Optimization of Fermentation Medium for Type b Haemophilus influenzae

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.