Teresa García-Martínez

Discrimination of sweet wines partially fermented by two osmo-ethanol-tolerant yeasts by gas chromatographic analysis and electronic nose.

Some special sweet wines are obtained by partial fermentation of musts from off-vine dried grapes containing large amounts of sugars. This process is very slow and subject to serious stop problems that can be avoided by using osmo-ethanol-tolerant yeasts. Musts containing 371g/l of sugars were partially fermented with selected Saccharomyces cerevisiae strains, X4 and X5, to 12% (v/v) and the wines obtained with X5 exhibited a higher volatile acidity but lower concentrations of higher alcohols, carbonyl compounds and polyols than those obtained with X4. A principal component analysis (PCA) of the data provided by an electronic nose (E-nose) afforded discrimination between fermented and unfermented musts, but not between wines obtained with X4 or X5. The PCA applied to the major volatile compounds and polyols shows similar results, but a clear discrimination between wines is obtained by removing the polyols glycerol and 2,3-butanediol from the PCA.

Volatile composition of partially fermented wines elaborated from sun dried Pedro Ximénez grapes.

In this work, we used a cell immobilisation system consisting of Penicillium chrysogenum fungi (GRAS) bound to the osmotolerant yeast strains Saccharomyces cerevisiae X4 and X5 for the partial fermentation of raisin musts. The resulting wines were compared with others obtained by partial fermentation of musts with free yeasts and with a traditionally produced sweet wine (i.e. without fermentation of the must). The analysis of volatile compounds grouped by aroma series showed the partially fermented musts had a more complex aroma than the traditional wine. Specially prominent among aroma series was that of ripe fruit, followed by the milky and chemical series. The volatiles with the greatest impact on wine aroma as assessed in terms of odour activity were ethyl hexanoate, ethyl octanoate, butyrolactone, isoamyl alcohols, acetaldehyde, ethyl acetate, 2,3-butanediol, acetoin and 2,3-butanedione. A cluster analysis according to the Ward method was performed to assess the similarity between the traditional sweet wine and those obtained by partial fermentation with free and immobilised yeasts revealed small differences between the wines obtained with free and immobilised yeasts, and marked differences between partially fermented and traditionally obtained sweet wine. The wines provided by immobilised yeasts were the most appreciated in the sensory analysis (especially those obtained with X4 yeasts).

Flor Yeast: New Perspectives Beyond Wine Aging

The most important dogma in white-wine production is the preservation of the wine aroma and the limitation of the oxidative action of oxygen. In contrast, the aging of Sherry and Sherry-like wines is an aerobic process that depends on the oxidative activity of flor strains of Saccharomyces cerevisiae. Under depletion of nitrogen and fermentable carbon sources, these yeast produce aggregates of floating cells and form an air–liquid biofilm on the wine surface, which is also known as velum or flor. This behavior is due to genetic and metabolic peculiarities that differentiate flor yeast from other wine yeast. This review will focus first on the most updated data obtained through the analysis of flor yeast with -omic tools. Comparative genomics, proteomics, and metabolomics of flor and wine yeast strains are shedding new light on several features of these special yeast, and in particular, they have revealed the extent of proteome remodeling imposed by the biofilm life-style. Finally, new insights in terms of promotion and inhibition of biofilm formation through small molecules, amino acids, and di/tri-peptides, and novel possibilities for the exploitation of biofilm immobilization within a fungal hyphae framework, will be discussed.

Application of a New Organic Yeast Immobilization Method for Sparkling Wine Production

The efficiency and effectiveness of a new immobilizing yeast method to produce sparkling wines was examined. The cell entrapment organic system, labeled biocapsules, was accomplished by the natural and spontaneous co-immobilization of a Saccharomyces cerevisiae strain and a filamentous fungus (Penicillium chrysogenum). The behavior of this immobilization method was compared with the activity of the same yeast strains in a free cell format and immobilized in calcium alginate beads. Two Saccharomyces cerevisiae strains were used as starters and two different base wines were tested. Metabolic kinetics of the organic biocatalyst during secondary fermentation, enological parameters, foam properties, and sensory profile of the produced sparkling wines after 10 months of aging were analyzed. Immobilization supports and base wine characteristics had an influence on fermentation kinetics. Unfermented sugars were found in some of the immobilized yeast batches. Enological parameters assessed in the final products did not show relevant enological differences, with the exception of calcium ion content, which was slightly higher in sparkling wines made with yeast immobilized in calcium alginate beads. The foaming properties of batches produced with yeast in biocapsules had similar or better values than those fermented with free cells. Discriminant analysis performed with the enological and foam data distinguished among the sparkling wines made with biocapsules and the other two yeast inoculum formats. A triangular test reported no significant differences among inoculating yeast forms in most of the batches. The results suggested that biocapsules might be a low cost, natural, and suitable yeast immobilization method for sparkling wine production.

Effects of ADH2 Overexpression in Saccharomyces bayanus during Alcoholic Fermentation

ABSTRACT The effect of overexpression of the gene ADH2 on metabolic and biological activity in Saccharomyces bayanus V5 during alcoholic fermentation has been evaluated. This gene is known to encode alcohol dehydrogenase II (ADH II). During the biological aging of sherry wines, where yeasts have to grow on ethanol owing to the absence of glucose, this isoenzyme plays a prominent role by converting the ethanol into acetaldehyde and producing NADH in the process. Overexpression of the gene ADH2 during alcoholic fermentation has no effect on the proteomic profile or the net production of some metabolites associated with glycolysis and alcoholic fermentation such as ethanol, acetaldehyde, and glycerol. However, it affects indirectly glucose and ammonium uptakes, cell growth, and intracellular redox potential, which lead to an altered metabolome. The increased contents in acetoin, acetic acid, and l-proline present in the fermentation medium under these conditions can be ascribed to detoxification by removal of excess acetaldehyde and the need to restore and maintain the intracellular redox potential balance.

Sweet wine production by two osmotolerant Saccharomyces cerevisiae strains.

The use of Saccharomyces cerevisiae to produce sweet wine is difficult because yeast is affected by a hyperosmotic stress due to the high sugar concentrations in the fermenting must. One possible alternative could be the coimmobilization of the osmotolerant yeast strains S. cerevisiae X4 and X5 on Penicillium chrysogenum strain H3 (GRAS) for the partial fermentation of raisin musts. This immobilized has been, namely, as yeast biocapsules. Traditional sweet wine (that is, without fermentation of the must) and must partially fermented by free yeast cells were also used for comparison. Partially fermented sweet wines showed higher concentration of the volatile compounds than traditionally produced wines. The wines obtained by immobilized yeast cells reached minor concentrations of major alcohols than wines by free cells. The consumption of specific nitrogen compounds was dependent on yeast strain and the cellular immobilization. A principal component analysis shows that the compounds related to the response to osmotic stress (glycerol, acetaldehyde, acetoin, and butanediol) clearly differentiate the wines obtained with free yeasts but not the wines obtained with immobilized yeasts.

Proteins involved in wine aroma compounds metabolism by a Saccharomyces cerevisiae flor-velum yeast strain grown in two conditions

A proteomic and exometabolomic study was conducted on Saccharomyces cerevisiae flor yeast strain growing under biofilm formation condition (BFC) with ethanol and glycerol as carbon sources and results were compared with those obtained under no biofilm formation condition (NBFC) containing glucose as carbon source. By using modern techniques, OFFGEL fractionator and LTQ-Orbitrap for proteome and SBSE-TD-GC-MS for metabolite analysis, we quantified 84 proteins including 33 directly involved in the metabolism of glycerol, ethanol and 17 aroma compounds. Contents in acetaldehyde, acetic acid, decanoic acid, 1,1-diethoxyethane, benzaldehyde and 2-phenethyl acetate, changed above their odor thresholds under BFC, and those of decanoic acid, ethyl octanoate, ethyl decanoate and isoamyl acetate under NBFC. Of the twenty proteins involved in the metabolism of ethanol, acetaldehyde, acetoin, 2,3-butanediol, 1,1-diethoxyethane, benzaldehyde, organic acids and ethyl esters, only Adh2p, Ald4p, Cys4p, Fas3p, Met2p and Plb1p were detected under BFC and as many Acs2p, Ald3p, Cem1p, Ilv2p, Ilv6p and Pox1p, only under NBFC. Of the eight proteins involved in glycerol metabolism, Gut2p was detected only under BFC while Pgs1p and Rhr2p were under NBFC. Finally, of the five proteins involved in the metabolism of higher alcohols, Thi3p was present under BFC, and Aro8p and Bat2p were under NBFC.

Proteins involved in flor yeast carbon metabolism under biofilm formation conditions

A lack of sugars during the production of biologically aged wines after fermentation of grape must causes flor yeasts to metabolize other carbon molecules formed during fermentation (ethanol and glycerol, mainly). In this work, a proteome analysis involving OFFGEL fractionation prior to LC/MS detection was used to elucidate the carbon metabolism of a flor yeast strain under biofilm formation conditions (BFC). The results were compared with those obtained under non-biofilm formation conditions (NBFC). Proteins associated to processes such as non-fermentable carbon uptake, the glyoxylate and TCA cycles, cellular respiration and inositol metabolism were detected at higher concentrations under BFC than under the reference conditions (NBFC). This study constitutes the first attempt at identifying the flor yeast proteins responsible for the peculiar sensory profile of biologically aged wines. A better metabolic knowledge of flor yeasts might facilitate the development of effective strategies for improved production of these special wines.

Differential Proteome Analysis of a Flor Yeast Strain under Biofilm Formation

Several Saccharomyces cerevisiae strains (flor yeasts) form a biofilm (flor velum) on the surface of Sherry wines after fermentation, when glucose is depleted. This flor velum is fundamental to biological aging of these particular wines. In this study, we identify abundant proteins in the formation of the biofilm of an industrial flor yeast strain. A database search to enrich flor yeast “biological process” and “cellular component” according to Gene Ontology Terminology (GO Terms) and, “pathways” was carried out. The most abundant proteins detected were largely involved in respiration, translation, stress damage prevention and repair, amino acid metabolism (glycine, isoleucine, leucine and arginine), glycolysis/gluconeogenesis and biosynthesis of vitamin B9 (folate). These proteins were located in cellular components as in the peroxisome, mitochondria, vacuole, cell wall and extracellular region; being these two last directly related with the flor formation. Proteins like Bgl2p, Gcv3p, Hyp2p, Mdh1p, Suc2p and Ygp1p were quantified in very high levels. This study reveals some expected processes and provides new and important information for the design of conditions and genetic constructions of flor yeasts for improving the cellular survival and, thus, to optimize biological aging of Sherry wine production.

Use of a flor velum yeast for modulating colour, ethanol and major aroma compound contents in red wine

The most important and negative effect of the global warming for winemakers in warm and sunny regions is the observed lag between industrial and phenolic grape ripeness, so only it is possible to obtain an acceptable colour when the ethanol content of wine is high. By contrast, the actual market trends are to low ethanol content wines. Flor yeast growing a short time under velum conditions, decreases the ethanol and volatile acidity contents, has a favorable effect on the colour and astringency and significantly changes the wine content in 1-propanol, isobutanol, acetaldehyde, 1,1-diethoxiethane and ethyl lactate. The Principal Component Analysis of six enological parameters or five aroma compounds allows to classify the wines subjected to different velum formation conditions. The obtained results in two tasting sessions suggest that the flor yeast helps to modulate the ethanol, astringency and colour and supports a new biotechnological perspective for red winemakers.