Abstract：
Aromatic compounds serve as the primary source of floral and fruity aromas in sauce-flavor (Maotai flavor) baijiu, constituting the skeleton components of its flavor profile. Nevertheless, the formation mechanism of these compounds and key aroma-producing enzymes in sauce-flavor Daqu (fermentation agent, SFD) remain elusive. Here, we combined metagenomics, metaproteomics, metabolomics, and key enzyme activity to verify the biosynthesis pathway of aromatic compounds and to identify key enzymes, genes, and characteristic microorganisms in SFD. The results showed that the later period of fermentation was critical for the generation of aromatic compounds in SFD. In-situ verification was conducted on the potential key enzymes and profiles in various metabolites, providing comprehensive evidence for the main synthetic pathways of aromatic compounds in SFD. Notably, our results showed that primary amine oxidase (PrAO) and aldehyde dehydrogenase (ALDH) emerged as two key enzymes promoting aromatic compound synthesis. Additionally, two potential key functional genes regulating aromatics generation were identified during SFD fermentation through correlation analysis between proteins and relevant metabolites, coupled with in vitro amplification test. Furthermore, original functional strains (Aspergillus flavus-C10 and Aspergillus niger-IN2) exhibiting high PrAO and ALDH production were successfully isolated from SFD, thus validating the results of metagenomics and metaproteomics analyses. This study comprehensively elucidates the pathway of aromatic compound formation in SFD at the genetic, proteomic, enzymatic, and metabolomic levels, providing new ideas for the investigation of key flavor substances in baijiu. Additionally, these findings offer valuable insights into the regulatory mechanisms of aromatic compounds generation.
摘要：
芳香化合物是酱香（茅台味）白酒中花香和果香的主要来源，构成其风味特征的骨架成分。然而，酱香大曲中这些化合物的形成机理和关键香气产生酶（发酵剂，SFD）仍然难以捉摸。这里,我们结合了宏基因组学，元蛋白质组学，代谢组学，和关键酶活性，以验证芳香族化合物的生物合成途径并确定关键酶，基因，和SFD中的特征性微生物。结果表明，发酵后期对SFD中芳香族化合物的产生至关重要。对各种代谢物中潜在的关键酶和谱进行了现场验证，为SFD中芳香族化合物的主要合成途径提供了全面的证据。值得注意的是,我们的结果表明，一级胺氧化酶（PrAO）和醛脱氢酶（ALDH）是促进芳香化合物合成的两个关键酶。此外,在SFD发酵过程中，通过蛋白质和相关代谢产物之间的相关性分析，确定了两个潜在的调节芳香族化合物生成的关键功能基因，结合体外扩增试验。此外，从SFD中成功分离出具有高PrAO和ALDH产量的原始功能菌株（黄曲霉C10和黑曲霉IN2），从而验证宏基因组学和元蛋白质组学分析的结果。本研究全面阐明了SFD中芳香族化合物形成的途径,蛋白质组学，酶，和代谢组学水平，为白酒关键风味物质的研究提供新思路。此外,这些发现为芳香族化合物生成的调控机制提供了有价值的见解。
