汽爆灭菌对固态发酵芽孢形成的影响及分子调控机制

Probiotics are environmental friendly alternatives to antibiotics. Viable cell number and sporulation rate are key parameters reflecting quality of Bacillus probiotics. A novel sterilization method by steam explosion for solid-state fermentation has been invented, which improves solid medium properties and increases viable cell number of Bacillus subtilis during fermentation effectively. However, in-depth understanding of whether and how does steam explosion sterilization affect sporulation is still lacking. This project aims to reveal influence on sporulation by steam explosion sterilization and regulate the sporulation in solid-state fermentation. Firstly, the characterization methods for solid medium physical and chemical properties would be systematically established, in order to reflect influence on solid medium by steam explosion and the dynamic changes of solid medium during fermentation process. Secondly, molecular biology parameters including gene expression levels of spo0A and sigF, E, G, K, phosphorylation level of Spo0A, and GTP concentration would be measured to compare molecular biology differences in fermentation sterilized by steam explosion and conventional thermal sterilization and then to analyze relationship between sporulation rate and the molecular biology parameters. Finally, principal component analysis (PCA) would be conducted based on operation parameters including sterilization, fermentation, characteristic parameters including solid medium properties, molecular biology, and target parameters including sporulation rate, viable cell number. It will clarify the key parameters affecting sporulation rate and provide guidance to study influence on sporulation by steam explosion sterilization. A systemic scheme for improving both sporulation rate and viable cell number would be proposed by using response surface method to optimize the key parameters. Based on the improved understanding of effect on sporulation by steam explosion sterilization and rational regulation, this project would provide the theoretical and technical direction for improving probiotics quality.

微生态制剂是抗生素的绿色替代品。活菌数及芽孢率为芽孢杆菌微生态制剂重要指标。前期研究建立了固态发酵汽爆灭菌新方法，有效改善基质特性，提高枯草芽孢杆菌活菌数。但汽爆灭菌对芽孢形成是否影响及机制如何，尚缺乏深入研究。本项目旨在阐明汽爆灭菌对固态发酵芽孢形成的影响机制，并指导调控。首先，建立培养基理化特性系统表征方法，揭示汽爆灭菌对基质影响及发酵过程基质变化。其次，建立spo0A，sigF、E、G、K基因表达量，Spo0A磷酸化，GTP水平等分子生物学表征体系，对比汽爆灭菌和常规灭菌发酵关键分子水平差异并研究其与芽孢率关系。最后，综合灭菌、发酵操作参数，基质、分子生物学特征参数及活菌数、芽孢率目标参数，运用主成分分析，厘定影响芽孢形成的关键参数并分析机制。对关键参数运用响应面法优化，建立提高芽孢率、活菌数工艺。本项目通过揭示汽爆灭菌影响芽孢形成机制并优化调控，将为提高微生态制剂品质提供理论指导。

微生态制剂是抗生素的绿色有效替代品。项目以枯草芽孢杆菌固态发酵生产微生态制剂为研究对象，为提高微生态制剂效价，从汽爆前处理及发酵过程分段式设计角度开展研究，主要研究结果如下：首先，建立了固态发酵过程基质理化性质以及芽孢形成关键基因表达系统表征体系，发现固态基质中毛细水比例对于菌体生长及芽孢形成具有重要影响。其次，探究了汽爆前处理对培养基性质以及枯草芽孢杆菌活菌数、芽孢数的系统影响，发现汽爆前处理一方面促进多糖解聚，生成更多葡萄糖、木糖；另一方面增加基质中毛细水比例，从而有效促进菌体生长。而随着活菌数增加以及对营养物质消耗，发酵后期形成不利于菌体生长的严峻环境，研究发现此时调控芽孢形成的关键基因spo0A开始快速表达，诱导芽孢形成。最后，基于枯草芽孢杆菌在不同发酵阶段生长和产芽孢规律，建立了两段式发酵过程，优化工艺参数为培养基Mn2+含量4.9mg MnSO4/g干重，分段时间48h，第二阶段温度47°C，以上参数中，第二阶段温度对发酵有效生物量影响最大。基于汽爆前处理以及分段发酵对枯草芽孢杆菌生长及芽孢形成的影响，系统优化发酵过程，枯草芽孢杆菌活菌数、芽孢数分别较常规发酵提高43.0%和27.4%，有效提高微生态制剂效价。项目研究成果为改善微生态制剂品质提供了理论依据和技术支持。