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.     

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.     

Aroma compounds in Japanese sweet rice wine (Mirin) screened by aroma extract dilution analysis (AEDA)

Thirty-nine key aroma compounds were newly identified or tentatively identified in the aroma concentrate of Japanese sweet rice wine (Mirin) by an aroma extract dilution analysis technique based on the 68 detected peaks. Among them, 3-(methylthio)propanal, 3-hydroxy-4,5-dimethyl-2(5H)-furanone, 3-methylbutanoic acid, 2-methylbutanoic acid, and 2-methoxy-4-vinylphenol were detected with the highest FD factors in this study.     

Acetaldehyde and ethanol are responsible for mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP) in flor yeasts

Flor yeasts grow and survive in fino sherry wine where the frequency of respiratory-deficient (petite) mutants is very low. Mitochondria from flor yeasts are highly acetaldehyde- and ethanol-tolerant, and resistant to oxidative stress. However, restriction fragment length polymorphism (RFLP) of mtDNA from flor yeast populations is very high and reflects variability induced by the high concentrations of acetaldehyde and ethanol of sherry wine on mtDNA. mtDNA RFLP increases as the concentration of these compounds also increases, but is followed by a total loss of mtDNA in petite cells. Yeasts with functional mitochondria (grande) are target of continuous variability, so that flor yeast mtDNA can evolve extremely rapidly and may serve as a reservoir of genetic diversity, whereas petite mutants are eventually eliminated because metabolism in sherry wine is oxidative.     

TASTE GROUP THRESHOLDS AND SENSORY EVALUATION OF SPANISH WINE VINEGARS

Wine vinegar is a product obtained from wine acidification which contains at least 5% by wt. of acetic acid, in general without any additives or colorings.
Aspects studied in this work include: the determination of the taste group thresholds (geometric mean of the individual best-estimate thresholds "BET") of two different acids (citric and acetic acids) in aqueous solution and spanish vinegars produced from table and sherry wines. The results obtained suggest that wine vinegar can be considered something more than just an acidulant agent.
In order to evaluate differences among wine vinegars, discriminant tests for twenty-five spanish vinegars (sherry, table and flavored vinegars) were applied. Six of the twelve attributes freely chosen by assessors allowed grouping of the spanish wine vinegars according to their sensory aspects.     

Genetic differentiation of the sherry yeasts Saccharomyces cerevisiae

Molecular genetic study of the yeast Saccharomyces cerevisiae isolated at various stages of sherry making (young wine, solera, and criadera) in various winemaking regions of Spain demonstrated that sherry yeasts diverged from the primary winemaking yeasts according to several physiological and molecular markers. All sherry strains independently of the place and time of their isolation carry a 24-bp deletion in the ITS 1 region of ribosomal DNA, whereas the yeasts of the primary winemaking lack this deletion. Molecular karyotypes of the sherry yeast from different populations were found very similar.     

Genetic Differentiation of the Sherry Yeasts <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Molecular and genetic studies of the yeast Saccharomyces cerevisiae isolated at distinct stages of sherry making (young wine, solera, and criadera) in various winemaking regions of Spain demonstrated that sherry yeasts diverged from primary winemaking yeasts according to several physiological and molecular markers. All sherry strains, regardless of the place and time of their isolation, carry a 24-bp deletion in the ITS1 region of ribosomal DNA, whereas the yeasts of primary winemaking lack this deletion. Molecular karyotypes of sherry yeasts from different populations were found to be very similar.     

Retention of Browning Compounds by Yeasts Involved in the Winemaking of Sherry Type Wines

Wine model solutions were used to study the ability of dehydrated yeasts to retain the brown products formed in the reaction between (+)-catechin and acetaldehyde. Saccharomyces cerevisiae races capensis and bayanus, two typical flor yeasts involved in the biological aging of sherry wines, had a higher capacity to retain coloured compounds than S. cerevisiae fermentative yeast. Of the flor yeasts, capensis exhibited a higher colour reduction capacity than bayanus. Such differences may account for the different rate at which browning compounds are removed at different times of year during the biological aging of wines.     

Continuous Production of Flor Sherry from New York State Wines

Flor sherry-like wines were produced continuously from New York State wines from Delaware or cold-pressed Concord grapes whose pigment contents were reduced with activated carbon. The course of flor fermentation was followed by total aldehyde analysis. Optimal flor production was observed at 18 to 20 C. Two continuous methods of fermentation were used. A glass column packed with ceramic saddles densely covered with yeasts gave good results, but required more careful management than the second method of submerged fermentation in a laboratory fermentor, which gave a higher sherry output and higher aldehyde production. With the laboratory fermentor, it was possible to obtain a sherry output of 22 liters per 24 hr with an aldehyde content of 300 to 500 mg per liter. The flor sherry produced by these methods required subsequent aging and fortification to the desired alcohol content.     

Authentication and identification of Saccharomyces cerevisiae‘flor’ yeast races involved in sherry ageing

Yeasts involved in velum formation during biological ageing of sherry wine have to date been classified into four races of Saccharomyces cerevisiae (beticus, cheresiensis, montuliensis, rouxii) according to their abilities to ferment different sugars. It has been proposed that race succession during biological ageing is essential for the development of the organoleptical properties of sherry wines. In this work we studied the physiological characteristics, the molecular differentiation and the phylogenetic relationships of the four races employing type and reference strains from culture collections and natural environments. Using restriction analysis of the ribosomal region that includes the 5.8S rRNA gene and internal transcribed regions (5.8S-ITS) we were able to differentiate 'flor' and non-'flor' S. cerevisiae yeast strains. However, no correlation between fermentation profile, mitochondrial DNA restriction analysis or chromosomal profiles and these races was found. Moreover, sequences of the D1/D2 domain of the 26S rRNA gene and the 5.8S-ITS region from these strains were analysed and no genetic differences were noted suggesting that 'flor' yeast cannot be grouped into four different races and the four races are identified as S. cerevisiae. Since the yeasts isolated from velum in sherry wine present a unique 5.8S rRNA pattern different from the rest of the Saccharomyces cerevisiae strains we propose that they should be included as a single race or variety inside the S. cerevisiae taxon.     

Continued exploration of biphenylsulfonamide scaffold as a platform for aggrecanase-1 inhibition

Design, synthesis and structure-activity relationship of a series of biphenylsulfonamido-3-methylbutanoic acid based aggrecanase-1 inhibitors are described. In addition to robust aggrecanase-1 inhibition, these compounds also exhibit potent MMP-13 activity. In cell-based cartilage explants assay compound 48 produced 87% inhibition of proteoglycan degradation at 10 μg/mL. Good pharmacokinetic properties were demonstrated by 46 with a half-life of 6h and bioavailability of 23%.     

Mitochondrial DNA loss caused by ethanol in Saccharomyces flor yeasts.

Saccharomyces flor yeasts proliferate at the surface of sherry wine, which contains over 15% (vol) ethanol. Since ethanol is a powerful inducer of respiration-deficient mutants, this alcohol has been proposed to be the source of the high diversity found in the mitochondrial genomes of flor yeasts and other wine yeasts. Southern blot analysis suggests that mitochondrial DNA (mtDNA) polymorphic changes are due to minor lesions in the mitochondrial genome. As determined in this work by pulsed-field gel electrophoresis, restriction analysis, and Southern blot analysis, ethanol-induced petite mutants completely lack mtDNA (rho zero). Ethanol-induced changes in the mitochondrial genome that could explain the observed mtDNA polymorphism in flor yeasts were not found. The transfer of two different mtDNA variants from flor yeasts to a laboratory strain conferred in both cases an increase in ethanol tolerance in the recipient strain, suggesting that mtDNAs are probably subjected to positive selection pressure concerning their ability to confer ethanol tolerance.     

Lignin-derived oak phenolics: a theoretical examination of additional potential health benefits of red wine

Lignin-derived phenolic compounds can be extracted from oak barrels during the aging of red wine, and it is hypothesized that these compounds may contribute to the health benefits of red wine by their antioxidant, radical-scavenging, or chemopreventive activities. Density functional calculations (B3LYP/6-311++G**) support the radical-scavenging abilities of the oak phenolics. Sinapaldehyde, syringaldehyde, syringol, and syringylacetone all have bond dissociation energies that are lower than resveratrol and comparable to the flavonoid catechin. Molecular docking studies of the oak phenolics with known resveratrol protein targets also show that these compounds dock favorably to the protein targets. Thus, lignin-derived oak phenolics, although found in small concentrations, may contribute to the beneficial antioxidant, chemopreventive, and cardioprotective effects of red wine.     

Nitrogen compounds in wine during its biological aging by two flor film yeasts: An approach to accelerated biological aging of dry sherry-type wines

Urea, ammonium, and free amino acid contents were quantified in biological aging of a young wine under two flor film forming yeast strains, Saccharomyces cerevisiae race capensis and S. cerevisiae race bayanus, and compared. Cell viability in the film was different for the two yeast strains. Thus, capensis maintained a much greater number of viable cells per surface area than bayanus and hence used greater amount of nitrogen compounds. The main source of nitrogen for the yeasts during the biological aging process was L-proline. The two yeast strains also differed in the amounts of assimilable nitrogen they utilized, in their preferences for amino acid consumption, and kinetics. To accelerate the aging process, the effect of controlled monthly aeration of the wine aged with capensis strain was investigated. The results revealed that short aeration did not appreciably increase the overall consumption of assimilable nitrogen, but consumption of some nitrogen compounds was accelerated (particularly L-proline, L-tryptophan, L-glutamic acid, ammonium ion, L-lysine, and L-arginine); the use of L-ornithine was inhibited; and GABA, L-methionine, and urea were depletes. Probably the aeration increases the aroma compounds, thereby producing wines with improved sensory properties. (c) 1997 John Wiley & Sons, Inc.     

Acid stress-mediated metabolic shift in Lactobacillus sanfranciscensis LSCE1

Lactobacillus sanfranciscensis LSCE1 was selected as a target organism originating from recurrently refreshed sourdough to study the metabolic rerouting associated with the acid stress exposure during sourdough fermentation. In particular, the acid stress induced a metabolic shift toward overproduction of 3-methylbutanoic and 2-methylbutanoic acids accompanied by reduced sugar consumption and primary carbohydrate metabolite production. The fate of labeled leucine, the role of different nutrients and precursors, and the expression of the genes involved in branched-chain amino acid (BCAA) catabolism were evaluated at pH 3.6 and 5.8. The novel application of the program XCMS to the solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) data allowed accurate separation and quantification of 2-methylbutanoic and 3-methylbutanoic acids, generally reported as a cumulative datum. The metabolites coming from BCAA catabolism increased up to seven times under acid stress. The gene expression analysis confirmed that some genes associated with BCAA catabolism were overexpressed under acid conditions. The experiment with labeled leucine showed that 2-methylbutanoic acid originated also from leucine. While the overproduction of 3-methylbutanoic acid under acid stress can be attributed to the need to maintain redox balance, the rationale for the production of 2-methylbutanoic acid from leucine can be found in a newly proposed biosynthesis pathway leading to 2-methylbutanoic acid and 3 mol of ATP per mol of leucine. Leucine catabolism to 3-methylbutanoic and 2-methylbutanoic acids suggests that the switch from sugar to amino acid catabolism supports growth in L. sanfranciscensis in restricted environments such as sourdough characterized by acid stress and recurrent carbon starvation.     

Wine flavor and aroma

The perception of wine flavor and aroma is the result of a multitude of interactions between a large number of chemical compounds and sensory receptors. Compounds interact and combine and show synergistic (i.e., the presence of one compound enhances the perception of another) and antagonistic (a compound suppresses the perception of another) interactions. The chemical profile of a wine is derived from the grape, the fermentation microflora (in particular the yeast Saccharomyces cerevisiae), secondary microbial fermentations that may occur, and the aging and storage conditions. Grape composition depends on the varietal and clonal genotype of the vine and on the interaction of the genotype and its phenotype with many environmental factors which, in wine terms, are usually grouped under the concept of “terroir” (macro, meso and microclimate, soil, topography). The microflora, and in particular the yeast responsible for fermentation, contributes to wine aroma by several mechanisms: firstly by utilizing grape juice constituents and biotransforming them into aroma- or flavor-impacting components, secondly by producing enzymes that transform neutral grape compounds into flavor-active compounds, and lastly by the de novo synthesis of many flavor-active primary (e.g., ethanol, glycerol, acetic acid, and acetaldehyde) and secondary metabolites (e.g., esters, higher alcohols, fatty acids). This review aims to present an overview of the formation of wine flavor and aroma-active components, including the varietal precursor molecules present in grapes and the chemical compounds produced during alcoholic fermentation by yeast, including compounds directly related to ethanol production or secondary metabolites. The contribution of malolactic fermentation, ageing, and maturation on the aroma and flavor of wine is also discussed.     

Correlation between acetaldehyde and ethanol resistance and expression of HSP genes in yeast strains isolated during the biological aging of sherry wines

In the production of sherry wines, the process of biological aging is essential for the development of their organoleptic properties. This process involves velum formation by "flor" yeasts. Several of these yeast strains have been isolated and characterized with regard to their genetic, physiological and metabolic properties. In this work, we studied their resistance to cold-, osmotic-, oxidative-, ethanol- and acetaldehyde-stress, and found, in most cases, a correlation between resistance to acetaldehyde stress and ethanol stress and isolation from "soleras." Moreover, gene expression analysis revealed induction of the heat shock protein (HSP) genes HSP12, HSP82, and especially HSP26 and HSP104, under acetaldehyde stress in most of the strains. In strain C, there was a clear correlation between resistance to ethanol and acetaldehyde, the high induction of HSP genes by these compounds and its presence as the predominant strain in most levels of several soleras.     

Determination of L- and D-amino acids in foodstuffs by coupling of high-performance liquid chromatography with enzyme reactors

Summary. A technique is described for the enantiomeric determination of L- and D-amino acids. It works on the principle that the separation efficiency of high-performance liquid chromatography is coupled with the specificity of enzymes and the sensitivity of electrochemical detection. After separation on a lithium cation-exchange column the amino acids are converted into keto acids and hydrogen peroxide under catalyzation of L- or D-amino acid oxidase. Hydrogen peroxide is detected amperometrically. The method has been tested by the analysis of beer, port, sherry, wine and fruit juice. A main emphasis was put onto the determination of D-alanine which can serve as an indicator for bacterial contamination. It is shown that a coupling of HPLC with enzyme reactors is a suitable technique for the rapid detection of this marker. Received April 14, 1999, Accepted September 15, 1999