## 4 Discussion
### Effect of Fermentation Gradient Temperature on Thiols and Esters Concentrations  
Our work investigated the impact of fermentation gradient temperature on the alteration of thiols and esters in Chardonnay. Based on what is known about fermentation temperature and how it affects the volatile composition of wine, in this case, thiols andesters, we attempted to increase the concentrations of both aroma families by employing fermentation gradient temperature (FG1 and FG2). Fermentation temperature plays an antagonistic effect on these aroma families, that is, while higher fermentation temperatures (\(\sim\)20 \({}^{\circ}\)C) are required to increase thiol concentrations in the wine, esters require lower fermentation temperatures (\(\sim\)13 \({}^{\circ}\)C) to allow for their retention [46; 47]. FG1 and FG2 clearly affected the concentrations of both free volatile thiols and yeast-derived esters (Table 3). Both 3 SH and its acetate 3 SHA showed significantly higher concentrations of FG1 and FG2 compared to the baseline fermentation (FG0). A minor increase of 0.01 ng/L was also observed for 4 MSP. Similar trends in concentration increase were obtained in Sauvignon Blanc fermented at temperatures between 18 \({}^{\circ}\)C and 24 \({}^{\circ}\)C for 3 SH, 3 SHA, and 4 MSP [23; 24]. [PERSON] et al. [23] measured volatile thiols during fermentation, and their results showed that higher thiol levels were obtained only at the beginning of fermentation, reaching their maximum concentrations at approximately 96 h of fermentation [23]. As fermentation progressed, the higher temperature ferment (23 \({}^{\circ}\)C and 28 \({}^{\circ}\)C) showed a decrease in volatile thiols due to evaporation, while the lower temperature fermentations (18 \({}^{\circ}\)C) maintained their levels of thiols. While we did not measure volatile thiols during fermentation in this study, we believe that thiol release from their precursors occurred in the early stages of fermentation as a result of yeast enzymatic activity. Additionally, the subsequent drop in temperature to 13 \({}^{\circ}\)C preserved thiol concentrations, thus allowing for higher levels of thiols in the final wines. White wines are normally fermented at lower temperatures, ranging from 11 to 25 \({}^{\circ}\)C, to preserve fresh and fruity characteristics [3; 23; 48]. White wines fermented at higher temperatures have lower concentrations of FAEE and acetate esters, mainly due to evaporative loss [46]. Other esters, such as ethyl octanoate and ethyl decanoate, have been found to increase at higher temperatures, and ethyl hexanoate concentrations were not impacted by fermentation temperature [48]. In general, ester concentrations in wine are mostly dependent on substrate, enzyme activity involved in the synthesis of those compounds, and ester hydrolysis. Any influences directly affecting these three factors will alter ester concentration in wine [23]. We believe that minimal evaporation loss occurred in the FG1 and FG2 wines due to the change in temperature. Both FG1 and FG2 resulted in higher concentrations of several ethyl and acetate esters compared to FG0, which was fermented at a constant 13 \({}^{\circ}\)C. The elevated temperatures at the beginning of the FG1 and FG2 fermentations accelerated the fermentation rate and yeast metabolism, resulting in greater synthesis of these compounds. Thus, the reduction in fermentation temperature to 13 \({}^{\circ}\)C was crucial to maintaining ester levels in the wine. Although there was no wine treatment fermented at a constant 20 \({}^{\circ}\)C, it is hypothesized that the volatile composition of this wine would be lower compared to FG1 and FG2 [44; 45]. Finally, FG2 obtained overall slightly lower concentrations of esters compared to FG1. The temperature change point for this wine was based on its sugar level (11.5 \({}^{\circ}\)Brix), which on average took place \(\sim\)24 h after FG1, depending on fermentation replicates (Table S12).