荧光定量PCR方法检测白酒发酵过程中Aspergillus tubingensis生物量

Detection and quantification the biomass of Aspergillus tubingensis in Chinese liquor fermentation process by real-time qPCR

摘要:

【目的】为了更好地分析霉菌在白酒发酵过程中的作用，需要快速准确地测定发酵过程中霉菌生物量的变化，本实验以白酒酿造中常用的塔宾曲霉(Aspergillus tubingensis)为例，建立一套快速准确定量塔宾曲霉生物量的方法。【方法】优化从酒醅中提取基因组的方法，设计和验证专一性引物，建立实时荧光定量PCR (Real-time quantitative PCR)方法，验证方法的有效性并应用于白酒发酵过程中塔宾曲霉生物量的检测。【结果】用原位机械破碎法提取酒醅中总基因组，其DNA的浓度能够达到1.060×105 ng/g酒醅；同时建立了一套快速准确测定固态基质中霉菌生物量的方法，并应用于白酒生产(制曲、堆积发酵和窖池发酵过程)中塔宾曲霉生物量的定量。【结论】实时荧光定量PCR方法能够快速准确地测定固态基质中霉菌的生物量，且检测限较低，对今后的相关研究具有借鉴意义。

关键词:荧光定量PCR，霉菌，固态发酵，塔宾曲霉，生物量

Abstract:

[Objective] In order to better understand the role of filamentous fungi in Chinese liquor fermentation process, a rapid and accurate method to monitor the change of fungal biomass is necessary. This study established a real-time qPCR method to detect and quantify Aspergillus tubingensis, which is widely used in Chinese liquor fermentation. [Methods] The methods of extracting genome from fermented grains have been optimized, the specific primers for A. tubingensis has also been designed and validated. [Results] The total DNA extracting from fermented grains can reach 1.060×105 ng/g by in situ mechanical crushing method. The applicability of real-time qPCR method to detect A. tubingensis in solid-state matrix has been validated in liquor fermentation process. [Conclusion] Real-time qPCR method can rapidly and accurately detect the fungal biomass in solid-state matrix, which provides a powerful tool for related researches.

Key words:real-time qPCR, filamentous fungi, SSF, Aspergillus tubingensis, biomass