基于高通量测序技术解析浓香型白酒中窖泥臭味物质4-甲基苯酚的来源

Detecting source of p-cresol in strong flavor Chinese liquor by high throughput sequencing

摘要:

【目的】研究浓香型白酒中主要窖泥臭味物质4-甲基苯酚的原料来源；解析窖泥菌群结构，从中分离得到产4-甲基苯酚的菌株，以明确4-甲基苯酚的微生物来源。【方法】运用气相色谱-质谱连用(GC-MS)技术对窖泥、糖化料和大曲中的4-甲基苯酚定性定量；运用高通量测序技术解析窖泥的菌群结构，并通过可培养技术从中筛选产4-甲基苯酚的微生物。【结果】糖化料和曲粉中4-甲基苯酚含量均低于检测限。窖泥中检测到4-甲基苯酚，其中窖底窖泥4-甲基苯酚含量达到24.24 μg/g。测定的窖泥菌群主要包括8个纲，其中Bacteroidia、Clostridia和Methanobacteria在上中底部窖泥中含量均高于8%，为主要优势纲。窖泥中含量在1%的属有11个，主要包括Clostridium、Aminobacterium、Methanobacterium、Methanobrevibacter等。经过分离筛选，窖泥中的Clostridium aminovalericum、Clostridium ultunense和Clostridium purinilyticum可产4-甲基苯酚，与窖泥高通量测序结果比对显示，含量都在0.20%以上。【结论】4-甲基苯酚主要来源于窖泥，窖泥微生物可代谢产生4-甲基苯酚。窖泥菌群结构复杂，窖池不同深度的菌群结构并不一致，其中Clostridia及Clostridium与4-甲基苯酚的变化规律相似，含量随深度增加而升高。从窖泥中筛选得到3株产4-甲基苯酚的菌株，3株菌都属于Clostridium。Clostridium在窖泥中的含量达到4.89%，其中筛选得到的3株菌在窖泥中的总含量接近1%，综上得出Clostridium是4-甲基苯酚的主要微生物来源。

Abstract:

[Objective] The aims of this study were to detect the source of p-cresol in strong flavor Chinese liquor; analyze the microbial community structure in pit mud, and separate strains producing p-cresol. [Methods] p-Cresol concentration in Daqu, pit mud and saccharogenic grains was detected by GC-MS. Microbial communities of different parts of pit muds were detected by highthroughput sequencing technology. Then strains that could produce p-cresol were separated. [Results] p-Cresol was only detected in pit mud, not in Daqu or saccharogenic grains. The concentration was 24.24 μg/g in bottom part of pit mud. The microbes mainly belonged to 8 classes in pit mud. Among them, Bacteroidia, Clostridia and Methanobacteria were all higher than 8%. The microbes mainly belonged to 11 genus, Clostridium, Aminobacterium, Methanobacterium, Methanobrevibacter, etc. After strains separation and volatile compounds detection, Clostridium aminovalericum, Clostridium ultunense and Clostridium purinilyticum could produce p-cresol, and were all higher than 0.20% in pit mud. [Conclusion] Clostridium was the primary microbial source of p-cresol.