一株产胞外多糖丹参内生真菌ZDH1-1的分离鉴定及其胞外多糖的提取工艺优化

Isolation and identification of endophytic fungi ZDH1-1 isolated from Salvia miltiorrhiza Bunge and optimization of extraction process of extracellular polysaccharide

目的对产胞外多糖的丹参内生真菌ZDH1-1进行菌种鉴定,并对其胞外多糖的提取工艺进行优化。方法采用形态鉴定结合ITS序列分析对内生真菌ZDH1-1进行鉴定;采用苯酚硫酸法对内生真菌ZDH1-1胞外多糖进行含量测定;在单因素试验基础上,以乙醇浓度、醇沉时间、醇沉温度为自变量,胞外多糖浓度为因变量,通过Box-Behnken设计对3个自变量各水平进行二项式拟合,优选内生真菌ZDH1-1胞外多糖的提取工艺。结果丹参内生真菌ZDH1-1胞外多糖的最佳提取条件为乙醇-真菌发酵液体积比4:1,乙醇沉淀时间10h,乙醇沉淀温度12℃,ZDH1-1胞外多糖浓度为4.50mg/m L,与预测值的偏差1.96%。结论优化的丹参内生真菌ZDH1-1胞外多糖提取工艺快速简便,稳定可靠,为丹参内生真菌多糖的开发利用提供参考。

Objective The endophytic fungus ZDH1 - 1 from Salvia militorrhiza Bge. was identified and optimized for the extrac- tion process of extracellular polysaccharide. Methods The endophytic fungus ZDH1 - 1 was identified by the morphological iden- tification and its sequence analysis ; the content of extraeellular polysacchafide was determined by phenol - sulfuric acid method. On the basis of single factor test, ethanol concentration, ethanol precipitation time, alcohol sink temperature as independent varia- bles and extracellular polysaccharide extraction rate as the dependent variable, and each level of three independent variables for binomial fitting through Box - Behnken design, extraction process of extracellular polysaccharide was optimized. Results The op- timal extraction process of extracellular polysaccharide from endophytic fungus ZDH1 - 1 was ethanol - broth volume ratio of 4 ： 1, the ethanol precipitation time of 10h, the ethanol precipitation temperature of 12 ℃. Under this condition, the polysaccharide content of ZDH1 - 1 was 4.5mg/mL, and the deviation was 1.96%. Conclusion Optimization of the extraction process of extra- cellular polysaccharide from endophytic fungus ZDH1 - 1 is fast and simple, stable and reliable, and can provide reference for the development and utilization of polysaccharide from endophytic fungi.