Cristina Alcalde-Eon

Development of a fractionation method for the detection and identification of oak ellagitannins in red wines.

During maturation and ageing in oak barrels wines improve their organoleptic properties. Ellagitannins can be released from wood to the wine and be involved in oxidation reactions and seem to influence the astringency and colour properties of the wine. Nevertheless, the ellagitannins levels are lower than those of other wine constituents and, consequently, they are not easily detected. This study has developed a two-step fractionation method consisting of a solid phase extraction in C-18 Sep-Pak cartridges followed by size exclusion chromatography in hand-packed Sephadex LH-20 minicolumn for the detection of oak ellagitannins in different types of wines. An HPLC method has also been developed which allows the separation of compounds with the same m/z ratios, facilitating the ellagitannin identification by means of the mass spectrometric analyses. The main oak ellagitannins (grandinin, vescalagin, roburin E and castalagin) were isolated, detected separately and identified in a spiked wine and in three real ones, proving the usefulness of the fractionation method.

Validation of a mass spectrometry method to quantify oak ellagitannins in wine samples.

Detection and individual quantification of oak wood ellagitannins in oak barrel aged red wine samples are difficult mainly due to their low levels and the similarity between their structures. In this work, a quantification method using mass spectrometry has been developed and validated to quantify wine ellagitannins after sample fractionation with a previously reported method. The use of an internal standard is a requirement to correct mass signal variability. (-)-Gallocatechin, among the different tested compounds, was the only one that proved to be a suitable internal standard making possible the accurate and individual quantification of the main oak wood ellagitannins. The developed methodology has been used to detect and quantify these ellagitannins in different Spanish commercial wines, proving its usefulness.

Color stabilization of red wines. A chemical and colloidal approach.

The effects of cold treatment and time on CIELAB color parameters and on anthocyanin and anthocyanin-derived pigments composition have been evaluated as has been the effectiveness of either an enological tannin or a mannoprotein (M) on their stabilization. With respect to color, hue (hab) was increased in the wines treated with both enological products. Furthermore, the color changes induced by cold treatment were lessened by the addition of these two enological products, although the protective effect was higher for the wines treated with M. The pigment analysis revealed higher percentages of anthocyanin-derived pigments in tannin and M-treated samples (in both cold treated and not) in relation to control ones. The addition of the enological tannin may favor the synthesis of anthocyanin-derived pigments, which are chemically more stable than native anthocyanins, whereas M seems to stabilize anthocyanin-derived pigments from a colloidal point of view, avoiding their aggregation and further precipitation.

Relationship between Agronomic Parameters, Phenolic Composition of Grape Skin, and Texture Properties of Vitis vinifera L. cv. Tempranillo.

The relationship between the agronomic parameters of grapevine and the phenolic composition of skin of Vitis vinifera L. cv. Tempranillo grapes was assessed. The physical and mechanical properties of berries and their skins were also determined and correlated to the chemical composition. Results showed a significant negative correlation between grapevine vigor-related parameters (such as leaf area and bunch weight) and anthocyanin composition, whereas the percentage (w/w) of seeds was negatively correlated with the amount of flavanols of grape skins. Texture properties of grape skins also showed an important relationship with chemical composition. Berry hardness showed a negative correlation with the coumaroyl-anthocyanin derivatives, but it was positively correlated to skin flavanic composition. Moreover, significant regressions with high coefficients of determination were found between phenolic composition and grapevine vigor-related and texture variables, thus pointing out that these parameters might be useful for estimating the phenolic composition of grape skins.

Hemisynthesis and Structural and Chromatic Characterization of Delphinidin 3-O-Glucoside–Vescalagin Hybrid Pigments

During red wine maturation in the presence of oak wood, reactions involving anthocyanins and ellagitannins might affect wine organoleptic properties such as color and astringency. In this work, the condensation reaction between myrtillin (delphinidin 3-O-glucoside) and vescalagin has been performed to determine the behavior of this anthocyanin in this kind of reaction and to assess the possible impact of such a reaction in wine color modulation. Two different hybrid pigments have been hemisynthetized and characterized by HPLC-DAD-MS and NMR spectroscopy. These pigments have been identified as 1-deoxyvescalagin-(1β→8)-myrtillin (major) and 1-deoxyvescalagin-(1β→6)-myrtillin (minor). The minor pigment could be formed both by the condensation reaction and by a regioisomerization process from the major pigment. Moreover, the chromatic properties of these pigments have been studied and compared to those of myrtillin. The hybrid pigments showed an important bathochromic shift (ca. 20 nm) in the maximum absorbance wavelength and lower molar absorption coefficients.

Schizanthus grahamii and Schizanthus hookeri. Is there any relationship between their anthocyanin compositions and their different pollination syndromes

Three different pollination syndromes (bee, hummingbird and moth-pollination syndromes) have been described for the different species of the genus Schizanthus Ruiz & Pav. (Solanaceae). Two closely related species from a phylogenetic point of view, Schizanthus grahamii and Schizanthus hookeri, show hummingbird and bee-pollination syndromes, respectively. One of the traits used to assign these pollination syndromes is the colour of the petals, which is red in S. grahamii and bluish-pink in S. hookeri. The objective of this work was to establish the differences in the anthocyanin composition of these two Schizanthus species that contribute to the different pollination syndrome. Identification of the pigments was carried out from the chromatographic and spectral data supplied by the HPLC-DAD-MS(n) analyses of the samples. Alkaline and acid hydrolyses were also performed in the extracts and in some isolated compounds in order to confirm the identities. First difference between these two species was found in the total anthocyanin content, which was about 13-fold higher in S. grahamii than in S. hookeri. Furthermore, the major peak was also different in both cases, thus explaining quantitative and qualitative colour differences between species. Delphinidin 3-O-rutinoside represented ca. 72% of the total area in S. grahamii and petanin (Petunidin 3-O-p-coumaroylrutinoside-5-O-glucoside) accounted for almost 62% of the total area in S. hookeri. The presence of the p-coumaroylrutinosyl moiety in the petanin makes the intramolecular copigmentation possible, thus conferring the bluish-pink hue to the petals of S. hookeri. Delphinidin 3-O-rutinoside is in turn responsible for the red colour exhibited by the petals of S. grahamii and along with other floral traits, responsible for the attraction of hummingbirds to the plant. Pigments structurally related to petanin, which have been often detected in other genus of Solanaceae, were detected in both species and in similar contents, supporting results from previous studies that pointed to a bee-pollinated common ancestor for both species that further specialised acquiring traits attractive to hummingbirds and among them, red colour of the petals which is mainly supplied by delphinidin 3-O-rutinoside.

Flavanol Quantification of Grapes via Multiple Reaction Monitoring Mass Spectrometry. Application to Differentiation among Clones of Vitis vinifera L. cv. Rufete Grapes

The determination of the detailed flavanol composition in food matrices is not a simple task because of the structural similarities of monomers and, consequently, oligomers and polymers. The aim of this study was the development and validation of an HPLC-MS/MS-multiple reaction monitoring (MRM) method that would allow the accurate and precise quantification of catechins, gallocatechins, and oligomeric proanthocyanidins. The high correlation coefficients of the calibration curves (>0.993), the recoveries not statistically different from 100%, the good intra- and interday precisions (<5%), and the LOD and LOQ values, low enough to quantify flavanols in grapes, are good results from the method validation procedure. Its usefulness has also been tested by determining the detailed composition of Vitis vinifera L. cv. Rufete grapes. Seventy-two (38 nongalloylated and 34 galloylated) and 53 (24 procyanidins and 29 prodelphinidins) flavanols have been identified and quantified in grape seed and grape skin, respectively. The use of HCA and PCA on the detailed flavanol composition has allowed differentiation among Rufete clones.

Enological Tannin Effect on Red Wine Color and Pigment Composition and Relevance of the Yeast Fermentation Products

Enological tannins are widely used in the winemaking process either to improve different wine characteristics (color stability, among others) or to compensate for low tannin levels. In this work, the influence of the addition of two different enological tannins, mainly composed of hydrolysable (ellagitannins) and condensed tannins, on the evolution of color and pigment composition of two different types of model systems containing the five main grape anthocyanins was studied. In addition, the effect of the addition of an enological tannin on the color and pigment composition of red wines made from Vitis vinifera L. cv Tempranillo grapes was also studied by high-performance liquid chromatography with diode array detection coupled to mass spectrometry (HPLC-DAD-MS). Results showed that, in model systems, the addition of the enological tannin favored the formation of anthocyanin-derived pigments, such as A-type and B-type vitisins and flavanol-anthocyanin condensation products, provided that the yeast precursors were previously supplied. Moreover, model systems containing the enological tannins were darker and showed higher values of chroma at the end of the study than control ones. The higher formation of these anthocyanin-derived pigments was also observed in the red wines containing the enological tannin. Moreover, these wine also showed lower lightness (L*) values and higher chroma (C*ab) values than control wines, indicating a higher stabilization of color.

Effect of the type of oak barrels employed during ageing on the ellagitannin profile of wines

Background and Aims Ellagitannins released from wood to wine during barrel ageing can affect wine colour and astringency. This work aimed to examine the influence that oak wood type (French or American) and age (first-fill or second-fill) of barrels has on the concentration of individual ellagitannins and their evolution during winemaking and ageing. Methods and Results The ellagitannin composition of red wines during ageing was determined by HPLC-MS/MS-multiple reaction monitoring. Differences in the extractability and stability of the individual ellagitannins were observed. Moreover, the ellagitannin composition of wines was determined by the type of barrel. Wines aged in new French oak barrels showed a concentration of ellagitannin about twofold higher than that aged in American or in second-fill French ones. The vescalagin/castalagin ratio was higher in wines aged in French oak barrels than that in wines aged in American barrels. Wine samples were grouped by discriminant analysis into three groups depending on the type of barrel employed during ageing. Conclusions The ellagitannin profile of aged red wines is strongly influenced by the type of oak barrel employed. Wines aged in new French oak barrels can be differentiated from those aged in second-fill French or in new American oak barrels by the ellagitannin concentration, whereas the vescalagin/castalagin ratio may assist in tracing the oak type (French or American) of the barrels. Significance of the Study The ellagitannin concentration along with the vescalagin/castalagin ratio in red wines aged in oak barrels can be employed for tracing the oak type of the barrels.