###### Abstract  
Nowadays, the use of sulfur dioxide (SO\({}_{2}\)) during the winemaking process is a controversial societal issue. In order to reduce its use, various alternatives are emerging, in particular bioprotection by adding yeasts, with different impacts on yeast microbiota in early winemaking stages. In this study, quantitative-PCR and metabarcoding high-throughput sequencing (HTS) were combined with MALDI TOF-MS to monitor yeast population dynamic and diversity in the early stages of red winemaking process without sulfites and with bioprotection by _Torulaspora delbrueckii_ and _Metschnikowia pulcherima_ addition. By using standard procedures for yeast protein extraction and a laboratory-specific database of wine yeasts, identification at species level of 95% of the isolates was successfully achieved by MALDI TOF-MS, thus confirming that it is a promising method for wine yeast identification. The different approaches confirmed the implantation and the niche occupation of bioprotection leading to the decrease of fungal communities (HTS) and _Hansenisporn uranium_ cultivable population (MALDI-TOF MS). Yeast and fungi diversity was impacted by stage of maceration and, to a lesser extent, by bioprotection and SO\({}_{2}\), resulting in a modification of the nature and abundance of the operational taxonomic units (OTUs) diversity. MALDI-TOF MS; oenological laboratory specific database; winemaking without SO\({}_{2}\) bioprotection; metabarcoding HTS; quantitative-PCR +
Footnote †: journal: Appl. sequencing (HTS). The Q-PCR method allows the population dynamics of targeted microorganisms to be analyzed, and was applied to characterize the must and wine microbial community [6; 7], and to study the impact of non-_Saccharomyces_ on wine quality [16] or the effect of oenological practices on grape must microbial populations [17]. High-throughput sequencing (HTS) methods allow relative abundance and biodiversity indices to be calculated from the sequences obtained. The HTS method has already been developed in oenology [18] using the 454 pyrosequencing method to study the microbial ecology of grape berries or in wine [19; 20]. However, this latter technology is no longer used and has now been surpassed by Illumina [21]. Traditionally, sequencing analysis of the internal transcribed spacer and 265 rDNA was used for yeast colonies identification at species level. For many years, matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has replaced phenotypic or genetic sequencing identification techniques in the medical environment to become a routine analysis technique [22; 23]. It is a fast, simple, accurate, and cost-efficient tool for the identification of microorganisms [24]. Furthermore, beverage sectors such as the beer industry have implemented this method for the identification of spoilage microorganisms or fermentative yeasts [25; 26; 27]. Cells are co-crystallized with the matrix in a way that yields a sufficient number of medium-sized ions in the mass spectra. The identification of microbiological samples by this method relies on the acquisition of mass fingerprints and subsequent comparison with a Biotyper database. In oenology, several studies have aimed to optimize protein extraction protocols, improve yeast identification by creating specific wine microorganism databases [28], or obtain finer identification by the extraction of high mass range moieties [29]. MALDI-TOF MS Biotyper has been used to differentiate oenological yeast at the genus or species level, such as _Saccharomyces_[30], to identify different groups of _Saccharomyces cerevisiae_[31; 32], and to describe the grape berry microbiota [33]. However, it has been applied little until now for describing the yeast diversity of a must or wine-related environment, and some wine yeast species have not yet been identified due to their absence from the Biotyper database [34]. Today, societal demand tends to reduce chemical inputs in the food industry, with no exception in oenology.