Inhibition of ALDH2 by quercetin glucuronide suggests a new hypothesis to explain red wine headaches.

The consumption of red wine induces headaches in some subjects who can drink other alcoholic beverages without suffering. The cause for this effect has been attributed to a number of components, often the high level of phenolics in red wine, but a mechanism has been elusive. Some alcohol consumers exhibit flushing and experience headaches, and this is attributed to a dysfunctional ALDH2 variant, the enzyme that metabolizes acetaldehyde, allowing it to accumulate. Red wine contains much higher levels of quercetin and its glycosides than white wine or other alcoholic beverages. We show that quercetin-3-glucuronide, a typical circulating quercetin metabolite, inhibits ALDH2 with an IC50 of 9.6 µM. Consumption of red wine has been reported to result in comparable levels in circulation. Thus, we propose that quercetin-3-glucoronide, derived from the various forms of quercetin in red wines inhibits ALDH2, resulting in elevated acetaldehyde levels, and the subsequent appearance of headaches in susceptible subjects. Human-subject testing is needed to test this hypothesis.

Wine Closure Performance of Three Common Closure Types: Chemical and Sensory Impact on a Sauvignon Blanc Wine.

A Napa Valley Sauvignon blanc wine was bottled with 200 each of a natural cork, a screw cap, and a synthetic cork. As browning is an index for wine oxidation, we assessed the brown color of each bottle with a spectrophotometer over 30 months. A random-effects regression model for longitudinal data on all bottles and closure groups found a browning growth trajectory for each closure group. Changes in the wine's browning behavior at 18 months and 30 months showed that the browning of the wine bottles appeared to slow down later in the storage period, especially for natural corks. The between-bottle variation was the highest for the natural cork. At 30 months, we separated the bottles by the extent of browning and samples were pulled from the high, mid, and low levels of browning levels for each closure. The degree of browning is inversely correlated with free SO2 levels ranging from 5 to 12 mg/L. However, a Quantitative Descriptive Analysis (QDA™) sensory panel could not detect any difference in their aroma and flavor profile between closure types regardless of browning level. Even low levels of free SO2 retain protection against strong oxidation aromas, and visual browning detected by spectrophotometer seemed to precede oxidative aroma and flavor changes of the aging Sauvignon blanc.

A novel method combining stable isotopic labeling and high-resolution mass spectrometry to trace the quinone reaction products in wines.

Quinone formation is a key initial step of wine oxidation. Nucleophiles sacrificially react with quinones to sustain color and aroma, but due to the complexity of wine, determining the identity of the constitutive nucleophile has been challenging. Here we apply a novel stable-isotope labelling approach combined with high-resolution mass spectrometry, using 13C6-labelled ortho-quinone. This allows for the specific detection of quinone reaction products with M and M + 6x peak feature-pairs in real wines. Analysis using MetExtract II successfully identified 225 quinone reaction suspects in negative mode in Sauvignon blanc wine, and 120 in Cabernet Sauvignon. Ten quinone reaction products with the most abundant peak areas were tentatively identified using a mass/structure workflow. It appears that sulfides largely quench quinones in white wines, whereas flavonoids are the dominant reactants in red wines. The latter result demonstrates how skin/seed extraction preserves red wine.

Acid complexation of iron controls the fate of hydrogen peroxide in model wine.

At a key branchpoint in wine oxidation, hydrogen peroxide reacts either with iron(II), leading to the Fenton oxidation of ethanol, or with sulfur dioxide, precluding oxidation. The fate of H2O2 was investigated in anoxic model wines with different pH and acid buffers. In the absence of SO2, anoxic conditions allowed the stoichiometric production of acetaldehyde from H2O2 despite iron(II) being limiting, indicating efficient iron redox cycling. Acetaldehyde production was faster at pH 4.0 than at pH 3.0, attributable largely to increased iron complexation. Citrate allowed the most rapid acetaldehyde formation, while the comparative effects of tartrate and malate were pH-dependent, likely due to differences in their iron-chelating abilities. The inclusion of SO2 greatly diminished acetaldehyde formation, but did not prevent it, and reduced the differential effects of pH and acid composition. Findings overall suggest management of wine acidity can significantly affect the rate and outcome of oxidation.

Evaluation of the potential of total proanthocyanidin content in feces as an intake biomarker.

Due to the health benefits associated with proanthocyanidins (PAs), it is useful to identify dietary PA biomarkers that can be determined by simple methods. Since increased levels of circulating PA metabolites are associated with increased fecal PA content, this study explores the spectrophotometric measurement of fecal PA content and its use as a biomarker of PA intake. To this end, fecal PA content was measured using an adaptation of Porter's spectrophotometric method in samples from a preclinical study and an observational study. In the former, excretion of 250-400 mg PA polymer equivalents/100 g feces was observed during supplementation and the day after, together with a significant association (p < 0.05) between PA intake and the excretion of both intact PAs and some PA metabolites, i.e., (+)-catechin, (-)-epicatechin and syringic acid. No relationship between intake and excretion was found in the observational study, either for the entire group (mean excretion of 240 ± 226 mg PA polymer equivalents/100 g feces) or after stratification into tertiles of consumption. In conclusion, the spectrophotometric determination of total PA content in feces proved to be a valid compliance marker in a preclinical study, but it was not associated with PA intake in free-living subjects. The potential of total PA excretion in observational studies, determined in fecal samples collected the day before dietary recall or in several fecal samples from the same subject, remains to be elucidated, as does a complete validation of the method proposed here.

Anthocyanin Addition Alters Tannin Extraction from Grape Skins in Model Solutions via Chemical Reactions.

Condensed tannin extraction and stable color formation are two of the cornerstones of red wine production. Without condensed tannin, red wine would lack the tactile feeling of astringency, and without the formation of modified pigments, it would lack color stability for long-term aging. To understand how malvidin-3,5-diglucoside interacts with condensed tannin under nonoxidative conditions, an experiment was designed conducting model-wine skin extractions of Sauvignon blanc grapes harvested at various dates of maturity. Monomeric malvidin-3,5-diglucoside was isolated from color concentrate and added during these extractions. Following a 72 h extraction, solutions were evaluated for recovery of monomeric anthocyanins, skin tannin concentration, skin tannin extractability, and impact of anthocyanins on condensed tannin size. Anthocyanins showed a significant impact on the extraction of flavan-3-ol material in the early stages of ripening that declined in the latter stages of ripening. Furthermore, anthocyanins significantly decreased the size of the condensed tannin extracted. These results suggest that anthocyanins are not only enhancing the extractability of condensed tannin but also readily incorporating into the polymeric material, leading to a decrease in the average molecular mass of the condensed tannin polymer. The extent of reaction in 72 h suggests that the rate of interflavan bond cleavage may be higher than previously reported and merits closer scrutiny.

Can Chemical Analysis Predict Wine Aging Capacity?

Oxidation is the limiting factor in wine aging, and recently some famous wines have exhibited unexpected premature oxidation. Antioxidant assays may provide a means to assess a wine's aging potential by measuring its capacity to chemically reduce reagent components. Correlations between antioxidant activity and wine components have the highest value with flavanols, notable for their catechol and phloroglucinol moieties. Both FRAP and DPPH based methods respond strongly to catechol groups, but these functional groups do not protect wine from oxidation. An ideal assay for wine aging capacity would respond selectively to thiols, phloroglucinol moieties, SO2 and other antioxidants capable of reducing quinones. A definitive test will be to compare the various assays against the shelf life of a number of commercial wines.

Normal-phase chromatographic separation of pigmented wine tannin by nano-HPLC quadrupole time-of-flight tandem mass spectrometry and identification of candidate molecular features.

BACKGROUND: As a wine ages, altered sensory properties lead to changes in perceived quality and value. Concurrent modifications of anthocyanin and tannin occur forming pigmented tannin, softening astringency and retaining persistent color. Wine tannin extracts of 1990 and 2010 vintages of Oakville Station Cabernet Sauvignon have been analyzed using normal-phase chromatography with tandem quadrupole time-of-flight mass spectrometry (QToF) to investigate the compositional differences in their pigmented tannin fractions. RESULTS: The older wine demonstrates much greater structural diversity and a range of more polar compounds, while the younger wine contains fewer observed ion peaks. Several hundred molecular features are observable, and, as expected, there is progression to higher molecular weights after long aging. Between 7% and 16% of molecular features could be matched to a database of anticipated pigmented tannin compounds. Many signals had multiple possible isomeric identities, but fragmentation to resolve their identity was stymied by low sensitivity of the tandem mass spectrometric capability provided by QToF, so isomeric disambiguation is incomplete. CONCLUSIONS: The chromatography displayed a high degree of resolution in aged wines, separating many of the known pigment types, including aldehyde bridged compounds, pyranoanthocyanins and direct condensation products among others, as well as resolving a great number of unknown compounds. Expanding our understanding of red wine pigments will lead to better wines as winemakers will be able to associate quality with particular wine pigment profiles once we can distinguish the relevant patterns in those pigments. © 2021 Society of Chemical Industry.

Yeasts Induce Acetaldehyde Production in Wine Micro-oxygenation Treatments.

Micro-oxygenation (Mox) is a common technique used to stabilize color and reduce harsh astringency in red wines. Here, we investigate the role of residual sugars, phenolics, SO2, and yeast on the oxidation of wine in three studies. In a Mox experiment, populations of yeasts emerged after the loss of SO2, and this was associated with sharp increases in oxygen consumption and acetaldehyde production. No acetaldehyde production was observed without the presence of yeast. In an oxygen saturation experiment, unfiltered wines, in particular those with residual sugar >3 g/L, consumed oxygen more quickly and produced more acetaldehyde than filtered wines. In a final experiment, the reincorporation of oxygen and glucose immediately after the completion of fermentation of an otherwise dry synthetic wine resulted in significant acetaldehyde production. These experiments highlighted the importance of yeast metabolism in determining a wine's response to Mox and suggested that the role of chemical oxidation to produce acetaldehyde during Mox may not be very important. It appears that control of microbial populations and residual sugar levels may be key to managing Mox treatments in winemaking, and production scale experiments should be conducted.

Effects of initial oxygenation on chemical and aromatic composition of wine in mixed starters of Hanseniaspora vineae and Saccharomyces cerevisiae.

The use of Saccharomyces and non-Saccharomyces yeast species as mixed starters has potential advantages over pure culture fermentation due to increased wine complexity based on modification of metabolites of oenological interest. In this work, the effects of initial oxygenation on fermentation performance, chemical and volatile composition of French Colombard wine fermented with Hanseniaspora vineae and Saccharomyces cerevisiae in sequential inoculations were investigated in 1 L flasks. Although dominated by S. cerevisiae at the middle-end of fermentation, initial aeration for 1 day boosted H. vineae populations, and allowed H. vineae to coexist longer with S. cerevisiae in mixed cultures compared to no aeration, and suppressed S. cerevisiae later in the fermentation, which resulted in extended fermentation time. More important, the major fermentation products and volatile compounds were significantly modified by aeration and different from no aeration fermentation. The wines produced by aeration of mixed fermentations were characterized with higher amounts of glycerol, lactic acid and acetate esters, and lower levels of ethanol, higher alcohol and ethyl fatty acid esters. The aeration had more potential to shape the quality of wines and diversify the aromatic characteristics relative to simple mixed inoculation, as indicated by PCA analysis. Our results suggested that the impact of early aeration on yeast physiology extends beyond the aeration phase and influences fermentation activity, chemical and aromatic compounds in the following anaerobic stage. The aeration for a short time during the cell growth stage in mixed fermentation is therefore a potential means to increase the aromatic diversity and quality of wine, possibly providing an alternative approach to meet the expectations of wine consumers for diverse aromatic qualities.

A quarter century of wine pigment discovery.

The red grape berry pigments, anthocyanins, were characterized in the early 20th century, but investigations of wine pigments were stymied during that era. The question of their identity was a major challenge for wine chemists. A number of techniques showed that the pigments were polymeric in nature. Some structures were postulated by mid-century based on reactions between anthocyanins and condensed tannin, and later postulated wine pigments were observed in model reactions. Some related compounds were then observed in wine. By the end of the 20th century, the ionization of non-volatiles for mass spectrometry opened the door to these compounds. In addition, a new class of compounds was observed, the pyranoanthocyanins, a product of fermentation and aging metabolites with anthocyanins. These compounds possess the pigment stability to SO2 and pH change that is characteristic of aged red wine. Aging experiments show a dynamic situation with shifts in the population of pigment classes over time. The very large number of diverse pigments explains why it has been so difficult to answer the century-old question of the structure of wine pigments. Our current understanding is founded on the use of mass spectral analysis using electrospray and related ionization techniques over the last 25 years. Future progress will rely on more sophisticated analysis of this very complex mixture of substances. © 2019 Society of Chemical Industry.

Red Wine Dryness Perception Related to Physicochemistry.

The responsibility of condensed tannins in the astringency sensation is well-known, even though the physical mechanism is unclear. The aims of the study are to go deeper into the relationships between the tannin structure and red wine dryness as well as evaluate the influence of the wine matrix and tannin concentration on the interaction with proteins and dryness perception. Condensed tannins were extracted from two red wines (cv. Cabernet Sauvignon and Pinot Noir) and added back to them and to a model wine at 0.5 g/L, prior to chemical characterization of their composition by reversed-phase high-performance liquid chromatography as well as their viscosity. The physical consequences of interactions between mucin, poly-l-proline, saliva, and wines were evaluated by turbidimetry. Descriptive analysis and time-intensity evaluations of dryness were carried out by a trained panel on all wines and model solutions. The mean degree of polymerization of Cabernet Sauvignon wine with or without tannins added was higher than that of Pinot Noir wine conditions. The turbidity of saliva and poly-l-proline with tannins added to Cabernet Sauvignon was higher than of tannins added to Pinot Noir wine. Cabernet Sauvignon wines were perceived dryer than Pinot Noir by panelists, and the dryness intensity of Cabernet Sauvignon wine conditions last longer than that of Pinot Noir or model wine conditions. The dryness of red wines was related to larger tannins, higher tannin concentration, and a greater turbidity with saliva. The dryness was not affected by the addition of tannins into wine probably as a result of the aroma/taste suppression effects and the presence of other components.

Adsorption and biotransformation of anthocyanin glucosides and quercetin glycosides by Oenococcus oeni and Lactobacillus plantarum in model wine solution.

BACKGROUND: Anthocyanins and flavonols play a significant role in contributing to wine color and mouthfeel, and the interaction of malolactic fermentation with these compounds is not well known. Here we investigated the adsorption of these compounds by Oenococcus oeni and Lactobacillus plantarum. RESULTS: Delphinidin-3-glucoside (D3G) was adsorbed the most, followed by malvidin-3-glucoside (M3G) and peonidin-3-glucoside (P3G) for both the bacterial species, while flavonols were not adsorbed. An increase in ß-glycosidase activity suggested that this enzyme breaks down the anthocyanin glucosides, providing sugars for growth. An average decline of approximately 65% in enzyme activity in the presence of substantial residual sugar was observed. The specific metabolic rates were found to be dependent on the class of anthocyanin and species / strain of the bacteria. Selective adsorption of anthocyanins and not the flavonol glycosides suggest that electrostatic interactions mediate the adsorption. Further, a breakdown of anthocyanins resulted in phloroglucinol aldehyde from the flavonoid A-ring and corresponding phenolic acids from the B-ring, i.e., gallic acid for D3G, syringic acid for M3G, and vanillic acid for P3G. CONCLUSIONS: The breakdown and adsorption of the anthocyanin glucosides can help explain the color loss and aroma changes, such as the appearance of syringic and vanillic acid, associated with malolactic fermentation. © 2019 Society of Chemical Industry.

Omics Forecasting: Predictive Calculations Permit the Rapid Interpretation of High-Resolution Mass Spectral Data from Complex Mixtures.

For some complex mixtures, chromatographic techniques are insufficient to separate the large numbers of compounds present. In addition, these mixtures often contain compounds with similar or identical molecular masses and shared fragmentation transitions. Advancements in mass spectrometry have provided more and more detailed molecular profiles with significant increases in resolution. This has led to a capacity to distinguish a very large number of compounds in complex mixtures, providing overwhelming data sets. The approach of calculating molecular formulas from a mass list has become more and more problematic as the number of signals has increased exponentially, to the point that it has become impossible to manually interpret the thousands of mass signals. The current approach is to calculate a list of possible formulas that fall within a specific mass error of the observed signal. Then, one must look for possible structures that can be derived from each entry on the list of formulas. However, an alternative approach is to anticipate the possible structures of a particular set of compounds, such as red wine pigments, and then compare the ion signals against a predicted list. To that end, starting with known wine pigment types, we have generated a set of expected wine pigment variants based on known derivatives of condensed tannin oligomers, anthocyanins, and fermentation products. After the ability to distinguish compounds by mass spectrometry was accounted for, over 1 million results were generated consisting of known and anticipated wine pigments. A comparison with a small sample of wine phenolic fractions show a large number of matches, suggesting that this approach may be helpful.

Combinatorics of proanthocyanidins in wine.

Condensed tannin are polymers comprised of procyanidin and prodelphinidin units and found in wine, chocolate, apples and many other foods. Current analytical methods to characterize these tannins provide aggregate functional results, such as quantified protein binding, important for its relation to astringency. A more detailed understanding of the constituents has become accessible via mass spectrometry (MS) with high resolution and tandem MS techniques. Analysis of wine tannin by these methods provides thousands of signals, far too many to assess using standard techniques. We propose targeted mass filtering using a table of predicted proanthocyanidin oligomers. Condensed tannin in wine is comprised of 4 constitutionally distinct subunits (catechin, catechin gallate, gallocatechin, and gallocatechin gallate), each of which has 4 stereoisomers. Accounting for all 16 subunits, there are over 5 million possible oligomers from monomer to decamer. Since mass spectroscopy is generally blind to stereoisomeric variations, the number of possible observable MS signals can be reduced to a list of 1000. By applying specialized combinatoric functions, a table for the now manageable compilation of possible proanthocyanidin oligomers has been created containing the compound subunit compositions, molecular formulae, and molecular ion signals for the interpretation of condensed tannin mass spectra. Mathematical formulae for the enumeration of possible compounds in any chemical system composed of polymers with discrete subunits resulted from this endeavor and are presented in general form with specific application to this system. As these condensed tannins react with anthocyanins, forming pigmented tannin, the table created here can also be the foundation of a database for the very complex red wine pigments. The system described here could also be applied to the analysis of proanthocyanidins in chocolate and other foods.

Oxygen exposure during red wine fermentation modifies tannin reactivity with poly-l-proline.

Red wines injected with nitrogen or oxygen during fermentation were used to identify the effect of gas exposure on tannin structure and reactivity with poly-l-proline. Tannin was purified from wine after fermentation and after three years of bottle storage. Tannin from nitrogen-treated wine had a lower percentage of galloylation and were less pigmented than tannin from oxygen-exposed wine. Self-aggregation of tannin was measured by nanoparticle tracking analysis and a larger particle size was observed for the oxidized treatment. The interaction of tannin and poly-l-proline was measured by isothermal titration calorimetry, and involved more hydrogen bonding than hydrophobic interactions in the case of nitrogen-treated wine tannin. Conversely, oxidized tannin was more hydrophobic and the association with poly-l-proline was entropy-driven due to a change of solvation. The results show meaningful changes in the structure and reactivity of tannin as a result of oxygen exposure during fermentation, which may impact astringency perception.

Acetaldehyde reactions during wine bottle storage.

Acetaldehyde is a major wine oxidation product. Here, three Cabernet Sauvignon wines, containing different levels of acetaldehyde from different micro-oxygenation (mOx) regimes, including yeast-mediated treatments, were aged under closures differing in oxygen ingress. Oxygen, phenolics, carbonyls and heterocyclic acetals were measured. Acetaldehyde levels at bottling was a significant factor in the phenolic compound profile after one year, with anthocyanins most affected, then flavonols, flavonoids and hydroxycinnamic acids, but there were negligible effects on benzoic acids. The effect of bottle closures with increased oxygen ingress had a similar trend. Increased acetaldehyde levels and oxygen ingress also yielded higher levels of the heterocyclic acetals from glycerol. These changes reflect aging, and suggest that managing mOx during production could be used to reduce the time needed to achieve some aged wine characteristics.

Tracing oxidation reaction pathways in wine using 13C isotopolog patterns and a putative compound database.

Quinones are key reactive electrophilic intermediates that are formed in abundance during wine oxidation and have a significant impact on wine character, altering color and flavor. They readily react with nucleophiles, such as SO2 and glutathione. Some nucleophiles have been reported to react with quinones in model wines, but many of the corresponding products have not been confirmed in real wines. Here, a stable isotope labeling approach employing 13C6 labeled ortho-quinone was evaluated in combination with an exact-mass list of putative products. The measurements were taken using high performance liquid chromatography coupled to time-of-flight mass spectrometry. Fourteen compounds were detected based on a match in the table plus an M+6 mass isotope pattern derived from the labeled quinone mixture. The presence of the mass label also establishes reaction pathways for the formation of these compounds, and suggests how wine catechols could react during wine oxidation, demonstrating the insight provided by this analytical technique.

Friction forces of saliva and red wine on hydrophobic and hydrophilic surfaces.

The physical aspect of human oral astringency perception - the mouthfeel - of red wine has not been quantitatively studied in depth. In this study, the interfacial friction/lubrication properties of saliva (mucin from bovine submaxillary glands or human saliva) with red wines (cv. Cabernet sauvignon and Pinot noir) were measured with a surface force apparatus (SFA). In SFA measurements sliding occurs between smooth, undamaged surfaces with a well-defined contact area and film thickness. The surfaces were either hard, hydrophilic mica or soft, hydrophobic PDMS-coated mica which mimic in-mouth conditions. Saliva was a better lubricant than mucin with the soft, hydrophobic surfaces. In addition, saliva's lubricity was 2.5 times better on the soft hydrophobic surfaces than hard hydrophilic surfaces. The addition of red wine with saliva further decreased friction and improved lubrication. Surprisingly, the coefficient of friction measured for red wine with saliva as the lubricant was higher for Pinot noir than Cabernet sauvignon wine. The aggregation and precipitation of salivary proteins by tannins is well known. The lower friction of high tannin Cabernet sauvignon compared to lower tannin Pinot noir was attributed to exclusion of these aggregates and depletion of more polymeric and protein material from the interfacial sliding region.