基于宏基因组学解析不同质量等级中温大曲微生物组的异质性

Metagenomics unveils the microbiome heterogeneity of medium-temperature Daqu with different quality grades

摘要:

【目的】中温大曲是浓香型白酒酿造中的糖化发酵剂和生香剂，目前其质量评价主要依赖于感官和理化特征。已有研究采用扩增子测序初步探索了大曲质量等级与微生物群落组成之间的关联性，但是微生物群落功能对大曲质量的影响尚缺乏深入研究。【方法】本研究采用宏基因组学解析和对比了不同质量等级大曲的微生物群落组成和潜在功能的差异，结合理化性质，聚焦分析了酶活、乳酸和乙酸代谢相关的关键酶基因丰度。【结果】两种等级大曲的真菌群落组成差异显著。一级大曲含有更高相对丰度的丝状真菌，主要分布在曲霉属(Aspergillus, 21.6%)、罗萨氏菌属(Rasamsonia, 6.8%)、拟青霉属(Paecilomyces, 5.0%)和篮状菌属(Talaromyces, 4.4%)等。一级大曲中与环境信息处理和细胞过程相关途径的基因相对丰度普遍显著高于二级大曲。一级大曲中薄层菌属(Hymenobacter)和曲霉属等提供了更高基因丰度的α-淀粉酶，使其具有显著较高的液化力。二级大曲则含有更高相对丰度的横梗霉属(Lichtheimia, 11.8%)、根霉属(Rhizopus, 13.4%)和毕赤酵母属(Pichia, 7.2%)以及乳酸菌类微生物，如伴生乳杆菌属(Companilactobacillus)、魏斯氏菌属(Weissella)和黏液乳杆菌属(Limosilactobacillus)等。二级大曲中与代谢相关途径的基因相对丰度显著较高，如碳水化合物代谢、能量代谢和核苷酸代谢等。二级大曲中较多的乳酸菌类群有利于提高乙醇脱氢酶基因丰度，同时，糖多孢菌属(Saccharopolyspora)、葡萄球菌属(Staphylococcus)、曲霉属和高温放线菌属(Thermoactinomyces)等提供了更多的羧酸酯酶基因丰度，分别使其具有较高的发酵力和酯化力。此外，二级大曲中较多的乳酸菌还可能产生更多的乳酸脱氢酶，降解乳酸，且更高基因丰度的乙酸代谢相关酶类可促进乙酸分解代谢，使得二级大曲酸度显著低于一级大曲。【结论】本研究在基因水平上研究了不同质量等级大曲酶活和酸度差异的微生物基础，可为建立完善的大曲质量评价体系以及理性地调控群落功能提供理论支撑。

Abstract:

[Objective] Medium-temperature Daqu is the saccharifying, fermentative, and aroma-producing agent used in the brewing of strong-flavor Baijiu. The current quality evaluation of medium-temperature Daqu mainly depends on sensory and physicochemical characteristics. Previous studies have employed amplicon sequencing to preliminarily explore the relationship between the quality grade of Daqu and the composition of microbial community. However, little is known about the impacts of microbial community functions on the quality of Daqu. [Methods] We employed metagenomics to analyze and compare microbial community composition and potential functions of medium-temperature Daqu with different quality grades. We then analyzed the activities and the gene abundance of key enzymes involved in lactate and acetate metabolism. [Results] The fungal community structure was different between two Daqu samples. Higher relative abundance of filamentous fungi, represented by Aspergillus (21.6%), Rasamsonia (6.8%), Paecilomyces (5.0%), and Talaromyces (4.4%), were observed in the first-grade Daqu (Grade_1). The relative abundance of genes involved in environmental information processing and cellular processes in Grade_1 was significantly higher than that in the second-grade Daqu (Grade_2). In Grade_1, Hymenobacter and Aspergillus contributed to the higher gene abundance of alpha-amylase, resulting in significantly higher liquefying activity. In Grade_2, Lichtheimia (11.8%), Rhizopus (13.4%), and Pichia (7.2%), and lactic acid bacteria such as Companilactobacillus, Weissella, and Limosilactobacillus had higher relative abundance. Moreover, the genes involved in metabolism such as carbohydrate metabolism, energy metabolism, and nucleotide metabolism had higher relative abundance in Grade_2. The more abundant lactic acid bacteria in Grade_2 was conducive to the higher gene abundance of alcohol dehydrogenase and Saccharopolyspora, Staphylococcus, Aspergillus, and Thermoactinomyces provided a higher gene abundance of carboxylesterase, enhancing the fermentative and esterifying activities, respectively. Furthermore, more lactic acid bacteria in Grade_2 might produce more lactate dehydrogenase to degrade lactic acid, and the higher gene abundance of acetate metabolism-related enzymes could promote acetate catabolism, which led to the significantly lower acidity in Grade_2 than in Grade_1. [Conclusion] We analyzed the microbial community functions of medium-temperature Daqu with different quality grades at the gene level and explored the potential microorganisms contributing to the differences in enzyme activities and acidity. The findings can underpin the establishment of a comprehensive Daqu quality evaluation system and the rational regulation of community functions.