葡萄酒发酵过程中标识Hanseniaspora菌群活力关键靶标分子筛选及细胞死亡过程解析

As widespread indigenous yeast which has the potential of application, Hanseniaspora was stressed and gradually lost its viability during the late stage of alcoholic fermentation. However, the death process of Hanseniaspora under stress remains unclear. The selection of key target molecules can accurately monitor the viability dynamic of Hanseniaspora population, which is meaningful for its application in mixed fermentation. Reverse transcription quantitative PCR (RT-qPCR) technique, quantitative PCR using propidium monoazide bromide treatment (PMA-qPCR) and fluorescence in situ hybridization (FISH) technique will be used in this project. Three regions including mRNA, rRNA and the integrity of cell membrane will be focused on to select target molecules. The selection of target molecules needs to test if they can differentiate live/dead cells and accurately quantify viable cells. Simulated wine mixed fermentation will be performed to verify whether the selected target molecules can monitor viable Hanseniaspora population. On the basis, the project will try to probe into the death process of Hanseniaspora cells. This project is designed to help in understanding the dynamic characteristic of the viability of Hanseniaspora population. The results of this project will be a guideline to better apply Hanseniaspora to wine mixed fermentation.

作为分布广泛、具有应用潜力的野生酵母，Hanseniaspora在酒精发酵后期处于被胁迫并逐渐失去活力的状态，但有关此胁迫死亡过程的认识尚不清楚。通过关键靶标分子的筛选，能够准确监控Hanseniaspora菌群活力的动态变化，对该菌在混合发酵中的应用具有重要意义。本项目拟采用反转录实时定量PCR技术（RT-qPCR）、叠氮溴化丙锭实时定量PCR技术（PMA-qPCR）和荧光原位杂交技术（FISH），从mRNA、rRNA和细胞膜完整性三个靶向部位中，筛选能够区分活/死细胞、能够定量表征活细胞的靶标分子，通过模拟葡萄酒混合发酵实验验证所选靶标分子对Hanseniaspora菌群活力的监控能力，并在此基础上探索Hanseniaspora细胞的死亡过程。本研究有助于理解葡萄酒发酵过程中Hanseniaspora菌群活力变化特征，为应用Hanseniaspora进行葡萄酒混合发酵提供理论依据。

有孢汉逊酵母属（Hanseniaspora）是葡萄醪和葡萄酒酒精发酵前期广泛存在的酵母菌，常见菌种为H. uvarum, H. opuntiae和H. guilliermondii等,有孢汉逊酵母属酵母具有高产乙酸乙酯、分泌酶提高品种香气、提高香气复杂性等葡萄酒应用潜力，但该酵母的发酵力和耐受性远远弱于酿酒酵母，因此目前主要以与酿酒酵母混合发酵方式用于相关葡萄酒酿造研究中。目前已经商业化的非酵母属酵母中尚不包含Hanseniaspora酵母，H. vineae等比较有应用潜力目前正在研究测试中。为有效监控非酵母属酵母在混合发酵中的贡献，需筛选能定量表征该酵母活细胞的靶标分子。本项目以中国本土分离筛选鉴定的酿酒酵母和Hanseniaspora酵母为研究对象，采用RT-qPCR技术、FISH技术和PMA-qPCR技术，从6个靶向mRNA、2个靶向rRNA、1个靶向DNA但表征细胞膜变化的9个靶标分子中，筛选能够区分活/死细胞、能够准确定量活细胞浓度的靶标分子，并应用筛选的靶标分子分析混合发酵过程中Hanseniaspora的细胞菌群活力变化。本项目从酿酒酵母纯发酵过程中制备酿酒酵母毒素粗提物，研究杀死Hanseniaspora酵母的条件，通过对比活细胞和死亡细胞的检测结果，发现mRNA和rRNA相对稳定，靶向二者的靶标分子基于RT-qPCR技术无法区分107 cells/mL细胞浓度下的活死细胞，而基于FISH技术和PMA-qPCR技术靶向rRNA和DNA的靶标分子可以有效区分。进一步的靶标分子定量化分析表明，靶向rRNA和DNA的量化分析效果优于mRNA。本项目选用靶向rRNA和DNA的靶标分子监控了混合发酵过程中Hanseniaspora酵母菌群活力的变化，为Hanseniaspora酵母在混合发酵中的应用提供有效的菌群监控技术。