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[目的]优化生防菌-枯草芽孢杆菌B579芽孢生成的发酵条件,提高芽孢浓度。[方法]采用两步控制策略,即发酵的第1阶段(菌体生长阶段,0～10h)促进细胞生长,发酵的第2阶段(菌体对数生长后期,芽孢生成阶段,10～30h)促进芽孢生成。分别考察了培养基初始葡萄糖浓度、发酵pH值、第2阶段摇床转速、第2阶段发酵温度对芽孢生成的影响。通过单因素试验确定其水平,在单因素实验基础上进行正交试验优化,确定获得高浓度芽孢的最佳发酵条件。[结果]培养基初始葡萄糖浓度是影响芽孢生成的显著因素,芽孢生成的最佳发酵条件是:培养基初始葡萄糖浓度5g/L,发酵pH7.0,第1阶段的发酵温度37℃,摇床转速180r/min,约10h后进入第2阶段,发酵温度提高到40℃,摇床转速调节为200r/min。发酵30h,最终获得的芽孢浓度为9.43×108CFU/ml,芽孢生成率为90.99%,分别是优化前的6.70倍和2.43倍。[结论]利用两步控制发酵策略可提高生防菌B579芽孢浓度,建立的发酵工艺为生防菌剂的大规模生产奠定了基础。
[Objective] The research aimed to optimize the fermentation conditions of biocontrol bacterium - Bacillus subtilis B579 and increase the spore concentration. [Method] The two-step control strategy, that is, the first stage of fermentation (cell growth stage, 0 ~ 10h) promoted cell growth. The second stage of fermentation (logarithmic growth phase, sporulation stage, 10-30h) Promote spore formation. The effects of initial glucose concentration, fermentation pH, stage 2 shaker rotation speed, and stage 2 fermentation temperature on sporulation were investigated. The single factor experiments to determine its level, based on the single factor experiments to optimize orthogonal test to determine the best fermentation conditions for obtaining high concentrations of spores. [Result] The initial glucose concentration in culture medium was a significant factor affecting the spore formation. The optimal fermentation conditions for spore formation were as follows: the initial glucose concentration of culture medium was 5 g / L, the fermentation pH was 7.0, the fermentation temperature of stage 1 was 37 ℃, The speed of 180r / min, about 10h after entering the second stage, the fermentation temperature was raised to 40 ℃, shaker speed adjusted to 200r / min. Fermentation 30h, the final concentration of spores obtained 9.43 × 108CFU / ml, spore formation rate was 90.99%, respectively, before the optimization of 6.70 times and 2.43 times. [Conclusion] The biocontrol bacterium B579 spore concentration could be increased by using two-step control fermentation strategy. The established fermentation process laid the foundation for the large-scale production of biocontrol agents.