Research Of Active Ingredient Produced By Bacillus Amyloliquefaciens B15

Grape disease is one of the most important problems in wine industry in China, causing different degrees of loss every year. According to statistics, the loss of wine industry in C hina each year caused by grape diseases is about 30% above. The lipopeptide is of small molecular weight, stable structure. Research shows that lipopeptide has surface activity,antibacterial and antifungal capacity, is a rare new antibiotics of great potential in medicine, food and daily necessities industry. Bacillus amyloliquefaciens, a member of Bacillus, is one of the first safety certificated bacteria approved by Food and Drug Administration(FDA). It is also a national free- inspection strain in the Genera l Principle of Biosafety for Microbial Fertilizer published by Chinese Ministry of Agriculture(NY-1109-2006).A bacterium B15, isolated from grape skin, showed a significant ability of inhibiting the molds. Then it was identified as B. amyloliquefacims by conventional biochemical identification 、 16 S r DN A and gyr B gene sequencing.The activity material of B. amyloliquefacims B15 was confirmed as lipopeptide. The extraction methods, structure of lipopeptide was studied, and quantitative analysis was performed, so is the antifungal activity of the different components which would lay good foundation for the development of biological pesticide.The main research content is as follows:1 The product of Bacillus amyloliquefaciens B15 was confirmed as lipopeptides after oil spreading and acid hydrolysis in situ—TLC.Three extraction methods of lipopeptide were compared. The extracting ratio using acid precipitation and ammonium sulfate precipitation extraction were higher than that extracted with methanol only.But the method of ammonium sulfate precipitation spends long time and the desalting step would be needed,the cost would be increased.So acid precipitation is the most suitable method for extracting lipppeptide.2 Matrix assisted laser ionization time of flight mass spectrometer(MALDI- TOF) was used for lipopeptide identification both fermentation broth and crude extractings. The [M + H] + values m/z 1065.5、1079.6、489.5、1534.6 and 1682.5.After acid precipitation crude lipopeptide was purificated by Sephadex LH-20.Three peak were got and one of them show inhibition against Botrytis cinerea.12 components were separated from the active material by preparation HPLC.6 of them are antifungal.Three components are identified as C14-16 iturin A, the other three groups C15-18 fengycin.Three types of iturin A also show inhibition against yeast,their antifungal capacity is relevant to the length of their fatty acid chain,the longer, the antifungal capacity stronger.3 A method to measure content of iturin A, surfactin and fengycin was set up by HPLC. The lipopeptide concentration and its antifungal capacity were studied in the fermentation process with the method. It was found that the concentration of surfactin and fengycin were in line with antifungal capacity.there are two main reasons, one is that fengycin is the main antifungal material secend is that surfactin can enhance fengycin antifungal capacity.4 finally the biological characteristics of lipopeptide produced by B15 were studied.Its minimum inhibition concentration(MIC) is 17 mg/L.The antifungal activity of lipopeptide have little loss within temperature,enzyme and p H from 2 to 10.But p H above 12 would cause big loss of antifungal capacity.By fluorescence microscope the effects of lipopeptide toward Botrytis cinerea was observed.Botrytis cinerea mycelium treated by lipopeptide inflated,its number decreased and its top swollen.