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.

Knowledge of the metabolism of functional enzymes is the key to accelerate the transformation and utilization of raw materials during high temperature Daqu (HTD) manufacturing. However, the metabolic contribution of raw materials-wheat is always neglected. In this research, the relationship between the metabolism of wheat and microorganisms was investigated using physicochemical and sequencing analysis method. Results showed that the process of Daqu generation was divided into three stages based on temperature. In the early stage, a positive correlation was found between Monascus, Rhizopus and glucoamylase metabolism (r > 0.8, p < 0.05). Meanwhile, the glucoamylase metabolism in wheat occupied 63.8 % of the total matrix at the day 4. In the middle to later stages, the wheat metabolism of proteases, α-amylases and lipases in gradually reached their peak. Additionally, Lactobacillus and α-amylases presented a positive correlation (r > 0.7, p < 0.05), and the α-amylases metabolism in wheat occupied 22.18 % of the total matrix during the same time period. More importantly, the changes of enzyme activity metabolic pathway in wheat and microorganism were reflected by respiratory entropy (RQ). Overall, these results guide the choice of substrate during Daqu production.

Even if fermented in the same qu-room, Daqu will form various microecologies. A gradual differentiation of microbial population niches was observed within different qu-layers, manifesting as variations in the abundance of dominant microorganisms including Bacillus, Saccharopolyspora, Weissella, Kroppenstedtia, Thermoascus, Thermomyces, Saccharomycopsis, and Rasamsonia. Moreover, distinctions were observed in the functional expression of microorganisms, including Aspergillus, Virgibacillus, Oceanobacillus, and Neurospora. The community in middle layer Daqu exhibited characteristics of high compactness and niche diversity, which facilitated efficient substrate utilization. During the initial phase, the upper Daqu community demonstrated heightened activity. However, in the middle and lower layers, fungi and bacteria respectively exhibited greater functional expression. The key environmental factors regulating the assembly of communities in the upper and middle layers were pH and temperature, respectively, and the lower was moisture and acidity. Notably, deterministic assembly exerted a stronger influence on fungi.

Chameleon-like microbes in the fermentation community are an internal factor that facilitate the transformation of the community to the corresponding homeostasis states under specific environmental conditions. High temperature daqu can form three typical microecologies during the preparation process, making it an ideal system for studying chameleon-like microbes. This study integrated multi-omic methods such as metaproteomics, and determined that Neurospora crassa, Aspergillus nidulans, Bacillus subtilis and Oceanobacillus iheyensis were chameleon-like microbes that regulated the metabolic differences of five-member heterocyclic amino acids in daqu, resulting in microecological differentiation. Synthetic microbial consortia consisting of the four chameleon-like microbes with (T6) and without (T4) the dominant functional bacteria Saccharopolyspora erythraea and Virgibacillus haloimitrificans were fermented under simulated in situ conditions. The community constructed by microorganisms with greater functional diversity (T6) was more robust, and its metabolome was more similar to the in situ system. When exposed to environmental disturbances, the functional diversity helped to maintain the community stability by increasing the dissimilarity of chameleon-like microbes in the community and forming different homeostasis.