## 3 Results
### M. pulcherrima Is Metabolically Active but Does Not Ferment Sugars in Lab-Scale Co-Fermentations  
To test the compatibility of the selected strain of _M. pulcherrima_ with _S. cerevisiae_ in sequential fermentation, we carried out a microvinification experiment using a frozen Chasselas must from the 2021 vintage. The enoological parameters of the must before viinification were 90 g/L of glucose, 105 g/L of fructose, 3.6 g/L of malic acid, 5.4 g/L total acidity, 230 mg/L of assimilable nitrogen, and a pH of 3.3. Therefore, in addition to a classical viniification with a commercial _S. cerevisiae_, we prepared a condition in which the _M. pulcherrima_ UASWS2926 VBI-A02 was present for 5 days before _S. cerevisiae_ inoculation. All fermentations were carried out in triplicate. Densitometry showed no significant changes in the condition of _M. pulcherrima_ during the first days of incubation (Figure 2). HPLC analysis showed a slight decrease in total sugars (120 g/L at time 0 versus 115 g/L at day 5). Therefore, we performed microbiological analysis using FCM to verify viability and metabolic activity. Our results showed the proliferative activity of _M. pulcherrima_ during the first days of incubation, which decreased after inoculation with _S. cerevisiae_ (Figure 3). Notably, we were able to discriminate the two co-fermenting yeasts using FCM by analyzing the combination of the metabolic marker CFDA and the nucleic acid marker Syto 41 (Figure S1). The metabolic activity of _M. pulcherrima_, defined by the mean fluorescence of the CFDA marker, followed a similar pattern (Figure 4). The profile of _S. cerevisiae_, both in single and co-fermentation, was similar to that obtained in other microvoinifications, namely rapid growth and high metabolic activity defined by the CFDA marker (Figures 3 and 4). To study the influence of the co-fermentation of _M. pulcherrima_/_S. cerevisiae_ versus the single fermentation of _S. cerevisiae_ on the main biochemical parameters, we performed HPLC analysis. Our results showed that the wine obtained after co-fermentation had significantly lower levels of tartaric acid and higher levels of glycerol than that obtained with single fermentation (Table 3). Overall, these data suggest that the _M. pulcherrima_ strain UASWS2926 VBI-A02 could play an active role and thus be used on a larger scale in Chasselas winemaking. Therefore, we prepared an experimental scheme for cellar-scale viinifications of Chasselas in the 2022 vintage.  
Figure 2: Densitometric analysis of bench-scale fermentations. SC single: _S. cerevisiae_ in single fermentation; MP: _M. pulcherrima;_ SC seq: _S. cerevisiae_ in sequential fermentation.  
Figure 4: CFDA fluorescence in _S. cerevisiae_ and _M. pulcherrina_ (in single or sequential bench-scale fermentation) obtained by FCM analysis. SC single: _S. cerevisiae_ in single fermentation; MP: _M. pulcherrina_; SC seq: _S. cerevisiae_ in sequential fermentation.  
\begin{table}
\begin{tabular}{c c c c c c c c c} \hline  & **Tartaric Acid** & **Malic Acid** & **Glucose** & **Fructose** & **Acetic Acid** & **Lactic Acid** & **Glycerol** & **Ethanol** \\  & **(g/L)** & **(g/L)** & **(g/L)** & **(g/L)** & **Acid** & **(g/L)** & **(g/L)** & **(g/L)** \\ \hline SC & 3.027 \(\pm\) 0.101 & 1.973 \(\pm\) 0.100 & \textless{}0.2 & \textless{}0.2 & 0.197 \(\pm\) 0.029 & \textless{}0.2 & 3.390 \(\pm\) 0.191 & 11.233 \(\pm\) 0.559 \\ \hline MP + SC & 2.527 \(\pm\) 0.108 * & 1.990 \(\pm\) 0.062 & \textless{}0.2 & \textless{}0.2 & 0.163 \(\pm\) 0.055 & \textless{}0.2 & 4.170 \(\pm\) 0.173 * & 11.320 \(\pm\) 0.230 \\ \hline \end{tabular}
\end{table}
Table 3: Comparison of oenological parameters after alcoholic fermentation between the single and co-fermentation. * \(p\) < 0.001 (two-way ANOVA with Sidak test).  
Figure 3: Kinetics of cell growth of _S. cerevisiae_ and _M. pulcherrina_ (in single or sequential bench-scale fermentation) obtained by FCM analysis. SC single: _S. cerevisiae_ in single fermentation; MP: _M. pulcherrina_; SC seq: _S. cerevisiae_ in sequential fermentation.