花生四烯酸产生菌高山被孢霉的高糖驯化研究

Domestication of the high-sugar-tolerant Mortierella alpina on arachidonic acid (ARA) production

摘要:

【目的】提高花生四烯酸(Arachidonic acid, ARA)产量, 克服ARA产生菌高山被孢霉(Mortierella alpina)在长期的保存及使用过程中易受到外界条件影响发生退化, 从而导致菌种耗糖量降低、影响菌种摄入营养的能力和不利于工业化生产的缺点。【方法】首先采用固体培养基驯化, 将菌种逐级涂布于梯度高糖PDA平板(含糖量分别为2%、5%、7%、10%和15%)培养, 挑选经固体驯化后能耐受10%高糖浓度平板的菌种, 转接到两种含不同氮源的梯度高糖(含糖量分别为3%、4%、5%和6%)液体培养基中进行驯化, 最后对驯化后的菌种进行2 L发酵罐放大实验。【结果】当培养基中以酵母粉为氮源时, 驯化后菌体的最高耗糖量由3 g/(L·d)提高到12 g/(L·d); 当培养基中以玉米浆为氮源时, 驯化后菌体的最高耗糖量由7 g/(L·d)提高到12 g/(L·d)。摇瓶驯化实验结果表明以玉米浆为氮源驯化的菌种发酵效果较好, 发酵罐实验结果显示菌体生物量为50 g/L, 总油脂为18 g/L, 目的产物ARA产量为8 g/L。相比未驯化之前的发酵结果, 生物量和总油脂含量提高了近3倍, ARA产量提高了近4倍。【结论】经过高糖驯化, 菌种的耗糖能力得到提高, 生物量、总油脂及ARA的产量也都有所增加, 从而可以使菌种在保存和使用过程中不易退化, 保持稳定。

Abstract:

[Objective] In order to improve arachidonia acid (ARA) production, and prevent the degeneration of ARA-producing strain Mortierella alpina in long-term culture preservation and cultivation, which could lead to low consumption rate of substrates like carbon source. [Methods] Mortierella alpina strain was first domesticated in high-sugar PDA plate with gradient sugar content (2%、5%、7%、10% and 15%). The strain which grew better in the solid medium containing 10% sugar was then selected and transferred to two liquid high-sugar media with different nitrogen sources to domesticate. The gradient sugar contents were 3%、4%、5% and 6%, respectivlely. Finally, the domesticated strain was used to produce ARA in a 2 L bioreactor. [Results] The experimental results show that the consumption of sugar increased from 3 g/(L·d) up to 12 g/(L·d) in the medium with yeast extract, and from 7 g/(L·d) up to 12 g/(L·d) in the medium with corn steep liquor. After cultivation for 144 h in a 2 L bioreactor, the yield of ARA reached 8 g/L, which was 4 times higher than that obtained with the original strain. [Conclusion] The domestication of the high-sugar-tolerant Mortierella alpina increased the consumption of sugar, biomass, total fatty acid and ARA production. The domesticated strain could maintain the high sugar resistance in the preserving process and turn to be more stable.