具群体感应淬灭作用地衣芽孢杆菌T-1株培养基及发酵条件的优化

Optimization of culture media and fermentation conditions of quorum sensing quenching bacteria Bacillus licheniformis T-1

【目的】优化具群体感应淬灭作用地衣芽孢杆菌(Bacillus licheniformis)T-1株的培养基及发酵条件,进一步提高其产孢量,为今后开发地衣芽孢杆菌制剂(抗菌益生菌)打下基础。【方法】在摇床发酵条件下,采用单因素试验筛选T-1株的最佳培养条件,并确定影响发酵菌液活菌含量的主要影响因素。根据Plackett-Burman试验结果,筛选出影响芽孢产量的2个最显著因素,以最陡爬坡试验确定其响应中心点和最佳浓度范围,然后通过Box-Behnken中心组合试验和响应面分析获得最佳发酵培养条件。【结果】T-1株的最适发酵培养条件为温度37℃、盐度0.5%和pH 6.0,在此条件下T-1株芽孢产量与培养时间呈正相关,在培养48~60 h期间其芽孢产量趋于稳定。在对T-1株芽孢产量影响较大的6个因子[X1(糖蜜)、X2(可溶性淀粉)、X3(酵母浸粉)、X4(CaCl2)、X5(摇床转速)和X6(接种量)]中,X2(可溶性淀粉)对T-1株芽孢产量的影响达显著水平(P<0.05),X5(摇床转速)的影响达极显著水平(P<0.01),其他因子的影响均不显著(P>0.05)。T-1株的最佳培养基为可溶性淀粉1.11%、酵母浸粉0.3%和CaCl20.5%,在摇床转速218 r/min的培养条件下,其芽孢产量较优化前采用初始基础培养基的芽孢产量提高6.25倍。【结论】具群体感应淬灭作用地衣芽孢杆菌T-1株在优化后的发酵培养条件下,其芽孢产量较优化前明显提高,且延长菌株生长的稳定期,加上优化筛选出的培养基是采用相对廉价且高效的原材料,有效节约了生产发酵成本,可在规模化生产上推广应用。

【Objective】To optimize the culture medium and fermentation conditions of Bacillus licheniformis T-1 strain with quorum induction quenching effect,further increase its spore production,lay the foundation for future development of Bacillus licheniformis preparations(antibacterial probiotics).【Method】The optimum culture condition of T-1 bacterial strain was screened and chosen in the way of single factor method under the shaking flask fermentation condition,and the main influencing factors affecting the live bacteria content of the fermentation broth were determined.According to the results of Plackett-Burman test,the two most significant factors affecting spore yield were screened.The steepest climbing test was used to determine the response center point and the optimal concentration range of the two factors,and then the box-Behnken center combination test and response surface were used to obtain the best fermentation culture conditions.【Result】The optimal culture temperature was 37℃,the optimum growth salinity was 0.5%and the optimum growth pH was 6.Under this condition,the spore formation was in positive correlation with culture time.The sporulation rate were stable between 48-60 h.Among the six factors that had great impact on the spore yield of T-1 strains[X1(molasses),X2(soluble starch),X3(yeast extract powder),X4(CaCl2),X5(shaker speed),and X6(inoculation amount)],the effect of X2(soluble starch)on the spore yield of T-1 strain reached a significant level(P<0.05),the effect of X5(shaker speed)reached an extremely significant level(P<0.01),and the effects of other factors were not significant(P>0.05).The optimal medium for T-1 strain was 1.11%soluble starch,0.3%yeast extract powder and 0.5%CaCl2.Under the culture conditions of 218 r/min shaker speed,the spore yield of the T-1 strain was higher than that of the original basic medium before optimization.The spore yield increased by 6.25 times.【Conclusion】Under the optimized fermentation conditions,B.licheniformis T-1 strain with quorum induction quenching can increase its spore yield greatly compared with the original method.Its stable period of growth also prolongs.Since the optimized media use cheap but high efficient raw materials,the fermentation costs can be reduced.This method can be applied in large-scale production in the future.