Identification And Fermentation Of Bacillus Cereus C332, Isolation And Characterization Of Its Antibacterial Substance

C332, a strain of antagonistic bacterium, was isolated from Mountain Tai soil. C332 strain shows strong inhibition against many pathogens, excellent fermentation characterization, and stability culture characterization.The strain was identified, The component of medium and ferment condition was optimized, The antibacterial was purified step by step and it`s physical chemical characterizations were studied. The results were as follows.Strain C332 was identified as bacillus cereus by study of phenotypic characteristics, physiological and biochemical properities and phylogenetic analysis of 16Sr DNA sequences. The sequence of 16S rDNA obtained in this study has been registered at GenBank database and its accession number was AY756511.In vitro, We testing the antagonistic spectrum of strain C332. Strain C332 shows inhibition against many pathogens such as Alternaria alternate, Phyllosticta thesefolia, Rhizoctonia solani, Fusarium oxysporum, Escherichia coli, Bacillus sp.et al.After one factor at a time experiment, response surface methodology was applied to optimize the culture medium of antibacterial production by strain C332.In the first optimization step, the influence of each factor was evaluated by first-order design. The significant factors at the analysis range were potato, glucose and beef-extract. Then a steepest ascent design was used to approach the optimal region of the medium composition. Finally, the optimal concentration of the medium was determined by central composite design and response surface analysis, and the study showed that glucose 0.99%, beef-extract 1.4%, potato 4.0%, MgSO4 0.3%, KH2PO4 0.2%, CaCO3 0.15% were the optimal medium composition.The production of antibacterial was improved by 208% compared with original medium, with the optimum ferment condition: Initial pH 5.0, Liquid volume 50 mL, Pre-inculation time 20 h, Temperature 30℃, Inculation volume 2%.Antibacterial produced by strain C332 was separated and purified with 20%~40% ammonium sulphate precipitation and column chromatography on CM-Sepharose, Sephadex G-50 and HPLC.Finally, we isolated the antibacterial for three single component by TLC with the Rf of 0.39, 0.53 and 0.72.We supposed the pure antibacterial should be a kind of substancewhich instable and often lose its activity in a single component. The crude antibacterial substance kept active under high temperature and pH 2.5~10.0, sensitive to protease, unsensitive under UV, harmless to silkworm.