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

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

Effects of transporting and processing Sauvignon blanc grapes on 3-mercaptohexan-1-ol precursor concentrations

The effects of different processing treatments on thiol precursor concentrations have been investigated through studies involving transportation of machine-harvested Sauvignon blanc fruit and assessment of different applications of antioxidants, along with juice preparation and enzyme inhibition experiments. The influence these trials had on 3-S-cysteinylhexan-1-ol (Cys-3-MH) and 3-S-glutathionylhexan-1-ol (Glut-3-MH) concentrations in juices is discussed. Very interesting findings included the large increase in precursor concentrations after transportation, particularly for Cys-3-MH, and the limited formation of Glut-3-MH when grape proteins were precipitated during processing. The various results provided information about the ability to modulate precursor concentrations depending on the processing technique employed. Additionally, a conjugated aldehyde, which is the obvious missing link between the reaction of (E)-2-hexenal and glutathione in the formation of Glut-3-MH, has been tentatively identified for the first time. Deuterium-labeled 3-S-glutathionylhexanal (Glut-3-MHAl) was produced through the addition of labeled (E)-2-hexenal to grapes, followed by grape crushing, and detected in the juice by HPLC-MS/MS, along with the corresponding labeled Glut-3-MH.     

Vitisin A content in Chilean wines from Vitis vinifera Cv. Cabernet Sauvignon and contribution to the color of aged red wines

Vitisin A was prepared from malvidin 3-glucoside and pyruvic acid in model wine medium, isolated by countercurrent chromatography, and purified by preparative high-performance liquid chromatography (HPLC). The synthesized compound was used as a reference standard to quantify vitisin A in Chilean wines from Vitis vinifera cv. Cabernet Sauvignon, including a vertical row of wines from the same vineyard over 16 years. Maximum vitisin A content was reached within the first year of storage. Importantly, up to half of the initial amount of vitisin A in young wines was still present in 15 year old wines. Although vitisin A was found to be much more stable as compared to other monomeric C-4 underivatized anthocyanins, it also slowly degrades after reaching its peak concentration. The "color activity concept" was applied to vitisin A, malvidin 3-glucoside, malvidin 3-(6' '-acetylglucoside), and polymeric pigments isolated by countercurrent chromatography in order to estimate their contribution toward the overall color expression of wines. It was found that vitisin A is only a minor contributor to the visually perceived color of aged red wines (color contribution approximately 5%). The major contributor is the polymeric fraction (color contribution approximately 70-90%).     

Sulfur aroma precursor present in S-glutathione conjugate form: identification of S-3-(hexan-1-ol)-glutathione in must from Vitis vinifera L. cv. Sauvignon blanc

When Sauvignon blanc or Gros Manseng grape must was percolated through an immobilized gamma-glutamyltranspeptidase column, there was a significant increase in the concentration of S-3-(hexan-1-ol)-L-cysteine, the precursor of 3-mercaptohexan-1-ol, a compound that contributes to the varietal aroma of wines made from these grapes. Low- and high-resolution liquid secondary ion mass sepectrometry (LSIMS) analyses established the presence of S-3-(hexan-1-ol)-glutathione in Sauvignon blanc must. The identification of this compound suggests that the S-3-(hexan-1-ol)-L-cysteine in grapes is produced by the catabolism of S-3-(hexan-1-ol)-glutathione. As is the case in other plant or animal organisms, S-glutathione conjugates may be involved in certain detoxification systems in vines.     

Effects on 3-mercaptohexan-1-ol precursor concentrations from prolonged storage of Sauvignon blanc grapes prior to crushing and pressing

Formation of wine thiol precursors is a dynamic process, which can be influenced by vineyard and winery processing operations. With the aim of increasing thiol precursor concentrations, a study of the effects of storing machine-harvested Sauvignon blanc grapes prior to crushing and pressing was undertaken on a commercial scale. 3-Mercaptohexan-1-ol (3-MH) precursors, 2-S-glutathionylcaftaric acid (grape reaction product, GRP), glutathione (GSH) and a number of C6 compounds were assessed at several time points during the experiment. The concentration of the cysteine precursor to 3-MH doubled within 8 h and tripled after 30 h while the GSH and cysteinylglycine precursors increased in concentration roughly 1.5 times. (E)-2-Hexenal and GSH levels decreased as thiol precursors, GRP and C6 alcohols increased during storage. Principal component analysis revealed that precursors contributed to most of the variation within the samples over the storage period, with additional influence, primarily from GSH and GRP, as well as (E)-2-hexenal and (Z)-3-hexen-1-ol. Early storage time points were associated with higher concentrations of GSH and some unsaturated C6 compounds while longer storage times were most closely associated with higher thiol precursor and GRP concentrations. This study provides a detailed overview of interactions related to thiol precursor formation on a commercial scale and highlights the ability to manipulate precursor concentrations prior to grape crushing.     

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

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

A novel method for quantification of 2-methyl-3-furanthiol and 2-furanmethanethiol in wines made from Vitis vinifera grape varieties

A rapid, easy method has been developed for isolating and quantifying 2-methyl-3-furanthiol (2M3F) in wines. Until now, it was not possible to quantify this highly odoriferous compound, with a smell reminiscent of cooked meat, in wine. The original aspect of this method is the specific release of volatile thiols using a cysteamine solution applied in reverse flow to sample percolation on the basis of a p-hydroxymercuribenzoate (pHMB)-volatile thiol conjugate formed by the direct addition of pHMB to 50 mL of wine. Purification of volatile thiols in wines is much faster and easier than our previous method. This method may also be used to assay 2-furanmethanethiol in wine. This thiol's strong aroma of roasted coffee has been shown to contribute to the "roast coffee" aroma of certain wines. Assaying 2M3F by this method showed that it was present in the wines analyzed (red and white Bordeaux, Loire Valley Sauvignon blanc, white Burgundy, and Champagne) at concentrations up to 100 ng/L, i.e., significantly above the olfactory perception threshold for this compound in model dilute alcohol solution.     

New insights on 3-mercaptohexanol (3MH) biogenesis in Sauvignon Blanc wines: Cys-3MH and (E)-hexen-2-al are not the major precursors

The molar conversion yield of Cys-3MH into 3MH, during alcoholic fermentation, was traced using a deuterated isotope of the precursor added to different Sauvignon Blanc musts. This yield is close to that found in synthetic media supplemented with synthetic Cys-3MH, that is, below 1%. Yet, this represents only 3-7% of the total 3MH production in wine. This clearly shows that Cys-3MH is a precursor of 3MH, but not the main one in the different musts tested. The contribution of ( E)-hex-2-enal, which has been suggested as another potential precursor of 3MH, was discarded as well, as shown using also a deuterated analogue. The third suggested precursor of 3MH is a glutathionyl-3MH (G-3MH), which upon proteolytic degradation could release Cys-3MH. The knockout of the OPT1 gene, which encodes the major glutathione transporter, reduces 3MH accumulation by a 2-fold factor in grape must as compared to the wild type strain. Consequently, it is deduced that major 3MH precursor(s) are transported into yeast via Opt1p, which is in favor of G-3MH being a 3MH precursor. This work opens the search for the major natural precursor(s) of 3MH in Sauvignon Blanc must.     

Assessing the aromatic potential of Cabernet Sauvignon and Merlot musts used to produce rose wine by assaying the cysteinylated precursor of 3-mercaptohexan-1-ol.

The development of a method for assaying S-3-(hexan-1-ol)-L-cysteine, the cysteinylated precursor of 3-mercaptohexan-1-ol (P-3MH), in must has made it possible to study its impact on the aromatic potential of Merlot and Cabernet Sauvignon grape varieties used to produce rose wines in Bordeaux. The original feature of this method is the purification of very small volumes of must (500 microL) containing P-3MH by affinity chromatography and gas-phase chromatography coupled with mass spectrometry of the purified precursor in trimethylsilylated derivative form. Assays of the cysteinylated precursor in Merlot and Cabernet Sauvignon grapes showed that it was mainly located in the grape skins (60%). Prolonged juice-skin contact increased the must's P-3MH content, and this phenomenon was more marked at higher temperatures. Assessment of the aromatic potential of must used to produce rose wines by chemical analysis of an S-cysteine conjugate is mentioned for the first time.     

Effect of soil type on wines produced from Vitis vinifera L. cv. Grenache in commercial vineyards

In recent years, the wine industry has become increasingly interested in the influence of the terroir characteristics on the features of a wine and, in particular, the mechanisms by which a soil influences wine quality. Among published papers on this topic, most merely describe the effect of the soil; few explain it. In this study were conducted a sensory evaluation and phenolic composition and stilbene concentration tests in order to analyze the effects of soil on wine. Significant differences were found in the results of the tests conducted on two vineyards during two consecutive harvests in 2004 and 2005. The results, in line with previous reports, show that the more fertile of the two vineyards, which was also the one with the greatest water-holding capacity, produced wines that presented significantly lower color intensity and shade, as well as lower total phenolic composition and a smaller quantity of hydroxycinnamic compounds. In 2004, these wines presented significantly higher trans-resveratrol content, due to a fungal attack that was favored by the vineyard's soil characteristics. Extreme drought conditions in 2005 had a marked impact on the characteristics of the wines, increasing wavelength measurements significantly and reducing stilbene concentrations. Finally, sensory evaluations revealed significant differences between the wines produced on the two vineyards in both years for five of the seven attributes evaluated. No significant differences were found from one year to the next between the wines produced from the same vineyard, indicating that the attributes of these wines were maintained despite markedly different vintage conditions.     

Determination of piceid and resveratrol in Spanish wines deriving from Monastrell (Vitis vinifera L.) grape variety

The presence of stilbenes in wine is becoming an important issue due to their claimed relation to a low incidence in coronary diseases and their increasing implication as cancer chemopreventive and neuroprotective agents. Total resveratrol content, quantified as glucoside and aglycone forms of resveratrol, has been determined in a survey of 45 Monastrell monovarietal Spanish red wine types (around 135 wine samples), belonging to Alicante and Bullas appellations. The average between ratio glucoside/aglycone forms of resveratrol in these wines was considerably high, ranging from 82 to 91% of resveratrol in its glycosidic form. This characteristic was observed in a high percentage of the studied wines, which were made under different winemaking procedures, and from different vintages (1995-2002). In addition, wines made using macerative fermentations with double amount of solid parts ("doble pasta") reached the highest levels of total stilbene content expressed as resveratrol equivalent, i.e., 30 mg/L (average of 18.8 mg/L). It can be concluded that high resveratrol glucoside concentration and low free isomer content can be considered characteristics of the Monastrell variety, as it happens to red wines deriving from other varieties grown at warm climates. This fact, also observed for other French and Portuguese red varieties, might play an important role in food habits involving these types of wines.     

New flavanol derivatives from grape (Vitis vinifera) byproducts. Antioxidant aminoethylthio-flavan-3-ol conjugates from a polymeric waste fraction used as a source of flavanols

A new family of antioxidants has been obtained from a residual fraction of polymeric polyphenols of grape origin. The integral exploitation of resources is important in any sustainable production scheme. Many byproducts and residues generated by the agroindustries contain polyphenols with potential application as preventative agents against cancer and cardiovascular diseases. Among these polyphenols oligomeric proanthocyanidins are particularly significant. The polymeric forms, considered of less interest because of their astringent properties, constitute the largest portion of the biologically active plant proanthocyanidins. The new compounds described here result from the breakdown of polymeric flavanols in the presence of cysteamine and bear an amino function, which facilitates their isolation from complex mixtures by cation-exchange gels or resins. In this way, valuable antioxidant molecules can be efficiently obtained from otherwise wasted polymers. The new molecules appear to be as effective as their underivatized counterparts (flavan-3-ols) as antioxidants.     

Determination of carotenoid profiles in grapes, musts, and fortified wines from Douro varieties of Vitis vinifera.

beta-Carotene and six xanthophylls (lutein, neoxanthin, violaxanthin, luteoxanthin, cryptoxanthin, and echinenone) have been identified and semiquantitatively or quantitatively determined in musts and port wines for the first time. An HPLC method was developed and compared with that of one based on thin layer cromatography with scanning densitometry. The most abundant carotenoids present in red grape varieties are beta-carotene and lutein. In wines, significant quantities of violaxanthin, luteoxanthin, and neoxanthin were found. This study was done with berries (skin and pulp), musts, and fortified wines. Some experiments were performed to follow carotenoid content from grapes to wines. Although the levels of beta-carotene and lutein found in fortified wines were lower than those found in musts, other xanthophylls, such as neoxanthin, violaxanthin, and luteoxanthin, exist in appreciable amounts in young ports.     

Detailed characterization of proanthocyanidins in skin, seeds, and wine of Shiraz and Cabernet Sauvignon wine grapes (Vitis vinifera)

The distribution of proanthocyanidin (PA) polymer lengths, proanthocyanidin concentration at each polymer length, and polymer composition were determined in the seed, skin, and wine of Shiraz and Cabernet Sauvignon grape berries grown in southeast Australia. PA was fractionated by semipreparative high performance liquid chromatography (HPLC) and analyzed by phloroglucinolysis and HPLC to report the degree of polymerization (DP), concentration, and composition at 11 DP values in seed and wine and 21 DP values in skin. In skin, the highest PA concentration was observed at a DP of 31 in Shiraz and 29 in Cabernet Sauvignon representing 15% of the total PA in both varieties. The distribution of seed PA had the highest concentration at a DP of 7 in Shiraz and 6 in Cabernet Sauvignon representing around 30% of the total PA. In the wine PA distribution, the highest concentration was observed at a DP of 11 in Shiraz and 9 in Cabernet Sauvignon representing around 26 and 32% of the distribution, respectively. A second peak in wine PA concentration was observed at the largest DP of 18 in Shiraz and 15 in Cabernet Sauvignon representing around 20% of the distribution. The composition in wine did not vary at different DP, but the proportion of epicatechin gallate varied in seed PA less than 4 DP. The proportion of epigallocatechin increased with increasing DP in skin PA. Wine PA had a DP range and composition similar to the distribution of skin PA between DP 4 and 18 suggesting that larger skin PAs are not extracted into wine. This study provides information that could be used to target the important PA fractions in grapes that need to be measured to understand (or predict) PA extraction into wine and eventual mouthfeel.     

Identification and quantitation of 3-S-cysteinylglycinehexan-1-ol (Cysgly-3-MH) in Sauvignon blanc grape juice by HPLC-MS/MS

Precursors to varietal wine thiols are a key area of grape and wine research. Several such precursors, in the form of odorless conjugates, have been closely studied in recent years. A new conjugate has now been identified as 3-S-cysteinylglycinehexan-1-ol (Cysgly-3-MH), being the dipeptide intermediate between cysteine and glutathione precursors of tropical thiol 3-mercaptohexan-1-ol (3-MH). Authentic Cysgly-3-MH was produced via enzymatic transformation of the glutathione conjugate and used to verify the presence of both diastereomers of Cysgly-3-MH in Sauvignon blanc juice extracts. Cysgly-3-MH was added into our HPLC-MS/MS precursor method, and the validated method was used to quantify this new analyte in a selection of Sauvignon blanc juice extracts. Cysgly-3-MH was found in the highest concentrations (10-28.5 μg/L combined diastereomer total) in extracts from berries that had been machine-harvested and transported for 800 km in 12 h. This dipeptide conjugate was much less abundant than the glutathione and cysteine conjugates in the samples studied. On the basis of the results, the new cysteinylglycine conjugate of 3-MH seemingly has a short existence as an intermediate precursor, which may explain why it has not been identified as a natural juice component until now.     

Analysis of grape Vitis vinifera L. DNA in must mixtures and experimental mixed wines using microsatellite markers

Because wine quality highly relies on the varietal composition of the must, the development of methods allowing the authentication of varieties in musts and wines would be of great value as a guarantee of quality. Microsatellite markers have already been applied to the authentication of grape juices (Faria, M. A.; Magalh?es, R.; Ferreira, M. A.; Meredith, C. P.; Ferreira Monteiro, F. J. Agric. Food Chem. 2000, 48, 1096-1100) and to the analysis of experimental wines (Siret, R.; Boursiquot, J. M.; Merle, M. H.; Cabanis, J. C.; This, P. J. Agric Food Chem. 2000, 48, 5035-5040). In the present paper, we accessed the usefulness of this technology for the analysis of must and wine mixtures. The detection limit of DNA mixtures was first estimated on DNA extracted from leaves: 4% of a foreign DNA can be detected. Analysis of must and wine mixtures (Chardonnay B/Clairette B and Syrah N/Grenache N) was performed on experimental fermentations. DNA was extracted along the fermentation process and analyzed using five microsatellite loci. The 70:30 (v/v) mixtures were successfully analyzed until the end of the fermentation. The applications of these results to commercial purposes are discussed.     

Influence of vine training and sunlight exposure on the 3-alkyl-2-methoxypyrazines content in musts and wines from the Vitis vinifera variety cabernet sauvignon

The influence of vine training and sunlight exposure on the 3-alkyl-2-methoxypyrazines contents in musts and wines was studied by means of two previously reported methods based on headspace solid-phase micro-extraction. Experimental samples were monitored throughout grape ripening and wine making. 3-Isobutyl-2-methoxypyrazine, 3-sec-butyl-2-methoxypyrazine and 3-isopropyl-2-methoxypyrazine were identified. The 3-isobutyl-2-methoxypyrazine content decreased throughout grape ripening in all of the sample types studied. After 1 day of maceration with the skins, there was an increase, but after racking, no further increase was observed. No significant differences between samples were found during grape ripening. Wines from goblet-trained vines, however, contained significantly less 3-isobutyl-2-methoxypyrazine. Clusters protected from sunlight since the beginning of the veraison resulted in wines with a significantly lower content of this compound than the control samples.     

Stilbenes and tyrosol as target compounds in the assessment of antioxidant and hypolipidemic activity of Vitis vinifera red wines from southern Brazil

The contents of stilbene monomers, cis-resveratrol, trans-resveratrol, cis-piceid, trans-piceid, and tyrosol, were quantified in Vitis vinifera red wines, cvs. Cabernet Franc, Merlot, Sangiovese, and Syrah, 2006 and 2007 vintages, from the Sa?o Joaquim region, a new grape-growing region at southern Brazil. Moreover, the effect of chronic consumption of these wines on the antioxidant and hypolipidemic activities was monitored in C57BL6 LDL receptor knockout mice and treated with a hypercholesterolemic diet. Red wines from this region had substantial levels of resveratrols (the predominant forms were glycoside and trans) and tyrosol. Biomonitoring of antioxidant and hypolipidemic activities in vivo revealed that consumption of these wines increased the antioxidant capacity and reduced the hypercholesterolemia and hypertriglyceridemia promoted by the hypercholesterolemic diet. Significant correlations were found between the increase of antioxidant capacity markers, the decrease of lipid levels promoted by wine consumption, and the contents of stilbenes and tyrosol, supporting the important biological activity of these compounds.     

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.     

葡萄ACO家族基因过量表达对番茄乙烯释放速率的影响

乙烯是启动和促进果蔬成熟和衰老的内源激素,ACC氧化酶(1-aminocyclopropane-1-carboxylic acid oxidase,ACO)是乙烯生物合成中的一个关键限速酶.葡萄ACO基因拥有一个基因家族,其中VvACO1和VvACO2基因在果实转色时期出现转录高峰,是葡萄果实发育过程中造成乙烯含量发生变化的关键基因.为验证葡萄(Vitis vinifera)ACO家族基因过量表达对番茄(Solanum locopersicum)乙烯释放速率的影响,本研究通过根癌农杆菌(Agrobacterium tumefaciens)介导法将葡萄ACO基因导入番茄基因组中,进行了PCR和RT-PCR分子检测,并对分子检测阳性的转基因番茄植株叶片的ACO酶活性和乙烯释放速率进行了分析.RT-PCR检测表明,在3个VvA CO1、4个VvACO2和6个VvACO3转基因番茄植株中目的基因均有一定的表达.所有转基因番茄植株的ACO酶活性和乙烯释放速率均比对照有所提高,其中以VvACO1基因的过量表达对叶片ACO酶活和乙烯释放速率增加的幅度最大,VvACO3基因的过量表达对果实的ACO酶活和乙烯释放速率增加的幅度最大,而转化VvACO2基因的番茄植株ACO酶活性和乙烯释放速率最低,比对照略有升高,且植株呈现矮化现象.本研究通过观察转基因番茄的生长状况以及测定转基因番茄的生理指标,探讨葡萄ACO家族基因的过量表达对番茄的影响,来预测葡萄ACO基因的初步功能.