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CRUE-CSIC (Acuerdos transformativos 2022), Saccharomyces cerevisiae is a highly fermentative species able to complete the wine fermentation. However, the interaction with other non-Saccharomyces yeasts can determine the fermentation performance of S. cerevisiae. We have characterised three rare non-Saccharomyces yeasts (Cyberlindnera fabianii, Kazachstania unispora and Naganishia globosa), studying their impact on S. cerevisiae fitness and wine fermentation performance. Using a wide meta-taxonomic dataset of wine samples, analysed through ITS amplicon sequencing, we show that about a 65.07% of wine samples contains Naganishia spp., a 27.21% contains Kazachstania spp., and only a 4.41% contains Cyberlindnera spp; in all cases with average relative abundances lower than 1% of total fungal populations. Although the studied N. globosa strain showed a limited growth capacity in wine, both K. unispora and C. fabianii showed a similar growth phenotype to that of S. cerevisiae in different fermentation conditions, highlighting the outstanding growth rate values of K. unispora. In mixed fermentations with S. cerevisiae, the three yeast species affected co-culture growth parameters and wine chemical profile (volatile compounds, polysaccharides and proteins). K. unispora DN201 strain presents an outstanding capacity to compete with S. cerevisiae strains during the first stage of wine fermentation, causing stuck fermentations in both synthetic and natural grape musts., Ministerio de Ciencia e Innovación (MICINN) (10.13039/ 501100011033)., Universidad Complutense de Madrid, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

The growing demand for wines with a more pronounced aromatic profile calls for low temperature alcoholic fermentations (10–15 °C). However, there are certain drawbacks to low temperature fermentations such as reduced growth rate, long lag phase and sluggish or stuck fermentations. The lipid metabolism of Saccharomyces cerevisiae plays a central role in low temperature adaptation. The aim of this study was to detect lipid metabolism genes involved in cold adaptation. To do so, we analyzed the growth of knockouts in phospholipids, sterols and sphingolipids, from the EUROSCARF collection S. cerevisiae BY4742 strain at low and optimal temperatures. Growth rate of these knockouts, compared with the control, enabled us to identify the genes involved, which were also deleted or overexpressed in a derivative haploid of a commercial wine strain. We identified genes involved in the phospholipid (PSD1 and OPI3), sterol (ERG3 and IDI1) and sphingolipid (LCB3) pathways, whose deletion strongly impaired growth at low temperature and whose overexpression reduced generation or division time by almost half. Our study also reveals many phenotypic differences between the laboratory strain and the commercial wine yeast strain, showing the importance of constructing mutant and overexpressing strains in both genetic backgrounds. The phenotypic differences in the mutant and overexpressing strains were correlated with changes in their lipid composition.

Wine can contain trace amounts of ethyl carbamate (EC), a carcinogen formed when ethanol reacts with carbamyl compounds such as citrulline. EC is produced from arginine by lactic acid bacteria (LAB), e.g., Lactobacillus and Pediococcus. Although the amounts of EC in wine are usually negligible, over the last few years there has been a slight butsteady increase, as climate change has increased temperatures and alcohol levels have become proportionately higher, both of which favor EC formation. In this study, resting cells of LAB were used to evaluate the effects of ethanol, glucose, malic acid, and low pH on the ability of non-oenococcal strains of these bacteria to degrade arginine and excrete citrulline. Malic acid was found to clearly inhibit arginine consumption in all strains. The relation between citrulline produced and arginine consumed was clearly higher in the presence of ethanol (10-12 %) and at low pH (3.0), which is consistent with both the decreased amount of ornithine produced from arginine and the reduction in arginine degradation. In L. brevis and L. buchneri strains isolated from wine and beer, respectively, the synthesis of citrulline from arginine was highest.

Wine can contain trace amounts of ethyl carbamate (EC), a carcinogen formed when ethanol reacts with carbamyl compounds such as citrulline. EC is produced from arginine by lactic acid bacteria (LAB), e.g., Lactobacillus and Pediococcus. Although the amounts of EC in wine are usually negligible, over the last few years there has been a slight butsteady increase, as climate change has increased temperatures and alcohol levels have become proportionately higher, both of which favor EC formation. In this study, resting cells of LAB were used to evaluate the effects of ethanol, glucose, malic acid, and low pH on the ability of non-oenococcal strains of these bacteria to degrade arginine and excrete citrulline. Malic acid was found to clearly inhibit arginine consumption in all strains. The relation between citrulline produced and arginine consumed was clearly higher in the presence of ethanol (10-12 %) and at low pH (3.0), which is consistent with both the decreased amount of ornithine produced from arginine and the reduction in arginine degradation. In L. brevis and L. buchneri strains isolated from wine and beer, respectively, the synthesis of citrulline from arginine was highest.

Mixed inoculation of non-Saccharomyces yeasts and S. cerevisiae is of interest for the wine industry for technological and sensory reasons. We have analysed how mixed inocula of the main non-Saccharomyces yeasts and S. cerevisiae affect fermentation performance, nitrogen consumption and volatile compound production in a natural Macabeo grape must. Sterile must was fermented in triplicates and under the following six conditions: three pure cultures of S. cerevisiae, Hanseniaspora uvarum and Candida zemplinina and the mixtures of H. uvarum:S. cerevisiae (90:10), C. zemplinina:S. cerevisiae (90:10) and H. uvarum:C. zemplinina:S. cerevisiae (45:45:10). The presence of non-Saccharomyces yeasts slowed down the fermentations and produced higher levels of glycerol and acetic acid. Only the pure H. uvarum fermentations were unable to finish. Mixed fermentations consumed more of the available amino acids and were more complex and thus better able to synthesise volatile compounds. However, the amount of acetic acid was well above the admissible levels and compromises the immediate application of mixed cultures.