## 4 Discussion

Table 3: Shannon index evaluated by MALDI-TOF MS.  
method also allows quantitative information to be obtained on relative abundances of a genus in particular [54], and biodiversity indices from OTU tables. Numerous studies targeting yeasts and fungi have previously been published [53; 55; 56; 57]. However, various biases have to be taken into account to interpret the data: (i) as for Q-PCR analysis, grape must may contain many PCR inhibitors [58; 59], (ii) taxa with low proportions in a community are underrepresented or have a low amplification reproducibility due to primer mismatches or PCR biases [54; 60], (iii) amplified DNA does not provide information as to whether yeast are physiologically active or dead, or may be active within the community. In our study, the most abundant OTUs were assigned to grape fungi, mainly _Aureobasidium_, _Botrytis_, and _Cladosporium_. These OTUs represented more than 75% of the total abundance, in line with previous results obtained with samples collected in grape must before the start of alcoholic fermentation [9; 10]. Grape must yeast diversity in the present study, as reported previously [19; 61; 62], was quite low compared to other matrices, such as sugar cane or the soil [53; 63; 64], but nonetheless richer than in traditional sourdoughs [65]. To gain further insight, it would be interesting to use the metagenomic approach to provide a more in-depth understanding, since it offers a non-targeted taxonomic study [66]. Illumina metabarcoding and MALDI-TOF MS allowed concordant yeast identification both at genus and species level, e.g., _Aureobasidium_, _Hansenispora_, _Metschnikowia_, and _Toruspora_, but with different abundances; _Aureobasidium_ was the most abundant OTU for Illumina metabarcoding, whereas it was detected only in one sample through cultivation, probably due to the use of biphenyl in the cultivation medium (LT). Inverse results were obtained for _Hansenispora_. _Saccharomyces_ was identified among isolates, but with a very low abundance in Illumina data, which is consistent with other studies based on high-throughput sequence analysis, reporting a near-absence of this genus [1; 6; 19]. Microbial culturomics, using multiple culture conditions and MALDI-TOF MS, was successfully applied recently to study human gut microbiota [67] and the plant prokaryotic microbiome [68]. In this study, only one medium was used to target yeasts. Higher combinations of various growth media (for example, specific fungi media) and higher number of isolates analyzed per sample dilution would offer a more in-depth estimation of microbial diversity [69]. Moreover, compared to Illumina metabarcoding, culturomics approaches make it possible to collect colonies related to microbial diversity, thus allowing collection enrichment and further phenotypic analysis. In oenology, non-_Saccharomyces_ yeast preparations are now proposed as bioprotection agents during the prefrementary stages. However, their impact on the microbial community and antiscite effectiveness have so far received only a few scientific demonstrations [14; 15]. Till now, only pure culture of non-_Saccharomyces_ yeast was studied. In the present study, the application of a mix of two species was considered. The ability of both bioprotective species _Torusalospora delbrueckii_ and _Metschnikowia pulcherrima_ to colonize the grape must during prefrementary stages was confirmed by the three methods. However, _Metschnikowia pulcherrima_ decreased during the prefrementary stages, whereas _Torusalpora delbrueckii_ remained stable. The addition of bioprotection led to a decrease in fungal communities, especially _Aureobasidium_ and _Botrytis_, the latter being considered a common grape pathogen. _Hansenispora uwarum_ is a major species in the grape must microbial community, which can have a negative effect on _Saccharomyces cerevisiae_ growth and even lead to delayed alcoholic fermentation [2]. It also produces unwanted metabolites, such as acetic acid, ethyl acetate, sulfur compounds, acetin, and biogenic amines [70; 71; 72].