## 3 Results and Discussion
### The Synthesizing and Degrading Metabolic Pathways of Key Flavor Chemicals  
Flavor chemicals fluctuated in the Baijiu fermentation process, and microorganisms contributed greatly to the synthesis of flavor chemicals (Figures 2 and 8). The essence of microbial metabolism is that for a specific flavor chemical, its concentration is a dynamic balance of microbial metabolic synthesis and degradation, and microbial metabolism is a biochemical reaction catalyzed by enzymes of related metabolic pathways [35]. In order to regulate the metabolism of related flavor chemicals by microorganisms in future studies, the metabolic network of related flavor chemicals was also analyzed, especially tetramethylpyrazine. The metabolic synthesis pathways of these chemicals were summarized together with the metabolic degradation pathways. To the best of our knowledge, all previous studies only focused on the metabolic synthesis pathway of tetramethylpyrazine, and this work proposed the degradation pathway of tetramethylpyrazine in Baijiu for the first time and could provide references for the subsequent regulation of the concentration of tetramethylpyrazine in the actual fermentation process using microbes. The ethyl ester metabolic synthesis pathway used starch and protein substances contained in fermented grains as the substrates, and starch was converted into the key metabolic intermediate pyruvate through glycolysis, pyruvate was converted to acetaldehyde by pyruvate decarboxylase, and acetaldehyde could be converted to acetic acid by aldehyde dehydrogenase, or to ethanol by alcohol dehydrogenase (Figure 9a). Pyruvate could also be converted to acetyl-CoA by 2-oxoacid oxidoreductase. The key metabolic intermediate acetyl-CoA served as the substrate for synthesizing medium and long chain fatty acids through the reverse \(\beta\)-oxidation pathway [36] and generated the precursors of flavor ethyl esters, such as hexanoic acid, octanoic acid, decanoic acid, and dodecanoic acid (Figure 9a). These acids were converted into the corresponding esters through an esterification reaction with alcohol (such as ethanol or butanol) (Figure 9a). Protein in grains could be hydrolyzed by a protease to produce amino acids, such as L-phenylalanine, and further converted to phenylpyruvic acid by amino acid transaminase, and finally produced phenylethanol and phenylacetic acid. Phenylethanol and phenylacetic acid could react with acetic acid and ethanol, respectively, to produce phenylacetic acid, ethyl ester, and acetic acid, phenylethyl ester (Figure 9a). Enzymes catalyzing esterification reactions by microorganisms from Baijiu had been reported in previous studies, such as carboxylesterase, and provided a theoretical basis for esterification using acids and alcohols as substrates [2]. In addition, short-chain fatty acid esters such as ethyl acetate could be produced through transesterification, which was also an important metabolic pathway to generate esters in Baijiu [37]. Meanwhile, enzymatic esterification was a reversible process, and microorganisms could also hydrolyze the ester bond through similar enzymes and then consume the acid or alcohol as the carbon source generated from ester hydrolysis [38]. Tetramethylpyrazine, as a health factor to alleviate cardiovascular and cerebrovascular diseases, has received great attention since its identification from Baijiu [39]. The metabolic synthetic pathway of tetramethylpyrazine has been revealed by microbial metabolism combined with a one-step chemical reaction (Figure 9b). The direct precursor  
Figure 9: The synthetic pathway of esters (**a**) and the synthetic and degrading pathway of tetramethylpyrazine (**b**). of tetramethylpyrazine is acetoin, catalyzed by \(\alpha\)-acetolactone decarboxylase (AlsD) using \(\alpha\)-acetolactone as the substrate, and \(\alpha\)-acetolactone was produced by \(\alpha\)-acetolactone synthetase (AlsS) using pyruvate acid as the substrate (Figure 9b).