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103~107CFU/mL;当体系中死菌与活菌的浓度比例高于10 000:1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 103-107 CFU/mL of the target strain in multiple matrices; when the concentration of dead microorganisms in the system was 104 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.
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