苏云金芽胞杆菌中华亚种CT-43菌株(Bacillus thuringien sissubsp.chinensis)是高产苏云金素(简称Thu)的野生菌株。但在产生Thu时总伴随有其他代谢物(简称ZZ)的合成。而且ZZ很难与Thu分离开。本实验通过比较不同的培养基,得到G培养基能抑制菌株CT-43合成ZZ。然后利用分批补料发酵的方法,通过正交试验设计,进一步抑制ZZ的合成,并提高Thu在有机萃取物中的含量。得到优化后的发酵工艺是:菌株CT-43在G培养基中培养至第20h,同时补柠檬酸钠和葡萄糖至终浓度分别为2.0g/L和16g/L,再继续发酵至第52h。通过此发酵工艺制备的Thu峰面积占初提物总峰面积的74.84%,比用LB培养基发酵所得提取物的29.85%提高了两倍多。为得到高纯度的Thu和更深入的研究创造了条件。最后通过液质联用技术证实,发酵工艺优化后,菌株CT-43发酵上清液中主要物质是Thu,几乎检测不到ZZ,而且优化后的Thu分子量并没有改变。
Bacillus thuringiensis subp.chinensis strain CT-43 highlyproduces thuringiensin(Thu).Meanwhile, other metabolizing product(ZZ) was synthesized, but was difficultly removed from Thu.In this study, various media were screened and the G medium was found to inhibit the ZZ synthesis.Based on the G medium, feed-batch fermentation and orthogonal arrays were combined to further inhibit ZZ synthesis and increase the yield of Thu.The optimal fermentation process was that, after strain CT-43 was cultured in G medium for 20 h, patching with 2.0 g/L tri-sodium citrate and 16 g/L glucose and culturing for additional 32 h, the peak area ratio of Thu could get 74.84% in the primary extract, which was 2.5 times for 29.85% of LB fermentation.These conditions could create a favorable fermentation process to produce more pure Thu and it will benefit for further research.Lastly, HPLC-MS was used to verify the produced Thu.Thu became the major element during the feed-batch fermentation, and no ZZ was detected, while Thu was not modified based on its molecular weight detection.