Analysis of ethylidene-bridged flavan-3-ols in wine

A method was developed to determine the amount of ethylidene-bridged flavan-3-ols in wine. The method was based upon the analysis of 2,2'-ethylidenediphloroglucinol (EDP), a product formed after acid-catalyzed cleavage of wine flavan-3-ols in the presence of excess phloroglucinol. In the developed analytical method, the wine was purified and concentrated using C18 solid-phase extraction before the phloroglucinolysis reaction was carried out. This procedure was used to quantify ethylidene-bridged flavan-3-ols in wine and the molar ratio between ethylidene-bridged linkages and native interflavan linkages. The method validation showed 9.2% repeatability. The recovery of the ethylidene-bridged flavanols in wine was 90% for concentrations up to 4.5 mg.L-1 of ethylidene-bridged linkages, and it decreases to 83% above and until the concentration reached 7.6 mg.L-1. Initial results showed that the concentration of ethylidene-bridged flavan-3-ols measured in wines was very low (less than 1.3 mg.L-1) and that they represented less than 1.3% of the total interflavonoid linkages on a molar ratio.     

Monomeric, oligomeric, and polymeric flavan-3-ol composition of wines and grapes from Vitis vinifera L. Cv. Graciano, Tempranillo, and Cabernet Sauvignon

The monomeric, oligomeric, and polymeric flavan-3-ol composition of wines, grape seeds, and skins from Vitis vinifera L. cv. Graciano, Tempranillo, and Cabernet Sauvignon has been studied using (1) fractionation by polyamide column chromatography followed by HPLC/ESI-MS analysis, (2) fractionation on C(18) Sep-Pak cartridges followed by reaction with vanillin and acid-catalyzed degradation in the presence of toluene-alpha-thiol (thiolysis). The content of monomers ((+)-catechin and (-)-epicatechin), procyanidin dimers (B3, B1, B4, and B2), trimers (T2 and C1), and dimer gallates (B2-3-O-gallate, B2-3'-O-gallate, and B1-3-O-gallate) ranged from 76.93 to 133.18 mg/L in wines, from 2.30 to 8.21 mg/g in grape seeds, and from 0.14 to 0.38 mg/g in grape skins. In wines, the polymeric fraction represented 77-84% of total flavan-3-ols and showed a mean degree of polymerization (mDP) value of 6.3-13.0. In grapes, the polymeric fraction represented 75-81% of total flavan-3-ols in seeds and 94-98% in skins and showed mDP values of 6.4-7.3 in seeds and 33.8-85.7 in skins. All the monomeric flavan-3-ols and oligomeric procyanidins found in wines were also present in seeds, although differences in their relative abundances were seen. The skin polymeric proanthocyanidins participated in the equilibration of the wine polymeric proanthocyanidin fraction, especially contributing to the polymer subunit composition and mDP.     

Influence of vine vigor on grape (Vitis vinifera L. Cv. Pinot Noir) and wine proanthocyanidins

The relationships between variations in grapevine (Vitis vinifera L. cv. Pinot noir) growth and resulting fruit and wine phenolic composition were investigated. The study was conducted in a commercial vineyard consisting of the same clone, rootstock, age, and vineyard management practices. The experimental design involved monitoring soil, vine growth, yield components, and fruit composition (soluble solids, flavan-3-ol monomers, proanthocyanidins, and pigmented polymers) on a georeferenced grid pattern to assess patterns in growth and development. Vine vigor parameters (trunk cross-sectional area, average shoot length, and leaf chlorophyll) were used to delineate zones within both blocks to produce research wines to investigate the vine-fruit-wine continuum. There was no significant influence of vine vigor on the amount of proanthocyanidin per seed and only minimal differences in seed proanthocyanidin composition. However, significant increases were found in skin proanthocyanidin (mg/berry), proportion of (-)-epigallocatechin, average molecular mass of proanthocyanidins, and pigmented polymer content in fruit from zones with a reduction in vine vigor. In the wines produced from low-vigor zones, there was a large increase in the proportion of skin tannin extracted into the wine, whereas little change occurred in seed proanthocyanidin extraction. The level of pigmented polymers and proanthocyanidin molecular mass were higher in wines made from low-vigor fruit compared to wines made from high-vigor fruit, whereas the flavan-3-ol monomer concentration was lower.     

Phenolic compounds and browning in sherry wines subjected to oxidative and biological aging

The composition in hydroxybenzoic and hydroxycinnamic acids, hydroxycinnamic esters, tyrosol, syringaldehyde, and flavan-3-ol derivatives of three different types of sherry wine obtained by aging of the same starting wine under different conditions was studied. So-called "fino" wine was obtained by biological aging under flor yeasts, "oloroso" wine by oxidative aging, and "amontillado" wine by a first stage of biological aging followed by a second oxidative step. On the basis of the results, the wines subjected to oxidative aging exhibited higher phenol contents, in addition to scarcely polar compounds absorbing at 420 nm that were absent in the wines obtained by biological aging. Taking into account that flavan-3-ol derivatives play an important role in wine browning, a model catechin solution was inoculated with flor yeast which, contrary to the findings of other authors in the absence of yeasts, formed no colored compounds. This different behavior may account for the resistance to browning of pale sherry wines in the presence of flor yeasts.     

Effect of SO2 concentration on polyphenol development during red wine micro-oxygenation

A Merlot wine in 15 L research tanks was subjected to micro-oxygenation at 10 mL O2 per liter of wine per month over a 16 week period with additions of 0, 50, 100, and 200 mg/L SO2. A large decrease in monomeric anthocyanins and flavan-3-ols was seen in wines with a lower concentration of SO2, coupled with an increase in nonbleachable pigments; an increase in tannin, measured using precipitation with methyl cellulose; and a greater size and red coloration of a proanthocyanidin extract obtained using Sephadex LH-20. These changes were largely suppressed in wines initially treated with 200 mg/L SO2 and occurred more slowly in wines stored in bottles in the absence of O2. The concentration of SO2 is shown to regulate the polyphenol chemistry involved in the formation of polymeric pigments and changes in tannin structure affecting wine astringency.     

Analysis of the oxidative degradation of proanthocyanidins under basic conditions

Proanthocyanidin isolates from grape (Vitis vinifera L. cv. Pinot noir) skin and seed underwent oxidative degradation in solution (10 g/L) under basic conditions while exposed to atmospheric oxygen. Degradation was monitored by reversed-phase HPLC following acid-catalyzed cleavage in the presence of excess phloroglucinol (phloroglucinolysis) and by high-performance gel permeation chromatography. All isolates degraded under these conditions and followed second-order kinetics for over 1 half-life, consistent with an oxidation reaction. The conversion of proanthocyanidins to known subunits (conversion yield) when measured by phloroglucinolysis showed a dramatic decline over the course of the reaction. With the exception of (+)-catechin extension subunits, all individual subunits decreased in concentration during the oxidation process, also following second-order kinetics for over 1 half-life. Skin proanthocyanidins degraded the fastest due to the presence of (-)-epigallocatechin extension subunits. Seed procyanidins were degraded with and without flavan-3-ol monomers. Flavan-3-ol monomers slowed the rate of seed procyanidin degradation. The mean degree of polymerization (mDP) determined by phloroglucinolysis indicated a large decrease in mDP as the reaction progressed; yet, by GPC, the size distribution of all proanthocyanidins changed little in comparison. The conversion yield could be an important parameter to follow when using phloroglucinolysis as a means for determining proanthocyanidin mDP, and when monitoring the oxidative degradation of proanthocyanidins.     

Orosensory-directed identification of astringent mouthfeel and bitter-tasting compounds in red wine

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.     

Characteristic phenolic composition of single-cultivar red wines of the Canary Islands (Spain)

The detailed phenolic composition of five single-cultivar (Baboso Negro, Listán Negro, Negramoll, Tintilla, and Vijariego Negro) young and aged (vintages 2005-2009) red wines of the Canary Islands has been determined by HPLC-DAD-ESI-MS(n). Despite the total monomeric anthocyanin content decreasing for older wines in each set of single-cultivar wines, the corresponding anthocyanin profiles remained almost unchanged. Although all wine anthocyanin profiles were dominated by malvidin 3-glucoside, their differentiation by grape cultivar was possible, with the exception of Listán Negro. In contrast, the total content of non-anthocyanin phenolics did not appreciably change within vintages but polymerization, hydrolysis, and isomerization reactions greatly modified the phenolic profiles. Aglycone-type flavonol profiles offered the best results for differentiation of the wines according to grape cultivar (Listán Negro and Negramoll; Baboso Negro and Vijariego Negro; and Tintilla). Within flavan-3-ols, the B-ring trihydroxylated monomers ((-)-epigallocatechin and (-)-gallocatechin) and also (-)-epicatechin provided additional cultivar differentiation. Hydroxycinnamic acid derivatives and stilbene profiles were very heterogeneous with regard to both grape cultivar and vintage and did not significantly contribute to wine differentiation, even when structure-type profiles were obtained, with the exception of Tintilla, which always appeared as the most different single-cultivar wines. Finally, most Canary Islands wines showed characteristic high contents of stilbenes, especially trans-resveratrol.     

Quantitative reconstruction of the nonvolatile sensometabolome of a red wine

The first comprehensive quantitative determination of 82 putative taste-active metabolites and mineral salts, the ranking of these compounds in their sensory impact based on dose-over-threshold (DoT) factors, followed by the confirmation of their sensory relevance by taste reconstruction and omission experiments enabled the decoding of the nonvolatile sensometabolome of a red wine. For the first time, the bitterness of the red wine could be demonstrated to be induced by subthreshold concentrations of phenolic acid ethyl esters and flavan-3-ols. Whereas the velvety astringent onset was imparted by three flavon-3-ol glucosides and dihydroflavon-3-ol rhamnosides, the puckering astringent offset was caused by a polymeric fraction exhibiting molecular masses above >5 kDa and was found to be amplified by the organic acids. The perceived sourness was imparted by l-tartaric acid, d-galacturonic acid, acetic acid, succinic acid, l-malic acid, and l-lactic acid and was slightly suppressed by the chlorides of potassium, magnesium, and ammonium, respectively. In addition, d-fructose and glycerol as well as subthreshold concentrations of glucose, 1,2-propandiol, and myo-inositol were found to be responsible for the sweetness, whereas the mouthfulness and body of the red wine were induced only by glycerol, 1,2-propandiol, and myo-inositol.     

Influence of different phenolic copigments on the color of malvidin 3-glucoside

The effectiveness of seven phenolic compounds (catechin, epicatechin, procyanidin B2, caffeic acid, p-coumaric acid, myricitrin, and quercitrin) as copigments of malvidin 3-glucoside, the major anthocyanin in red wines from Vitis vinifera, using a copigment/pigment molar ratio of 1:1 was assayed in model wine solutions under the same conditions (pH=3.6, 12% ethanol). The stability of the copigment-pigment complexes formed was studied during a storage period of 60 days at 25 degrees C. Tristimulus colorimetry was applied for color characterization of the copigmentation process, and HPLC-DAD-MS was used to monitor changes in the composition of the samples. Copigmentation has been found to occur in all cases despite the low copigment/pigment molar ratio used, although the effect was different depending on the compound. Flavan-3-ols appeared as the less effective copigments, procyanidin B2 being even worse than monomeric flavanols, whereas flavonols behaved as the best ones. These latter copigments also induced the most statistically significant bathochromic shift in lambdamax. In the colorimetric analysis, it was observed that the lightness L* of the copigmented solutions increased with time, chroma C*ab decreased, and the hue hab increased. The copigments that produced a greater increase in the hue angle were the monomeric flavan-3-ols, which therefore showed to be the least protective cofactors against browning of the anthocyanin solution during the storage. With the time of storage, the formation of new pigments was observed in the solutions containing flavanols (xanthylium structures) and hydroxycinnamic acids (pyranoanthocyanins), which explains some of the color modifications produced in these solutions. Another relevant observation was that the stability of the anthocyanin was not much improved by most of the assayed copigments, since quite similar degradation rates were observed in the presence and absence of those cofactors.     

Adsorption of phenolic compounds and browning products in white wines by yeasts and their cell walls

Dehydrated yeast cells at variable concentrations were used as fining agents to decrease the color of white wines with two different degrees of browning (0.153 and 0.177 au, measured at 420 nm). Both wines showed a linear decrease of browning with increasing yeast concentration. However, in terms of efficiency, the yeasts exhibited a higher color lightening at greater concentrations acting on the darker wine. This suggests a preferential retention of some types of yellow-brown compounds that could increase their concentrations at the higher degree of browning. To confirm the role of yeast cell walls in the retention of browning compounds and to evaluate their potential use as fining agents, they were applied at variable concentrations to a browned wine (0.175 au). The cell walls were found to be the active support for the adsorption of browning compounds, but their efficiency was much lower than that of an equivalent amount of the yeast cells from which they were obtained. Finally, HPLC determinations of low-molecular-weight phenolic compounds showed flavan-3-ol derivatives to be significantly retained by both yeasts and their cell walls.     

Quantitative analysis of red wine tannins using Fourier-transform mid-infrared spectrometry

Tannin content and composition are critical quality components of red wines. No spectroscopic method assessing these phenols in wine has been described so far. We report here a new method using Fourier transform mid-infrared (FT-MIR) spectroscopy and chemometric techniques for the quantitative analysis of red wine tannins. Calibration models were developed using protein precipitation and phloroglucinolysis as analytical reference methods. After spectra preprocessing, six different predictive partial least-squares (PLS) models were evaluated, including the use of interval selection procedures such as iPLS and CSMWPLS. PLS regression with full-range (650-4000 cm(-1)), second derivative of the spectra and phloroglucinolysis as the reference method gave the most accurate determination for tannin concentration (RMSEC = 2.6%, RMSEP = 9.4%, r = 0.995). The prediction of the mean degree of polymerization (mDP) of the tannins also gave a reasonable prediction (RMSEC = 6.7%, RMSEP = 10.3%, r = 0.958). These results represent the first step in the development of a spectroscopic methodology for the quantification of several phenolic compounds that are critical for wine quality.     

Effect of ripeness and postharvest storage on the phenolic profiles of Cherries (Prunus avium L.)

The phenolic compounds hydroxycinnamates, anthocyanins, flavonols, and flavan-3-ols of sweet cherry cultivars Burlat, Saco, Summit, and Van harvested in 2001 and 2002 were quantified by HPLC-DAD. Phenolics were analyzed at partially ripe and ripe stages and during storage at 15 +/- 5 degrees C (room temperature) and 1-2 degrees C (cool temperature). Neochlorogenic and p-coumaroylquinic acids were the main hydroxycinnamic acid derivatives, but chlorogenic acid was also identified in all cultivars. The 3-glucoside and 3-rutinoside of cyanidin were the major anthocyanins. Peonidin and pelargonidin 3-rutinosides were the minor anthocyanins, and peonidin 3-glucoside was also present in cvs. Burlat and Van. Epicatechin was the main monomeric flavan-3-ol with catechin present in smaller amounts in all cultivars. The flavonol rutin was also detected. Cultivar Saco contained the highest amounts of phenolics [227 mg/100 g of fresh weight (fw)] and cv. Van the lowest (124 mg/100 g of fw). Phenolic acid contents generally decreased with storage at 1-2 degrees C and increased with storage at 15 +/- 5 degrees C. Anthocyanin levels increased at both storage temperatures. In cv. Van the anthocyanins increased up to 5-fold during storage at 15 +/- 5 degrees C (from 47 to 230 mg/100 g of fw). Flavonol and flavan-3-ol contents remained quite constant. For all cultivars the levels of phenolic acids were higher in 2001 and the anthocyanin levels were higher in 2002, which suggest a significant influence of climatic conditions on these compounds.     

Quantitative determination of sulfur-containing wine odorants at sub parts per billion levels. 2. Development and application of a stable isotope dilution assay

[(2)H(10)]-4-Mercapto-4-methylpentan-2-one (d(10)-1), [(2)H(2)]-3-mercaptohexan-1-ol (d(2)-2), and [(2)H(5)]-3-mercaptohex-1-yl acetate (d(5)-3), deuterated analogues of impact odorants of wines, were used to determine quantitatively the natural compounds in white wines (Muscadet, Sauvignon, and Bacchus) with a stable isotope dilution assay using gas chromatography coupled either with ion trap tandem mass spectrometry (GC-ITMS-MS) or with atomic emission detection monitored on sulfur-selective acquisition (GC-AED). The thiol compounds were recovered from wines by liquid-liquid extraction, then purified from the wine extracts by covalent chromatography, and analyzed. The quantitative determination of 4-mercapto-4-methylpentan-2-one 1 in the wines that were analyzed was performed better with GC-AED than with GC-ITMS-MS under the conditions that were used. However, the detection limit of the method was higher than the odor threshold of 4-mercapto-4-methylpentan-2-one 1 in wine (5 vs 0.8 ng/L). The levels of this compound in the Sauvignon and Bacchus wines were much higher than its odor threshold, but it was not detectable in the Muscadet wines. On the contrary, GC-ITMS-MS was much more sensitive than GC-AED for detection of 3-mercaptohexan-1-ol 2 and 3-mercaptohex-1-yl acetate 3, and the detection limits were much lower than their odor thresholds in wine. The former compound was detected in all of the Muscadet wines that were analyzed at levels always higher than its odor detection threshold, while the latter occurred at levels higher than its odor threshold in only one Muscadet wine.     

Aggregation of a proline-rich protein induced by epigallocatechin gallate and condensed tannins: effect of protein glycosylation

Astringency is one of the most important organoleptic qualities of numerous beverages, including red wines. It is generally thought to originate from interactions between tannins and salivary proline-rich proteins (PRPs). In this work interactions between a glycosylated PRP, called II-1, and flavan-3-ols were studied in aqueous solutions and at a colloidal level, by dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS). The flavan-3-ols were a monomer, epigallocatechin gallate (EGCG), and polymerized flavan-3-ol fractions extracted from grape seeds. In aqueous solutions containing EGCG and protein II-1, protein aggregation took place when protein concentration and the EGCG/protein ratio exceeded a threshold. The aggregates had a small size, comparable with the dimensions of protein monomers, and formed stable dispersions (no phase separation). Most proteins remained free in solution. This behavior is in sharp contrast with the phase separation observed for nonglycoslated PRP in the same conditions. Moreover, this slight aggregation of II-I in the presence of EGCG was disrupted by the addition of 12% ethanol. Increasing the flavan-3-ol molecular weight strongly enhanced II-I/tannin aggregation: the threshold was at a lower protein concentration (0.2 mg/mL) and a lower tannin/protein ratio. Still, in most cases, and in contrast with that observed with a nonglycosylated PRP, the aggregates remained of discrete size and stable. Only at low ethanol content (2%) did the addition of tannin polymers finally lead to phase separation, which occurred when the molar ratio of tannins to proteins exceeded 12. This systematic effect of ethanol confirmed the strong effect of cosolvents on protein/tannin interactions.     

Relationship between red wine grade and phenolics. 2. Tannin composition and size

Commercial red wines ( Vitis vinifera L. cv. Shiraz) produced during the 2009 vintage underwent winemaker assessment for allocation grade soon after production. The wines were then subjected to phenolic analysis to measure wine color (total anthocyanin, SO(2) nonbleachable pigment, and wine color density) and tannins (concentration, composition, and average degree of polymerization). A positive relationship was found between wine phenolic concentration and projected bottle price. Tannin compositional analysis suggested that there was specifically a relationship between wine grade and skin-derived tannins. These results suggest that maximization of skin tannin concentration and/or proportion is related to an increase in projected wine bottle price.     

Effect of fungicide residues on the aromatic composition of white wine inoculated with three Saccharomyces cerevisiae strains

The effects of three fungicide residues (cyprodinil, fludioxonil, and pyrimethanil) on the aromatic composition (acids, alcohols, and esters) of Vitis vinifera white wines (var. Airén) inoculated with three Saccharomyces cerevisiae strains (syn. bayanus, cerevisiae, and syn. uvarum) are studied. The aromatic exponents were extracted and concentrated by adsorption-thermal desorption and were determined by gas chromatography using a mass selective detector. The addition of the three fungicides at different doses (1 and 5 mg/L) produces significant differences in the acidic fraction of the aroma, especially in the assays inoculated with S. cerevisiae, although the final contents do not exceed the perception thresholds. The lower quality wines, according to isomeric alcohol content [(Z)-3-hexen-1-ol and 3-(methylthio)propan-1-ol] are those obtained by inoculation with S. cerevisiae(syn. bayanus) and addition of cyprodinil. The addition of fungicides in the assays inoculated with S. cerevisiae (syn. bayanus) produces an increase in the ethyl acetate and isoamyl acetate contents, which causes a decrease in the sensorial quality of the wine obtained.     

Influence of ethanol content on the extent of copigmentation in a cencibel young red wine

The effect of ethanol content on the copigmentation of a Cencibel young red wine was studied, by means of ethanol elimination and reconstitution of the initial volume with different ethanol proportions. The reference wine (14.0% ethanol, 0.40 g/L volatile acidity) showed a bathochromic shift of 4 nm and a color enhancement (DeltaColor) of 41%, lower than that found for the reconstituted wine with the same ethanol content (53%). This discrepancy could be attributable to the loss of acetic acid during the ethanol elimination step. DeltaColor was 95% for the reconstituted wine without ethanol and decreased until 18% for the reconstituted wine with 22% ethanol. Copigmentation was important for reconstituted wines with ethanol contents typical for table red wine, showing DeltaColor between 53 and 57%. An increase in ethanol content in reconstituted wines was accompanied by an increase in the pH value. Perceivable changes in color (DeltaE > 1) followed every increase in ethanol content.     

Phenolic and volatile composition of wines made from Vitis vinifera cv. Muscat lefko grapes from the island of Samos

A study of the phenolic and volatile composition of wines produced from the white cultivar Muscat lefko from the island of Samos was conducted. Dry, fortified, naturally sweet wines and mistelles (aged and nonaged) have been studied. The phenolic components (flavan-3-ols, hydroxycinnamates, and flavonols) were measured by high-performance liquid chromatography after solid phase extraction (SPE). The terpenes (free and glycosidically linked) were determined by the use of gas chromatography-mass spectrometry (GC-MS) after SPE. The fermentation aroma components were analyzed by GC-MS after liquid-liquid extraction. It was found that the dry wines contained lower amounts of most of the phenolics and higher quantities of terpenes and fermentation aroma compounds than the sweet wines. The aged mistelle wines contained lower levels of coutaric and caftaric acids, higher concentrations of the free acids, and markedly fewer free and bound terpenes and fermentation aroma components compared to the other sweet wines. The naturally sweet wine contained relatively increased amounts of phenolics, 2,3-butanediol, and glycosidically linked terpenes.