高浓度啤酒花苦味酸诱导酵母应激进入活的不可培养状态的机制研究

With the increasing variety of consumer tastes, the special beers, including hoppy beers, becomes more and more popular. Our previous study found that yeast cells can gradually form the VBNC state in the presence of high concentration of hop bitter acids, which may lead to a significant decrease in yeast activity in the later stage of fermentation. So far, the VBNC state has been extensively studied in bacteria, but the researches on the VBNC yeast are still scarce. Therefore, it is necessary to study the induction and molecular mechanism of yeast in VBNC state. In this project, firstly in order to obtain the regulation of the formation of VBNC yeast cells, the different concentrations of iso-α-acid are used to treat yeast cells, and the difference of cell physiological and metabolic characteristics at different stages of state transformation was compared. Secondly, based on RNA sequencing the changes of gene expression profiles between the normal cells and VBNC cells of beer yeast are observed at the transcriptome level. Thirdly, the structural analysis and functional prediction of differential genes are carried out by bioinformatics. After the key functional genes are identified, the study is attempting to further determine their function using the gene knock out technique. Finally the genes and their regulation mechanism involved in the induction of VBNC yeast by hop bitter acids will be revealed. This work is expected to provide a theoretical basis for further development of the formation mechanism of VBNC state in yeast, and propose new solutions for solving the problem of insufficient yeast activity during fermentation of hoppy beers.

随着消费者口味越来越多样化，苦味型啤酒等特色啤酒逐步受到大众青睐。申请人前期研究证实，在高浓度啤酒花苦味酸（异α-酸）作用下酵母逐渐进入活的不可培养（VBNC）状态，从而解释为何高苦味啤酒发酵后期酵母活力会显著下降。目前关于VBNC研究多集中于细菌，对酵母等真核生物的报道还相当匮乏。因此，亟需深入解析酵母应激形成VBNC状态的分子机理。本课题拟采用不同浓度异α-酸诱导啤酒酵母进入VBNC状态，比较状态转变过程的细胞生理及代谢特性变化，以明确VBNC酵母的形成规律；基于RNA测序，从转录组水平分析不同状态酵母的基因表达谱差异；运用生物信息学对差异基因进行结构分析和功能预测，定位调控VBNC形成的关键功能因子；利用实时荧光定量PCR和基因敲除技术对调控基因进行功能验证。研究结果将为揭示苦味酸诱导酵母VBNC状态产生的内在机制奠定理论基础，为解决高苦味啤酒发酵时酵母活力减弱的问题提供新思路。

目前，关于活的不可培养（VBNC）状态研究多集中于细菌，对酵母等真核生物的报道还相当匮乏。而且，在高浓度啤酒花苦味酸（异α-酸）作用下酵母被证实可逐渐进入VBNC状态，从而导致高苦味啤酒发酵后期酵母活力显著下降。因此，本项目采用不同浓度异α-酸诱导啤酒酵母进入VBNC状态，比较状态转变过程的细胞生理及代谢特性变化，以明确VBNC酵母的形成规律；从转录组水平分析不同状态酵母的基因表达谱差异，并运用生物信息学对差异基因进行结构分析和功能预测，定位调控VBNC形成的关键功能因子；利用实时荧光定量PCR和基因敲除技术对调控基因进行功能验证。主要研究结果包括：1）啤酒酵母经300mg/L异α-酸处理2h后完全进入VBNC状态；添加过氧化氢酶可使VBNC酵母复苏，且酶活力为2500 U/mL过氧化氢酶的复苏效果最好；2）与正常状态相比，VBNC细胞形态差异不显著，且VPNC酵母麦汁发酵效率显著低于正常酵母，酒精产量较低，生产的啤酒中芳香醇和酯明显不足，但复苏后酵母与正常酵母相比，在发酵性能和风味形成方面没有观察到显著差异；3）转录组学分析进一步表明，在VBNC细胞中参与碳水化合物和氨基酸代谢、DNA复制、细胞分裂的基因下调，而TCA循环、ABC转运体、有机酸代谢和氧化还原酶活性增加，此外，Yap1基因的缺失可抑制了酵母细胞进入VBNC状态。综上，细胞存活能力增强，细胞分裂受到抑制，代谢活性降低等可能是导致酵母在啤酒花苦味酸诱导下应激进入VBNC状态的内在原因。研究结果揭示了苦味酸诱导酵母VBNC状态产生的内在机制，为解决高苦味啤酒发酵时酵母活力减弱的问题提供新思路。