Medium Optimization And The Antifungal Substances Purification Of Bacillus Amyloliquefaciens BW-13

With the increasing infection of phytopathogenic fungus,the research and development for new antifungal agent is becoming more and more important.In our previous works,four model strains(S.cerevisiae Whu2a,CHT003,CHT020,Afksl),in which CHT003 and CHT020 are defective mutants in genes of chitin synthase and ?fks1 is defective mutant in genes of glucan sythase.Bacillus amyloliquefaciens BW-13 was screened for its great antifungal activities and special effect on S.cerevisiae CHT003.In order to increase the yield of antifungal substance produced by BW-13,the medium was optimized by single factor and uniform design.And then the fermentation conditions were also optimized.The optimized medium and fermentation conditions were as follows:corn starch 40 g/L,peptone 6.7 g/L,manganese chloride 5 mg/L,initial pH 5.0,liquid volume 30mL in 250 mL flask,inoculums concentration 4%,incubation temperature 28 ?,shaking revolution 200 r/min,cultural time 144 h.The antifungal substance of BW-13 fermentation broth was increased to 8.33 times compared with the control.The physical and chemical properties of the antifungal substance were further analysed by the paper electrophoresis,paper chromatography and TLC.The paper electrophoresis and paper chromatography demonstrated that the antifungal substance have subacidity and hydrophilic property.The antifungal substance could be separated by TCL with butanol:acetic acid:water(3:1:1),and the spots could be colorated by ninhydrin.The above characteristics of antifungal substance are similar to some of peptides antifungals.To purify the antifungal substance,hydrochloric acid was chosed to precipitate the proteins or other impurities from the broth of BW-13,and then macroporous resin S-8 chromatography was taken as the second step to remove the pigments,and finally the cation ion exchange resin HZ-011 chromatography was used to refine the antifungal substance with the eluent of 0.3 mol/L ammonia water.The partially purified antifungal substance were tested by UV-scanning and RP-HPLC anylisis,and one peak with purity of 84.86%was dectected at 220 nm.However,in activity test,three peaks were detected.This was concerned with the result of TLC,in which also three spots of antifungal substances were detected.Althogh the antifungal substance still need to be purified,this work would be great useful for the further study of antifungal substance by BW-13.