One of the alternatives to SO₂ as an antimicrobial is the use of bioprotection yeasts, which colonize the medium preventing the proliferation of undesirable microorganisms. In this work, the bioprotective effect of a mixed inoculum formed by Torulaspora delbrueckii/Lachancea thermotolerans during fermentation was evaluated. For this purpose, fermentations were carried out using this mixed inoculum and the populations of yeasts, lactic bacteria and acetic bacteria, and the physical–chemical parameters of the wines obtained were studied. The results were compared with those obtained in spontaneous fermentation with and without SO₂. The different fermentation strategies caused a differentiation in the yeast species present during fermentation. Regarding populations of lactic acid bacteria, results showed that the effect of the addition of the mixed inoculum was comparable to that exerted by SO₂. On the other hand, due to the high sensitivity of acetic acid bacteria to SO₂, the sulfite vinifications showed a lower population of acetic acid bacteria in the early stages of fermentation, followed by the vinifications with the mixed inoculum.

Nitrogen supplementation in musts or during the alcoholic fermentation is a common practice to promote fermentations. In this study, the impact of the supplementation of two different sources of nitrogen during Tempranillo red wine elaboration was studied. Mineral and organic nitrogen was added after the exponential yeast growth phase and during winemaking, examining its impact on the alcoholic and malolactic fermentation development, on the aromatic wine composition and on the nitrogenous wine composition. The nitrogen supplementation did not provide neither significant advantages in kinetics and fermentations time, nor differences in the chemical wine composition. The aromatic composition of the wines improved with the addition of inorganic nitrogen, although its organoleptic evaluation was not favored. Moreover, the concentration of amino acids in wines increased slightly after the malolactic fermentation and significantly during the stabilization time, especially with organic nitrogen addition. However, the synthesis of biogenic amines did not increase in wines neither after the malolactic fermentation, nor after the storage period.

The use of non-Saccharomyces yeasts in sequential fermentations with S. cerevisiae has been proposed to improve the organoleptic characteristics involved in the quality of wine. The present study set out to select a non-Saccharomyces inoculum from the D.O.Ca. Rioja for use in winemaking. Strains included in the study belonged to Torulaspora delbrueckii, Lachancea thermotolerans, Metschnikowia pulcherrima, Zygosaccharomyces bailii, Williopsis pratensis, Debaryomyces hansenii, Pichia kluyveri, Sporidiobolus salmonicolor, Candida spp., Cryptococcus spp. and two mixed inocula of Lachancea thermotolerans-Torulaspora delbrueckii in a 30/70 ratio. In the first stage of the process, SO₂ resistance and presence of enzymatic activities related to wine aroma and wine color and fining (esterase, esterase-lipase, lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, β-glucosidase, pectinase, cellulose, xylanase and glucanase) were studied. In the later stages, selection criteria such as fermentative behavior, aroma compound production or influence on phenolic compounds were studied in laboratory scale vinifications. Taking into account the results obtained in the different stages of the process, a mixed inoculum of Lachancea thermotolerans-Torulaspora delbrueckii in a 30/70 ratio was finally selected. This inoculum stood out for its high implantation capacity, the production of compounds of interest such as glycerol and lactic acid and the consequent modulation of wine acidity. Given these characteristics, the selected inoculum is suitable for the production of quality wines.