无乳链球菌荚膜多糖的粗提及多糖含量测定条件优化

Optimization for Extraction and Content Determination of Capsular Polysaccharide fromStreptococcus agalactiae

为提取无乳链球菌荚膜多糖,并对苯酚-硫酸法测定多糖含量条件进行优化。离心收集发酵上清液,超滤浓缩,经800mL/L乙醇沉淀提取无乳链球菌荚膜多糖;采用单因素试验对苯酚硫酸法测定条件进行优化,并对测定方法的可靠性进行验证。结果表明,从1L发酵液中提取到含量为38.51%的荚膜多糖0.36g;测定的最佳条件为:2mL样品液中,加入1 mL 50 mL/L的苯酚,5 mL浓硫酸,混匀后80℃作用20min,在1.5h内测定485nm处的光密度。在该条件下,平均加样回收率为99.96%,相对标准偏差为0.40%。说明从无乳链球菌发酵上清液中成功提取到荚膜多糖,建立了苯酚硫酸法测定荚膜多糖的最优条件。

This study was aimed to isolate capsular polysaccharide from Streptococcus agalactiae,and to establish optimal conditions for determination of capsular polysaccharide （CP）.Bacterial ceils were removed by centrifugation,and the culture fluid was concentrated over ultrafiltration system.Crude CP was precipi- tated by bringing the solution to a concentration of 800 gL/L ethanol.Single factor test was preformed to investigate optimal conditions of phenol-sulfuric acid method, and reliability of the method was also evaluated.The results showed that 0.36 g crude polysaccharide was isolated from one liter broth of S.agalactiae, and the polysaccharide content was 38.51%.The optimal conditions for determination of CP was established：add 1 mL 50 mL/L phenol solution into 2 mL polysaccharide solution; then 5 mL sulfuric acid was added,and incubated for 20 min at 80℃; All tubes were allowed to cool down to room temperature before reading the absorbances at 485 nm.Under the optimal conditions,the average recoveries of the method was 99.96 %, the relative standard deviation was 0.40%.S.agalactiae CP was isolated from culture supernatant, and the optimal conditions for determination of CP was established.