丝状真菌Glarea lozoyensis SIIA-F1108产纽莫康定B_0发酵条件的研究

Optimization of pneumocandin B_0 production by filamentous fungus Glarea lozoyensis SIIA-F1108

【目的】采用响应面法优化丝状真菌Glarea lozoyensis SIIA-F1108发酵生产纽莫康定B_0培养基,提高发酵产量;通过氮源优化,降低发酵液菌体浓度,改善发酵过程的溶氧水平。【方法】采用Plackett-Burman设计和响应面法进行培养基优化,筛选出对纽莫康定B_0产量具有显著影响的因素;通过最陡爬坡实验及Box-Behnken设计,并利用Design-Expert软件对实验数据进行回归分析,得到优化的发酵培养基配方;通过对优化培养基中氮源组分进行全因子实验,最终得到高产量和低菌体浓度发酵培养基。【结果】实验数据表明:甘露醇、脯氨酸和葡萄糖对纽莫康定B_0产量影响最大;最佳浓度分别为甘露醇167.3 g/L、脯氨酸26.1 g/L、葡萄糖28.5 g/L。采用优化后的培养基进行摇瓶发酵,纽莫康定B_0产量达到了1 840 mg/L,较优化前提高了42%,与预测结果一致。用硫酸铵部分替换棉籽饼粉后,发酵液菌体浓度降低,在100 L发酵罐上对优化后的结果做了进一步的验证,纽莫康定B_0产量达到1 980 mg/L。【结论】模型预测值与实验值有较高吻合度,具备较高可信度和显著性,发酵产量提高了42%,响应面实验设计和分析方法能够有效地用于丝状真菌Glarea lozoyensis SIIA-F1108产纽莫康定B_0发酵培养基进行优化。通过调整培养基中的氮源组成,降低了发酵液菌体浓度,改善了发酵过程的溶氧水平。

[Objective] The culture media for the production of pneumocandin B_0 by the filamentous fungus Glarea lozoyensis SIIA-F1108 were optimized using the response surface methodology（RSM）. The nitrogen source of the culture was optimized to reduce the volume concentration of mycelium and improve the level of dissolved oxygen in fermentation process. [Methods] Through Plackett-Burman design and RSM, the factors that had significant effects on the production of pneumocandin B_0 were screened. Afterwards, the path of steepest ascent and the Box-Behnken design were adopted for further optimization, and the optimal concentration levels and the relationships among these factors was found out by quadratic regression model equation with Design-Expert statistic methods. The high-yield and low-viscosity of fermentation medium was obtained by carrying out the whole factor experiment with nitrogen source components. [Results] Mannitol, proline and glucose were found to have greatest influence on the production of pneumocandin B_0, and the optimal concentrations were 167.3, 26.1 and 28.5 g/L, respectively. Under the optimal conditions, the production of pneumocandin B_0 reached 1 840 mg/L in three baches of shake flask experiments, with an increase of 42% compared with the ordinary culture. The pneumocandin B_0 production of 100 L fermentor achieved 1 980 mg/L under the optimal conditions. The viscosity of the fermentation broth was reduced by partly replacing the cottonseed meal with（NH_4）_2SO_4. [Conclusion] The production of pneumocandin B_0, which was in agreement with the prediction, was increased by 42% compared with the previous condition. The RSM method was effective for optimizing the composition of medium for the production of pneumocandin B_0 byfilamentous fungus Glarea lozoyensis SIIA-F1108. After replacing the cottonseed meal with（NH_4）_2SO_4, the viscosity of the fermentation broth was reduced and the level of dissolved oxygen was improved in fermentation process.