红曲黄酒传统酿造过程中优势细菌活菌检测方法的构建及应用

Construction and Application of Detection Method for Viable Dominant Bacteria during Traditional Brewing of Hong Qu Glutinous Rice Wine

PMA结合qPCR技术的分子检测手段是一种用于检测活性微生物的有效方法。以红曲黄酒传统酿造体系中的6种优势细菌:巴氏葡萄球菌、肠膜明串珠菌、戊糖片球菌、乳酸乳球菌亚种、植物乳杆菌和干酪乳杆菌为研究对象,设计和合成相应的特异性引物用于实时荧光定量PCR检测,并优化PMA处理条件,通过绘制标准曲线构建6种优势细菌的PMA-qPCR定量检测方法。结果显示:6种特异性引物有效可行,最优的PMA处理条件为PMA处理浓度50μmol/L,交联曝光时间5 min。将构建的PMA-qPCR活菌检测方法应用于红曲黄酒传统酿造过程,发现戊糖片球菌、肠膜明串珠菌和乳酸乳球菌乳亚种是传统酿造体系中最主要的3种细菌。本研究构建的PMA-qPCR活菌检测方法可用于实时定量检测红曲黄酒传统酿造过程中优势细菌及其动态变化,阐明不同细菌在红曲黄酒酿造过程的作用机制。

The molecular detection method of qPCR combined with PMA is an effective method for the detection of active microorganisms. This study aimed to develop PMA-qPCR method for the six dominant bacteria（including Staphylococcus pasteuri, Leuconostoc mesenteroides, Pediococcus pentosaceus, Lactococcus lactis subsp. lactis, Lactobacillus plantarum and Lactobacillus casei） during the traditional brewing of Hong Qu glutinous rice wine. Firstly, specific primers were designed and synthesized for real-time quantitative PCR analysis, and PMA treatment conditions were further optimized. Then, the PMA-qPCR method for the quantitative detection of six dominant bacteria was constructed through the construction of standard curves. The results showed that the six designed and synthesized specific primers were effective and feasible; the optimal PMA treatment condition was PMA concentration 50 μmol/L and cross linking exposure time 5 min. The constructed PMA-qPCR assay for the live bacteria was applied to the traditional brewing process of Hong Qu glutinous rice wine. Pediococcus pentosaceus, Leuconostoc mesenteroides and Lactococcus lactis subsp.lactis were determined to be the three dominant bacteria during traditional brewing system. The constructed PMA-qPCR assay for viable bacteria could be used to detect the dominant bacteria and their dynamic changes timely and quantitatively during the traditional brewing of Hong Qu glutinous rice wine, which is helpful to elucidate the mechanism of action of different bacteria during the traditional brewing process of Hong Qu glutinous rice wine.