高效棉酚降解枯草芽孢杆菌M-4菌株的常压室温等离子体诱变及发酵工艺优化

Mutagenesis of Bacillus subtilis M-4 strain with high gossypol degradation by atmospheric and room temperature plasma and optimization of fermentation process

摘要:

【背景】棉粕中游离棉酚的存在制约了棉粕作为饲料蛋白源的利用，棉酚的微生物降解问题成为研究热点。本实验室前期发现枯草芽孢杆菌(Bacillus subtilis) M-4菌株具有较强的降解棉酚能力，而且已经应用于棉粕脱毒工业。【目的】进一步提高枯草芽孢杆菌M-4菌株降解棉粕中棉酚的能力，扩大棉粕在养殖业的应用领域。【方法】采用常压室温等离子体诱变(atmospheric and room temperature plasma，ARTP)技术对菌株M-4进行诱变，以液体培养条件下的棉酚降解率为初筛指标，筛选获得正向突变株。以棉粕固体发酵条件下的棉酚降解率为复筛指标，测定初筛获得的正向突变株实际降解棉粕中游离棉酚的能力。以棉酚残存量和棉酚降解率为检测指标，采用单因素试验优化固体条件下棉粕发酵条件，获得突变株发酵棉粕的最适工艺参数。【结果】初筛获得正向突变株19株。复筛得到一株高效的突变株MY-4-17，在棉粕固体发酵条件下其棉酚降解率高达97.15%，比菌株M-4的棉酚降解率提高了2.55%。经过5次传代培养，突变株MY-4-17的遗传稳定性良好。突变株MY-4-17发酵棉粕的最适工艺参数为：碳源选用4%玉米粉，物料含水量控制在45%−50%，菌剂使用量为1.0×10¹³ CFU/t，发酵时间13−15 d。此工艺下发酵棉粕，可将棉粕中的棉酚含量由980 mg/kg降至22 mg/kg以下，降解率达97.75%以上。【结论】采用ARTP技术诱变选育的突变株可以有效降解棉粕中的棉酚，为棉粕发酵菌剂的开发与生产提供参考。

Abstract:

[Background] Free gossypol in cottonseed meal restricts the application of the meal as a dietary protein resource, and thus the degradation of gossypol has become a research hotspot. We previously found that Bacillus subtilis M-4 can degrade gossypol and it has been used in the industrial detoxification of cottonseed meal. [Objective] To improve the ability of M-4 to degrade gossypol in cottonseed meal and expand the application of cottonseed meal in breeding industry. [Methods] We employed atmospheric and room temperature plasma (ARTP) for mutagenesis of M-4, and preliminarily screened the forward mutants with the index of gossypol degradation rate in liquid medium. Furthermore, according to the degradation rate of gossypol in solid fermentation medium, the free gossypol-degrading ability of the forward mutants was evaluated. In addition, based on gossypol residue and degradation rate of gossypol, the solid fermentation conditions for cottonseed meal were optimized by single-factor experiment and the optimal fermentation parameters were obtained. [Results] A total of 19 forward mutants were screened out and MY-4-17 was identified to be most efficient. In solid medium, the gossypol degradation rate of MY-4-17 reached 97.15%, 2.55% higher than that of M-4. MY-4-17 showed genetic stability after 5 passages. The optimal fermentation conditions for cottonseed meal with MY-4-17 are as follows: 4% corn flour as sugar source, water content in raw material of 45%–50%, MY-4-17 dose at 1.0×10¹³ CFU/t, and fermentation duration of 13–15 days. Under such conditions, the content of gossypol in cottonseed meal was reduced from 980 mg/kg to 22 mg/kg and the degradation rate was over 97.75%. [Conclusion] The mutant screened by ARTP can effectively degrade the gossypol in cottonseed meal. The result can serve as a reference for the development and production of fermentation agent for cottonseed meal.