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Obtain an antagonistic bacteria (strain number: C332) is separated from the soil, Titan, preliminary studies: The results showed that this strain has a strong antagonistic action on a variety of plant and animal pathogenic fungi strains excellent fermentation characteristics, stable nature of the fermentation broth, with a certain application prospects. In this study, the strain identified flask fermentation produces anti-bacterial active substance formulations and conditions of optimum fermentation confrontation the bacteria active substances isolated and purified, and study some of its physical and chemical properties. The main findings are as follows. Observed by cell morphology characteristics, physiological and biochemical characteristics, of 16S rDNA sequences and their phylogenetic tree analysis, identification C332 strain Bacillus cereus (Bacillus cereus). 16S rDNA sequences of the strains have been registered in GenBank accession number AY756511. The in vitro antagonistic tests showed that the C332 strain of tobacco brown spot disease (Alternaria alternata), round tea leaf spot pathogen (Phyllosticta thesefolia), cotton Blight (Rhizoctonia solani) and Fusarium (Fusarium oxysporum) Fusarium oxysporum (Fusarium oxysporum f.sp . vasinfectum), E. coli (Escherichia coli), black chest septicemia (Bacillus sp.), etc. a variety of plant and animal pathogens having antagonistic action. Single single-factor method (one factor at a time) on the C332 strain fermentation medium formula of the substance of the anti-bacterial activity and fermentation conditions, further fermentation medium optimized by response surface method, first used linear regression medium components were evaluated trial design, filter out the impact of three main factors of production: beef extract, glucose and potatoes. Based on the test results steepest ascent was used to approach the maximum production area, the last with optimal concentration of central composite design and response surface analysis to determine the main influencing factor. Optimized fermentation conditions: initial pH 5.0, medium volume 50mL, inoculum size 2%, fermentation temperature 30 ° C, seed age 20h. Optimum medium: glucose 0.99%, beef extract 1.4%, potatoes 4.0% of MgSO4 0.3%, KH2PO4 0.2%, CaCO3 0.15%. Use the formula and conditions for shake flask fermentation, an increase of 2.08 times the the fermentation broth titer initial medium. The salting by 20% to 40% ammonium sulfate fractionation, C332 strain generated by the CM-Sepharose ion exchange column chromatography on Sephadex G-50 column chromatography, high performance liquid chromatography, and thin layer chromatography on the anti-bacterial active substance The separated and purified by silica gel G thin layer chromatography (TLC) will eventually antibacterial substance separation is Rf value respectively 0.39,0.53,0.72 3 kinds of active ingredients. 3 effective components in the single-component state unstable performance. Crude anti-bacterial active substance was pH2.5 ~ 10.0 stability, high temperature, protease-sensitive, not sensitive to ultraviolet, non-toxic to silkworm security.
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