枯草芽孢杆菌HAB-1产生抗菌物质的最优发酵条件

Optimization of Fermentation Conditions for Bacillus subtilis HAB-1 to Produce Antimicrobial Substances

为筛选适合Bacillus subtilis HAB-1生长和抑菌物质产生的最佳培养基和最佳培养条件,采用摇瓶培养法和平板对峙法,通过单因子试验筛选适合菌株HAB-1生长的碳源、氮源、金属离子及最佳培养条件。结果表明,经摇瓶发酵培养筛选到适合菌株HAB-1生长及其拮抗物质产生的最佳碳源为蔗糖和玉米粉,最佳氮源为酵母粉和麸皮,最佳金属离子是K+。用正交试验法确定了使HAB-1具有较强抗菌活性的最优培养基为ZY,其各组分的质量浓度为:蔗糖10 g·L-1,玉米粉30 g·L-1,酵母粉30 g·L-1,麸皮40 g·L-1,KH2PO41 g·L-1;在确定适合该菌株的培养基组分基础上,对其培养条件进行优化试验,结果表明,初始pH为6.0,培养温度为37℃,250 mL摇瓶装液量为60 mL,接种量的体积分数为2%,培养时间为24 h,转速为190r·min-1时为该菌株的最佳培养条件,获得菌株HAB-1活菌数最多且对橡胶树炭疽菌的抑菌活性最强,活菌数和抑菌直径分别为2.6×1010·mL-1和34.0 mm,较NB培养的HAB-1提高了123.81倍和1.10倍。

An attempt was made to determine the optimal medium for Bacillus subtilis HAB-1 growth and the best fermentation conditions for Bacillus subtilis HAB-1 to produce anti-microbial substances. Suitable C source, N source and metal ions were screened for the medium and the cultural conditions determined for the strain HAB- 1 through single factor test with the methods of shake flask culture and plate confrontation. The optimal carbon source was sucrose and corn flour, the optimal nitrogen was yeast extract and bran, and the optimal metal ions were K＋ Orthogonal experiment showed that HAB-1 had the strong anti-microbial activity against pathogen when cultured in the medium ZY. This medium contained 10 g L-1 sucrose, 30 g L-1 corn flour, 30 g L-1 yeast extract, 40 g L-1 bran, and 1 g L-1 KH2PQ. The culture conditions for this medium were further opti- mized. The optimal cultural conditions of this medium were pH 6.0, the fermentation temperature 37 ℃, the medium volume 60 mL in a 250 mL flask, the inoculation volume ratio 2% （ V/V）, the fermentation time 24 h, and the rpm 190 r min-1 Under these culturing conditions, the strain HAB-1 had the largest viable count and the highest antimicrobial activity against anthracnose pathogen in rubber tree. The viable count and inhibition zone diameter were 2.6×1010/mL and 34.0 mm, respectively, 123.81 times and 1.10 times higher than those of the strain HAB-1 cultured in NB.