In situ analysis and structural elucidation of sainfoin (Onobrychis viciifolia) tannins for high-throughput germplasm screening

A rapid thiolytic degradation and cleanup procedure was developed for analyzing tannins directly in chlorophyll-containing sainfoin ( Onobrychis viciifolia ) plants. The technique proved suitable for complex tannin mixtures containing catechin, epicatechin, gallocatechin, and epigallocatechin flavan-3-ol units. The reaction time was standardized at 60 min to minimize the loss of structural information as a result of epimerization and degradation of terminal flavan-3-ol units. The results were evaluated by separate analysis of extractable and unextractable tannins, which accounted for 63.6-113.7% of the in situ plant tannins. It is of note that 70% aqueous acetone extracted tannins with a lower mean degree of polymerization (mDP) than was found for tannins analyzed in situ. Extractable tannins had between 4 and 29 lower mDP values. The method was validated by comparing results from individual and mixed sample sets. The tannin composition of different sainfoin accessions covered a range of mDP values from 16 to 83, procyanidin/prodelphinidin (PC/PD) ratios from 19.2/80.8 to 45.6/54.4, and cis/trans ratios from 74.1/25.9 to 88.0/12.0. This is the first high-throughput screening method that is suitable for analyzing condensed tannin contents and structural composition directly in green plant tissue.     

Ripening-induced changes in grape skin proanthocyanidins modify their interaction with cell walls

Proanthocyanidins were isolated from the skins of Cabernet Sauvignon grapes at different stages of grape development in order to study the effect of proanthocyanidin modification on the interaction with grape cell wall material. After veraison, the degree of proanthocyanidin polymerization increased, and thereafter was variable between 24 and 33 subunits as ripening progressed. Affinity of skin cell wall material for proanthocyanidin decreased with proanthocyanidin ripeness following veraison. A significant negative relationship (R2=0.93) was found for average proanthocyanidin molecular mass and the proportion of high molecular mass proanthocyanidin adsorbed by skin cell wall material. This indicated that as proanthocyanidin polymerization increased, the affinity of a component of high molecular mass proanthocyanidins for skin cell wall material declined. This phenomenon was only associated with skin proanthocyanidins from colored grapes, as high molecular mass proanthocyanidins of equivalent subunit composition from colorless mutant Cabernet Sauvignon grapes had a higher affinity for skin cell wall material.     

Composition and cellular localization of tannins in grape seeds during maturation

Cell walls were isolated from seeds of grape berries (Vitis vinifera L.), and proanthocyanidin composition was determined over the course of ripening for different levels of vine water status. During the ripening period the tannins from the cell walls were always more polymerized than those from the inner part of the cell. At maturity this difference becomes more significant compared to véraison, due to a significant increase in the mean degree of polymerization of the cell wall tannins. The tannin composition was typical of grape seed tannins and was quite similar in the two cell fractions studied, but the epicatechin gallate proportion was significantly higher in the cell wall fraction. There were no significant effects of water deficit on composition and polymerization of seed tannins.     

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.     

Influence of grape maturity and maceration length on color, polyphenolic composition, and polysaccharide content of cabernet sauvignon and tempranillo wines

The aim of this paper was to study how maturity and maceration length affect color, phenolic compounds, polysaccharides, and sensorial quality of Cabernet Sauvignon and Tempranillo wines at three stages of grape ripening. Ripeness increased color extractability, phenolic compounds, and polysaccharide concentrations. Moreover, the proanthocyanidin mean degree of polymerization (mDP) and the percentage of prodelphinidins also increased with maturity, whereas the percentage of galloylation decreased. In general, wines from riper grapes contain higher proportions of skin proanthocyanidins. Color and anthocyanin concentration decreased when the maceration was longer, whereas polysaccharide and proanthocyanidin concentrations did the opposite. It was also detected that the mDP and the percentage of prodelphinidins decreased when the maceration was extended, whereas the percentage of galloylation increased. These data seem to indicate that proanthocyanidin extraction from seeds is clearly increased throughout the maceration time.     

Quantitative analysis of red wine tannins using Fourier-transform mid-infrared spectrometry

Tannin content and composition are critical quality components of red wines. No spectroscopic method assessing these phenols in wine has been described so far. We report here a new method using Fourier transform mid-infrared (FT-MIR) spectroscopy and chemometric techniques for the quantitative analysis of red wine tannins. Calibration models were developed using protein precipitation and phloroglucinolysis as analytical reference methods. After spectra preprocessing, six different predictive partial least-squares (PLS) models were evaluated, including the use of interval selection procedures such as iPLS and CSMWPLS. PLS regression with full-range (650-4000 cm(-1)), second derivative of the spectra and phloroglucinolysis as the reference method gave the most accurate determination for tannin concentration (RMSEC = 2.6%, RMSEP = 9.4%, r = 0.995). The prediction of the mean degree of polymerization (mDP) of the tannins also gave a reasonable prediction (RMSEC = 6.7%, RMSEP = 10.3%, r = 0.958). These results represent the first step in the development of a spectroscopic methodology for the quantification of several phenolic compounds that are critical for wine quality.     

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.     

1H nuclear magnetic resonance-based metabolomic characterization of wines by grape varieties and production areas

(1)H NMR spectroscopy was used to investigate the metabolic differences in wines produced from different grape varieties and different regions. A significant separation among wines from Campbell Early, Cabernet Sauvignon, and Shiraz grapes was observed using principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA). The metabolites contributing to the separation were assigned to be 2,3-butanediol, lactate, acetate, proline, succinate, malate, glycerol, tartarate, glucose, and phenolic compounds by PCA and PLS-DA loading plots. Wines produced from Cabernet Sauvignon grapes harvested in the continental areas of Australia, France, and California were also separated. PLS-DA loading plots revealed that the level of proline in Californian Cabernet Sauvignon wines was higher than that in Australian and French Cabernet Sauvignon, Australian Shiraz, and Korean Campbell Early wines, showing that the chemical composition of the grape berries varies with the variety and growing area. This study highlights the applicability of NMR-based metabolomics with multivariate statistical data sets in determining wine quality and product origin.     

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.     

Characterization of Vitis vinifera L. Cv. Carménère grape and wine proanthocyanidins

A formal compositional study of the proanthocyanidins of Vitis vinifera L. cv. Carménère was conducted in this work. We first characterized the polymeric proanthocyanidins of Carménère skins, seeds, and wines. In addition, the wine astringency was analyzed and compared with Cabernet Sauvignon. Although Carménère wines had a higher proanthocyanidin concentration and mean degree of polymerization than Cabernet Sauvignon wines, the former wines were perceived as less astringent. The low seed/skin proportion in Carménère wines as compared to other varieties, as evidenced by the reduced number of seeds per berry and the higher amount of epigallocatechin subunits of Carménère wine proanthocyanidins, could explain this apparent paradox.     

Variations in the profile and content of anthocyanins in wines made from cabernet sauvignon and hybrid grapes

To detect adulteration of wine, it has been proposed that the ratio of acetylated to p-coumaroylated conjugates of nine characteristic anthocyanins can be used to determine whether a wine is derived from Cabernet Sauvignon or hybrid grapes. If the ratio is >3, then a wine is classified as being derived from Cabernet Sauvignon grapes. This test has significant commercial implications as it is being used to decide whether Cabernet Sauvignon-labeled wines are genuine and can be imported into Germany. To assess whether this is a valid approach, 24 wines were analyzed, 4 of which were made from hybrids and 20 from Cabernet Sauvignon, with vintages ranging from 1993 to 2000. Only 13 of the Cabernet Sauvignon wines contained all nine of the "characteristic" anthocyanins, and the ratio of acetylated to p-coumaroylated derivatives varied from 1.2 to 6.5. It is evident that the use of the anthocyanin ratio method is flawed and that examination of the whole anthocyanin profile and/or investigation of the proportion of monoglucoside and acetylated anthocyanins is a better approach to distinguish between hybrid and Cabernet Sauvignon wines.     

Proanthocyanidin composition in the seed coat of lentils (Lens culinaris L.)

Lentils (Lens culinaris L.) are a popular food in many countries. However, little is known about their phenolic composition. Because polyphenols in lentils are located essentially in their seed coat, the objective of this work was to study the composition of proanthocyanidins, the major group of polyphenols, in this part of the tissue. The use of C(18) Sep-Pak cartridges permitted the fractionation of lentil seed coat extract into monomer, oligomer, and polymer proanthocyanidin fractions. Subsequent thiolysis of oligomer and polymer fractions followed by HPLC analysis allowed the mean degree of polymerization (mDP) and the structural composition of proanthocyanidins to be determined. A fractionation of lentil seed coat extracts on a polyamide column followed by HPLC and HPLC-DAD-MS analyses was used to identify the individual proanthocyanidins. The results showed that the major monomeric flavan-3-ol was (+) catechin-3-glucose, with lesser amounts of (+)-catechin and (-)-epicatechin. In the oligomer fraction, various dimer, trimer, and tetramer proanthocyanidins constituted of catechin, gallocatechin, and catechin gallate units were identified, and several procyanidins and prodelphinidins from pentamers to nonamers constitute the polymer fraction. The most abundant proanthocyanidins in the seed coat of lentils are the polymers (65-75%), with a mDP of 7-9, followed by the oligomers (20-30%), with a mDP of 4-5.     

NMR, ESI/MS, and MALDI-TOF/MS analysis of pear juice polymeric proanthocyanidins with potent free radical scavenging activity

The structure of a polymeric proanthocyanidin fraction isolated from pear juice was characterized by NMR, ESI/MS, and MALDI-TOF/MS analyses, and its antioxidant activity was investigated using the DPPH free radical scavenging method. The results obtained from 13C NMR analysis showed the predominance of signals representative of procyanidins. Typical signals in the chemical shift region between 70 and 90 ppm demonstrated the exclusive presence of epicatechin units. The results obtained through negative ESI/MS analysis showed singly and doubly charged ions corresponding to the molecular mass of procyanidins with a degree of polymerization up to 22. The spectra obtained through MALDI-TOF/MS analysis revealed the presence of two series of tannin oligomers. Supporting the observations from NMR spectroscopy, the first series consists of well-resolved tannin identified as procyanidin polymers units with chain lengths of up to 25. A second series of monogalloyl flavan-3-ols polymers with polymerization degree up to 25 were also detected. This is the first mass spectrometric evidence confirming the existence of galloylated procyanidin oligomers in pear fruits. Within each of these oligomers, various signals exist suggesting the presence of several oligomeric tannins. The antioxidant properties of the polymeric fraction were investigated through reduction of the DPPH free radical, and the results obtained showed that the polymeric fraction exhibited a higher antioxidant power compared to those of (+)-catechin and B3 procyanidin dimer.     

Tissue-specific and developmental modifications of grape cell walls influence the adsorption of proanthocyanidins

Cell wall material from Vitis vinifera L. cv. Cabernet Sauvignon grape skin and flesh was isolated at different stages of grape maturity to determine whether developmental changes in cell wall composition in different tissue types influence the binding of proanthocyanidins (PAs). Trends in cell wall adsorption of, and selectivity for, PAs were determined using two skin PAs that differed in their average molecular masses. Flesh cell walls consistently bound a higher amount of PA than those from skin. Key structural differences that reduced PA adsorption in skin cell walls by comparison with flesh cell walls were endogenously higher concentrations of insoluble PA, Klason lignin, and lower cell wall-bound protein. These differences may confer reduced flexibility and porosity of skin cell walls relative to flesh cell walls. Analysis of skin and flesh cell wall properties revealed that the onset of ripening was associated with a loss of type I arabinogalactan and galacturonic acid, which indicated a degradation of pectin within the cell wall. Flesh cell walls consistently bound PAs of larger molecular mass, and changes in PA adsorption properties after the onset of ripening were minor. For skin cell walls, adsorption of PA was lowest immediately following solubilization of galacturonic acid, and high molecular mass PAs were poorly bound. As ripening progressed, PAs of higher molecular mass were selectively adsorbed by skin cell walls, which indicates that ongoing cell wall remodeling during ripening may confer an increased porosity within the skin cell wall matrix, resulting in a greater adsorption of PA within a permeable structure.     

Phenolic compositions of grapes and wines from cultivar cabernet sauvignon produced in chile and their relationship to commercial value

The phenolic composition of wine depends on, among other factors, the grapes used to make it. In this sense, knowledge of the chemical composition of grapes and its association with the resulting wines is an important tool to determine if there is a relationship between the phenolic composition of grapes and the price that these wines obtain in the market. For this purpose, grape skins and seeds from the cultivar Cabernet Sauvignon from the central region of Chile, in 2009 and 2010 vintages from two ripening points, were subjected to chemical and phenolic analyses, as were the wines made from these grapes. Grapes and the corresponding wines from three retail price wine categories, U.S. $6-8, U.S. $28-30, and U.S. $150-160, were evaluated. No differences were found across the price categories in the chemical analysis of grapes. Berry skins and wines from the higher price categories presented a higher concentration only of total tannins, and the differences in their concentrations were only among the different fractions of proanthocyanidins in the skins, seeds, and wines; there were no differences in their proportions. A seasonal effect influenced the concentrations of certain compounds in grapes and led to a decrease in the concentration of total phenols, total tannins, and total anthocyanins between sampling dates as harvesting moved toward the common commercial grape harvest in Chilean viticulture.     

Relationship between red wine grade and phenolics. 2. Tannin composition and size

Commercial red wines ( Vitis vinifera L. cv. Shiraz) produced during the 2009 vintage underwent winemaker assessment for allocation grade soon after production. The wines were then subjected to phenolic analysis to measure wine color (total anthocyanin, SO(2) nonbleachable pigment, and wine color density) and tannins (concentration, composition, and average degree of polymerization). A positive relationship was found between wine phenolic concentration and projected bottle price. Tannin compositional analysis suggested that there was specifically a relationship between wine grade and skin-derived tannins. These results suggest that maximization of skin tannin concentration and/or proportion is related to an increase in projected wine bottle price.     

Toward the authentication of wines of Nemea denomination of origin through cleaved amplified polymorphic sequence (CAPS)-based assay

In the present study, we developed a cleaved amplified polymorphic sequence (CAPS)-based assay as a first attempt to detect fraud in grapevine musts with a long-term objective to establish an analytical methodology to authenticate wines of Nemea denomination of origin (Agiorgitiko). The analytical assay makes use of a single nucleotide polymorphism that discriminates Agiorgitiko and Cabernet Sauvignon varieties. The latter grape variety is one of the major adulterants for Nemea wines. Agiorgitiko grapevine must was spiked with Cabernet Sauvignon in several ratios (v/v) from 50 down to 10%, and the subsequent mixes were subjected to alcoholic microfermentation. DNA was extracted from all mixture samples up to the end of the fermentation process and was subjected to the CAPS assay. Both standard agarose gel and lab-on-a-chip capillary electrophoresis illustrated the ability of the method to detect the presence of Cabernet Sauvignon down to 10% throughout the whole fermentation process.     

Study of wine tannin oligomers by on-line liquid chromatography electrospray ionization mass spectrometry.

Thiolysis of a wine tannin fraction yielded trihydroxylated flavanol units (as previously observed in grape skins) in addition to the well-known procyanidins (dihydroxylated units), usually described in the literature for grape condensed tannins. To determine how they occur in condensed tannins, the wine fraction was analyzed by liquid chromatography coupled to electrospray ionization mass spectrometry. Thus, various series of ion peaks containing a variable number of trihydroxylated units were detected as monocharged ions from dimers up to pentamers. From pentamers, oligomers were found as doubly charged ions. Heptamer species corresponded to the highest mass detected. These results showed that wine condensed tannins consist of, besides procyanidins, mixed tri- and dihydroxylated flavanol units and also of pure trihydroxylated flavanol units. These new data should be taken into account to interpret organoleptic properties of wines.