## 4 Discussion
The kinetics of the sequential inoculation experiment followed those already described in the literature, with proliferation of _M. pulcherrima_ followed by a decrease after inoculation of S. cerevisiae [18; 35]. Interestingly, our data show that the sequential fermentation had a lower concentration of _S. cerevisiae_ compared to single fermentations (inoculated with the same amount of _S. cerevisiae_), even if the metabolic activity as detected by CFDA was comparable. Although _M. pulcherrima_ is not considered a high nitrogen-demanding yeast, we could speculate that _M. pulcherrima_ UASWS2926 VBI-A02 in the sequential fermentation consumed a certain amount of nitrogen that couldhave been used by _S. cerevisiae_, resulting in a reduction in the total biomass [36]. At the end of alcoholic fermentation, we then measured the main parameters using HPLC. Our analysis showed a clear increase in the glycerol content in the sequential fermentation and not in the single fermentation, a result that is a general characteristic of sequential fermentation of _M. pulcherrina_/_S. cerevisiae_[35; 37; 38]. Interestingly, a significant decrease in tartaric acid content without changes in pH (not shown) was observed. This phenomenon, also observed by others [38], remains to be explained. After evaluating the feasibility of SF at the bench scale, we moved to the cell scale to prepare an experiment on Chasselas winemaking with _M. pulcherrina_ UASWS2926 VBI-A02. In addition to SF and the single inoculum of _S. cerevisiae_ (CC condition), we prepared a PDC condition. Our results at cellar scale generally recapitulate the findings of bench-scale fermentations, especially in cell kinetics, suggesting the appropriateness of our model. Interestingly, in the PDC condition, we did not observe the classical peak of CFDA at the beginning of the fermentation process. Sugar consumption continued, but cell activity was low. Most likely, since they had already undergone many cell divisions, the yeasts in PDC were less active than their \"fresh\" counterparts (_S. cerevisiae_ in CC or SF conditions) but were still able to maintain fermentative activity. In the SF, even if we started with a similar inoculum in terms of cell concentrations (around 1 \(\times\) 10\({}^{6}\) cell/mL), we did not observe a similar degree of proliferation of _M. pulcherrina_ compared to bench-scale viifications. For example, at the day of _S. cerevisiae_ inoculation, we observed 14 \(\times\) 10\({}^{6}\)_M. pulcherrina_ cells/mL at bench scale and only 2 \(\times\) 10\({}^{6}\)_M. pulcherrina_ cells/mL at cellar scale. The day after, the ratio of _M. pulcherrina_/_S. cerevisiae_ was 5:1 at bench scale, whereas in the cellar scale it was 1:1. This may explain the biochemical differences between the two experiments at the end of alcoholic fermentation, especially the lack of glycerol production in the cellar scale. Thus, at least hypothetically, a low number of _M. pulcherrina_ cells were not able to influence _S. cerevisiae_ metabolism toward increased glycerol production [39]. The proliferation of _M. pulcherrina_ may have been limited by endogenous populations in the must prior to inoculation with _S. cerevisiae_. In fact, after molecular analysis, we found that all the pre-inoculation conditions contained a microbial community that included yeast-like fungi such as _A. pululans_ and other yeasts such as _S. bacillaris_ and _H. uvarum_. Interestingly, after inoculation with _M. pulcherrina_ UASWS2926 VBI-A02, the relative abundance of these microorganisms was reduced, especially the most \"risky\" from a spoilage perspective (in particular, they increase volatile acidity), such as _Hanseniaspora_. This bioprotective effect exerted by _M. pulcherrina_ is currently being studied extensively from the perspective of a continuous reduction in chemical inputs, such as sulfites [40]. However, it is important to point out that bioprotection is usually carried out in must clarification/maccation steps at low temperatures, conditions that favor biocontrol by _M. pulcherrina_[41; 42].