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.

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.

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.

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.

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.

Yeast alter micro-oxygenation of wine: oxygen consumption and aldehyde production.

BACKGROUND: Micro-oxygenation (MOx) is a common winemaking treatment used to improve red wine color development and diminish vegetal aroma, amongst other effects. It is commonly applied to wine immediately after yeast fermentation (phase 1) or later, during aging (phase 2). Although most winemakers avoid MOx during malolactic (ML) fermentation, it is often not possible to avoid because ML bacteria are often present during phase 1 MOx treatment. We investigated the effect of common yeast and bacteria on the outcome of micro-oxygenation. RESULTS: Compared to sterile filtered wine, Saccharomyces cerevisiae inoculation significantly increased oxygen consumption, keeping dissolved oxygen in wine below 30 µg L-1 during micro-oxygenation, whereas Oenococcus oeni inoculation was not associated with a significant impact on the concentration of dissolved oxygen. The unfiltered baseline wine also had both present, although with much higher populations of bacteria and consumed oxygen. The yeast-treated wine yielded much higher levels of acetaldehyde, rising from 4.3 to 29 mg L-1 during micro-oxygenation, whereas no significant difference was found between the bacteria-treated wine and the filtered control. The unfiltered wine exhibited rapid oxygen consumption but no additional acetaldehyde, as well as reduced pyruvate. Analysis of the acetaldehyde-glycerol acetal levels showed a good correlation with acetaldehyde concentrations. CONCLUSION: The production of acetaldehyde is a key outcome of MOx and it is dramatically increased in the presence of yeast, although it is possibly counteracted by the metabolism of O. oeni bacteria. Additional controlled experiments are necessary to clarify the interaction of yeast and bacteria during MOx treatments. Analysis of the glycerol acetals may be useful as a proxy for acetaldehyde levels. © 2017 Society of Chemical Industry.

The PI3K/Akt pathway is involved in procyanidin-mediated suppression of human colorectal cancer cell growth.

Colorectal cancer (CRC) has the third highest incidence worldwide. Epidemiological studies showed that the consumption of fruit and vegetables containing procyanidins (PCA), polymers of flavan-3-ols, is associated with lower CRC risk. However, the molecular mechanisms supporting this positive association are unclear. This study investigated the capacity of PCA with different degrees of polymerization to reduce CRC cell growth, characterizing the underlying mechanisms. Compared to the monomer ((-)-epicatechin) and the trimer, the hexamer (Hex) was the most active at reducing CRC cell viability. Hex caused a concentration- (2.5-50 µM) and time- (24-72 h) dependent decrease in the viability of six human CRC cell lines in culture. Hex caused CRC apoptotic Caco-2 cell death within 24 h, as evidenced by caspase 3 and caspase 9 activation, DNA fragmentation, and changes in nuclear morphology/staining. Hex-induced apoptosis occurs through the mitochondrial pathway, as evidenced by an increased Bad mitochondrial translocation, and cytochrome c release from the mitochondria to the cytosol. Hex also arrested the Caco-2 cell cycle at G2 /M phase and upregulated genes involved in autophagy. Mechanistically, in Caco-2 cells Hex inhibited the PI3K/Akt signaling pathway, causing the downstream downregulation of proteins involved in the regulation of cell survival (Bad, GSK-3ß). Accordingly, the Akt inhibitor MKK-2206 decreased Bad and GSK-3ß phosphorylation. MKK-2206 decreased cell growth, having an additive effect with Hex. In conclusion, our results show that large PCA can inhibit CRC cell growth via the Akt kinase pathway, demonstrating a mechanism to explain the epidemiological evidence linking PCA-rich diets with lower CRC risk. © 2016 Wiley Periodicals, Inc.

Direct Analysis of Free and Sulfite-Bound Carbonyl Compounds in Wine by Two-Dimensional Quantitative Proton and Carbon Nuclear Magnetic Resonance Spectroscopy.

Recent developments that have accelerated 2D NMR methods and improved quantitation have made these methods accessible analytical procedures, and the large signal dispersion allows for the analysis of complex samples. Few natural samples are as complex as wine, so the application to challenges in wine analysis look promising. The analysis of carbonyl compounds in wine, key oxidation products, is complicated by a multitude of kinetically reversible adducts, such as acetals and sulfonates, so that sample preparation steps can generate complex interferences. These challenges could be overcome if the compounds could be quantified in situ. Here, two-dimensional ((1)H-(1)H) homonuclear and heteronuclear ((13)C-(1)H) single quantum correlations (correlation spectroscopy, COSY, and heteronuclear single quantum coherence, HSQC) nuclear magnetic resonance spectra of undiluted wine samples were observed at natural abundance. These techniques achieve simultaneous direct identification and quantitation of acetaldehyde, pyruvic acid, acetoin, methylglyoxal, and α-ketoglutaric acid in wine with only a small addition of D2O. It was also possible to observe and sometimes quantify the sulfite, hydrate, and acetal forms of the carbonyl compounds. The accuracy of the method was tested in wine samples by spiking with a mixture of all analytes at different concentrations. The method was applied to 15 wine samples of various vintages and grape varieties. The application of this method could provide a powerful tool to better understand the development, evolution, and perception of wine oxidation and insight into the impact of these sulfite bound carbonyls on antimicrobial and antioxidant action by SO2.

Influence of closure, phenolic levels and microoxygenation on Cabernet Sauvignon wine composition after 5 years' bottle storage.

BACKGROUND: Wine aging is generally limited by the amount of oxidation, which is dependent on the amount of oxygen entering via the closure. Cabernet Sauvignon wine is well known for its high concentration of tannin, making it an ideal red wine for aging. The impact of closure type after 5 years' bottle aging has been investigated on a 2007 Cabernet Sauvignon red wine, treated with or without polyvinylpolypyrrolidone (PVPP) and micro-oxygenation (Mox). Two oxygen transfer rate (OTR) conditions (16 and 5 µg per day) into 375 mL bottles were obtained by using different synthetic stoppers. RESULTS: Color was evaluated by UV-visible spectrophotometry, carbonyls by 2,4-dinitrophenylhydrazine derivatization, phenolics by high-performance liquid chromatography and sulfur dioxide by the aspiration method. Closure type strongly influenced color parameters involving SO2 bleaching and some phenolics, particularly quercetin, were affected, but there was little effect on carbonyls other than acetaldehyde. PVPP treatment afforded wines with the lowest levels of phenolics and color density, but highest acetaldehyde. Few effects of Mox could be detected. CONCLUSIONS: Closure OTR strongly affects sulfur dioxide levels - the primary antioxidant in wine - in aged wine, but phenolic levels substantially alter the secondary reactions of oxidative aging.

Untargeted profiling of tracer-derived metabolites using stable isotopic labeling and fast polarity-switching LC-ESI-HRMS.

An untargeted metabolomics workflow for the detection of metabolites derived from endogenous or exogenous tracer substances is presented. To this end, a recently developed stable isotope-assisted LC-HRMS-based metabolomics workflow for the global annotation of biological samples has been further developed and extended. For untargeted detection of metabolites arising from labeled tracer substances, isotope pattern recognition has been adjusted to account for nonlabeled moieties conjugated to the native and labeled tracer molecules. Furthermore, the workflow has been extended by (i) an optional ion intensity ratio check, (ii) the automated combination of positive and negative ionization mode mass spectra derived from fast polarity switching, and (iii) metabolic feature annotation. These extensions enable the automated, unbiased, and global detection of tracer-derived metabolites in complex biological samples. The workflow is demonstrated with the metabolism of (13)C9-phenylalanine in wheat cell suspension cultures in the presence of the mycotoxin deoxynivalenol (DON). In total, 341 metabolic features (150 in positive and 191 in negative ionization mode) corresponding to 139 metabolites were detected. The benefit of fast polarity switching was evident, with 32 and 58 of these metabolites having exclusively been detected in the positive and negative modes, respectively. Moreover, for 19 of the remaining 49 phenylalanine-derived metabolites, the assignment of ion species and, thus, molecular weight was possible only by the use of complementary features of the two ion polarity modes. Statistical evaluation showed that treatment with DON increased or decreased the abundances of many detected metabolites.

The anthocyanin metabolites gallic acid, 3-O-methylgallic acid, and 2,4,6-trihydroxybenzaldehyde decrease human colon cancer cell viability by regulating pro-oncogenic signals.

Anthocyanins are a class of polyphenols abundant in the skins of red grapes, and have been shown to have anti-cancer effects in models of colon cancer [Cooke et al. Int J Cancer 2006;119:2213-2220; Jing et al. J Agric Food Chem 2008;56:9391-9398]. Gut microflora metabolize anthocyanins to phenolic acids and aldehydes. These metabolites may explain the relationship between anthocyanin consumption and reduced incidence of colorectal cancer (CRC). Previously, gallic acid (Gal), 3-O-methylgallic acid (Megal), and 2,4,6-trihydroxybenzaldehyde (THBA) were found to decrease Caco-2 cell viability to a larger extent than other anthocyanin metabolites. To better understand the potential anti-CRC action of these compounds, this paper investigated their capacity to modulate the cell cycle, and induce apoptotic cell death. Dividing Caco-2 cells were incubated for 24-72 h in the presence of 10-100 µM Gal, Megal, THBA, and malvidin-3-glucoside (M3g). THBA reduced cell viability only at 100 µM, while Gal and Megal (10-100 µM) caused a time- and dose-dependent decrease in cell viability. After 72 h incubation, the metabolites caused cell cycle arrest at G0 /G1 . The activation of the apoptotic pathway by Megal, Gal, and THBA was evidenced by the activation of caspase-3. However, only Megal and Gal caused DNA fragmentation and nuclear condensation. Megal, Gal, and THBA inhibited transcription factors NF-κB, AP-1, STAT-1, and OCT-1 which are known to be activated in CRC. In conclusion, the anti-cancer effects of Megal and Gal occurs as a consequence of both the inhibition of cell proliferation and induction of apoptosis. The inhibition of transcription factors that promote cell proliferation and survival can in part underlie the observed effects.

Napa Valley Cabernet Sauvignon Proanthocyanidin Changes During Fruit Ripening: A Multi-Appellation Survey.

In 2015, an experiment was designed to investigate the distribution and variance of in winegrape flavonoids across the ripening phase in the Napa Valley. This Cabernet Sauvignon experiment was intended to evaluate the polyphenol differences across Napa Valley in order to understand parameters controlling "proanthocyanidin activity." This method has shown promise in understanding proanthocyanidin (PA) astringency based on size distribution, pigmentation, conformation, and composition. Results from whole berry partial extractions showed that seed PA material was driving PA activity early in the ripening phase, while the formation of the pigmented polymer led to a decrease later in the growing season. Multivariate analysis showed that the main drivers of changes across the ripening phase were the molecular masses of PAs and the amount of pigmentation. Given the high amount of variability seen in the experiment between sites in such a small geographical area, the results suggest that manipulation of PA activity may be possible in the vineyard, perhaps explaining variations in wine mouthfeel attributes between locations. These results can be used to develop furthermore controlled experiments targeting the variables responsible for PA activity changes.

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.

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.

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.

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.

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.

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.