens HE with good inhibitory effect on A.hydrophila was isolated in our laboratory at the early stage.In order to explore the antibacterial composition of B.amyloliquefaciens HE against A.hydrophila and the safety of its application in aquaculture,the following studies were carried out in this paper.To clarify the active components of B.amyloliquefaciens HE fermentation liquid,and to determinate the physicochemical properties of B.amyloliquefaciens HE fermentation liquid,lipopeptide synthesis related genes were detected by PCR.Four pairs of primer amplification products were cloned,sequenced and BLAST analyzed,and UPLC-Q-TOF-MS was used to further analyze its active substance composition.The results showed that the fermentation broth of the strain had surface activity and antibacterial activity,and had certain tolerance to high temperature,acid,alkali and protease.The HE genome of B.amyloliquefaciens contains lipopeptide synthesis related genes of ituA,fenB,sfP and mycB.The crude lipopeptide extract contains the molecular weight of three kinds of lipopeptide antibiotics,namely Surfactin,Iturin and Fengycin.Based on the above results,it was speculated that the fermentation broth of B.amyloliquefaciens contained three types of lipopeptide antibiotics,namely Surfactin,Iturin and Fengycin.In order to understand the antimicrobial effect of lipopeptide produced by HE strain on A.hydrophila,the minimum inhibitory concentration?MIC?and minimum bactericidal concentration?MBC?of lipopeptide against Aeromonas hydrophila were determined by double dilution method.The effects of lipopeptide on growth curve and bactericidal kinetics of Aeromonas hydrophila were determined.The antimicrobial effect of lipopeptide was also discussed from the microscopic structure.The results showed that the MIC and MBC of lipopeptide against A.hydrophila were both 137.97?g/mL.The antimicrobial effect of lipopeptide on A.hydrophila decreased with the increase of bacterial concentration,but increased with the increase of lipopeptide concentration or the extension of the action time.Lipopeptide can cause cell membrane collapse and porosity of A.hydrophila.The safety of HE strain was evaluated by acute toxicity test of HE strain and its metabolite lipopeptide on fish,as well as the effect test of HE strain on aquaculture water environment in integrated aquaculture model.The results showed that HE bacteria had high biological safety to grass carp,gibel carp and silver carp,but the lipopeptides produced by HE bacteria had low toxicity to grass carp(500 mg/kg<LD₅₀<5000 mg/kg).Adding HE bacteria with a concentration of 1×10¹¹ cfu/m³ every seven days could significantly reduce ammonia nitrogen,total nitrogen,total phosphorus and chemical oxygen demand in the aquaculture water for a long time,but increase nitrite?p<0.05?.It was found that the diversity of water microflora in the treatment group was significantly reduced.Bacterial diversity in the tanks was greatest in the un-inoculated tanks and Proteobacteria,Actinobacteria,Cyanobacteria,Verrucomicrobia and Bacteroidetes dominated.In the presence of added B.amyloliquefaciens,Proteobacteria and Bacteroidetes levels increased while Actinobacteria decreased.At the class level,there were also differences between the treatment group and the control group.At the genus level,the two groups of bacillus were at the same level.As above,we conclude that supplementation with B.amyloliquefaciens did not multiply in large numbers and could cause alterations of the microbial composition in intergrated culture system.Functional genes detected by 16SRDNA showed no significant difference in the self-purification ability between treatment group and control group?P>0.05?.