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Yeast isolates from commercial red wines were characterized with regards to tolerances to molecular SO(2), ethanol, and temperature as well as synthesis of 4-ethyl-phenol/4-ethyl-guaiacol in grape juice or wine. Based on rDNA sequencing, nine of the 11 isolates belonged to Dekkera bruxellensis (B1a, B1b, B2a, E1, F1a, F3, I1a, N2, and P2) while the other two were Candida pararugosa (Q2) and Pichia guilliermondii (Q3). Strains B1b, Q2, and Q3 were much more resistant to molecular SO(2) in comparison to the other strains of Dekkera. These strains were inoculated (10(3)-10(4)cfu/ml) along with lower populations of Saccharomyces (<500 cfu/ml) into red grape juice and red wine incubated at two temperatures, 15 degrees C and 21 degrees C. Although Saccharomyces quickly dominated fermentations in grape juice, B1b and Q2 grew and eventually reached populations >10(5)cfu/ml. In wine, Q3 never entered logarithmic growth and quickly died in contrast to Q2 which survived >40 days after inoculation. B1b grew well in wine incubated at 21 degrees C while slower growth was observed at 15 degrees C. Neither Q2 nor Q3 produced 4-ethyl-phenol or 4-ethyl-guaiacol, unlike B1b. However, lower concentrations of volatile phenols were present in wine incubated at 15 degrees C compared to 21 degrees C.

The combination of QTL mapping studies of synthetic lines and association mapping studies of natural diversity represents an opportunity to throw light on the genetically based variation of quantitative traits. With the positional information provided through quantitative trait locus (QTL) mapping, which often leads to wide intervals encompassing numerous genes, it is now feasible to directly target candidate genes that are likely to be responsible for the observed variation in completely sequenced genomes and to test their effects through association genetics. This approach was performed in grape, a newly sequenced genome, to decipher the genetic architecture of anthocyanin content. Grapes may be either white or colored, ranging from the lightest pink to the darkest purple tones according to the amount of anthocyanin accumulated in the berry skin, which is a crucial trait for both wine quality and human nutrition. Although the determinism of the white phenotype has been fully identified, the genetic bases of the quantitative variation of anthocyanin content in berry skin remain unclear. A single QTL responsible for up to 62% of the variation in the anthocyanin content was mapped on a Syrah x Grenache F(1) pseudo-testcross. Among the 68 unigenes identified in the grape genome within the QTL interval, a cluster of four Myb-type genes was selected on the basis of physiological evidence (VvMybA1, VvMybA2, VvMybA3, and VvMybA4). From a core collection of natural resources (141 individuals), 32 polymorphisms revealed significant association, and extended linkage disequilibrium was observed. Using a multivariate regression method, we demonstrated that five polymorphisms in VvMybA genes except VvMybA4 (one retrotransposon, three single nucleotide polymorphisms and one 2-bp insertion/deletion) accounted for 84% of the observed variation. All these polymorphisms led to either structural changes in the MYB proteins or differences in the VvMybAs promoters. We concluded that the continuous variation in anthocyanin content in grape was explained mainly by a single gene cluster of three VvMybA genes. The use of natural diversity helped to reduce one QTL to a set of five quantitative trait nucleotides and gave a clear picture of how isogenes combined their effects to shape grape color. Such analysis also illustrates how isogenes combine their effect to shape a complex quantitative trait and enables the definition of markers directly targeted for upcoming breeding programs.

The aim of this work was to evaluate the fate of ochratoxin A (OTA) content from must to wine during the red wine making process in a pilot scale vinification. The study was done using musts obtained from two red grape varieties (Bonarda and Tempranillo) artificially contaminated with two OTA levels. A duplicate set of tanks of 100 I each was established for each must (Bonarda and Tempranillo). The fermentations were initiated by inoculation of two Saccharomyces spp. strains having different fermentation performance. The must from the Tempranillo variety was spiked with 6 microg/I of OTA while that from the Bonarda variety with 0.3 microg/I of the toxin. Samples were collected at different stages of the process. Performance of the alcoholic and malolactic fermentations was monitored. Titratable and volatile acidity, pH, ethanol, sugar and SO2 concentrations were determined following standard methods proposed by the Office International de la Vigne et du Vin (OIV). OTA analysis was done by HPLC. Detection and quantification limits were 0.01 and 0.1 ng/ml, respectively. The OTA levels during the vinification trials dropped to an average of about 86.5%. The type of Saccharomyces strains used showed no effect on toxin reduction.

The impact of mixed cultures of Hanseniaspora osmophila and Saccharomyces cerevisiae with different initial yeast ratios on wine composition has been examined. The mixed culture significantly affected sugar consumption, the main enological parameters and ester concentrations, with the exception of glycerol, isoamyl acetate and diethyl succinate levels. Remarkably, in wines obtained with mixed cultures the concentration of 2-phenylethyl acetate was approximately 3- to 9-fold greater than that produced by S. cerevisiae pure culture. Moreover sensory evaluation revealed a stronger fruity character in wines fermented with mixed cultures than in control wines. Independently of the mixed culture used, all wines showed concentrations of acetic acid and ethyl acetate within the ranges described for wines. Our data suggest that a mixed culture of H. osmophila and S. cerevisiae can be used as a tool to increase 2-phenylethyl acetate in wine and that its concentration can be controlled by modulating the initial yeast ratio in the culture.

We report the effect of vine variety on the absorption of metals from soil and follow the variety from wine through juice, verifying which metals could be used to assess wine provenance. Eleven metals were determined by atomic absorption spectroscopy in 32 soils, 16 grapes juices, and 18 wines sampled from a single vineyard having four red grape varieties (Cabernet Sauvignon, Bonarda, Malbec, and Syrah). The K nearest neighbor method allows us to distinguish among different soils, juices, and wines. Linear discriminant analysis affords descriptors to point out differences, mainly Mg, Mn, Ca, K, and Na. Data analysis evidenced that some elements have equivalent concentrations in soil, juice, and wine, while others did not. Canonical analysis shows good correlation between grape juice and wine with their provenance soil. We suggest using Mg as a marker of wine provenance, while Mn could be used to evaluate differences between wine varieties associated with plant physiology.

The aroma compositions of 25 premium Spanish red wines have been screened by quantitative gas chromatography-olfactometry and have been related to the quality scores of the wines. The study has shown that up to 65 odorants can be present in the aroma profiles of those wines, 32 of which have been detected in less than half of the samples. One new odorant is reported for the first time in wine [(Z)-2-nonenal], and only 11 odorants, most of them weak and infrequent, remain unknown. Quality was not positively correlated with any single compound or with any olfactometric vector built by the summation of odorants with similar odors. However, an olfactometric vector built by the summation of the olfactometric scores of defective odorants, such as 2-methoxy-3,5-dimethylpyrazine, 4-ethylphenol, 3-ethylphenol, 2,4,6-trichloroanisole, and o-cresol was significant and negatively related to quality. Quality could be satisfactorily explained by a simple partial least-squares model (79% explained variance in cross-validation) with just three X-variables: the aforementioned defective vector, a second vector grouping 9 other compounds with negative aroma nuances, and the fruity vector, grouping 15 compounds with fruit-sweet descriptors. This result shows that the quality of these red wines is primarily related to the presence of defective or negative odorants, and secondarily to the presence of a relatively large number of fruit-sweet odorants. Remarkably, only in a few low-quality samples could defective aroma nuances be detected, which suggests that defective and negative odorants exert a strong aroma suppression effect on fruity aroma.

The aim of this work was to study the variability of 36 Saccharomyces cerevisiae wild strains isolated from different grape varieties and from two very distant zones, located in Northern and Southern Italy. The strains were differentiated on the basis of parameters of technological interest, such as resistance to antimicrobial compounds frequently present in wine, and the production of volatile aromatic compounds (VOC), determined by SPME procedure in the experimental wines obtained by inoculated fermentations. The VOC profile allowed to differentiate the yeasts in function of isolation area: S. cerevisiae isolated from Southern Italy grapes were able to produce more volatile compounds than those from Northern Italy. The compounds synthesized by all the yeasts, besides the ethanol, were 3-methyl-1-butanol and ethyl acetate. The production of acids during the alcoholic fermentation was a characteristic of Southern yeast strains. The screening of S. cerevisiae strains for technological parameters, such as sulphur dioxide, copper and ethanol resistance or hydrogen sulphide production, revealed similar behaviour for sulphur dioxide resistance among Northern and Southern S. cerevisiae strains. Copper resistance and sulphur dioxide production were correlated to isolation area: S. cerevisiae "Northern" strains showed higher copper resistance and lowest hydrogen sulphide production than that exhibited from "Southern" strains.

Saccharomyces cerevisiae is the main yeast species responsible for wine fermentation; however, its presence during maturing or barrel-ageing can sometimes result in a reduction in the quality of wine by refermentation. In this work, we developed a quantitative real-time PCR (QPCR) for the rapid detection and quantification of S. cerevisiae in wine. The primers and the hydrolysis probe (TaqMan) were designed from the sequence of a DNA fragment present only in S. cerevisiae and absent in other wine yeasts obtained from an RAPD-PCR analysis. The QPCR developed was highly reproducible, allowing the specific detection and quantification of this yeast in artificially contaminated wines, with a detection limit of 78 CFU/mL. Furthermore, the usefulness of the QPCR developed was evaluated through the quantification of the yeast in wine samples obtained from vineyards, confirming the quantitative capacity of the method. The methodology developed was specific, fast and a sensitive tool for the detection and enumeration of S. cerevisiae cells in wine.

The main objective of this study was to investigate the effect of different Saccharomyces cerevisiae wine yeast strains on the concentration of aroma-enhancing volatile thiols and fermentation metabolites in Sauvignon Blanc wine. Seven commercial wine yeast strains were selected based on their putative ability to modulate the concentrations of the fruity volatile thiols, 4-mercapto-4-methylpentan-2-one (4MMP), 3-mercapto-hexanol (3MH) and 3-mercapto-hexylacetate (3MHA). Each of these yeasts was used to produce Sauvignon Blanc wines under controlled conditions, in triplicate, in 20-L quantities. The levels of 4MMP, 3MH and 3MHA in these wines were quantified using the p-hydroxymercuribenzoate method. In addition, a total of 24 volatile yeast-derived fermentation aroma compounds were also quantified using headspace solid-phase microextraction coupled with gas chromatography mass spectrometry (HS-SPME-GC-MS). Formal sensory analysis was conducted by 12 trained assessors and, additionally, a panel of 24 experienced wine industry professionals assessed the wines and ranked them in order of preference. The results indicated that the yeast strains varied significantly in terms of their capabilities to (i) produce volatile thiols and fermentation metabolites; and (ii) to modulate the varietal characters of Sauvignon Blanc wine. Yeast strains that produced the highest levels of volatile thiols were responsible for wines with the highest perceived intensity of fruitiness, and these wines were preferred by the tasting panels. While the 'green' characters in Sauvignon Blanc wines can be manipulated through vineyard management, the 'tropical fruity' characters appear to be largely dependent on the wine yeast strain used during fermentation. Therefore, the choice of yeast strain offers great potential to modulate wine aroma profiles to definable styles and predetermined consumer market specifications.

A haploid sake yeast strain derived from the commercial diploid sake yeast strain Kyokai no. 7 showed better characteristics for sake brewing compared to the haploid laboratory yeast strain X2180-1B, including higher production of ethanol and aromatic components. A hybrid of these two strains showed intermediate characteristics in most cases. After sporulation of the hybrid strain, we obtained 100 haploid segregants of the hybrid. Small-scale sake brewing tests of these segregants showed a smooth continuous distribution of the sake brewing characteristics, suggesting that these traits are determined by multiple quantitative trait loci (QTLs). To examine these sake brewing characteristics at the genomic level, we performed QTL analysis of sake brewing characteristics using 142 DNA markers that showed heterogeneity between the two parental strains. As a result, we identified 25 significant QTLs involved in the specification of sake brewing characteristics such as ethanol fermentation and the production of aromatic components.

The consumption of citrate by the malolactic bacterium Oenococcus oeni changes the aromatic profile of wines due to the production of volatile compounds such as diacetyl and acetic acid. In this study, the expression of genes related to citrate utilization in the O. oeni strain PSU-1 was investigated to further understand the role of this metabolic pathway in the adaptation to wine environment and its impact on organoleptic qualities. Different conditions of ethanol content (0% and 10%) and pH (3.5 and 4.0) were assayed to evaluate the transcriptional response to both these stress factors. In the presence of ethanol, metabolic and transcriptional behavior was different than the observed when ethanol was absent. The expression of citrate pathway genes was mainly affected by ethanol, while pH showed a lower effect. Among the studied genes, citE, ackA and alsD were the genes revealing a distinctive transcriptional response. The differences observed in gene expression were in correlation with the different content of end products such as acetic acid and diacetyl. The increment of gene expression observed in the presence of ethanol at low pH suggests the participation of citrate metabolism in the response to stress conditions.

The possibility of using front-face fluorescence spectroscopy to characterize red wines was investigated, and a tentative identification of their main fluorescent components was attempted. Fifty-seven red wine samples from different origins were included in the present study. Their fluorescence excitation-emission matrices (EEMs) were registered directly on 3-mL aliquots of untreated samples. The assayed excitation and emission ranges were 245-340 and 300-500 nm, respectively. The set of 57 EEMs was analyzed by means of parallel factor analysis (PARAFAC). Thus, the spectral excitation and emission profiles of possible "pure" fluorescence components (PARAFAC loadings) and the relative contribution of each component to the individual EEMs (PARAFAC scores) were obtained. The red wine system contained four main fluorescence components, and the excitation and emission loadings had maxima at the wavelength pairs 260/380, 275/323, 330/410, and 280/364 nm, respectively. A tentative identification of fluorophores was done by matching PARAFAC score values with HPLC measurements on the same 57 samples, as well as fluorescence measurements on pure compounds typically present in red wine. It was found that the third component was highly correlated with concentrations of catechin and epicatechin. When the PARAFAC score values were plotted against each other, they did to some extent discriminate the wines according to origin (country) and grape variety.

Wine contamination with ochratoxin A (OTA) is due to the attack of wine grapes by ochratoxigenic Aspergillus carbonarius and Aspergillus spp. section Nigri. Four A. pullulans strains, AU14-3-1, AU18-3B, AU34-2, and LS30, are resistant to and actively degrade ochratoxin A in vitro. The less toxic ochratoxin alpha and the aminoacid L-beta-phenylalanine were the major degradation products, deriving from the cleavage of the amide bond linking these two moieties of OTA. The same strains were studied further as biocontrol agents of A. carbonarius on wine grapes in laboratory experiments. Three of the four strains significantly prevented infections by A. carbonarius. Berries pretreated with the biocontrol agents and infected with A. carbonarius contained lower amounts of OTA as compared to the untreated infected control berries. Two of these strains were shown to degrade OTA to ochratoxin alpha in fresh grape must, but the mechanisms of the decrease of OTA accumulation in infected berries pretreated with the biocontrol agents remain to be elucidated. Assessment of one strain carried out in the vineyard during the growing season of 2006 showed that the tested strain was an effective biocontrol agent, reducing both severity of Aspergillus rots and OTA accumulation in wine grapes. To our knowledge this is the first report describing the positive influence of biocontrol agents on OTA accumulation in this crop species.

In winemaking, spontaneous grape must fermentations have been replaced by inoculation of commercial active dry wine yeast (ADWY). Yeast rehydration is the key to avoiding stuck and sluggish fermentations. Despite the importance of this step, not enough is known about what this process implies for winemaking as a whole or about what kind of practices could help to improve it. The main aim of this study is to determine the best yeast rehydration conditions for ensuring good cell viability and vitality before inoculation into the must. The experimental rehydration media in this study can be divided into four groups: carbon and nitrogen compounds, metallic ions, oxidant and antioxidant agents, and membrane fluidity agents. We studied the biochemical and biophysical behaviour of ADWY after rehydration in the various media under oenological conditions, i.e. incubation at 37 degrees C for 30 min. The viability of rehydrated yeast cells was evaluated by plating, and assessed by fluorescence microscopy and flow cytometry. The vitality of rehydrated cells was estimated by indirect impedance. The rehydrating solution complemented with magnesium provided the best vitality rate because the time taken to reach the activity threshold was cut by two thirds. This improvement was also illustrated by the less time needed to stop the leakage of intracellular compounds during the rehydration process.

Aims: The toxicity of the fungicide pyrimethanil on the growth of wine yeasts was evaluated using in vivo and in vitro experimentation., Methods and Results: The effect of pyrimethanil in the must was studied during the spontaneous wine fermentation of three consecutive vintages and by the cultivation of Hanseniaspora uvarum and Saccharomyces cerevisiae yeasts in a liquid medium. The residues of the fungicide were measured using gas chromatography-mass spectrometry system and the sugar concentration in the must using HPLC-RI. Molecular and standard methods were used for identifying the yeast species. Although the pyrimethanil residues in grapes were below the maximum residue limits, they significantly affected the reduced utilization of sugars in the first days of fermentation. Its residues controlled the growth of H. uvarum during the fermentation and during in vitro cultivation as well., Conclusions: The fungicide pyrimethanil had an effect on the course and successful conclusion of spontaneous wine fermentation that was correlated with the initial concentration of yeasts in the must., Significance and Impact of the Study: The impact of pyrimethanil on the indigenous mixed yeast flora in fermenting must was investigated for the first time. The results showed that its residues might play an important role in the growth and succession of yeast during spontaneous wine fermentation.

Utilization of phenolic acids, including gallic acid, coumaric acid, caffic acid, cinnamic acid, and ferulic acid, for methanol reduction in wine was investigated. Enzyme activities of pectinesterase and pectin lyase decreased significantly when 0.1 mg/L of gallic acid, coumaric acid, caffic acid, cinnamic acid, or ferulic acid was added. However, no inhibition on polygalacturonase activity was observed when 0.5 mg/L of phenolic acid was added. Methanol content in commercial pectic enzyme (CPE) group increased from 11.53 +/- 1.34 to 56.67 +/- 3.75 ppm in the final products. Adding gallic acid or coumaric acid with CPE inhibited the increase of methanol production. In addition, when 0.2 mg/L of phenolic acid (gallic acid or coumaric acid) was added, the amount of total phenolic acid released from CPE + gallic acid or CPE + coumaric acid groups became higher than CPE group by approximately 466 and 539 mg/L, respectively. In conclusion, the values of lightness, red content, yellow content, total pigment, and total phenolic acid increased in the presence of gallic acid or coumaric acid with CPE, suggesting that adding gallic acid or coumaric acid into winemaking process is a potential method for reducing methanol content, improving wine quality, as well as increasing healthy compounds in wine production.

More than 800 aromatic compounds have been identified in wine, some of them at the ng/l level. Wine, therefore, constitutes a very complex matrix, from which it is difficult to isolate a specific aroma character. Gas chromatography-olfactometry (GC-O) applied to wine extracts is used to characterize odor-active zones that are often treated in a hierarchical way by Aroma Extract Dilution Analysis (AEDA). The aromatic impact of the volatiles is evaluated, generally by determining perception thresholds. This methodology has provided convincing results concerning wine flavors, but it does have its limitations. For instance, data on beta-damascenone have demonstrated that these methods could reach their limits for this volatile, in particular, because of the non-quantitative representation of aroma extracts of wines, and because of the difficulty to accurately determine the perception threshold in wines for a compound already present. For beta-damascenone, we have shown that its very low detection threshold with GC-O, its wide range, and its dependence on the composition of the medium resulted in overestimating its direct impact on the aroma of wine. Another way to facilitate the characterization of aromatic compounds was, therefore, investigated. High-Performance Liquid Chromatography (HPLC) methods were developed for the analysis of wine extracts. From an aromatic extract, 25 fractions with various flavors were thus obtained, and reverse-phase methodology was used for the selection and characterization of red- and black-fruit aromas in red wines.

The enantiomers of sotolon, a flavor compound typical of oxidized white wines, were separated by preparative HPLC to determine their perception thresholds and distribution in wines. The enantiomeric ratios of chiral sotolon were evaluated in several dry white wines using gas chromatography and a chiral column (beta-cyclodextrin) connected to a 2 m precolumn (BP20). The perception threshold of (S)-sotolon (0.8 microg/L) in model wine solution was 100 times lower than that of the (R) form (89 microg/L), indicating that (S)-sotolon contributes to the characteristic aroma of prematurely aged dry white wines. Both enantiomers are detected in white wines. Analysis of commercial dry white wines from various vintages and origins revealed three types of distribution patterns: the racemic form, an excess of R, and an excess of S. The proportions found in these wines may be partially explained by the slow racemization kinetics (20 months) of optically active sotolon.

This work describes the influence of yeast population on the chemical characteristics of wine obtained by spontaneous and inoculated fermentation of must from Vitis vinifera Lado, a minor white grapevine autochthonous to Galicia (NW Spain). The study was carried out for two consecutive years. The results showed that musts derived from Lado presented a high acidity though the potential alcohol level was acceptable. The genetic diversity of S. cerevisiae strains isolated from spontaneous fermentations was low, probably due to must characteristics, although these did not interfere with the implantation of the commercial strains used. Analyses showed that the wines subsequently produced had high alcoholic levels and very high acidities (pH 3.0) as was expected from must composition. Wines obtained from spontaneous fermentations had a lower alcohol content but higher total acidity than those from inoculated fermentations. Monovarietal wines produced from Lado were poorly evaluated in sensorial tests because of their unbalanced structure and sourness; however, when they were mixed with other autochthonous white varieties with less acidity, the resulting wines were well accepted.

Application of sequential solvent extraction, followed by HPLC combined with the taste dilution analysis, enabled the localization of the most intense velvety astringent, drying, and puckering astringent, as well as bitter-tasting, compounds in red wine, respectively. Isolation of the taste components involving gel adsorption chromatography, ultrafiltration, and synthesis revealed the identification of 26 sensory-active nonvolatiles, among which several hydroxybenzoic acids, hydroxycinnamic acids, flavon-3-ol glycosides, and dihydroflavon-3-ol rhamnosides as well as a structurally undefined polymeric fraction (>5 kDa) were identified as the key astringent components. In contradiction to literature suggestions, flavan-3-ols were found to be not of major importance for astringency and bitter taste, respectively. Surprisingly, a series of hydroxybenzoic acid ethyl esters and hydroxycinnamic acid ethyl esters were identified as bitter compounds in wine. Taste qualities and taste threshold concentrations of the individual wine components were determined by means of a three-alternative forced-choice test and the half-mouth test, respectively.

The present article aims to review research papers that focus on the parietal adsorption activity of wine yeast and on its contribution to the enhancement of wine safety and quality. There is a great diversity among yeasts for their parietal adsorption activity: the outermost layer of the cell wall has a chemical composition that notably varies from yeast to yeast. Parietal mannoproteins can contain phosphate, pyruvate, or glucuronic acid, which impart negative charges, modifying the electrostatic and ionic interactions with wine components. The following could give a good reason to propose a specific selection of wine yeasts based on their parietal adsorption activity to improve wine safety and quality: (a) ochratoxin A content of wines is greatly reduced by expressly selected yeasts, sequestering the toxin during winemaking; (b) yeast influences concentration and composition of phenolic compounds in wine, above all by adsorbing them on cell wall; (c) among grape pigments, anthocyanins, in particular, may be adsorbed by yeast cell wall; and (d) yeast can also interact with wine colour producing anthocyanin-beta-d-glucosidase, pyruvic acid, and acetaldehyde or releasing mannoproteins and different polysaccharides. Genomic strategies could also be used to obtain a further enhancement of the adsorption/non-adsorption activity of wine yeasts. Based on winemaking requirements and on parietal adsorption activity, a specific selection of yeasts might be performed: (a) to protect wine colour during red winemaking, (b) to remove residual wine colour during white winemaking, (c) to selectively remove ochratoxin A, and (d) to protect phenolic compounds responsible for antioxidant activity.

Numerous studies have used a J-shaped or U-shaped curve to describe the relationship between alcohol use and total mortality. The nadir of the curves based on recent meta-analysis suggested optimal benefit at approximately half a drink per day. Fewer than 4 drinks per day in men and fewer than 2 per day in women appeared to confer benefit. Reductions in cardiovascular death and nonfatal myocardial infarction were also associated with light to moderate alcohol intake. Although some studies suggested that wine had an advantage over other types of alcoholic beverages, other studies suggested that the type of drink was not important. Heavy drinking was associated with an increase in mortality, hypertension, alcoholic cardiomyopathy, cancer, and cerebrovascular events, including cerebrovascular hemorrhage. Paradoxically, light-to-moderate alcohol use actually reduced the development of heart failure and did not appear to exacerbate it in most patients who had underlying heart failure. Numerous mechanisms have been proposed to explain the benefit that light-to-moderate alcohol intake has on the heart, including an increase of high-density lipoprotein cholesterol, reduction in plasma viscosity and fibrinogen concentration, increase in fibrinolysis, decrease in platelet aggregation, improvement in endothelial function, reduction of inflammation, and promotion of antioxidant effects. Controversy exists on whether alcohol has a direct cardioprotective effect on ischemic myocardium. Studies from our laboratory do not support the concept that alcohol has a direct cardioprotective effect on ischemic/reperfused myocardium. Perhaps the time has come for a prospectively randomized trial to determine whether 1 drink per day (or perhaps 1 drink every other day) reduces mortality and major cardiovascular events.

2-Isopropyl-3-methoxypyrazine (IPMP) is a grape-derived component of wine flavor in some wine varieties as well as the causal compound of the off-flavor known as ladybug taint (LBT), which occurs when Harmonia axyridis beetles are incorporated with the grapes during juice and wine processing. The main objective of this study was to obtain robust estimates of the orthonasal (ON) and retronasal (RN) detection thresholds (DTs) for IPMP in wines of differing styles. The ASTM E679 ascending forced choice method of limits was used to determine DTs for 47 individuals in 3 different wines--Chardonnay, Gewürztraminer, and a red wine blend of Baco Noir and Marechel Foch. The group best estimate thresholds (BETs) obtained for IPMP (ng/L) were Chardonnay, ON: 0.32; Gewürztraminer, ON: 1.56, RN: 1.15, and red wine blend, ON: 1.03, RN: 2.29. A large variation in individual DTs was observed. Familiarity with LBT was inversely correlated with DTs for Gewürztraminer, and no difference in thresholds was observed between winemakers and nonwinemakers. We conclude that the human DT for IPMP is extremely low and influenced significantly by wine style and evaluation mode. We recommend against the reporting of single-threshold values for wine flavor compounds, and encourage the determination of consumer rejection thresholds for IPMP in wine.

The ACE inhibitory activity (IACE) and the oxygen radical absorbance capacity (ORAC-FL) values of yeast peptides isolated from a model wine during accelerated autolysis of Saccharomyces cerevisiae have been studied. Samples were taken at 6, 24, 48, 121, and 144 h of autolysis. Peptide concentration increased throughout autolysis process. Peptides were fractionated into 2 fractions: F1, constituted by hydrophilic peptides, and F2, containing hydrophobic peptides. Both IACE activity and ORAC-FL values increased during 121 h of autolysis, then decreased afterward. Peptide fraction F2 was the main fraction involved in IACE activity and ORAC-FL.

Higher-quality Touriga Nacional (TN) wines are characterized by a fruity-citric aroma described as sweet and fresh citrus evoking the bergamot fruit (Citrus bergamia). In fact, "bergamot-like" descriptor is currently employed to rate higher quality TN wines. The aim of this work was to identify among volatile compounds present in bergamot fruit extracts (mainly terpenes) which of them contributes the most to the bergamot overall perception, and relate these data with the volatile composition of TN wines. The identification of the most important descriptors was done by sensory analysis. Among 18 descriptors 3 were selected: bergamot-like aroma, orange like, and violet. A GCO of a typical TN wine extract allows the identification of 3 related odorant zones ZO1, ZO2, and ZO3 related with bergamot-like aroma. Using AEDA, ZO2 was confirmed to be one of the most important odorant zones. Using AEDA the presence of linalool and linalyl acetate was confirmed. A similarity test was performed with a non-TN wine added with linalool and linalyl acetate alone or in combinations. The highest similarity value was observed when linalool (SV = 5.9) was added. In fact, results obtained from the analysis of several red wines from different varieties show that terpenols are present in higher amounts in wines coming from TN variety, which proves that these compounds can be the clue to the varietal aroma of TN wines.

Although the use of starter cultures in winemaking has improved the reproducibility and predictability of wine quality, the main drawback to this practice is the lack of the typical traits of wines produced by spontaneous fermentation. In this study, we identified for the first time the yeast population occurring during spontaneous fermentation of the Catalanesca white grape, a variety from Campania (Italy). Yeasts were identified using molecular tools: PCR-DGGE and partial sequence analysis of the 26S rRNA gene from isolates. Eighteen different species belonging to 11 different genera were identified. Hanseniaspora spp., Issatchenkia spp. and Candida spp. were the dominant yeasts during the early stages of fermentation, while the middle and end phases were dominated by Saccharomyces cerevisiae. Four species of Issatchenkia spp., rarely isolated from wine fermentation, were found in this study accounting for the 33.5% of the total isolates. The RAPD-PCR screening of the isolates followed by partial rRNA gene sequencing proved to be a very effective approach to first differentiate the isolates and then identify yeast species involved in a wine making procedure. The results show very high yeast diversity in this natural wine fermentation and also highlight the possibility of considering interesting autochthonous strains for starter selection.

Spontaneous fermentations are still conducted by several wineries in different regions of Argentina as a common practice. Native Saccharomyces strains associated with winery equipment, grape and spontaneous fermentations of Malbec musts from "Zona Alta del Río Mendoza" region (Argentina) were investigated during 2001 and 2002 in the same cellar. Low occurrence of Saccharomyces on grapes and their limited participation during fermentation were confirmed. Strain sequential substitution during fermentation was observed. Between 30% and 60% of yeast population at the end of fermentation was coming from yeasts already present in the winery. A stable and resident Saccharomyces micro-flora in the winery was confirmed. It exhibited a dynamic behaviour during season and between years. Commercial strains were found during fermentation in different percentages, but their presence on winery equipment was low. The present work represents a first approach to winery yeast and spontaneous fermentation Saccharomyces population dynamics in an important viticultural region from Argentina that has never been characterized before. The results obtained have an important significance for the local industry, showing for the first time the real situation of the microbial ecology of alcoholic fermentation in an industrial winery from Mendoza, Argentina.

The effect of yeasts on wine flavor response is of primary importance. The genus Saccharomyces, and mainly the species Saccharomyces cerevisiae, is responsible for alcoholic fermentation. Recently, several novel yeast isolates from wines have been described as hybrid yeasts between S. cerevisiae x S. kudriavzevii. We have analyzed their influence on two grape musts (Macabeo and Tempranillo) in fermentations conducted at four different temperatures (14, 18, 22 and 32 degrees C) by studying volatile compound production, sugar assimilation and other characteristics influencing the enological properties of wine caused by the impact of yeast. Hybrid yeasts behave particularly well at 14, 18 and 22 degrees C and the commercial strain of S. cerevisiae (T73) is better adapted at higher temperatures. Regarding the production of glycerol, acetic acid and malic acid, the hybrids display moderate behavior and concerning aromatic compound production, they are greater producers of higher alcohols. The behavior displayed by these hybrids in the fermentations studied in this work leads us to conclude that the use of hybrid strains can constitute an advantage in wine making.

Evolution of the microbial composition during the production of Vinsanto wine was investigated under different fermentation conditions to determine their impact on the yeast population and the wine sensorial characteristics. Fermentations were carried out according to the traditional process in 50-l barrels. Different fermentation conditions were applied (yeast inoculum, "mother sediment" addition and temperature) to standardise the Vinsanto production process. Fermentations and products were monitored over the aging period by chemical, microbial and sensory evaluation. The low temperature at the beginning of the fermentations under traditional cellar conditions results in prolonged survival of the non-Saccharomyces yeast. In contrast, Saccharomyces yeast populations dominated throughout the fermentation when the temperature of the cellar was maintained at a constant 16-18 degrees C. Results indicate that inoculation with a commercial yeast strain and fermentation temperature strongly influence the evolution of Vinsanto wine. The "mother sediment" seems to have no direct role as a microbiological starter in Vinsanto production but, despite this, it does have a strong influence on the sensory attributes of the Vinsanto wine. Our work highlights the importance of managing the fermenting microflora to improve the sensorial characteristics of Vinsanto wine.

Saccharomyces cerevisiae is the main yeast responsible for alcoholic fermentation of grape juice during wine making. This makes wine strains of this species perfect targets for the improvement of wine technology and quality. Progress in winemaking has been achieved through the use of selected yeast strains, as well as genetic improvement of wine yeast strains through the sexual and pararexual cycles, random mutagenesis and genetic engineering. Development of genetically engineered wine yeasts, their potential application, and factors affecting their commercial viability will be discussed in this review.

The effect of the application of two fungicides (cyprodinil alone and a mixture of cyprodinil and fludioxonil) on the mycoflora of grapes, especially ochratoxigenic fungi, was studied. Different doses and application times were analyzed. Grape mycobiota was isolated and identified, and the classification of black aspergilli was carried out. We found that 81.7% of the isolates belonged to Aspergillus niger aggregate and 18.3% to Aspergillus carbonarius. The ability to produce ochratoxin A (OTA) was studied on Czapek yeast extract agar (CYA) medium in 238 isolates. Most A. carbonarius (97.2%) produced detectable amounts of OTA, while only 2.9% of the A. niger aggregate were OTA producers. Most of the isolates (58%) produced less than 2.5 microg OTA per g of CYA. That, together with the highest levels of black aspergilli detected near harvest, proved the reported theory that they are primarily responsible for OTA in grapes. The fungicides studied had a significant effect on black aspergilli in three of the four vineyards sampled, as the natural increase of black aspergilli when approaching harvest was in general lower in all the fields treated with fungicides as compared to the control treatment. A mixture of cyprodinil (37.5%) and fludioxonil (25%) applied at veraison and 21 days before harvest was the most effective treatment to prevent black aspergilli in grapes, together with a single application of this mixture at veraison followed by an application of cyprodinil (50%) 21 days before harvest. No OTA was detected in musts (n=112) produced from either the control treatment or the treated grapes.

The influence of six fungicides (famoxadone, fenhexamid, fluquinconazole, kresoxim-methyl, quinoxyfen, trifloxystrobin) on the yeast content in harvested grapes has been studied along with their effect on it during the wine-making process. Two treatments were carried out with authorized formulates at the manufacturer doses. The first was carried out under good agricultural practices, obeying the security times, and the second one under critical conditions, applied on the day of harvesting. The grapes were harvested two hours after the application. During the wine-making process, samples were taken at 1, 5, 12 and 20 days after the start. The levels observed in the control sample (1.9 log CFU/cm2) were similar to previous studies. The counts in all treated samples were higher than the control sample, without adverse effect of the pesticides on the yeast count even in the grapes treated on the day of harvest. As with the observations on grapes, no effect of the pesticides on the fermentation count was observed. All counts on the treated samples were higher than the control sample. The treatment on the day of harvest did not have any effect on the fermentation count, with superior results being obtained for all the pesticides.

Interspecific hybrids obtained from flocculent Saccharomyces cerevisiae and non-flocculent Saccharomyces uvarum (S. bayanus var. uvarum) are flocculent to a higher degree and have the capacity to ferment well at both low and high temperatures, from 6 to 36 degrees C. These interspecific hybrids generate minor compounds of fermentation in intermediate quantities with respect to the parent strains and produce high-quality sparkling wines, which are not inferior to those of the parent strains. For these reasons, interspecific strains are particularly adapted to production of sparkling wines that are re-fermented in bottles.

The ochratoxigenic mycobiota of grapes intended for liqueur wines from four Spanish vineyards were studied. The specific wine-making technology of these wines requires overripening of the grapes on the vine or extended post-harvest exposure of the grapes in the sun. In every vineyard, samples were taken at three different developmental stages: veraison, harvesting time and after over-ripening. With the maturation of the berries there was a clear increase of Aspergillus spp. In the last sampling time studied, they were isolated from the 90.3% of the plated berries. Black aspergilli (mainly A. niger aggregate and A. carbonarius) were predominant among the different Aspergillus spp. isolated and constituted 98.5% of the total Aspergillus strains isolated. At harvesting time and after over-ripening, the percentage of colonized berries with A. carbonarius exceeded that of Aspergillus niger aggregate. Due to their low frequency of isolation, Penicillium spp. and Aspergillus spp. outside black aspergilli are not an important source of ochratoxin A in grapes for liqueur wine production. On the contrary, 98.5% of the A. carbonarius isolates screened were able to produce ochratoxin A. Although the possible participation of different ochratoxin A-producing species may occur, our results confirm that A. carbonarius is the most important source of ochratoxin A in liqueur wines, increasing its occurrence along the ripening of grapes.

We report the detection of several fungicide residues (Dichlofluanid, Benomyl, Iprodione, Procymidone and Vinclozolin) in red and white bottled wines from two Portuguese wine-producing zones. Studies were done in order to evaluate the active compound transfer percentage from grapes to the final product along fermentation process. We also investigated their effects in Saccharomyces and non-Saccharomyces yeasts as well as their influence on the physical, chemical and organoleptic wine properties. All the tested fungicides had a negative effect on in vitro yeast growth, with Dichlofluanid and Benomyl being the most toxic. Zygosaccharomyces rouxii and Saccharomyces cerevisiae were the most resistant yeasts while Rhodotorula glutinis was the most susceptible. Microvinification experiments carried out in the presence of fungicides produced wine samples with unaltered physical, chemical and organoleptic properties, though Benomyl, a slight delay in the initiation of fermentation process was observed.

To analyse the yeast population diversity during wine fermentations, specific fluorescein-labelled oligonucleotide probes targeted to the D1/D2 region of the 26S rRNA of different yeast species known to occur frequently in this environment were designed and tested with reference strains. The probes were then used to identify wine must isolates and to follow, in combination with plate counts, the evolution of yeast populations in two winery fermentations of white and red grape musts. In both cases, a high diversity of non-Saccharomyces yeast species was detected, including Candida stellata, Hanseniaspora uvarum, H. guilliermondii, Kluyveromyces marxianus, K. thermotolerans and Torulaspora delbrueckii. Some of these species (e.g., K. marxianus, K. thermotolerans and T. delbrueckii) were present in significant amounts during the tumultuous fermentation stage, despite the predominance of Saccharomyces cerevisiae cells following the inoculation of the wine musts with a starter strain. To further clarify the yeast population dynamics at the late phase of the fermentations, and because winery conditions do not allow a reliable control of experimental variables, strains isolated from the industrial musts were used to conduct two laboratory microvinifications in synthetic grape juice, using different ratios of S. cerevisiae/non-Saccharomyces in the inocula. Under these conditions, the results were similar to those obtained in the winery, showing a yeast profile with mixed species throughout the first fermentation stage, i.e. until about 40-50% of the total sugar was consumed. Non-Saccharomyces yeasts were outgrown by S. cerevisiae only after ethanol reached concentrations around 4-5% (v/v), which argues in favour of a potential important role of non-Saccharomyces in the final organoleptic characteristics of the wine.

Xynomavro and Muscat Hamburg grapes were fermented spontaneously, producing red and white wine, respectively. The indigenous yeast flora, isolated at three phases of these fermentations, was studied. One hundred and ten strains were isolated, eighty-four of which were identified as Saccharomyces cerevisiae. Six of these strains were selected for their satisfactory oenological properties to be tested as starters in wine fermentations. The quality of the produced wines was evaluated after determination of their aromatic compounds and sensory analysis. S. cerevisiae strain BT4, as well as the AM4+BM3 combination, were considered the best as they produced wines characterized by fruity aromas and were highly accepted among panelists.

In agricultural sciences, breeding strategies have historically been used to select new, optimized plant varieties or animal breeds. Similar strategies are possible for genetic improvement of wine yeasts. We optimized 11 relevant enological traits in a single clone using successive hybridization and segregation steps. A hybrid obtained by crossing two parent strains derived from commercial wine yeasts showed that some of the traits were readily optimized. Dominance/recessivity, heterosis and transgression were observed among 51 segregating progeny. On the basis of this information, all the optimal characters from both parents were combined in a single strain following two targeted sexual crosses. This article presents a powerful methodology for obtaining a single wine strain with numerous fermentative qualities that does not produce off-flavors.

The post-harvest mycobiota of dried grapes, used in Friuli Venezia Giulia (Northern-East Italy) for the production of "passito" dessert wines, was investigated in order to detect potential ochratoxin A (OTA)-producers. Five grape cultivars were analysed and only isolates belonging to the genera Aspergillus and Penicillium were evaluated. No Aspergillus spp. was found while 379 strains of Penicillium spp. were isolated. Four strains produced UV fluorescent metabolites on grape juice agar and synthetic liquid media as observed by thin-layer chromatography (TLC). Three of these resulted OTA producers when analyzed by high pressure liquid chromatography (HPLC), following immunoaffinity column purification. According to the results of morphological examinations and ribosomal DNA sequencing, the OTA producer strains did not belong to the species P. verrucosum or P. nordicum. The corresponding passito wines did not contain OTA.

Amine and organic acid composition of Aszú wines from the Tokaj region of Hungary, nonbotrytized Hungarian wines from different regions, and foreign botrytized wines were analyzed by high-performance liquid chromatography. Hungarian and foreign wines (36 Hungarian and seven foreign botrytized wines) were compared in different ways by calculation of ratios of given amine compounds, analyses of variance, principal component, and discriminant analysis. In wines, putrescine and in some samples 3-methyl-butylamine and/or phenyl ethylamine were found in remarkable concentrations, while in botrytized wines four other amines were verified in high concentration. Good separation between Aszú and foreign botrytized wines was found by calculation of the amine component's ratio. The first two principal components of the principal component analysis accounted for 77 and 84% of the total variance in the data of amines and acids, respectively. The component scores of samples grouped according to Aszú, foreign botrytized, and nonbotrytized wines. Linear discriminant analysis was used for differentiation of Aszú, foreign botrytized, and normal wines. Using nine amines and two acids as variables, the correct classification was 97.6%. On the basis of results, an objective evaluation method can be elaborated for quality control in order to protect the authenticity and origin of wine specialties made from botrytized grapes.

Lactic acid bacteria play an important role in wine-making by undertaking the malolactic fermentation, yet little information is available on other aspects of their physiology, such as their profile of external enzymatic activities. In this study we sought evidence for the existence and action of glycosidase enzymes in wine isolates of Oenococcus oeni. This group of enzymes is of interest because of their potential for liberation of grape-derived aroma compounds from their natural glycosylated state. This comprehensive study reveals that these bacteria produce glycosidases that might be important in wine-making. Strains did not necessarily hydrolyse all substrates tested, but rather were grouped according to substrate specificity. Thus a subset comprising strains 2, 5 and 16 possessed high cumulative activities against beta-d- and alpha-d-glucopyranoside substrates, while a group comprising strains 4, 21 and 22 was noted for superior hydrolysis of beta-d-xylopyranoside, alpha-l-rhamnopyranoside and alpha-l-arabinofuranoside substrates. Key physico-chemical inhibitors of analogous systems from other microorganisms were seen to produce variable responses across the strains investigated here. Accordingly, several strains retained significant hydrolytic activity at typical wine pH values ( approximately 3.0-4.0), residual glucose and fructose contents (up to 20 g/L), and ethanol contents (up to 12%). These findings highlight the potential of O. oeni as a useful alternative source of glycosidase enzymes for use in wine-making.

Wines by yeast mutants producing polygalacturonase in high glucose concentration, from Saccharomyces wine-making strains, had higher filterability and more concentrated anthocyanin contents than that of their parent strains. These results suggest that the clarification process was improved at a lower cost by the low viscosity and that high-quality wines result from the increase in the anthocyanin contents.

Penicillium was used to immobilize Saccharomyces cerevisiae, without using physico-chemical external supports, to form yeast biocapsules. The biocapsules, once the Penicillium was killed by the ethanol produced, were used in a grape must fermentation. Must fermentation was carried out for 160 h with the biocapsules and for 300 h with free yeast cells. Acetaldehyde (84 vs. 63 mg/l), isobutanol (217 vs. 194 mg/l), L: -proline (7.7 vs. 6.5 mM: ) and aspartic acid (0.42 vs. 0 mM: ) in final wine were higher with the biocapsules than with free cells.

The amperometric biosensor based on lactate oxidase for determination of lactate has been developed, and two methods of immobilization of lactate oxidase on the surface of industrial screen-printed platinum electrodes SensLab were compared. A sensor with immobilized in the Resydrol polymer lactate oxidase by the method of physical adsorption is characterized of narrow dynamic range and greater response value in comparison with a biosensor based on immobilised in poly(3,4-ethylenedioxythiophene) lactate oxidase by the method of electrochemical polymerization. Operational stability of the biosensor developed was studied and it was shown, that the immobilization method does not influence their stability. The analysis of the lactate in wine and during wine fermentation has been conducted. High correlation of the data obtained by means of amperometric lactate biosensor and a standard method of an ionic chromatography has been shown. The developed biosensor could be applied in the food industry for the control and optimization of the wine fermentation process, and quality control of wine.

In recent decades, science and food technology have contributed at an accelerated rate to the introduction of new products to satisfy nutritional, socio-economic and quality requirements. With the emergence of modern molecular genetics, the industrial importance of Saccharomyces cerevisiae, is continuously extended. The demand for suitable genetically modified (GM) S. cerevisiae strains for the biofuel, bakery and beverage industries or for the production of biotechnological products (e.g. enzymes, pharmaceutical products) will continuously grow in the future. Numerous specialised S. cerevisiae wine strains were obtained in recent years, possessing a wide range of optimised or novel oenological properties, capable of satisfying the demanding nature of modern winemaking practise. The unlocking of transcriptome, proteome and metabolome complexities will contribute decisively to the knowledge about the genetic make-up of commercial yeast strains and will influence wine strain improvement via genetic engineering. The most relevant advances regarding the importance and implications of the use of GM yeast strains in the wine industry are discussed in this mini-review. In this work, various aspects are considered including the strategies used for the construction of strains with respect to current legislation requirements, the environmental risk evaluations concerning the deliberate release of genetically modified yeast strains, the methods for detection of recombinant DNA and protein that are currently under evaluation, and the reasons behind the critical public perception towards the application of such strains.

A molecularly-imprinted polymer (MIP) was prepared by electropolymerization of pyrrole (Py) onto a stainless steel frit, using ochratoxin A (OTA) as the template, in order to make a micro solid phase preconcentration (microSPP) device. The OTA template was removed with 1% triethylamine (TEA) in methanol. Compared to non-imprinted polypyrrole (PPy), the molecularly-imprinted polypyrrole (MIPPy) enhanced the selective binding of OTA. The percentage recovery improved from 0 to 40% when the OTA sample solution was acidified with 1 M HCl (1% by volume). At a flow rate of 0.2 mL/min, maximum OTA binding was reached in 6 min after a total loading of 3.2 ng OTA. Final elution of the OTA was analyzed by high performance liquid chromatography (HPLC) with fluorescence detection, using 20:80 v/v acetonitrile-ammonia buffer (NH4Cl/NH3, 20 mM, pH 9.2) as the mobile phase. The MIPPy-microSPP-HPLC results clearly demonstrated that the MIPPy-microSPP device afforded selective preconcentration of OTA from red wine samples, at OTA concentration levels as low as 0.05 ppb, prior to HPLC analysis.