肺炎球菌无动物源性成分培养工艺及纯化新工艺探索

目的:比较无动物源性成分培养基和动物源性培养基培养肺炎球菌的差异;摸索不使用有机溶剂的肺炎球菌多糖纯化新工艺。方法:用无动物源性的原料替代肺炎发酵培养基中动物源性的成分,考察菌体密度和多糖收量的变化;采用柱层析纯化工艺收获肺炎球菌多糖,测定各阶段核酸、蛋白、多糖含量。结果:与疫苗生产所采用动物源性发酵培养基相比,无动物源性发酵培养基收获的1,7F,19A型菌体密度分别提高了1倍左右;发酵液中多糖含量分别提高了48%,50%和200%;多糖收量提高了30%～90%。纯化新工艺收获的17F,18C,19F精糖,核酸和蛋白的含量均远远低于企业的注册标准,HPLC检测多糖纯度分别为99.24%,98.92%和99.02%。结论:无动物源性成分发酵培养基适合于肺炎球菌生长,不仅增加了多糖产量,也提高疫苗的安全性;纯化新工艺能够收获合格的肺炎精制多糖。

Research on animal-free medium of Streptococcus pneumoniae and new purification process of pneumococcal polysaccharide

Objective: To compare the difference between animal-free medium and traditional animal medium used for culturing Streptococcus pneumonia fermentation;To explore a new purification process for pneumococcal polysaccharide without using harmful organic.Methods: Animal based material was replaced by animal-free material,comparing the changes in cell concentration and the polysaccharide output;column chromatography was used to purify the pneumococcal polysaccharide,and determine the concentration of polysaccharide,protein and nucleic acids in each process.Results: For serotype 1,7F,19A,cell concentration was increased about 1 times,polysaccharide concentration in lysate grew 48%~200%,and the output grew 30%~90% when using animal-free medium.When the new purification proces was used to prepare the polysaccharides of pneumococcal serotyp 17F,18C,and 19F,their purity were 99.24%,98.92% and 99.02%,respectively.The contents of protein and nucleic acids in purified polysaccharides were far less than the standards which was determined by HPLC combined with RI detector.Conclusion: Cell concentration,polysaccharide output and the vaccine safety improved a lot when animal-free medium was used for fermentation of Streptococcus pneumonia;New purification process can be used to harvest qualified polysaccharide.