Aims Twenty-five enological yeasts belonging to nine different species (Candida zeylanoides, Cryptococcus uzbekistanensis, Debaryomyces hansenii, Lachancea thermotolerans, Metschnikowia pulcherrima, Torulaspora delbrueckii, Williopsis pratensis, Zygosaccharomyces bailii and Saccharomyces cerevisiae) were screened for aroma formation and fermentative behaviour as part of a non-Saccharomyces yeast selection programme. Methods and Results Pure cultures were inoculated in pasteurized grape juice in order to perform alcoholic fermentations. Some non-Saccharomyces species did not ferment, others did not get established and none of them completed alcoholic fermentations. The physico-chemical parameters of the wines and the abundance of aromatic compounds at the end of alcoholic fermentation highlighted the notable differences in the aroma-forming ability and fermentative behaviour of the different non-Saccharomyces species, but not within clones. Conclusions Lower diversity was detected within non-Saccharomyces species than that reported in S. cerevisiae with regard to enological behaviour and aromatic profiles. Metschnikowia pulcherrima and L. thermotolerans are the two species with higher possibilities to become an inoculum. Significance and Impact of the Study Few significant differences were found within clones of the same species, but very important parameters in wine quality, such as volatile acidity, ethyl acetate and acetoin, which would justify selection programmes within those species. The results also demonstrated that T. delbrueckii and L. thermotolerans are two close species in their aromatic profiles.

This study was aimed to measure the impact of the application of a bio-fungicide against Botrytis cinerea on the microbiota involved in the alcoholic fermentation (AF) of Tempranillo Rioja wines. For this purpose, a bio-fungicide composed of the biological control bacterium Bacillus subtilis QST713 was applied to the vineyard. The microbial diversity was analyzed from grape biofilm to wine. Impact on microbial diversity was measured employing indexes assessed with the software PAST 3.10 P.D. Results were compared to non-treated samples and to samples treated with a chemical fungicide mainly composed by fenhexamid. Overall, the impact of the biological-fungicide (bio-fungicide) on the microbial diversity assessed for grape biofilm and for musts was not remarkable. Neither of the tested fungicides enhanced the growth of any species or acted against the development of any microbial groups. The bio-fungicide had no significant impact on the wine microbiota whereas the chemical fungicide caused a reduction of microbial community richness and diversity. Although environmental threats might generate a detriment of the microbial species richness, in this study the tested bio-fungicide did not modify the structure of the microbial community. Indeed, some of the Bacillus applied at the grape surface, were detected at the end of the AF showing its resilience to the harsh environment of the winemaking; in contrast, its impact on wine quality during aging is yet unknown.

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.

Yeast selection for the wine industry in Spain started in 1950 for the understanding of the microbial ecology, and for the selection of optimal strains to improve the performance of alcoholic fermentation and the overall wine quality. This process has been strongly developed over the last 30 years, firstly on Saccharomyces cerevisiae, and, lately, with intense activity on non-Saccharomyces. Several thousand yeast strains have been isolated, identified and tested to select those with better performance and/or specific technological properties. The present review proposes a global survey of this massive ex-situ preservation of eukaryotic microorganisms, a reservoir of biotechnological solutions for the wine sector, overviewing relevant screenings that led to the selection of strains from 12 genera and 22 species of oenological significance. In the first part, the attention goes to the selection programmes related to relevant wine-producing areas (i.e. Douro, Extremadura, Galicia, La Mancha and Uclés, Ribera del Duero, Rioja, Sherry area, and Valencia). In the second part, the focus shifted on specific non-Saccharomyces genera/species selected from different Spanish and Portuguese regions, exploited to enhance particular attributes of the wines. A fil rouge of the dissertation is the design of tailored biotechnological solutions for wines typical of given geographic areas.