## 3 Results and Discussion
### Production of Minor Volatile Compounds  
After 24 h of fermentation, 101 compounds were identified (Table S1), which were distributed into the following chemical families: acetal (1), acids (6), alcohols (20), aldehydes (10), ketones (7), esters (24), terpenes (14), pyrazines (11), and others (8), including phenols, furans, lactones, pyrroles, and sulfur compounds. These results are summarized in the heat maps shown in Figure 4.  
The most representative group of compounds was higher alcohols. In general, the YPDA medium produced more alcohol than the M2 medium. The Sc/Td, Sc/Zb, and ternary co-cultures may have fewer higher alcohols than the Td/Zb co-culture, which showed a more significant number of higher alcohols within group M2. Nevertheless, it was also observed that each yeast strain was affected differently by the nutritional composition of the two media. For instance, _T. delbrueckii_ produced the highest quantity of higher alcohols (5.197 \(\pm\) 0.286 mg/L), even more so than in co-cultures in nutritionally rich YPDA medium. Conversely, this yeast produced the lowest amount (1.652 \(\pm\) 0.32 mg/L) in nutritionally poor M2 medium. The nutritional content and variable loss of viability could explain the changes in the levels of some alcohols, such as 2-phenylethanol. This alcohol is related to cell-cell communication, which is dependent on cell density and the nutritional status of the environment [52]. Therefore, the lower production of higher alcohols in general, but particularly 2-phenyl ethanol, 2-methyl-1-propanol, and 1-propanol, could be related to the loss of viability of _T. delbrueckii_. Figure 4: Semi-qualitative analysis of volatile compounds in M2 and YPDA media (**A**) Cluster heatmap of volatile compounds produced by monocultures in both media. (**B**) Cluster heatmap of volatile compounds produced by co-culture in both media. The production of aldehydes and ketones is related to the ability of yeasts to carry out oxidation-reduction reactions to achieve the biotransformation of alcohols to their respective ketones or aldehydes and back [53]. Ketone production was higher in _Z. bailii_ monocultures, regardless of the culture medium. In contrast, the production of aldehydes was better in the _Z. bailii_ monoculture, but only in the M2 medium. Zygosaccharomyces bailii stands out for its high production of 2-heptanone and its corresponding alcohol, 2-heptanol. These compounds have previously been reported to contribute to the flavor of Maotai liquor by providing fruity and spicy notes, such as cinnamon aroma (2-heptanone) and fruity flavor (2-heptanol) [54]. Many pyrazines were identified, mainly in the YPDA medium. It is explained because the yeast extract is rich in this type of compound [43]. Therefore, these compounds originate in the components of the culture medium rather than from yeast metabolism. The production of ethyl esters was poor in M2 medium, especially in monocultures of _T. delbrueckii_ and _Z. bailii_. Instead, in the YPDA medium, the production of esters was better in all fermentations than in the _S. cerevisiae_ monoculture. This result suggests a synergistic effect on ester production mediated by the yeast species involved. The production of esters has been related to the presence of precursors (fatty acids) and to the activity of the enzymes acetyl-CoA carboxylase and fatty acid synthase complex [55]. The fermentation time at which the measurements were performed (24 h) may be related to the abundance of esters because these usually occur in the late stages of fermentation [56]. This could be the case for M2 fermentation, where incomplete fermentation produces poor ester production. The Sc/Zb co-culture in the YPDA medium obtained the highest number of terpen among all fermentations. However, the other fermentations in the YPDA medium showed terpene production below that obtained in the M2 medium. Terpene compounds originate from raw materials and are generally bound to carbohydrates; therefore, yeasts must express glucosidase enzymes to release these compounds [57].