Novel antioxidant reactions of cinnamates in wine

Plant-derived polyphenolic compounds have received much attention for their ability to sequester high-energy free radicals in a great variety of food-related and biological systems, protecting those systems from oxidative change. The ability of these compounds to scavenge free radicals has always been attributed to their phenolic functionality, from which a hydrogen atom can be easily abstracted. In this study, the cinnamates and the ubiquitous hydroxycinnamates were found to equally suppress the formation of oxidation products in wine exposed to the Fenton reaction (catalytic Fe(II) with hydrogen peroxide). Mechanistic investigations led to the unexpected discovery that the α,β-unsaturated side chain of cinnamic acids could efficiently trap 1-hydroxyethyl radicals, representing a newly discovered mode of antioxidant radical scavenging activity for these broadly occurring compounds in a food system. The proposed pathway is supported by prior fundamental studies with radiolytically generated radicals.     

Glyceraldehyde bridging between flavanols and malvidin-3-glucoside in model solutions

Hydroxyl radicals (.OH) seem to have an important role in the oxidation of wine constituents and the production of important electrophilic aldehydes and ketones. In this experiment, glyceraldehyde, a .OH oxidation product of glycerol, recently described in wine, reacts with (+)-catechin, (-)-epicatechin, and malvidin-3-glucoside (Mv3gl), in model solutions, yielding new condensed phenolic compounds. The adduct compounds formed were separated by means of reversed phase liquid chromatography and detected and characterized using UV-vis and electrospray ionization mass spectrometry. Flavanol-flavanol and anthocyanin-flavanol adducts linked with glyceraldehyde yielded compounds with m/z ratios for their main ions, in positive ion mode, of 653.2 for the (+)-catechin dimer or the (-)-epicatechin dimer and 855.5 for Mv3gl/(+)-catechin or Mv3gl/(-)-epicatechin dimers. The possible occurrence of these compounds in wine is suggested, and the potential role of these and related reactions in wine aging is discussed.     

Antioxidant action of glutathione and the ascorbic acid/glutathione pair in a model white wine

Glutathione was assessed individually, and in combination with ascorbic acid, for its ability to act as an antioxidant with respect to color development in an oxidizing model white wine system. Glutathione was utilized at concentrations normally found in wine (30 mg/L), as well as at concentrations 20-fold higher (860 mg/L), the latter to afford ascorbic acid (500 mg/L) to glutathione ratios of 1:1. The model wine systems were stored at 45 °C without sulfur dioxide and at saturated oxygen levels, thereby in conditions highly conducive to oxidation. Under these conditions the results demonstrated the higher concentration of glutathione could initially provide protection against oxidative coloration, but eventually induced color formation. In the period during which glutathione offered a protective effect, the production of xanthylium cation pigment precursors and o-quinone-derived phenolic compounds was limited. When glutathione induced coloration, polymeric pigments were formed, but these were different from those found in model wine solutions without glutathione. In the presence of ascorbic acid, high concentrations of glutathione were able to delay the decay in ascorbic acid and inhibit the reaction of ascorbic acid degradation products with the wine flavanol compound (+)-catechin. However, on depletion, the glutathione again induced the production of a range of different polymeric pigments. These results highlight new mechanisms through which glutathione can offer both protection and spoilage during the oxidative coloration of a model wine.     

Sulfate--a candidate for the missing essential factor that is required for the formation of protein haze in white wine

Protein haze formation in white wine is dependent on the presence of both wine protein and other unknown wine components, termed factor(s) X. The ability to reconstitute protein haze upon heating artificial model wine solutions (500 mg/L thaumatin, 12% ethanol, 4 g/L tartaric acid) to which candidate components were added was employed to identify factor(s) X. No protein haze was formed in the absence of additives. The individual or combined addition of caffeic acid, caftaric acid, epicatechin, epigallocatechin-O-gallate, gallic acid, or ferulic acid at typical white wine concentrations did not generate protein haze. However, PVPP fining of commercial wines resulted in a reduction in protein haze, suggesting that phenolic compounds may play a modulating role in haze formation. To elucidate the nature of the unknown factor(s) wine was fractionated and fractions were back-added to model wine and tested for their essentiality. Wine fractions were generated by ultrafiltration, reverse-phase chromatography, and mixed-mode anion-exchange and reverse-phase chromatography. The only purified fraction containing the essential component(s) was free of phenolic compounds, and analysis by mass spectrometry identified sulfate anion as the dominant component. Reconstitution with KHSO4 using either commercially available thaumatin or wine proteins confirmed the role of sulfate in wine protein haze formation. The two main wine proteins, thaumatin-like protein and chitinase, differed in their haze response in model wines containing sulfate. Other common wine anions, acetate, chloride, citrate, phosphate, and tartrate, and wine cations, Fe(2+/3+) and Cu(+/2+), when added at typical white wine concentrations were not found to be essential for protein haze formation.     

Formation pathways of ethyl esters of branched short-chain fatty acids during wine aging

The particular behavior during wine aging of fermentative branched fatty acid ethyl esters, related to yeast nitrogen metabolism, compared that of their straight-chain analogues, related to yeast lipid metabolism, was first checked in 1-5 year aged Muscadet wines. Quantitative SIDA measurements showed that the levels of the former increased, whereas those of the latter decreased. Then, three hypothetical pathways suggested in the literature to explain these variations of branched esters were investigated. Two Muscadet and Sylvaner wines were spiked with levels of deuterated isobutanoic acid and its ethyl ester, similar to those of their natural analogues, then they were submitted to model aging. Quantitative SIDA measurements on the formation of these natural and labeled ethyl esters from the corresponding acids revealed that the behavior of the natural and labeled compounds were similar. The acid levels were much higher than the ester levels in the initial young wine, and a significant upward trend of their esterification ratios to those of the acid-ester equilibrium was observed with aging. Thus, this equilibrium proved to be the most effective in generating the branched fatty acid ethyl esters during wine aging. In contrast, the formation of these acids by Strecker-type degradation of wine amino acids in the conditions of the model aging or by hydrolysis of their glycoconjugates proved to be ineffective.     

Ascorbic acid-induced browning of (+)-catechin in a model wine system

The ability of ascorbic acid to induce browning of (+)-catechin in a model wine system has been studied. A significant increase in absorbance at 440 nm was observed over 14 days when ascorbic acid was incubated at 45 degrees C with (+)-catechin in a model wine base. The onset of browning was delayed for about 2 days, although the length of the lag period was dependent on the amount of molecular oxygen in the headspace of the reaction system. The lag period was not observed when a preoxidized solution of ascorbic acid was used, suggesting that a product of ascorbic acid oxidation is responsible for the onset of browning. Hydrogen peroxide, when added directly to (+)-catechin in the model system, was not capable of producing the same degree of browning as that generated by ascorbic acid. Liquid chromotography evidence is presented to show that different reaction products are produced by ascorbic acid and hydrogen peroxide.     

Aroma compound sorption by oak wood in a model wine

Oak wood used for wine barrels was immersed into a model wine containing eight aroma compounds (e.g., aromatic and terpene alcohols, ethyl esters, and aldehyde), for which activity coefficients in water and model wine were determined using the mutual solubility measurement. A mass balance of these volatiles considering their reactivity in model wine was established. For most of the studied aroma compounds, and mainly for linalool and ethyl octanoate, a sorption behavior into wood was reported for the first time. This phenomenon was selective and could not be related to the solubilities in model wine and hydrophobicities of the studied aroma compounds, suggesting that acid-base and polar characteristics of wood were more involved in this sorption mechanism. This study has also shown that the level of sorption is a function of the ratio of wood surface area/solution volume.     

Sorption behavior of volatile phenols at the oak wood/wine interface in a model system

The sorption in a model system of aroma compounds of enological interest (mixture of the eight derivatives from guaiacol, 4-ethylphenol, and whiskylactone) onto wood was investigated to assess the influence of wood on the concentration of these volatiles during the aging of wine. To evaluate the influence of the solubility of aroma compounds in sorption phenomena, this parameter was determined for each volatile compound in model wine at 10 and 25 degrees C. The solubility is significantly higher in the model wine than in water and remains constant in the range of temperatures studied, except for guaiacol and vanillin. Kinetic and equilibrium sorptions were investigated. Sorption kinetics showed that the sorption equilibrium for all aroma compounds was reached after 6-7 days. From the study of the individual sorption isotherms, two distinct kinds of sorption behavior were observed depending on the presence or not of an ethylenic para substituent conjugated to the phenyl ring. K(ww) partition coefficients between the wood and the model wine were determined, which exhibited an unusual positive variation with temperature.     

Model aging and oxidation effects on varietal, fermentative, and sulfur compounds in a dry botrytized red wine

From harvest until wine arrives to the consumer, oxygen plays a crucial role in the definition of the final aroma. In the present research, the effect of the model oxidative aging on a dry red Botrytis wine, such as Italian Amarone, was considered. Amarone wine was submitted to model oxidative aging and then analyzed with two different approaches (SPE-GC-MS and HS-SPME/GC-MS). The same sampling plan was adopted to study the model aging of the same Amarone wine in anaerobic conditions. The HS-SPME/GC-MS method was applied to investigate for the first time the effect of the oxidative aging on a vast number of fermentative sulfur compounds. This research highlighted peculiar evolutions for several volatile compounds. In particular, benzaldehyde showed a sensitive increment during the oxidative aging, with a rate much higher than that reported for non-Botrytis red wines. On the other hand, several sulfides (dimethyl sulfide, 3-(methylthio)-1-propanol, etc.) disappeared after just 15 days of oxidative aging. A wine oxidation marker such as 3-(methylthio)-propanal was not found in any of the oxidized wines; conversely methionol-S-oxide was tentatively identified. This evidence has not been mentioned in the literature. A possible involvement of grape withering process and Botrytis in these mechanisms was supposed: a dry red wine, produced from the same but without any grape withering process and Botrytis infection (e.g., Bardolino wine), was submitted to oxidative aging and analysis. This red wine showed an evolution similar to those reported in the literature for dry red wines but significantly different from the Amarone wine.     

Approaches to wine aroma: release of aroma compounds from reactions between cysteine and carbonyl compounds in wine

Under conditions close to those of wine, that is, low pH, aqueous medium, and low temperatures, this work examines the role of carbonyl (acetoin and acetol) and dicarbonyl (glyoxal, methylglyoxal, diacetyl, and pentane-2,3-dione) compounds associated with cysteine in the formation of odorous products. In particular, thiazole, 4-methylthiazole, 2-acetylthiazole, and trimethyloxazole and two sulfur and oxygenated heterocyclic compounds, 2-furanmethanethiol and thiophene-2-thiol, are examined. For thiophene-2-thiol, the reactional mechanism is proposed. Attempts were made to detect these compounds in wines from various origins. Certain molecules were identified for the first time in wine.     

Inhibition of trypsin by condensed tannins and wine

Phenolic compounds are abundant vegetable secondary metabolites in the human diet. The ability of procyanidin oligomers and wine polyphenols to inhibit trypsin activity was studied using a versatile and reliable in vitro method. The hydrolysis of the chromogenic substrate N-benzoyl-d,l-arginine-p-nitroanilide (BApNA) by trypsin was followed by spectrophotometry in the presence and absence of condensed tannins and wine. A clear relationship between the degree of polymerization of procyanidins and enzymatic inhibition was observed. Trypsin activity inhibition was also detected in several types of wine. In general, the inhibition increased with the concentration of phenolic compounds in wines. These results may be relevant when considering these compounds as antinutritional factors, thereby contributing to a reduced absorption of nutrients.     

Isomeric influence on the oxidative coloration of phenolic compounds in a model white wine: comparison of (+)-catechin and (-)-epicatechin

The reactions of (+)-catechin and (-)-epicatechin with glyoxylic acid were studied in a model white wine solution. When the reactions were performed in darkness at 45 degrees C, the (-)-epicatechin concentration decreased more rapidly than that of (+)-catechin, and the (-)-epicatechin sample had twice the 440 nm absorbance of the (+)-catechin sample after the 14 day incubation period. The main pigments generated were identified as xanthylium cation pigments regardless of the isomeric character of the phenolic compound. Using a combination of absorbance and ion current data, the xanthylium cation pigments generated from (-)-epicatechin were found to have combined molar absorptivity coefficients 1.8 times that of the xanthylium cation pigments generated from (+)-catechin. The implication of these results on the development of an index of white wine oxidation susceptibility is discussed.     

Concurrent phenomena contributing to the formation of the aroma of wine during aging in oak wood: an analytical study

Red wine was stored in different oak barrels or in stainless steel, and samples were taken for two years to determine 79 aroma compounds. Aging in oak affects 41 compounds. The type of wood affects 11 compounds. At least seven different processes seem to take place concurrently in aroma evolution, and five such processes, affecting 37 compounds, are linked to the oak cask. These are extraction from the wood, oxidation of wine alcohols and amino acids, microbiological formation of ethyl phenols, sorption processes, and condensation of acetaldehyde with polyphenols. The wood can release linear gamma- and delta-lactones, beta-damascenone, and ionones. Some compounds are released very fast from wood, which suggests they lie in the external part of the wood. Some extraction profiles are too complex to be explained by physical processes. Finally, the levels of 2,5-dimethyl-4-hydroxy-3(2H)-furanone and 2-ethyl-5-methyl-4-hydroxy-3(2H)-furanone increase even in the reference wine, which suggests the presence of a precursor.     

Changes during storage in conventional and ecological wine: phenolic content and antioxidant activity

Polyphenol content, free radical scavenging capacity, and changes during storage over 7 months in the dark were studied in ecological and conventional red and white wines. In red wines, the most changeable components during storage were the anthocyanins since during storage anthocyanins content decreased 88% in conventional wine and 91% in ecological wine. Initially, the total flavonol contents of the conventional and ecological red wines were 163.88 +/- 2.69 and 153.58 +/- 1.71 mg/L, respectively, and no significant variations occurred during storage. No differences in hydroxycinnamic acid derivatives content between conventional and ecological red and white wines were observed. The flavonol level in white wines was very low, as expected since these compounds are found in grape skin. The initial antioxidant activity was 5.37 +/- 0.14 and 5.82 +/- 0.31 mM equivalents Trolox for conventional and ecological red wines, respectively; no significant differences were observed (p = 0.2831), and these values were 7-8 times higher than the antioxidant activity observed in conventional and ecological white wine. In contrast with other studies, the total concentrations of phenolic compounds in conventional and ecological red and white wines were not related to antioxidant activity (p > 0.05). In red wines, no significant differences were observed in the antioxidant activity of ecological and conventional red wine (p = 0.28), while in white wine significant differences were observed in the antioxidant activity between conventional and ecological white wine (p = 0.006).     

Red wine antioxidants bind to human lipoproteins and protect them from metal ion-dependent and -independent oxidation

Plant-derived polyphenols may exert beneficial effects on atherosclerosis and cardiovascular diseases, in part, because of their antioxidant properties. In this study we compared the effects of unbound (free) and lipoprotein-associated red wine components on in vitro antioxidant protection of human low-density lipoprotein (LDL). Preincubation of LDL (1 mg protein/mL) with 0-2.5% (v/v) red wine for 3 h at 37 degrees C followed by gel filtration to remove unbound red wine components resulted in a dose-dependent, up to 4-fold increase in LDL-associated antioxidant capacity (measured as Trolox equivalents). Similar results were obtained with high-density lipoprotein (HDL) and bovine serum albumin (BSA). Furthermore, LDL was subjected to oxidation by copper and aqueous peroxyl radicals (2,2'-azobis[2-amidinopropane] dihydrochloride, AAPH). Under both types of oxidative stress, LDL-associated and free red wine components significantly decreased oxidation of the lipoprotein's protein moiety (assessed by tryptophan fluorescence) and lipid moiety (assessed by thiobarbituric acid-reactive substances and conjugated dienes). Similar protective effects of red wine components were observed against HDL oxidation. In contrast, red wine exerted a pro-oxidant effect on copper-induced oxidation of BSA tryptophan residues, while protecting them from AAPH-induced oxidation. Ascorbate strongly enhanced the protective effect of red wine against copper-induced LDL oxidation, and had an additive effect against AAPH-induced oxidation. Our data indicate that red wine components bind to LDL and HDL and protect these lipoproteins from metal ion-dependent and -independent protein and lipid oxidation.     

Ellagic acid and ellagitannins affect on sedimentation in muscadine juice and wine

A mechanism for the formation of water-insoluble sediments in wines and juices made from red and white muscadine grapes (Vitis rotundifolia) was investigated as a function of processing methodology and storage. Sediments are considered quality defects in muscadine grape products, and their presence may influence consumer acceptability and expansion of retail markets. Processing regimes included both hot (70 degrees C) and cold (25 degrees C) press techniques for wine or juice production, and fermentations in contact with grape skins for 3, 5, and 7 days. Relationships between free ellagic acid (FE), total ellagitannins (ET), and total ellagic acid (TE) concentrations were evaluated initially in each product and in sediments that formed during storage for 50 and 120 days at 20 degrees C. Processing techniques influenced initial concentrations of these compounds and the extent of sediment formation. Following storage, juices generally had higher concentrations of FE in sediments compared to wines, but sedimentation was independent of initial FE or TE concentrations. Decreases in ET were observed for hot-pressed juice and skin-fermented wines after storage indicating their hydrolysis during storage and possible contribution to FE in sediments. However, quantitative analysis of the collected sediments revealed that no more than 12% FE by weight was actually present in the sediments, with the remainder consisting of either unidentified compounds or conjugated forms of ellagic acid. This work elucidated a potential mechanism for the presence of FE in muscadine wine and juice sediments through ellagitannin hydrolysis and suggests that sedimentation from mechanisms other than ellagic acid precipitation may also contribute to wine and juice quality.     

Investigations on anthocyanins in wines from Vitis vinifera cv. pinotage: factors influencing the formation of pinotin A and its correlation with wine age

Pinotage red wines were found to contain a reaction product of malvidin 3-glucoside and caffeic acid, the so-called pinotin A. A total of 50 Pinotage wines from the vintages 1996-2002 were analyzed for the content of pinotin A, malvidin 3-glucoside, caffeic acid, and caftaric acid. Statistical analyses were performed to reveal variations in the content of these compounds and to determine the factors that influence pinotin A formation during wine aging. An exponential increase of the concentration of this aging product was observed with prolonged storage time. The most rapid synthesis of pinotin A was observed in 2.5-4 year old wines, although at this age malvidin 3-glucoside is already degraded to a large extent. This phenomenon is explained by the increased ratio of caffeic acid/malvidin 3-glucoside, which strongly favors the formation of pinotin A and makes side reactions less likely. Pinotin A formation proceeds as long as a certain level of malvidin 3-glucoside is maintained in the wines. In wines >5-6 years old degradation or polymerization of pinotin A finally exceeds the rate of its de novo synthesis.     

Analysis of wine components in Cynthiana and Syrah wines

Red wine is composed of a complex matrix of compounds that can interfere with analysis. A high-performance liquid chromatography (HPLC) procedure was developed to efficiently analyze organic acids, sugars, glycerol, and ethanol in Cynthiana (Vitis aestivalis) wine. Standard laboratory procedures (pH, titratable acidity, and color attributes) and HPLC were found reproducible for Cynthiana wine. HPLC recovery efficiency was determined by analysis of spiked and unspiked samples (model, Cynthiana, and Syrah (Vitis vinifera) wines). Although recovery of components was greater in the model wine, recovery in Cynthiana and Syrah wine was comparable. The HPLC procedure was further compared to commercial rapid enzyme analysis tests using model, Cynthiana, and Syrah wines. HPLC analyses were more accurate than enzymatic tests for determining components in the model, Cynthiana, and Syrah wines. Considering the complexity of the wines analyzed, reproducibility and recovery of the HPLC procedure was demonstrated and showed improvement and precision when compared to existing methods.     

Synthesis and stable isotope dilution assay of ethanethiol and diethyl disulfide in wine using solid phase microextraction. Effect of aging on their levels in wine

Ethanethiol and diethyl disulfide (DEDS) most often occurred at levels above their olfactive threshold in wines with nauseous sulfur-linked smells. As ethanethiol is very oxidizable and chemically reactive, a stable isotopic dilution analysis of both ethanethiol and its disulfide in wines using solid phase microextraction and GC-MS was developed. The latter involved the determination of the proportion of DEDS formed by oxidation of the thiol during the analysis conditions, which was obtained by the use of two differently labeled disulfide standards. An original synthesis of labeled ethanethiol standards in conditions minimizing oxidation was developed, and the corresponding labeled diethyl disulfides were obtained from these thiols. This analytical method was used to follow the levels of these sulfur compounds during aging in a young red wine spiked with ethanethiol and added with enological tannins, with or without oxygen addition. The total levels of these two sulfur compounds were shown to decrease steadily after 60 days of aging, up to 83%. The effect of oxygen sped this decrease, but the effect of enological tannins was very slight. Residual ethanethiol was detected in its disulfide form from approximately 36% in the nonoxygenated wines to 69% in the oxygenated samples.     

Roles of grape thaumatin-like protein and chitinase in white wine haze formation

Grape chitinase was found to be the primary cause of heat-induced haze formation in white wines. Chitinase was the dominant protein in a haze induced by treating Sauvignon blanc wine at 30 °C for 22 h. In artificial wines and real wines, chitinase concentration was directly correlated to the turbidity of heat-induced haze formation (50 °C for 3 h). Sulfate was confirmed to have a role in haze formation, likely by converting soluble aggregates into larger visible haze particles. Thaumatin-like protein was detected in the insoluble fraction by SDS-PAGE analysis but had no measurable impact on turbidity. Differential scanning calorimetry demonstrated that the complex mixture of molecules in wine plays a role in thermal instability of wine proteins and contributes additional complexity to the wine haze phenomenon.