## 3 Results
### Yeast and Fungi Community during the Early Stages of Winemaking without Sulfites
#### 3.2.2 Yeasts and Fungal Diversity
Overall, the number of observed OTUs decreased and \(\alpha\) diversity increased (\"Shannon\" and \"invSimpson\" index) during prefermentary maceration, whatever the treatment considered. The drop in the number of observed OTUs was particularly marked for the without SO\({}_{2}\) (0) and SO\({}_{2}\) treatments. The \"Shannon\" index and \"InvSimpson\" index were significantly higher for the BP and the SO\({}_{2}\) treatment than for without SO\({}_{2}\) for the first two stages (vatting and 24 h of maceration (24 h)). Table 2 presents the explained variance by \"Stage\" (vatting, 24 h and 48 h of maceration) and \"Treatment\" (BP, 0, SO\({}_{2}\)) factor and the combination of the two factors for biodiversity indices. The \"Stage\" factor accounted for the higher percentage of variance (ANOVA \(p\) < 0.05) for all the biodiversity indices and explained 50% of the variance on average. \"Treatment\" significantly impacted the number \"Observed\" and the \"InvSimpson\" index. The interaction between these two parameters did not show any significant impact whatever the biodiversity index considered. MALDI-TOF-MS was applied on yeast isolates from samples collected during prefermentary stages and start of alcoholic fermentation. \begin{table}
\begin{tabular}{c c c c c} \hline \hline  & **Observed** & **Chao1** & **Shannon** & **InvSimpson** \\ \hline Stage & 52.70\% ** & 53.29\% ** & 44.90\% * & 53.96\% ** \\ Treatment & 19.13\% * & 11.25\% & 13.77\% & 22.79\% * \\ Treatment*Stage & 14.48\% & 19.67\% & 4.90\% & 6.19\% \\ Residuals & 13.64\% & 15.78\% & 36.33\% & 17.05\% \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Percentage of variance explained by treatment and stage factors for different biodiversity indices (significance codes for \(p\) value: ** 0.01 and * 0.05). Figure 3: (**A**) Relative genus abundances based on the taxonomic assignation of high-quality 18S rDNA reads of _Ascomyca_ phylum from must samples at prefermentary stages (treatment: without SO\({}_{2}\) (0), SO\({}_{2}\) (SO\({}_{2}\)), bioprotection (BP); stages: vatting, 24 h prefermentary maceration, 48 h prefermentary maceration). (**B**) Relative genus abundances in “Others” category for each sample. (Mean of biological replicates). Levels of the total yeast population by plating method on LT medium are given in Table 14. Total yeast populations were from 8.3 \(\times\) 10\({}^{2}\) to 2.3 \(\times\) 10\({}^{3}\) CFU/mL for the vatting stage, except for the bioprotection treatment where the population was logically higher (3.4 \(\times\) 10\({}^{5}\) to 4.8 \(\times\) 10\({}^{5}\) CU/mL) due to non-_Saccharomyces_ addition. Total yeast populations were generally stable during the prefermentary stages and reached 7.7 \(\times\) 10\({}^{6}\) to 4 \(\times\) 10\({}^{7}\) at the beginning of the alcoholic fermentation. A total of 60 colonies per treatment and 180 colonies per stage (4) were isolated from LT medium and subcloning on YPG medium, resulting in 683 clones to be analyzed by MALDI-TOF MS for species identification (Table 14), instead of the 720 planned. Five isolates did not grow after subcloning and only eleven isolates were isolated from one sample, and this sample was therefore not considered for further analyses. Some isolates (26) were not identified as their profiles did not find a match in the database and, finally, 657 isolates (95%) were identified successfully at species level by MALDI-TOF MS, among which were ten different species (Figure 5). _Metschnikowia pulcherrima, Torulaspora delbrueckii, Aureobasidium pullulans_, and _Hanseniagpora uvarum_ were previously reported as OTUs by HTS, whereas _Kluyveromyces lactis, Saccharomyces cerevisiae, Whickerhamomyces anomulus_, and _Lachancea thermotolerans_ were identified among the \"Others\". _Candida guillemondii_ and _Cryptococcus fluscescens_ were not identified by HTS.