Study On The Mutation Of Bacillus Amyloliquefaciens BI₂ And Fermentation Of Antifungal Substance

Bacillus amyloliquefaciens BI2 is an efficient antagonistic strain,which can effectively inhibit more than twenty species of plant pathogens.This paper mainly studied mutation breeding and the optimization of fermentation medium and conditions.The condition optimization of Bacillus spores proliferation as well as isolation of the antifungal substance produced by BI2 were also studied.The specific results are as follows:The ARTP system was used to mutate BI2 with different times.Six hundred and twenty one strains producing significant inhibition zones were selected from primary screen.16 strains generating bigger inhibition zones were isolated,from which 5 mutants with high inhibition rates were determined after rescreening.A mutant strain named BI2-012 yielding antifungal substance stably was identified genetic stability experiment,whose inhibition ratio increased 22.70%than the original strain.In order to optimize the fermentation medium and conditions of yielding antifungal substance by the mutant strain,the optimal carbon source and the optimal nitrogen source were determined through single factor experiment.The optimized medium results were as following:yeast powder 1.5%,monosodium glutamate 2%,sodium nitrate 0.8%,soluble starch 0.17%,maltose 0.33%,Tween-80 0.06%,NaCl 0.5%;the optimal culture conditions was:medium 60 mL/250 mL,culture temperature 30 ?,inoculums size 2%,fermentation for 36 h,shaking speed 200 r/min,pH 7.5.The inhibition rate increased 11.70%tthan the original medium and conditions.In order to obtain the largest number of Bacillus spores,0.015%Mn2+ was added to the medium when mutant strain cultured 12 h.The optimal condition for spores proliferation was showed as following:medium 65 mL/250 mL,shaking speed 220 r/min,inoculums size 4%,the culture temperature was modified to 37 ? from 30 ? after cultivating 24 h.When fermentation process was stopped at 48 h,the number of Bacillus spores reached to 1.20X 109 cfu/mL,which increased 63.50%than before optimizing.The fermentation broth was treateded by membrane filtration,decolorizing technique,ion exchange chromatography.Antagonistic substance was obtained by flash chromatography after evaporating.Two analysis methods were developed:(1)C18 column was used to analyse antifungal substance with adding 5 mmol/L tetrabutyl ammonium bisulfate to the liquid phase.(2)The analytical method of using HILIC column with adding nothing to liquid phase can be also enlarged for preparation.