Commercial Wine Yeast Strains Research Articles

Isolation and characterization of Saccharomyces cerevisiae mutants with increased cell wall chitin using fluorescence-activated cell sorting.

Yeast cell wall chitin has been shown to bind grape pathogenesis-related chitinases that are the primary cause of protein haze in wines, suggesting that yeast cell walls may be applied for haze protection. Here, we present a high-throughput screen to identify yeast strains with high cell wall chitin using a reiterative enrichment strategy and fluorescence-activated cell sorting of cells labelled with either GFP-tagged chitinase or Calcofluor white. To assess the validity of the strategy, we first used a pooled deletion strain library of Saccharomyces cerevisiae. The strategy enriched for deletion mutants with genes that had previously been described as having an impact on chitin levels. Genes that had not previously been linked to chitin biosynthesis or deposition were also identified. These genes are involved in cell wall maintenance and/or membrane trafficking functions. The strategy was then applied to a mutagenized population of a commercial wine yeast strain, S. cerevisiae EC1118. Enriched mutant strains showed significantly higher cell wall chitin than the wild type and significantly reduced the activity of chitinases in synthetic model wine, suggesting that these strains may be able to reduce haze formation in wine.

Chemical and Chromatic Effects of Commercial Wine Yeast Strains (Saccharomyces spp.) on ‘Dolgo’ Crabapple Rosé Cider

Chemical and Chromatic Effects of Commercial Wine Yeast Strains (Saccharomyces spp.) on ‘Dolgo’ Crabapple Rosé Cider

Different Wines from Different Yeasts? "Saccharomyces cerevisiae Intraspecies Differentiation by Metabolomic Signature and Sensory Patterns in Wine".

Alcoholic fermentation is known to be a key stage in the winemaking process that directly impacts the composition and quality of the final product. Twelve wines were obtained from fermentations of Chardonnay must made with twelve different commercial wine yeast strains of Saccharomyces cerevisiae. In our study, FT-ICR-MS, GC-MS, and sensory analysis were combined with multivariate analysis. Ultra-high-resolution mass spectrometry (uHRMS) was able to highlight hundreds of metabolites specific to each strain from the same species, although they are characterized by the same technological performances. Furthermore, the significant involvement of nitrogen metabolism in this differentiation was considered. The modulation of primary metabolism was also noted at the volatilome and sensory levels. Sensory analysis allowed us to classify wines into three groups based on descriptors associated with white wine. Thirty-five of the volatile compounds analyzed, including esters, medium-chain fatty acids, superior alcohols, and terpenes discriminate and give details about differences between wines. Therefore, phenotypic differences within the same species revealed metabolic differences that resulted in the diversity of the volatile fraction that participates in the palette of the sensory pattern. This original combination of metabolomics with the volatilome and sensory approaches provides an integrative vision of the characteristics of a given strain. Metabolomics shine the new light on intraspecific discrimination in the Saccharomyces cerevisiae species.

Metabolic flux sampling predicts strain-dependent differences related to aroma production among commercial wine yeasts

BackgroundMetabolomics coupled with genome-scale metabolic modeling approaches have been employed recently to quantitatively analyze the physiological states of various organisms, including Saccharomyces cerevisiae. Although yeast physiology in laboratory strains is well-studied, the metabolic states under industrially relevant scenarios such as winemaking are still not sufficiently understood, especially as there is considerable variation in metabolism between commercial strains. To study the potential causes of strain-dependent variation in the production of volatile compounds during enological conditions, random flux sampling and statistical methods were used, along with experimental extracellular metabolite flux data to characterize the differences in predicted intracellular metabolic states between strains.ResultsIt was observed that four selected commercial wine yeast strains (Elixir, Opale, R2, and Uvaferm) produced variable amounts of key volatile organic compounds (VOCs). Principal component analysis was performed on extracellular metabolite data from the strains at three time points of cell cultivation (24, 58, and 144 h). Separation of the strains was observed at all three time points. Furthermore, Uvaferm at 24 h, for instance, was most associated with propanol and ethyl hexanoate. R2 was found to be associated with ethyl acetate and Opale could be associated with isobutanol while Elixir was most associated with phenylethanol and phenylethyl acetate. Constraint-based modeling (CBM) was employed using the latest genome-scale metabolic model of yeast (Yeast8) and random flux sampling was performed with experimentally derived fluxes at various stages of growth as constraints for the model. The flux sampling simulations allowed us to characterize intracellular metabolic flux states and illustrate the key parts of metabolism that likely determine the observed strain differences. Flux sampling determined that Uvaferm and Elixir are similar while R2 and Opale exhibited the highest degree of differences in the Ehrlich pathway and carbon metabolism, thereby causing strain-specific variation in VOC production. The model predictions also established the top 20 fluxes that relate to phenotypic strain variation (e.g. at 24 h). These fluxes indicated that Opale had a higher median flux for pyruvate decarboxylase reactions compared with the other strains. Conversely, R2 which was lower in all VOCs, had higher median fluxes going toward central metabolism. For Elixir and Uvaferm, the differences in metabolism were most evident in fluxes pertaining to transaminase and hexokinase associated reactions. The applied analysis of metabolic divergence unveiled strain-specific differences in yeast metabolism linked to fusel alcohol and ester production.ConclusionsOverall, this approach proved useful in elucidating key reactions in amino acid, carbon, and glycerophospholipid metabolism which suggest genetic divergence in activity in metabolic subsystems among these wine strains related to the observed differences in VOC formation. The findings in this study could steer more focused research endeavors in developing or selecting optimal aroma-producing yeast stains for winemaking and other types of alcoholic fermentations.

Stress Resistance and Adhesive Properties of Commercial Flor and Wine Strains, and Environmental Isolates of Saccharomyces cerevisiae

Flor strains of Saccharomyces cerevisiae represent a special group of yeasts used for producing biologically aged wines. We analyzed the collection of commercial wine and flor yeast strains, as well as environmental strains isolated from the surface of grapes growing in vineyards, for resistance to abiotic stresses, adhesive properties, and the ability to form a floating flor. The degree of resistance of commercial strains to ethanol, acetaldehyde, and hydrogen peroxide was generally not higher than that of environmental isolates, some of which had high resistance to the tested stress agents. The relatively low degree of stress resistance of flor strains can be explained both by the peculiarities of their adaptive mechanisms and by differences in the nature of their exposure to various types of stress in the course of biological wine aging and under the experimental conditions we used. The hydrophobicity and adhesive properties of cells were determined by the efficiency of adsorption to polystyrene and the distribution of cells between the aqueous and organic phases. Flor strains were distinguished by a higher degree of hydrophobicity of the cell surface and an increased ability to adhere to polystyrene. A clear correlation between biofilm formation and adhesive properties was also observed for environmental yeast isolates. The overall results of this study indicate that relatively simple tests for cell hydrophobicity can be used for the rapid screening of new candidate flor strains in yeast culture collections and among environmental isolates.

Nitrogenous Compound Utilization and Production of Volatile Organic Compounds among Commercial Wine Yeasts Highlight Strain-Specific Metabolic Diversity.

ABSTRACTGenetic background and environmental conditions affect the production of sensory impact compounds by Saccharomyces cerevisiae. The relative importance of the strain-specific metabolic capabilities for the production of volatile organic compounds (VOCs) remains unclear. We investigated which amino acids contribute to VOC production and whether amino acid-VOC relations are conserved among yeast strains. Amino acid consumption and production of VOCs during grape juice fermentation was investigated using four commercial wine yeast strains: Elixir, Opale, R2, and Uvaferm. Principal component analysis of the VOC data demonstrated that Uvaferm correlated with ethyl acetate and ethyl hexanoate production, R2 negatively correlated with the acetate esters, and Opale positively correlated with fusel alcohols. Biomass formation was similar for all strains, pointing to metabolic differences in the utilization of nutrients to form VOCs. Partial least-squares linear regression showed that total aroma production is a function of nitrogen utilization (R2 = 0.87). We found that glycine, tyrosine, leucine, and lysine utilization were positively correlated with fusel alcohols and acetate esters. Mechanistic modeling of the yeast metabolic network via parsimonious flux balance analysis and flux enrichment analysis revealed enzymes with crucial roles, such as transaminases and decarboxylases. Our work provides insights in VOC production in wine yeasts.IMPORTANCE Saccharomyces cerevisiae is widely used in grape juice fermentation to produce wines. Along with the genetic background, the nitrogen in the environment in which S. cerevisiae grows impacts its regulation of metabolism. Also, commercial S. cerevisiae strains exhibit immense diversity in their formation of aromas, and a desirable aroma bouquet is an essential characteristic for wines. Since nitrogen affects aroma formation in wines, it is essential to know the extent of this connection and how it leads to strain-dependent aroma profiles in wines. We evaluated the differences in the production of key aroma compounds among four commercial wine strains. Moreover, we analyzed the role of nitrogen utilization on the formation of various aroma compounds. This work illustrates the unique aroma-producing differences among industrial yeast strains and suggests more intricate, nitrogen-associated routes influencing those aroma-producing differences.

Fermentation of Sauvignon blanc grape marc extract yields important wine aroma 3-sulfanylhexan-1-ol (3SH)

Grape marc, comprised of grape stems, seeds and skins, is an underutilised and potentially problematic waste material produced by the wine industry in often overwhelming quantities. This work aims to showcase this abundant waste material, for the first time, as a potentially valuable technological tool for improving wine quality through the production of the aroma compound 3-sulfanylhexan-1-ol (3SH) during fermentation.A scaled-up eco-friendly solid-liquid extraction method, using 100% water, to extract thiol precursor compounds 3-S-cysteinylhexan-1-ol (Cys-3SH) and 3-S-glutathionylhexan-1-ol (GSH-3SH) from Sauvignon blanc grape marc was undertaken. Quantitation of the vacuum-dried crude extract dissolved in model wine, using liquid chromatography tandem mass spectrometry (LC-MS/MS), showed that the extract contained 1.9 μg Cys-3SH and 11.46 μg GSH-3SH per gram of grape marc extract. Addition of low (14.6 g L−1) and high (58.3 g L−1) quantities of grape marc extract to synthetic grape medium inoculated with a commercial wine yeast strain, yielded appreciable concentrations of 3SH in finished wines (average of 216 ng L−1 for the low addition and 1244 ng L−1 for the high addition). Interestingly, the grape marc extract was found to accelerate the fermentation rate of the yeast, rather than cause inhibition or toxicity.

Saccharomyces cerevisiae nutrient signaling pathways show an unexpected early activation pattern during winemaking

BackgroundSaccharomyces cerevisiae wine strains can develop stuck or sluggish fermentations when nutrients are scarce or suboptimal. Nutrient sensing and signaling pathways, such as PKA, TORC1 and Snf1, work coordinately to adapt growth and metabolism to the amount and balance of the different nutrients in the medium. This has been exhaustively studied in laboratory strains of S. cerevisiae and laboratory media, but much less under industrial conditions.ResultsInhibitors of such pathways, like rapamycin or 2-deoxyglucose, failed to discriminate between commercial wine yeast strains with different nutritional requirements, but evidenced genetic variability among industrial isolates, and between laboratory and commercial strains. Most signaling pathways involve events of protein phosphorylation that can be followed as markers of their activity. The main pathway to promote growth in the presence of nitrogen, the TORC1 pathway, measured by the phosphorylation of Rps6 and Par32, proved active at the very start of fermentation, mainly on day 1, and ceased soon afterward, even before cellular growth stopped. Transcription factor Gln3, which activates genes subject to nitrogen catabolite repression, was also active for the first hours, even when ammonium and amino acids were still present in media. Snf1 kinase was activated only when glucose was exhausted under laboratory conditions, but was active from early fermentation stages. The same results were generally obtained when nitrogen was limiting, which indicates a unique pathway activation pattern in winemaking. As PKA remained active throughout fermentation, it could be the central pathway that controls others, provided sugars are present.ConclusionsWine fermentation is a distinct environmental situation from growth in laboratory media in molecular terms. The mechanisms involved in glucose and nitrogen repression respond differently under winemaking conditions.

Selection of low nitrogen demand yeast strains and their impact on the physicochemical and volatile composition of mead.

Mead is an ancient alcoholic beverage produced through the fermentation of a diluted solution of honey. Due to the peculiar and varied composition of honey, mead production faces several problems, such as slow or stuck fermentations mainly due to the low nitrogen concentration, lack of uniformity of the final product and the production of unpleasant aromas. In this context, this work aimed to select low nitrogen-demand yeast strains and evaluate their potential for the production of mead. Therefore, among 21 commercial wine yeast strains, 5 were selected based on their fermentative behavior at low assimilable nitrogen concentrations. The selected strains were further evaluated for their contributions in meads produced with limited nitrogen availability, and the results showed significant differences on some physicochemical parameters like biomass production, residual sugars, glycerol concentration, and fermentative rate. Moreover, meads obtained with selected strains differed in the concentration of several volatile compounds. The volatile compounds concentration and the principal component analysis based on odor activity values allowed separating strains into three groups. In general, S. cerevisiae var bayanus strains (QA23, Spark, and AWRI-R2) were the largest producers of aromatic compounds, particularly those with floral and fruity descriptors. The selection of yeast strains with low nitrogen-demand and different volatile compounds production can be explored by mead makers to limit fermentation problems and obtain characteristic products.

Grape pomace fermentation and cell wall degradation by Kluyveromyces marxianus Y885

Kluyveromyces marxianus Y885, an endopolygalacturonase (EPG) producing yeast, was evaluated for its capacity to hydrolyse the cell wall polymers of grape pomace as well as its general fermentation capabilities on this substrate. Small scale batch fermentations on autoclaved pomace inoculated with Y885 delivered 10 g/l ethanol and 7.7 g/l glycerol, the latter 14% more than a commercial wine yeast strain. When comparing the hydrolysis of the pomace cell wall by the Kluyveromyces EPG with that of a commonly used pectolytic commercial enzyme preparation, the polymer analysis confirmed that both the Y885 EPG and the commercial enzyme preparation primarily degraded surface exposed pectin polymers, while the hemicellulose-cellulose backbone was unchanged. High levels of galacturonic acid (GalA) monomers were generated by the commercial enzyme, whereas the Y885 EPG mostly liberated (polymeric) GalA-containing fragments together with low quantities of monomers. The Y885 strain produced strong pectinase activity throughout the fermentation process, whereas pectinolytic activity levels rapidly declined for the commercial enzyme after addition. The data obtained shows the evolution of pectin degradation during fermentation and provide compelling evidence that Y885 can utilise/valorise the grape pomace, and that the secreted enzymes (including an EPG) can effectively unravel the structural components of the grape cell walls.

A new chromosomal rearrangement improves the adaptation of wine yeasts to sulfite.

Sulfite-generating compounds are widely used during winemaking as preservatives because of its antimicrobial and antioxidant properties. Thus, wine yeast strains have developed different genetic strategies to increase its sulfite resistance. The most efficient sulfite detoxification mechanism in Saccharomyces cerevisiae uses a plasma membrane protein called Ssu1 to efflux sulfite. In wine yeast strains, two chromosomal translocations (VIIItXVI and XVtXVI) involving the SSU1 promoter region have been shown to upregulate SSU1 expression and, as a result, increase sulfite tolerance. In this study, we have identified a novel chromosomal rearrangement that triggers wine yeast sulfite adaptation. An inversion in chromosome XVI (inv-XVI) probably due to sequence microhomology, which involves SSU1 and GCR1 regulatory regions, increases the expression of SSU1 and the sulfite resistance of a commercial wine yeast strain. A detailed dissection of this chimeric SSU1 promoter indicates that both the removed SSU1 promoter sequence and the relocated GCR1 sequence contribute to SSU1 upregulation and sulfite tolerance. However, no relevant function has been attributed to the SSU1-promoter-binding transcription factor Fzf1. These results unveil a new genomic event that confers an evolutive advantage to wine yeast strains.

The Impact of Changes in Environmental Conditions on Organic Acid Production by Commercial Wine Yeast Strains

Acidity is one of the primary sensory elements in wine, and the balance of sugar and acidity is probably the strongest element affecting wine appreciation. However, little is known about how yeast strains and fermentation conditions will affect the production of fermentation-derived acids, including acetic, succinic and pyruvic acid. This study employs a multifactorial experimental design to provide a better understanding of how individual or simultaneous changes in environmental parameters such as pH, sugar and temperature influence the production of individual organic acids during fermentation in several yeast strains in synthetic must. Certain changes in environmental factors led to conserved trends between strains and treatments. Strains produced higher succinic acid levels when temperature was increased. Significant strain-dependent differences were observed when sugar concentrations were varied for both strains: the combinatorial impact of high initial sugars and fermentation temperature was more pronounced when increased pyruvic acid production was observed in yeast strain VIN13. On the other hand, while combinatorial influences are evident, higher sugar fermentation settings were largely characterised by high acetic acid concentrations for both strains. It is clear that simultaneous changes in sugar, pH and temperature affect organic acid trends in a variable manner, depending on the particular combination of environmental parameters and yeast strain. The study provides valuable information regarding the manner in which initial must parameters and environmental conditions throughout fermentation may affect wine acidity. Since many of these parameters can be controlled at least in part during the winemaking process, the data provide important background information for oenological strategies that aim to optimise the acid balance of wines.

The Impact of Single Amino Acids on Growth and Volatile Aroma Production by Saccharomyces cerevisiae Strains.

Nitrogen availability and utilization by Saccharomyces cerevisiae significantly influence fermentation kinetics and the production of volatile compounds important for wine aroma. Amino acids are the most important nitrogen source and have been classified based on how well they support growth. This study evaluated the effect of single amino acids on growth kinetics and major volatile production of two phenotypically different commercial wine yeast strains in synthetic grape must. Four growth parameters, lag phase, maximum growth rate, total biomass formation and time to complete fermentation were evaluated. In contrast with previous findings, in fermentative conditions, phenylalanine and valine supported growth well and asparagine supported it poorly. The four parameters showed good correlations for most amino acid treatments, with some notable exceptions. Single amino acid treatments resulted in the predictable production of aromatic compounds, with a linear correlation between amino acid concentration and the concentration of aromatic compounds that are directly derived from these amino acids. With the increased complexity of nitrogen sources, linear correlations were lost and aroma production became unpredictable. However, even in complex medium minor changes in amino acid concentration continued to directly impact the formation of aromatic compounds, suggesting that the relative concentration of individual amino acids remains a predictor of aromatic outputs, independently of the complexity of metabolic interactions between carbon and nitrogen metabolism and between amino acid degradation and utilization pathways.

Impact of Yeast Breeding for Elevated Glycerol Production on Fermentative Activity and Metabolite Formation in Chardonnay Wine

Glycerol in wine originates mainly as a by-product during fermentation by yeast and is thought to add to the body and smooth mouth-feel. We evaluated the properties of Chardonnay wine produced using various wine yeast strains of Saccharomyces cerevisiae and hybrid strains that were bred to produce elevated glycerol concentrations in laboratory trial experiments. The wine yeast strains (commercial strains or strains from culture collections) produced a mean glycerol and ethanol concentration of 4.38 and 101.2 g/L (12.8% v/v; n=26) respectively, whereas the glycerol and ethanol concentrations in wine made using the hybrid strains was 7.18 g/L and 96.0 g/L (12.2% v/v; n=15). Considerable variability in the glycerol-producing ability of the wine yeast and hybrid strains was apparent. Coupled to the higher glycerol levels formed by the hybrid strains, acetic acid, volatile acidity, acetoin, acetaldehyde and 2,3-butanediol levels were higher than the levels produced by the wine yeast strains. The levels of some of these metabolites were strongly linked to elevated glycerol production. The hybrid strains fermented the Chardonnay grape juice more slowly than the wine yeast strains, but in most instances dryness was achieved. The concentrations of miscellaneous metabolites (alcohols, acids and esters) were in most instances similar in the wine made with the wine yeast strains and hybrid strains, indicating that the breeding of yeast to produce higher glycerol levels has a minor influence on the production of these compounds. In a wine production experiment one hybrid yeast strain producing elevated glycerol levels yielded a Chardonnay wine with a better or equivalent body than wine made with commercial wine yeast strains, although the aroma and general quality were worse. These results suggest that further breeding and selection might yield yeast strains for fermentation that improves the body of wine without impacting on the overall balance of wine.

Flocculation, Pseudohyphal Development and Invasive Growth in Commercial Wine Yeast Strains

Flocculation of Saccharomyces cerevisiae cells at the end of alcoholic fermentation is an important phenomenon in winemaking, especially in the production of bottle-fermented sparkling wine. Most wine yeast strains do not flocculate during the fermentation process and it is unknown whether they contain the necessary genes to flocculate and whether these genes are expressed adequately under wine-making conditions. These genes include the FLO1 flocculin gene which is one of the most important genes to confer the ability of yeast cells to flocculate and the MUC1 gene (subsequently also cloned as FLO11) encoding a mucin-Iike protein which was previously shown not only to play a key role in pseudohyphal development and invasive growth, but also to be involved in flocculation in S. cerevisiae. Together with MUC1, the involvement of FL08 (encoding a transcriptional activator of FLO1) and TUPI in flocculation, pseudohyphal development and invasive growth indicates that these processes might somehow be linked. Therefore, in order to construct wine yeast strains that are able to flocculate, 25 commercial wine yeast strains were investigated for their ability to flocculate, form pseudohyphae and invade solid media. Twenty-one of these strains were able to penetrate into agar media and different degrees of pseudohyphal and invasive growth were observed. The average length of cells and pseudohyphae and the efficiency of invasive growth varied among these strains. Two of the strains are known to flocculate in wine, while three other strains could be induced to aggregate to a limited degree in glycerol-ethanol medium. Southern blot analyses revealed the presence of homologous DNA sequences in all of the 25 strains using DNA fragments of FL08, MSS1O and MSS11 (encoding transcriptional activators of MUC1), FLO1 and MUC1 as probes. Using Northern blot analysis, FLO1 transcripts were detected in only one of the strains that showed constitutive flocculation in all the growth media tested. MUCJ transcripts of varying sizes could be detected in most of the strains. From these results it is clear that MUC1 does not primarily confer the phenotype of flocculation and that FLO1 (flocculation) and MUC1 (pseudohyphal differentiation, invasive growth and flocculation) are not co-regulated. We therefore suggest that MUC1, as opposed to FLO11, be retained as the most appropriate designation of this gene encoding the S. cerevisiae mucin-like protein.

The Value of Long-Chain Fatty Acid Analysis, Randomly Amplified Polymorphic DNA and Electrophoretic Karyotyping for the Characterization of Wine Yeast Strains

Wine yeast strains of Saccharomyces had previously been classified into several different species or varieties. This classification system was based mainly on sugar fermentation and assimilation patterns. Subsequently, most of these species were reclassified as Saccharomyces cerevisiae. The assignment of the majority of wine yeast strains to a single species does, however, not imply that all stains of S. cerevisiae are equally suitable for wine fermentation. These physiological strains of S. cerevisiae differ significantly in their fermentation performance and their ability to contribute to the final bouquet and quality of the various types of wine and distillates. Therefore, to ensure strain authenticity, security and proper strain management, it is of cardinal importance to have reliable taxonomic techniques available to identify and characterize individual strains of commercial cultures. In this study, 18 commercial wine yeast strains were characterized in order to evaluate and compare three taxonomic techniques, namely long-chain fatty acid analysis, randomly amplified polymorphic DNA (RAPD) and electrophoretic karyotyping. As a single identification technique, electrophoretic karyotyping seems to be the most useful method for routine fingerprinting of wine yeast strains. However, we propose that the combined use of these three techniques provides the most reliable means of differentiating amongst commercial wine yeast strains.

The Impact of Yeast Genetics and Recombinant DNA Technology on the Wine Industry - A Review

Despite our limited knowledge of genetic make-up of commercial wine yeasts and fact thatthe advantages of genetic manipulation of wine yeasts have not yet been demonstrated in practice, wine industry has to realise that the name of game is recombinant DNA and that pace of progress is fast. The technology is so powerful that it now enables manipulation of genome in ways hard to imagine only a decade ago. In this review we highlight importance of wine yeast to wine industry and necessity for well-planned breeding programmes. First, we summarise reliable taxonomic methods that are useful as diagnostic techniques in such breeding strategies. Second, we emphasise complexity of genetic features of commercial wine-yeast strains. Third, we review genetic techniques available and point out potential of these techniques (individually and in combination) in strain-development programmes. Finally, we attempt to stimulate interest in genetic engineering of wine yeasts by discussing a few potential targets of strain development. The impact of yeast genetics and recombinant DNA technology on wine industry promises to be impressive.

Construction of low-ethanol\u2013wine yeasts through partial deletion of the Saccharomyces cerevisiae PDC2 gene

We propose an alternative GMO based strategy to obtain Saccharomyces cerevisiae mutant strains with a slight reduction in their ability to produce ethanol, but with a moderate impact on the yeast metabolism. Through homologous recombination, two truncated Pdc2p proteins Pdc2pΔ344 and Pdc2pΔ519 were obtained and transformed into haploid and diploid lab yeast strains. In the pdc2Δ344 mutants the DNA-binding and transactivation site of the protein remain intact, whereas in pdc2Δ519 only the DNA-binding site is conserved. Compared to the control, the diploid BY4743pdc2Δ519 mutant strain reduced up to 7.4% the total ethanol content in lab scale-vinifications. The residual sugar and volatile acidity was not significantly affected by this ethanol reduction. Remarkably, we got a much higher ethanol reduction of 10 and 15% when the pdc2Δ519 mutation was tested in a native and a commercial wine yeast strain against their respective controls. Our results demonstrate that the insertion of the pdc2Δ519 mutation in wine yeast strains can reduce the ethanol concentration up to 1.89% (v/v) without affecting the fermentation performance. In contrast to non-GMO based strategies, our approach permits the insertion of the pdc2Δ519 mutation in any locally selected wine strain, making possible to produce quality wines with regional characteristics and lower alcohol content. Thus, we consider our work a valuable contribution to the problem of high ethanol concentration in wine.

Malic Acid in Wine: Origin, Function and Metabolism during Vinification

The production of quality wines requires a judicious balance between the sugar, acid and flavour components of wine. L-Malic and tartaric acids are the most prominent organic acids in wine and play a crucial role in the winemaking process, including the organoleptic quality and the physical, biochemical and microbial stability of wine. Deacidification of grape must and wine is often required for the production of well-balanced wines. Malolactic fermentation induced by the addition of malolactic starter cultures, regarded as the preferred method for naturally reducing wine acidity, efficiently decreases the acidic taste of wine, improves the microbial stability and modifies to some extent the organoleptic character of wine. However, the recurrent phenomenon of delayed or sluggish malolactic fermentation often causes interruption of cellar operations, while the malolactic fermentation is not always compatible with certain styles of wine. Commercial wine yeast strains of Saccharomyces are generally unable to degrade L-malic acid effectively in grape must during alcoholic fermentation, with relatively minor modifications in total acidity during vinification. Functional expression of the malolactic pathway genes, i.e. the malate transporter (mae1) of Schizosaccharomyces pombe and the malolactic enzyme (mleA) from Oenococcus oeni in wine yeasts, has paved the way for the construction of malate-degrading strains of Saccharomyces for commercial winemaking.

Seasonal Variation of Indigenous Saccharomyces cerevisiae Strains Isolated from Vineyards of the Western Cape in South Africa

There is strong support for the use of naturally-occurring Saccharomyces cerevisiae strains that improve the sensory quality of wines and reflect the characteristics of a given region. Contrary to popular belief, S. cerevisiae is found at very low numbers on healthy, undamaged grapes and is rarely isolated from intact berries. The majority of studies on the population kinetics and geographic distribution of indigenous S. cerevisiae strains have not adequately focused on the variation in their numbers over a longer period of time. This paper discusses the results obtained in the first phase of a comprehensive research programme aimed at assessing how the natural population dynamics of S. cerevisiae are affected over the long term by abiotic factors. Indigenous strains of S. cerevisiae were aseptically isolated from eight sites in four areas in the coastal regions of the Western Cape, South Africa, during 1995 through 1998. Thirty colonies per site were isolated and the S. cerevisiae strains were characterised by electrophoretic karyotyping. Strain numbers per site varied over the four-year study period. Weather conditions resulting in severe fungal infestations and heavy applications of chemical sprays dramatically reduced the numbers of S. cerevisiae strains recovered during 1997. A return to normal weather patterns in 1998 resulted in a gradual recovery of the indigenous population. Indications are that some of the strains isolated are widespread in the study area and may represent yeasts typical of the area. Commercial wine yeast strains were recovered in only a few instances and the likelihood that commercial yeasts will eventually replace the natural yeast microflora in vineyards seems remote.