基于PMA-CELL-qPCR的葡萄酒发酵中酿酒酵母活菌计数方法的开发与应用  

Development and application of Saccharomyces cerevisiae viable cell quantitative method in wine fermentation based on PMA-CELL-qPCR

作　　者：卫博 王杰[1] 陈亦新 王春光 陈卓君 张柏林[1] 朱保庆[1] WEI Bo;WANG Jie;CHEN Yixin;WANG Chunguang;CHEN Zhuojun;ZHANG Bolin;ZHU Baoqing(Beijing Key Laboratory of Forestry Food Processing and Safety,College of Biological Sciences and Technology,Beijing Forestry University,Beijing 100083,China)

机构地区：[1]北京林业大学,林业食品加工与安全北京市重点实验室,生物科学与技术学院,北京100083

出　　处：《食品与发酵工业》2022年第17期79-86,共8页Food and Fermentation Industries

基　　金：国家自然科学基金项目(31471834);北京林业大学大学生创新创业训练计划项目(G202010022092,G202010022093)。

摘　　要：实时监测发酵过程中的微生物动态对葡萄酒的质量控制至关重要。开发了一种将荧光染料叠氮溴化丙啶(propidium monoazide,PMA)与CELL-qPCR相结合的方法,不需要提取DNA,即可以高效、快速地检测葡萄酒发酵过程中酿酒酵母的动态变化。针对酿酒酵母开发的PMA-CELL-qPCR计数方法对葡萄酒环境下的细胞破壁处理、PMA处理浓度、PMA检测灵敏度和样品预处理等多种条件进行了优化,结果表明,优化后的方法可以准确定量目标菌株10~3~10~7CFU/mL;当体系中死菌与活菌的浓度比例高于10000∶1时,检测结果存在0.5 lg CFU/mL的误差;用优化的PMA-CELL-qPCR方法监测赤霞珠葡萄酒发酵过程中酿酒酵母的动态变化,结果与平板计数法一致。研究开发的PMA-CELL-qPCR对酿酒酵母的活菌定量方法具有定量准确、操作简单等优势,可以作为准确监测葡萄酒和其他果酒中微生物动态变化的手段。Real-time monitoring of microbial dynamics during fermentation is essential for wine quality control.A method could distinguish between dead and live microbes for Saccharomyces cerevisiae was developed,which combines the fluorescent dye propidium monoazide(PMA)with CELL-qPCR.It also could detect the quantity of microbes efficiently and rapidly without DNA extraction during wine fermentation.The method was optimized for cell wall breakage,PMA treatment concentration,PMA detection sensitivity and multiple conditions of sample pretreatment in wine environment.The results showed that the optimum method can accurately quantify 10~3-10~7 CFU/mL of the target strain in multiple matrices;when the concentration of dead microorganisms in the system was 10~4 times higher than that of live microorganisms,there was an error of 0.5 lg CFU/mL;the optimized PMA-CELL-qPCR method was used to monitor the dynamic changes of S.cerevisiae during the fermentation of Cabernet Sauvignon wine were consistent with the plate count method.In conclusion,the live bacteria quantification method developed for PMA-CELL-qPCR in S.cerevisiae wines is accurate in quantification and simple in operation,and can be used as a mean to accurately monitor microbial dynamics in wine and other fruit wines.

关 键 词：叠氮溴化丙啶 CELL-qPCR 酿酒酵母 活菌定量 葡萄酒 

分 类 号：TS262.6[轻工技术与工程—发酵工程]