Isolation And Identification Of Antimicrobial Substance Produced By Bacillus Subtilis FmbJ

Recently, an increasing number of people are focusing on exploring the natural antimicrobial substance which is broad spectrum antibacterial, safer and more stable. Natural antimicrobial substance can be divided into three kinds according to the sources originated from animal, plant and microorganism. Microorganism is becoming the predominant source for natural antimicrobial substance as they could be easily cultivated during a short period with low cost.At present, bacteriocin and lipopeptide produced by Bacillus subtilis had been throughly investigated and already been used in plant disease control and pharmacy industry. Several antimicrobial substances from Bacillus subtilis fmbJ have been identified by our lab. This study mainly focused on isolation and structural identification of natural antimicrobial substance from Bacillus subtilis fmbJ. This research provided theories about antimicrobial substance application in the food processing and preservation. The main conclusions are as follow:1. Establishing the isolation and purification methods of the antimicrobial substance. The crude extracts were isolated from the metabolic products using silica gel column chromatography and Sephadex LH-20. Then, the purified substance was obtained by preparative HPLC. In the process of silica gel column chromatography, the effect factors of mobile phase, sample load and flow rate were optimized. The optimal conditions were as below:flow rate 0.9 ml/min, sample load 1 ml, chloroform/methanol(V/V=1;97/3;19/ 1;93/7) as eluting solvent.2. The inhibitory test showed that the antimicrobial substance I exhibited a strong antagonistic activity against Penicillium meleagrinum. The antimicrobial substanceⅡinhibited gram-positive bacteria such as Staphylococcus aureus and Bacill us cereus.3. Identifying the antimicrobial substance I with method of ESI-MS. The result displayed that the molecular weight of antimicrobial substance I was 1534D, on base of the characteristic fragment m/z 994,1108, the antimicrobial substance I of m/z 1535 is identif- ed as FengycinB.4. Identifying the antimicrobial substance II by use of ES1-MS, IR and NMR. The result of IR indicated that the infrared spectrum of the antimicrobial substance II was similar to acetyl spiramycin. These data of 3350 cm-1(OH),1728 cm-1(CO),1580 cm-1 (HC=CH-CH=CH),1030 cm-1 and 1110 cm-1 (C-O-C) suggested that antimicrobial substance II has macrolide residues with the hydroxyl and the acyl group.ESI-MS demonstrated that the molecular weight of antimicrobial substance II was 982D. MS/MS analysis showed that the antibacterial substanceⅡshared the same characteristic fragment of m/z 755,596and 582 with acetyl spiramycinⅢ, which indicated that the both substances had the same structure.The 1H-NMR and 13C-NMR of the antimicrobial substances II were close to acetyl spiramycinⅢ. The proton signals and carbon signals of forosamine, mycaminose, isovalerylmycarose unit and macrolide residues could be easily found in the 1H-NMR and 13C-NMR. Summarizing all above analysis,the antimicrobial substancesⅡwas identified to be 4"-isovaleryl-spiramycin.