b型流感嗜血杆菌发酵培养基的优化

Optimization of Fermentation Medium for Type b Haemophilus influenzae

目的分析b型流感嗜血杆菌(Type b Haemophilus influenzae,Hib)不同培养基发酵液中蛋白质、核酸含量及不同培养基添加物对发酵产物的影响,优化Hib发酵培养基。方法设计不同配方的Hib摇瓶培养基,经摇瓶培养,检测发酵液上清中的蛋白质及核酸含量,选择合适的基础培养基,并在此基础上进行发酵罐培养,检测不同添加物(磷酸盐、钠盐、金属离子等)对Hib多糖复合物收获量、菌体形态及发酵液颜色的影响,并发酵3批,进一步检测多糖复合物中蛋白质和核酸的含量。结果培养基介质不同,其发酵上清液中的蛋白质和核酸含量也不同,2号培养基(主要成分为酸水解酪素粉和酵母浸粉)发酵液蛋白质和核酸含量较低且稳定;培养基中不同添加物对Hib菌体形态、发酵液颜色及多糖复合物的收获量、质量和性状均产生不同的影响,其中b培养基发酵液菌体形态、多糖复合物收获量及质量均较好;3批发酵培养液中分别约有7%～10%的蛋白质和34%～42%的核酸被沉淀至多糖复合物中。结论选择2号培养基为Hib基础培养基,b培养基为发酵培养基,本实验为Hib疫苗培养基的优化和多糖抗原的纯化提供了实验依据。

Objective To analyze the protein and nucleic acid contents in fermentation supernatant of type b Haemophilus influenzae（Hib） using various media as well as the effect of additives in medium on fermentation product,and optimize the fermentation medium.Methods The media with various formula for shake-flask culture of Hib were designed,and the protein and nucleic acid contents in culture supernatant were determined,based on which an appropriate basal medium was selected for culture of Hib in fermenter,and the effects of various additives（such as phosphate,sodium salt,and metal ions） on the recovery of polysaccharide complex,morphology and color of fermentation supernatant of Hib were analyzed.Three batches of Hib were fermented by using the selected medium and further determined for the protein and nucleic acid contents in polysaccharide complex.Results The protein and nucleic acid contents in fermentation supernatant using various media were different,which were low and stable in those using medium No.2 mainly consisting of casamino acid powder and yeast extract powder.The additives in medium showed different effects on the morphology,color of fermentation supernatant as well as recovery,quality and property of polysaccharide complex.The fermentation supernatant of Hib using medium b was of good morphology as well as high recovery and quality of polysaccharide complex.In the three batches of fermentation liquids,about 7% ～ 10% of proteins and 34% ～ 42% of nucleic acids were precipitated into the polysaccharide complex.Conclusion Medium No.2 was selected as basal medium,and medium b as fermentation medium for Hib.The study provided an experimental basis for optimization of medium and purification of polysaccharide antigen of Hib vaccine.