蝉生球孢虫草液体发酵培养基的优化及其菌丝多糖对免疫细胞功能的影响

Optimization of the fermentation medium for the liquid culture of Cordyceps bassiana and the effect of mycelial polysaccharides on immune cells

目的:对药用真菌球孢虫草Cordyceps bassiana Bs1112菌株的液体培养进行优化,提取纯化的菌丝多糖并进行免疫功效的评估。方法:采用Plackett-Burman设计和响应面分析获得的优化培养基组分,通过凝胶渗透色谱法(GPC)色谱分析菌丝多糖的重均相对分子质量;采用流式细胞术检测球孢虫草多糖对人体关键免疫调节细胞(NKT细胞)的CD3+CD56+双阳性影响,并以肝癌细胞Hep G2为靶细胞评估经球孢虫草多糖处理后的NKT细胞的杀伤率。结果:响应面分析优化获得培养基组分比例,多糖含量最高可达913.5 mg/L。经凝胶渗透色谱法色谱分析发现菌丝多糖的重均相对分子质量为2212815 u。不同质量浓度球孢虫草多糖处理对CD3+CD56+NKT细胞的体外增殖产生抑制作用,但增强了该细胞对Hep G2细胞的杀伤力。结论:条件优化后获取的球孢虫草菌丝多糖对人体NKT细胞具有免疫调节作用。

Aims:The liquid medium of Cordyceps bassiana Bs1112,teleomorph(sexually reproducing stage)of medical fungi Beauveria bassiana,was optimized.The mycelial polysaccharide was extracted and purified;and the immune efficacy was evaluated.Methods:The Plackett-Burman design and the response surface method were used for medium formula.The weight-average molecular weight of mycelial polysaccharide was measured by GPC chromatographic analysis.Furthermore,the CD3+CD56+of cord blood-derived NKT-like cells was detected by flow cytometry after they were Cordyceps polysaccharide-treated;and the hepatoma cell Hep G2 was used as the target cell to evaluate the killing rate of NKT cells treated with Cordyceps polysaccharide.Results:The highest content of mycelial polysaccharide could reach 913.5 mg/L;and the weight-average molecular weight of mycelial polysaccharide was 2212815 u by GPC chromatographic analysis.Additionally,the polysaccharide efficacy evaluation suggested that different C.bassiana polysaccharide concentration had a significant inhibitory effect on the proliferation of CD3+CD56+NKT-like cells in vitro.However,NKT cells treated with purified polysaccharide enhanced the CD3+CD56+ratio with the increasing concentration.Conclusions:The mycelial polysaccharide of C.bassiana obtained after optimized conditions has an immunomodulatory effect on human NKT-like cells.