蛇足石杉内生真菌2F09P03B产石杉碱甲发酵条件的研究

Study on fermentation condition for production of huperzine A from endophytic fungus 2F09P03B of Huperzia serrata

目的研究发酵条件对蛇足石杉支顶孢属内生真菌2F09P03B石杉碱甲生成的影响,并探讨发酵条件的优化。方法通过测定2F09P03B生长曲线、石杉碱甲积累曲线和培养液pH值随时间的变化情况,了解该菌株的培养特性;考察不同碳源和氮源对石杉碱甲积累的影响。在单因素试验的基础上,利用正交试验优化培养基碳源、氮源组成;考察MgSO4、KH2PO4、NaCl、CaSO4对石杉碱甲生成的影响,确定无机盐组成;优化接种量、发酵时间、培养温度、摇床转速等发酵工艺条件。结果菌株2F09P03B的最佳培养基组成为:马铃薯150g/L,玉米粉30g/L,麦芽糖25g/L,酵母膏10g/L,麸皮30g/L,KH2PO41.5g/L,MgSO40.5g/L,NaCl0.3g/L,CaSO40.3g/L,pH自然;最佳发酵工艺条件为:接种量8%,发酵时间10d,培养温度28℃,摇床转数140r/min。在此条件下,发酵液中石杉碱甲含量为8.17μg/L,最高达到8.32μg/L,约为其干重的0.07%左右。结论发酵条件对菌株2F09P03B石杉碱甲产量有明显影响。优化后的发酵条件可使该菌株石杉碱甲产量明显提高,有望成为石杉碱甲新药源。

Objective To investigate the effects of fermentation conditions on the production of huperzine A from an endophytic fungus 2F09P03B of huperzia serrata. Methods Growth curve, huperzine A accumulation curve, and the pH value in culture fluid at different fermentation time of the 2F09P03B was analyzed to investigate its characteristics for culture. The effects of carbon and nitrogen sources on the accumulation of huperzine A was studied, and the the composition of carbon and nitrogen sources was optimized through orthogonal experiment based on the result of single-factor analysis. The effects of MgSO4, KH2PO4, NaCl, and CaSO4 on the accumulation of huperzine A was investigated by orthogonal experiment to determine the composition of inorganic salts. The fermentation conditions including cultural temperature, ratational speed of rocking bed, the quantity of inoculation, fermentation time was optimized by another orthogonal experiment. Results The optimum medium composition for strain 09P03B was as follows: potato extract 150 g/L, yeast extract 10 g/L, maltose 25 g/L, corn flour 30 g/L, wheat bran 30 g/L, KH2PO4 1.5 g/L, MgSO4 0.5 g/L, NaCl 0.3 g/L, CaSO4 0.3 g/L, with natural pH. The optimum fermentation conditions included quantity of inoculation 8%, fermentation time 10 d, cultural temperature 28 ℃, ratational speed of rocking bed 140 r/min. Under these conditions, the production of huperzine A from the strain 2F09P03B amounted to 8.17 μg/L, with a maximal value of 8.32 μg/L, which was about 0.07% of its dry weight. Conclusions The fermentation condition markedly affects the production of huperzine A from the strain 2F09P03B. Since the optimized fermentation condition significantly increases the production, it can be a new medicinal resources of huperzine A.