Selección de levaduras no-Saccharomyces para la elaboración de vinos tintos de calidad

Abstract

The spontaneous alcoholic fermentation of wine is a microbial process resulting from the sequential action of different non-Saccharomyces and Saccharomyces yeasts. The use of selected cultures of S. cerevisiae in fermentations is a widespread practice in the oenological industry that has contributed to better control of vinifications, but also to a simplification of the microbial communities that participate in fermentations, leading to a loss of complexity and certain standardization of the wines. To avoid the homogeneity of the wines inoculated with commercial strains of S. cerevisiae, the use of selected non-Saccharomyces yeasts in sequential fermentations with S. cerevisiae has been proposed. This practice allows obtaining some of the advantages of spontaneous fermentations such as greater organoleptic complexity, but with greater control of the process. The aim of this work was to select non-Saccharomyces yeast strains for its use as inoculums in the production of quality red wines. The study began with 103 isolates from different oenological ecosystems of the D.O.Ca. Rioja. In an initial stage, the clonal characterization and the study of the SO2 resistance of the strains was carried out, as well as the killer activity and possible interactions with other yeasts present in vinifications, such as S. cerevisiae and B. bruxellensis. The next step was the characterization of the strains based on their ability to produce enzymes of oenological interest. 13 enzymatic activities related to the aroma and colour and clarification of the wines were evaluated. The results showed high intraspecific variability, which highlighted the importance of selection at clonal level. In the next stage of the selection process, the oenological behaviour and the production of aromatic compounds of the strains in pure fermentations were studied. Significant differences were detected in the parameters analysed between the different non-Saccharomyces yeast species studied. In the case of the strains belonging to the same species, differences were found in a smaller number of parameters. However, among them were included parameters with a great impact on the quality of the wine, such as volatile acidity. The results also showed that none of the studied strains was able to complete the fermentation, which indicated that their use in winemaking required sequential inoculation with S. cerevisiae. For this reason, the following tests evaluated the characteristics of the strains in sequential inoculations with S. cerevisiae. Moreover, it was detected that two of the L. thermotolerans strains grew together with T. delbrueckii, so it was decided to include this combination as a mixed inoculum in the following assays. In the next stage of the process, the evolution of the aromatic composition was evaluated, as well as the effect on the composition of anthocyanins and stilbenes of the wines sequentially fermented with non-Saccharomyces/S. cerevisiae. The implantation controls of the inoculated non-Saccharomyces yeasts showed that T. delbrueckii and L. thermotolerans strains and the mixed inocula constituted by those same species remained for a longer time in the medium. In addition, it was observed that the mixed inocula maintained the initial proportions during the first days of fermentation. At the end of the fermentation, only the wines inoculated with T. delbrueckii and L. thermotolerans, yeasts that remained the longest in the medium, differed from the others due to their aromatic profile. Regarding the analyses related to colour, the results showed that the wines fermented sequentially with M. pulcherrima, T. delbrueckii, Z. bailii and C. zeylanoides presented higher levels of total anthocyanins and higher values of the total polyphenol index. (TPI) with respect to those found in wines made only with S. cerevisiae. In the last stage of the selection process, the oenological behaviour of the selected yeasts was evaluated in wines of three grape varieties: Tempranillo, Grenache and Graciano. Most of the differences due to the inoculation strategy were detected in Tempranillo variety. The changes produced in the characteristics of the wine could be due to the greater implantation and permanence of the inocula during the fermentation in this variety. The non-Saccharomyces strains used in the inocula had been originally isolated in Tempranillo, which explains their pre-adaptation to the must characteristics. Throughout the selection process, the inoculum formed by T. delbrueckii and L. thermotolerans had shown a greater capacity to implant in the medium during fermentation, remaining for a longer time in the tanks. On the other hand, the combination of the characteristics of the two strains that constitute the inoculum allowed the release of compounds of interest for the quality of wine such as ethyl lactate, glycerol or lactic acid. Likewise, the wines made with this combination of yeasts showed higher contents of vitisin B, relevant for color stability.This inoculum also showed interesting enzymatic activities to improve the colour and aroma of wine. Considering its positive characteristics for the quality of the wines, and since there is no inoculum composed of these non-Saccharomyces yeast species on the market, the mixed inoculum of T. delbrueckii and L. thermotolerans could have the potential to be commercialized, which is why it is the one that has finally been selected in this study.