Changes in gluconic acid, polyols and major volatile compounds in sherry wine during aging with submerged flor yeast cultures

The traditional biological process by which sherry wines are aged can be accelerated by using submerged Saccharomyces cerevisiae var. capensis (G1) strain cultures previously grown in glycerol. The used controlled shaking conditions raise the acetaldehyde, acetoin, and meso 2,3-butanediol contents in the wine, and increases the consumption of gluconic acid by flor yeast relative to traditional biological aging under flor yeast velum.     

Yeast population dynamics during the fermentation and biological aging of sherry wines

Molecular and physiological analyses were used to study the evolution of the yeast population, from alcoholic fermentation to biological aging in the process of "fino" sherry wine making. The four races of "flor" Saccharomyces cerevisiae (beticus, cheresiensis, montuliensis, and rouxii) exhibited identical restriction patterns for the region spanning the internal transcribed spacers 1 and 2 (ITS-1 and ITS-2) and the 5.8S rRNA gene, but this pattern was different, from those exhibited by non-flor S. cerevisiae strains. This flor-specific pattern was detected only after wines were fortified, never during alcoholic fermentation, and all the strains isolated from the velum exhibited the typical flor yeast pattern. By restriction fragment length polymorphism of mitochondrial DNA and karyotyping, we showed that (i) the native strain is better adapted to fermentation conditions than commercial strains; (ii) two different populations of S. cerevisiae strains are involved in the process of elaboration, of fino sherry wine, one of which is responsible for must fermentation and the other, for wine aging; and (iii) one strain was dominant in the flor population integrating the velum from sherry wines produced in González Byass wineries, although other authors have described a succession of races of flor S. cerevisiae during wine aging. Analyzing all these results together, we conclude that yeast population dynamics during biological aging is a complex phenomenon and differences between yeast populations from different wineries can be observed.     

Impact of fermentation rate changes on potential hydrogen sulfide concentrations in wine

The correlation between alcoholic fermentation rate, measured as carbon dioxide (CO2) evolution, and the rate of hydrogen sulfide (H2S) formation during wine production was investigated. Both rates and the resulting concentration peaks in fermentor headspace H2S were directly impacted by yeast assimilable nitrogenous compounds in the grape juice. A series of model fermentations was conducted in temperature-controlled and stirred fermentors using a complex model juice with defined concentrations of ammonium ions and/or amino acids. The fermentation rate was measured indirectly by noting the weight loss of the fermentor; H2S was quantitatively trapped in realtime using a pre-calibrated H2S detection tube which was inserted into a fermentor gas relief port. Evolution rates for CO2 and H2S as well as the relative ratios between them were calculated. These fermentations confirmed that total sulfide formation was strongly yeast strain-dependent, and high concentrations of yeast assimilable nitrogen did not necessarily protect against elevated H2S formation. High initial concentrations of ammonium ions via addition of diammonium phosphate (DAP) caused a higher evolution of H2S when compared with a non-supplemented but nondeficient juice. It was observed that the excess availability of a certain yeast assimilable amino acid, arginine, could result in a more sustained CO2 production rate throughout the wine fermentation. The contribution of yeast assimilable amino acids from conventional commercial yeast foods to lowering of the H2S formation was marginal.     

Kinetics of citrate uptake in growing cells of Leuconostoc spp.

Abstract Several yeast strains of the species Saccharomyces cerevisiae, S. bayanus and S. paradoxus , first identified by hybridization experiments and measurements of DNA/DNA homology, were characterized using polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis of the MET2 gene. There was no exception to the agreement between this method and classical genetic analyses for any of the strains examined, so PCR/RFLP of the MET2 gene is a reliable and fast technique for delimiting S. cerevisiae and S. bayanus . Enological strains classified as S., bayanus , S. chevalieri , and S. capensis gave S. cerevisiae restriction patterns, whereas most S. uvarum strains belong to S. bayanus . Enologists should no longer use the name of S. bayanus for S. cerevisiae Gal strains, and should consider S. bayanus as a distinct species.     

Assimilable nitrogen utilisation and production of volatile and non-volatile compounds in chemically defined medium by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> wine yeasts

Surveys conducted worldwide have shown that a significant proportion of grape musts are suboptimal for yeast nutrients, especially assimilable nitrogen. Nitrogen deficiencies are linked to slow and stuck fermentations and sulphidic off-flavour formation. Nitrogen supplementation of grape musts has become common practice; however, almost no information is available on the effects of nitrogen supplementation on wine flavour. In this study, the effect of ammonium supplementation of a synthetic medium over a wide range of nitrogen values on the production of volatile and non-volatile compounds by two high-nitrogen-demand wine fermentation strains of Saccharomyces cerevisiae was determined. To facilitate this investigation, a simplified chemically defined medium that resembles the nutrient composition of grape juice was used. Analysis of variance revealed that ammonium supplementation had significant effects on the concentration of residual sugar, L-malic acid, acetic acid and glycerol but not the ethanol concentration. While choice of yeast strain significantly affected half of the aroma compounds measured, nitrogen concentrations affected 23 compounds, including medium-chain alcohols and fatty acids and their esters. Principal component analysis showed that branched-chain fatty acids and their esters were associated with low nitrogen concentrations, whereas medium-chain fatty esters and acetic acid were associated with high nitrogen concentrations.     

De novo synthesis of monoterpenes by Saccharomyces cerevisiae wine yeasts

This paper reports the production of monoterpenes, which elicit a floral aroma in wine, by strains of the yeast Saccharomyces cerevisiae. Terpenes, which are typical components of the essential oils of flowers and fruits, are also present as free and glycosylated conjugates amongst the secondary metabolites of certain wine grape varieties of Vitis vinifera. Hence, when these compounds are present in wine they are considered to originate from grape and not fermentation. However, the biosynthesis of monoterpenes by S. cerevisiae in the absence of grape derived precursors is shown here to be of de novo origin in wine yeast strains. Higher concentration of assimilable nitrogen increased accumulation of linalool and citronellol. Microaerobic compared with anaerobic conditions favored terpene accumulation in the ferment. The amount of linalool produced by some strains of S. cerevisiae could be of sensory importance in wine production. These unexpected results are discussed in relation to the known sterol biosynthetic pathway and to an alternative pathway for terpene biosynthesis not previously described in yeast.     

Nitrogen availability of grape juice limits killer yeast growth and fermentation activity during mixed-culture fermentation with sensitive commercial yeast strains.

The competition between selected or commercial killer strains of type K2 and sensitive commercial strains of Saccharomyces cerevisiae was studied under various conditions in sterile grape juice fermentations. The focus of this study was the effect of yeast inoculation levels and the role of assimilable nitrogen nutrition on killer activity. A study of the consumption of free amino nitrogen (FAN) by pure and mixed cultures of killer and sensitive cells showed no differences between the profiles of nitrogen assimilation in all cases, and FAN was practically depleted in the first 2 days of fermentation. The effect of the addition of assimilable nitrogen and the size of inoculum was examined in mixed killer and sensitive strain competitions. Stuck and sluggish wine fermentations were observed to depend on nitrogen availability when the ratio of killer to sensitive cells was low (1:10 to 1:100). A relationship between the initial assimilable nitrogen content of must and the proportion of killer cells during fermentation was shown. An indirect relationship was found between inoculum size and the percentage of killer cells: a smaller inoculum resulted in a higher proportion of killer cells in grape juice fermentations. In all cases, wines obtained with pure-culture fermentations were preferred to mixed-culture fermentations by sensory analysis. The reasons why killer cells do not finish fermentation under competitive conditions with sensitive cells are discussed.     

Growth and fermentation patterns of Saccharomyces cerevisiae under different ammonium concentrations and its implications in winemaking industry

AIMS: To study the effects of assimilable nitrogen concentration on growth profile and on fermentation kinetics of Saccharomyces cerevisiae. METHODS AND RESULTS: Saccharomyces cerevisiae was grown in batch in a defined medium with glucose (200 g l(-1)) as the only carbon and energy source, and nitrogen supplied as ammonium sulphate or phosphate forms under different concentrations. The initial nitrogen concentration in the media had no effect on specific growth rates of the yeast strain PYCC 4072. However, fermentation rate and the time required for completion of the alcoholic fermentation were strongly dependent on nitrogen availability. At the stationary phase, the addition of ammonium was effective in increasing cell population, fermentation rate and ethanol. CONCLUSIONS: The yeast strain required a minimum of 267 mg N l(-1) to attain complete dryness of media, within the time considered for the experiments. Lower levels were enough to support growth, although leading to sluggish or stuck fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings reported here contribute to elucidate the role of nitrogen on growth and fermentation performance of wine yeast. This information might be useful to the wine industry where excessive addition of nitrogen to prevent sluggish or stuck fermentation might have a negative impact on wine stability and quality.     

Production of fermentation aroma compounds by Saccharomyces cerevisiae wine yeasts: effects of yeast assimilable nitrogen on two model strains

The contribution of yeast fermentation metabolites to the aromatic profile of wine is well documented; however, the biotechnological application of this knowledge, apart from strain selection, is still rather limited and often contradictory. Understanding and modeling the relationship between nutrient availability and the production of desirable aroma compounds by different strains must be one of the main objectives in the selection of industrial yeasts for the beverage and food industry. In order to overcome the variability in the composition of grape juices, we have used a chemically defined model medium for studying yeast physiological behavior and metabolite production in response to nitrogen supplementation so as to identify an appropriate yeast assimilable nitrogen level for strain differentiation. At low initial nitrogen concentrations, strain KU1 produced higher quantities of esters and fatty acids whereas M522 produced higher concentrations of isoacids, gamma-butyrolactone, higher alcohols and 3-methylthio-1-propanol. We propose that although strains KU1 and M522 have a similar nitrogen consumption profile, they represent useful models for the chemical characterization of wine strains in relation to wine quality. The differential production of aroma compounds by the two strains is discussed in relation to their capacity for nitrogen usage and their impact on winemaking. The results obtained here will help to develop targeted metabolic footprinting methods for the discrimination of industrial yeasts.     

Regulation of hydrogen sulfide liberation in wine-producing Saccharomyces cerevisiae strains by assimilable nitrogen.

Saccharomyces cerevisiae wine-producing yeast cultures grown under model winemaking conditions could be induced to liberate hydrogen sulfide (H2S) by starvation for assimilable nitrogen. The amount of H2S produced was dependent on the yeast strain, the sulfur precursor compound, the culture growth rate, and the activity of the sulfite reductase enzyme (EC 1.8.1.2) immediately before nitrogen depletion. Increased H2S formation relative to its utilization by metabolism was not a consequence of a de novo synthesis of sulfite reductase. The greatest amount of H2S was produced when nitrogen became depleted during the exponential phase of growth or during growth on amino acids capable of supporting short doubling times. Both sulfate and sulfite were able to act as substrates for the generation of H2S in the absence of assimilable nitrogen; however, sulfate reduction was tightly regulated, leading to limited H2S liberation, whereas sulfite reduction appeared to be uncontrolled. In addition to ammonium, most amino acids were able to suppress the liberation of excess H2S when added as sole sources of nitrogen, particularly for one of the strains studied. Cysteine was the most notable exception, inducing the liberation of H2S at levels exceeding that of the nitrogen-depleted control. Threonine and proline also proved to be poor substitutes for ammonium. These data suggest that any compound that can efficiently generate sulfide-binding nitrogenous precursors of organic sulfur compounds will prevent the liberation of excess H2S.     

The vinification of partially dried grapes: a comparative fermentation study of Saccharomyces cerevisiae strains under high sugar stress

AIMS: The study of the fermentation performance of Saccharomyces cerevisiae strains under high sugar stress during the vinification of partially dried grapes. METHODS AND RESULTS: Microvinification of partially dried grape must with sugar concentration of 35 degrees Brix was performed using four commercial strains to carry out alcoholic fermentation. A traditional red vinification without nutrients addition was applied. Yeasts displayed different efficiency to convert sugar in ethanol and varied in glycerol yield. Sugar consumption and ethanol level were attested at 80-87% and 143.5-158.0 g l(-1) respectively. High correlation between sugar and assimilable nitrogen consumption rate was observed. Statistical treatment of data by principal component analysis highlighted the different behaviours that strains exhibited in regard to the production of higher alcohols and other compounds important to wine quality. CONCLUSIONS: Saccharomyces cerevisiae strains displayed appreciable capability to overcome osmotic stress and to yield ethanol fermenting high sugar concentration grape must in winemaking condition. SIGNIFICANCE AND IMPACT OF THE STUDY: The results provided insights on the strain contribution to wine quality subordinate to stress condition. This investigation is of applicative interest for winemaking and processing industry that use high sugar concentration musts.     

Genetic and phenotypic diversity of Saccharomyces sensu stricto strains isolated from Amarone wine

Individual yeast strains belonging to the Saccharomyces sensu stricto complex were isolated from Amarone wine produced in four cellars of the Valpolicella area (Italy) and characterized by conventional physiological tests and by RAPD-PCR and mtDNA restriction assays. Thirteen out of 20 strains were classified as Saccharomyces cerevisiae (ex S. cerevisiae p.r. cerevisiae and p.r. bayanus) and the remaining as Saccharomyces bayanus (ex S. cerevisiae p.r. uvarum). RAPD-PCR method proved to be a fast and reliable tool for identification of Saccharomyces sensu stricto strains and also gave intraspecific differentiation. Restriction analysis of mtDNA permitted to distinguish S. cerevisiae and S. bayanus species and to discern polymorphism among S. cerevisiae isolates. The assessment of the phenotypic diversity within the isolates by gas-chromatographic analysis of secondary fermentation products was explored. Small quantities of isobutanol were produced by most of the strains and higher amounts by some S. cerevisiae strains with phenotypes Gal^- and Mel^-; all S. bayanus strains produced low amounts of amilyc alcohols. From this study it appears that each winery owns particular strains, with different genetic and biochemical characteristics, selected by specific environmental pressures during the Amarone winemaking process carried out at low temperature in presence of high sugar content.     

Transcriptional profiling of wine yeast in fermenting grape juice: regulatory effect of diammonium phosphate

The nitrogen composition of grape musts affects fermentation kinetics and production of aroma and spoilage compounds in wine. It is common practice in wineries to supplement grape musts with diammonium phosphate (DAP) to prevent nitrogen-related fermentation problems. Laboratory strains of Saccharomyces cerevisiae preferentially use rich nitrogen sources, such as ammonia, over poor nitrogen sources. We used global gene expression analysis to monitor the effect of DAP addition on gene expression patterns in wine yeast in fermenting Riesling grape must. The expression of 350 genes in the commercial wine yeast strain VIN13 was affected; 185 genes were down-regulated and 165 genes were up-regulated in response to DAP. Genes that were down-regulated encode small molecule transporters and nitrogen catabolic enzymes, including those linked to the production of urea, a precursor of ethyl carbamate in wine. Genes involved in amino acid metabolism, assimilation of sulfate, de novo purine biosynthesis, tetrahydrofolate one-carbon metabolism, and protein synthesis were up-regulated. The expression level of 86 orphan genes was also affected by DAP.     

Discrepancy in glucose and fructose utilisation during fermentation by Saccharomyces cerevisiae wine yeast strains

While unfermented grape must contains approximately equal amounts of the two hexoses glucose and fructose, wine producers worldwide often have to contend with high residual fructose levels (>2 gl(-1)) that may account for undesirable sweetness in finished dry wine. Here, we investigate the fermentation kinetics of glucose and fructose and the influence of certain environmental parameters on hexose utilisation by wine yeast. Seventeen Saccharomyces cerevisiae strains, including commercial wine yeast strains, were evaluated in laboratory-scale wine fermentations using natural Colombard grape must that contained similar amounts of glucose and fructose (approximately 110 gl(-1) each). All strains showed preference for glucose, but to varying degrees. The discrepancy between glucose and fructose utilisation increased during the course of fermentation in a strain-dependent manner. We ranked the S. cerevisiae strains according to their rate of increase in GF discrepancy and we showed that this rate of increase is not correlated with the fermentation capacity of the strains. We also investigated the effect of ethanol and nitrogen addition on hexose utilisation during wine fermentation in both natural and synthetic grape must. Addition of ethanol had a stronger inhibitory effect on fructose than on glucose utilisation. Supplementation of must with assimilable nitrogen stimulated fructose utilisation more than glucose utilisation. These results show that the discrepancy between glucose and fructose utilisation during fermentation is not a fixed parameter but is dependent on the inherent properties of the yeast strain and on the external conditions.     

The wine yeast strain-dependent expression of genes implicated in sulfide production in response to nitrogen availability

Sulfur metabolism in S. cerevisiae is well established, but the mechanisms underlying the formation of sulfide remain obscure. Here we investigated by real time RT-PCR the dependence of expression levels of MET3, MET5/ECM17, MET10, MET16 and MET17 along with SSU1 on nitrogen availability in two wine yeast strains that produce divergent sulfide profiles. MET3 was the most highly expressed of the genes studied in strain PYCC4072, and SSU1 in strain UCD522. Strains behaved differently according to the sampling times, with UCD522 and PYCC4072 showing the highest expression levels at 120h and 72h, respectively. In the presence of 267mg assimilable N/l, the genes were more highly expressed in strain UCD522 than in PYCC4072. MET5/ECM17 and MET17 were only weakly expressed in both strains under any condition tested. MET10 and SSU1 in both strains, but MET16 only in PYCC4072, were consistently up-regulated when sulfide production was inhibited. This study illustrates that strain genotype could be important in determining enzyme activities and therefore the rate of sulfide liberation. This linkage, for some yeast strains, of sulfide production to expression levels of genes associated to sulfate assimilation and sulfur amino acid biosynthesis could be relevant for defining new strategies for genetic improvement of wine yeasts.     

The role of GAP1 gene in the nitrogen metabolism of Saccharomyces cerevisiae during wine fermentation

Aim:  The aim of this study was to analyse the relevance of the general amino acid permease gene ( GAP1 ) of the wine yeast Saccharomyces cerevisiae on nitrogen metabolism and fermentation performance.
Methods and Results:  We constructed a gap1 mutant in a wine strain. We compared fermentation rate, biomass production and nitrogen consumption between the gap1 mutant and its parental strain during fermentations with different nitrogen concentrations. The fermentation capacity of the gap1 mutant strain was impaired in the nitrogen-limited and -excessive conditions. The nitrogen consumption rate between the wild strain and the mutant was different for some amino acids, especially those affected by nitrogen catabolite repression (NCR). The deletion of GAP1 gene also modified the gene expression of other permeases.
Conclusions:  The Gap1 permease seems to be important during wine fermentations with low and high nitrogen content, not only because of its amino acid transporter role but also because of its function as an amino acid sensor.
Significance and Impact of the Study:  A possible biotechnological advantage of a gap1 mutant is its scarce consumption of arginine, whose metabolism has been related to the production of the carcinogenic ethyl carbamate.     

Mitochondrial DNA polymorphism of the yeast Saccharomyces bayanus var. uvarum

Genetic relationships among forty-one strains of Saccharomyces bayanus var. uvarum isolated in different wine regions of Europe and four wild isolates were investigated by restriction analysis (RFLP) of mitochondrial DNA (mtDNA) with four restriction endonucleases, AluI, DdeI, HinfI and RsaI. No clear correlation between origin and source of isolation of S. bayanus var. uvarum strains and their mtDNA restriction profiles was found. On the whole, the mtDNA of S. bayanus var. uvarum is much less polymorphic than that of S. cerevisiae. This observation is in good agreement with results obtained by electrophoretic karyotyping. Unlike wine S cerevisiae, strains of S. bayanus var. uvarum display a low level of chromosome length polymorphism.     

Relationship of low lysine and high arginine concentrations to efficient ethanolic fermentation of wheat mash.

Very high gravity wheat mashes containing 20 or more grams of carbohydrates per 100 mL were fermented completely by Saccharomyces cerevisiae, even though these mashes contained low amounts of assimilable nitrogen. Supplementation of wheat mashes with various amino acids or with yeast extract, urea, or ammonium sulfate reduced the fermentation time. However, lysine or glycine added as single supplements, inhibited yeast growth and fermentation. With lysine, yeast growth was severely inhibited, and a loss of cell viability as high as 80% was seen. Partial or complete reversal of lysine-induced inhibition was achieved by the addition of a number of nitrogen sources. All nitrogen sources that relieved lysine-induced inhibition of yeast growth also promoted uptake of lysine and restored cell viability to the level observed in the control. They also increased the rate of fermentation. Experiments with minimal media showed that for lysine to be inhibitory to yeast growth, assimilable nitrogen in the medium must be in growth-limiting concentrations or totally absent. In the presence of excess nitrogen, lysine stimulated yeast growth and fermentation. Results indicate that supplementing wheat mash with other nitrogen sources increases the rate of fermentation not only by providing extra nitrogen but also by reducing or eliminating the inhibitory effect of lysine on yeast growth.     

Changes in volatile compounds and aromatic series in sherry wine with high gluconic acid levels subjected to aging by submerged flor yeast cultures

Volatile compounds of sherry wine containing gluconic acid under aging by submerged flor yeast cultures were analyzed. The aroma profile was obtained by grouping the compounds in nine aromatic series. The balsamic, fatty, herbaceous and empyreumatic series increased significantly as consequence of the increase of pantolactone, acids (butanoic, 2-methylbutanoic and 3-methylbutanoic), methionol and gamma-butyrolactone compounds, respectively. The decrease of higher alcohols promoted solvent series diminished. These changes are consistent with those observed in the production of commercial sherry wine using traditional biological aging.