## 3 Results
### Yeast and Fungi Community during the Early Stages of Winemaking without Sulfites  
Merlot N. (_Vitis vinifera_ L.) grapes collected in 2018 were separated into three batches according to the following treatments: bioprotection (BP), SO\({}_{2}\) at 50 mg/L, and without SO\({}_{2}\) (0). Grape must was collected at four different stages (vatting, 24 h, 48 h, beginning of alcoholic fermentation), thus resulting in 18 samples for further analysis. Chemical analysis of the Merlot grape must and wine at the end of the alcoholic fermentation are given in Table 13; no significant differences from one modality to another were noticed for grape must enological parameters, except for the total SO\({}_{2}\) that was logically higher for the SO\({}_{2}\) modality comparing with bioprotection and without SO\({}_{2}\) modalities. The wine analysis showed no significant difference concerning the acetic acid content except for the residual sugars for the without sulfites modality (Table 13).  
.2.1 Population Dynamics of _Hanseniapora_ spp. and Non-_Saccharomyces_ Yeasts Used as Bioprotection  
Three yeast species and genera (_Torulaspora delbrueckii_, _Metschnikowia pulcherrina_, and _Hanseniapora_ spp.) were targeted to monitor population dynamics using Q-PCR. Results are given in Figure 2 and Table 14. Population levels of _Hanseniapora_ spp. were relatively stable during the prefermetary stages, ranging from 2.8 \(\times\) 10\({}^{2}\) to 1.2 \(\times\) 10\({}^{4}\) cells/mL, whatever the modality considered, and then increased during the start of the alcoholic fermentation reaching 1.4-1.2 \(\times\) 10\({}^{6}\) cells/mL. Levels of indigenous populations of _Torulaspora delbrueckii_ and _Metschnikowia pulcherrina_ (with SO\({}_{2}\) and without SO\({}_{2}\) treatment) were low and below the detection limit of Q-PCR (<100 cells/mL), except for duplicates at the vatting stage without SO\({}_{2}\) (_Metschnikowia pulcherrina_ at 3.9 \(\times\) 10\({}^{2}\) cells/mL and 5.8 \(\times\) 10\({}^{2}\) cells/mL, respectively) and the start of alcoholic fermentation. Population levels of both species inoculated as the bioprotection treatment at the vatting stage confirmed the effective implantation of _Torulaspora delbrueckii_ and _Metschnikowia pulcherrina_, with averages of 5.7 \(\times\) 10\({}^{4}\) and 4.4 \(\times\) 10\({}^{6}\) cells/mL, respectively. _Torulaspora delbrueckii_ population levels were relatively stable during the prefermetary stages, whereas the _Metschnikowia pulcherrina_ population decreased to 3.4 \(\times\) 10\({}^{4}\) cells/mL on average after 48 h of maceration and then increased to 1.2 \(\times\) 10\({}^{6}\) at the start of alcoholic fermentation.  
\begin{table}
\begin{tabular}{c c c c} \hline
**Genus** & **Species** & \
\begin{tabular}{c} **Number of Strains in** \\ **Biotyper DB** \\ \end{tabular} &
\begin{tabular}{c} **Number of Strains in** \\ **OLS DB** \\ \end{tabular} \\ \hline _Aureobusidium_ & _pullulans_ & 3 & 2 \\ _Brettanomyces_ & _acidodurans_ & 0 & 1 \\ _Brettanomyces_ & _bruxellensis_ & 5 & 15 \\ _Candida_ & _cantarelli_ & 0 & 2 \\ _Starmerella_ & _bacillus_ & 0 & 2 \\ _Hanseniaspora_ & _uvarum_ & 8 & 2 \\ _Lanchancea_ & _thermotolerans_ & 3 & 1 \\ _Metschnikowia_ & _pulcherima_ & 4 & 2 \\ _Pichia_ (_Candida_) & _guiliermondii_ & 14 & 1 \\ _Pichia_ & _kluyveri_ & 1 & 1 \\ _Pichia_ (_Candida_) & _membantifaciens_ & 2 & 1 \\ _Saccharomyces_ & _cerevisiae_ & 13 & 5 \\ _Shizosacharromyces_ & _japonicus_ & 0 & 1 \\ _Shizosacharromyces_ & _octsporus_ & 0 & 1 \\ _Shizosacharromyces_ & _pombe_ & 4 & 3 \\ _Torulaspora_ & _delbrueckii_ & 5 & 2 \\ _Zygosacacharromyces_ & _buili_ & 3 & 1 \\ \hline \end{tabular}
\end{table}
Table 1: Numbers of strains per species in the Biotyper database (Biotyper DB) and the oenological laboratory specific database (OLS DB).