Studies On Optimization Of Fermentation Conditions And Purification Of The Antifungal Substance Produced By Bacillus Megaterium Strain196

The antifungal substance was isolated and purified from Bacillus megaterium strain196after optimized its optimal fermentation conditions of producing antagonistic substance. The structure of the compound was identified by MS and MS/MS and its characteristics and antimicrobial activity were tested. The results were showed as follows:1. The optimal carbon, nitrogen source and salt source for the production of the antimicrobial substance by Bacillus megaterium strain B196were sugar, peptone and NaCl,respectively on the basis single factor experiment. The orthogonal experiment results showed that the composition of the best medium was10g/L sugar,20g/L peptone,7g/L NaCl,5g/L K2HPO4. The optimal fermentation conditions was temperature27℃, incubation time5d, inoculum volume1%, medium volumed40mL/200mL, initial pH7.0.2. An antimicrobial substance purified from Bacillus megaterium strain B196showed positive reaction to biuret and negative reaction to ninhydrin, but it showed positive reaction to ninhydrin when it was hydrolyzed, which indicated that the substance could be ring peptide. It contains Asx, Glx, Pro, Ser, Tyr in a molar ratio of nearly3:1:1:1:1. The molecular weights of the antimicrobial substance was1042.5927Da, and the typical b and y fragments which were exactly similar to Iturin A2were obtained by the MALDI-TOF-MS coupled with collision-induced dissociation(CID). Therefore, the antimicrobial substance was identified as Iturin A2.3. When the antimicrobial substance was treated at90℃for60min and at121℃for20min respectively, its antibacterial activity against Rhizoctonia solani reduced by40.45%and 60.45%. The antibacterial activity of the antimicrobial substance was stable at pH2to10, but reduced at pH12. Proteinase K, trypsin, papain and pepsin protease could not affect the activity of antimicrobial substance.The activity of the antimicrobial substance on7plant pathogenic fungi was tested by mycelial growth rate test. The result showed that7plant pathogenic could be inhibited by the antimicrobial substance and the growth of Pyricularia grisea was inhibited strongly The antimicrobial substance could also inhibit the growth of Xanthomonas oryzae pv. oryzicola, X. oryzae pv. Oryzae, and Ralstonia solanacearum tested by inhibition zone method, but it could not inhibit the growth of Pseudomonas syringae pv. glycinea, P. syringae pv. tomato and Pectobacterium carotovora subsp. Crotovora.4. The antimicrobial substance purified from strain196had good control efficacy against Rhizoctonia solani in vitro. With the concentration of40μg/mL,80μg/mL and160μg/mL, its control efficacies were84.01%,97.47%,100%, respectively.