利用响应面法优化α-糖苷酶抑制剂发酵培养基

Medium optimization of α-glucosidase inhibitors production by response surface analysis

摘要:

【目的】采用响应面法对戈壁三素链霉菌PW409发酵合成α-糖苷酶抑制剂的培养基进行优化。【方法】采用Plackett-Burman法筛选影响α-糖苷酶抑制剂产生的关键因素，用最陡爬坡试验逼近关键因素的最大响应区域, 采用Box-Behnken设计以及响应面分析法, 得到各因素的最佳浓度，通过液相色谱-串联质谱法(LC-MS/MS)对发酵液中α-糖苷酶抑制剂进行定量分析。【结果】发酵培养基中可溶性淀粉、KNO3和K2HPO4的浓度对α-糖苷酶抑制剂的产量影响较大。优化后的培养基组成为: 可溶性淀粉9.01 g/L, KNO3 11.0 g/L, K2HPO4 0.32 g/L, MgSO4?7H2O 0.50 g/L, FeSO4?7H2O 0.01 g/L, pH 7.5。【结论】在此优化条件下, 链霉菌PW409发酵液对麦芽糖苷酶的半数抑制浓度IC50为22 mg/L, 抑制活性较优化前提高了近10倍。发酵液中的1-脱氧野尻霉素含量可达7.84 mg/L, 较优化前提高了668倍, 米格列醇的含量可达0.94 mg/L, 较优化前提高了10倍。

Abstract:

[Objective] In order to improve production of α-glucosidase inhibitors from Streptomyces gobitricini PW409, response surface methodology was used to optimize the fermentation medium. [Methods] A Plackett-Burman design was used to evaluate the influence of seven factors firstly. Then, the path of steepest ascent and the Box-Behnken design were adopted for further optimization, the optimum concentration levels and the relationships among these fac-tors was found out by quadratic regression model equation with Design-Expert statistic methods. The concentrations of α-glucosidase inhibitors in the fermentation broth were detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS). [Results] The concentration of soluble starch, KNO3 and K2HPO4 played important roles in influencing the content of inhibitors. The optimal fermentation medium is: soluble starch 9.01 g/L, KNO3 11 g/L, K2HPO4 0.32 g/L, MgSO4?7H2O 0.50 g/L, FeSO4?7H2O 0.01 g/L, pH 7.5. [Conclusion] Under such conditions, the half maximal inhibitory concentration (IC50) of fermentation broth to maltase glucoamylase was 22 mg/L, inhibitory activity increased nearly 10 times. The concentration of 1-deoxynojirimycin and miglitol were increased to 7.84 mg/L and 0.94 mg/L, which was 668 and 10 times higher than before, respectively.