Isolation And Properties Of Antimicrobial Substance Produced By Bacillus Subtilis

Bacillus subtilis can produce many kinds of antimicrobial substances,which have wide application fields in medicine,feed additive,food additive,detergent,oil exploitation,environmental protection and others.A mutant strain B.subtilis D14-24R(his~-),histidine sensitive strain,was obtained from B.subtilis 168 in the previous work by way of UV-induced mutation.B.subtilis D14-24R can grow only with low histidine concentration.While no growth in the absence of histidine and growth was inhibited in high histidine concentration.In this study,B.subtilis D14-24R was used to explore the mechanism of histidine-depended effect.The relationship between histidine concentration and growth/inhibitory effect,and the culture conditions of antimicrobial substance(AT)accumulation were carried out.The target AT was tried to separate and purify.And properties of the AT were studied.The main results are as follows:(1)B.subtilis D14-24R performs both histidine auxotrophic and histidine utilizing defective(leaky defective),which didn't grow on minimal medium without histidine and can grow with appropriate concentration of histidine supplied.But in the presence of high concentration of histidine(>2 g/L),AT was accumulated and the bacterial growth was inhibited.(2)Two-step culture method was established to prepare AT:bacteria was inoculated into LB medium,cell was collected and transferred into histidine supplemental medium,AT could be accumulated up to 400 u/mL after 36 h.(3)A one-step culture method was established to prepare AT,and medium formulations were optimized as:glucose 15 g/L,tryptone 20 g/L,histidine 2 g/L,yeast extract 5 g/L and NaCl 10g/L.The fluid volume is 90 mL/500 mL,and the culture temperature was 37?.AT could be accumulated up to 1600 u/mL after 18-24 h.The yield of AT in one-step culture method was 4times higher than that in two-step fermentation method.The method was easy to manipulate and beneficial to the separation and purification for AT.(4)The adsorption capacity of activated carbon materials to AT was tested.Among them,the powder activated carbon could extract AT completely from the culture medium,but the desorption was difficult,no suitable organic solvent has been found for desorption.Seven kinds of organic solvents were used to extract AT.Target AT crude product was obtained by trichloromethane extraction and rotatory distillation.(5)The antibacterial test was carried out on 33 strains of microorganism with AT crude preparation,and AT inhibited 10 of 24 strains of G~+bacteria.There were no inhibitory effect on G~—bacteria,Yeast and Mould.AT was stable during pH 6-12 and unstable as pH<4;AT was stable under 60?,and unstable above 60?;Its antibacterial activity was not affected by protease digestion.