Laterosporus Bacillus Bl-1 Production Of Antimicrobial Substances Fermentation Conditions, And Antimicrobial Substances Isolated And Purified

Bacillus laterosporus BL-1 was inhibitory to Gibberella sanbinetti, Fusarium graminearum Magnaporthe grisea and many other phytopathogenic fungi. Bacillus laterosporus BL-1 can produce antibacterial protein, but its production is low in BL-1 fermentation liquid of normal medium. In order to make further exploitation of the BL-1 strain, the process parameter and medium in shaking flask for BL-1 fermentation was optimized, and the antibacterial active component from BL-1 fermentation liquid was purified.A total of 11 factors were tested for their function of improving antibiotics yield of BL-1 by single-factor test method. The results showed that the optimum process parameter in shaking flask on fermentation consist of initial pH of 8.0, 30℃of culture, loadage 90ml/250ml, 180r/min of rotation, and 48h of fermentation time.The results also indicated that glucose is the best carbon source for the antibiotics productivity of BL-1, and beef extract is the best nitrogen source, in addition, univalent ions as Cl+ and divalent ions as Mn2+ stimulated BL-1 antibacterial active component biosynthesis. Hereby, an uniform design test was applied to optimize above 4 factors. The follow regression model equation and optimized medium were obtained according to regression analysis of the data from uniform design experiment: Y=20.972+2.583X1-1.278X2-3.689X3-123.453X4-0.301X12+0.150X22+0.596X32+324.968X42optimum culture medium includes, glucose 5.27(g/L),beef extract 13.10(g/L),NaCl 5.60(g/L),MnSO4 0.35(g/L)。The model was validated experimentally by the value of 31mm, and the deviation was less than 10% compared with theoretical value.The determination of bioactivity of this antagonistic component showed that its inhibitant activity depressed evidently when treated with 45℃for 30 minutes. and its inhibitant activity tossed under 50℃for 30 minutes. All those indicated that the antagonistic component could not endure high temperature. The result also showed that the antagonistic component was sensitive to Trypsin,Proteinase K and caroid . Its active pH range was from PH 6.0 to8.0.Crude proteins were extracted from precipitation of the strain BL-1 fermentation liquid with ammonium sulphate at 70% saturation. Through SDS-PAGE, antagonistic activity tracing, CM-Sepharose Fast Flow column chromatography,Sephadex G25 and Sephadex G50 column chromatography, the antifungal protein was prepared and purified. The purified protein was demonstrated to be one single protein band by SDS-PAGE with a molecular weight of 55KD.