“Wine yeast biodiversity during spontaneous fermentation in response to environmental stress”

“Natural wines”, nowadays, could appear an evocative recall to what wine was like before. During the ages, science allowed winemakers to produce wines with the full control of vinification condition, obtained mainly by addition of exogenous components, supplements and microorganisms. Contrarily, “natural wine” come back to basics and simplicity as invasive operations are avoided; but, on the other hand, a deeper knowledge and monitoring of winemaking process is required, in order to obtain high quality products without exogenous substances. Natural wine has been produced by spontaneous fermentation of must by yeasts originated from grapes and winery equipment. The wide variety of natural yeasts reflects the biodiversity, which is still under-exploited despite the large use of commercial Saccharomyces cerevisiae in most grape musts. During fermentation, several strains compete in the same fermenting must, and the dominance of Saccharomyces cerevisiae takes place when it overcomes all the others. The aim was to investigate Saccharomyces cerevisiae diversity and its technological behavior in two biodynamic wine farms in Tuscany. Autochthonous S. cerevisiae from Syrah fermentations were isolated and molecularly characterized in 2009 and 2013 harvests. Saccharomyces cerevisiae strains were isolated starting from Syrah musts with zero or low levels of sulphite added, according with the tendency of natural wines producers to abait every invasive operation during wine making. Samples were collected in different steps of winemaking. ITS-PCR method confirmed isolates belong to Saccharomyces sensu stricto complex. The multiplex PCR amplification of microsatellite loci (SC8132X, YOR267C and SCPTSY7) discriminated S. cerevisiae strains. Genetically diverse S. cerevisiae strains were subsequently subjected to technological characterization. Micro-fermentations were set up to study fitness and quality traits of biotypes characterized. Weight loss kinetics were measured and chemical analyses were performed. Collected Saccharomyces cerevisiae strains were tested also in order to verify the effect of low sulphite additions on biodiversity pursuing information about stress-adaptation mechanisms that allow the survival in the challenging environment of fermenting must. Sulphite addition may act as a selective factor to induce the presence and the activity of strains with diverse features about sulphite metabolism. The addition of different dose of sulphite induced a considerable variability among strains. However, some biotypes exhibited tolerance to certain concentration and only one biotype was classified as resistant to a determinate concentration. In addition, biotypes were subjected to an other environmental stress factor such as ethanol. The increasing of levels of ethanol limited considerably the activity of selected strains, but some of them highlighted a positive tolerance to various ethanol concentrations. During the harvest of 2013, the alcoholic fermentation was conducted inoculating a biotype characterized previously, which expressed good fermentative performance and a multi-strains culture of other S. cerevisiae strains characterized in the same harvest (2009) in a biodynamic farm from a workgroup of Edmund Mach Foundation of San Michele all'Adige (TN). The results of 2009 highlighted peculiar behaviors of 13 biotypes (characterized from four hundred isolates) that dominate the spontaneous fermentations. Not surprisingly, some biotypes were also found in fermentations of 2013, demonstrating yeasts are ubiquitous in that environment. Sensorial analyses of wine, originated by biotypes fermentation, revealed certain diversity in aroma and flavor traits, but also in the structure. Overall, this work suggests the consolidate relationship existing between autochthonous yeasts and the terroir, where yeasts are ubiquitous. Furthermore, autochthonous yeasts surely contribute to the unique identity of wine. In addition, this work suggests that the use of isolated and characterized autochthonous yeast strains could help the winemaking process, where the vintage could be inadequate to develop a particular product such as the wine of a specific wine farm.