A new class of blue anthocyanin-derived pigments isolated from red wines

Two newly formed anthocyanin-derived pigments that revealed unique spectroscopic features, showing maximum absorption in their UV-vis spectra at 575 nm, were isolated by TSK Toyopearl HW-40 (S) gel column chromatography and semipreparative HPLC from an aged Port red wine. Further characterization by ESI/MS and NMR ((1)H, gCOSY) showed them to belong to a new class of pigments described here for the first time, the structure of which consisted of a pyranoanthocyanin moiety linked to a flavanol by a vinyl bridge. The extended conjugation of the pi electrons throughout all the pigment molecule is likely to confer a higher stability on it and is probably the origin of the intense blue color. The formation of these pigments was found to arise from the reaction between anthocyanin-pyruvic acid adducts and vinyl-flavanol adducts.     

Isolation and structural characterization of new anthocyanin-derived yellow pigments in aged red wines

Two newly formed yellow pigments that revealed unique spectral features were detected and isolated from an aged Port red wine by TSK Toyopearl HW-40(s) gel chromatography and characterized by UV-visible spectrophotometry, 1H NMR and 13C NMR, and mass spectrometry (LC-ESI/MS). The UV-vis spectra of these pigments showed maximum absorption at 478 nm that is significantly hypsochromically shifted from those of original grape anthocyanins and other pyranoanthocyanins, exhibiting a more yellow hue in acidic solution. The structures of these pigments correspond to methyl-linked pyranomalvidin 3-glucoside and its respective coumaroyl glucoside derivative. They were shown to arise from the reaction between acetoacetic acid and genuine grape anthocyanins. Isolation and NMR identification using 1D and 2D NMR techniques are reported for the first time for this new family of anthocyanin-derived yellow pigments occurring in red wines.     

Identification of anthocyanin-flavanol pigments in red wines by NMR and mass spectrometry

Three newly formed Port wine pigments were isolated by Toyopearl HW-40(s) gel chromatography and semipreparative HPLC. Furthermore, the pigments were identified by mass spectrometry (LC/MS) and NMR techniques (1D and 2D). These anthocyanin-derived pigments showed UV-visible spectra different from those of the original grape anthocyanins. These pigments correspond to malvidin 3-glucoside linked through a vinyl bond to either (+)-catechin, (-)-epicatechin, or procyanidin dimer B3 [(+)-catechin-(+)-catechin]. NMR data of these pigments are reported for the first time.     

Occurrence of pyranoanthocyanins in sparkling wines manufactured with red grape varieties

The anthocyanin pigments in rosé (Vitis vinifera cv. Garnacha) and blanc de noir (V. vinifera cv. Monastrell) base and sparkling wines were studied by LC-DAD/ESI-MS. Anthocyanins of grape origin and pyranoanthocyanins resulting from C-4/C-5 cycloaddition of the former ones with pyruvic acid, acetaldehyde, 4-vinylphenol, 4-vinylguaiacol, and 4-vinylcatechol were identified in the different wines. Rosé wines presented a higher total pigment content than blanc de noir wines. Pyranoanthocyanins represented 68.9-76.0% of total pigment content in rosé wines and 49.4-60.7% in blanc de noir wines. Malvidin 3-glucoside-pyruvate was the most abundant pigment in both rosé and blanc de noir base wines. Important qualitative and quantitative changes were observed in terms of the anthocyanin and pyranoanthocyanin pigments after the second (bottle) fermentation and 9 months of aging on yeast lees, but not after a further time (3-9 additional months) of aging on lees. Evaluation of the wine color characteristics was consistent with a greater color stability for the rosé sparkling wines that could be associated with the high content, structural diversity, and spectroscopic features of the pyranoanthocyanins present in these wines.     

Isolation and structural characterization of new acylated anthocyanin-vinyl-flavanol pigments occurring in aging red wines

Three newly formed pigments were detected and isolated from a 2-year-old Port wine through TSK Toyopearl HW-40(S) gel column chromatography and characterized by UV-visible spectrophotometry, NMR, and mass spectrometry (ESI/MS). (1)H NMR and (13)C NMR data for these pigments obtained using 1D and 2D NMR techniques (COSY, NOESY, gHSQC, and gHMBC) are reported for the first time. The structure of the pigments was found to correspond to the vinyl cycloadducts of malvidin 3-coumaroylglucoside bearing either a procyanidin dimer or a flavanol monomer ((+)-catechin or (-)-epicatechin). Additionally, conformational analysis was performed for one of these newly formed pigment using computer-assisted model building and molecular mechanics. A chemical nomenclature is proposed to unambiguously name this new family of anthocyanin-derived pigments.     

Pathway leading to the formation of anthocyanin-vinylphenol adducts and related pigments in red wines

On the basis of observations from Vitis vinifera cv. Pinotage wines and experiments performed in model wine medium, a new chemical pathway responsible for the formation of anthocyanin-vinylphenol adducts in red wines is described. Until now, these pigments have been considered to be reaction products of anthocyanins and vinylphenols, the latter being generated during fermentation by enzymatic decarboxylation of the respective cinnamic acids. The mechanism of the novel pathway, involving intact hydroxycinnamic acid and anthocyanin, is explained. Only cinnamic acids with electron-donating substituents on the aromatic ring, such as coumaric acid, ferulic acid, caffeic acid, and sinapic acid, undergo this conversion, as they stabilize an intermediately formed carbenium ion. Decarboxylation and oxidation of the pyran moieties are the final steps in the generation of the corresponding 4-vinylphenol, 4-vinylguaiacol, 4-vinylcatechol, and 4-vinylsyringol adducts of anthocyanins in red wine.     

Evolution and stability of anthocyanin-derived pigments during Port wine aging.

For three years, the evolution of the three major anthocyanidin monoglucosides (malvidin 3-glucoside, malvidin 3-acetylglucoside, and malvidin 3-coumaroylglucoside) and their anthocyanin-pyruvic acid adducts was monitored in Port wines stored in oak barrels. The degradation reactions of all pigments followed first-order kinetics in all the wines studied. The degradation rate constants of the anthocyanin-pyruvic acid adducts were much lower than those of the anthocyanidin monoglucosides. The results of both anthocyanins and pyruvic acid adducts show that acylation on the sugar moiety of all the pigments decreased their stability in wine. The levels of malvidin 3-glucoside-pyruvic acid adduct and its acylated forms increased right after wine fortification with wine spirit before starting to decrease around 100 days. The initial formation of anthocyanin-pyruvic acid adducts was concurrent with the degradation of anthocyanidin monoglucosides.     

Role of polyphenols in copper complexation in red wines.

Four monovarietal red wines, Tinta Roriz, Tinta Barroca, Touriga Nacional, and Touriga Francesa, were studied for their complexometric properties toward copper. For the purpose, ion selective electrode potentiometric titrations were carried out, and the data were treated by two methods suitable for heterogeneous ligands: the Scatchard plot, which provided the complexation capacity (CC(total) = maximum number of moles of sites per liter), and the differential equilibrium function (DEF), which provided the conditional stability constants of the sites effectively involved in the metal complexation (K(DEF)) at a given titration point. The different wines displayed similar properties: CC(total) ranged from 4.5x10(-3) (Tinta Barroca) to 4.9x10(-3) M (Touriga Francesa), and log K(DEF) values were between 4.2 and 6.5. The range of log(|Cu|(bound)/CC(total)) = log theta embraced in the titration was between -0.2 and -1.9. With regard to the natural levels of copper, from 4.4x10(-7) (Tinta Roriz) to 1.3x10(-6) M (Touriga Nacional), and ligands (CC(total)) in the wines, it can be expected that copper will be strongly complexed in the studied wines (log K(DEF) > 6). Studies carried out, in parallel, for solutions of isolated seed tannins and skin polyphenols (mainly anthocyans) showed that their contribution to the CC(total) of the wines was <40% for tannins and <2% for skin polyphenols. However, skin polyphenols were shown to be much stronger copper ligands (log K(DEF) = 6.7, log theta = -1.5) than tannins (log K(DEF) = 4.5, log theta = -1.5).     

Effect of red grapes co-winemaking in polyphenols and color of wines

The red grapes co-winemaking effect on phenolic fraction and wine color has been studied for the first time, where Monastrell was comacerated and cofermentated with Cabernet Sauvignon and Merlot. Changes in the relative abundance of anthocyanins were observed as well as hyperchromic shifts at 530 and 620 nm; these effects remain constant after aging. Co-winemaking also favored copigmentation, giving way to more stable anthocyanins and facilitating their polymerization. With regard to color evolution, the mixture of Monastrell with Merlot grapes was more appropriate than with Cabernet Sauvignon for aging wines in oak barrels. The extent of copigmentation was more important in young wines than in aged wines. This is mainly due to the self-anthocyanin monomer reactions in the case of young wines, whereas in aged wines copigmentation is mainly due to the reaction between the anthocyanins and other polyphenolic cofactors. Discriminant analysis showed the possibility of differentiating wines according to the aging time and the type of wine, with color parameters (color intensity, OD 620 nm, and OD 520 nm) being the most important discrimination variables in the first case and petunidin-3-glucoside and peonidin-3-glucoside contents in the second case.     

Multivariate analysis of antioxidant power and polyphenolic composition in red wines

It has been demonstrated that the dietary intake of compounds having antioxidant activity is very important, and various chemical, biological, and electrochemical methods have been proposed to evaluate the antioxidant power of compounds such as polyphenols. Wine, although nonessential, has a high polyphenol content up to 2-3 g/L in red wines obtained by traditional maceration. The polyphenol content of wines is usually evaluated by the Folin reagent, which provides an appropriate response to the requirements of wine manufacturers. Because the presence of individual polyphenols may be evaluated by HPLC, more or less selective methods toward the various chemical classes of polyphenols have been developed. An HPLC method set up recently was applied to evaluate how individual polyphenols contributed to the overall antioxidant power (AOP) of 60 Italian red wines, trying to identify the effect that individual compounds may have on the total AOP. Application of the multivariate analysis allowed us to detect some determining compounds such as gallic acid and some flavonols. On the basis of the correlation between two traditional chemical methods, namely the total polyphenol determination by the Folin reagent and the flavanol determination by the condensation reaction with p-(dimethylamino)-cinnamaldehyde, it was shown that the use of these two merely chemical methods is well correlated (r = 0.83 and 0.87) to an AOP evaluation of red wines.     

Stable free radicals and peroxyl radical trapping capacity in red wines

Thirty-two experimental red wines, obtained from eight cultivars and aged in bottles for 2 and 7 years, were examined for the presence of stable free radicals (SFR), for the peroxyl radical trapping capacity (PRTC), and for the concentrations of some important polyphenol families. Aging significantly increases SFR, polyphenol polymers with n > or = 5 (HMWP), and PRTC and is accompanied by a strong decrease of free anthocyanins. Multivariate regression analyses show that HMWP and SFR are independently associated with PRTC while HMWP and anthocyanins are independently associated with the formation of SFR. These results indicate that polymeric polyphenols generated from anthocyanins and proanthocyanidins during wine aging are able to convert highly reactive free radicals into nonreactive radicals through electron delocalization. The strict correlation between SFR and antioxidant activity that we found suggests that these characteristics are related to the functional properties of food.     

Application of an ELISA to the determination of benalaxyl in red wines

The applicability of an ELISA for detection and quantification of benalaxyl in red wine samples is described. The study of the influence of this matrix on the reliability of the assay indicates that red wine samples require a rapid and simple cleanup step before ELISA assay. Recovery and precision of the method were evaluated by spiking red wine samples with benalaxyl in the 0.5-24 ng/mL range. Benalaxyl can be determined with good accuracy and precision up to 0. 5 ng/mL in starting red wine samples (detection limit of 0.13 ng/mL). No false negative or positive results were obtained. Authentic red wine samples were analyzed by ELISA and by RP-HPLC. The amounts of benalaxyl found by ELISA were in good agreement with RP-HPLC analysis.     

Varietal differentiation of red wines in the Valencian region (Spain)

Ninety-one young varietal wines from the Valencian community (Spain), made from Cabernet Sauvignon, Tempranillo, Monastrell, and Bobal grapes, were tested on the basis of 33 variables: 9 conventional parameters, 10 alcohols and polyols, and 14 esters. Discriminant analysis was used to identify and explain the differences among samples, as well as to determine whether it is possible or not to differentiate among varieties. This differentiation (100% of the samples) has been possible due to the new discriminant analysis based on only 11 main variables: total acidity, cis-3-hexenol, methanol, glycerol, 2,3-butanediol, isobutyric alcohol, 1-pentanol, acetaldehyde, ethyl propionate, ethyl decanoato, and gamma-butyrolactone, which allow differentiating 100% of the 1994 vintage and 97% of the 1995 vintage.     

Free radical scavenging capacity in the aging of selected red Spanish wines

Free radical scavenging capacity by the DPPH(*) method and main physicochemical properties, polyphenols content by HPLC, color by a tristimulus colorimeter, and UV-vis spectra in the aging of selected red Spanish wines, were studied. As the wines age, they become darker (lower lightness, L) and increase their hue angle (lower red color) as well as the ratio of absorbance at 420 nm to that at 520 nm. Main polyphenolics identified in the samples were tannic acid, oenin, and gallic acid. The antiradical efficiency of the samples increased during aging, which could be related to an increase in the tannic acid concentration shown by the following correlationship: EC(50) = 1/(0.18 + 0.0011[tannic acid](mg/L)) with a correlation coefficient of 0.744.     

Flavonol profiles of Vitis vinifera red grapes and their single-cultivar wines

The main flavonols found in seven widespread Vitis vinifera red grape cultivars include the 3-glucosides and 3-glucuronides of myricetin and quercetin and the 3-glucosides of kaempferol and isorhamnetin. In addition, the methoxylated trisubstituted flavonols, laricitrin and syringetin, were predominantly found as 3-glucosides. As minority flavonols, the results suggest the detection of the 3-galactosides of kaempferol and laricitrin, the 3-glucuronide of kaempferol, and the 3-(6' '-acetyl)glucosides of quercetin and syringetin. The flavonol profiles based on the eight above-mentioned flavonols allowed the cultivar differentiation of the grape samples. With regard to flavonol biosynthesis in the berry skin, quercetin 3-glucuronide predominated at véraison, followed by quercetin 3-glucoside, and only trace amounts of trisubstituted flavonols were detected. The proportion of quercetin 3-glucoside remained almost constant during berry ripening, whereas the proportion of quercetin 3-glucuronide decreased and the other flavonols, especially myricetin 3-glucoside, increased their importance. In wines, flavonol 3-glycosides coexisted with their corresponding free aglycones released by hydrolysis. The presence of laricitrin, syringetin, and laricitrin 3-glucoside in red wines is reported here for the first time. The extent of hydrolysis was widely variable among wines made from the same grape cultivar, and the results suggest the influence of the type of aglycone and glycoside on the rate of hydrolysis. Due to hydrolysis, the differentiation of single-cultivar wines gave acceptable results only when aglycone-type flavonol profiles were used.     

Wheat gluten used as a clarifying agent of red wines.

Bovine spongiform encephalopathy caused a situation of crisis leading the public and winemakers to lose their confidence in the use of gelatin as a fining agent and to reject animal proteins in general. Therefore, we started the search for a substitute for gelatin and egg protein by comparing gluten with these fining treatments currently used. This study concerned the fining of a Burgundy red wine (Rully, Controlled Appellation). For 6 g/hL, enzymatically hydrolyzed glutens (EHG) gave better efficiencies than deamidated glutens. The efficiency of the egg proteins treatment was situated between those of the hydrolyzed glutens and deamidated glutens. For 12 and 18 g/hL, turbidities of the wine treated by five glutens were 67 to 86% less than that of the control wine. Better results were obtained with egg proteins for short kinetics particularly. Wine fining with gluten was always better than gelatin treatments. The differences between the five glutens became very small when the dose incorporated in the wine increased. The volumes of lees generated by fining with gluten are situated between the values obtained with egg proteins and gelatin. After fining, immunodetection with gluten polyclonal antibodies failed to detect residual deamidated gluten.     

Terpene compounds as possible precursors of 1,8-cineole in red grapes and wines

While the contribution of 1,8-cineole to the aroma of wine has been reported, it is a matter of controversy that the vineyards producing such wines are surrounded by Eucalyptus trees, which may contribute their essence to the grapes. However, experimental information presented in this paper suggests that 1,8-cineole can be produced by chemical transformation of limonene and alpha-terpineol, and this process may be responsible for the occurrence of Eucalyptus-like aroma in Tannat wines from vines not grown in the vicinity of Eucalyptus trees. A mechanism for the chemical transformation of these aroma compounds is proposed.     

Effect of pesticide residues on the aromatic composition of red wines.

The influence of pesticide residues on the aromatic composition (major and minor volatiles) of red wines made from Vitis vinifera (Monastrell var.) was studied by comparing the concentration of aromas in wines made from grapes subjected (or not) to phytosanitary treatment with chlorpyrifos, fenarimol, mancozeb, metalaxyl, penconazole, and vinclozolin, according to the agricultural practice of the area. The analytical determination of the major volatiles was made by gas chromatography using a flame ionization detector, while the minor volatiles were determined by adsorption-thermal desorption gas chromatography using a mass selective detector. There were significant differences between the ethyl acetate, methanol, isobutanol, and diethylacetal levels of the control wine and that containing chlorpyrifos residues, although only the ethyl acetate exceeded the olfactory threshold. With regard to the minor volatiles, significant differences were detected in the concentrations of some esters, aldehydes, and acids. However, only isoamyl acetate exceeded the olfactory threshold in wines containing residues of chlorpyrifos, fenarimol, and vinclozolin.     

Differentiation of young red wines based on chemometrics of minor polyphenolic constituents

For reasons of examining any possible discrimination of young red wines based on selected, minor polyphenols, a survey was carried out including 35 samples originating from three Hellenic native and three international Vitis vinifera cultivars from various regions of Greece. All samples were experimental wines vinified and stored under identical conditions, in an effort to minimize the effect of different winemaking technologies as well as aging. The polyphenols analyzed belonged to two categories: benzoic acid derivatives, including gallic acid, protocatechuic acid, vanillic acid, and syringic acid, and stilbenes, including astringin, piceid, and resveratrol (all trans isomers). Data handling employing discriminant analysis (DA) yielded very satisfactory categorization of samples in terms of both cultivar and geographical region of origin. This outcome was discussed with regard to the value of certain minor polyphenols that could serve as characteristic indices for discrimination of varietal red wines, after appropriate implementation of chemometrics.