C型产气荚膜梭菌合成培养基使用参数及培养毒素浓缩工艺优化

摘要

To define the parameters in the synthesized culture medium for Clostridum peofringens C type and the ultrafiltration of cultured toxin, the experiment compared the toxin activity of Clostridum peofringens CVCC60102 cultured in the synthesized medium prepared with different pH, sterilisation temperature, water type, also under condition of different culture time and temperature, and designed two different methods to concentrate the toxin according to the molecular weight and the MWCO ( Molecular Weight Cut Off) of the uhrafihration membrane, and compared the harvest ratio and activity of toxin obtained by the previous two methods. Results showed that the optimal synthesized culture medium should be prepared with deionic water at the most suitable pH of 8.0 ~ 8.4 and autoclaved for 30 min at 116 ~C. For the strain (CVCC60102) of Clostridum perfringens, the toxic activity reached 500- 1000 MLD/mL after growth for 18 hours. The toxicity did not increase with even longer growth time. The highest level of toxicity would be reached when cultured at 36 or 37 ℃. When concentrated by the 10 ku MWCO membrane, the harvest ratio of toxin was 68%, and the discarded effluent resulted in 2/2 lethal ratio on rat injected by 0.2 mL in vein. While by 8 ku MWCO, the harvest ratio was 80% and the discarded effluent had no lethal activity. Therefore, the ultrafiltration with membrane of 8 ku MWCO was suitable for the concentration of the cultured toxin. The above results provided the data support for the application of the synthesized culture medium for Clostridum pefringens C type, and these would be consulted for the manufactural application of corporations.%确定C型产气英膜梭菌合成培养基使用参数及建立配套的毒素浓缩工艺。比较不同pH值、灭菌温度、配制用水的合成培养基及其不同培养时间和温度下培养产气荚膜梭菌CVCC60102株的毒力；按毒素的分子量及超滤膜的截留分子量设计并比较2种不同浓缩工艺的毒素收获率及透出液的毒力。结果表明，pH值为8．0～8．4、灭菌方式为116℃30min、配制用水为去离子水时产毒最佳，毒力达到500—1000MLD／mL；合成培养基培养产气英膜梭菌CVCC60102株18h后毒力达500—1000MLD／mL，随着时间的延长，毒力不再增强；培养温度为36℃或37℃时，产毒效果最佳；不同截留分子量的超滤膜浓缩，10ku截留分子量的收获率为68％，其透出液静脉接种0．2mL，小鼠2／2死亡；而8ku收获率达80％，其透出液静脉接种0．2mL，小鼠0／2死亡。因此8ku截留分子量膜包适宜对C型菌培养毒素的浓缩。上述结果为C型产气荚膜梭菌合成培养基的应用提供了数据支撑。