肺炎链球菌培养及其荚膜多糖的制备

The Cultivation of Streptococcus Pneumoniae andPreparation of Capsular Polysaccharide

肺炎链球菌荚膜多糖是肺炎疫苗的有效组分,采用自制培养基以15L立瓶和100L发酵罐对流行肺炎链球菌进行培养,探讨其生长和荚膜多糖产生情况.结果表明肺炎链球菌以上述两种方式大量培养均生长良好.采用7～9h的培养物上清液经超滤浓缩,以乙醇沉淀法制备荚膜多糖,所获多糖基本符合《欧洲药典》规定要求.立瓶培养的6B、18C、19F及23F荚膜多糖产量分别达112mg/L、123mg/L、115mg/L及103mg/L,这些数据均高于欧洲专利Yavordios所述72mg/L的产量.用立瓶培养时多糖收量较低的1、5和14型(收量分别为40mg/L、43mg/L及25mg/L)肺炎链球菌进行发酵罐培养的多糖收量与立瓶培养的相似.

The capsular polysaccharide was currently used as an effective antigen in pneumococcal vaccines. Streptococcus pneumoniae serotypes 1, 5, 6B, 14, 18C, 19F and 23F were cultivated in 15 L bottles using self-made liquid medium by shaking; serotypes 1, 5 and 14 Streptococus pneumoniae were also cultivated in a 100 L bioreactor under stirring conditions to explore the growth of tested strains and the production of capsular polysaccharide. The results showed that the good bacterial growth was observed in the above mentioned cultivated modes. The logarithmic growth can be exhibited after cultivated for 5～6 hours. The supernatants of culture were used to extract pneumococcal polysaccharide by ethanol precipitation. After isolating and purifying of bacterial polysaccharide(PS), the quality levels of the most bacterial PSs were coincident with the requirements of European Pharmacopoeia. The PS yields of types 6B, 18C, 19F and 23F were 112 mg/L, 123 mg/L, 115 mg/L and 103 mg/L separatively, which had 1.5-fold increase in yield as compared with those described in the published patent[Yavordios and Cousin(1983) European Patent 0 071515 A1]. The PSs yields of types 1, 5 and 14 strains in cultivation of 15 L bottles were 40 mg/L, 43 mg/L and 25 mg/L separatively. The PSs yields in bioreactor cultivation were similar to those in bottle cultivation.