In search for flavonoid and colorimetric varietal markers of Vitis vinifera L. cv Rufete wines.

The better adaptation of autochthonous grape varieties to environmental changes is increasing the interest on them. Previous studies on Vitis vinifera L. cv Rufete, the flagship of DOP "Sierra de Salamanca", demonstrated its phenolic potential to produce quality wines. The present study aims at discovering flavonoid (HPLC-DAD-MSn) and colorimetric (CIELAB) varietal markers in commercial Rufete wines (RW) that could be employed to discriminate them from those made with other varieties or mixtures. Compared to Tempranillo wines (TW), RW showed lower phenolic contents, with lower proportions of anthocyanins and flavonols but greater of flavanols. Principal Component Analysis applied to flavonoid and chromatic variables allowed varietal separation of the samples along PC2, which was mostly driven by %total prodelphinidins, %oligomeric procyanidins and C ab * (greater in TW) and by %monomeric procyanindins and L* (greater in RW) and, additionally, by the proportions of A-type vitisins, acetaldehyde-related pigments, quercetin 3-O-glucuronide and Quercetin/Myricetin ratio (all greater in RW). The results of the present study show that the typicity of Rufete grapes is quite preserved in the wines made with up to 95% of Rufete and 5% of Tempranillo grapes or wines and independently of the enological practices of the different wineries.

Role of Oak Ellagitannins in the Synthesis of Vitisin A and in the Degradation of Malvidin 3-O-Glucoside: An Approach in Wine-Like Model Systems.

Recent studies highlight the influence that oak ellagitannins can have on wine astringency and color. Direct reactions between flavanols or anthocyanins with vescalagin have been reported to occur, but participation of these compounds in the formation of other types of derivatives has only been suggested but not demonstrated. This study aims at evaluating, in wine-like model systems, the possible different roles of the main oak ellagitannins, castalagin and vescalagin, alone or combined, in the synthesis of vitisin A and in the degradation of malvidin 3-O-glucoside. In the presence of pyruvic acid, the anthocyanin disappeared mainly as a result of the synthesis of vitisin A, whereas in its absence, degradation reactions prevailed. In general, ellagitannins increased the synthesis of vitisin A, decreased the total content of degradation products, and changed the degradation profile, with differences observed between castalagin and vescalagin. The results of the study revealed that the fate of malvidin 3-O-glucoside is conditioned by the presence of ellagitannins.

Effects of different industrial processes on the phenolic composition of white and brown teff (Eragrostis tef (Zucc.) Trotter).

Teff is currently being incorporated into a range of foodstuffs, especially gluten-free flakes and extruded products. The main objective of this work was to assess the effects of different treatments (industrial milling, formation of flakes and extrusion) on the phenolic composition of two types of teff grains differing in their colour (white and brown). Fifty-nine phenolic compounds were detected by HPLC-DAD-MSn. C-glycosyl flavones accounted for more than 90% of the total phenolic contents in both teff types. White teff mostly contained apigenin-derived flavones (86-92%), whereas luteolin derivatives prevailed in brown teff (91-94%). The industrial processes, mainly flaking and extrusion, caused marked changes in the phenolic composition, some of which were dependent on the teff type. In both teff types, processing changed the phenolic profiles similarly by increasing C-monoglycosyl flavones and decreasing acylated derivatives. However, the total content was increased in flakes and extruded products made only from brown teff grains.

Monitoring the effects and side-effects on wine colour and flavonoid composition of the combined post-fermentative additions of seeds and mannoproteins.

One of the main consequences of the advancement of harvest date associated to global climate change is that the phenolic maturity of grapes can be delayed in relation to their technological maturity. As a consequence, wines made from these grapes can be poor in phenolic compounds or possess an unbalanced phenolic composition, affecting their global quality. The combined post-fermentative addition of seeds and an astringency-modulator mannoprotein (MP) might be a potential strategy to solve this problem, since seeds might supply flavanols and improve wine chemical stability and the mannoprotein might modulate the changes induced in astringency by the addition of seeds and improve wine colloidal stability. The present study aimed at monitoring at different moments of winemaking and ageing the effects and side-effects of this combined strategy on the detailed flavanol, flavonol and anthocyanin compositions and on colour of wines made from Syrah grapes. Seeds were obtained from Pedro Ximénez overripe grapes. Flavanol composition and flavonol and anthocyanin compositions were determined by HPLC-MSn-MRM and HPLC-DAD-MSn analyses, respectively. Colour changes caused by these additions were studied from CIELAB parameters as well as the ability of these techniques to protect colour from bleaching agents, such as SO2. In general, the addition of seeds initially increased the levels of flavanols and anthocyanins. However, during bottle ageing a reduction in the levels of flavanols, flavonols and anthocyanins could be observed in seed treated wines, which might be related to the greater formation of flavanol aggregates associated with greater levels of flavanols. This effect was partially solved for most of the flavonoids studied with the additional MP treatment. Treated and control wines showed colour differences that were visible to human eye, although they were reduced over time. At the end of the study, treated wines showed colour parameters corresponding to younger wines than those observed in control wines. A greater resistance against SO2 bleaching was also observed in treated wines, which can be mainly associated to the greater percentages of polymeric pigments caused by seed treatment and to the improvement of the colloidal stability of SO2-resistant pigments caused by the addition of the MP.

Stability of petal color polymorphism: the significance of anthocyanin accumulation in photosynthetic tissues.

BACKGROUND: Anthocyanins are the primary source of colour in flowers and also accumulate in vegetative tissues, where they have multiple protective roles traditionally attributed to early compounds of the metabolic pathway (flavonols, flavones, etc.). Petal-specific loss of anthocyanins in petals allows plants to escape from the negative pleiotropic effects of flavonoid and anthocyanins loss in vegetative organs, where they perform a plethora of essential functions. Herein, we investigate the degree of pleiotropy at the biochemical scale in a pink-white flower colour polymorphism in the shore campion, Silene littorea. We report the frequencies of pink and white individuals across 21 populations and underlying biochemical profiles of three flower colour variants: anthocyanins present in all tissues (pink petals), petal-specific loss of anthocyanins (white petals), and loss of anthocyanins in all tissues (white petals). RESULTS: Individuals lacking anthocyanins only in petals represent a stable polymorphism in two populations at the northern edge of the species range (mean frequency 8-21%). Whereas, individuals lacking anthocyanins in the whole plant were found across the species range, yet always at very low frequencies (< 1%). Biochemically, the flavonoids detected were anthocyanins and flavones; in pigmented individuals, concentrations of flavones were 14-56× higher than anthocyanins across tissues with differences of > 100× detected in leaves. Loss of anthocyanin pigmentation, either in petals or in the whole plant, does not influence the ability of these phenotypes to synthesize flavones, and this pattern was congruent among all sampled populations. CONCLUSIONS: We found that all colour variants showed similar flavone profiles, either in petals or in the whole plant, and only the flower colour variant with anthocyanins in photosynthetic tissues persists as a stable flower colour polymorphism. These findings suggest that anthocyanins in photosynthetic tissues, not flavonoid intermediates, are the targets of non-pollinator mediated selection.

Addition of Mannoproteins and/or Seeds during Winemaking and Their Effects on Pigment Composition and Color Stability.

The increase of temperature can cause a decoupling of sugar and anthocyanin accumulation in grapes, leading to wines poor in color. Different enological techniques are nowadays under study to overcome this problem. Among them, the present study has evaluated the color and pigment composition modifications caused by the addition during Syrah winemaking of either Pedro Ximénez seeds (intended to increase chemical stability) or/and two different mannoproteins (color-protective and astringency-modulator) to increase colloidal stability. Color and pigment composition modifications were assessed from CIELAB color parameters and from HPLC-DAD-MS n results before and after cold-treatment (used to force colloidal instability). The addition of both seeds and mannoproteins increased color stability against cold and, additionally, against SO2-bleaching in the case of mannoproteins. However, the initial pigment composition and color of the samples were differently affected by these additions, being clearly affected (Δ E* ab > 3) in the cases of seeds and with the astringency-modulator mannoprotein and hardly modified with the other one.

Effect of the presence of different oak ellagitannins in their own disappearance under oxidative or inert atmosphere.

The disappearance of the C-glycosidic ellagitannins over time can occur even in absence of oxygen and their disappearance rate seems to be affected by the presence of other ellagitannins in the media. The objective of this work was to study the influence of the presence of other ellagitannins and/or oxygen on the individual evolution of the main oak ellagitannins in simple model systems in order to understand their behaviours in more complex media, such as wine. In all the studied conditions, vescalagin disappeared faster than castalagin, highlighting its greater reactivity. Oxygen increased the individual disappearance rate, as also occurred when more than one type of ellagitannin was present, above all if the additional ellagitannin(s) contained the same conformation in C1 as vescalagin. Experimental data were fitted to a kinetic model considering both the oxygen-dependent and oxygen-independent reactions, making possible the comparison between individual compounds in different scenarios.

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.

An Approach to the Study of the Interactions between Ellagitannins and Oxygen during Oak Wood Aging.

During the aging of red wine in oak wood barrels, or in alternative aging systems, interactions between the compounds released from wood, the compounds of the wine, and oxygen can take place. The main objective of the present work was to study oxygen-ellagitannin interactions by monitoring their levels in three model systems, all containing the same amounts of French oak chips and differing only in the oxygen content: total absence, only the oxygen released from the chips, and air-saturated (model systems F, OW, and OS, respectively). This study has highlighted the influence of oxygen in the ellagitannins' evolution and the relevance of the oxygen trapped into the oak chips, reporting for the first time the kinetics of oxygen release to the model wine. Furthermore, the indirect contribution of oxygen to the ellagitannins' disappearance by boosting autoxidative reactions has also been pointed out. Vescalagin seems to be the ellagitannin most affected by the initial oxygen levels.

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.

Anthocyanins of the anthers as chemotaxonomic markers in the genus Populus L.. Differentiation between Populus nigra, Populus alba and Populus tremula.

Three main species of Popululs L. (Salicaceae) have been reported to occur in the Iberian Peninsula: Populus nigra L., Populus alba L. and Populus tremula L. The degree of pilosity of the bracts of the male catkins is a key character for their differentiation. The anthers of these poplar species possess anthocyanins that provide them a red colouration. Since these poplars are wind-pollinated and, consequently, do not need to attract pollinators, anthocyanins in the anthers might be acting as photoprotectors, shielding pollen grains from excessive sunlight. In order to verify this hypothesis, the first objective of this study was to establish if there is any relationship between the degree of pilosity of the bracts (related to the physical shading of the pollen grains) and the levels and types of anthocyanins in the anthers of these three species. This study also aimed to check the usefulness of the anthocyanins of the anthers as chemotaxonomic markers, through the study of the differences in the anthocyanin composition between these poplar species. Anthocyanins were identified from the data supplied by HPLC-DAD-MS(n) analyses. Seventeen different compounds, including mono-, di- and triglycosides and anthocyanin-derived pigments (F-A(+) dimers) have been identified. Cyanidin 3-O-glucoside was the major compound in all the samples (>60% of the total content), which may be in accordance with the photoprotective role proposed for them. However, qualitative and quantitative differences were detected among samples. Cyanidin and delphinidin 3-O-sambubiosides have been detected only in the anthers of P. tremula as well as cyanidin 3-O-(2″-O-xyloxyl)rutinoside, making them valuable chemotaxonomic markers for this species. Hierarchical Cluster and Principal Components Analyses (HCA and PCA) carried out with the anthocyanin percent composition data have allowed a separation of the samples that is in accordance with the initial classification of the samples made from the morphological characters of the specimens. Furthermore, these analyses have revealed intraspecific differences among samples that point out to different clones or varieties of a same species.

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.

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.

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-(1ß→8)-myrtillin (major) and 1-deoxyvescalagin-(1ß→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.

Thermodynamic and kinetic properties of a new myrtillin-vescalagin hybrid pigment.

During red wine maturation in contact with oak wood, C-glucosidic ellagitannins can react with anthocyanins, leading to new pigments. In this work the thermodynamic and kinetic constants of the network pH-dependent equilibrium of a new myrtillin (delphinidin 3-O-glucoside)-vescalagin hybrid pigment (1-deoxyvescalagin-(1ß→8)-myrtillin) have been determined by UV-visible absorption and stopped-flow experiments and compared to those determined for myrtillin. The vescalagin substitution at the C-8' center of myrtillin entails important variations in the pigment behavior upon pH changes. The hybrid pigment showed lower pK'a and pKa values and a much higher value of Kt. As a consequence, at moderately acidic pH values (4 < pH < 6), the percentage of the hemiketal is much lower and the quinoidal base and the (E)-chalcone represent higher percentages relative to those for myrtillin. Therefore, the hybrid pigment can provide in slightly acidic or neutral solutions an exceptionally different color compared to that of myrtillin.

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.

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.

Identification of dimeric anthocyanins and new oligomeric pigments in red wine by means of HPLC-DAD-ESI/MSn.

High-pressure liquid chromatography-diode array detector-electrospray ionisation/ion trap mass spectrometry (HPLC-DAD-ESI/MS(n)) analyses carried out in red wine fractions revealed the existence of dimeric anthocyanins (A-A(+)), previously detected in grape skin, and allowed the detection and identification, for the first time, of other derived oligomers. The structure of these compounds was characterised according to their MS(n)(n = 1-4) fragmentation patterns. The newly detected oligomers consisted of a flavanol, (epi)catechin or (epi)gallocatechin, linked through its C(4) position to the nucleophilic positions of the upper unit of a dimeric anthocyanin (F-A-A(+)). All the compounds contained malvidin as one of the anthocyanin subunits, whereas the other anthocyanin moiety could be either delphinidin, cyanidin, petunidin, peonidin or malvidin. With the fractionation method employed, the dimeric anthocyanins eluted in the same fractions as the monomeric anthocyanins. However, the new F-A-A(+) oligomers were found in the same fractions as F-A(+) dimers, which might indicate a structural similarity between both types of compounds. ESI/MS(n) analyses, coupled or not to HPLC, showed to be a useful and powerful tool for detecting and identifying these oligomers in wines, which usually elute from reversed-phase columns as humps and/or are overlapped by the peaks of other compounds. The detection of these oligomeric pigments in wine has provided more information about the complex pigments responsible for the elevation of the base line observed in the chromatograms of wines and has also revealed that oligomeric anthocyanins can take part in the reactions usually undergone by monomeric anthocyanins.

Aging effect on the pigment composition and color of Vitis vinifera L. Cv. Tannat wines. Contribution of the main pigment families to wine color.

Red wines made from Vitis vinifera L. cv. Tannat grapes are known to possess high contents of tannins and intense color, features that are responsible for the originality of these wines. This work aimed to study the evolution of the pigment composition and CIELAB color parameters as Tannat wines become older, as well as to establish the contribution to wine color of the main pigment families. Tannat wines produced in Uruguay from grapes of the same vineyard in six consecutive vintages (1998-2003) and Tannat grapes of the 2003 harvest were analyzed by means of HPLC-DAD-MS and UV-vis spectrometric techniques. The correlations between the different pigment families and the CIELAB parameters revealed the importance of the variations of the percentage, found in anthocyanins and flavanol-anthocyanin acetaldehyde-mediated condensation products (decrease) and pyranoanthocyanins and direct condensation products (increase), in the modification of the color from purple-red hues to more orange-red ones. The color suffered qualitative rather than quantitative changes, that is, the hue (h*ab) increased, whereas the chroma (C*ab) and lightness (L) did not show a defined trend with time.

Anthocyanins in berries of Maqui (Aristotelia chilensis (Mol.) Stuntz).

The anthocyanin composition of berries of Maqui [Aristotelia chilensis (Mol.) Stuntz] was determined by HPLC with photodiode array and MS detection. Eight pigments corresponding to the 3-glucosides, 3,5-diglucosides, 3-sambubiosides and 3-sambubioside-5-glucosides of delphinidin and cyanidin were identified, the principal anthocyanin being delphinidin 3-sambubioside-5-glucoside (34% of total anthocyanins). The average total anthocyanin content was 137.6 +/- 0.4mg/100g of fresh fruit (211.9 +/- 0.6 mg/100g of dry fruit). The relative high anthocyanin content and the important presence of polar polyglycosylated derivatives makes the fruits of A. chilensis an interesting source of anthocyanin extracts for food and pharmaceutical uses.