Research On The Fermentation Media Of Bacillus Subtillus B-332 And The Purification Of Its Antifungal Substances

Rice blast is one of the most severe diseases on rice. It can cause 30% reduction of the world rice field. Now the products used to control the Magnaporthe grisea are mainly chemical pesticides. To maintain the sustainable development of environment and reduce the damage of the chemical pesticides to human and environment, developing a bio-pesticide that can control Magnaporthe grisea is indispensable. In this paper, the effective substances produced by strain B-332 and the flask fermentation condition of the strain were explored. The results were as follows:B-332 strain of bacillus subtilis has great bio-control potential, which can strongly inhibit the germination of the spores of Magnaporthe grisea and deform the bud.The effective substances were gained by the following procedures : amplification of the strain through fermentation-centrifuge-crude extraction by iso-electricity method. The effective substances presented a peak under the UV wavelength of 272.9nm.The content measurement of it was established.The crude effective substances were separated and purified through HPLC-MS. Five components had a control effect on the Magnaporthe grisea. The molecular weights of the five components were defined by MC analysis and they belonged to Bacillomycin D.Three methods to count the number of the spores were compared. The result showed that Gram dye is the best one, which can distinguish clearly the spore and body.The optimization of the fermentation process of the Bacillus Subtilis B-332 strain laid a solid foundation for the manufactory production. In terms of media, carbon source, nitrogen source and inorganic soil were considered. The optimization experiment targeted at the principal components was conducted and the optimized media components were established: bean cake powder 6.0%, sucrose 2.5%, (NH₄)₂SO₄0.7%, Sodium citrate 0.3%;In terms of cultivation condition, the inoculation age and content were first explored, and the loading content, temperature and rotate speed were optimized afterwards. With the combination of the optimized media and fermentation condition, the metabolism parameters of the fermentation process were assessed. The rule of change was identified; the end point of the fermentation period is 72h, when the spore number reached 1.43×10¹¹/mL,which improved 34.48 times compared with the original condition.The producing level of the antibiotics reached 213mg/ml.