Antagonism Of The Lipopeptide Metabolites Produced By Bacillus Amyloliquefaciens Strain SWB16Against Beauveria Bassiana And Prelimary Optimization Of Its Fermation

Bacillus is a kind of very important biocontrol microorganism resources, which has been developed into many kinds of microbial fungicides. At present the number of spores was used as the quality evaluation index for this kind of fungicides, while most manufacturers added the antibacterial substance after concentration and drying in the formulation process of fermentation broth without qualitative and quantitative analysis. Because of diversity of the antibacterial mechanism of Bacillus, including competition, antagonism, bacteriolysis, induced resistance, is the comprehensive functions, and the type and content of antibacterial substances varied as the differences of strains and the fermentation condition, so based on only the spores, it was not accurate enough to evaluate this kind of microbial pesticides quality, thus only by qualitative and quantitative of spores and antifungal activity analysis, the quality of this microbial pesticides can be accurately and comprehensively evaluated.This paper selects the endophytic bacteria Bacillus amyloliquefaciens SWB16, and performed the fermentation of the strain, combined the previous research results, the lipopeptide antibiotics was exracted by the specific method of extraction, and treated Bacillus amyloliquefaciens SWB16fermentation supernatant solution, obtaining the methanol extract. On the basis of the fermentation extracts, the following experimental study were peformed:1. A Beauveria bassiana strain matained in our laboratory was selected to examine the activity of methanol extract for fermentation products, and through antagonistic test, inhibition activity to Beauveria bassiana conidial germination and mycelial growth was determined, under the treatment of the methanol extract, the condia of Beauveria bassiana could not germinate or ruptured, and it resulted in the mycelial ablation, plasma leaks, mycelium distorted or forming a vesicle structure; furthermore, some studies show that the active products is well tolerated to temperature, ultraviolet, protease K, which is a major component of strain SWB16fermentation broth exhibiting growth inhibition to Beauveria bassiana.2. The quantitative and qualitative analysis of the crude extract activity was performed by using HPLC, LC-MS technology, via the comparison of molecular weight of the active products components with theoretical value of relative molecular mass of the lipopeptide, and combined with the experimental phenomena observed in the previous experiments, suggesting the active extracts contains three lipid peptide antibiotics scilicet, iturins, fengycins and surfactins. Among them, the iturins contains, C14-IturinA, C15-IturinA, C16-IturinA and C17-IturinA; the fengycins class is not determined only by LC-MS technology, probably they also contain two homologues, C15-FengycinA, C16-FengycinA, C17-FengycinA, C18-FengycinA and C14-FengycinB, C15-FengycinB, C16-FengycinB, C17-FengycinB; type Surfactins may contain three homologs, namely C13-SurfactinA, C14-SurfactinA, C15-SurfactinA and C14-SurfactinB, C15-SurfactinB, C16-SurfactinB, C13-SurfactinC, C14-SurfactinC, C15-SurfactinC. The relative quality of the total three lipid peptide antibiotics reached77.216%of crude extracts.3. The fermentation conditions of strain SWB16were optimized through single-factor test and the optimum fermentation conditions of SWB16is preliminarily determined, namely the initial pH at7,5%inoculums, fermentation temperature of30℃, fermentation time for36h,60ml liquid volume (250ml triangle flask), the rotate speed at190rpm. Most of lipopeptide antibiotics precipitated from fermentation supernatant at pH3. In the optimum fermentation conditions and precipitation conditions, Bacillus amyloliquefaciens SWB16was fermented and the final yield of lipopeptide antibiotics obtained reached0.072g/100ml.