Genetic improvement of non-conventional Torulaspora delbrueckii for traditional sparkling winemaking by mixing for eventual hybridization with Saccharomyces cerevisiae.

Non-conventional yeasts such as Torulaspora delbrueckii (Td) have been proposed for sparkling winemaking. Unfortunately, this yeast has poor efficiency in completing wine fermentation as compared to Saccharomyces cerevisiae (Sc). New mutants with increased resistance to SO2, ethanol, and high CO2 pressure were previously isolated from spore clones of Td. Although these mutants showed improved capability for base wine fermentation, there is still room for genetic improvement of Td yeasts until the fermentative capacity of Sc is achieved. As an alternative approach, yeast mixture for eventual hybridization of Td with Sc was assayed in this study. The new yeast mixture clones (Sc-mixed Td) showed an intermediate phenotype between both parent yeasts for some relevant biotechnological properties, such as resistance to SO2, ethanol, copper, high CO2 pressure, and high temperature, as well as flocculation potential. These properties varied depending on the specific Sc-mixed Td clone. Several mixture clones showed improved capability for base wine fermentation as compared to the Td parent strain, approaching the fermentation capability of the Sc parent strain. The organoleptic quality of sparkling wine was also improved by using some mixture clones and this improved quality coincided with an increased amount of acetate and ethyl esters. The genetic stability of some Sc-mixed Td clones was good enough for commercial yeast production and winery applications.

Genetic Improvement of Torulaspora delbrueckii for Wine Fermentation: Eliminating Recessive Growth-Retarding Alleles and Obtaining New Mutants Resistant to SO2, Ethanol, and High CO2 Pressure.

The use of Torulaspora delbrueckii has been repeatedly proposed to improve a wine's organoleptic quality. This yeast has lower efficiency in completing wine fermentation than Saccharomyces cerevisiae since it has less fermentation capability and greater sensitivity to SO2, ethanol, and CO2 pressure. Therefore, the completion of fermentation is not guaranteed when must or wine is single-inoculated with T. delbrueckii. To solve this problem, new strains of T. delbrueckii with enhanced resistance to winemaking conditions were obtained. A genetic study of four wine T. delbrueckii strains was carried out. Spore clones free of possible recessive growth-retarding alleles were obtained from these yeasts. These spore clones were used to successively isolate mutants resistant to SO2, then those resistant to ethanol, and finally those resistant to high CO2 pressure. Most of these mutants showed better capability for base wine fermentation than the parental strain, and some of them approached the fermentation capability of S. cerevisiae. The genetic stability of the new mutants was good enough to be used in industrial-level production in commercial wineries. Moreover, their ability to ferment sparkling wine could be further improved by the continuous addition of oxygen in the culture adaptation stage prior to base wine inoculation.

Using Torulaspora delbrueckii killer yeasts in the elaboration of base wine and traditional sparkling wine.

For still wines, killer strains of Torulaspora delbrueckii can be used instead of non-killer strains to improve this species' domination during must fermentation, with an ensured, reliable impact on the final wine quality. The present work analysed the usefulness of these killer yeasts for sparkling-wine making. After the first fermentation, the foaming capacity of T. delbrueckii base wines was very low compared to Saccharomyces cerevisiae base wines. Significant positive correlations of foaming parameters were found with the amounts of C4-C16 ethyl esters and proteins, and negative with some anti-foaming alcohols produced by each yeast species. There were, however, no evident positive effects of polysaccharides on those parameters. The organoleptic quality of the T. delbrueckii base wines was judged inappropriate for sparkling-wine making, so that the following second-fermentation experiments only used a single assemblage of S. cerevisiae base-wines. While second fermentation was completed with inoculation of S. cerevisiae (both alone and mixed with T. delbrueckii) to yield dry sparkling wines with high CO2 pressure, single inoculation with T. delbrueckii did not complete this fermentation, leaving sweet wines with poor CO2 pressure. Yeast death due to CO2 pressure was much greater in T. delbrueckii than in S. cerevisiae, making any killer effect of S. cerevisiae over T. delbrueckii irrelevant because no autolysed cells were found during the first days of mixed-inoculated second fermentation. Nonetheless, the organoleptic quality of the mixed-inoculated sparkling wines was better than that of wines single-inoculated with S. cerevisiae, and showed no deterioration in foam quality. This seemed mainly to be because T. delbrueckii increased the amounts of ethyl propanoate and some acids (e.g., isobutyric and butanoic), alcohols (e.g., 3­ethoxy­1­propanol), and phenols (e.g., 4­vinylguaiacol). For these sparkling wines, no significant correlations between foaming parameters and aroma compounds were found, probably because the differences in foaming parameter values among these wines were fairly small. This is unlike the case for the base wines for which there were large differences in these parameters, which facilitated the analysis of the influence of aroma compounds on base-wine foamability.

Influence of the dominance of must fermentation by Torulaspora delbrueckii on the malolactic fermentation and organoleptic quality of red table wine.

Torulaspora delbrueckii can improve wine aroma complexity, but its impact on wine quality is still far from being satisfactory at the winery level, mainly because it is easily replaced by S. cerevisiae yeasts during must fermentation. New T. delbrueckii killer strains were selected to overcome this problem. These strains killed S. cerevisiae yeasts and dominated fermentation better than T. delbrueckii non-killer strains when they were single-inoculated into crushed red grape must. All the T. delbrueckii wines, but none of the S. cerevisiae wines, underwent malolactic fermentation. Putative lactic acid bacteria were always found in the T. delbrueckii wines, but none or very few in the S. cerevisiae wines. Malic acid degradation was the greatest in the wines inoculated with the killer strains, and these strains reached the greatest dominance ratios and had the slowest fermentation kinetics. The T. delbrueckii wines had dried-fruit/pastry aromas, but low intensities of fresh-fruit aromas. The aroma differences between the T. delbrueckii and the S. cerevisiae wines can be explained by the differences that were found in the amounts of some fruity aroma compounds such as isoamyl acetate, ethyl hexanoate, ethyl octanoate, and some lactones. This T. delbrueckii effect significantly raised the organoleptic quality scores of full-bodied Cabernet-Sauvignon red wines inoculated with the killer strains. In particular, these wines were judged as having excellent aroma complexity, mouth-feel, and sweetness.

Using mixed inocula of Saccharomyces cerevisiae killer strains to improve the quality of traditional sparkling-wine.

The quality of traditional sparkling-wine depends on the aging process in the presence of dead yeast cells. These cells undergo a slow autolysis process thereby releasing some compounds, mostly colloidal polymers such as polysaccharides and mannoproteins, which influence the wine's foam properties and mouthfeel. Saccharomyces cerevisiae killer yeasts were tested to increase cell death and autolysis during mixed-yeast-inoculated second fermentation and aging. These yeasts killed sensitive strains in killer plate assays done under conditions of low pH and temperature similar to those used in sparkling-wine making, although some strains showed a different killer behaviour during the second fermentation. The fast killer effect improved the foam quality and mouthfeel of the mixed-inoculated wines, while the slow killer effect gave small improvements over single-inoculated wines. The effect was faster under high-pressure than under low-pressure conditions. Wine quality improvement did not correlate with the polysaccharide, protein, mannan, or aromatic compound concentrations, suggesting that the mouthfeel and foaming quality of sparkling wine are very complex properties influenced by other wine compounds and their interactions, as well as probably by the specific chemical composition of a given wine.

Effects of new Torulaspora delbrueckii killer yeasts on the must fermentation kinetics and aroma compounds of white table wine.

Torulaspora delbrueckii is becoming widely recommended for improving some specific characteristics of wines. However, its impact on wine quality is still far from satisfactory at the winery level, mostly because it is easily replaced by Saccharomyces cerevisiae-like yeasts during must fermentation. New T. delbrueckii killer strains were here isolated and selected for winemaking. They killed S. cerevisiae yeasts and were able to dominate and complete the fermentation of sterile grape must. Sequential yeast inoculation of non-sterile white must with T. delbrueckii followed by S. cerevisiae did not ensure T. delbrueckii dominance or wine quality improvement. Only a single initial must inoculation at high cell concentrations allowed the T. delbrueckii killer strains to dominate and complete the must fermentation to reach above 11% ethanol, but not the non-killer strains. None of the wines underwent malolactic fermentation as long as the must had low turbidity and pH. Although no statistically significant differences were found in the wine quality score, the S. cerevisiae-dominated wines were preferred over the T. delbrueckii-dominated ones because the former had high-intensity fresh fruit aromas while the latter had lower intensity, but nevertheless nice and unusual dried fruit/pastry aromas. Except for ethyl propanoate and 3-ethoxy-1-propanol, which were more abundant in the T. delbrueckii-dominated wines, most of the compounds with fresh fruit odor descriptors, including those with the greatest odor activity values (isoamyl acetate, ethyl hexanoate, and ethyl octanoate), were more abundant in the S. cerevisiae-dominated wines. The low relative concentrations of these fruity compounds made it possible to detect in the T. delbrueckii-dominated wines the low-relative-concentration compounds with dried fruit and pastry odors. An example was γ-ethoxy-butyrolactone which was significantly more abundant in these wines than in those dominated by S. cerevisiae.

Characterization, ecological distribution, and population dynamics of Saccharomyces sensu stricto killer yeasts in the spontaneous grape must fermentations of southwestern Spain.

Killer yeasts secrete protein toxins that are lethal to sensitive strains of the same or related yeast species. Among the four types of Saccharomyces killer yeasts already described (K1, K2, K28, and Klus), we found K2 and Klus killer yeasts in spontaneous wine fermentations from southwestern Spain. Both phenotypes were encoded by medium-size double-stranded RNA (dsRNA) viruses, Saccharomyces cerevisiae virus (ScV)-M2 and ScV-Mlus, whose genome sizes ranged from 1.3 to 1.75 kb and from 2.1 to 2.3 kb, respectively. The K2 yeasts were found in all the wine-producing subareas for all the vintages analyzed, while the Klus yeasts were found in the warmer subareas and mostly in the warmer ripening/harvest seasons. The middle-size isotypes of the M2 dsRNA were the most frequent among K2 yeasts, probably because they encoded the most intense K2 killer phenotype. However, the smallest isotype of the Mlus dsRNA was the most frequent for Klus yeasts, although it encoded the least intense Klus killer phenotype. The killer yeasts were present in most (59.5%) spontaneous fermentations. Most were K2, with Klus being the minority. The proportion of killer yeasts increased during fermentation, while the proportion of sensitive yeasts decreased. The fermentation speed, malic acid, and wine organoleptic quality decreased in those fermentations where the killer yeasts replaced at least 15% of a dominant population of sensitive yeasts, while volatile acidity and lactic acid increased, and the amount of bacteria in the tumultuous and the end fermentation stages also increased in an unusual way.

A new wine Saccharomyces cerevisiae killer toxin (Klus), encoded by a double-stranded rna virus, with broad antifungal activity is evolutionarily related to a chromosomal host gene.

Wine Saccharomyces cerevisiae strains producing a new killer toxin (Klus) were isolated. They killed all the previously known S. cerevisiae killer strains, in addition to other yeast species, including Kluyveromyces lactis and Candida albicans. The Klus phenotype is conferred by a medium-size double-stranded RNA (dsRNA) virus, Saccharomyces cerevisiae virus Mlus (ScV-Mlus), whose genome size ranged from 2.1 to 2.3 kb. ScV-Mlus depends on ScV-L-A for stable maintenance and replication. We cloned and sequenced Mlus. Its genome structure is similar to that of M1, M2, or M28 dsRNA, with a 5'-terminal coding region followed by two internal A-rich sequences and a 3'-terminal region without coding capacity. Mlus positive strands carry cis-acting signals at their 5' and 3' termini for transcription and replication similar to those of killer viruses. The open reading frame (ORF) at the 5' portion codes for a putative preprotoxin with an N-terminal secretion signal, potential Kex2p/Kexlp processing sites, and N-glycosylation sites. No sequence homology was found either between the Mlus dsRNA and M1, M2, or M28 dsRNA or between Klus and the K1, K2, or K28 toxin. The Klus amino acid sequence, however, showed a significant degree of conservation with that of the product of the host chromosomally encoded ORF YFR020W of unknown function, thus suggesting an evolutionary relationship.

A low-cost procedure for production of fresh autochthonous wine yeast.

A low-cost procedure was designed for easy and rapid response-on-demand production of fresh wine yeast for local wine-making. The pilot plant produced fresh yeast culture concentrate with good microbial quality and excellent oenological properties from four selected wine yeasts. The best production yields were obtained using 2% sugar beet molasses and a working culture volume of less than 60% of the fermenter capacity. The yeast yield using 2% sugar grape juice was low and had poor cell viability after freeze storage, although the resulting yeast would be directly available for use in the winery. The performance of these yeasts in commercial wineries was excellent; they dominated must fermentation and improved its kinetics, as well as improving the physicochemical parameters and the organoleptic quality of red and white wines.

Wine yeast molecular typing using a simplified method for simultaneously extracting mtDNA, nuclear DNA and virus dsRNA.

Quick and accurate methods are required for the identification of industrial, environmental, and clinical yeast strains. We propose a rapid method for the simultaneous extraction of yeast mtDNA, nuclear DNA, and virus dsRNA. It is simpler, cheaper, and faster than the previously reported methods. It allows one to choose among a broad range of molecular analysis approaches for yeast typing, avoiding the need to use of several different methods for the separate extraction of each nucleic acid type. The application of this method followed by the combined analysis of mtDNA and dsRNA (ScV-M and W) is a highly attractive option for fast and efficient wine yeast typing.

Sulfometuron resistance as a genetic marker for yeast populations in wine fermentations.

Winemaking with selected yeasts requires simple and cheap techniques to monitor the yeast population dynamics. We obtained new sulfometuron (smr) resistant mutants, easy to detect by replica-plate assay, from selected wine yeasts. The mutations were dominant and were located at the ilv2 locus that encodes for acetolactate synthase enzyme. The mutants were genetically stable and maintained the killer phenotype of the parent yeast strain. They were genetically improved by elimination of recessive growth-retarding alleles followed by spore clone selection according to the must fermentation kinetics and the organoleptic quality of the wine. Some mutants were tested in industrial winemaking and were easily monitored during must fermentation using a simple plate assay. They accounted for more than 95% of the total yeasts in the must, and the resulting wine had as good a quality as those made with standard commercial wine yeasts.